Patent application title: Materials and Methods for Producing Alkaloids
Inventors:
IPC8 Class: AC12P1718FI
USPC Class:
1 1
Class name:
Publication date: 2021-06-03
Patent application number: 20210164006
Abstract:
The invention generally relates to methods of producing loline alkaloids
or precursors thereof, expression constructs, and host cells useful for
producing loline alkaloids or precursors thereof, and methods for
producing loline alkaloids or precursors thereof in a host cell.Claims:
20. A host cell able to produce more of at least one loline alkaloid or
precursor thereof, than does a control cell, as a result of the host cell
being transformed or modified to comprise at least one polynucleotide
selected from the group consisting of: i) a polynucleotide encoding a
polypeptide comprising the sequence of any one of SEQ ID NO:1, 12, 13 and
61 or a variant thereof with at least 40% identity to any one of SEQ ID
NO:1, 12, 13 and 61 with at least one of activity of a gamma-class PLP
enzyme and an activity substantially equivalent to that of a lolC gene
product, ii) a polynucleotide encoding a polypeptide comprising the
sequence of any one of SEQ ID NO:2, 14, 15 and 62 or a variant thereof
with at least 40% identity to any one of SEQ ID NO:2, 14, 15 and 62 with
at least one of activity of an alpha-class PLP enzyme and activity
substantially equivalent to that of the lolD gene product, iii) a
polynucleotide encoding a polypeptide comprising the sequence of any one
of SEQ ID NO:3, 16, 17 and 63 or a variant thereof with at least 40%
identity to of any one of SEQ ID NO:3, 16, 17 and 63 with at least one of
monooxygenase activity and activity substantially equivalent to that of
the lolF gene product, iv) a polynucleotide encoding a polypeptide
comprising the sequence of any one of SEQ ID NO:4, 18 and 19 or a variant
thereof with at least 40% identity of any one of SEQ ID NO:4, 18 and 19
with at least one of amino acid bridging activity and activity
substantially equivalent to that of the lolA gene product, v) a
polynucleotide encoding a polypeptide comprising the sequence of any one
of SEQ ID NO:5, 20, 21 and 64 or a variant thereof with at least 40%
identity to of any one of SEQ ID NO:5, 20, 21 and 64 with at least one of
activity of an alpha-class PLP enzyme and activity substantially
equivalent to that of the lolT gene product, vi) a polynucleotide
encoding a polypeptide comprising the sequence of of any one of SEQ ID
NO:6, 22, 23 and 65 or a variant thereof with at least 40% identity to of
any one of SEQ ID NO:6, 22, 23 and 65 with at least one of activity of a
non-heme iron dioxygenase and activity substantially equivalent to that
of the lolE gene product, vii) a polynucleotide encoding a polypeptide
comprising the sequence of any one of SEQ ID NO:7, 24, 25 and 66 or a
variant thereof with at least 40% identity to any one of SEQ ID NO:7, 24,
25 and 66 with at least one of activity of a non-heme iron dioxygenase
and activity substantially equivalent to that of the lolO gene product,
viii) a polynucleotide encoding a polypeptide comprising the sequence of
any one of SEQ ID NO:8, 26 and 27 or a variant thereof with at least 40%
identity to any one of SEQ ID NO:8, 26 and 27 with activity substantially
equivalent to that of the lolU gene product, ix) a polynucleotide
encoding a polypeptide comprising the sequence of SEQ ID NO:9 or 28 or a
variant thereof with at least 40% identity to SEQ ID NO:9 or 28 with at
least one of N-Methyltransferase activity and activity substantially
equivalent to that of the lolM gene product, x) a polynucleotide encoding
a polypeptide comprising the sequence of any one of SEQ ID NO:10, 29 and
67 or a variant thereof with at least 40% identity to any one of SEQ ID
NO:10, 29 and 67 with at least one of acetamidase activity and activity
substantially equivalent to that of the lolN gene product, and xi) a
polynucleotide encoding a polypeptide comprising the sequence of SEQ ID
NO:11 or 30 or a variant thereof with at least 40% identity to SEQ ID
NO:11 or 30 with at least one of cytochrome P450 monooxygenase activity
and activity substantially equivalent to that of the lolP gene product.
21. The host cell of claim 20 wherein the host cell is able to produce more of at least one loline alkaloid, than does a control cell, as a result of the host cell being transformed or modified to comprise the at least one polynucleotide.
22. The host cell of claim 20 wherein the host cell is tolerant of endogenous (3-amino-3-carboxypropyl) proline (ACPP) production.
23. The host cell of claim 22 wherein the host cell has been pre-selected for tolerance to cellular ACPP.
24. The host cell of claim 20 wherein the host cell prior to modification or transformation, is able to convert exo-1-aminopyrrolizidine (1-AP) to exo-1-acetamido-pyrrolizidine (AcAP).
25. The host cell of claim 24 wherein the host cell prior to modification or transformation, has been pre-selected for the ability to convert 1-AP to AcAP.
26. The host cell of claim 20 wherein the host cell is transformed or modified to comprise at least the polynucleotides of i), ii), iii), v) and vii).
27. The host cell of claim 20 wherein the host cell is not transformed or modified to comprise the polynucleotide of vi).
28. The host cell of claim 20 wherein the host cell is not transformed or modified to comprise the polynucleotide of viii).
29. A method for producing a host cell that produces at least one loline alkaloid or precursor thereof, the method comprising modifying or transforming a host cell to comprise at least one polynucleotide as defined in claim 20.
30. The method of claim 29 wherein the host cell produces more of at least one loline alkaloid, than does a control cell, as a result of the host cell being transformed or modified to comprise the at least one polynucleotide.
31. The method of claim 29 wherein the host cell is tolerant of endogenous (3-amino-3-carboxypropyl) proline (ACPP) production.
32. The method of claim 29 further comprising the step of pre-selecting the host cell for tolerance to cellular ACPP.
33. The method of claim 29 wherein the host cell prior to modification or transformation, is able to convert exo-1-aminopyrrolizidine (1-AP) to exo-1-acetamido-pyrrolizidine (AcAP).
34. The method of claim 29 wherein the host cell prior to modification or transformation, has been pre-selected for the ability to convert 1-AP to AcAP.
35. The method of claim 29 further comprising the step of pre-selecting the host cell for said ability to convert 1-AP to AcAP.
36. The method of claim 29 wherein the host cell is transformed or modified to comprise at least the polynucleotides of i), ii), iii), v) and vii).
37. A method for producing at least one loline alkaloid or a precursor thereof, the method comprising culturing the host cells of claim 1 under conditions conducive to the production of the at least one loline alkaloid or precursor thereof, by the host cells.
38. The method of claim 37 wherein the host cell produces at least one loline alkaloid.
Description:
FIELD OF THE INVENTION
[0001] The present invention relates to materials and methods of producing loline alkaloids or precursors thereof.
BACKGROUND TO THE INVENTION
[0002] Loline alkaloids are produced symbiotically during infection of grasses by Epichloe species fungal endophytes. These endophytes are considered to be bioprotective, conferring pest, and possibly drought and disease protection to the symbionts of which they form part.
[0003] Lolines are potent broad spectrum insecticidal alkaloids with no observed toxicity to animals. These fungal secondary metabolites are major contributors to the bioprotective pest tolerance conferred on cool season grasses by Epichloe endopytes.
[0004] A robust and/or scalable method for preparing loline alkaloids in the absence of a symbiotic relationship is not presently available.
[0005] There is a need for a method to produce loline alkaloids in fungi that do not natively produce lolines in order to extract and use lolines as a natural pesticide.
[0006] It is an object of the present invention to provide improved materials and methods for producing loline alkaloids or precursors thereof, and/or at least provide the public with a useful choice.
SUMMARY OF THE INVENTION
Host Cell
[0007] In one aspect, the invention provides a host cell modified or transformed to comprise at least one polynucleotide selected from the group consisting of:
[0008] i) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:1, 12, 13 and 61 or a variant thereof with at least 40% identity to any one of SEQ ID NO:1, 12, 13 and 61 with at least one of activity of a gamma-class PLP enzyme and an activity substantially equivalent to that of a lolC gene product,
[0009] ii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:2, 14, 15 and 62 or a variant thereof with at least 40% identity to any one of SEQ ID NO:2, 14, 15 and 62 with at least one of activity of an alpha-class PLP enzyme and activity substantially equivalent to that of the lolD gene product,
[0010] iii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:3, 16, 17 and 63 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:3, 16, 17 and 63 with at least one of monooxygenase activity and activity substantially equivalent to that of the lolF gene product,
[0011] iv) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:4, 18 and 19 or a variant thereof with at least 40% identity of any one of SEQ ID NO:4, 18 and 19 with at least one of amino acid bridging activity and activity substantially equivalent to that of the lolA gene product,
[0012] v) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:5, 20, 21 and 64 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:5, 20, 21 and 64 with at least one of activity of an alpha-class PLP enzyme and activity substantially equivalent to that of the lolT gene product,
[0013] vi) a polynucleotide encoding a polypeptide comprising the sequence of of any one of SEQ ID NO:6, 22, 23 and 65 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:6, 22, 23 and 65 with at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolE gene product,
[0014] vii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:7, 24, 25 and 66 or a variant thereof with at least 40% identity to any one of SEQ ID NO:7, 24, 25 and 66 with at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolO gene product,
[0015] viii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:8, 26 and 27 or a variant thereof with at least 40% identity to any one of SEQ ID NO:8, 26 and 27 with activity substantially equivalent to that of the lolU gene product,
[0016] ix) a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:9 or 28 or a variant thereof with at least 40% identity to SEQ ID NO:9 or 28 with at least one of N-Methyltransferase activity and activity substantially equivalent to that of the lolM gene product,
[0017] x) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:10, 29 and 67 or a variant thereof with at least 40% identity to any one of SEQ ID NO:10, 29 and 67 with at least one of acetamidase activity and activity substantially equivalent to that of the lolN gene product, and
[0018] xi) a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:11 or 30 or a variant thereof with at least 40% identity to SEQ ID NO:11 or 30 with at least one of cytochrome P450 monooxygenase activity and activity substantially equivalent to that of the lolP gene product.
[0019] In one embodiment the host cell is modified or transformed to comprise at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11 polynucleotides selected from i) to xi)
[0020] In a further embodiment the host cell is modified or transformed to comprise the polynucleotides of i), ii), iii), v) and vii). That is the host cell is modified or transformed to comprise a lolC, lolF, lolD, lolT and a lolO gene, or polynucleotides encoding a lolC, lolF, lolD, lolT and a lolO gene product.
[0021] In a further embodiment the host cell contains more copies of the at least one polynucleotide than does a control cell.
[0022] In a further embodiment the host cell is not modified or transformed to comprise the polynucleotide of vi). That is the host cell is not modified or transformed to comprise a lolE gene, or a polynucleotide encoding a lolE gene product.
[0023] In a further embodiment the host cell is not modified or transformed to comprise the polynucleotide of viii). That is the host cell is not modified or transformed to comprise a lolU gene, or a polynucleotide encoding a lolU gene product.
[0024] In a further embodiment the host cell is not modified or transformed to comprise the polynucleotides of vi) or viii). That is the host cell is not modified or transformed to comprise a lolE or a lolU gene, or polynucleotides encoding a lolE or a lolU gene product.
[0025] In one embodiment the host cell produces more of at least one loline alkaloid or precursor thereof, than does a control cell.
[0026] In a further embodiment the host cell produces more of at least one loline alkaloid or precursor thereof, than does a control cell, as a result of the host cell being transformed or modified to comprise at least one polynucleotide.
[0027] In a further embodiment the control cell has not been modified or transformed to comprise the at least one polynucleotide.
[0028] In a further embodiment the control cell is of the same species or strain as the host cell that has been modified or transformed to comprise the at least one polynucleotide
Host Cell where Polynucleotide is Part of an Expression Construct
[0029] In a further embodiment the at least one polynucleotide is part of an expression construct.
[0030] In a further embodiment the at least one polynucleotide is operably linked to a promoter.
[0031] In a further embodiment the at least one polynucleotide is operably linked to a terminator.
[0032] In a further embodiment the at least one polynucleotide is operably linked to a promoter and a terminator.
Expression Construct
[0033] In a further aspect, the invention provides an expression construct comprising at least one polynucleotide selected from the group consisting of:
[0034] i) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:1, 12, 13 and 61 or a variant thereof with at least 40% identity to any one of SEQ ID NO:1, 12, 13 and 61 with at least one of activity of a gamma-class PLP enzyme and an activity substantially equivalent to that of a lolC gene product,
[0035] ii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:2, 14, 15 and 62 or a variant thereof with at least 40% identity to any one of SEQ ID NO:2, 14, 15 and 62 with at least one of activity of an alpha-class PLP enzyme and activity substantially equivalent to that of the lolD gene product,
[0036] iii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:3, 16, 17 and 63 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:3, 16, 17 and 63 with at least one of monooxygenase activity and activity substantially equivalent to that of the lolF gene product,
[0037] iv) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:4, 18 and 19 or a variant thereof with at least 40% identity of any one of SEQ ID NO:4, 18 and 19 with at least one of amino acid bridging activity and activity substantially equivalent to that of the lolA gene product,
[0038] v) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:5, 20, 21 and 64 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:5, 20, 21 and 64 with at least one of activity of an alpha-class PLP enzyme and activity substantially equivalent to that of the lolT gene product,
[0039] vi) a polynucleotide encoding a polypeptide comprising the sequence of of any one of SEQ ID NO:6, 22, 23 and 65 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:6, 22, 23 and 65 with at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolE gene product,
[0040] vii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:7, 24, 25 and 66 or a variant thereof with at least 40% identity to any one of SEQ ID NO:7, 24, 25 and 66 with at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolO gene product,
[0041] viii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:8, 26 and 27 or a variant thereof with at least 40% identity to any one of SEQ ID NO:8, 26 and 27 with activity substantially equivalent to that of the lolU gene product,
[0042] ix) a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:9 or 28 or a variant thereof with at least 40% identity to SEQ ID NO:9 or 28 with at least one of N-Methyltransferase activity and activity substantially equivalent to that of the lolM gene product,
[0043] x) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:10, 29 and 67 or a variant thereof with at least 40% identity to any one of SEQ ID NO:10, 29 and 67 with at least one of acetamidase activity and activity substantially equivalent to that of the lolN gene product, and
[0044] xi) a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:11 or 30 or a variant thereof with at least 40% identity to SEQ ID NO:11 or 30 with at least one of Cytochrome P450 monooxygenase activity and activity substantially equivalent to that of the lolP gene product.
[0045] In one embodiment the expression comprises at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11 polynucleotides selected from i) to xi).
[0046] In a further embodiment the expression construct comprises at least the polynucleotides of i), ii), iii), v) and vii). That is the construct comprises at least a lolC, lolF, lolD, lolT and a lolO gene, or at least polynucleotides encoding a lolC, lolF, lolD, lolT and a lolO gene product.
[0047] In a further embodiment the at least one polynucleotide is operably linked to at least one promoter.
[0048] In a further embodiment the expression construct does not comprise the polynucleotide of vi). That is the expression construct does not comprise a lolE gene, or a polynucleotide encoding a lolE gene product.
[0049] In a further embodiment the expression construct does not comprise the polynucleotide of viii). That is the expression construct does not comprise a lolU gene, or a polynucleotide encoding a lolU gene product.
[0050] In a further embodiment the expression construct does not comprise the polynucleotides of vi) or viii). That is the expression construct does not comprise a lolE or a lolU gene, or polynucleotides encoding a lolE or a lolU gene product.
Host Cell Comprising the Construct
[0051] In a further aspect the invention provides a host cell comprising at least one construct of the invention.
[0052] Those skilled in the art will understand that the desired complement of lol genes or polynucleotides may be present in one or multiple constructs that are transformed into the host.
[0053] In a preferred embodiment the at least one polynucleotide, or expression construct, is stably incorporated into the genome of the host cell.
Host Cell is Tolerant of ACPP Production
[0054] In a further embodiment the host cell is tolerant of endogenous (3-amino-3-carboxypropyl)proline (ACPP) production.
[0055] In a further embodiment the host cell has been pre-selected for tolerance to said level of cellular ACPP.
[0056] In a further embodiment the host cell has been pre-selected for tolerance of endogenous ACPP production by transformation with at least one polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:1 or a variant thereof with at least 40% identity to SEQ ID NO:1 with at least one of activity of a gamma-class PLP enzyme and activity substantially equivalent to that of a lolC gene product.
[0057] In a further embodiment the host cell was supplied, or fed, with O-acetyl-L-homoserine (OAH) during selection. Preferably the host cell was supplied, or fed, with non-limiting amounts of OAH during selection.
[0058] In a further embodiment the host cell was supplied, or fed, with L-proline during selection. Preferably the host cell was supplied, or fed, with non-limiting amounts of L-proline during selection.
[0059] In a further embodiment the host cell is tolerant to at least 0.2 mM, more preferably at least 0.4 mM, more preferably at least 0.6 mM, more preferably at least 0.8 mM, more preferably at least 1 mM, more preferably at least 1.2 mM, more preferably at least 1.4 mM, more preferably at least 1.6 mM, more preferably at least 1.8 mM, more preferably at least 2 mM, more preferably at least 2.2 mM, more preferably at least 2.4 mM, more preferably at least 2.6 mM, more preferably at least 2.8 mM, more preferably at least 3 mM, more preferably at least 3.2 mM, more preferably at least 3.4 mM, more preferably at least 3.6 mM, more preferably at least 3.8 mM, more preferably at least 4 mM, more preferably at least 4.2 mM, more preferably at least 4.4 mM, more preferably at least 4.6 mM, more preferably at least 4.8 mM, more preferably at least 5 mM, more preferably at least 5.2 mM, more preferably at least 5.4 mM, more preferably at least 5.6 mM, more preferably at least 5.8 mM, more preferably at least 6 mM, more preferably at least 6.2 mM, more preferably at least 6.4 mM, more preferably at least 6.6 mM, more preferably at least 6.8 mM, more preferably at least 7 mM, more preferably at least 7.2 mM, more preferably at least 7.4 mM, more preferably at least 7.6 mM, more preferably at least 7.8 mM, more preferably at least 8 mM ACPP in the growth medium.
[0060] In a further embodiment host cell is tolerant of a level of cellular ACPP that is toxic to a control cell of the same strain or species.
Host Cell can Convert 1-AP to AcAP
[0061] In a further embodiment the host cell, prior to modification or transformation, is able to convert exo-1-aminopyrrolizidine (1-AP) to exo-1-acetamido-pyrrolizidine (AcAP).
[0062] In a further embodiment the host cell, prior to modification or transformation, has been pre-selected for the ability to convert 1-AP to AcAP.
[0063] In a further embodiment the host cell has been pre-selected by measuring AcAP production in the host cell.
[0064] In a further embodiment the host cell was supplied, or fed, with 1-AP during selection. Preferably the host cell was supplied, or fed, with non-limiting amounts 1-AP during selection.
[0065] In a further embodiment the selected host cell can produce at least 0.005 milligrams (mg), preferably 0.01 milligrams (mg), more preferably 0.02 milligrams (mg), more preferably 0.03 milligrams (mg), more preferably 0.04 milligrams (mg), 0.05 milligrams (mg), more preferably at least 0.1 mg, more preferably at least 0.15 mg, more preferably at least 0.2 mg, more preferably at least 0.25 mg, more preferably at least 0.3 mg, more preferably at least 0.35 mg, more preferably at least 0.4 mg, more preferably at least 0.45 mg, more preferably at least 0.5 mg, more preferably at least 0.75 mg, more preferably at least 1 mg, more preferably at least 1.5 mg, more preferably at least 2 mg of AcAP per gram (g) of cellular biomass.
Host Cell Type
[0066] In a further embodiment the cell is from a fungal species.
[0067] In a further embodiment the cell is from a bacterial species.
[0068] In a further embodiment the cell is from the subkingdom Dikarya.
[0069] In a further embodiment the cell is from a phylum selected from Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Glomeromycota, Ascomycota and Basidiomycota or a subphylum incertae sedis selected from Mucoromycotina, Kickxellomycotina, Zoopagomycotina and Entomophthoromycotina.
[0070] In a further embodiment the cell is from an order selected from Mucorales, Hypocreales, Eurotiales, Sebacinales and Saccharomycetales.
[0071] In a further embodiment the cell is from a genus selected from Metarhizium, Epichloe, Saccharomyces, Kluveromyces, Trichoderma, Aspergillus, Beauveria, Pichia, Penicillium, Serendipita, Umbelopsis, Neurospora, Epicoccum, Sarocladium, Balansia, Fusarium, Alternaria, Ustilago, Sebacina, Glomus and Rhizopus.
[0072] In a further embodiment the cell is from the species Metarhizium robertsii. In a further embodiment the cell is from the species Trichoderma reesei. In a further embodiment the cell is from the species Aspergillus niger. In a further embodiment the cell is from the species Aspergillus nidulans. In a further embodiment the cell is from the species Aspergillus oryzae. In a further embodiment the cell is from the species Beauveria bassiana. In a further embodiment the cell is from the species Saccharomyces cerevisiae. In a further embodiment the cell is from the species Pichia pastoris. In a further embodiment the cell is from the species Kluveromyces marxianus. In a further embodiment the cell is from the species Epichloe festucae. In a further embodiment the cell is from the species Epichloe typhina. In a further embodiment the cell is from the species Penicllium chrysogenum. In a further embodiment the cell is from the species Penicillium paxilli. In a further embodiment the cell is from the species Penicillium expansum. In a further embodiment the cell is from the species Serendipita indica. In a further embodiment the cell is from the species Umbelopsis isabellina. In a further embodiment the cell is from the species Neurospora crassa. In a further embodiment the cell is from the species Epicoccum italicum. In a further embodiment the cell is from the species Sarocladium zeae. In a further embodiment the cell is from the species Fusarium verticillioides. In a further embodiment the cell is from the species Ustilago maydis.
[0073] In one embodiment, the cell is a fungal cell other than a yeast cell.
[0074] In one embodiment the cell is a yeast cell.
[0075] In a further embodiment the cell is from a non-Epichloe uncinata fungal species.
Fermentation Suitable Host Cells
[0076] In a further embodiment the cell is from species or strain of fungi that is tractable to use in fermentation. In a further embodiment the cell is from a species or strain of fungi capable of a specific growth rate (.mu.h.sup.-1) of at least 0.01, preferably 0.02, more preferably at least 0.03, more preferably at least 0.04, more preferably at least 0.05, more preferably at least 0.1, more preferably at least 0.15, more preferably at least 0.2, more preferably at least 0.25, more preferably at least 0.3, more preferably at least 0.35, more preferably at least 0.4, more preferably at least 0.45.
[0077] In a one embodiment the host cells suitable for fermentation is from a phylum selected from: Ascomycota and Basidiomycota or subphylum incertae sedis Mucoromycotina. In one embodiment the host cells suitable for fermentation are from a genus selected from: Aspergillus, Beauveria, Epichloe, Neurospora, Epicoccum, Sarocladium, Kluveromyces, Metarhizium, Penicillium, Pichia, Rhizopus, Saccharomyces, Serendipita, Trichoderma, and Umbelopsis.
[0078] In a one embodiment the host cells suitable for fermentation is from a species selected from: Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Beauveria bassiana, Epichloe festucae, Epichloe typhina, Epicoccum italicum, Metarhizium robertsii, Penicillium expansum, Penicillium chrysogenum, Penicillium paxilli, Saccharomyces cerevisiae, Kluveromyces marxianus, Pichia pastorus, Rhizopus oryzae, Rhizopus stolonifer, Rhizopus microsporus, Serendipita indica, Trichoderma reesei, Neurospora crassa, Sarocladium zeae and Umbelopsis isabellina.
Method for Producing a Host Cell for Producing at Least One Loline Alkaloid, or Precursor Thereof
[0079] In a further aspect the invention provides a method for producing a host cell for producing at least one loline alkaloid or precursor thereof, the method comprising modifying or transforming a host cell to comprise at least one polynucleotide as herein described.
[0080] In a further embodiment the host cell is produced by transforming a cell to comprise at least one polynucleotide or construct as herein described.
Method for Producing at Least One Loline Alkaloid or a Precursor Thereof
[0081] In a further aspect the invention provides a method for producing at least one loline alkaloid or a precursor thereof, the method comprising culturing host cells of the invention, or produced by a method of the invention, under conditions conducive to the production of the at least one loline alkaloid or precursor thereof, by the host cells.
[0082] In one embodiment the method further comprises separating, purifying, fractionating or isolating the at least one loline alkaloid or precursor thereof.
[0083] In a further embodiment the host cells are cultured in the presence of at least one loline alkaloid precursor.
[0084] In various embodiments the method comprises maintaining the host cells in the presence of at least one of:
[0085] (a) an effective amount of proline or a biosynthetic precursor thereof,
[0086] (b) an effective amount of O-acetyl-L-homoserine (OAH) or a biosynthetic precursor thereof,
[0087] (c) an effective amount of (3-amino-3-carboxypropyl)proline (ACPP) or a biosynthetic precursor thereof,
[0088] (d) an effective amount of exo-1-aminopyrrolizidine (1-AP) or a biosynthetic precursor thereof,
[0089] (e) an effective amount of exo-1-acetamido-pyrrolizidine (AcAP) or a biosynthetic precursor thereof, or
[0090] (f) any combination of two or more of (a) to (e) above.
[0091] In one embodiment the method comprises maintaining the host cells, or a culture thereof, at a temperature of from about 15.degree. C. to about 35.degree. C.
[0092] In a further embodiment the method comprises maintaining the host cells, or a culture thereof, at a temperature of from about 15.degree. C. to about 40.degree. C.
[0093] In one embodiment the method comprises maintaining the host cells, or a culture thereof, at for at least about 1 day, at least about 3 days, at least about 4 days, at least about 7 days or at least about 10 days.
[0094] In one embodiment the method comprises maintaining the host cells, or a culture thereof, in a bioreactor.
[0095] In one exemplary embodiment the method is a method of producing one or more loline alkaloids, comprising:
[0096] i) providing a culture comprising a host cell of the invention,
[0097] ii) maintaining the culture for at least about 1 day at a temperature of from about 15.degree. C. to about 40.degree. C. in the presence of one or more of the following:
[0098] (a) an effective amount of proline or a biosynthetic precursor thereof,
[0099] (b) an effective amount of O-acetyl-L-homoserine (OAH) or a biosynthetic precursor thereof,
[0100] (c) an effective amount of (3-amino-3-carboxypropyl)proline (ACPP) or a biosynthetic precursor thereof,
[0101] (d) an effective amount of exo-1-aminopyrrolizidine (1-AP) or a biosynthetic precursor thereof,
[0102] (e) an effective amount of exo-1-acetamido-pyrrolizidine (AcAP) or a biosynthetic precursor thereof, or
[0103] (f) any combination of two or more of (a) to (e) above, and
[0104] iii) separating the one or more loline alkaloids from the culture, or at least partially purifying or isolating the one or more loline alkaloids, thereby to provide the one or more loline alkaloids.
[0105] In one embodiment the purification or isolation is achieved via filtration and/or column purification.
[0106] In one aspect the invention provides a method for conferring the ability to produce a loline alkaloid or a precursor thereof on an organism, the method comprising transforming the organism with an expression construct of the invention.
[0107] In one embodiment the organism, prior to transformation, does not produce the loline alkaloid or precursor thereof.
[0108] The cell as herein described may be part of an organism. Thus reference to a cell or host cell can be used interchangeably with reference to an organism or host organism.
[0109] In one embodiment the loline alkaloid or precursor thereof is toxic to a pest.
[0110] In one embodiment the pest is an insect.
[0111] The term "insect" includes, but not limited to, aphids, mealybugs, whiteflies, moths, butterflies, psyllids, thrips, stink bugs, rootworms, weevils, leafhoppers and fruit flies, such as Myzus persicae (green peach aphid), Aphis gossypii Glover (melon/cotton aphid), Rhopalosiphum maidis (corn leaf aphid), Aphis glycines Matsumura (soybean aphid), Brevicoryne brassicae (cabbage aphid), Anasa tristis (squash bug); Pseudococcus longispinus (long tailed mealybug), Pseudococcus calceolariae (scarlet mealybug), Pseudococcus viburni (obscure mealybug), Planococcus citri (Citrus mealybug); Trialeurodes vaporariorum (greenhouse whitefly), Bemisia tabaci (silverleaf whitefly); Plutella xylostella (diamondback moth), Citripestis sagittiferella (citrus fruit moth), Helicoverpa armigera (tomato fruitworm or corn earworm), Pectinophora gossypiella (pink bollworm), Phthorimaea operculella (potato tuber moth), Amyelois transitella (Navel orangeworm), Cydia pomonella (codling moth), Cnephasia jactatana (black-lyre leafroller), Epiphyas postvittana (light-brown apple moth), Grapholita molesta (oriental fruit moth), Ostrinia furnacalis (Asian corn borer), Ostrinia nubilalis (European corn borer), Scirpophaga excerptalis (sugarcane top borer) Diatraea saccharalis (sugarcane borer), Chilo plejadellus (rice stalk borer), Earias vitella (spotted bollworm), Earias insulana (spiny bollworm), Spodoptera frugiperda (fall armyworm), Spodoptera litura (tobacco cutworm), Melittia cucurbitae (squash vine borer), Teia anartoides (painted apple moth), Trichoplusia ni (Cabbage looper); Pieris rapae (white butterfly); Bactericera cockerelli (tomato/potato psyllid), Diaphorina citri (Asian citrus psyllid), Trioza erytreae (African citrus psyllid); Thrips obscuratus (flower thrips), Heliothrips haemorrhoidalis (greenhouse thrips), Thrips tabaci (onion thrips), Frankliniella williamsi (Maize thrip); Halyomorpha halys (brown marmorated stink bug), Oebalus pugnax (rice stink bug), Diabrotica virgifera virgifera (western corn rootworm), Diabrotica barberi (northern corn rootworm), Diabrotica undecimpunctata howardi (southern corn rootworm), Diabrotica virgifera zeae (Mexican corn rootworm); Pempheres affinis (cotton stem weevil); Nephotettix virescens (green leafhopper), Nilaparvata lugens (brown planthopper); Bactrocera tryoni (Queensland fruit fly).
[0112] In one embodiment the pest is a non-insect pest.
[0113] In one embodiment the pest is a nematode.
[0114] The term "nematode" includes but is not limited to root-knot nematodes (Meloidogyne species), cyst nematodes (Heterodera and Globodera species), lesion nematodes (Pratylenchus species), reniform nematodes (Rotylenchulus reniformis), lance nematodes (Hoplolaimus species) and stem and bulb nematodes (Ditylenchus species).
[0115] Preferred LOL genes for use in various aspects and embodiments of the invention are lolC, lolD, lolF, lolT and lolO.
[0116] In certain embodiments the host cells and organisms are not transformed to express lolE. In certain embodiments the host cells and organisms and are not transformed to express lolU. In certain embodiments the host cells and organisms are not transformed to express lolE or lolU.
[0117] Any one or more of the following embodiments may relate to any of the aspects described herein or any combination thereof.
[0118] In will be appreciated that the polynucleotide may be an allelic variant, degenerate sequence, homologue or orthologue of the specified nucleotide sequences.
[0119] In various embodiments the variant polypeptide has at least 40%, more preferably at least 41%, more preferably at least 42%, more preferably at least 43%, more preferably at least 44%, more preferably at least 45%, more preferably at least 46%, more preferably at least 47%, more preferably at least 48%, more preferably at least 49%, more preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% amino acid identity with the specified polypeptide sequences.
[0120] In various embodiments the polynucleotide variant comprises one or more alternative codons that code for the eventual translation of a polypeptide having at least 40%, more preferably at least 41%, more preferably at least 42%, more preferably at least 43%, more preferably at least 44%, more preferably at least 45%, more preferably at least 46%, more preferably at least 47%, more preferably at least 48%, more preferably at least 49%, more preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% amino acid identity with the specified polypeptide sequences.
[0121] In various embodiments the polynucleotide variant having at least 40%, more preferably at least 41%, more preferably at least 42%, more preferably at least 43%, more preferably at least 44%, more preferably at least 45%, more preferably at least 46%, more preferably at least 47%, more preferably at least 48%, more preferably at least 49%, more preferably at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% nucleotide sequence identity to the specified polynucleotide sequence.
[0122] Polynucleotide and polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs for e.g., the Needleman-Wunsch global alignment program (Needleman and Wunsch, 1970). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice, et al., 2000) which can be obtained from the World Wide Web at http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi.ac.uk/emboss/align/.
[0123] Alternatively, the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps (Huang, 1994).
[0124] A preferred method for calculating polynucleotide and polypeptide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin, et al., 1998).
[0125] In one embodiment the genome of the untransformed (wild type) host cell does not prior to transformation with a construct of the invention comprise the one or more LOL genes. In another embodiment the genome of the untransformed (wild type) host cell does not prior to transformation with a construct of the invention comprise a gene homologous to the one or more LOL genes. In another embodiment the untransformed (wild type) host cell or does not prior to transformation with a construct of the invention express the one or more LOL genes.
[0126] In one embodiment the host cell, expression construct or polynucleotide comprises and/or expresses at least the
[0127] a) lolC gene,
[0128] b) lolC and lolD genes,
[0129] c) lolC, lolD and lolT genes,
[0130] d) lolC, lolD, lolT and lolF genes,
[0131] e) lolC, lolD, lolT, lolF and lolA genes,
[0132] f) lolC, lolD, lolT, lolF and lolE genes,
[0133] g) lolC, lolD, lolT, lolF, lolE and lolA genes,
[0134] h) lolC, lolD, lolT, lolF, and lolO genes,
[0135] i) lolC, lolD, lolT, lolF, lolA and lolO genes,
[0136] j) lolC, lolD, lolT, lolF, lolE and lolO genes,
[0137] k) lolC, lolD, lolT, lolF, lolA, lolE and lolO genes,
[0138] I) lolC, lolD, lolF, lolT, lolN and lolM genes,
[0139] m) lolC, lolD, lolF, lolT, lolA, lolN and lolM genes,
[0140] n) lolC, lolD, lolF, lolT, lolE, lolN and lolM genes,
[0141] o) lolC, lolD, lolF, lolT, lolE, lolA, lolN and lolM genes,
[0142] p) lolC, lolD, lolF, lolT, lolN, lolM and lolP genes,
[0143] q) lolC, lolD, lolF, lolT, lolA, lolN, lolM and lolP genes,
[0144] r) lolC, lolD, lolF, lolT, lolE, lolN, lolM and lolP genes, or
[0145] s) lolC, lolD, lolF, lolT, lolA, lolE, lolN, lolM and lolP genes.
[0146] t) lolC, lolD, lolF, lolT, lolA, lolE, lolN, lolM, lolP and lolU genes.
[0147] In one embodiment the one or more LOL genes are derived from Epichloe uncinata, Epichloe festucae, Epichloe coenophiala, Epichloe amarillans, Epichloe glyceriae, Epichloe canadensis, Epichloe brachyelytri, Epichloe aotearoae, Epichloe siegelli, Aktinsonella hypoxylon, or Penicillium expansum.
[0148] In one embodiment the expression construct, genome or polynucleotide comprises, or the host cell expresses a gene encoding a heterologous acetyltransferase.
[0149] In another embodiment the genome of the untransformed (wild type) host cell comprises an endogenous acetyltransferase. In another embodiment the host cell expresses an endogenous acetyltransferase.
Promoters
[0150] In one embodiment one or more of the LOL genes are operably linked to a constitutive promoter. In various embodiments the promoter is the histone H3 promoter, the GAPDH promoter, the pna2/tpi hybrid promoter (Aspergillus nidulans or Aspergillus niger), the gpdA promoter (Metarhizium, Aspergillus, or Serendipita), the mbfA promoter, the trpC promoter (Aspergillus nidulans), the hexokinase-1 promoter (Metarhizium robertsii), the class I hydrophobin promoter (Beauveria bassiana) or any other constitutive promoter described herein.
[0151] In one embodiment one or more of the LOL genes are operably linked to an inducible promoter. In various embodiments the promoter is the alcA promoter, the alcR promoter, amyB promoter, the gas promoter, the glaA promoter, the niiA promoter, the cbhI promoter, the ctr4 promoter, the thiA promoter or any other inducible promoter described herein.
[0152] Those skilled in the art will understand that the different LOL genes may be operably linked to and/or expressed under the control of different promoters and/or terminators.
Loline Alkaloids
[0153] In one embodiment the loline alkaloid is selected from the group comprising N-acetylnorloline (NANL), norloline, loline, N-acetylloline (NAL), N-methylloline (NML), N-formylloline (NFL) and a combination of any two or more thereof.
[0154] In one embodiment the loline alkaloid or precursor thereof is selected from the group comprising N-acetylnorloline (NANL), norloline, loline, N-acetylloline (NAL), N-methylloline (NML), N-formylloline (NFL), (3-amino-3-carboxypropyl)proline (ACPP), exo-1-aminopyrrolizidine (1-AP), exo-1-acetamido-pyrrolizidine (AcAP), and a combination of any two or more thereof.
[0155] In one embodiment the loline alkaloid is selected from the group comprising N-acetylnorloline (NANL), norloline, loline, N-methylloline (NML), N-formylloline (NFL) and a combination of any two or more thereof.
[0156] In one embodiment the loline alkaloid or precursor thereof is selected from the group comprising N-acetylnorloline (NANL), norloline, loline, N-methylloline (NML), N-formylloline (NFL), (3-amino-3-carboxypropyl)proline (ACPP), exo-1-aminopyrrolizidine (1-AP), exo-1-acetamido-pyrrolizidine (AcAP), and a combination of any two or more thereof.
[0157] Other aspects of the invention may become apparent from the following description which is given by way of example only and with reference to the accompanying drawings.
[0158] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7).
[0159] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
[0160] In this specification, where reference has been made to external sources of information, including patent specifications and other documents, this is generally for the purpose of providing a context for discussing the features of the present invention.
[0161] Unless stated otherwise, reference to such sources of information is not to be construed, in any jurisdiction, as an admission that such sources of information are prior art or form part of the common general knowledge in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0162] The invention will now be described by way of example only and with reference to the drawings in which:
[0163] FIG. 1 a) shows four transformant M. robertsii ARSEF 23 isolates cultured on M100 medium containing phosphinothricin observed under visible light, b) the same four transformant isolates observed under blue light. Note that isolate no. 1 is not fluorescent while isolates 2, 3, 4 fluoresce green.
[0164] FIG. 2 shows an agarose gel photograph showing PCR amplification results with three sets of primers. Labelled in each gel are lanes carrying gDNA of isolates 17, 18, 19, 21, 22, 26, 29, and 30, which gave the correct band size indicating the presence of the Epichloe lolC gene, and the lane with gDNA of isolate 11, which went through the transformation process, but does not carry Epichloe lolC. pPH3-lolC and Epichloe gDNA were used as positive controls for lolC. M. robertsii ARSEF 23 (Mr Ma23) and water were the negative controls.
[0165] FIG. 3 shows an agarose gel photograph showing results of the PCR of cDNA of M. robertsii ARSEF 23 (Mr Ma23), transformant isolates and controls. PCR was done on undiluted and 1:10 diluted cDNA. Note the size difference between the band produced by gDNA (330 bp) and cDNA (285 bp).
[0166] FIG. 4 (a) shows a typical extracted chromatogram for m/z 217 of M. robertsii ARSEF 23 and isolate 11, a transformant that lacks E. festucae lolC, but carries the same selectable marker as the E. festucae lolC transformants; (b) shows a typical extracted chromatogram for m/z 217 of four transformant isolates, all of which carry E. festucae lolC. Note the presence of the third peak that corresponds to ACPP in (b).
[0167] FIG. 5 shows the loline biosynthetic pathway (modified from (Pan, et al., 2014)). Condensation of L-proline and O-acetylhomoserine is the first committed step of the pathway resulting in formation of ACPP. The next chemically detectable intermediates are 1-AP and AcAP. NANL is the first fully cyclized intermediate that will then be converted to the array of lolines in the chemical arsenal against insects. lolU and lolE, genes present in the loline cluster, but with unknown function, are not shown here.
[0168] FIG. 6 shows a comparison of chromatograms of chemically-synthesized AcAP standard to the AcAP produced by B. bassiana `Bb CT3` transformant carrying lolCDFA1TEU. Top: chromatogram of AcAP produced by Bb CT3 transformant; bottom: chromatogram of chemically-synthesized AcAP standard. Both chromatograms exhibit the same retention time and transition (169>110), confirming the biologically-produced AcAP is identical to the chemical standard
[0169] FIG. 7 shows a comparison of daughter ion scans of chemically-synthesized AcAP standard to the AcAP produced by Bb CT3 transformant. Top: daughter ion scan of chemically-synthesized AcAP standard, bottom: daughter ion scan of AcAP produced by Bb CT3 transformant. While more background noise is present in the biologically-produced AcAP sample due to its relatively low concentration, the scans show the compounds fragment the same, confirming the chemically- and biologically-produced AcAP are the same compound.
[0170] FIG. 8 shows a chromatogram of NANL detected in Bb H15, the first heterologous host expressing the first eight genes of the loline production pathway, thus successfully producing NANL, the first fully cyclized intermediate of the loline production pathway.
[0171] FIG. 9. Chromatograms of loline pathway intermediates detected in Bb H15 transformant. Top to bottom: Chromatograms showing the peaks corresponding to ACPP, 1-AP, and AcAP. All these intermediates occur prior to NANL, which was also detected in the same sample.
[0172] FIG. 10 shows the relative expression of loline genes (vs. actin) in selected B. bassiana transformants. Bb CT3: transformant carrying lolCDFA1TEU, produced AcAP when fed with 2 mM ACPP. No AcAP was observed in SDB+30 mM proline+2 mM OAH, 25K, 4 dpi
(i.e. without added ACPP). BbCT does not carry a functional copy of lolO, thus relative expression of lolO is given as 0.0; Bb H15: transformant carrying lolCDFA1TEOU, produced NANL (0.385 .mu.M) when grown in SDB+30 mM proline+2 mM OAH, 25.degree. C., 4 dpi. Note relatively less expression of lolF and lolO compared to the rest of the loline genes; Bb O16: transformant generated by transforming Bb H15 parent with an additional copy of lolO controlled by the Fl1 histone H3 promoter and glaA terminator. O16 produced NANL (average=1.231 .mu.M) when grown under the same conditions as Bb H15.
[0173] FIG. 11 shows a box and whiskers graph of AcAP amount (mg) produced per gram of dry biomass after 72 hours fermentation in presence of 1-AP.
[0174] FIG. 12 shows a box and whiskers graph of specific growth rate (h.sup.-1) during 1-AP feeding experiment, E. uncinata specific growth rate is provided as a reference.
DETAILED DESCRIPTION OF THE INVENTION
[0175] The present invention is in part directed to host cells transformed with, having a genome comprising, or transformed with an expression construct comprising, one or more heterologous LOL genes, and methods of producing such host cells. The invention is also directed to recombinant methods for producing one or more loline alkaloids or precursors thereof by culturing a host cell described herein, and methods for producing, or conferring the ability to produce, one or more loline alkaloids to a host cell or organism. The lolines and precursors there of produced by the host cell, and methods of the invention are useful for controlling pests.
[0176] The present inventors have for the first time demonstrated production of lolines from heterologous expression of genes in the lolines biosynthetic pathway. This is the first pyrrolizidine alkaloid to be produced in any heterologous host.
[0177] As far as the inventors are aware, although there are numerous publications on lolines and loline genes, there is no publication reporting or even considering the production of lolines in a heterologous host.
[0178] While there have been some reports of heterologous expression of individual LOL genes, these studies relate to assessing gene function rather than any attempt to produce lolines.
[0179] Loline genes have only been reported in Epichloe, Atkinsonella hypoxylon and Penicillium expansum. In Penicillium and Atkinsonella the products of the LOL gene cluster are only predicted, and the cluster is missing some Epichloe LOL gene equivalents. Thus, there is no evidence to suggest that production of the lolines by Epichloe outside of Epichloe itself is possible.
[0180] Furthermore, ACPP, the product of the LoIC enzyme, is reported to be toxic even to the producer fungus, even when applied at relatively low amounts of 4 mM (Faulkner, et al., 2006). Heterologous expression of lolC (performed to attempt to complement a cystathionine synthase mutant) in Aspergillus nidulans was lost after a single subculture (Spiering, et al., 2005). In addition, attempts to express lolC in E. coli were unsuccessful (Schardl, et al., 2007) and the authors state that these results "suggest that lolC or its enzyme product is toxic to cells".
[0181] The toxicity of lolC or its enzyme product, thus makes the applicants successful production of lolines via the expression of LOL genes including lolC all the more surprising. The applicant's invention therefore additionally provides for pre-selection of strains tolerant to ACPP for use in the heterologous production of lolines.
[0182] In addition, the applicants have surprisingly shown that none of the reported LOL genes, perform the step of converting 1-AP to AcAP in heterologous hosts in their experiments. However, the applicants have surprisingly shown that this step can be performed by endogenous enzymatic activity present in some strains. The applicant's invention therefore additionally provides pre-selection of strains capable of performing the conversion of 1-AP to AcAP for use in the heterologous production of lolines.
1. Definitions
[0183] The term "and/or" can mean "and" or "or".
[0184] The term "comprising" as used in this specification means "consisting at least in part of". When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.
[0185] The term "polynucleotide(s)," as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, genes, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments. The term also incudes fragments of polypeptides.
[0186] A "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides.
[0187] The term "polypeptide", as used herein, encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. Polypeptides of the present invention, or used in the methods of the invention, may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term also incudes fragments of polypeptides.
[0188] A "fragment" of a polypeptide is a subsequence of the polypeptide that in some embodiments performs a function/activity of and/or influences three-dimensional structure of the polypeptide.
[0189] As used herein the term "gene" refers to a polynucleotide sequence or its complement that is involved in producing a polypeptide, including regions preceding (leader) and following (trailer) the coding sequence, and introns between individual coding sequence (exons). It also includes to a codon-optimised polynucleotide sequence of the native gene.
[0190] The term "constitutive promoter", as used herein, refers to a promoter that is not regulated and is active in all conditions in the host cell resulting in continuous transcription of its associated gene.
[0191] The term "inducible promoter", as used herein, refers to a promoter that is regulated and is active in the host cell only in response to specific stimuli resulting in transcription of its associated gene.
[0192] A "LOL gene" refers to any of the genes that encode an enzyme involved in catalysing a reaction in the loline biosynthetic pathway, as summarised in FIG. 5 and Table 1 and elsewhere in the specification. This includes any of lolC, lolA, lolT, lolO, lolE, lolN, lolM, lolP, lolU, lolD, or lolF, and/or any of the following polynucleotide sequences: SEQ ID NO. 31 to 60 and 68 to 74. The terms also encompass variants of these polynucleotide sequences as herein defined.
[0193] In various embodiment the term "LOL gene" encompasses a polynucleotide selected from the group consisting of:
[0194] i) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:1, 12, 13 and 61 or a variant thereof with at least 40% identity to any one of SEQ ID NO:1, 12, 13 and 61 with at least one of activity of a gamma-class PLP enzyme and an activity substantially equivalent to that of a lolC gene product,
[0195] ii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:2, 14, 15 and 62 or a variant thereof with at least 40% identity to any one of SEQ ID NO:2, 14, 15 and 62 with at least one of activity of an alpha-class PLP enzyme and activity substantially equivalent to that of the lolD gene product,
[0196] iii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:3, 16, 17 and 63 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:3, 16, 17 and 63 with at least one of monooxygenase activity and activity substantially equivalent to that of the lolF gene product,
[0197] iv) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:4, 18 and 19 or a variant thereof with at least 40% identity of any one of SEQ ID NO:4, 18 and 19 with at least one of aspartokinase activity and activity substantially equivalent to that of the lolA gene product,
[0198] v) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:5, 20, 21 and 64 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:5, 20, 21 and 64 with at least one of activity of an alpha-class PLP enzyme and activity substantially equivalent to that of the lolT gene product,
[0199] vi) a polynucleotide encoding a polypeptide comprising the sequence of of any one of SEQ ID NO:6, 22, 23 and 65 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:6, 22, 23 and 65 with at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolE gene product,
[0200] vii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:7, 24, 25 and 66 or a variant thereof with at least 40% identity to any one of SEQ ID NO:7, 24, 25 and 66 with at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolO gene product,
[0201] viii) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:8, 26 and 27 or a variant thereof with at least 40% identity to any one of SEQ ID NO:8, 26 and 27 with activity substantially equivalent to that of the lolU gene product,
[0202] ix) a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:9 or 28 or a variant thereof with at least 40% identity to SEQ ID NO:9 or 28 with at least one of N-Methyltransferase activity and activity substantially equivalent to that of the lolM gene product,
[0203] x) a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:10, 29 and 67 or a variant thereof with at least 40% identity to any one of SEQ ID NO:10, 29 and 67 with at least one of acetamidase activity and activity substantially equivalent to that of the lolN gene product, and
[0204] xi) a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:11 or 30 or a variant thereof with at least 40% identity to SEQ ID NO:11 or 30 with at least one of cytochrome P450 monooxygenase activity and activity substantially equivalent to that of the lolP gene product.
[0205] In one embodiment a lolC gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:1, 12, 13 and 61 or a variant thereof with at least 40% identity to any one of SEQ ID NO:1, 12, 13 and 61.
[0206] Preferably the lolC gene or variant thereof has at least one of the activity of a gamma-class PLP enzyme encodes and an activity substantially equivalent to that of a lolC gene product.
[0207] In a further embodiment the lolD gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:2, 14, 15 and 62 or a variant thereof with at least 40% identity to any one of SEQ ID NO:2, 14, 15 and 62.
[0208] Preferably the polypeptide or variant thereof has at least one of the activity of an alpha-class PLP enzyme, and an activity substantially equivalent to that of the lolD gene product,
[0209] In a further embodiment the lolF gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:3, 16, 17 and 63 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:3, 16, 17 and 63.
[0210] Preferably the polypeptide or variant thereof has at least one of monooxygenase activity, and activity substantially equivalent to that of the lolF gene product,
[0211] In a further embodiment the lolA gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:4, 18 and 19 or a variant thereof with at least 40% identity of any one of SEQ ID NO:4, 18 and 19.
[0212] Preferably the polypeptide or variant thereof has at least one of amino acid bridging activity and activity substantially equivalent to that of the lolA gene product.
[0213] In a further embodiment the lolT gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:5, 20, 21 and 64 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:5, 20, 21 and 64.
[0214] Preferably the polypeptide or variant thereof has at least one of activity of an alpha-class PLP enzyme and activity substantially equivalent to that of the lolT gene product,
[0215] In a further embodiment the lolE gene comprises a polynucleotide encoding a polypeptide comprising the sequence of of any one of SEQ ID NO:6, 22, 23 and 65 or a variant thereof with at least 40% identity to of any one of SEQ ID NO:6, 22, 23 and 65.
[0216] Preferably the polypeptide or variant thereof has at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolE gene product,
[0217] In a further embodiment the lolO gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:7, 24, 25 and 66 or a variant thereof with at least 40% identity to any one of SEQ ID NO:7, 24, 25 and 66.
[0218] Preferably the polypeptide or variant thereof has at least one of activity of a non-heme iron dioxygenase and activity substantially equivalent to that of the lolO gene product.
[0219] In a further embodiment the lolU gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:8, 26 and 27 or a variant thereof with at least 40% identity to any one of SEQ ID NO:8, 26 and 27.
[0220] Preferably the polypeptide or variant thereof has activity substantially equivalent to that of the lolU gene product.
[0221] In a further embodiment the lolM gene comprises a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:9 or 28 or a variant thereof with at least 40% identity to SEQ ID NO:9 or 28.
[0222] Preferably the polypeptide or variant thereof has at least one of N-Methyltransferase activity and activity substantially equivalent to that of the lolM gene product,
[0223] In a further embodiment the lolN gene comprises a polynucleotide encoding a polypeptide comprising the sequence of any one of SEQ ID NO:10, 29 and 67 or a variant thereof with at least 40% identity to any one of SEQ ID NO:10, 29 and 67.
[0224] Preferably the polypeptide or variant thereof has at least one of acetamidase activity and activity substantially equivalent to that of the lolN gene product.
[0225] In a further embodiment the lolP gene comprises a polynucleotide polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO:11 or 30 or a variant thereof with at least 40% identity to SEQ ID NO:11 or 30.
[0226] Preferably the polypeptide or variant thereof has at least one of cytochrome P450 monooxygenase activity and activity substantially equivalent to that of the lolP gene product.
[0227] A "LOL gene product" refers to the polypeptide product of any of the genes, i.e. the encoded enzyme, involved in catalysing a reaction in the loline biosynthetic pathway, as summarised in FIG. 5 and Table 1 and elsewhere in the specification. The terms include any of lolC, to/A, lolT, lolO, lolE, lolN, lolM, lolP, lolU, lolD, or lolF polypeptides, and/or any of the following polypeptide sequences: SEQ ID NO. 1 to 30 and 61 to 67. The terms also encompass variants of these polypeptide sequences as herein defined.
[0228] The term "heterologous" as used herein with reference to a gene or a polynucleotide or polypeptide sequence transformed into or expressed by a host cell or fungus generally means a gene, or a polynucleotide or polypeptide sequence that is not encoded or expressed naturally by the wild type or native host cell or fungus.
[0229] The term "heterologous" as used herein with reference to polynucleotides, promoters and terminators, means that such heterologous sequences are not found operably linked to one another in wild type cells in nature. Thus, for example if a promoter is heterologous to the polynucleotide the promoter and polynucleotide are not found operably linked to one another in wild type cells in nature.
[0230] The term "host cell" as used herein refers to a fungal cell line cultured as a unicellular entity, which can be, or has been, used as a recipient for LOL genes, and/or expression constructs bearing one or more LOL genes, and/or which can be, or has been, transformed with or subjected to homologous recombination to integrate one or more heterologous LOL genes into the host cell genome. The term includes the progeny of the original host cell which has been transformed or subjected to homologous recombination. It will be appreciated that the progeny of a parent host cell may not be entirely identical in morphology or in genomic or total DNA complement to the original parent.
[0231] The term "plant" as used herein encompasses not only whole plants, but extends to plant parts, cuttings as well as plant products including roots, leaves, flowers, seeds, stems, callus tissue, nuts and fruit, bulbs, tubers, corms, grains, cuttings, root stock, or scions, and includes any plant material whether pre-planting, during growth, and at or post-harvest. Plants that may benefit from the application of the present invention cover a broad range of agricultural and horticultural crops, including crops produced using organic production systems.
[0232] The term "plant" includes those from any plant species. Such species include gymnosperm species, angiosperm species, and plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants. Such species include those that are used as fodder or forage crops, ornamental plants, food crops, row crops, horticultural crops, fruit crops, vegetable crops, biofuel crops, timber crops, and other trees or shrubs. Such species may be selected from the following: Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape, kale]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macro tyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., SaNx sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum betaceum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, and Ziziphus spp., among others.
[0233] The term "loline alkaloid precursor" as used herein refers to compounds produced as intermediates in the loline biosynthetic pathway. Loline alkaloid precursors may comprise the product of reactions catalysed by the enzymatic product of expression of one or more LOL genes.
[0234] The term modified, modify, and grammatical variations thereof, with respect to modifying host cells, or fungi to comprise a polynucleotide, include editing the endogenous genome of the host cells or fungi.
[0235] Methods for modifying endogenous genomic DNA sequences are known to those skilled in the art. Such methods may involve the use of sequence-specific nucleases that generate targeted double-stranded DNA breaks in genes of interest. Examples of such methods include: zinc finger nucleases (Curtin, et al., 2011, Sander, et al., 2011), transcription activator-like effector nucleases or "TALENs" (Cermak, et al., 2011, Mahfouz, et al., 2011, Li, et al., 2012), and LAGLIDADG homing endonucleases, also termed "meganucleases" (Tzfira, et al., 2012).
[0236] Targeted genome editing using engineered nucleases such as clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, is an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. A modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells (Sander and Joung, 2014). The technique is applicable to fungi (Nodvig, et al., 2015).
[0237] The term "substantially equivalent" with reference to any given gene product, enzyme, protein, or polypeptide having activity substantially equivalent to that of any given LOL gene product, preferably means that the gene product, enzyme, protein, or polypeptide is capable of fulfilling the role of the LOL gene product as summarised in Table 1 below, and/or has the enzymatic activity listed in Table 1 below.
[0238] The term "control", "controlling", "biocontrol" or "biological control" are used interchangeably herein to refer to reduction in growth, growth rate, development, feeding rate, reproduction or number of pests, particularly plant pests, and/or reducing the severity of, or eliminating, symptoms of such pests, particularly symptoms in plants caused by such pests.
[0239] The term "(s)" following a noun contemplates the singular or plural form, or both.
2. Loline Alkaloid Biosynthesis
[0240] Loline alkaloids are produced symbiotically during infection of grasses by endophytes, particularly Epichloe endophytes (which, following a nomenclature realignment, now includes the previously separate anamorph Neotyphodium spp.). These endophytes are considered to be bioprotective, conferring pest, and possibly drought and disease protection to the symbionts of which they form part. Lolines are potent insecticidal compounds and contribute a substantial amount of the bioprotective benefit conferred by Epichloe species that produce them.
[0241] Loline alkaloids are 1-aminopyrrolizidines having an oxygen bridge between C2 and C7. The various loline alkaloid variants, namely N-acetylnorloline (NANL), norloline, loline, N-acetylloline (NAL), N-methylloline (NML), N-formylloline (NFL) are differentiated by substituents on the primary amine.
[0242] A loline alkaloid biosynthesis pathway has been proposed as shown in FIG. 5.
[0243] A loline alkaloid biosynthetic gene cluster has only been identified in fungi belonging to two clades in Pezizomycotina, namely Sordariomycetes (only Epichloe species and Atkinsonella hypoxylon) and Eurotiomycetes (only Penicillium expansum). The Epichloe LOL gene cluster comprises eleven genes, referred to as the LOL genes herein, encoding key enzymes in the loline alkaloid biosynthesis pathway. Homologs to seven of these genes have been reported in P. expansum. The LOL genes are summarised in Table 1 below.
TABLE-US-00001 TABLE 1 Summary of LOL genes. Predicted encoded Polypeptide Polynucleotide Gene enzymatic activity Proposed Role SEQ ID NO: SEQ ID NO: Species lolC gamma-class Formation of 1 31 E. festucae PLP ACPP from OAH and proline lolD alpha-class Decarboxylation 2 32 E. festucae PLP of pyrrolodinium ion lolF Monooxygenase Oxidative 3 33 E. festucae decarboxylation of ACPP to form pyrrolodinium ion lolA Amino acid Increasing the 4 34 E. festucae binding levels of OAH lolT alpha-class Cyclisation of 5 35 E. festucae PLP pyrrolodinium ion(s) to form 1-AP lolE Nonheme iron Not clear 6 36 E. festucae dioxygenase lolO Nonheme iron Formation of the 7 37 E. festucae dioxygenase C2-C7 ether bridge in AcAP to form NANL lolU Not clear Not clear 8 38 E. festucae lolM N- Methylation of 9 39 E. festucae Methyl- norloline to form transferase loline, and of loline to form NML lolN Acetamidase Deacetylation of 10 40 E. festucae NANL to form norloline lolP Cytochrome Oxygenation of 11 41 E. festucae P450 NML to form monooxygenase NFL lolC1 gamma-class Formation of 12 42 E. uncinata PLP ACPP from OAH and proline lolC2 gamma-class Formation of 13 43 E. uncinata PLP ACPP from OAH and proline lolD1 alpha-class Decarboxylation 14 44 E. uncinata PLP of pyrrolodinium ion lolD2 alpha-class Decarboxylation 15 45 E. uncinata PLP of pyrrolodinium ion lolF1 Monooxygenase Oxidative 16 46 E. uncinata decarboxylation of ACPP to form pyrrolodinium ion lolF2 Monooxygenase Oxidative 17 47 E. uncinata decarboxylation of ACPP to form pyrrolodinium ion lolA1 Amino acid Increasing the 18 48 E. uncinata binding levels of OAH lolA2 Amino acid Increasing the 19 49 E. uncinata binding levels of OAH lolT1 alpha-class Cyclisation of 20 50 E. uncinata PLP pyrrolodinium ion(s) to form 1-AP lolT2 alpha-class Cyclisation of 21 51 E. uncinata PLP pyrrolodinium ion(s) to form 1-AP lolE1 Nonheme iron Not clear 22 52 E. uncinata dioxygenase lolE2 Nonheme iron Not clear 23 53 E. uncinata dioxygenase lolO1 Nonheme iron Formation of the 24 54 E. uncinata dioxygenase C2-C7 ether bridge in AcAP to form NANL lolO2 Nonheme iron Formation of the 25 55 E. uncinata dioxygenase C2-C7 ether bridge in AcAP to form NANL lolU1 Not clear Not clear 26 56 E. uncinata lolU2 Not clear Not clear 27 57 E. uncinata loIM N-Methyl- Methylation of 28 58 E. uncinata transferase norloline to form loline, and of loline to form NML lolN Acetamidase Deacetylation of 29 59 E. uncinata NANL to form norloline lolP Cytochrome Oxygenation of 30 60 E. uncinata P450 NML to form monooxygenase NFL lolC gamma-class Formation of 61 68 P. expansum PLP ACPP from OAH and proline lolD alpha-class Decarboxylation 62 69 P. expansum PLP of pyrrolodinium ion lolF Monooxygenase Oxidative 63 70 P. expansum decarboxylation of ACPP to form pyrrolodinium ion lolT alpha-class Cyclisation of 64 71 P. expansum PLP pyrrolodinium
3. Expression Constructs and Host Cells
[0244] The host cells described herein comprise a genome encoding, expressing or having been transformed with one or more LOL genes or expression construct of the invention.
[0245] In various embodiments the expression construct of the invention comprises two or more or three or more LOL genes.
[0246] In various embodiments the one or more LOL genes are operably linked to one or more regulatory elements that control the transcription, translation or expression of the gene in a host cell transformed with the expression construct. The one or more regulatory elements may be contiguous with the one or more LOL genes or act in trans or at a distance to control the gene of interest.
[0247] Suitable regulatory elements include appropriate transcription initiation, termination, promoter and enhancer sequences, or RNA processing signals such as splicing or polyadenylation signals.
[0248] Examples of suitable promoters for use in fungal host cells include promoters which are homologous or heterologous to the host cell. Furthermore, suitable promoters for use in the expression constructs of the invention include constitutive promoters, regulatable promoters, inducible promoters or repressible promoters. The promoter may be derived from a gene of the host cell, or a promoter derived from the genes of other fungi, viruses or bacteria. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating expression constructs using genetic constructs comprising the LOL genes of the sequences described herein.
[0249] In embodiments where the expression construct comprises two or more LOL genes, or where the host cell comprises or has been transformed with two or more LOL genes, each gene may be under the control of the same promoter or different promoters.
[0250] In various embodiments the method comprises transforming the host cell with two or more, three or more, or four or more expression constructs of the invention.
[0251] Host cells may be transformed using suitable methods known in the art for achieving heterologous gene expression in fungi and/or yeast. Choice of transformation method will depend on the species and form of the host cell, and the number of expression constructs and or LOL genes to be transformed.
[0252] In one embodiment the method comprises transforming the host cell with an expression vector so that the one or more LOL genes is integrated into the genome of the host cell via homologous or non-homologous recombination.
[0253] In various embodiments the host cell comprises protoplasts, spheroplasts, spores or conidia.
[0254] In one embodiment the method comprises transforming the host cell using polyethylene glycol (PEG)-mediated transformation. Other suitable transformation methods include electroporation, Agrobacterium tumefaciens-mediated transformation, biolistic transformation, or non-PEG-mediated spheroplast transformation.
[0255] An exemplary method that may be used to achieve homologous recombination of one or more LOL genes into a fungal host cell genome using sequential transformations is that described by Chiang and co-workers (Chiang, et al., 2013).
[0256] Briefly, for genes that are very large and difficult to amplify by PCR, two smaller transforming fragments may be created that fuse by homologous recombination in vivo to reconstruct the full-length coding sequences under the control of a single promoter. Two or more LOL genes may be integrated into the host cell genome using sequential transformations. Each gene or transforming fragment carries a selectable marker to enable selection of transformants that are have been transformed with the gene. Marker recycling may be used so that many genes may be transferred easily into the host cell.
4. Production of Pyrrolizidine Alkaloids
In Vitro
[0257] Exemplary methods to produce and at least partially purify and/or isolate one or more of the loline alkaloids or of the invention are described herein. These include the at least partial purification and/or isolation of one or more loline alkaloids from a culture of one or more species, or from culture media or culture supernatants and the like obtained therefrom.
[0258] In one embodiment the method comprises maintaining a culture of host cells in the presence of one or more loline alkaloid precursors. For example, in various embodiments the culture is maintained in the presence of one or more of
[0259] a) an effective amount of proline or a biosynthetic precursor thereof,
[0260] b) an effective amount of O-acetyl-L-homoserine (OAH) or a biosynthetic precursor thereof,
[0261] c) an effective amount of (3-amino-3-carboxypropyl)proline (ACPP) or a biosynthetic precursor thereof,
[0262] d) an effective amount of aspartic acid or a biosynthetic precursor thereof,
[0263] e) an effective amount of exo-1-acetamido-pyrrolizidine (AcAP) or a biosynthetic precursor thereof,
[0264] f) an effective amount of exo-1-aminopyrrolizidine (1-AP) or a biosynthetic precursor thereof, or
[0265] g) any combination of two or more of (a) to (f) above.
[0266] Choice of culture conditions, including duration of culturing, temperature and/or culture media will depend upon the particular characteristics of the host cell.
[0267] The invention consists in the foregoing and also envisages constructions of which the following gives examples only and in no way limit the scope thereof.
EXAMPLES
Example 1: Heterologous Expression of lolC in Heterologous Hosts to Produce (3-amino-3-carboxypropyl)proline (ACPP)
BACKGROUND
[0268] The Epichloe LOL gene cluster, consisting of 11 genes, has been reported to be required for loline biosynthesis (Spiering, et al., 2005, Pan, et al., 2014). Epichloe lolC was predicted to encode the enzyme that catalyses the first committed step of the loline pathway--the condensation of L-proline with the 3-amino-3-carboxypropyl group from O-acetyl-L-homoserine (Faulkner, et al., 2006). The biosynthetic intermediate produced by this reaction has a dose-dependent effect in E. uncinata--it is toxic to cells when fed at 4 mM, but results in the enrichment of N-formylloline when fed to cultures at 2 mM. P. expansum, the only species outside the Epichloe Glade known to carry the loline genes, has homologs to Epichloe lolC, D, F, T, E, O, N. There are no published studies characterizing P. expansum lolC (Pe lolC), but based on its similarity to Epichloe lolC, applicants expect that Pe lolC also encodes an enzyme that catalyzes the same reaction as Epichloe lolC (see Table 2 for amino acid percent identity between E. festucae and P. expansum loline gene products).
[0269] The applicants considered that heterologous expression of lolC in fungi which do not possess the LOL gene cluster nor produce lolines, is an important first step towards transforming and expressing selected LOL genes in a non-Epichloe fungus, particularly in light of ACPP's reported toxicity. As initial proof of concept, Epichloe festucae lolC (Ef lolC) was expressed in M. robertsii ARSEF 23 and surprisingly the applicants were able to demonstrate that ACPP was produced. This is described in detail below. The applicants further individually expressed Ef lolC and Pe lolC with successful ACPP production in B. bassiana, A. niger, and T. reesei. S. indica was successfully transformed with Pe lolC, the next step will be confirming experession of lolC. A full summary of expression results of lolC and ACPP production in heterologous hosts is given in Table 5.
TABLE-US-00002 TABLE 2 Amino acid percent identity between proteins encoded by E. festucae and P. expansum loline genes Gene Amino acid percent identity lolC 63.2% lolD 57.6% lolF 56.9% lolT 61.7% lolE 52.0% lolO 52.5% lolN 45.0%
Protocol
[0270] A plasmid carrying E. festucae lolC gene (SEQ ID NO: 31) fused to the constitutively expressed histone H3 promoter (pPH3-loC) and a plasmid carrying phosphinothricin resistance and green fluorescence (pBAR-GFP) were co-transformed into competent protoplasts of M. robertsii strain ARSEF 23. Transformants carrying pBAR-GFP were selected based on their ability to grow on regeneration medium containing phosphinothricin and green fluorescence. Genomic DNA was extracted from the selected transformants, and presence of pPH3-lolC was confirmed through amplification of Ef lolC in three sets of polymerase chain reactions (PCR).
[0271] Two parallel approaches were taken in order to analyse Ef lolC expression and activity in M. robertsii. Firstly, RNA was extracted from four transformed M. robertsii isolates carrying Ef lolC, an isolate that was subjected to the transformation process but does not carry lolC, and the parental strain M. robertsii ARSEF 23, with the aim of observing Ef lolC transcription via RNA extraction, cDNA synthesis, and PCR. Secondly, the same strains (above) were grown on M100 medium, freeze-dried, and analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for presence of (3-amino-3-carboxypropyl)proline (ACPP), the suggested product of condensation of L-proline and O-acetyl-L-homoserine (OAH), in the transformant mycelium or medium, indicating the activity of the Ef lolC-encoded enzyme.
Results
[0272] Co-Transformation and Identification of Ef lolC Transformants
[0273] Thirty (30) transformant isolates that exhibited both phosphinothricin resistance and green fluorescence (a representative set of transformants shown in FIG. 1) were obtained from the co-transformation and selection processes.
[0274] Three sets of PCRs, which were done using three different primer combinations on genomic DNA (gDNA) of a subset of the transformants isolated above, identified and confirmed transformants carrying the pPH3-lolC (FIG. 2). Of the transformant isolates that gave a positive result indicating presence of the lolC gene in all three PCRs, isolates 17, 18, 21, and 26 were used for RNA extraction and chemical analysis. Parent strain M. robertsii ARSEF 23 and transformant isolate 11, which did not produce a band in all three PCRs were used as negative controls in the same experiments.
Transcription of the Ef lolC Gene
[0275] Total RNA extracted from M. robertsii ARSEF 23 and transformant isolates 11, 17, 18, 21, 26 were used for complementary DNA (cDNA) synthesis. In a PCR done using two primers designed to complement two consecutive exons that were separated by an intron, cDNA of transformant isolates 17, 18, 21, 26, produced a 245 bp band, while the pPH3-lolC and gDNA produced a 330 bp band. The size difference between the bands produced by cDNA and gDNA matched the size of the intron, indicating Ef lolC was transcribed to RNA and correctly spliced in transformant isolates 17, 18, 21, and 26. M. Robertsii ARSEF 23 and isolate 11, both not carrying Ef lolC, as well as controls done with water and without any reverse transcriptase (RT) enzyme during cDNA synthesis did not give any bands (FIG. 3).
Chemical Analysis of Ef lolC Transformants
[0276] When analysed at m/z 217 (the protonated mass of ACPP), parental strain M. robertsii ARSEF 23 and transformant isolate 11, which did not carry pPH3-lolC, produced two peaks (FIG. 4a). Transformant isolates 17, 18, 21, and 26, carrying pPH3-lolC, gave three peaks (FIG. 4b). This result was consistent across all biological replicates of each isolate. The growth medium (M100) and the fungicide Sporekill.TM. (ICA International Chemicals Ltd., active ingredient Didecyldimethylammonium chloride), which was used to make the cultures non-viable, did not produce any peaks (see Table 3 for a list of peak areas for all samples).
[0277] Fragmentation analysis of the third peak present in the chromatograms of Ef lolC-expressing isolates matched those with a compound structurally similar to ACPP. This confirmed that the Ef lolC transcript was translated to its functional enzymatic form, which catalysed the condensation of proline and OAH to produce ACPP.
TABLE-US-00003 TABLE 3 Peak areas for the growth medium (M100), fungicide (Sporekill), and three biological replicates each for M. robertsii ARSEF 23 and transformant isolates 11, 17, 18, 21, and 26. RT 2.32 [(3-amino-3- carboxypropyl) RT 0.98 RT 1.51 proline] Sample Peak Area Peak Area Peak Area Sporekill 10% 0 0 0 M100 0 0 0 Ma23-1 1170000 717000 0 Ma23-2 887000 472000 0 Ma23-3 1260000 733000 0 C11-1 1860000 1130000 0 C11-2 2150000 1520000 0 C11-3 1610000 993000 0 C17-1 2060000 1520000 999000 C17-2 1110000 583000 869000 C17-3 1430000 835000 913000 C18-1 1040000 650000 209000 C18-2 1020000 393000 242000 C18-3 1230000 787000 209000 C21-1 1100000 634000 5160000 C21-2 1350000 799000 4920000 C21-3 1120000 660000 5640000 C26-1 1050000 573000 2270000 C26-2 1340000 668000 2430000 C26-3 1790000 1150000 3690000 [Note the inability to quantify the amount of (3-amino-3-carboxypropyl)proline as this analysis was performed in the absence of an authentic standard]
SUMMARY
[0278] The E. festucae lolC gene was heterologously expressed in M. robertsii. This was evidenced by the presence of Ef lolC in transformant gDNA, the presence of the correctly-spliced transcript, and the isolation of ACPP--the suggested end product of the reaction catalysed by the enzyme encoded by Ef lolC. This experimental report marks the first-ever documentation of successful stable heterologous expression of lolC in a non-Epichloe fungus, confirms its role in producing ACPP and demonstrates the potential for producing subsequent steps in the loline biosynthetic pathway, which was in doubt due to the previously suggested toxicity of ACPP.
Example 2: Production of Lolines and Loline Intermediates in Heterologous Hosts
Introduction
[0279] Lolines, which consist of a variable combination of N-acetylnorloline (NANL), N-formylloline (NFL), and N-acetylloline (NAL) in nature, are produced in planta by many endophytic Epichloe species (e.g. E. festucae E2368, E. glyceriae E2772, E. uncinata E167). These alkaloids protect their grass hosts from herbivorous insects, but do not show toxicity towards mammals (Jackson, et al., 1996, Wilkinson, et al., 2000, Schardl, et al., 2013). Blankenship and co-workers (Blankenship, et al., 2001) demonstrated that E. uncinata was able to make lolines in culture, which gave evidence that lolines--and the genes responsible--were of fungal origin. An as yet unidentified plant-encoded acetyl transferase converts loline to NAL, a later `decorative` step that increases the diversity of lolines (Pan, et al., 2014).
[0280] In Epichloe, eleven genes (lolC, D, F, A, T, E, O, U, M, N, P) collectively known as the LOL gene cluster, are reported to be required for loline biosynthesis (see FIG. 5 and (Pan, et al., 2014)). Of these, lolC encodes the enzyme that catalyses the first committed step of the loline pathway--the condensation of primary metabolites L-proline and O-acetyl-L-homoserine (OAH), as previously predicted (Faulkner, et al., 2006, Schardl, et al., 2007). However, this was only conclusively demonstrated for the first time in a heterologous host by the applicants in Example 1.
[0281] Expression of the eleven genes in the Epichloe LOL cluster was predicted to lead to production of NFL, the end-point of the biosynthetic pathway. Alternatively, eight genes are predicted to be required for production of NANL, which can be converted to an array of lolines. While not predicted to be required for NANL biosynthesis per se, the additional expression of lolA is predicted to increase loline biosynthesis via increasing the biological availability of the precursor molecule OAH.
[0282] Outside the Epichloe Glade, the loline genes have only been reported in P. expansum (Ballester, et al., 2015, Marcet-Houben and Gabaldon, 2016), wherein the products of the LOL gene cluster have only been predicted. In addition, the P. expansum LOL gene cluster is missing some Epichloe LOL gene equivalents and has homologs of seven of the Epichloe loline genes (lolC, D, F, T, E, O, N).
[0283] Production of NANL in a heterologous host in culture vs. production in Epichloe species, is industrially advantageous because of (1) the ability to use a fast-growing heterologous host in a fermenter for continuous mass production of lolines (vs. very little production per day of lolines in vitro and only during the stationary phase by the slow-growing E. uncinata), and (2) the ability to bias production towards individual loline analogues. Lolines have also proven to be very complex to produce via synthetic chemistry (Faulkner, et al., 2006, Cakmak, et al., 2011), thus favouring production via biological means.
[0284] The heterologous hosts tested in the reported experiments are (1) E. festucae Fl1, an Epichloe strain that does not possess the loline cluster; (2) B. bassiana strain K4B1, an insect pathogen which is also fermentation-compatible; (3) A. niger strain ATCC 1015, commonly used for industrial fermentation; (4) T. reesei strain RUT-C30 (ATCC 56765), commonly used for industrial fermentation; (5) M. robertsii ARSEF 23, an insect pathogen; (6) Neurospora crassa, a model fungus commonly used for genetic research; (7) S. cerevisiae, a model fungus commonly used for genetic research and industrial fermentation; (8) S. indica, a plant protective endophyte with a broad Angiosperm host range; and (9) U. isabellina, an endophyte. Fungi listed (1)-(7) belong to phylum Ascomycota, while (8) is a member of the phylum Basidiomycota and (9) is a Mucoromycota.
Materials and Methods
Fungal Strains
[0285] Strains used in this study and method of genetic transformation are listed in Table 4.
[0286] All strains are stored at the Biotelliga laboratory at the University of Auckland, Auckland, New Zealand.
TABLE-US-00004 TABLE 4 Fungal strains, media, and transformation methods used in the current study Fungal Method of genetic strain Media used transformation A. niger Wild type and Protoplast transformation (ATCC 1015) hygromycin selection: (standard methods) potato dextrose (PD); Phosphinothricin selection: M100 B. bassiana Wild type: Sabouraud Same as for A. niger K4B1 dextrose (SD); Sulfonyl urea, phosphinothricin, hygromycin selection: Czapeks Dox (CD); Geneticin selection: PD E. festucae Epichloe M100 Fl1 M. robertsii Wild type and ARSEF 23 phosphinothricin selection: M100 N. crassa Wild type: Vogel's Spore electroporation (ICMP 7781) medium N (VM); (Navarro-Sampedro, Phosphinothricin et al., 2007) selection: fructose/glucose/ sorbose (FGS) medium S. cerevisiae Wild type: yeast (Gietz and Schiestl, 2007) BY4743 pepetone dextrose (ATCC 201390) (YPD), G418, uracil auxotroph selection: yeast synthetic defined medium (SD) S. indica Wild type and Protoplast transformation (ATCC 204458) hygromycin selection: (Zuccaro, et al., 2009) and Aspergillus complete Electroporation of medium (ACM); hyphal fragments For growth immediately (Yadav, et al., 2010) prior to and selection after electroporation: Aspergillus minimal medium T. reesei Wild type and Protoplasts generated as RUT-C30 hygromycin selection: described (Penttila, et al., (ATCC 56765) PD; 1987, Gruber, et al., 1990) Phosphinothricin transformed by standard selection: M100 protoplast transformation methods U. isabellina Wild type and (Zhang, et al., 2007) ICMP 22148 hygromycin selection: PD
Genetic Constructs
[0287] A detailed list of transformation constructs is given in Table 11. In brief, all transformed Epichloe loline genes (SEQ ID NO:31 to 33 and 35 to 41) were cloned from E. festucae E2368, except for pBTL10 that, in addition to E. festucae lolD and lolF, contains E. uncinata lolA1 coding sequence (from `wild type lolA1`--SEQ ID NO 48), pBTL11 that, in addition to E. festucae lolC, lolD and lolF, contains E. uncinata lolA1 coding sequence (from `wild type lolA1`--SEQ ID NO 48), pBTL15 that, in addition to E. festucae lolC, contains E. uncinata lolA1 coding sequence (from `wild type lolA1`--SEQ ID NO 48), and pBTL57 that contains E. uncinata lolA1 coding sequence (from `wild type lolA1`--SEQ ID NO 48). Modified open reading frames [i.e. exons only, codon optimized for Neurospora crassa (using the Codon Optimization Tool at the Integrated DNA Technologies website https://sg.idtdna.com/CodonOpt), and with an HA tag] were also used in some cases. All transformed Penicillium loline genes (SEQ ID NO:68 to 71 and 73) were cloned from P. expansum ICMP 8595. Loline genes were transformed either coupled with a constitutive promoter or with a constitutive promoter and terminator. In a few cases, the gene encoding the selectable marker was present in the same plasmid as the loline genes. In most, however, the appropriate selectable marker was co-transformed with the loline gene constructs. All transformants were selected in media with appropriate selection. PCR screening was done to test for the presence of the loline genes on gDNA preparations done according to standard DNA extraction (miniprep gDNA extraction).
RNA Extraction and qPCR
[0288] RNA was extracted from fungal mycelia using the TRIzol.RTM. reagent (Life Technologies) according to the manufacturer's protocol. RNA was either DNased with DNase I recombinant (Roche) and used for cDNA synthesis with iScript.TM. (Biorad) or was DNased and cDNA synthesised using the iScriPt.TM. gDNA Clear cDNA Synthesis kit (Biorad). qPCR was done as per standard Biotelliga laboratory protocol using SsoAdvanced.TM. Universal SYBR Green Supermix (Biorad).
Chemical Analysis
[0289] Production of relevant compounds (ACPP, 1-AP, AcAP, and lolines) by transformed fungi were detected with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The values of detected compounds are represented in micromolar (.mu.M) and were calculated using the following formula:
Concentration in .mu.M=(Concentration in .mu.g/ml.+-.molecular weight).times.1000
[0290] The molecular weights of the compounds are: 216 (ACPP), 126 (1-AP), 168 (AcAP), 182 (NANL), 154 (loline), and 182 (NFL).
Results
[0291] Expression of lolC Results in Production of ACPP in Heterologous Hosts
[0292] Of the non-Epichloe fungi tested, A. niger, B. bassiana, M. robertsii, and T. reesei produced ACPP constitutively in culture upon expression of Ef lolC (Table 5). A. niger, B. bassiana, and T. reesei also produced ACPP constitutively in culture upon expression of Pe lolC (Table 5). Pe lolC was not attempted to be transformed into M. robertsii. The ACPP of biological origin was identical to chemically synthesised ACPP. It was observed in typical extracted chromatograms for the protonated mass of ACPP (m/z 217) in transformants with lolC, compared to wild type and a transformant not carrying lolC, but with the same selectable marker (FIG. 4), N. crassa and S. cerevisiae did not produce ACPP although transcription of lolC was detected.
TABLE-US-00005 TABLE 5 Summary of observations of expression of lolC and ACPP production in heterologous hosts Summary of transformed Plasmid Transcription ACPP Fungus gene of interest number of lolC detected A. niger lolC pBTL6 Yes 149-322 .mu.M ATCC 1015 (from E. festucae E2368) A. niger lolC pBTL74 Yes 212-913 .mu.M ATCC 1015 (from P. expansum ICMP 8595) B. bassiana lolC pBTL6 Yes 10-1390 .mu.M K4B1 (from E. festucae E2368) B. bassiana lolC pBTL74 Yes 476-1844 .mu.M K4B1 (from P. expansum ICMP 8595) M. robertsii lolC pBTL6 Yes 7-180 .mu.M ARSEF 23 (from E. festucae E2368) N. crassa lolC pBTL6 Yes No ACPP ICMP 7781 (from E. festucae E2368) detected N. crassa lolC Transformed Yes No ACPP ICMP 7781 (from E. festucae E2368, exons as PCR detected only, codon optimized for product N. crassa, C-terminal HA tag) S. cerevisiae lolC pBTL14 No No ACPP BY4743 (from E. festucae E2368, exons detected only, codon optimized for N. crassa,C-terminal HA tag removed) S. cerevisiae lolC pBTL13 Yes No ACPP BY4743 (from E. festucae E2368, exons detected only, codon optimized for S. cerevisiae, C-terminal HA tag) S. cerevisiae lolC (from E. festucae Modified No No ACPP BY4743 E2368, exons only, codon pBTL13 detected optimized for S. cerevisiae, C-terminal HA tag removed) T. reesei lolC pBTL6 Yes 2-17 .mu.M RUT-C30 (from E. festucae E2368) T. reesei lolC pBTL74 Yes 4-878 .mu.M RUT-C30 (from P. expansum ICMP 8595)
Expression of Epichloe Loline Pathway Genes Results in Loline Production in Heterologous Hosts
[0293] Based on their ability to produce ACPP upon expression of lolC, heterologous hosts A. niger, B. bassiana, and M. robertsii, along with E. festucae Fl1, were selected as candidate heterologous hosts for expression of lolCDFA1TEOU--or subsets of genes thereof. The lol genes transformed were obtained from E. festucae E2368, E. uncinata AR1006 and/or P. expansum ICMP 8595. The industrial A. niger strain ATCC 1015, was transformed with Epichloe lolD, F, T, A1, O. Cultures were supplemented with 1 mM ACPP, and upon feeding, successfully produced 0.385 .mu.M NANL (Table 6).
[0294] Transformation of Epichloe lolCDFA1TEU to B. bassiana resulted in production of 0.179 .mu.M AcAPwhen fed with 2 mM ACPP (see FIGS. 6 and 7 for comparison of AcAP produced by the Epichloe lolCDFA1TEU-carrying transformant Bb CT3 to chemically synthesised AcAP). This may be indicative of the selection of a transformant with relatively weak gene expression, which may have been caused by exclusion of transformants with `good` gene expression due to toxicity of pathway intermediates to the host. Transformation of B. bassiana with Epichloe lolCDFA1TEOU under selected promoters and terminators (see Table 11 for details of constructs) resulted in production of 0.385 .mu.M NANL (see FIG. 8 for chromatogram), as well as the full complement of detectable intermediates post-ACPP (see FIG. 9 for chromatograms). Expression of Epichloe lolCDFA1TEOUMNP in B. bassiana resulted in NFL and loline (see Table 6 for detailed summary of results). Gene expression levels of transformant isolate Bb4-18-H15 (`Bb H15`) showed possible bottlenecks in the pathway due to relatively `low` expression of lolF and lolO (FIG. 10). lolF and/or lolO, each under E. festucae Fl1 histone H3 promoter, shown to result in relatively high expression levels in our previous experiments, and followed by the g/aA terminator, were transformed into Bb H15. Transformants carrying the additional copy of lolO (named isolate Bb 016) produced more NANL than parent Bb H15. When fed with 30 mM proline, 4 mM OAH, 2 mM alpha-ketoglutaric acid and 0.25 mM iron [in ammoniumiron(II)sulfate hexahydrate (Pan, et al., 2018)], this isolate, Bb016 produced the hightest amount of NANL observed in any heterologous host system to-date (5.49 .mu.M NANL at 4 days post inoculation). Analysis of gene expression of the two isolates showed increased expression in all loline genes in Bb O16 compared to Bb H15 (FIG. 10).
TABLE-US-00006 TABLE 6 Summary observations of expression of Epichloe loline pathway genes and lolines and/or intermediates production in hosts tested positive for ACPP with lolC expression Summary of Lolines and/or transformed Plasmid Transcription intermediates Fungus genes of interest number of lol genes detected A. niger Ef lolDFTO, pBTL38, Transcripts of 0.385 .mu.M ATCC Eu lolA1 pBTL32, Ef lolDFTO NANL (in 1015 and Pe lolC pBTL40, detected. cultures fed pBTL57, Pe lolC gene with 1 mM pBTL33 didn't ACPP) integrate. B. Ef lolCDFTEOU pBTL11, Yes, except 0.179 .mu.M bassiana and Eu lolA1 pBTL12 for lolO (in AcAP (in K4B1 pBTL12), cultures fed which had a with 2 mM SNP and ACPP, but thereby not in cultures truncated grown with and non- precursors functional. proline and OAH). See FIGS. 6 and 7 for chromatograms comparing synthetic vs. biologically- produced AcAP. B. Ef lolCDFTEOU pBTL15, Yes 0.385 .mu.M bassiana and Eu lolA1 pBTL16, NANL K4B1 pBTL17 B. Ef pBTL15, Yes 0.055 .mu.M NFL, bassiana lolCDFTEOUMNP pBTL16, 0.065 .mu.M loline K4B1 and Eu lolA1 pBTL17, pBTL18 E. Ef lolDFTEOU pBTL15, Yes 0.22 .mu.M NANL festucae and Eu lolA1 pBTL16, (in cultures fed Fl1 pBTL17 with 1.6 mM ACPP)
Expression of P. expansum 101 Genes Result in Loline Production in Heterologous Hosts
[0295] Heterologous hosts A. niger and B. bassiana, which were capable of producing ACPP by expression of Pe lolC (Table 5), were transformed with Epichloe lolDFTA1, or P. expansum lolDFTO, henceforth Pe lolDFTO, or subsets of these genes thereof. All genes in transformants were confirmed to be expressed using qRT-PCR. Both A. niger and B. bassiana successfully produced loline pathway intermediates ACPP, 1-AP, and AcAP in the transformant strains expressing the Pe lolCDFTO genes (see Table 7 for details). However, in both A. niger and B. bassiana, transformants did not produce any detectable levels of NANL despite producing relatively high amounts of the precursor intermediate AcAP. This was unexpected as these transformants were expressing Pe lolO .gtoreq.1-fold relative to actin, compared to previous transformants which produced detectable levels of NANL with even .ltoreq.1-fold expression of Ef lolO (relative to actin). This may indicate that, at least in heterologous systems, Pe lolO is less efficient than Ef lolO in converting AcAP to NANL. Therefore, Ef lolO was transformed to B. bassiana transformants already expressing Pe lolCDFTO gene array. Biological triplicates of 17 transformants that resulted from transformation of Ef lolO to the Pe CDFTO background were screened for Ef lolO and Pe lolO expression levels. From these, 12 transformants with the highest Ef lolO/Pe lolO expression were selected for testing gene expression of the remaining loline genes. (see Table 8 for expression of Pe lolC, D, F, T, O and Ef lolO in biological triplicates of the 12 transformants). Variable gene expression further supported selection against toxic intermediate production in this system and highlighted the requirement for careful selection of transformants with appropriate gene expression ratios. A transformant (no. 17 in Table 8), which did not express Pe lolC, but expressed all five subsequent genes, was fed with 2 mM ACPP and produced 3.63 .mu.M NANL.
TABLE-US-00007 TABLE 7 Summary observations of expression of P. expansum lol genes and lolines and/or intermediates production in hosts tested positive for ACPP with Pe lolC expression Summary of transformed Lolines and/or genes of Plasmid Transcription intermediates Fungus interest number of lol genes detected A. niger Pe lolCDFTO pBTL74, Yes 610 .mu.M ACPP, ATCC pBTL78, 450 .mu.M 1-AP, 7.3 1015 pBTL77, .mu.M AcAP (in the pBTL76, transformant pBTL75 with the most loline pathway output); NANL not detected A. niger Pe lolC, Ef pBTL74, Yes 385 .mu.M ACPP, 45 ATCC lolDFT and pBTL38, .mu.M 1-AP, 18 .mu.M 1015 Eu lolA1 pBTL32, AcAP (in the pBTL40, transformant pBTL57 with the most loline pathway output) B. bassiana Pe lolCDFTO pBTL74, Yes 1892 .mu.M ACPP, K4B1 pBTL78, 35 .mu.M 1-AP, 68 pBTL77, .mu.M AcAP (in the pBTL76, transformant pBTL75 with the most loline pathway output); NANL not detected. B. bassiana Pe lolCDFTO pBTL74, Yes 3.63 .mu.M NANL K4B1 and Ef lolO pBTL78, (in a pBTL77, transformant pBTL76, expressing Pe pBTL75, lolDFTO and Ef pBTL33 lolO fed with 2 mM ACPP).
TABLE-US-00008 TABLE 8 Gene expression in biological triplicate cultures of B. bassiana transformants positive for Ef lolO in Pe lolC,D,F,T,O parent background Fold change (relative to act) Isolate Triplicate Pe Pe Pe Pe Pe Ef Chemical number ID lolC lolD lolF lolT lolO lolO analysis 5 5a 0.07 13.91 0.23 0.05 3.58 6.61 Not 5b 2.40 11.70 0.36 0.24 2.73 3.88 processed 5c 3.59 19.18 3.58 0.35 2.51 2.84 for chemical analysis since all gene expressed but low expression of lolF,T. 7 7a 15.24 30.73 0.00 0.00 3.77 3.17 Not 7b 12.94 34.60 0.00 0.00 3.77 3.60 processed 7c 17.15 25.39 0.00 0.00 3.39 4.85 for chemical analysis since Pe lolF, T not expressed. 8 8a 12.52 34.72 52.31 4.10 3.36 1.49 2142 .mu.M 8b 8.33 2.84 31.88 5.33 4.13 2.53 ACPP, 73 8c 9.03 13.58 41.65 3.16 4.57 3.10 .mu.M 1-AP and 4 .mu.M AcAP. NANL not detected. 9 9a 16.06 12.03 0.00 0.00 3.72 4.56 Not 9b 23.27 9.19 0.00 0.00 6.41 7.00 processed 9c 11.48 37.46 0.02 0.00 4.80 4.01 for chemical analysis since Pe lolF, T not expressed. 14 14a 24.59 0.00 5.40 0.00 1.45 15.88 Not 14b 6.77 0.00 6.69 0.00 1.15 5.85 processed 14c 4.96 0.00 5.06 0.00 0.99 4.59 for chemical analysis since Pe lolD, T not expressed. 15 15a 22.36 8.70 0.00 4.47 4.85 8.58 Not 15b 12.44 7.98 0.00 2.65 3.32 6.98 processed 15c 19.75 19.14 0.00 2.44 4.99 7.73 for chemical analysis since Pe lolF not expressed. 17 17a 0.00 21.16 10.04 0.93 4.58 5.11 Only Pe 17b 0.00 29.66 8.85 0.75 4.10 4.90 lolC not 17c 0.00 56.08 30.17 0.82 9.27 4.73 expressed, hence fed 2 mM ACPP. 147 .mu.M 1-AP, 24 .mu.M AcAP and 3.63 .mu.M NANL. 19 19a 11.88 12.05 0.00 0.00 2.82 3.18 Not 19b 26.58 15.92 0.00 0.00 3.97 3.63 processed 19c 21.68 38.78 0.00 0.00 4.35 2.71 for chemical analysis since Pe lolF, T not expressed. 20 20a 0.00 0.00 0.00 0.00 5.36 13.89 Not 20b 0.00 0.00 0.00 0.00 5.20 9.33 processed 20c 0.00 0.00 0.00 0.00 4.96 12.60 for chemical analysis since Pe lolC,D,F, T not expressed. 22 22a 32.89 59.77 24.56 5.50 3.89 5.47 2463 .mu.M 22b 46.22 103.09 44.81 3.54 6.78 3.93 ACPP and 22c 21.07 37.53 11.43 4.05 3.84 4.00 11 .mu.M 1- AP. AcAP and NANL not detected. 24 24a 0.00 0.00 0.00 0.00 11.75 11.41 Not 24b 0.00 0.00 0.00 0.00 4.72 4.82 processed 24c 0.00 0.00 0.00 0.00 10.70 6.46 for chemical analysis since Pe lolC,D,F, T not expressed. 25 25a 24.01 57.00 31.40 0.00 3.66 1.83 Not 25b 48.38 129.60 97.61 0.00 6.61 3.06 processed 25c 31.34 183.34 87.54 0.00 7.87 2.84 for chemical analysis since Pe lolT not expressed.
A Native N-Acetyltransferase of the Heterologous Host Converts 1-AP to AcAP
[0296] Epichloe lolCDFTEOUA1 were transformed to heterologous hosts in accordance with the general scientific consensus that the former seven genes from Epichloe are necessary and sufficient to produce NANL, and that lolA, while not necessary for NANL production, would increase precursor OAH levels resulting in a good flux of intermediates through the pathway. However, existing experimental evidence does not fully clarify the function and necessity of lolD, lolF, lolU, and lolE for NANL production (see Table 9 for summary and references to experimental evidence of loline gene functions).
TABLE-US-00009 TABLE 9 Putative functions of loline pathway genes and their role in the loline pathway. Predicted function (as listed on Putative role of the Reference for Gene (Schardl, et al., encoded enzyme in experimental name 2013)) the loline pathway evidence loLA Amino acid Increasing the levels of Current study binding OAH lolC gamma-class PLP Formation of ACPP Current study enzyme from proline and OAH lolD alpha-class PLP Decarboxylation of Not available enzyme pyrrolodinium ion lolF Monooxygenase Oxidative Not available decarboxylation of ACPP to form pyrrolodinium ion lolT alpha-class PLP Cyclisation of (Zhang, et al., 2009, enzyme pyrrolodinium Pan, et al., 2014) ion(s) to form 1-AP lolE Nonheme iron Not clear Not available dioxygenase lolO Nonheme iron Formation of the C2-C7 (Pan, et al., 2014, dioxygenase ether bridge to form Pan, et al., 2018) NANL lolU Unknown Unknown Not available lolN Acetannidase Deacetylation of NANL (Pan, et al., 2014) to form norloline lolM N- Methylation of norloline (Pan, et al., 2014) Methyltransferase to form loline, and of loline to form NML lolP Cytochrome P450 Oxygenation of NML to (Spiering, et al., monooxygenase form NFL 2008) Note only the references which give experimental proof of gene function are listed.
[0297] The roles of lolF and lolD are well supported by biochemical theory that the pyrrolodinium ions, which are the putative products of LoID- and LoIF-catalysed reactions, are likely to be highly unstable compounds not able to be synthesised for authentic standards or detected by targeted LC-MS/MS (D. Rennison, personal communication, 2016). Therefore, the applicants focused on clarifying the function of lolE and lolU.
[0298] lolU was considered by the applicants to be a possible candidate for the acetylation of 1-AP to AcAP, based on the identification of an HMM signature of CoA-dependent acyltransferases superfamily in InterPro (https://www.ebi.ac.uk/interpro/) and the top hit to acetyltransferase in Swiss-MODEL structural analysis (https://swissmodel.expasy.org/). When fed with 2 mM 1-AP, AcAP was detected in two independent B. bassiana transformants which were expressing E. festucae E2368 lolU, as well as in two independent transformants without lolU. No AcAP was observed in the growth medium with 2 mM 1-AP control. No statistical difference in 1-AP acetylation was observed between lolU-containing and non-containing B. bassiana K4B1. To further explore this preliminary data that showed that an N-acetyltransferase gene native to the host is capable of the acetylation of 1-AP to AcAP, a 1-AP feeding assay was done using a range of fungi. No ACPP or hydroxy-AcAP was detected in any culture. However, wild type strains of all fungi tested, except for E. festucae Fl1, S. cerevisiae and S. indica, were capable of acetylating 1-AP to AcAP (see Table 10 for details). All other Epichloe strains tested (E. uncinata AR1006 and E. festucae E2368), and an Fl1 strain carrying Ef lolDFTEOU were capable of the acetylation however, which may indicate that the Fl1 strain tested may have converted 1-AP to AcAP, albeit less efficiently--and thus at levels less than the detection threshold. While the ability of wild type fungi to convert 1-AP to AcAP without lolU is not conclusive evidence that lolU plays a role in acetylation of 1-AP to AcAP in the native system, it is proof that a native acetyl-transferase, perhaps universally present across Kingdom Fungi, has the ability to convert 1-AP to AcAP.
TABLE-US-00010 TABLE 10 Production of 1-AP to AcAP by different fungi Species Strain 1-AP fed (.mu.M) AcAP detected (.mu.M) A. niger ATCC 1015 1000 10.99 A. niger ATCC 1015 1000 7.15 B. bassiana K4B1 1000 9.97 B. bassiana K4B1 1000 8.63 B. bassiana K4B1 lolU3 1000 8.52 B. bassiana K4B1 lolU3 1000 15.45 B. bassiana K4B1 lolU20 500 9.03 B. bassiana K4B1 lolU20 500 6.28 E. festucae Fl1 1000 <0.25 E. festucae Fl1 1000 <0.25 E. festucae E2368 500 2.38 E. festucae E2368 500 0.48 E. festucae Fl1 l0l9 500 0.95 E. festucae Fl1 l0l9 500 1.41 E. uncinata AR1006 500 0.88 E. uncinata AR1006 500 0.88 E. nigrum SF7849 1000 11.57 E. nigrum SF7850 1000 6.79 P. expansum ICMP 8595 1000 8.11 P. expansum ICMP 8595 1000 5.41 R. solani ICMP 17586 500 7.92 R. solani ICMP 17586 500 7.16 Rhizopus sp. 1000 8.1 Rhizopus sp. 1000 14.58 S. zeae EBTL 218 500 22.94 S. zeae EBTL 219 500 19.22 T. reesei ATCC 56765 1000 32.44 T. reesei ATCC 56765 1000 28.86 S. indica ATCC 204458 1000 <3 S. indica ATCC 204458 1000 <3 U. isabellina ICMP 22148 1000 70 U. isabellina ICMP 22148 1000 82 K. marxianus Y-1008 1000 310 K. marxianus Y-1008 1000 224 S. cerevisiae BY4743 1000 <3 S. cerevisiae BY4743 1000 <3
[0299] Epichloe lolE, the only other nonheme iron dioxygenase besides lolO present in the Clavicipitaceae loline cluster, is suggested to be `not absolutely required` in the ether bridge formation due to unpublished observations with a lolE knockout mutant (alluded to in (Pan, et al., 2014)). However, recently it was reported that LolE has no role in vitro in the two oxygenation steps required to form NANL from AcAP (Pan, et al., 2018). But, there is no published data to provide conclusive evidence to whether LolE plays a role in vivo in one of the two oxygenation steps that are required to form NANL from AcAP. To this end, B. bassiana transformants expressing either lolE, lolO, or lolE and lolO together (`lolEO`) were obtained, fed with 0.8 mM 1-AP, and the mycelial fraction was analysed for compounds of interest. All transformants as well as wild type B. bassiana fed with 1-AP produced AcAP. However, the intermediate generated by hydroxylation of AcAP (hydroxy-AcAP) and NANL were only detected in lolO and lolEO transformant cultures. No AcAP, hydroxy-AcAP, or NANL was detected in the growth medium with 0.8 mM 1-AP control. There was no correlation of lolE expression with hydroxy-AcAP levels in lolEO cultures (R.sup.2=0.0009).
Discussion
[0300] The study of the current Example sought to achieve three main goals: (1) produce ACPP in heterologous fungal hosts by expression of the lolC gene; (2) produce NANL in heterologous hosts selected due to their ability to form ACPP; and (3) understand the requirement for lolE and lolU in the Clavicipitaceae loline production pathway.
[0301] When attempting to produce ACPP by expression of Epichloe lolC, the applicants observed that the native E. festucae gene with the introns and exons expressed under a constitutive promoter consistently resulted in a detectable lolC transcript in all filamentous fungi tested. Transcription of the gene led to ACPP production in culture in B. bassiana, M. robertsii, and the industrial strains A. niger and T. reesei (E. festucae Fl1 was not tested with the E. festucae lolC cassette). Although transcripts were detected in N. crassa, no ACPP was detected. The modified lolC--i.e., Ef lolC exons only, codon-optimzed for N. crassa--resulted in variable levels of transcription in the tested hosts and was unsuccessful in producing ACPP in all cases. The highest amount of ACPP observed in a heterologous host expressing only Ef lolC was 1390 .mu.M, produced by a B. bassiana transformant. Expression of Pe lolC was attempted only in A. niger, B. bassiana, and T. reesei, and resulted in successful transcription of Pe lolC and ACPP production in all three cases. The highest amount of ACPP observed in a heterologous host expressing Pe lolC only was 1844 .mu.M, produced by a B. bassiana transformant. The maximum amount of ACPP observed in any transformant to-date is 2463 .mu.M, which was produced by a B. bassiana transformant carrying Pe lolCDFTO and Ef lolO. It is noteworthy that endogenous ACPP amounts .gtoreq.3 mM has not been observed in any heterologous host tested so far. This may be due to the dose-dependent cytotoxic effect of ACPP, which has been reported for E. uncinata in previous literature (Faulkner, et al., 2006).
[0302] The applicants successfully produced NANL-loline-NFL, and NANL by itself, as well as the full array of chemically detectable pathway intermediates in B. bassiana via expression of Epichloe lolCDFA1TEOUMNP and Epichloe lolCDFA1TEOU, respectively. To the best of the applicant's knowledge, this is the first report of heterologous production of the full loline pathway in a non-native host. NANL was also produced in transformants expressing Epichloe lolDFTA10 in the industrial A. niger strain ATCC 1015, those expressing Ef lolDFTEOU in E. festucae strain Fl1, and those expressing the Pe lolDFTO-Ef lolO combination in B. bassiana strain K4B1. The latter three cultures were all fed 1 or 2 mM ACPP as they all lacked lolC. All detectable loline pathway intermediates upto NANL, were produced by A. niger and B. bassiana transformants that were expressing Pe lolCDFTO as well, although detectable levels of NANL was absent. The Epichloe loline genes transformed to the heterologous hosts originated from E. uncinata AR1006 (Eu lolA1) and/or E. festucae E2368 (Ef lolCDFTEOUMNP), which has currently been observed to produce lolines in planta only. All Penicillium lol genes are from P. expansum, the only Penicillium species in which the lolgenes have been reported to-date. To the best of the applicant's knowledge, the current study is also the first report of successful heterologous expression of any Pe lol gene.
[0303] Analysis of loline gene expression data showed possible bottlenecks in expression of genes such as lolF and loll). Re-transformation of lolO under the E. festucae Fl1 histone H3 promoter that was previously seen to produce relatively `high` gene expression, increased the NANL level from 0.385 .mu.M in Bb H15 to 1.209 .mu.M in Bb O16. A further increase in production in the BbO16 strain was achieved by feeding with iron (in the form of ammoniumiron(II)sulfate hexahydrate) and 2-oxoglutarate, which have been shown to bias the oxygenation reaction catalyzed by lolO-encoded enzyme towards NANL (Pan, et al., 2018).
[0304] The applicants saw that both wild type B. bassiana without any loline genes and B. bassiana lolU transformants accumulated AcAP when fed with 1-AP. This is consistent with the previous observation that there was no obvious change in the loline alkaloid profile of lolU RNAi transformants (Pan, 2014). Therefore, the applicants conclude that lolU, although a putative acetyl transferase, is not exclusively responsible for the acetylation of 1-AP to AcAP, and that a native gene of the heterologous host is capable of this conversion. The applicants also observed no lolU expression in transformant Bb O16, although it produced NANL. Collectively, these observations make the necessity of lolU in the loline pathway questionable.
[0305] Production of NANL in A. niger ATCC 1015 strain is of significance as it is a strain well-suited- and commonly used--in fermentation industry. B. bassiana, the heterologous host which has produced the highest levels of lolines in the systems tested so far, is reported as a generalist systemic endophyte. Suitability when using as a spray treatment, lifespan in soil vs. foliage, effect on plant growth, and ability to form a loline-producing but non-sporulating mutant all remain basic questions to be answered.
[0306] Based on our observations, a two-step screen, which consists of checking the ability of a selected fungus to (1) produce ACPP through expression of lolC and (2) convert 1-AP to AcAP without any transgenes, is proposed when selecting a heterologous host for expression of lolines. If a fungus is capable of both, and is fermentation compatible and/or is an established endophyte, it may be an optimal heterologous host for loline production. The best experimental approach should be then determined based on the ACPP- and 1-AP-feeding experiment results and availability of selectable markers for the selected fungus. If a fungus with a high potential as an endophyte or fermentation compatibility is capable of producing ACPP, but is unable to convert 1-AP to AcAP, it may be useful to check if it could accumulate `high` levels of 1-AP. If so, it may be used in a sequential fermentation, where the 1-AP formed is converted to the next products of the pathway by a co-cultivated host. Alternatively, if a gene that can convert 1-AP to AcAP is identified, it can be expressed in the fungus along with the other loline genes, thus giving it the ability to make the desired end product. On the other hand, if a fungus is capable of the 1-AP to AcAP conversion, but cannot produce ACPP, it may be useful to determine if ACPP-toxicity has led to the mortality of ACPP-producers and if so, an approach which transforms other desired loline genes prior to transformation of lolC, and transformation of lolC last could be considered. A case-by-case review of risks vs. benefits for each scenario is recommended.
[0307] Following the experiments of this example, the applicants postulate that, contrary to previous expectation for requirement of seven genes, expression of just five genes --lolC, lolD, lolF, lolT, lolO--in a heterologous host should result in the production of NANL--the first fully cyclized loline intermediate of the pathway.
TABLE-US-00011 TABLE 11 Detail of loline gene constructs used in the current application Alternative Transgene Promoter loline (Predicted) Name name promoter(s) organism gene Gene function Gene origin Gene modifications pBTL2 pBAR-GFP Translation Aureobasidium None Not applicable Not applicable Not applicable elongation factor pullulans 1a pBTL4 pUC57- Glyceraldehyde 3- M. robertsii ARSEF lolC .gamma.-class PLP E. festucae Codon-optimised for N. lolCDF phosphate 23 cystathionine E2368 crassa, CDS, C terminal synthase HA tag lolD PLP-containing E. festucae Codon-optimised for N. ornithine E2368 crassa, CDS, C terminal decarboxylase HA tag lolF FAD-containing E. festucae Codon-optimised for N. monooxygenase E2368 crassa, CDS, C terminal HA tag pBTL5 pBargfp- Glyceraldehyde 3- M. robertsii ARSEF lolC .gamma.-class PLP E. festucae Codon-optimised for N. lolCDF phosphate 23 cystathionine E2368 crassa, CDS, C terminal synthase HA tag Glyceraldehyde 3- M. robertsii ARSEF lolD PLP-containing E. festucae Codon-optimised for N. phosphate 23 ornithine E2368 crassa, CDS, C terminal decarboxylase HA tag Glyceraldehyde 3- M. robertsii ARSEF lolF FAD-containing E. festucae Codon-optimised for N. phosphate 23 monooxygenase E2368 crassa, CDS, C terminal HA tag pBTL6 pH3-lolC Histone H3 E. festucae Fl1 lolC .gamma.-class PLP E. festucae none cystathionine E2368 synthase pBTL8 pChuk4:lolC TDH3 (same as S. cerevisiae lolC .gamma.-class PLP E. festucae Codon-optimised for N. glyceraldehyde 3- BY4743 cystathionine E2368 crassa, CDS phosphate) synthase pBTL9 pUC57- Translation E. festucae E2368 lolT PLP-containing E. festucae T2A peptides between T2A::TEO elongation factor pyrolizidinase E2368 lolT and lolE, and 1a between lolE and lolO.Codon-optimized for N. crassa, CDS Translation E. festucae E2368 lolE oxidoreductase E. festucae T2A peptides between elongation factor E2368 lolT and lolE, and 1a between lolE and lolO.Codon-optimized for N. crassa, CDS Translation E. festucae E2368 lolO non-heme iron E. festucae T2A peptides between elongation factor oxygenase E2368 lolT and lolE, and 1a between lolE and lolO.Codon-optimized for N. crassa, CDS pBTLTOPO1 TOPO Translation lolT PLP-containing E. festucae Codon-optimised for N. synthetic lolT elongation factor pyrolizidinase E2368 crassa, CDS gblock 1a pBTLTOPO2 TOPO Translation lolE oxidase E. festucae Codon-optimised for N. synthetic lolE elongation factor E2368 crassa, CDS gblock 1a pBTLTOPO3 TOPO Translation lolO non-heme iron E. festucae Codon-optimised for N. synthetic lolO elongation factor oxygenase E2368 crassa, CDS gblock 1a WT-DFA Glyceraldehyde 3- M. robertsii ARSEF lolD PLP-containing E. festucae none phosphate 23 ornithine E2368 decarboxylase Glyceraldehyde 3- E. festucae Fl1 lolF FAD-containing E. festucae none phosphate monooxygenase E2368 Histone H3 E. festucae Fl1 lolAl Amino acid E. uncinata CDS binding AR1006 pBTL11 WT-CDFA Translation M. robertsii ARSEF lolC .gamma.-class PLP E. festucae none elongation factor 23 cystathionine E2368 1a synthase Glyceraldehyde 3- M. robertsii ARSEF lolD PLP-containing E. festucae none phosphate 23 ornithine E2368 decarboxylase Glyceraldehyde 3- E. festucae Fl1 lolF FAD-containing E. festucae none phosphate monooxygenase E2368 Histone H3 E. festucae Fl1 lolAl Amino acid E. uncinata CDS binding AR1006 pBTL12 WT-TEOU Hexokinase-1 M. robertsii ARSEF lolT PLP-containing E. festucae none 23 pyrolizidinase E2368 Histone H3 M. robertsii ARSEF lolE oxidase E. festucae none 23 E2368 Glyceraldehyde 3- A. nidulans lolO non-heme iron E. festucae Contains SNP phosphate oxygenase E2368 Translation M. robertsii ARSEF lolU 15-O- E. festucae none elongation factor 23 acetyltransferase E2368 1a pBTL13 pChuk4: lolC TDH3 (same S. cerevisiae lolC .gamma.-class PLP E. festucae Codon-optimised for s. (Sc, with HA glyceraldehyde 3- BY4743 cystathionine E2368 cerevisiae, CDS, C tag) phosphate) synthase terminal HA tag pBTL14 pChuk4: lolC TDH3 (same S. cerevisiae lolC .gamma.-class PLP E. festucae Codon-optimised for N. (Sc, without glyceraldehyde 3- BY4743 cystathionine E2368 crassa, CDS HA tag) phosphate) synthase pBTL15 pCA Translation M. robertsii ARSEF lolA1 Amino acid E. uncinata CDS elongation factor 23 binding AR1006 1a Histone H3 E. festucae Fl1 lolC .gamma.-class PLP E. festucae none cystathionine E2368 synthase pBTL16 pDFT Histone H3 E. festucae Fl1 lolD PLP-containing E. festucae none ornithine E2368 decarboxylase Histone H3 B. bassiana K4B1 lolF FAD-containing E. festucae none monooxygenase E2368 Translation M. robertsii ARSEF lolT PLP-containing E. festucae none elongation factor 23 pyrolizidinase E2368 1a pBTL17 pEOU Histone H3 E. festucae Fl1 lolE oxidase E. festucae none E2368 Histone H3 B. bassiana K4B1 lolO non-heme iron E. festucae none oxygenase E2368 Translation M. robertsii ARSEF lolU 15-O- E. festucae none elongation factor 23 acetyltransferase E2368 1a pBTL18 pMNP Histone H3 E. festucae Fl1 lolM N- E. festucae none methyltransferase E2368 Histone H3 B. bassiana K4B1 lolN acetamidase E. festucae none E2368 Translation M. robertsii ARSEF lolP cytochrome P450 E. festucae none elongation factor 23 E2368 1a pBTL32 plolFnew Histone H3 E. festucae Fl1 lolF FAD-containing E. festucae none monooxygenase E2368 pBTL33 plolOnew Histone H3 E. festucae Fl1 lolO non-heme iron E. festucae none oxygenase E2368 pBTL38 plolD Histone H3 E. festucae Fl1 lolD PLP-containing E. festucae none ornithine E2368 decarboxylase pBTL40 plolT Translation M. robertsii ARSEF lolT PLP-containing E. festucae none elongation factor 23 pyrolizidinase E2368 1a pBT7L55 lolU Class I B. bassiana K4B1 lolU 15-O- E. festucae none overexpression Hydrophobin acetyltransferase E2368 pBTL56 lolO Class I B. bassiana K4B1 lolO non-heme iron E. festucae none overexpression Hydrophobin oxygenase E2368 pBTL57 plolA Translation M. robertsii ARSEF lolA1 Amino acid E. uncinata CDS elongation factor 23 binding AR1006 1a pBTL58 plolC Histone H3 E. festucae Fl1 lolC .gamma.-class PLP E. festucae none cystathionine E2368 synthase pBTL59 plolE Histone H3 E. festucae Fl1 lolE oxidase E. festucae none E2368 pBTL60 plolO Histone H3 B. bassiana K4B1 lolO non-heme iron E. festucae none oxygenase E2368 pBTL61 plolU Translation M. robertsii ARSEF lolU 15-O- E. festucae none elongation factor 23 acetyltransferase E2368 1a pBTL62 plolM Histone H3 E. festucae Fl1 lolM N- E. festucae none methyltransferase E2368 pBTL63 plolN Histone H3 B. bassiana K4B1 lolN acetamidase E. festucae none E2368 pBTL64 plolP Translation M. robertsii ARSEF lolP cytochrome P450 E. festucae none elongation factor 23 E2368 1a pBTL71 pFI1H3/lolT/ Histone H3 E. festucae Fl1 lolT PLP-containing E. festucae none glaA pyrolizidinase E2368 pBTL74 pPe-lolC Histone H3 E. festucae Fl1 lolC .gamma.-class PLP P. expansum none cystathionine ICMP 8595 synthase pBTL75 pPe-lolO Histone H3 E. festucae Fl1 lolO non-heme iron P. expansum none oxygenase ICMP 8595 pBTL76 pPe-lolT Histone H3 E. festucae Fl1 lolT PLP-containing P. expansum none pyrolizidinase ICMP 8595 pBTL77 pPe-lolF Histone H3 E. festucae Fl1 lolF FAD-containing P. expansum none monooxygenase ICMP 8595 pBTL78 pPe-lolD Histone H3 E. festucae Fl1 lolD PLP-containing P. expansum none ornithine ICMP 8595 decarboxylase pBTL80 P.sub.PIGPD-hph- Translation S. indica lolC .gamma.-class PLP P. expansum none ter.sub.glaA elongation factor cystathionine ICMP 8595 1a synthase pBTL81 WT-TEOU Hexokinase-1 M. robertsii ARSEF lolT PLP-containing E. festucae none 23 pyrolizidinase E2368 Histone H3 M. robertsii ARSEF lolE oxidase E. festucae none 23 E2368 Glyceraldehyde 3- A. nidulans lolO non-heme iron E. festucae none phosphate oxygenase E2368 Translation M. robertsii ARSEF lolU 15-O- E. festucae none elongation factor 23 acetyltransferase E2368 1a
Example 3: Screening Multiple Taxa for their Ability to Endogenously Generate AcAP from 1-AP
Introduction/Summary
[0308] The protein encoded by lolU was considered a possible candidate for the acetylation of 1-AP to AcAP, based on its structural similarity to N-acetyltransferases. However, when fed with 2 mM 1-AP, AcAP was detected in two independent B. bassiana transformants which were expressing E. festucae E2368 lolU, as well as in two independent transformants without lolU. No AcAP was observed in the growth medium with 2 mM 1-AP control. This indicated that an N-acetyltransferase gene native to the host was capable of the acetylation of 1-AP to AcAP. As this gene is yet unidentified, the ability to convert 1-AP to AcAP is currently a required characteristic for a given fungus to successfully produce NANL. Therefore, a screen of multiple fungal taxa for the ability to convert 1-AP to AcAP was proposed. This screen has been done with a set of fungi from phylum Ascomycota, to which the native loline producer Epichloe species belong, and a fungus from unplaced subphylum Mucoromycota (formerly Zygomycota). All strains tested, except Epichloe festucae strain Fl1 were able to acetylate 1-AP to form AcAP.
Materials and Methods
Fungal Strains
[0309] All fungal strains (Table 12) were sub-cultured onto potato dextrose agar (PDA) from original or glycerol stocks where possible. All subsequent sub-cultures were also performed with PDA.
TABLE-US-00012 TABLE 12 Fungal strains used in this study No. Species name Strain Class Source 1 Aspergillus niger ATCC 1015 Eurotiomycetes U.S.A 2 Beauveria bassiana K4B1 Sordariomycetes New Zealand 3 Beauveria bassiana K4B1 ::lolU3 Sordariomycetes New Zealand 4 Beauveria bassiana K4B1 ::lolU20 Sordariomycetes New Zealand 5 Epichlo{umlaut over (e)} festucae Fl1 Sordariomycetes U.S.A 6 Epicoccum sp. SF7849 Dothidiomycetes New Zealand 7 Metarhizium robertsii ARSEF 23 Sordariomycetes U.S.A 8 Penicillium expansum ICMP 8595 Eurotiomycetes Spain 9 Rhizoctonia solani ICMP 17586 Agaricomycetes New Zealand 10 Rhizopus sp. Mucoromycotina New Zealand 11 Sarocladium spp. BTL-E218 Sordariomycetes New Zealand 12 Trichoderma reesei ATCC 56765 Sordariomycetes U.S.A
Establishing Optimal Media for all Fungal Strains
[0310] A preliminary growth study for six wild type strains of fungi was performed using two complex media, PD broth (PDB) and sabouraud dextrose broth (SDB). The strains used were A. niger, B. bassiana, E. festucae, M. robertsii, P. expansum, and T. reesei.
[0311] A small square of fungi (approximately 0.5 cm.sup.2) was added to 1 ml of MilliQ water in a sterile bead beating tube. The mycelial suspension was macerated in a tissue homogenizer for 30 seconds at 4,000 RPM. 40 .mu.l of this mycelium was then added to four 50 ml Falcon tubes containing 2 ml of SDB or PDB in duplicate.
[0312] After one day the cultures were observed and growth was recorded. Those with significant biomass were fed with 22 .mu.l of 60% w/v ethanol. Three days post-inoculation, the cultures were again observed and growth noted. Overall growth was measured using a subjective scale.
Fungal Cultures and Inoculation
[0313] All fungi, excluding numbers 5 and 7, were inoculated in batches, based on qualitative observations of their growth. There are three groups, with fast, medium, and slow growing species. Each fungus was correspondingly inoculated so that the feeding of 1-AP could all be performed at the same time for all species. A small square of fungi approximately 1 cm.sup.2) was added to 1 ml of MilliQ water in a sterile bead beating tube. This tube was then bead beaten in a tissue homogenizer for 30 seconds at 4,000 RPM. 40 .mu.l of this mycelium was then added to five 50 ml Falcon tubes containing 2 ml of SDB; duplicate treatment and triplicate treatment cultures. The lids were loosely fitted on the tubes and secured with sellotape. These tubes were then incubated in an upright rack at 25.degree. C. with shaking at 125 RPM in the dark. Four media-only controls were also incubated in these conditions.
Feeding of 1-AP
[0314] 1-AP solution sufficient for the required number of treatments to give a final concentration of 2 mM was run through a vacuum pump for three minutes to concentrate it, leaving a solution containing roughly 60% w/v ethanol. This 1-AP solution was then fed in equal amounts to the treatment cultures, and an equivalent volume of 60% w/v ethanol added to the control tubes. All tubes were incubated in the same conditions for 48-72 h before harvesting.
Harvesting of Chemical Samples
[0315] One ml of mycelium and broth was macerated by bead-beating in a Precellys homogenizer, then filtered through a 0.2 .mu.m syringe filter. Filtrates were then analysed for loline intermediates via LC-MS/MS.
Biomass Measurements
[0316] For each control tube, the dry biomass was determined; the filter papers were allowed to incubate at room temperature for several days before weighing.
Results
Biomass Measurement
[0317] Individual weights of a set of labelled filter papers were recorded. Mycelium was sterilized holding .gtoreq.20 min in 10% Prevail.RTM., and was added to the corresponding filter paper and was incubated at 60.degree. C., overnight. As some of these papers had brown charred sections were lighter than expected, they were left on the bench for three more days and were weighed again. The average dry biomass weight for all species ranged between 22 and 52 mg. The highest biomass was produced by A. niger ATCC 1015, and the lowest by E. festucae Fl1 and Rhizopus spp.
Chemistry Analysis
[0318] Initially, eight different strains were analysed to test for their endogenous ability to convert 1-AP to AcAP. Of these eight, seven were found to be able to perform this conversion, with the exception being E. festucae Fl1. Roughly 0.5-3 percent of 1-AP was converted to AcAP. The highest average amount of AcAP measured was 267 .mu.M by for K. marxianus; and the lowest was 9.1 .mu.M by A. niger ATCC 1015 (Table 10).
[0319] Chemistry samples for B. bassiana K4B1 lolU #20, R. solani ICMP 17586, and Sarocladium spp. BTL-E218 were harvested, but not analysed.
Discussion
[0320] In the current screen conducted, all fungal species tested, except E. festucae Fl1 strain, were capable of acetylating 1-AP to AcAP. The amount of AcAP produced by different strains was low but still highly variable among species, with the production of AcAP by T. reesei in this study the highest detected from any 1-AP feeding study or produced by heterologous hosts in any instance (FIG. 11). This preliminary analysis supports the utility of a screen for 1-AP acetylation in identifying a suitable heterologous host for loline production.
REFERENCES
[0321] Ballester, A. R., Marcet-Houben, M., Levin, E., Sela, N., Selma-Lazaro, C., Carmona, L., et al. (2015) Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity, Mol Plant Microbe Interact 28: 232-248.
[0322] Blankenship, J. D., Spiering, M. J., Wilkinson, H. H., Fannin, F. F., Bush, L. P., and Schardl, C. L. (2001) Production of loline alkaloids by the grass endophyte, Neotyphodium uncinatum, in defined media, Phytochemistry 58: 395-401.
[0323] Cakmak, M., Mayer, P., and Trauner, D. (2011) An efficient synthesis of loline alkaloids, Nat Chem 3: 543-545.
[0324] Cermak, T., Doyle, E. L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., et al. (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting, Nucleic Acids Res 39: e82.
[0325] Chiang, Y. M., Oakley, C. E., Ahuja, M., Entwistle, R., Schultz, A., Chang, S. L., et al. (2013) An efficient system for heterologous expression of secondary metabolite genes in Aspergillus nidulans, J Am Chem Soc 135: 7720-7731.
[0326] Curtin, S. J., Zhang, F., Sander, J. D., Haun, W. J., Starker, C., Baltes, N. J., et al. (2011) Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases, Plant Physiol 156: 466-473.
[0327] Faulkner, J. R., Hussaini, S. R., Blankenship, J. D., Pal, S., Branan, B. M., Grossman, R. B., and Schardl, C. L. (2006) On the sequence of bond formation in loline alkaloid biosynthesis, Chembiochem 7: 1078-1088.
[0328] Gietz, R. D., and Schiestl, R. H. (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method, Nat Protoc 2: 31-34.
[0329] Gruber, F., Visser, J., Kubicek, C. P., and de Graaff, L. H. (1990) The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG-negative mutant strain, Curr Genet 18: 71-76.
[0330] Huang, X. (1994) On global sequence alignment, Comput Appl Biosci 10: 227-235.
[0331] Jackson, J. A., Varney, D. R., Petroski, R. J., Powell, R. G., Bush, L. P., Siegel, M. R., et al. (1996) Physiological responses of rats fed loline and ergot alkaloids from endophyte-infected tall fescue, Drug Chem Toxicol 19: 85-96.
[0332] Jeanmougin, F., Thompson, J. D., Gouy, M., Higgins, D. G., and Gibson, T. J. (1998) Multiple sequence alignment with Clustal X, Trends Biochem Sci 23: 403-405.
[0333] Li, T., Liu, B., Spalding, M. H., Weeks, D. P., and Yang, B. (2012) High-efficiency TALEN-based gene editing produces disease-resistant rice, Nat Biotechnol 30: 390-392.
[0334] Mahfouz, M. M., Li, L., Shamimuzzaman, M., Wibowo, A., Fang, X., and Zhu, J. K. (2011) De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks, Proc Natl Acad Sci USA 108: 2623-2628.
[0335] Marcet-Houben, M., and Gabaldon, T. (2016) Horizontal acquisition of toxic alkaloid synthesis in a clade of plant associated fungi, Fungal Genet Biol 86: 71-80.
[0336] Navarro-Sampedro, L., Olmedo, M., and Corrochano, L. M. (2007) How to transform Neurospora crassa by electroporation. http://www.fgsc.net/neurosporaprotocols/How%20to%20transform%20Nc%20by%20- electroporation.pdf.
[0337] Needleman, S. B., and Wunsch, C. D. (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins, J Mol Biol 48: 443-453.
[0338] Nodvig, C. S., Nielsen, J. B., Kogle, M. E., and Mortensen, U. H. (2015) A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi, PLoS One 10: e0133085.
[0339] Pan, J. (2014) Ether bridge formation and chemical diversification in loline alkaloid biosynthesis. PhD thesis. Lexington, Ky., USA: University of Kentucky
[0340] Pan, J., Bhardwaj, M., Faulkner, J. R., Nagabhyru, P., Charlton, N. D., Higashi, R. M., et al. (2014) Ether bridge formation in loline alkaloid biosynthesis, Phytochemistry 98: 60-68.
[0341] Pan, J., Bhardwaj, M., Nagabhyru, P., Grossman, R. B., and Schardl, C. L. (2014) Enzymes from fungal and plant origin required for chemical diversification of insecticidal loline alkaloids in grass-Epichloe symbiota, PLoS One 9: e115590.
[0342] Pan, J., Bhardwaj, M., Zhang, B., Chang, W. C., Schardl, C. L., Krebs, C., et al. (2018) Installation of the Ether Bridge of Lolines by the Iron- and 2-Oxoglutarate-Dependent Oxygenase, LolO: Regio- and Stereochemistry of Sequential Hydroxylation and Oxacyclization Reactions, Biochemistry 57: 2074-2083.
[0343] Penttila, M., Nevalainen, H., Ratto, M., Salminen, E., and Knowles, J. (1987) A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei, Gene 61: 155-164.
[0344] Rice, P., Longden, I., and Bleasby, A. (2000) EMBOSS: the European Molecular Biology Open Software Suite, Trends Genet 16: 276-277.
[0345] Sander, J. D., Dahlborg, E. J., Goodwin, M. J., Cade, L., Zhang, F., Cifuentes, D., et al. (2011) Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA), Nat Methods 8: 67-69.
[0346] Sander, J. D., and Joung, J. K. (2014) CRISPR-Cas systems for editing, regulating and targeting genomes, Nat Biotechnol 32: 347-355.
[0347] Schardl, C. L., Grossman, R. B., Nagabhyru, P., Faulkner, J. R., and Mallik, U. P. (2007) Loline alkaloids: Currencies of mutualism, Phytochemistry 68: 980-996.
[0348] Schardl, C. L., Young, C. A., Hesse, U., Amyotte, S. G., Andreeva, K., Calie, P. J., et al. (2013) Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci, PLoS Genet 9: e1003323.
[0349] Spiering, M. J., Faulkner, J. R., Zhang, D. X., Machado, C., Grossman, R. B., and Schardl, C. L. (2008) Role of the LoIP cytochrome P450 monooxygenase in loline alkaloid biosynthesis, Fungal Genet Biol 45: 1307-1314.
[0350] Spiering, M. J., Moon, C. D., Wilkinson, H. H., and Schardl, C. L. (2005) Gene clusters for insecticidal loline alkaloids in the grass-endophytic fungus Neotyphodium uncinatum, Genetics 169: 1403-1414.
[0351] Tzfira, T., Weinthal, D., Marton, I., Zeevi, V., Zuker, A., and Vainstein, A. (2012) Genome modifications in plant cells by custom-made restriction enzymes, Plant Biotechnol J 10: 373-389.
[0352] Wilkinson, H. H., Siegel, M. R., Blankenship, J. D., Mallory, A. C., Bush, L. P., and Schardl, C. L. (2000) Contribution of fungal loline alkaloids to protection from aphids in a grass-endophyte mutualism, Mol Plant Microbe Interact 13: 1027-1033.
[0353] Yadav, V., Kumar, M., Deep, D. K., Kumar, H., Sharma, R., Tripathi, T., et al. (2010) A phosphate transporter from the root endophytic fungus Piriformospora indica plays a role in phosphate transport to the host plant, J Biol Chem 285: 26532-26544.
[0354] Zhang, D. X., Stromberg, A. J., Spiering, M. J., and Schardl, C. L. (2009) Coregulated expression of loline alkaloid-biosynthesis genes in Neotyphodium uncinatum cultures, Fungal Genet Biol 46: 517-530.
[0355] Zhang, X., Li, M., Wei, D., Wang, X., Chen, X., and Xing, L. (2007) Disruption of the fatty acid Delta6-desaturase gene in the oil-producing fungus Mortierella isabellina by homologous recombination, Curr Microbiol 55: 128-134.
[0356] Zuccaro, A., Basiewicz, M., Zurawska, M., Biedenkopf, D., and Kogel, K. H. (2009) Karyotype analysis, genome organization, and stable genetic transformation of the root colonizing fungus Piriformospora indica, Fungal Genet Biol 46: 543-550.
INDUSTRIAL APPLICATION
[0357] The expression constructs, host cells, and methods of the invention have utility for many agricultural, horticultural, medical and veterinary applications, such as providing horticulturalists with a useful means of controlling plant pests, and providing therapies for the treatment or prevention of insect infection or infestation in humans or non-human animals.
[0358] Claims 1-19 (canceled)
Sequence CWU
1
1
741473PRTEpichloe festucae 1Met Thr Val Asp Thr Ile Thr Ser Thr Ser Asn
Gly Asn Gln Asp Val1 5 10
15Pro Lys Glu Phe Phe Leu Lys Glu Phe Glu Thr Gln Leu Leu His Val
20 25 30Gly Arg Phe Pro Asp Ile Leu
Gly Ser Cys Ala Val Pro Val Tyr Ser 35 40
45Ser Ala Ala Phe Glu Phe Asn Ser Val Ala His Gly Ala Arg Leu
Leu 50 55 60Asn Leu Thr Gln Phe Gly
Asn Ile Tyr Ser Arg Phe Thr Asn Pro Thr65 70
75 80Val Asn Val Leu Gln Asn Arg Leu Ala Gly Leu
Glu Gly Gly Val Ala 85 90
95Ala Cys Ala Val Ala Ser Gly Ser Ala Ala Val Val Val Thr Val Met
100 105 110Ala Leu Ala Gly Val Gly
Asp Asn Phe Val Ser Ser Phe His Val His 115 120
125Ala Gly Thr Phe His Gln Phe Glu Ser Leu Ala Lys Gln Met
Gly Ile 130 135 140Glu Cys Arg Phe Val
Lys Ser Arg Asp Pro Ala Asp Phe Ala Ala Ala145 150
155 160Ile Asp Asp Lys Thr Lys Phe Val Trp Leu
Glu Thr Ile Ser Asn Pro 165 170
175Gly Asn Val Ile Leu Asp Leu Glu Ala Val Ser Met Val Cys His Thr
180 185 190Lys Gly Ile Pro Leu
Ile Cys Asp Asn Thr Phe Gly Cys Ala Gly Tyr 195
200 205Phe Cys Arg Pro Ile Asn His Gly Val Asp Ile Val
Val His Ser Ala 210 215 220Thr Lys Trp
Ile Gly Gly His Gly Thr Thr Val Gly Gly Ile Ile Val225
230 235 240Asp Gly Gly Thr Phe Asp Trp
Gly Gln His Pro Asp Arg Phe Pro Gln 245
250 255Phe His Asp Pro Arg Thr Arg Leu Trp Glu Arg Phe
Ser Arg Arg Ala 260 265 270Phe
Ala Val Arg Cys Gln Phe Glu Ile Leu Arg Asp Thr Gly Ser Thr 275
280 285Leu Ser Ala Pro Ala Ala Gln Gln Leu
Leu Val Gly Leu Glu Ser Leu 290 295
300Ala Val Arg Cys Glu Arg His Ala Gln Asn Ala Ala Lys Ile Ala Asp305
310 315 320Trp Leu Arg Glu
His Pro Leu Val Ala Trp Val Ser Tyr Val Gly His 325
330 335Pro Asn His Pro Asp Tyr Gln Gly Ala Leu
Lys Tyr Leu Lys Arg Gly 340 345
350Phe Gly Ser Val Ile Cys Phe Gly Leu Arg Gly Gly Phe Glu Ala Gly
355 360 365Ala Leu Phe Cys Asp Ala Leu
Lys Met Val Ile Thr Thr Thr Asn Leu 370 375
380Gly Asp Ala Lys Thr Leu Ile Leu His Pro Ala Ser Thr Thr His
Glu385 390 395 400His Phe
Arg Ser Glu His Arg Ala Glu Ala Gly Val Thr Asp Asp Met
405 410 415Ile Arg Leu Ser Val Gly Ile
Glu Gln Ile Asn Asp Ile Lys Ala Asp 420 425
430Phe Glu Gln Ala Phe Glu Gln Val Leu Arg Gly Lys Lys Ser
Leu Arg 435 440 445Lys Pro Cys Ile
Gly Lys Ile Leu Leu Gln Asp Glu Ile Asn Glu Asp 450
455 460Leu Phe Gly Pro Ser Ala Cys Arg Thr465
4702418PRTEpichloe festucae 2Met Ala Thr Ala Val Arg Glu Ala Phe Glu
Asn His Val Lys Leu Val1 5 10
15Glu Ser Arg Asn Ser Pro Gly His Val Leu Ala Ser Ser Glu Ala Ser
20 25 30Phe Phe Val Ala Asp Leu
Asn Asp Val Val Arg Lys Trp Ala Ala Trp 35 40
45Lys Glu Ala Leu Pro Asp Ala Thr Pro Phe Phe Ala Val Lys
Ser Ser 50 55 60Tyr Asp Arg Arg Leu
Ile Gln Thr Leu Ala Thr Cys Gly Ala Gly Phe65 70
75 80Asp Cys Ala Ser Ala Glu Glu Ile Glu Leu
Thr Leu Ser Leu Gly Ile 85 90
95Gly Ala Glu Arg Ile Ile Phe Thr His Pro Cys Lys Pro Val Ser Ser
100 105 110Leu Gly Leu Cys Arg
Lys Leu Gly Ile Thr Leu Ile Thr Phe Asp Asn 115
120 125Glu Cys Glu Leu Arg Lys Leu His His His Tyr Pro
Glu Ala Gln Thr 130 135 140Val Leu Arg
Ile Phe Ala Asp Asp Pro Thr Asn Ala Asp Pro Leu Gly145
150 155 160Thr Lys Phe Gly Ala Ala Arg
Asn Asp Phe Asp Gly Leu Val Arg Leu 165
170 175Val Lys Glu Leu Asn Met Gln Leu Ala Gly Ala Ser
Phe His Ala Ala 180 185 190Pro
Ser Val Ala Val Asp Ala Ala Ala Tyr Val Arg Gly Ile Arg Asp 195
200 205Ala Ala Glu Val Phe Ala Arg Ala Arg
Gln Val Gly Leu Asn Pro Thr 210 215
220Val Leu Asp Ile Gly Gly Gly Tyr Thr Asp Ser Thr Phe Gln Gln Ile225
230 235 240Ala Gly Ala Val
Arg Pro Ala Ile Ala Glu Cys Phe Thr Ser Gln Val 245
250 255Gly Glu Gly Arg Leu Arg Ile Leu Ala Glu
Pro Gly Thr Leu Phe Ser 260 265
270Cys Ser Pro Phe Tyr Leu Ala Val Lys Val Val Ala Arg Arg Val Asn
275 280 285Ala Thr Ala Phe Gly His Glu
Pro Ala Thr Arg Leu Tyr Ile Asn Asp 290 295
300Gly Ile Tyr Ser Asn Phe Met Met Arg Phe Ile Val Asn Met Thr
Phe305 310 315 320Ser Pro
Thr Ala Val Ile Arg Glu Gly Val Trp His Asp Gln Ala Asp
325 330 335His Thr Met Arg Gly Glu Ala
Cys Ser Leu Trp Gly Arg Ser Cys Asp 340 345
350Ser Asn Asp Cys Ile Asn Arg Asp Cys Arg Leu Gly Met Lys
Val Gly 355 360 365Val Gly Asp Trp
Leu Val Phe Lys Asp Met Gly Ala Tyr Thr Thr Val 370
375 380Cys Asn Thr Thr Phe Asn Gly Phe Thr Ser Ser Asn
His Thr Ile Tyr385 390 395
400Leu Glu Pro Thr Gln Val Asp Lys Ala Gln Ser Thr Phe Glu Gln Leu
405 410 415Ala
Ile3540PRTEpichloe festucae 3Met Thr Leu Thr Asn Leu Asp Val Ile Val Val
Gly Ala Gly Phe Ser1 5 10
15Gly Ile Leu Ala Ala His Arg Leu Arg Lys Leu Gly Phe Arg Val Gln
20 25 30Gly Phe Glu Arg Gln Glu Arg
Leu Gly Gly Val Trp Arg Glu Asn Ala 35 40
45Tyr Pro Gly Ala Ala Val Asp Ser Leu Phe Pro Phe Tyr Gln Phe
Tyr 50 55 60Asp Ala Glu Leu Leu Gln
Asp Trp Glu Trp Gly Glu Gln Phe Pro Thr65 70
75 80Arg Ala Glu Met Leu Arg Tyr Phe Asp His Val
Asp Lys Arg Trp Glu 85 90
95Ile Ser Thr Ser Phe Glu Phe Gly Val Ser Val Ser Ala Ala Arg Tyr
100 105 110Ser Glu Thr Thr Gln Arg
Trp Thr Val Ser Leu Glu Asp Gly Arg Arg 115 120
125Ala Glu Ala Gln Trp Phe Ile Pro Ala Val Gly Phe Ser Ser
Val Leu 130 135 140Asn Ile Pro Lys Ile
Pro Gly Met Ser Arg Phe Arg Gly Pro Ile Tyr145 150
155 160His Thr Ala Lys Trp Pro His Asp Ala Val
Ser Met Arg Gly Lys Arg 165 170
175Val Ala Val Ile Gly Thr Gly Pro Ser Gly Val Gln Ile Ile Gln Ala
180 185 190Val Gly Lys Ile Ala
Lys Ala Met Thr Ile Phe Gln Gln Ser Pro Cys 195
200 205Leu Thr Leu Arg Lys Tyr Gly Ser Pro Ser Gln Thr
Ala Thr Ala Leu 210 215 220Cys Met Arg
Pro Asp Asp His Arg Glu Ala Leu Arg Leu Gly Leu Gln225
230 235 240Thr Ser Asn Gly Phe Gly Tyr
Val Ile Arg Asp Gln Asp Thr Leu Asp 245
250 255Val Pro Ile Glu Glu Arg Asn His Phe Tyr Gln Gln
Arg Tyr Leu Ala 260 265 270Gly
Gly Trp Ala Phe Trp Met Ala Gly Phe Arg Asp Leu Cys Gln Asn 275
280 285Ile Gln Ala Asn Arg Asp Ala Tyr Asp
Phe Trp Ala Arg Arg Thr Arg 290 295
300Ala Arg Ile Ser Asp Val Thr Lys Arg Glu Leu Leu Val Pro Gln Ile305
310 315 320Pro Pro Phe Ala
Phe Gly Ile Lys Arg Pro Cys Leu Glu Glu Asp Leu 325
330 335Tyr Glu Val Met Asp Gln Pro His Val Lys
Val Ile Asp Ile Ser Asn 340 345
350Gln Gln Ile Glu Leu Ile Thr Glu Thr Gly Ile Arg Val His Gly Gln
355 360 365Thr Val Glu Cys Glu Ala Ile
Ile Phe Ala Thr Gly Phe Gly Asp Glu 370 375
380Ala Ser Gly Leu Arg Ser Leu His Ile Arg Gly Arg Asn Gly Ile
Arg385 390 395 400Leu Glu
Asp Ala Trp Ser Asp Gly Val Glu Ser His Leu Gly Met Ala
405 410 415Ile His Ser Phe Pro Asn Met
Phe Phe Leu Tyr Gly Pro Gln Cys Pro 420 425
430Thr Leu Leu Val Asn Ser Pro Ala Val Ile Thr Val Gln Val
Glu Trp 435 440 445Leu Cys Glu Ile
Ile Ser Arg Cys Gln Gln Ala Gly Ile Tyr Gln Leu 450
455 460Glu Ala Thr Ser Lys Ser His Cys Gln Trp Glu Lys
Lys Met Ser Leu465 470 475
480Leu Trp Asp Lys Thr Leu Tyr His Thr His Ala Arg Lys Ser Lys Thr
485 490 495Thr Ala Ala Ala Asn
Lys Glu Glu Lys Thr Trp Val Gly Gly Leu Ile 500
505 510Leu Tyr Arg Arg Glu Leu Glu Asn Cys Leu Ala Asn
Asn Leu Glu Gly 515 520 525Phe Gln
Ala Trp His Val Glu Glu Ala Ala Leu Leu 530 535
5404209PRTEpichloe festucae 4Met Leu Asp Glu Ser Pro Met Arg Lys
Gly Asp Ser Val Ser Asn Asp1 5 10
15Gln Gly Asn Pro Glu Ser Asn Ala Ser Val Ser Ile His Gln Gln
Asn 20 25 30Gln Ile Ile Thr
Cys Val Ser Pro Asp Pro Val Cys Pro Asn Ala Ile 35
40 45Arg Ile Lys Arg Asp Ile Val Ile Val Arg Leu Arg
Pro Val Glu Ser 50 55 60Cys Pro Gly
Tyr Arg Phe Phe Arg Arg Val Phe Glu Thr Leu Glu Lys65 70
75 80Trp Gln Leu Gln Val Asp Met Phe
Ser Thr Ser Leu Gly Arg Ile Thr 85 90
95Leu Ala Leu Gly Ala Ala Ala Leu Gln Ala Gly Ile Gly Asp
Ser Cys 100 105 110Ser Ala Arg
Asn Asp Met Met Ser Arg Asp Leu Met His Gly Met Gln 115
120 125Lys Leu Leu Pro Asp Asp His Ile Glu Leu Phe
Pro His Met Thr Ile 130 135 140Ile Ser
Val Val Glu His Pro Ser Arg Arg Met Ala Gly His Ile Phe145
150 155 160Ala Thr Met Asp Ala Asn Asp
Ile Pro Thr Val Met Ile Ser His Asp 165
170 175Ala Ala Arg Leu Gly Ile Ala Cys Ala Ile Ser Glu
Gln Tyr Thr Ala 180 185 190Lys
Ala Leu Cys Val Phe Glu Gln Cys Leu Phe Arg Tyr Ser Leu Thr 195
200 205His5454PRTEpichloe festucae 5Met Thr
Val Asn Ser Lys Arg Ile Pro Phe Gly Lys Pro Met Leu Glu1 5
10 15Ala Phe Cys Met Asp Pro Glu Tyr
Thr Asn Leu Asn Ser Ser Ser Cys 20 25
30Gly Ser Trp Pro Lys Val Val Ser Lys Gln Ile Arg Asp Tyr Trp
Ser 35 40 45Leu Leu Glu Ala Gln
Pro Asp Leu Phe Ser Glu Phe Ser Gln Gly Leu 50 55
60Val Leu Gln Glu Ala Arg Leu Gly Leu Ala Arg Leu Val His
Ala Ala65 70 75 80Val
Ser Glu Cys Val Leu Val Ser Asn Val Thr Thr Gly Ile Phe Thr
85 90 95Val Leu Tyr Asn Gln Ala Phe
Glu Glu Arg Asp Val Val Val Thr Leu 100 105
110Ser Thr Thr Tyr Gly Ala Ile Asp His Gly Ile Thr Ser Leu
Ala Glu 115 120 125Ala Arg Pro Phe
Lys Thr Arg Arg Val Glu Phe Glu Leu Pro Thr Thr 130
135 140Gly Glu Lys Ile Val Ser Arg Phe Glu Thr Thr Met
Ala Gln Ile Arg145 150 155
160Ala Glu Gly Leu Arg Pro Arg Leu Ala Ile Leu Glu Thr Ile Val Ser
165 170 175Ile Pro Ala Val Arg
Met Pro Phe Glu Asp Leu Leu Arg Val Cys Gln 180
185 190Lys Glu Gly Ile Met Thr Leu Val Asp Gly Ala His
Ser Val Gly Gln 195 200 205Phe Glu
Val Asn Leu Gln Glu Leu Gln Pro Asp Phe Phe Val Ser Asp 210
215 220Cys His Lys Trp Leu Phe Val Pro Arg Pro Cys
Ala Phe Leu Tyr Val225 230 235
240Ala Glu Arg Asn Gln His Met Met Arg Ser Ala Ile Pro Thr Ser Phe
245 250 255Gly Phe Ile Pro
Lys Asn Gly Asn Ser Arg Leu Pro Leu Trp Ser Gln 260
265 270Met Val Ser Ala Ser Glu Thr Ala Ser Ser Phe
Glu Thr Leu Phe Ala 275 280 285Tyr
Thr Ala Thr Ser Asp Asn Met Pro His Leu Cys Ile Pro Thr Ala 290
295 300Leu Arg Phe Arg Arg Asp Val Cys Gly Gly
Glu Thr Ala Ile Tyr Glu305 310 315
320Tyr Ile Lys Trp Leu Ala Thr Glu Gly Gly Asp Lys Val Ala Asn
Ile 325 330 335Leu Gln Thr
Glu Val Leu Glu Glu Pro Ser Leu Gly Ala Gly Val Asp 340
345 350Gly Gln Met Arg Asp Cys Gly Ile Val Thr
Val Arg Leu Pro Leu Ala 355 360
365Ile Ala Thr Gly Pro Ser Thr Ala Pro Ala His Val Pro Met Pro Gly 370
375 380Gly Ala Leu Thr Glu Lys Glu Val
Gly Pro Ala Val Arg Tyr Leu Thr385 390
395 400Lys Ala Leu Ala Glu Arg Tyr Lys Thr Trp Ile Pro
Ile Ile Asp Tyr 405 410
415Arg Gly Trp Ile Trp Ala Arg Leu Cys Ala Gln Val Tyr Leu Glu Val
420 425 430Ser Asp Phe Glu Met Ala
Gly Asn Ala Leu Lys Gly Ile Cys Glu Glu 435 440
445Ile Leu Ser Arg Glu Arg 4506257PRTEpichloe festucae
6Met Ile Ala Ala Ser Ser Pro His Ser Gly Val Val Ser Ala Glu Asp1
5 10 15Ile Glu Phe Tyr Gln Ala
Asn Gly Tyr Leu Arg Leu Pro Gln Glu Ala 20 25
30His Gly Leu Phe Asp Asp Leu Ala Lys Leu Gln Val Trp
Val Ala Lys 35 40 45Ile Ser Gln
Trp Gly Leu Glu Thr Gly Lys Trp Arg His Tyr Tyr Glu 50
55 60Thr Thr Asn Gly Lys His Leu Leu Trp Gly Thr Glu
Lys Leu Met Glu65 70 75
80Tyr His Ala Pro Met Arg Asp Leu Ile Ala Gly Asp Ala Pro Leu Ala
85 90 95Leu Leu Lys Ser Leu Thr
Gly Lys Asp Met Val Val Phe Lys Asp Glu 100
105 110Ile Gly Trp Lys Leu Pro Gly Gly Lys Gly Ala Val
Pro His Leu Asp 115 120 125Arg Pro
Ala Tyr Ser Met Phe Ala Pro Glu Phe Ile Glu Ile Met Ile 130
135 140Ala Val Asp Ala His Thr Val Glu Asn Gly Cys
Leu Gln Phe Val Pro145 150 155
160Gly Ser His Lys Glu Ala Val Pro Ile Ser Ala Asp Gly Arg Ile Ala
165 170 175Ser Ala Trp Leu
Glu Gly Lys Glu Phe Ile Pro Met Val Leu Asp Pro 180
185 190Gly Asp Val Leu Ile Phe Asn Glu Ser Met Ala
His Arg Leu Glu Pro 195 200 205Asn
Lys Thr Asp Gln Arg Arg Ala Ala Val Phe Gly Thr Tyr His Phe 210
215 220Asp Leu Ser Gln Pro Asp Leu Arg Asp Lys
Phe Tyr Ala His Arg Leu225 230 235
240Ile His Ser Pro Pro Glu Asn Ala Trp Val Glu Thr Val Gly Ala
Gln 245 250
255Thr7363PRTEpichloe festucae 7Met Thr Val Thr Asn Lys Pro Val Lys Pro
Ala Asn Val Pro Val Met1 5 10
15Asp Phe Glu Ala Ile His Ala Ser Val Gly Asn Glu Arg Lys Lys Tyr
20 25 30Leu Arg Gln Leu Asp Glu
Ala Trp Ser His His Gly Ala Ile Tyr Val 35 40
45Ile Asn His Ser Ile Gly Thr Glu Thr Leu Glu Glu Ala Phe
Ala Trp 50 55 60Cys Lys Lys Phe Phe
Asp Leu Pro Leu Ala Val Lys Asn Ser Val His65 70
75 80Ile Pro Pro Asp Val Ser Lys His Phe Gln
Gly Trp Thr Gly Thr Gly 85 90
95Glu Ala Ile Ser Ser Gln Gly Val Trp Asp Pro Asp Glu Ile Glu Arg
100 105 110Leu Arg Lys Glu Met
Pro Thr Glu Leu Lys Glu Ala Met Glu Leu Gln 115
120 125Asp Pro Cys Gly Thr Tyr Pro Pro Gly Leu Pro Asp
Leu Asn Leu Val 130 135 140Glu Gln His
Leu Pro Gly Tyr Leu Asp Phe Leu Lys Lys Trp Phe Ala145
150 155 160Ala Cys Tyr Arg Gln Ser Leu
Gln Asn Met Arg Leu Val Cys Glu Ile 165
170 175Leu Gly Met Glu Asp Leu Asp Tyr Ile Gly Lys Lys
Phe Ala Pro Arg 180 185 190His
Met Ser Thr His Ser Thr Trp Asn Tyr Phe Leu Gly Gln Pro Val 195
200 205Ser Gln Leu Ala Arg Gly Ser Ala Asn
Arg Leu Asn Ala His Thr Asp 210 215
220Tyr Cys Gln Phe Thr Met Leu Phe Gln Asp Met Val Gly Gly Leu Glu225
230 235 240Leu His Asp Tyr
Glu Glu Asp Ile Tyr Arg Pro Val Pro Pro Ile Lys 245
250 255Gly Ala Met Ile Val Gln Val Gly Asp Leu
Leu Glu Lys Gln Thr Asn 260 265
270Gly Arg Trp Arg Ser Ala Leu His Arg Val Thr Ala Pro Ser Arg Tyr
275 280 285Met Tyr Glu Gly Ser Ala Gly
Asp Asn Asp Glu Leu Val Gln Arg Tyr 290 295
300Ser Leu Val Phe Phe Gly His Leu Asn Leu Asp Glu Met Ile Glu
Pro305 310 315 320Leu Pro
Gly Cys Glu Lys Pro Gly Lys Trp Ser Thr Leu Glu Trp Lys
325 330 335Asp Arg Met Thr Ala Gly Gln
Trp Leu Ala Arg Arg Val Ala Leu Glu 340 345
350Tyr Glu Arg Lys Lys Thr Ala Ala Thr Val Met 355
3608496PRTEpichloe festucae 8Met Val Ser Ala Gly Asn Cys Ala
Ile Val Ala Pro Gly Trp Thr Arg1 5 10
15Ser Glu Asp Gly Thr Leu Thr Arg Pro Leu Asp Leu Val Glu
Asn Trp 20 25 30Leu Leu Ala
Arg Ile Gln Arg Ala Asn Thr Pro Pro Gly Arg Glu Ala 35
40 45Glu Gly Leu Thr Tyr Lys Leu Lys Leu Arg Leu
Pro His Asp Ile Asp 50 55 60Asp Pro
Ile Pro Tyr Leu Arg Arg Ala Trp Leu Val Phe Arg Tyr Val65
70 75 80Gln Pro Leu Ile Gly Ala Ile
Tyr Pro Pro Tyr Ser Glu Arg Asp Glu 85 90
95Thr Gly Arg Tyr Leu Val Thr Val Pro Pro Met Asp Pro
Glu Glu Trp 100 105 110Leu Arg
Leu Ser Phe His Val Asn Gln Gly Ser Gln Ala Val Phe Arg 115
120 125Asp Val Asp Asp Ala Gly Lys Ile Phe Arg
Pro Arg Glu Thr Ala Met 130 135 140Ala
Tyr Trp Phe Pro Ser Ser Ser Thr Leu Val Ile Arg Ser Thr His145
150 155 160Leu Arg Phe Asp Ala Val
Gly Leu Tyr Lys Ala Thr Asn Thr Phe Met 165
170 175Leu Gly Leu Glu Ser Val Phe Arg Leu Gly Leu Asp
Ala Asn Leu Tyr 180 185 190Cys
Tyr Thr Thr Asp Val Lys Gln Pro Ser Leu Pro Pro Gly Ile Asp 195
200 205Tyr Ile Leu Gly Phe Pro Pro Gln Glu
Thr Pro Val Ser His Arg Val 210 215
220Glu Arg Ala Val Asp Glu Leu Met Arg His Trp His His Gly Leu Tyr225
230 235 240Ser Leu Ser Leu
Pro Val Arg Glu Gly Ser Glu Asp Ala Ala Pro Ala 245
250 255Asn Thr Gln His Leu Val Thr Leu Phe Asp
Glu Pro Thr Leu Glu Ala 260 265
270Ile Val Ala Gly Cys Lys Lys Leu Gly Val Ser Val Ser Ala Ala Val
275 280 285His Ala Ser Ile Val Arg Val
Trp Ala Ser Phe Pro Gln Gln Gln His 290 295
300Thr Gly Ala Arg Asn Asn Met Leu Ile Pro Leu Val Ala Asn Leu
Arg305 310 315 320Pro Leu
Leu Asp Pro Lys Trp Val Val Pro Asp Tyr Ala Leu Ser Leu
325 330 335Cys Ile Phe Val Val Pro Phe
Cys Leu Thr Gly Gly Phe Glu Asp Leu 340 345
350Thr Gln Arg Leu Gly Ala Val Tyr Ser Arg Asp Leu Ser Ala
Leu Pro 355 360 365Ser Asp Pro Ala
Gly Asp Pro Val Ser Phe Leu Glu Leu Leu Pro Leu 370
375 380Tyr Asp Ser Gly Glu Ala Ala Phe Leu Gly Ser Leu
Pro Val Ala Gly385 390 395
400Cys Pro Pro Phe Arg Val Pro Asn Leu Ser Ser Leu Gly Val Leu Glu
405 410 415Arg Tyr Leu Ala Arg
Ala Tyr Gly Lys Arg Gly Ala Gln Ala Pro Val 420
425 430Cys Glu Ile Glu Asp Val Ala Leu Val Asn Ala Thr
Thr Asp Pro Thr 435 440 445Ile Glu
Phe Gln Leu Phe Thr Phe Arg Gly Thr Met Arg Leu Tyr Leu 450
455 460Tyr Tyr Asn Asp Ala Tyr Tyr Thr Glu Asp Phe
Leu Ala Pro Val Met465 470 475
480Glu Met Val Arg Asp Ser Leu Leu Gln Glu Leu Gly Leu Gly Arg Ser
485 490 4959279PRTEpichloe
festucae 9Met Thr Val Asn Ser Ser Val Lys Gln Glu Tyr Asp Ala Gln Ala
Ala1 5 10 15Ile Tyr Asp
Gly Tyr Met Asp Arg Pro Ser Gly Val Ile Glu Arg Gln 20
25 30Leu Phe Thr Ala Ala Leu Gly Asn Cys Thr
Gly Leu Thr Val Leu Asp 35 40
45Leu Gly Gly Gly Thr Gly Leu Lys Ala Arg Glu Ala Ala Asp Ala Gly 50
55 60Ala Ser Ala Val Asp Val Ile Asp Leu
Ser Pro Glu Met Met Arg Val65 70 75
80Gly Arg Asp Ala Glu Gln Ala Gly Pro Arg Arg Gly Lys Asp
Ile Leu 85 90 95Arg Trp
Tyr Glu Gly Asp Val Thr Ser Ala Asp Leu Val Glu Thr Leu 100
105 110Pro Gly Leu Arg Gly Pro Tyr Asp Leu
Val Ile Val Gly Trp Thr Phe 115 120
125Asp His Ala His Asn Arg Ala Gln Leu Glu Ala Met Trp His Asn Ala
130 135 140Val Val Arg Leu Lys Pro Ser
Thr Gly Arg Leu Leu Val Val Arg Asn145 150
155 160Gly Asp Pro Arg Ser Pro Ala Val Thr Gly Gly Arg
Tyr Gly Ile Arg 165 170
175Tyr Ala Gly His Val Pro Ile Pro Gly Gly Phe Arg Phe Arg Asp Gln
180 185 190Met Ile Arg Trp Gly Gly
Gly Gly Gln Gln Gln Gly Thr Lys Pro Asp 195 200
205Gln Phe Glu Ile Leu Ala Asp Tyr Glu Thr Thr Ala Leu Glu
Val Met 210 215 220Tyr Ser Gly Ser His
Glu Met Tyr His Gln Phe Gly Leu Thr Asp Ile225 230
235 240Arg Thr Gln Pro Tyr Glu Glu Thr Ala Ala
Val Arg Ala Asp Pro Ala 245 250
255Phe Trp Ala Gln Phe Leu Glu Asn Pro Cys Leu Ala Val Val Thr Ala
260 265 270Arg Lys Met Gly Lys
Val Glu 27510572PRTEpichloe festucae 10Met Ser Asn Met Arg Ala Thr
Ile Ala Gly Gly Ala Arg Trp Gln Glu1 5 10
15Val Ala Ala Asp Cys Gln Gln His Arg Asp Ala Thr Val
Thr Lys Ile 20 25 30His Pro
Pro Ile Pro Asp Thr Gln Ala Leu Glu Ser Leu Phe Ala Ser 35
40 45Gly Asp Pro Arg Asp Val Ser Ser Ile Pro
Thr Leu Val Leu Ser Glu 50 55 60Gly
Glu Leu Ala Ile Thr Ser Ala Asn Val Glu Asp Leu Val Pro Arg65
70 75 80Leu Ala Ser Gly Glu Trp
Ser Ala Ser Thr Val Leu Lys Ala Phe Leu 85
90 95Arg Arg Ala Ala Leu Ala Gln Arg Leu Val Asn Cys
Val Thr Glu Met 100 105 110Leu
Ser Glu Thr Ala Leu Lys Arg Ala Ala Glu Leu Asp Glu His Leu 115
120 125Ala Val His Gly Lys Pro Ile Gly Pro
Leu His Gly Val Pro Ile Ser 130 135
140Val Lys Glu His Ile Ala Met Lys Gly Leu Asp Val Asn Gly Gly Tyr145
150 155 160Val Ser Glu Val
Gly Arg Val Ala Glu Glu Asp Ala Leu Ile Leu Asn 165
170 175Ile Leu Arg Asp Ala Gly Ala Ile Phe Tyr
Val Arg Thr Thr Glu Pro 180 185
190Gln Ser Ser Met His Leu Glu Thr Ser Ser Ser Leu Tyr Gly Glu Thr
195 200 205Val Asn Pro Phe Asn Thr Thr
Leu Thr Ser Gly Gly Ser Ser Gly Gly 210 215
220Glu Gly Ala Ile Ile Ala Met Arg Gly Ser Val Leu Gly Val Gly
Ser225 230 235 240Asp Ile
Gly Gly Ser Ile Arg Ser Pro Ala His Cys Asn Gly Ile Phe
245 250 255Gly Phe Lys Pro Thr Thr Gly
Arg Leu Pro Thr Leu Gly Trp Phe Ala 260 265
270Leu Met Val Gly Ser Glu Ala Ile His Ala Thr Thr Gly Pro
Leu Ser 275 280 285Thr Ser Ile Glu
Gly Leu Arg Leu Phe Thr Lys Thr Leu Leu Asp Ala 290
295 300Lys Pro Trp Leu Gln Asp Pro Ser Leu Thr Pro Met
Glu Trp Arg Asp305 310 315
320Met Ser Thr Ala Phe Ala Gly Arg Arg Leu Lys Val Ala Val Met Trp
325 330 335Asp Asp Gly Val Val
Lys Pro His Pro Pro Val Thr Arg Ala Leu Lys 340
345 350Ser Val Val Glu Asp Leu Lys Lys Ser Glu Lys Ile
Glu Val Val Asp 355 360 365Trp Lys
Pro Trp Lys His Asp Leu Ala Trp Ser Ile Ile Ala Gly Leu 370
375 380Tyr Phe Cys Asp Gly Gly Ala Gln Leu Asn Ala
Ala Phe Glu Ala Ala385 390 395
400Lys Glu Pro Leu Arg Pro Leu Ser His Trp Ile Leu Lys Glu Asn Pro
405 410 415His Val Lys His
His Ser Ile Ala Ser Leu Trp Ser Ala Cys Ala Glu 420
425 430Arg Asp Ala Tyr Arg Leu Lys Tyr Ala Glu Leu
Trp Asn Asp Thr Ala 435 440 445Lys
Gly Gly Gly Gly Pro Val Asp Val Ile Leu Cys Pro Ala Gly Pro 450
455 460Gly Ala Ala Pro Lys Leu Asn Thr Ser Arg
Tyr Trp Gly Tyr Thr Ala465 470 475
480Gln Trp Asn Leu Leu Asp Tyr Pro Ala Val Val Phe Pro Thr Gly
Asp 485 490 495Ile Val Ser
Val Glu Lys Asp Gly Ala Ala Gly Glu Gln Gly Gly Gly 500
505 510Asp Pro Ala Ser Gly Ala Asp Leu Asp Asn
Trp Ser Leu Trp Thr Glu 515 520
525His Gly Ala Glu Gly Tyr Ser Asn Ala Pro Leu Ala Leu Gln Leu Val 530
535 540Ala Arg Arg Cys Asp Asp Glu Lys
Leu Leu His Ala Leu Glu Met Val545 550
555 560Met Lys Glu Ala Gly Leu Ala Thr Glu Leu Val Gly
565 57011496PRTEpichloe festucae 11Met Asp
Leu Thr Gln Phe Lys Thr Ala Gly Ile Val Trp Pro Thr Val1 5
10 15Ala Ala Met Ala Ile Ser Tyr Ile
Leu Leu Ser Ser Phe Leu Ser Trp 20 25
30Tyr Arg Leu Arg His Ile Pro Gly Pro Phe Leu Ala Ser Ile Ser
Ser 35 40 45Leu Trp Asn Val Leu
Asn Ile Val Thr Gly Arg Thr Ser Pro Val Leu 50 55
60Glu Lys Leu Pro Gly Lys Tyr Gly Pro Met Val Arg Thr Gly
Pro Asn65 70 75 80Tyr
Val Leu Thr Asp Asp Ala Glu Ile Leu Arg His Val Asn Gly Ala
85 90 95Arg Ser Thr Tyr Pro Arg Asn
Gly Trp Tyr Glu Gly Phe Lys Val Asp 100 105
110Glu His Asp His Met Gly Ser His Ile Asp Thr Ser Val His
Asp Ala 115 120 125Ile Lys Ser Lys
Val Ile Gly Gly Tyr Asn Gly Lys Asp Gly Ile Asp 130
135 140Leu Glu Gly Ala Ile Gly Ser Gln Val Lys Thr Leu
Val Ser Glu Ile145 150 155
160Arg Arg Arg His Leu Gly Gln Pro Val Asp Phe Ser Arg Leu Met Arg
165 170 175Gln Met Ala Leu Asp
Ala Ile Thr Ala Val Ala Phe Gly Glu Ala Leu 180
185 190Gly Phe Leu Thr Ala Glu Asp Gly Asp Val Phe Gly
Tyr Val Ser Ala 195 200 205Val Asp
Lys Met Leu Thr Tyr Leu Thr Leu Ala Ser Asp Leu Pro Ile 210
215 220Val Arg Ser Phe Val Arg Ser Arg Arg Met Ala
Pro Ala Val Arg Cys225 230 235
240Val Leu Ala Tyr Thr Gly Ile Gly Arg Met Leu Asn His Thr Arg Arg
245 250 255Val Val Ala Glu
Arg Tyr Ala Ala Asp Asp Pro Gly Lys Gly Asp Met 260
265 270Thr Ala Ser Phe Ile Arg Lys Gly Leu Thr Gln
Ile Glu Cys Glu Gly 275 280 285Glu
Ser His Leu Gln Leu Ile Ala Gly Ala Asp Thr Ala Val Thr Val 290
295 300Leu Arg Ser Thr Leu Leu Tyr Ile Met Thr
Thr Pro Arg Val Tyr Thr305 310 315
320Arg Leu Lys Ala Glu Ile Lys Ala Ala Val Asp Ala Gly Glu Val
Val 325 330 335Glu Val Ile
Thr Met Ala Gln Ala Gln Gly Leu Pro Tyr Leu Gln Ala 340
345 350Val Val Leu Glu Gly Phe Arg Met Arg Pro
Ala Val Val Tyr Gly His 355 360
365Phe Lys Ser Val Pro Ala Gly Gly Asp Thr Leu Pro Asn Gly Val Arg 370
375 380Leu Pro Ala Gly Thr Ala Ile Ala
Pro Asn Tyr Ile Ala Leu Thr Arg385 390
395 400Arg Thr Asp Val Tyr Gly Ala Asp Val Asp Leu Phe
Arg Pro Glu Arg 405 410
415Phe Leu Asp Ala Glu Pro Ala Lys Arg His Glu Met Glu Arg Ala Met
420 425 430Asp Leu Asn Phe Gly Leu
Gly Arg Trp Gln Cys Ala Gly Arg Asn Ile 435 440
445Ala Leu Met Glu Met Asn Lys Val Phe Phe Glu Leu Leu Arg
His Phe 450 455 460Asp Leu Gln Ile Val
Tyr Pro Gly Lys Ala Trp Asp Glu Tyr Thr Gly465 470
475 480Val Val Tyr Ser Gln His Asn Met Trp Val
Gln Ile Thr Glu Ser Ser 485 490
49512473PRTEpichloe uncinata 12Met Thr Val Asp Thr Ile Thr Ser Thr
Ser Asn Gly Asn Gln Asp Val1 5 10
15Pro Lys Glu Phe Phe Pro Lys Glu Phe Glu Thr Gln Leu Leu His
Val 20 25 30Gly Arg Phe Pro
Asp Ile Leu Gly Ser Cys Ala Val Pro Val Tyr Ser 35
40 45Ser Ala Ala Phe Glu Phe Asn Ser Val Ala His Gly
Ala Arg Leu Leu 50 55 60Asn Leu Thr
Gln Phe Gly Asn Ile Tyr Ser Arg Phe Thr Asn Pro Thr65 70
75 80Val Asn Val Leu Gln Asn Arg Leu
Ala Gly Leu Glu Gly Gly Val Ala 85 90
95Ala Cys Ala Val Ala Ser Gly Ser Ala Ala Val Val Val Thr
Val Met 100 105 110Ala Leu Ala
Gly Val Gly Asp Asn Phe Val Ser Ser Phe His Val His 115
120 125Ala Gly Thr Phe His Gln Phe Glu Ser Leu Ala
Lys Gln Met Gly Ile 130 135 140Glu Cys
Arg Phe Val Lys Ser Arg Asp Pro Ala Asp Phe Ala Ala Ala145
150 155 160Ile Asp Asp Lys Thr Lys Phe
Val Trp Leu Glu Thr Ile Ser Asn Pro 165
170 175Gly Asn Val Ile Leu Asp Leu Glu Ala Val Ser Met
Val Cys His Thr 180 185 190Lys
Gly Ile Pro Leu Ile Cys Asp Asn Thr Phe Gly Cys Ala Gly Tyr 195
200 205Phe Cys Arg Pro Ile Asn His Gly Val
Asp Ile Val Val His Ser Ala 210 215
220Thr Lys Trp Ile Gly Gly His Gly Thr Thr Val Gly Gly Val Ile Val225
230 235 240Asp Gly Gly Thr
Phe Asp Trp Gly Gln His Pro Asp Arg Phe Pro Gln 245
250 255Phe His Asp Pro Arg Thr Arg Leu Trp Glu
Arg Phe Ser Arg Arg Ala 260 265
270Phe Ala Val Arg Cys Gln Phe Glu Ile Leu Arg Asp Thr Gly Ser Thr
275 280 285Leu Ser Ala Pro Ala Ala Gln
Gln Leu Leu Val Gly Leu Glu Ser Leu 290 295
300Ala Val Arg Cys Glu Arg His Ala Gln Asn Ala Ala Lys Ile Ala
Asp305 310 315 320Trp Leu
Arg Glu His Pro Leu Val Ala Trp Val Ser Tyr Val Gly His
325 330 335Pro Asn His Pro Asp His Gln
Gly Ala Leu Lys Tyr Leu Lys Arg Gly 340 345
350Phe Gly Ser Val Ile Cys Phe Gly Leu Arg Gly Gly Phe Glu
Ala Gly 355 360 365Ala Leu Phe Cys
Asp Ala Leu Lys Met Val Ile Thr Thr Thr Asn Leu 370
375 380Gly Asp Ala Lys Thr Leu Ile Leu His Pro Ala Ser
Thr Thr His Glu385 390 395
400His Phe Ser Ser Glu His Arg Ala Glu Ala Gly Val Thr Asp Asp Met
405 410 415Ile Arg Leu Ser Val
Gly Ile Glu Gln Ile Lys Asp Ile Lys Ala Asp 420
425 430Phe Glu Gln Ala Phe Glu Gln Val Leu Arg Gly Lys
Lys Ser Leu Arg 435 440 445Lys Pro
Cys Ile Gly Lys Ile Leu Leu Gln Asp Glu Ile Asn Glu Asp 450
455 460Leu Phe Gly Pro Ser Ala Cys Arg Thr465
47013473PRTEpichloe uncinata 13Met Thr Val Asp Thr Ile Thr Ser
Thr Ser Asn Gly Asn Gln Asp Val1 5 10
15Pro Lys Glu Phe Leu Pro Ile Glu Phe Glu Thr Gln Leu Leu
His Leu 20 25 30Gly Arg Phe
Pro Asp Ile Leu Gly Ser Cys Ala Val Pro Val Tyr Ser 35
40 45Ser Ala Ala Phe Glu Phe Asn Ser Val Ala His
Gly Ala Arg Leu Leu 50 55 60Asn Leu
Thr Gln Phe Gly Asn Ile Tyr Ser Arg Phe Thr Asn Pro Thr65
70 75 80Val Asn Val Leu Gln Asn Arg
Leu Ala Gly Leu Glu Gly Gly Val Ala 85 90
95Ala Cys Gly Val Ala Ser Gly Ser Ala Ala Val Val Val
Thr Val Met 100 105 110Ala Leu
Thr Gly Val Gly Asp Asn Phe Val Ser Ser Phe His Val His 115
120 125Ala Gly Thr Phe His Gln Phe Asp Ser Leu
Ala Lys Gln Met Gly Ile 130 135 140Glu
Cys Arg Phe Val Lys Ser Arg Asp Pro Ala Asp Phe Ala Ala Ala145
150 155 160Ile Asp Asp Lys Thr Lys
Phe Val Trp Leu Glu Thr Ile Ser Asn Pro 165
170 175Gly Asn Val Ile Leu Asp Leu Glu Ala Val Ser Thr
Val Cys His Thr 180 185 190Lys
Gly Ile Pro Leu Ile Cys Asp Asn Thr Phe Gly Cys Ala Gly Tyr 195
200 205Phe Cys Arg Pro Ile Asp His Gly Val
Asp Ile Val Val His Ser Ala 210 215
220Thr Lys Trp Ile Gly Gly His Gly Thr Thr Val Gly Gly Ile Ile Val225
230 235 240Asp Gly Gly Thr
Phe Asp Trp Gly Gln His Pro Asp Arg Phe Pro Gln 245
250 255Phe His Asp Pro Arg Thr Arg Leu Trp Glu
Arg Phe Ser Arg Arg Ala 260 265
270Phe Ala Val Arg Cys Gln Phe Glu Ile Leu Arg Asp Thr Gly Ser Thr
275 280 285Leu Ser Ala Pro Ala Ala Gln
Gln Leu Leu Val Gly Leu Glu Ser Leu 290 295
300Ala Val Arg Cys Glu Arg His Ala Gln Asn Ala Ala Lys Ile Ala
Asp305 310 315 320Trp Leu
Arg Glu Tyr Pro Leu Val Ala Trp Val Ser Tyr Val Gly His
325 330 335Pro Asn His Pro Asp His Gln
Gly Ala Leu Lys Tyr Leu Lys Arg Gly 340 345
350Phe Gly Ser Val Ile Cys Phe Gly Leu Arg Gly Gly Phe Glu
Ala Gly 355 360 365Ala Leu Phe Cys
Asp Ala Leu Lys Met Val Ile Thr Thr Thr Asn Leu 370
375 380Gly Asp Ala Lys Thr Leu Ile Leu His Pro Ala Ser
Thr Thr His Glu385 390 395
400His Phe Ser Ser Glu His Arg Ala Glu Ala Gly Val Thr Asp Asp Met
405 410 415Ile Arg Leu Ser Val
Gly Ile Glu Gln Ile Lys Asp Ile Lys Ala Asp 420
425 430Phe Glu Gln Ala Phe Lys Gln Val Leu Arg Gly Lys
Lys Ser Leu Arg 435 440 445Lys Pro
Cys Ile Gly Lys Ile Leu Met Gln Asp Glu Ile Asn Glu Asp 450
455 460Leu Phe Gly Pro Ser Ala Cys Arg Thr465
47014420PRTEpichloe uncinata 14Met Ala Thr Val Val Arg Glu Ala
Phe Glu Asn His Val Lys Leu Val1 5 10
15Glu Ser Arg Asn Ser Pro Gly His Val Leu Ala Ser Ser Glu
Ala Ser 20 25 30Phe Phe Val
Ala Asp Leu Asn Asp Ile Val Arg Lys Trp Ala Ala Trp 35
40 45Lys Lys Ala Leu Pro Asp Val Thr Pro Phe Phe
Ala Val Lys Ser Ser 50 55 60Tyr Asp
Arg Arg Leu Ile Gln Thr Leu Ala Thr Cys Gly Ala Gly Phe65
70 75 80Asp Cys Ala Ser Val Glu Glu
Ile Glu Leu Ile Leu Ser Leu Gly Ile 85 90
95Gly Ala Glu Arg Ile Val Phe Thr His Pro Cys Lys Pro
Val Ser Ser 100 105 110Leu Gly
Leu Cys Arg Lys Leu Gly Ile Thr Leu Ile Thr Phe Asp Asn 115
120 125Glu Cys Glu Leu Arg Lys Leu His His His
Tyr Pro Glu Ala Gln Thr 130 135 140Val
Leu Arg Val Phe Ala Asp Asp Pro Thr Asn Ala Asp Pro Leu Gly145
150 155 160Thr Lys Phe Gly Ala Ala
Arg Glu Asp Ile Asp Gly Leu Val Arg Leu 165
170 175Val Lys Glu Leu Asn Met Lys Leu Ala Gly Ala Ser
Phe His Ala Ala 180 185 190Pro
Ser Val Ala Val Asp Ala Ala Ala Tyr Val Arg Gly Ile Arg Asp 195
200 205Ala Ala Glu Val Phe Ala Arg Ala Arg
Arg Val Gly Leu Asn Pro Thr 210 215
220Val Leu Asp Ile Gly Gly Gly Tyr Thr Asp Ser Thr Phe Gln Gln Ile225
230 235 240Ala Gly Ala Val
Arg Pro Ala Ile Ala Glu Cys Phe Lys Ser Gln Val 245
250 255Val Glu Gly Arg Leu Arg Ile Leu Ala Glu
Pro Gly Thr Leu Phe Ser 260 265
270Cys Ser Pro Phe Tyr Leu Ala Val Lys Val Val Ala Arg Arg Arg Asn
275 280 285Ala Ala Ala Phe Gly Asn Glu
Pro Ala Thr Arg Leu Tyr Ile Asn Asp 290 295
300Gly Ile Tyr Ser Asn Phe Met Met Arg Phe Ile Val Asn Met Thr
Phe305 310 315 320Ser Pro
Val Ala Val Ile Arg Lys Gly Val Trp Tyr Asp Gln Thr Glu
325 330 335Gln Thr Met Arg Arg Glu Ala
Cys Ser Leu Trp Gly Arg Ser Cys Asp 340 345
350Ser Asn Asp Cys Ile Asn Arg Asp Cys Arg Leu Asp Pro Glu
Val Gly 355 360 365Val Gly Asp Trp
Leu Val Phe Lys Asp Met Gly Ala Tyr Thr Thr Val 370
375 380Cys Asn Thr Thr Phe Asn Gly Phe Thr Ser Ser Asn
His Thr Ile Tyr385 390 395
400Ile Glu Pro Thr Gln Val Asp Lys Ala Gln Ser Thr Phe Glu Gln Leu
405 410 415Glu Leu Ala Ile
42015415PRTEpichloe uncinata 15Met Thr Thr Val Val Arg Glu Ala Phe
Glu Asn His Val Lys Leu Val1 5 10
15Glu Ser Arg Asn Ser Pro Gly His Val Leu Ala Ser Ser Glu Ala
Ser 20 25 30Phe Phe Val Ala
Asp Leu Asn Asp Val Val Arg Lys Trp Ala Ala Trp 35
40 45Lys Glu Ala Leu Pro Asp Val Thr Pro Phe Phe Ala
Val Lys Ser Ser 50 55 60Tyr Asp Arg
Arg Leu Ile Gln Thr Leu Ala Thr Cys Gly Ala Gly Phe65 70
75 80Asp Cys Ala Ser Thr Glu Glu Ile
Glu Leu Ile Leu Ser Leu Gly Ile 85 90
95Gly Ala Glu Arg Ile Ile Phe Thr His Pro Cys Lys Pro Val
Ser Ser 100 105 110Leu Gly Leu
Cys Arg Lys Leu Gly Ile Thr Leu Ile Thr Phe Asp Asn 115
120 125Glu Cys Glu Leu Arg Lys Leu His His His Tyr
Pro Glu Ala Gln Thr 130 135 140Val Leu
Arg Val Phe Ala Asp Asp Pro Thr Asn Ala Asp Pro Leu Gly145
150 155 160Thr Lys Phe Gly Ala Ala Arg
Asp Asp Phe Asp Gly Leu Val Arg Leu 165
170 175Val Lys Glu Leu Asn Met Gln Leu Ala Gly Ala Ser
Phe His Ala Ala 180 185 190Pro
Ser Val Ala Val Asp Ala Ala Ala Tyr Val Arg Gly Ile Arg Asp 195
200 205Thr Ala Glu Val Phe Ala Arg Ala Arg
Gln Val Gly Leu Asn Pro Thr 210 215
220Val Leu Asp Ile Gly Gly Gly Tyr Thr Asp Ser Thr Phe Gln Gln Ile225
230 235 240Ala Gly Ala Val
Arg Pro Ala Ile Ala Glu Cys Phe Lys Ser Glu Val 245
250 255Gly Glu Gly Arg Leu Arg Ile Leu Ala Glu
Pro Gly Thr Leu Phe Ser 260 265
270Cys Ser Pro Phe Tyr Leu Ala Val Lys Val Val Ala Arg Arg Val Asn
275 280 285Ala Thr Ala Phe Gly His Glu
Pro Ala Thr Arg Leu Tyr Ile Asn Asp 290 295
300Gly Ile Tyr Ser Asn Phe Met Met Arg Phe Ile Val Asn Met Thr
Phe305 310 315 320Ser Pro
Ala Ala Val Ile Arg Glu Gly Val Trp His Asp Gln Ala Asp
325 330 335His Thr Met Arg Gly Glu Ala
Cys Ser Leu Trp Gly Arg Ser Cys Asp 340 345
350Ser Asn Asp Cys Ile Asn Arg Asp Cys Arg Leu Gly Pro Glu
Val Arg 355 360 365Val Gly Asp Trp
Leu Val Phe Lys Asp Met Gly Ala Tyr Thr Thr Val 370
375 380Cys Asn Thr Thr Phe Asn Gly Phe Thr Ser Ser Asn
His Thr Ile Tyr385 390 395
400Leu Glu Pro Gly Thr His Pro Ser Arg Arg Ser Pro Val Asp Leu
405 410 41516540PRTEpichloe uncinata
16Met Thr Leu Thr Asn Leu Asp Val Ile Val Val Gly Ala Gly Phe Ser1
5 10 15Gly Ile Leu Ala Val Tyr
Arg Leu Arg Lys Leu Gly Phe Arg Val Gln 20 25
30Gly Phe Glu Arg Gln Glu Arg Leu Gly Gly Val Trp Arg
Glu Asn Ala 35 40 45Tyr Pro Gly
Ala Ala Val Asp Ser Leu Phe Pro Phe Tyr Gln Phe Tyr 50
55 60Asp Ala Glu Leu Leu Gln Asp Trp Glu Trp Val Glu
Gln Phe Pro Thr65 70 75
80Arg Ala Glu Met Leu Arg Tyr Phe Asp His Val Asp Lys Arg Trp Glu
85 90 95Ile Ser Ala Ser Phe Glu
Phe Gly Val Ser Val Ser Ala Ala Arg Tyr 100
105 110Ser Glu Thr Thr Gln Arg Trp Thr Val Ser Leu Glu
Asp Gly Arg Arg 115 120 125Ala Glu
Ala Arg Trp Phe Ile Pro Ala Val Gly Phe Ser Ser Val Leu 130
135 140Asn Ile Pro Arg Ile Pro Gly Met Ser Arg Phe
Arg Gly Pro Ile Tyr145 150 155
160His Thr Ala Lys Trp Pro His Asp Ala Val Ser Met Arg Gly Lys Arg
165 170 175Val Ala Val Ile
Gly Thr Gly Pro Ser Gly Val Gln Ile Ile Gln Ser 180
185 190Val Gly Lys Ile Ala Lys Ala Met Thr Ile Phe
Gln Gln Ser Pro Cys 195 200 205Leu
Thr Leu Arg Lys Tyr Gly Ser Pro Ser Gln Thr Ala Thr Ala Leu 210
215 220Cys Met Arg Pro Asp Asp His Arg Glu Ala
Leu Arg Leu Gly Leu Gln225 230 235
240Thr Ser Asn Gly Phe Gly Tyr Val Pro Arg Asp Gln Asp Thr Leu
Asp 245 250 255Val Pro Ile
Glu Glu Arg Asn His Phe Tyr Gln Gln Arg Tyr Leu Ala 260
265 270Gly Gly Trp Ala Phe Trp Met Ala Gly Phe
Arg Asp Leu Cys Gln Asn 275 280
285Ile Gln Ala Asn Arg Asp Ala Tyr Asp Phe Trp Ala Arg Arg Thr Arg 290
295 300Ala Arg Ile Ser Asp Val Ala Lys
Arg Glu Leu Leu Val Pro Gln Ile305 310
315 320Pro Ser Phe Ala Phe Gly Ile Lys Arg Pro Cys Leu
Glu Glu Asp Leu 325 330
335Tyr Glu Val Met Asp Gln Pro His Val Lys Val Ile Asp Ile Ser Asn
340 345 350Gln Gln Ile Glu Leu Ile
Thr Glu Thr Gly Ile Arg Val His Gly Gln 355 360
365Thr Val Glu Cys Glu Ala Ile Ile Leu Ala Thr Gly Phe Gly
Asp Glu 370 375 380Ala Ser Gly Leu Arg
Ser Leu His Ile Arg Gly Arg Asn Gly Ile Arg385 390
395 400Leu Glu Asp Ala Trp Ser Asp Gly Val Glu
Ser His Leu Gly Met Ala 405 410
415Ile His Ser Phe Pro Asn Met Val Ile Leu Tyr Gly Pro Gln Cys Pro
420 425 430Thr Leu Leu Val Asn
Ser Pro Ala Val Ile Thr Val Gln Val Glu Trp 435
440 445Leu Cys Glu Ile Ile Ala Arg Cys Gln Gln Ala Gly
Ile Cys Gln Leu 450 455 460Glu Ala Thr
Ser Lys Ser His Cys Gln Trp Glu Arg Lys Met Ser Leu465
470 475 480Leu Trp Asp Lys Thr Leu Tyr
His Thr His Ala Arg Lys Ser Lys Lys 485
490 495Thr Ala Glu Ala Asn Lys Glu Glu Lys Thr Trp Val
Gly Gly Leu Ile 500 505 510Leu
Tyr Arg Arg Glu Leu Glu Asn Cys Leu Ala Asn Asn Leu Glu Gly 515
520 525Phe Gln Ala Trp His Val Glu Glu Thr
Gly Leu Leu 530 535
54017540PRTEpichloe uncinata 17Met Thr Leu Thr Asn Leu Asp Ala Ile Val
Val Gly Ala Gly Phe Ser1 5 10
15Gly Ile Leu Ala Val Tyr Arg Leu Arg Lys Leu Gly Phe Arg Val Gln
20 25 30Gly Phe Glu Arg Gln Glu
Arg Leu Gly Gly Val Trp Arg Glu Asn Ala 35 40
45Tyr Pro Gly Ala Ala Val Asp Ser Leu Phe Pro Phe Tyr Gln
Phe Tyr 50 55 60Asp Ala Glu Leu Leu
Gln Asp Trp Glu Trp Gly Glu Gln Phe Pro Thr65 70
75 80Arg Ala Glu Met Leu Arg Tyr Phe Asp His
Val Asp Lys Arg Trp Glu 85 90
95Ile Ser Ala Ser Phe Glu Phe Gly Val Ser Val Ser Ala Ala Arg Tyr
100 105 110Ser Glu Thr Thr Gln
Arg Trp Thr Val Thr Leu Glu Asp Gly Arg Arg 115
120 125Ala Glu Ala Arg Trp Phe Ile Pro Ala Val Gly Phe
Ser Ser Val Leu 130 135 140Asn Ile Pro
Arg Ile Pro Gly Met Ser Arg Phe Arg Gly Ala Ile Tyr145
150 155 160His Thr Ala Lys Trp Pro His
Asp Ala Val Ser Met Arg Gly Lys Arg 165
170 175Val Ala Val Ile Gly Thr Gly Pro Ser Gly Val Gln
Ile Ile Gln Ser 180 185 190Val
Gly Lys Ile Ala Lys Ala Met Thr Ile Phe Gln Gln Ser Pro Cys 195
200 205Leu Thr Leu Arg Lys Tyr Gly Ser Pro
Asn Gln Thr Ala Thr Ala Leu 210 215
220Cys Met Arg Pro Asp Asp His Arg Glu Ala Leu Arg Leu Gly Leu Gln225
230 235 240Thr Ser Asn Gly
Phe Gly Tyr Val Pro Arg Asp Gln Asp Thr Leu Asp 245
250 255Val Pro Ile Glu Glu Arg Asn His Phe Tyr
Gln Gln Arg Tyr Leu Ala 260 265
270Gly Gly Trp Ala Phe Trp Met Ala Gly Phe Arg Asp Leu Cys Gln Asn
275 280 285Ile Gln Ala Asn Arg Asp Ala
Tyr Asp Phe Trp Ala Arg Arg Thr Arg 290 295
300Ala Arg Ile Gly Asp Val Thr Lys Arg Glu Leu Leu Val Pro Gln
Ile305 310 315 320Pro Ser
Phe Ala Phe Gly Ile Lys Arg Pro Cys Leu Glu Glu Asp Leu
325 330 335Tyr Glu Ile Met Asp Gln Pro
His Val Lys Ile Ile Asp Ile Ser Asn 340 345
350Gln Gln Ile Glu Leu Ile Thr Glu Thr Ser Ile Arg Val His
Gly Gln 355 360 365Thr Val Glu Cys
Glu Ala Ile Ile Phe Ala Thr Gly Phe Gly Asp Glu 370
375 380Ala Ser Gly Leu Arg Ser Leu His Ile Arg Gly Arg
Asn Gly Ile Arg385 390 395
400Leu Glu Asp Ala Trp Ser Asp Gly Val Glu Ser His Leu Gly Met Ala
405 410 415Ile His Ser Phe Pro
Asn Met Phe Phe Leu Tyr Gly Pro Gln Cys Pro 420
425 430Thr Leu Leu Val Asn Ser Pro Ala Val Ile Thr Val
Gln Val Glu Trp 435 440 445Leu Cys
Glu Ile Ile Ser Lys Cys Gln Gln Ala Gly Ile Cys Gln Leu 450
455 460Glu Ala Thr Ser Lys Ser His Cys Gln Trp Glu
Lys Lys Met Ser Leu465 470 475
480Leu Trp Asp Lys Thr Leu Tyr His Thr His Ala Arg Lys Ser Lys Lys
485 490 495Thr Ala Glu Ala
Asn Lys Glu Glu Lys Thr Trp Val Gly Gly Leu Ile 500
505 510Leu Tyr Arg Arg Glu Leu Glu Asn Cys Leu Ala
Asn Asn Leu Glu Gly 515 520 525Phe
Gln Ala Trp Tyr Val Glu Glu Thr Ala Leu Leu 530 535
54018209PRTEpichloe uncinata 18Met Leu Asp Glu Ser Pro Met
Arg Lys Gly Asp Ser Val Ser Asn Asp1 5 10
15Gln Ser Asn Pro Glu Ser Asn Ala Ser Val Ser Ile His
Gln Gln Asn 20 25 30Gln Ile
Ile Thr Cys Val Ser Pro Gly Pro Val Cys Pro Asn Ala Ile 35
40 45Glu Ile Lys Arg Asp Ile Val Ile Val Arg
Leu Arg Pro Val Glu Ser 50 55 60Cys
Pro Gly Tyr Arg Phe Phe Arg Arg Val Phe Glu Thr Leu Glu Lys65
70 75 80Trp Gln Leu Gln Val Asp
Met Phe Ser Thr Ser Leu Gly Arg Ile Thr 85
90 95Leu Ala Leu Gly Ala Ala Ala Leu Gln Ala Gly Ile
Ser Asp Ser Cys 100 105 110Ser
Ala Arg Asn Asp Met Met Ser Arg Asp Leu Met His Gly Met Gln 115
120 125Lys Leu Leu Pro Asp Asp His Ile Glu
Leu Phe Pro His Met Ala Ile 130 135
140Ile Ser Val Val Gly His Pro Ser Arg Arg Met Ala Gly His Ile Phe145
150 155 160Ala Thr Met Asp
Ala Asn Asp Ile Pro Thr Val Met Ile Ser His Asp 165
170 175Ala Ala Arg Leu Gly Ile Ala Cys Ala Ile
Ser Glu Gln Tyr Thr Ala 180 185
190Lys Ala Leu Cys Val Phe Glu Gln Cys Leu Phe Arg Tyr Ser Leu Thr
195 200 205His19210PRTEpichloe uncinata
19Met Leu Asp Glu Ser Pro Met Arg Lys Gly Asn Ser Val Ser Asn Asp1
5 10 15Gln Gly Asn Pro Glu Ser
Asn Ala Ser Val Ser Ile His Gln Gln Asn 20 25
30Gln Ile Ile Thr Cys Ala Ser Pro Gly Pro Val Cys Pro
Asn Ala Ile 35 40 45Gly Ile Lys
Arg Asp Ile Val Val Val Arg Leu Arg Pro Val Lys Ser 50
55 60Cys Pro Asp Tyr Arg Phe Phe Arg Arg Val Phe Glu
Thr Leu Glu Lys65 70 75
80Trp Gln Leu Gln Val Asp Met Phe Ser Thr Ser Leu Gly Arg Ile Thr
85 90 95Leu Ala Leu Gly Ala Ala
Ala Leu Gln Ala Gly Ile Gly Asp Ser Cys 100
105 110Ser Ala Arg Asn Asp Met Met Ser Arg Asp Leu Met
His Gly Met Gln 115 120 125Lys Leu
Leu Pro Asp Asp His Ile Leu Glu Leu Phe Pro His Met Ala 130
135 140Ile Ile Ser Val Val Gly His Pro Ser Arg Arg
Ile Ala Gly His Ile145 150 155
160Phe Ala Thr Met Asp Ala Asn Asp Ile Leu Thr Val Met Ile Ser His
165 170 175Asp Ala Ala Arg
Leu Gly Ile Ala Cys Val Ile Ser Glu Gln His Thr 180
185 190Ala Lys Ala Leu Gly Val Phe Glu Gln Cys Leu
Phe Arg Tyr Ser Leu 195 200 205Thr
His 21020454PRTEpichloe uncinata 20Met Thr Val Asn Ser Lys Arg Ile Pro
Phe Gly Lys Pro Met Leu Glu1 5 10
15Ala Phe Cys Met Asp Pro Glu Tyr Thr Asn Leu Asn Ser Ser Ser
Cys 20 25 30Gly Ser Trp Pro
Lys Val Val Ser Lys Gln Ile Arg Asp Tyr Trp Ser 35
40 45Leu Leu Glu Ala Gln Pro Asp Leu Phe Ser Glu Phe
Ser Gln Gly Leu 50 55 60Val Leu Gln
Glu Ala Arg Ile Gly Leu Ala His Leu Val His Ala Ala65 70
75 80Val Ser Glu Cys Val Leu Val Ser
Asn Val Thr Thr Gly Ile Phe Thr 85 90
95Val Leu Tyr Asn Gln Ala Phe Glu Glu Arg Asp Val Val Val
Thr Leu 100 105 110Ser Thr Thr
Tyr Gly Ala Ile Asp His Gly Ile Thr Ser Leu Ala Glu 115
120 125Thr Arg Pro Phe Lys Thr Arg Arg Val Glu Phe
Glu Leu Pro Thr Thr 130 135 140Gly Gln
Lys Ile Val Ser Gln Phe Glu Thr Ala Met Ala Gln Ile Arg145
150 155 160Ala Asp Gly Leu Arg Pro Arg
Leu Ala Ile Leu Glu Thr Ile Val Ser 165
170 175Ile Pro Ala Val Arg Met Pro Phe Glu Asp Leu Leu
Arg Val Cys Gln 180 185 190Lys
Glu Gly Ile Met Thr Leu Val Asp Gly Ala His Ser Val Gly Gln 195
200 205Phe Glu Val Asn Leu Gln Glu Leu Gln
Pro Asp Phe Phe Val Ser Asp 210 215
220Cys His Lys Trp Leu Phe Val Pro Arg Pro Cys Ala Val Leu Tyr Val225
230 235 240Ala Glu Arg Asn
Gln His Met Met Arg Ser Val Ile Pro Thr Ser Phe 245
250 255Gly Phe Ile Pro Lys Asn Gly Asn Ser Arg
Leu Pro Leu Trp Ser Gln 260 265
270Met Val Ser Ala Ser Glu Thr Ala Ser Ser Phe Glu Thr Leu Phe Ala
275 280 285Tyr Thr Ala Thr Ser Asp Tyr
Met Pro His Leu Cys Ile Pro Ala Ala 290 295
300Leu Arg Phe Arg Arg Asp Val Cys Gly Gly Glu Ala Ala Ile Tyr
Glu305 310 315 320Tyr Ile
Lys Trp Leu Ala Lys Glu Gly Gly Asp Lys Ile Ala Asp Ile
325 330 335Leu Gln Thr Glu Val Leu Glu
Glu Pro Gly Leu Gly Ala Gly Val Asp 340 345
350Gly Gln Met Arg Asn Cys Gly Ile Val Thr Val Arg Leu Pro
Leu Ala 355 360 365Ile Ala Thr Gly
Pro Ser Thr Ala Pro Ala His Val Pro Gly Gly Ala 370
375 380Leu Thr Glu Lys Glu Val Gly Pro Ala Val Arg Tyr
Leu Thr Lys Ala385 390 395
400Leu Ala Glu Arg Tyr Lys Thr Trp Ile Pro Ile Ile Asp Tyr Arg Gly
405 410 415Trp Ile Trp Ala Arg
Leu Cys Ala Gln Val Tyr Leu Glu Val Ser Asp 420
425 430Phe Glu Met Ala Gly Asn Ser Leu Lys Val Ile Cys
Glu Glu Ile Leu 435 440 445Asn Arg
Glu Met Gly Gln 45021464PRTEpichloe uncinata 21Met Thr Val Asn Ser Lys
Arg Ile Pro Phe Gly Lys Pro Met Leu Glu1 5
10 15Ala Phe Cys Met Asp Pro Glu Tyr Thr Asn Leu Asn
Ser Ser Ser Cys 20 25 30Gly
Ser Trp Pro Lys Val Val Ser Lys Gln Ile Arg Asp Tyr Trp Ser 35
40 45Leu Leu Glu Ala Gln Pro Asp Leu Phe
Ser Glu Phe Tyr Gln Gly Leu 50 55
60Val Leu Gln Glu Ala Arg Leu Gly Leu Ala Arg Leu Val His Ala Ala65
70 75 80Val Ser Glu Cys Val
Leu Val Ser Asn Val Thr Thr Gly Ile Phe Thr 85
90 95Val Leu Tyr Asn Gln Glu Phe Glu Glu Arg Asp
Val Val Val Thr Leu 100 105
110Ser Thr Thr Tyr Gly Ala Ile Asp His Gly Ile Thr Ser Leu Ala Glu
115 120 125Thr Arg Ser Phe Lys Thr Arg
Arg Val Glu Phe Glu Leu Pro Thr Thr 130 135
140Gly Glu Lys Ile Val Ser Gln Phe Glu Thr Thr Ile Ala Gln Ile
Arg145 150 155 160Ala Lys
Gly Leu Arg Pro Arg Leu Ala Ile Leu Glu Thr Ile Val Ser
165 170 175Ile Pro Ala Val Arg Met Pro
Phe Glu Asp Leu Leu Arg Val Cys Gln 180 185
190Lys Glu Cys Ile Met Thr Leu Val Asp Gly Ala His Ser Val
Gly Gln 195 200 205Phe Glu Val Asn
Leu Gln Glu Leu His Pro Asp Phe Phe Val Ser Asp 210
215 220Cys His Lys Trp Leu Phe Val Pro Arg Pro Cys Ala
Phe Leu Tyr Val225 230 235
240Ala Glu Arg Asn Gln His Met Met Arg Ser Ala Ile Pro Thr Ser Phe
245 250 255Gly Phe Ile Pro Lys
Asn Gly Asn Ser Gln Leu Pro Leu Trp Ser Gln 260
265 270Met Val Ser Ala Asn Gly Thr Ala Ser Ser Phe Glu
Thr Leu Phe Ala 275 280 285Tyr Thr
Ala Thr Ser Asp Asn Met Pro His Leu Cys Ile Pro Thr Ala 290
295 300Leu Arg Phe Arg Arg Asp Val Cys Gly Gly Glu
Ala Ala Ile Tyr Glu305 310 315
320Tyr Ile Lys Trp Leu Ala Lys Glu Gly Gly Asp Lys Val Ala Glu Ile
325 330 335Leu Gln Thr Glu
Val Leu Glu Glu Pro Gly Leu Gly Ala Gly Ala Asp 340
345 350Gly Gln Met Arg Asp Cys Gly Ile Val Thr Val
Arg Leu Pro Leu Ala 355 360 365Ile
Ala Thr Gly Pro Ser Thr Ala Pro Ala His Val Pro Gly Gly Ala 370
375 380Leu Thr Glu Lys Glu Val Gly Pro Ala Val
Arg Tyr Leu Thr Lys Ala385 390 395
400Leu Ala Asp Arg Tyr Lys Thr Trp Ile Pro Ile Ala Asp Cys Arg
Gly 405 410 415Trp Ile Trp
Ala Arg Leu Cys Ala Gln Val Tyr Leu Glu Val Ser Asp 420
425 430Phe Glu Met Ala Gly Asn Ala Leu Lys Val
Ile Cys Glu Glu Ile Leu 435 440
445Ser Arg Glu Met Gly Gln Glu Ile Ser Asp Ser Tyr Arg Trp His Asp 450
455 46022256PRTEpichloe uncinata 22Met
Thr Ala Ala Ser Ser Pro His Pro Gly Val Ser Ala Glu Asp Ile1
5 10 15Glu Phe Tyr Gln Ala Asn Gly
Tyr Leu Arg Leu Pro Gln Glu Ala His 20 25
30Gly Leu Phe Asp Asp Leu Ala Lys Leu Gln Ala Trp Val Ala
Glu Ile 35 40 45Ser Gln Trp Gly
Leu Glu Thr Gly Lys Trp Arg His Tyr Tyr Glu Thr 50 55
60Thr Asn Gly Lys His Leu Leu Trp Gly Thr Glu Lys Leu
Met Glu Tyr65 70 75
80His Ala Pro Met Arg Asp Leu Ile Ala Gly Glu Ala Pro Leu Thr Leu
85 90 95Leu Lys Ser Leu Thr Gly
Lys Asp Met Val Val Phe Lys Asp Glu Ile 100
105 110Gly Trp Lys Leu Pro Gly Gly Lys Gly Ala Val Pro
His Leu Asp Arg 115 120 125Pro Ala
Tyr Ser Met Phe Ala Pro Glu Phe Ile Glu Ile Met Ile Ala 130
135 140Val Asp Ala His Thr Val Glu Asn Gly Cys Leu
Gln Phe Val Pro Gly145 150 155
160Ser His Lys Glu Ala Val Pro Ile Ser Ala Asp Gly Arg Ile Ala Ser
165 170 175Ala Trp Leu Glu
Gly Lys Glu Phe Ile Pro Met Val Leu Asp Pro Gly 180
185 190Asp Val Leu Ile Phe Asn Glu Ser Met Ala His
Arg Leu Asp Pro Asn 195 200 205Lys
Thr Asp Gln Arg Arg Ala Ala Val Phe Gly Thr Tyr His Phe Asp 210
215 220Arg Ser Gln Pro Asp Leu Arg Asp Lys Phe
Tyr Ala His Arg Leu Ile225 230 235
240His Ser Pro Pro Glu Asn Ala Trp Val Glu Thr Val Glu Ala Gln
Thr 245 250
25523256PRTEpichloe uncinata 23Met Thr Ala Ala Ser Ser Pro His Pro Gly
Val Ser Ala Glu Asp Ile1 5 10
15Glu Phe Tyr Gln Ala Asn Gly Tyr Leu Arg Leu Pro Gln Glu Ala His
20 25 30Gly Leu Phe Asp Asp Leu
Ala Lys Leu Gln Val Trp Val Ala Glu Ile 35 40
45Ser Gln Trp Gly Leu Glu Thr Gly Lys Trp Arg His Tyr Tyr
Glu Thr 50 55 60Thr Asn Gly Lys His
Leu Leu Trp Gly Thr Glu Lys Leu Met Glu Tyr65 70
75 80His Ala Pro Met Gln Asp Leu Ile Ser Gly
Glu Ala Pro Leu Ala Leu 85 90
95Leu Lys Ser Leu Thr Gly Lys Asp Met Val Val Phe Lys Asp Glu Ile
100 105 110Gly Trp Lys Leu Pro
Gly Gly Lys Gly Ala Val Pro His Leu Asp Arg 115
120 125Pro Ala Tyr Ser Met Phe Ala Pro Glu Phe Ile Glu
Ile Met Ile Ala 130 135 140Val Asp Ala
His Thr Val Glu Asn Gly Cys Leu Gln Phe Val Pro Gly145
150 155 160Ser His Lys Glu Ala Ala Pro
Ile Ser Ala Asp Gly Arg Ile Ala Ser 165
170 175Ala Trp Leu Glu Gly Lys Glu Phe Ile Pro Met Val
Leu Asp Pro Gly 180 185 190Asp
Val Leu Ile Phe Asn Glu Ser Met Ala His Arg Leu Glu Pro Asn 195
200 205Lys Thr Asp Gln Arg Arg Ala Ala Val
Phe Gly Thr Tyr His Phe Asp 210 215
220Leu Ser Gln Pro Asp Leu Arg Asp Lys Phe Tyr Ala His Arg Leu Ile225
230 235 240His Ser Pro Pro
Glu Asn Ala Trp Val Glu Lys Val Gly Ala Gln Thr 245
250 25524362PRTEpichloe uncinata 24Met Thr Val
Thr Asn Lys Pro Val Lys Pro Ala Asn Val Pro Val Met1 5
10 15Asp Phe Glu Ala Ile His Ala Ser Val
Gly Asn Glu Arg Lys Lys Tyr 20 25
30Leu Arg Gln Leu Asp Glu Ala Trp Ser His His Gly Ala Ile Tyr Val
35 40 45Ile Asn His Ser Ile Gly Thr
Arg Thr Leu Glu Glu Ala Phe Ala Trp 50 55
60Cys Lys Lys Phe Phe Asp Leu Pro Leu Ala Val Lys Asn Ser Val His65
70 75 80Ile Pro Pro Asp
Val Ser Lys His Phe Gln Gly Trp Thr Gly Thr Gly 85
90 95Glu Ala Ile Ser Ser Gln Gly Val Trp Asp
Pro Asp Glu Ile Glu Arg 100 105
110Leu Arg Lys Glu Thr Pro Thr Glu Leu Lys Glu Ala Met Glu Leu Gln
115 120 125Asp Pro Cys Gly Thr Tyr Pro
Pro Gly Ala Pro Asp Leu Asn Leu Val 130 135
140Glu Gln His Leu Pro Gly Phe Leu Asp Phe Leu Lys Lys Trp Phe
Ala145 150 155 160Ala Cys
Tyr Lys Gln Ser Leu Gln Asn Met Arg Leu Val Cys Glu Ile
165 170 175Leu Gly Met Glu Asp Leu Asp
Tyr Ile Gly Lys Lys Phe Glu Pro Arg 180 185
190His Met Ser Thr His Ser Thr Trp Asn Tyr Phe Leu Gly Gln
Pro Val 195 200 205Ser Gln Leu Ala
Ser Gly Ser Ala Asn Arg Leu Asn Ala His Thr Asp 210
215 220Tyr Cys Gln Phe Thr Met Leu Phe Gln Asp Met Val
Gly Gly Leu Glu225 230 235
240Leu His Asp Tyr Glu Glu Asp Ile Tyr Arg Pro Val Pro Pro Ile Lys
245 250 255Gly Ala Met Ile Val
Gln Val Gly Asp Leu Leu Glu Lys Gln Thr Asn 260
265 270Gly Arg Trp Arg Ser Ala Leu His Arg Val Thr Ala
Pro Ser Arg Tyr 275 280 285Met Tyr
Glu Gly Ser Ala Gly Asp Asp Asp Glu Leu Val Gln Arg Tyr 290
295 300Ser Leu Val Phe Phe Gly His Leu Asn Leu Asp
Glu Met Ile Glu Pro305 310 315
320Leu Pro Gly Cys Glu Lys Gln Gly Lys Trp Ser Thr Leu Glu Trp Lys
325 330 335Asp Arg Met Thr
Ala Gly Gln Trp Leu Ala Arg Arg Val Ala Leu Glu 340
345 350Tyr Glu Arg Lys Thr Ala Ala Thr Val Met
355 36025362PRTEpichloe uncinata 25Met Thr Val Thr Asn
Lys Pro Val Glu Pro Ala Asn Val Pro Val Met1 5
10 15Asp Phe Glu Ala Ile His Ala Ser Val Gly Asn
Glu Arg Lys Glu Tyr 20 25
30Leu Arg Gln Leu Asp Glu Ala Trp Ser His His Gly Ala Val Tyr Val
35 40 45Ile Asn His Ser Ile Gly Thr Glu
Thr Leu Glu Glu Ala Phe Val Trp 50 55
60Cys Lys Lys Phe Phe Asp Leu Pro Leu Ala Val Lys Asn Ser Val His65
70 75 80Ile Pro Pro Asp Val
Ser Lys His Phe Gln Gly Trp Thr Gly Thr Gly 85
90 95Glu Ala Ile Ser Ser Gln Gly Val Trp Asp Pro
Asp Glu Ile Glu Arg 100 105
110Leu Arg Lys Glu Met Pro Thr Glu Leu Lys Glu Ala Met Glu Leu Gln
115 120 125Asp Pro Cys Gly Thr Tyr Pro
Pro Gly Asn Pro Asp Leu Asn Leu Val 130 135
140Glu Gln His Leu Pro Gly Tyr Leu Asp Phe Leu Lys Lys Trp Phe
Ala145 150 155 160Ala Cys
Tyr Lys Gln Ser Leu Gln Asn Met Arg Leu Val Cys Glu Ile
165 170 175Leu Gly Met Glu Asp Leu Asp
Tyr Ile Gly Lys Lys Phe Glu Pro Arg 180 185
190His Met Ser Thr His Ser Thr Trp Asn Tyr Phe Leu Gly Gln
Pro Val 195 200 205Ser Gln Leu Ala
Ser Gly Ser Ser Asn Arg Leu Asn Ala His Thr Asp 210
215 220Tyr Cys Gln Phe Thr Met Leu Phe Gln Asp Met Val
Gly Gly Leu Glu225 230 235
240Leu His Asp Tyr Glu Glu Asp Ile Tyr Arg Pro Val Pro Pro Ile Lys
245 250 255Gly Ala Met Ile Val
Gln Val Gly Asp Leu Leu Glu Lys Gln Thr Asn 260
265 270Gly Arg Trp Arg Ser Ala Leu His Arg Val Thr Ala
Pro Ser Arg Tyr 275 280 285Met Tyr
Gly Gly Ser Pro Gly Gly Asp Asp Glu Leu Val Gln Arg Tyr 290
295 300Ser Leu Val Phe Phe Gly His Leu Asn Leu Asp
Glu Met Ile Lys Pro305 310 315
320Leu Pro Gly Cys Glu Lys Pro Gly Lys Trp Ser Thr Leu Glu Trp Lys
325 330 335Asp Leu Met Thr
Ala Gly Gln Trp Leu Ala Arg Arg Val Ala Leu Glu 340
345 350Tyr Glu Arg Lys Thr Ala Ala Thr Val Met
355 36026495PRTEpichloe uncinata 26Met Ala Ser Pro Gly
Asn His Ala Ile Val Ala Pro Gly Trp Thr Arg1 5
10 15Ser Glu Asp Gly Ser Leu Thr Arg Pro Leu Asp
Leu Val Glu Asn Trp 20 25
30Leu Leu Ala Arg Ile Gln Arg Ala Asn Thr Pro Pro Gly Arg Glu Ala
35 40 45Glu Gly Leu Thr Tyr Lys Leu Lys
Leu Arg Leu Pro Gln Asp Ile Asp 50 55
60Asp Pro Ile Pro Tyr Leu Arg Arg Ala Trp Leu Val Phe Arg Tyr Val65
70 75 80Gln Pro Leu Ile Gly
Ala Ile Tyr Pro Pro Tyr Ser Glu Arg Asp Glu 85
90 95Thr Gly Arg Tyr Leu Val Thr Val Pro Leu Met
Asp Pro Glu Glu Trp 100 105
110Leu Arg Leu Ser Phe His Val Asn Gln Gly Thr Gln Ala Val Phe Arg
115 120 125Asp Val Asp Asp Ala Gly Lys
Ile Phe Gln Pro Arg Pro Thr Ala Met 130 135
140Ala Tyr Trp Phe Pro Pro Ser Ser Thr Leu Ile Ile Arg Ser Thr
His145 150 155 160Leu Arg
Phe Asp Ala Val Gly Ile Tyr Lys Ala Thr Asn Thr Phe Met
165 170 175Leu Gly Leu Glu Ser Val Phe
Arg Leu Gly Leu Asp Ala Asn Leu Asp 180 185
190Cys Tyr Thr Thr Asp Val Lys Gln Pro Ser Leu Pro Pro Gly
Ile Asp 195 200 205Tyr Ile Leu Gly
Phe Pro Pro Gln Glu Thr Pro Val Pro His Arg Val 210
215 220Glu Arg Ala Val Asp Glu Leu Met Arg His Trp His
His Gly Leu Tyr225 230 235
240Ser Leu Ser Leu Pro Val Arg Glu Gly Ser Glu Asp Ala Ala Pro Ala
245 250 255Asn Thr Gln His Leu
Val Thr Leu Phe Asp Glu Pro Thr Leu Glu Ala 260
265 270Ile Val Ala Gly Cys Lys Lys Leu Gly Val Ser Val
Ser Ala Ala Val 275 280 285His Ala
Ser Ile Val Arg Val Trp Ala Ser Phe Pro Gln Gln Gln His 290
295 300Thr Gly Ala Arg Asn Met Leu Ile Pro Leu Val
Ala Asn Leu Arg Pro305 310 315
320Leu Leu Asp Pro Lys Trp Val Val Pro Asp Tyr Ala Leu Gly Leu Cys
325 330 335Ile Phe Val Val
Pro Phe Cys Leu Thr Gly Gly Phe Glu Asp Leu Thr 340
345 350Gln Arg Leu Gly Ala Val Tyr Ser Arg Asp Leu
Ser Ala Leu Pro Ser 355 360 365Asp
Pro Ala Gly Asp Pro Val Ser Phe Leu Glu Leu Leu Pro Leu Tyr 370
375 380Glu Ser Arg Glu Ala Ala Phe Leu Gly Ser
Leu Pro Val Ala Gly Cys385 390 395
400Pro Pro Phe Arg Val Pro Asn Leu Ser Ser Leu Gly Val Leu Glu
Arg 405 410 415Tyr Leu Ala
Arg Ala Tyr Gly Lys Lys Gly Ala Gln Ala Pro Val Cys 420
425 430Glu Ile Glu Asp Val Ala Leu Val Asn Ala
Thr Thr Asp Pro Thr Ile 435 440
445Glu Phe Gln Leu Phe Thr Phe Arg Gly Thr Met Arg Leu Tyr Leu Tyr 450
455 460Tyr Asn Asp Ala Tyr Tyr Thr Glu
Asp Phe Leu Ala Pro Val Met Glu465 470
475 480Met Val Arg Asp Ser Leu Leu Gln Glu Leu Gly Leu
Gly Gly Ser 485 490
49527506PRTEpichloe uncinata 27Met Val Ser Ala Gly Asn His Ala Ile Val
Ala Leu Gly Trp Thr Thr1 5 10
15Ser Glu Asp Gly Thr Leu Thr Arg Pro Leu Asp Leu Val Glu Asn Trp
20 25 30Leu Leu Ala Arg Ile Gln
Arg Ala Asn Thr Pro Pro Gly Arg Glu Ala 35 40
45Glu Gly Leu Thr Tyr Lys Leu Lys Leu Arg Leu Pro Gln Asp
Ile Asp 50 55 60Asp Pro Ile Pro Tyr
Leu Arg Arg Ala Trp Leu Val Phe Arg Tyr Val65 70
75 80Gln Pro Leu Ile Gly Ala Ile Tyr Pro Pro
Tyr Ser Glu Arg Asp Glu 85 90
95Thr Gly Arg Tyr Leu Val Thr Val Pro Pro Met Asp Pro Glu Glu Trp
100 105 110Leu Arg Leu Ser Phe
His Val Asn Gln Gly Ser Gln Ala Val Phe Arg 115
120 125Asp Val Asp Asp Ala Gly Met Ile Phe Arg Pro Arg
Pro Thr Ala Met 130 135 140Ala Tyr Trp
Phe Pro Pro Ser Ser Thr Leu Val Ile Arg Ser Thr His145
150 155 160Leu Arg Phe Asp Ala Val Gly
Leu Tyr Lys Ala Thr Asn Thr Phe Met 165
170 175Leu Gly Leu Glu Ser Val Phe Arg Leu Gly Leu Asp
Ala Asn Leu Asp 180 185 190Cys
Tyr Thr Thr Asp Val Lys Gln Pro Ser Leu Pro Pro Gly Ile Asp 195
200 205Tyr Ile Leu Gly Phe Pro Pro Gln Glu
Thr Pro Val Ser His Arg Val 210 215
220Gly Cys Ala Val Asp Glu Leu Met Arg His Trp His His Gly Leu Tyr225
230 235 240Ser Leu Ser Leu
Pro Val Arg Glu Gly Ser Glu Asp Ala Ala Pro Ala 245
250 255Asn Thr Gln His Met Val Thr Leu Phe Asp
Glu Pro Thr Leu Glu Ala 260 265
270Ile Val Ala Gly Cys Lys Glu Leu Gly Val Ser Val Ser Ala Ala Val
275 280 285His Ala Ser Ile Val Arg Val
Trp Ala Ser Phe Pro Gln Gln Gln His 290 295
300Thr Gly Ala Arg Asn Met Leu Ile Pro Leu Val Ala Asn Leu Arg
Pro305 310 315 320Leu Leu
Asp Pro Lys Trp Val Val Pro Asp Tyr Ala Leu Ser Leu Cys
325 330 335Ile Phe Val Val Pro Phe Cys
Leu Thr Gly Gly Phe Glu Asp Leu Thr 340 345
350Gln Arg Leu Gly Ala Val Tyr Ser Arg Asp Leu Ser Ala Leu
Pro Ser 355 360 365Asp Ser Ala Gly
Asp Pro Val Ser Phe Leu Glu Leu Leu Pro Leu Tyr 370
375 380Asp Ser Gln Glu Ala Ala Phe Leu Gly Ser Leu Pro
Val Ala Gly Cys385 390 395
400Pro Pro Phe Arg Val Pro Asn Leu Ser Ser Leu Gly Val Leu Glu Arg
405 410 415Tyr Leu Ala Arg Ala
Tyr Gly Gln Lys Gly Ala Gln Ala Pro Val Cys 420
425 430Glu Ile Glu Asp Val Ala Leu Val Asn Ala Thr Thr
Asp Pro Thr Ile 435 440 445Glu Phe
Gln Leu Phe Thr Phe Arg Gly Thr Met Arg Leu Tyr Leu Tyr 450
455 460Tyr Asn Asp Ala Tyr Tyr Thr Glu Asp Phe Leu
Ala Ser Val Met Glu465 470 475
480Met Val Arg Glu Ser Leu Leu Gln Glu Leu Gly Leu Asp Gly Ser Glu
485 490 495Ser Ser Glu Gly
Leu Asp Pro Lys Glu Ala 500
50528279PRTEpichloe uncinata 28Met Thr Val Asn Ser Ser Val Lys Gln Glu
Tyr Asp Ala Gln Ala Ala1 5 10
15Ile Tyr Asp Gly Tyr Met Asp Arg Pro Ser Gly Val Ile Glu Arg Gln
20 25 30Leu Phe Thr Ala Ala Leu
Gly Asn Cys Thr Gly Leu Thr Val Leu Asp 35 40
45Leu Gly Gly Gly Thr Gly Leu Lys Ala Arg Glu Ala Ala Asp
Ala Gly 50 55 60Ala Ser Ala Val Asp
Val Ile Asp Leu Ser Pro Glu Met Met Arg Val65 70
75 80Gly Arg Asp Ala Glu Gln Ala Gly Pro Arg
Arg Gly Lys Asp Ile Leu 85 90
95Arg Trp Tyr Glu Gly Asp Val Thr Arg Ala Asp Leu Val Glu Thr Leu
100 105 110Pro Gly Leu Arg Gly
Pro Tyr Asp Leu Val Ile Val Gly Trp Thr Phe 115
120 125Asp His Ala His Asp Arg Ala Gln Leu Glu Ala Met
Trp His Asn Ala 130 135 140Val Val Arg
Leu Lys Leu Gly Thr Gly Arg Leu Leu Val Val Arg Asn145
150 155 160Gly Asp Pro Arg Ser Pro Ala
Val Thr Gly Gly Arg Tyr Gly Ile Arg 165
170 175Tyr Ala Asp His Val Pro Ile Pro Gly Gly Phe Arg
Phe Arg Asp Gln 180 185 190Met
Ile Arg Trp Gly Gly Gly Gly Gln Lys Gln Gly Lys Asn Pro Asp 195
200 205Gln Phe Glu Ile Leu Ala Asp Tyr Glu
Cys Thr Ala Leu Glu Val Met 210 215
220Tyr Ser Gly Ser His Glu Met Tyr His Gln Phe Gly Leu Thr Asp Ile225
230 235 240Arg Val Gln Pro
Tyr Glu Glu Thr Ala Ala Val Arg Ala Asp Pro Ala 245
250 255Phe Trp Ala Gln Phe Leu Glu Asn Pro Cys
Leu Ala Val Val Thr Ala 260 265
270Arg Lys Met Gly Met Val Glu 27529578PRTEpichloe uncinata 29Met
Ser Asn Ile Arg Ala Thr Ile Ala Gly Gly Ala Arg Trp Gln Glu1
5 10 15Val Ala Ala Asp Cys Gln Gln
His Arg Asp Ala Thr Ile Ala Lys Ile 20 25
30His Pro Pro Ile Pro Asp Thr Gln Ala Leu Glu Ser Leu Phe
Ala Gly 35 40 45Gly Asp Pro Arg
Asp Val Leu Ser Ile Pro Thr Leu Val Leu Thr Lys 50 55
60Gly Glu Leu Ala Ile Thr Ser Ala Asn Val Glu Asp Leu
Val Pro Arg65 70 75
80Leu Ala Ser Gly Glu Trp Ser Ala Ser Thr Val Leu Lys Ala Phe Leu
85 90 95Arg Arg Ala Ala Leu Ala
Gln Arg Leu Val Asn Cys Val Thr Glu Met 100
105 110Leu Ser Glu Thr Ala Leu Lys Arg Ala Ala Glu Leu
Asp Glu His Leu 115 120 125Ala Val
His Gly Lys Pro Ile Gly Pro Leu His Gly Val Pro Ile Ser 130
135 140Val Lys Glu His Ile Ala Met Lys Gly Leu Asp
Val Asn Gly Gly Tyr145 150 155
160Val Ser Glu Val Gly Arg Val Ala Glu Glu Asp Ala Leu Ile Leu Gly
165 170 175Ile Leu Arg Asp
Ala Gly Ala Ile Phe Tyr Val Arg Thr Thr Glu Pro 180
185 190Gln Ser Ser Met His Leu Glu Thr Ser Ser Ser
Leu Tyr Gly Glu Thr 195 200 205Val
Asn Pro Phe Asn Thr Thr Leu Thr Ser Gly Gly Ser Ser Gly Gly 210
215 220Glu Gly Ala Ile Ile Ala Met Arg Gly Ser
Val Leu Gly Val Gly Ser225 230 235
240Asp Ile Gly Gly Ser Ile Arg Ser Pro Ala His Cys Asn Gly Ile
Phe 245 250 255Gly Phe Lys
Pro Thr Ala Gly Arg Leu Pro Thr Leu Gly Trp Phe Ala 260
265 270Leu Met Val Gly Ser Glu Ala Ile His Ala
Thr Thr Gly Pro Leu Ser 275 280
285Thr Ser Ile Glu Gly Leu Trp Leu Phe Thr Lys Thr Leu Leu Asp Ala 290
295 300Lys Pro Trp Leu Gln Asp Pro Ser
Leu Thr Pro Met Glu Trp Arg Asp305 310
315 320Met Ser Thr Ala Phe Ala Gly Arg Arg Leu Lys Val
Ala Val Met Trp 325 330
335Asp Asp Gly Val Val Lys Pro His Pro Pro Val Thr Arg Ala Leu Lys
340 345 350Ser Val Val Glu Ala Leu
Lys Lys Ser Glu Lys Ile Glu Val Val Asp 355 360
365Trp Lys Pro Trp Lys His Asp Leu Ala Trp Ser Ile Ile Ala
Gly Leu 370 375 380Tyr Phe Cys Asp Gly
Gly Ala Gln Leu Asn Ala Ala Phe Glu Ala Ala385 390
395 400Lys Glu Pro Leu Arg Pro Leu Ser His Trp
Ile Leu Lys Glu Asn Pro 405 410
415His Val Lys His His Ser Ile Ala Ser Leu Trp Ser Ala Cys Ala Glu
420 425 430Arg Asp Ala Tyr Arg
Leu Lys Tyr Ala Glu Leu Trp Asn Asp Thr Ala 435
440 445Lys Gly Gly Gly Gly Pro Val Asp Val Ile Leu Cys
Pro Ala Gly Pro 450 455 460Gly Ala Ala
Pro Lys Leu Asn Thr Ser Arg Tyr Trp Gly Tyr Thr Ala465
470 475 480Gln Trp Asn Leu Leu Asp Tyr
Pro Ala Val Val Phe Pro Thr Gly Asp 485
490 495Ile Val Ser Val Glu Lys Asp Gly Ala Ala Gly Glu
Gln Gly Gly Gly 500 505 510Asp
Pro Ala Ser Gly Ala Asp Leu Asp Asn Trp Ser Leu Trp Thr Glu 515
520 525His Gly Ala Glu Gly Tyr Ser Asn Ala
Pro Leu Thr Leu Gln Leu Val 530 535
540Ala Arg Arg Tyr Asp Asp Glu Lys Leu Leu His Ala Leu Glu Met Val545
550 555 560Met Lys Glu Ala
Gly Leu Pro Thr Glu Leu Val Gly Arg Ser Arg Gly 565
570 575Pro Ala30496PRTEpichloe uncinata 30Met
Asp Leu Thr Gln Phe Asn Thr Ala Gly Ile Val Trp Pro Thr Val1
5 10 15Ala Ala Ile Ala Ile Ser Tyr
Ile Leu Leu Ser Ser Phe Leu Ser Trp 20 25
30Tyr Arg Leu Arg His Ile Pro Gly Pro Phe Leu Ala Ser Ile
Ser Ser 35 40 45Leu Trp Asn Val
Leu Asn Ile Val Thr Gly Arg Thr Ser Pro Val Leu 50 55
60Glu Lys Leu Pro Gly Lys Tyr Gly Pro Leu Val Arg Thr
Gly Pro Asn65 70 75
80Tyr Val Leu Thr Asp Asp Ala Glu Ile Leu Arg His Val Asn Gly Ala
85 90 95Arg Ser Thr Tyr Pro Arg
Asn Gly Trp Tyr Glu Gly Phe Lys Val Asp 100
105 110Glu His Asp His Met Gly Ser His Ile Asp Thr Ser
Val His Asp Ala 115 120 125Ile Lys
Ser Lys Val Ile Gly Gly Tyr Asn Gly Lys Asp Gly Ile Asp 130
135 140Leu Glu Gly Ala Ile Gly Ser Gln Val Lys Thr
Leu Val Ser Glu Ile145 150 155
160Arg Arg Arg His Leu Gly Gln Pro Val Asp Phe Ser Arg Leu Met Arg
165 170 175Gln Met Ala Leu
Asp Ala Ile Thr Ala Val Ala Phe Gly Glu Ala Leu 180
185 190Gly Phe Leu Thr Ala Glu Asp Gly Asp Val Phe
Gly Tyr Val Ser Ala 195 200 205Val
Asp Lys Met Leu Thr Tyr Leu Thr Leu Ala Ser Asp Leu Pro Val 210
215 220Val Arg Ser Val Val Arg Ser Arg Arg Met
Ala Pro Ala Val Arg Cys225 230 235
240Val Leu Ala Tyr Thr Gly Ile Gly Arg Met Leu Asn His Thr Arg
Arg 245 250 255Val Val Ala
Glu Arg Tyr Ala Ala Asp Asp Pro Gly Lys Gly Asp Met 260
265 270Thr Ala Ser Phe Ile Arg Lys Gly Leu Thr
Gln Ile Glu Cys Glu Gly 275 280
285Glu Ser His Leu Gln Leu Ile Ala Gly Ala Asp Thr Ala Val Thr Val 290
295 300Leu Arg Ser Thr Leu Leu Tyr Ile
Met Thr Thr Pro Arg Val Tyr Thr305 310
315 320Arg Leu Lys Ala Glu Ile Lys Ala Ala Val Asp Ala
Gly Glu Val Val 325 330
335Glu Val Ile Thr Met Ala Gln Ala Gln Arg Leu Pro Tyr Leu Gln Ala
340 345 350Val Val Leu Glu Gly Phe
Arg Met Arg Pro Ala Val Val Tyr Gly His 355 360
365Phe Lys Ser Val Pro Ala Gly Gly Asp Thr Leu Pro Asn Gly
Val Arg 370 375 380Leu Pro Ala Gly Thr
Ala Ile Ala Pro Asn Tyr Ile Ala Leu Thr Arg385 390
395 400Arg Ala Asp Val Tyr Gly Ala Asp Val Asp
Leu Phe Arg Pro Glu Arg 405 410
415Phe Leu Asp Ala Glu Pro Ala Lys Arg His Glu Met Glu Arg Ala Met
420 425 430Asp Leu Asn Phe Gly
Leu Gly Arg Trp Gln Cys Ala Gly Arg Asn Ile 435
440 445Ala Leu Met Glu Met Asn Lys Val Phe Phe Glu Leu
Leu Arg His Phe 450 455 460Asp Leu Gln
Ile Leu Tyr Pro Gly Lys Ala Trp Asp Glu Tyr Thr Gly465
470 475 480Val Val Tyr Ser Gln His Asn
Met Trp Val Gln Ile Thr Glu Ser Ser 485
490 495311808DNAEpichloe festucae 31atgacagtag atacgattac
ttcgacttct aacgggaacc aagatgttcc aaaggaattc 60ttcctaaaag aattcgaaac
tcagcttctc catgttgggt aggttatcgc gcctcatatg 120acggtcctct atcccaaaac
taatcagttt tcggttcaag ccggttcccg gacattttag 180gcagttgcgc ggtgcctgta
tacagttcgg cagtaagatc aagccatcgc ctcctcgaaa 240cccaccaact atgccacatg
cactgacctt tttttttcct ttttgcgcat cttaataggc 300ctttgagttc aacagcgttg
cccacggtgc gcgtcttcta aacttgacgc agttcggcaa 360catctacagc cgcttcacca
atgtttgtct ctttctcctc ctccctcatg actgcttttc 420cccaacgagg acacaagcta
ataaaacaca accaaccctt cacagcccac cgtcaatgta 480ttgcaaaatc gactggccgg
actggaagga ggcgtcgctg cttgtgccgt cgcatccggc 540tctgctgctg tagtcgtgac
ggtaatggcc ctcgcaggcg ttggcgacaa cttcgtctca 600tcctttcacg ttcatgctgg
cactttccac cagttcgaga gtttagccaa gcagatgggc 660atcgagtgcc gctttgtgaa
gtctcgagac cctgcagact ttgcggcggc catcgacgac 720aagaccaagt tcgtctggct
tgagaccatc agcaaccctg gcaacgtaat actagacctt 780gaggcagtct cgatggtctg
ccacaccaag ggcattcctt tgattgttag tatcccaatg 840aatactgtcc gtcccatagg
ggggttgggg ctaaaattag gggggatggg gttcccatcc 900aagattttag tgcgataaca
cctttggctg tgccgggtac ttttgtcgtc ccatcaacca 960cggcgtcgat atcgtcgttc
actcggccac caagtggatc ggcggccacg gcactacggt 1020aggcggtatc atcgtcgacg
gcggtacctt tgactggggc cagcacccgg atcggttccc 1080ccagttccat gatccacgga
cgcgactctg ggaacgcttt tcccgtcggg cgtttgctgt 1140ccgctgccag tttgagatcc
tgcgtgatac ggggagcacc ctcagcgctc ctgcggccca 1200gcagctgctg gttggcctcg
aatcccttgc cgtgcgatgc gagcgccacg cgcagaatgc 1260ggccaagatt gctgactggc
tgcgtgagca tcccctcgtg gcctgggtca gctatgttgg 1320tcacccgaac caccccgatt
accagggagc gctcaagtac ctcaagcgag gctttggctc 1380ggtcatctgt tttggtctac
gggggggttt cgaggcaggt gccctgttct gcgatgcgtt 1440gaagatggtc atcaccacta
ccaagtgcgt aattaaacta gccccttctt ctcccgagag 1500agcatcgttc tgccattcta
acctcgcttt gcagcctggg ggatgccaag accctgatcc 1560tccatcccgc ctcgactact
catgagcact tcaggtccga acatcgagct gaggctggcg 1620tcacagatga tatgatcagg
ctgtctgtgg gtattgagca gatcaatgat atcaaggccg 1680acttcgagca ggcctttgag
caagtgcttc ggggtaagaa gagtcttcgt aagccttgca 1740tcggaaagat tctcttgcag
gatgagatca atgaagactt atttggacct tcagcttgtc 1800gtacgtaa
1808321516DNAEpichloe
festucae 32atggccacag ccgtacgaga agcatttgag aaccatgtca agctggtcga
gagtcggaac 60tcgcccggcc atgtgcttgc ttcttctgag gcctccttct ttgttgccga
cttgaacgac 120gttgttcgta agtgggcggc gtggaaggaa gctctcccag atgccacccc
tttttttggt 180acgtcttcaa ttcccccccc catttcatgt ctcactaagc tgggaagtcc
cgtccgagga 240tccgaatcaa ttgacttgga ggcagccgtg aaaagcagct atgatcgacg
gctgatccag 300actctggcca cctgtggagc cggatttgac tgtgcctcgg cagaggagat
tgagttgacc 360ctgtccttgg gcattggcgc agaacgaatc atcttcactc acccgtgcaa
gcccgtctcc 420tccctcgggc tgtgccgcaa gcttgggatc acgctcatca ctttcgacaa
tgaatgtgag 480ctccgtaagc tccaccatca ctatcccgaa gctcagaccg tcctccgcat
tttcgccgac 540gacccaacca atgccgatcc tttgggtacc aagtttggcg ccgcgcggaa
cgactttgat 600ggactcgtcc gtctggttaa ggagttgaac atgcagctgg ccggcgccag
ctttcatgca 660ggtgcgtctc gctcccggta ccttggctgc atgcagctag ctaacggcgg
caattccacg 720ccctgttcta gcccctagcg tcgccgtcga tgcagctgca tacgtacggg
gcatccggga 780cgcagctgag gtcttcgcgc gggcccgcca ggtcgggctg aaccctacgg
tgctggatat 840cggcggcggc tacactgact cgacgtttca acagattgca ggggcggtca
ggccggcgat 900tgccgagtgc ttcacgtcgc aagtgggcga gggacgcctg cgcatccttg
cggagccggg 960gactctcttc tcctgcagcc cgttctatct agcagtcaag gttgtcgcgc
ggagggtgaa 1020cgccactgcg tttgggcatg agccagccac gcgtctctac atcaacgacg
gcatctacag 1080caacttcatg atgcgtttca tcgtcaacat gaccttctcg cccacggccg
tcatccggga 1140gggtgtgtgg cacgatcagg cggatcatac gatgcgcggc gaggcgtgct
ctctttgggg 1200tcggagctgc gactccaacg actgtatcaa cagggattgc cggcttggta
tgaaagtggg 1260ggtcggggac tggcttgtct tcaaagacat ggggggtgag cgtttcccct
tttccccttt 1320ttccttttcg tttttgggtc cctgtggagg agagtggcat acatgtatgt
aggagctaac 1380catgcatggt gtggcagcct acacaacggt atgcaacacc accttcaatg
gcttcaccag 1440ctccaaccac acaatttacc tggaacccac ccaagtcgac aaagcccagt
cgacctttga 1500acagttggcc atctga
1516331748DNAEpichloe festucae 33atgacattga ccaatttgga
cgtaatcgtc gttggcgctg gtttttcagg tattctcgct 60gcccacaggt gagtctacgt
tgccatccaa gacacgaata gagtagagag gtaggactga 120actatgacta aagactgcga
aagctcggat ttcgagtcca agggtttgag cgccaggaac 180gtcttggagg tgtctggcgc
gagaacgctt atccgggagc agcagtcgac agcctgtttc 240ccttctacca gttctatgat
gcggagcttc tccaagattg ggaatgggga gagcaatttc 300ccacccgtgc agagatgctc
agatattttg accacgtgga caagcgatgg gaaatctcta 360ctagttttga gtttggcgtt
tcggtttccg cggcccggta ctcggaaact acccagagat 420ggaccgtctc tttagaagat
ggcagaagag ccgaggcgca atggttcatt ccagctgtag 480ggttttcgtc cgttctcaat
atccccaaga ttccaggaat gtctcgattc cgcggtccca 540tctaccacac cgcaaaatgg
cctcatgacg ctgtcagtat gcgcggtaag agggtggctg 600tcatcggaac ggggccaagc
ggagttcaga tcatccaagc tgtgggtaag atagccaagg 660ccatgacgat atttcagcag
tctccatgcc tcactctacg caagtacggc agcccgagcc 720agacggcaac ggcactttgc
atgagacccg acgaccacag agaagcccta cgacttggac 780tgcagacttc gaacggtttc
ggctacgtga tccgtgacca ggacacgctg gatgttccaa 840tagaggagcg aaaccatttt
tatcagcagc gctatctggc gggaggctgg gctttctgga 900tggctgggtt ccgggatctg
tgccagaaca tccaagccaa ccgagatgcg tatgatttct 960gggctcggcg gacacgagcg
agaatcagcg atgtgaccaa acgggagctc ctggtgcctc 1020agattccacc ttttgccttt
ggtatcaagc gaccctgttt ggaagaggat ctttacgagg 1080tcatggacca accccatgtg
aaggttattg acatcagcaa ccagcaaatc gagttaatta 1140cagagacagg tatccgcgtt
catgggcaga cagttgaatg cgaagccatc atttttgcca 1200ccgggtttgg cgacgaggca
agcgggctca ggagtcttca tatcagaggc cggaatggca 1260tccgtttaga agatgcctgg
tccgatggtg tcgagtcgca tctcggaatg gccattcatt 1320cattccccaa catgttcttc
ctctatggac cccagtgtcc tacacttctg gtcaactccc 1380ccgcggtcat cactgttcag
gtagagtggt tgtgcgaaat catctcaagg tgccaacagg 1440cgggcattta tcaacttgag
gcgacttcca agtcccactg ccagtgggag aagaagatga 1500gcctactttg ggacaagaca
ctctaccata cacatgcacg caaaagcaag acaacagctg 1560cggctaacaa ggaagagaaa
acttggtagg ttgcacaagg gctcttgtcg ctttgttctg 1620tggactgctc taacaaaccg
gccagggttg gggggttgat tctgtatcgt cgcgagctgg 1680aaaactgtct ggctaacaac
ctggaggggt ttcaagcatg gcatgtagag gaggcggctc 1740ttctgtga
174834698DNAEpichloe festucae
34atgctcgatg aaagcccgat gcggaagggt gattctgtga gcaacgacca aggcaacccg
60gagagcaatg catctgtttc aatccatcag cagaatcaaa tcatcacctg tgtctcccca
120gacccagtct gccccaatgc gatacgaatc aagcgtgaca ttgtgatcgt gagactgcgg
180cccgtcgaaa gttgtccagg ctatcgcttc tttcgccgag tctttgagac actagagaaa
240tggcagctgc aagtcgacat gttctcaacc agtctggggc gaataaccct ggcgctgggg
300gccgcagcgc tgcaagccgg cattggtgac tcatgcagtg ccagaaacga catgatgagc
360cgggatctca tgcacggcat gcagaagctg ctcccggacg atcacataga gctctttcct
420cacatgacca tcatctcggt cgttgaacac ccgagccgac gaatggccgg ccacatcttc
480gccaccatgg atgccaatga tattcccacg gtcatgattt cgcacggtat ggcctcaatc
540ctttcccgtt tccatagcaa ataatcgtcc cattgttctg accagcatgt tcagacgccg
600ccaggctcgg tatagcctgt gccatctcgg agcagtacac tgctaaagct ttgtgcgtct
660tcgagcaatg cttattccgg tactccttga cacattga
698351518DNAEpichloe festucae 35atgacagtga acagcaagcg tatcccattc
ggcaagccta tgctggaggc tttctgcatg 60gatccggaat acaccaacct caattcttgt
cagttgctcg tcacacgatg tgcccgtttc 120agtttccatg tgactgtatc tgaactaaca
cgatgtggtc gtagcatctt gtgggtcatg 180gcccaaggtg gtgagcaagc agatcagaga
ttactggtcc ctgctggagg cccagcccga 240cttgttctcc gagttttccc aaggcttggt
gctgcaggag gcacgtctcg gcctcgctcg 300tctcgttcac gccgccgtct cggagtgcgt
cctcgtctcc aacgtcacta ctggtatctt 360caccgtcctt tacaaccagg catttgagga
acgagacgtc gtggtgactc tatcaactac 420ttacggtgcc atcgaccatg gcatcacttc
cttggccgag gcgcggccct tcaaaacacg 480tagggtggag tttgagctcc ccacgacggg
cgaaaagatt gtgtctcggt ttgagactac 540catggcccaa atcagggctg agggtctacg
cccgcgccta gcaattctag agacgatagt 600gagcatccct gctgttagga tgccgtttga
agacttgctg cgcgtatgcc agaaggaagg 660catcatgacg ttggtcgacg gggcgcatag
cgtgggccag tttgaggtca atctccagga 720gctgcagccc gacttcttcg tctctgattg
ccacaagtat gcagatccaa cgtcacgcac 780gcccttgtaa tacggtgatt ccatgctgat
actctacgat ttcactttac taggtggcta 840tttgttcctc gaccttgtgc tttcttatac
gttgccgagc gcaaccagca catgatgcgc 900tccgccatcc cgacctcttt cggatttatt
cccaagaatg gcaactctcg acttccccta 960tggtcgcaga tggtcagtgc cagcgaaacg
gcgtcttcgt tcgagacact gttcgcctac 1020acagccacga gcgataacat gccccatctg
tgcatcccga ccgccctccg cttcaggcgg 1080gacgtctgtg gtggcgagac ggcaatttat
gagtatataa agtggctcgc tacagagggt 1140ggtgacaagg tcgccaatat tcttcagaca
gaggtcttgg aggagcctag tctcggggcc 1200ggggtagatg gccagatgag agactgcggc
atcgtgacag tgcgactccc tttggccatt 1260gccacgggcc cgtccactgc cccagctcac
gtgccgatgc cgggcggcgc tctgacggag 1320aaagaggtcg gcccagcagt tcgttacttg
acaaaggctc tggcggaaag atacaagacc 1380tggataccca tcatcgacta ccgcggatgg
atatgggcta gactctgtgc gcaagtatac 1440ttggaggtca gtgattttga gatggccggc
aatgctctca agggaatatg cgaagagata 1500ctcagcaggg agaggtga
151836853DNAEpichloe festucae
36atgatcgctg cttcttcccc tcactcaggc gtcgtctctg cagaggacat cgaattttac
60caagccaacg gatatcttcg cttgccccaa gaggctcacg gcctgttcga cgacttggca
120aagctgcagg tatgggtggc aaaaatctcc cagtggggcc tggaaacagg gaaatggcga
180cattattacg agacgacgaa tggcaagcat cttctctggg ggacggagaa gctcatggaa
240tatcacgcgc ccatgcgaga cctgattgcc ggcgatgcac ctctcgcact gctcaagtcg
300ctgacgggca aagacatggt ggtcttcaag gacgagatag ggtggaaact cccaggcggg
360aagggggcgg tccctcacct cgaccggccc gcgtactcca tgtttgcccc cgagttcatc
420gagatcatga tcgccgtcga tgcccatacg gtcgagaatg gttgtttgca atttgtgcca
480ggctctcaca aggaggcagt cccgatctcg gccgacggcc gcattgcatc ggcgtggcta
540gagggcaagg aattcatccc catggtcctc gatccgggcg acgtcttgat cttcaacgag
600agcatggccc atcggttgga gcctaacaag acggaccaaa gacgtgcagc tgtctttggc
660acctaccact ttgacctgtc ccagcccgac ctgcgggaca aattctacgc ccaccggctc
720atccacagcc ccccggaaaa cggtaaggct tttccttggc cagatgatat ttgcatgttt
780ggaggccaat gctaacatga tgcgtgacca atctcacgta gcctgggttg aaacagtggg
840agcgcagact tga
853371184DNAEpichloe festucae 37atgacggtaa caaacaagcc tgttaagcct
gctaatgtgc cagtgatgga ctttgaggca 60atccatgcca gtgtcgggaa tgagcgcaag
aaatacttgc gacagctcga cgaggcatgg 120agccatcacg gagccatcta cgttattaat
cacagtattg gcactgagac gctcgaggaa 180gcattcgcct gggtaagtag ctggcctggt
tactcaagaa tgggctggca ttcctcatgc 240aagttaggag cgctaaatga tcttgtgtgc
cttttgaaat gcagtgcaag aagttttttg 300acctgcctct ggcggtcaag aactcggttc
acatcccacc tgacgtatcc aagcatttcc 360agggctggac gggcacaggt gaggccatct
cctcgcaggg cgtctgggat cccgacgaga 420tcgagaggct ccgcaaggag atgccgacgg
agctcaaaga ggccatggag ctgcaggacc 480cttgcggaac gtaccccccg ggcctcccag
atctaaactt ggtggagcag catctcccgg 540gctatctcga cttcttgaag aagtggttcg
cggcctgcta caggcaatct ctccagaaca 600tgcggctcgt gtgcgaaatc ctcgggatgg
aggatttgga ttacattgga aaaaagtttg 660cgccgcgcca catgagcacc cactcaacct
ggaactactt cctcgggcag cccgtttcac 720agctggccag gggatccgcg aaccggctca
acgcgcatac ggactactgc cagttcacca 780tgctcttcca ggacatggtc ggggggcttg
agctgcacga ctacgaggaa gacatttatc 840gacctgtgcc tccgatcaag ggggctatga
ttgttcaagt tggggacctg cttgagaagc 900agaccaacgg cagatggcgg agcgccctcc
accgcgtcac ggcgccgagc cggtacatgt 960acgaaggaag tgccggtgac aatgacgagc
tggtgcagcg ctactcgctc gtcttcttcg 1020gacacttgaa tctggacgag atgatcgaac
ctctgccggg ctgcgagaag ccaggaaagt 1080ggagcacgct cgagtggaag gatcggatga
cggcagggca gtggctggcc cgccgagttg 1140ctcttgagta tgagcgcaag aagacagcag
caacggtcat gtag 1184381491DNAEpichloe festucae
38atggtcagtg cagggaattg tgccattgtc gctcccggat ggaccagatc agaagacggc
60actctcactc gccccctcga cttggtggag aactggctgc ttgcccgtat tcaacgcgcc
120aacacgcctc ctggtcgtga agccgagggc ctcacctaca agctcaaatt acgactgccc
180cacgacattg atgaccccat cccctacctc cgccgagctt ggcttgtttt tcgatatgtc
240caaccgctca taggcgcaat ctatccgccc tattcagaac gagatgagac agggcggtac
300ctggttacgg ttcccccaat ggatcctgag gagtggctgc ggctaagttt ccacgtaaac
360caagggagcc aggccgtttt cagggacgtg gacgatgccg gaaagatctt tcgacctcgt
420gaaacggcaa tggcctactg gtttccttcg tcgtcgacct tggttattcg cagcactcac
480ctccgatttg acgctgtggg actatacaag gcgacaaaca ccttcatgct cggtcttgag
540tcagtctttc gtctcggcct cgacgccaac ctatattgct acactaccga tgtcaaacaa
600ccgtccctcc cgcccggcat cgactatatt ctggggttcc cgccccagga aacacccgtc
660tcacaccgcg tggagcgtgc cgtcgatgag ctgatgcggc attggcatca tggtttgtac
720agcttgtctc tccctgtgcg tgaggggtcc gaggacgctg cgcctgccaa cacccaacac
780ctggtgacct tattcgatga gccgacgctt gaggccatcg tggcgggttg caagaagctg
840ggtgtgagcg tctcggccgc cgtgcacgcg agtattgtcc gcgtctgggc ctcattccct
900caacagcagc acactggagc gcgcaacaac atgctcattc ccctcgtcgc gaacctgcgt
960ccacttctgg accccaaatg ggtggttccg gactacgcac ttagcctctg tatctttgtc
1020gtgccgttct gtctcacggg tggctttgag gacctcacgc aacgtctggg tgctgtttac
1080tcgcgagatc tgtcggcgct gccctcagac ccggcaggcg accctgtgag ctttctcgag
1140ttgctgccgc tgtatgacag cggggaggcc gctttccttg gctccttgcc cgttgccggc
1200tgtccgccct tccgggtacc caacctcagt agcctcggcg tgttggagag atatctagcg
1260cgtgcgtacg ggaaaagggg ggcacaagcc cctgtttgtg agatcgaaga cgtagccctc
1320gtcaatgcaa cgactgatcc aaccatcgag ttccagttat ttaccttccg cggcactatg
1380cggctgtact tgtactacaa tgatgcatac tatacggaag actttctggc tcccgtcatg
1440gagatggtgc gcgacagtct tctccaggag ctaggactcg gtagaagtta g
149139840DNAEpichloe festucae 39atgacggtga atagcagcgt gaagcaggag
tacgacgccc aagccgccat ctacgatggc 60tacatggacc ggcccagcgg cgtgatcgag
cggcagctct tcacggcagc actcggcaac 120tgcacagggc tgacggtgct cgacctgggc
ggcgggacgg ggctcaaggc gcgcgaggcg 180gccgacgcag gagcgtcagc cgtcgacgtg
atcgacctgt cgcccgagat gatgcgggtg 240ggccgagatg cggagcaggc gggccctcgc
cgcggaaaag acattctccg gtggtacgag 300ggcgacgtga caagcgccga cctcgtcgag
acgctgccgg gactccgcgg gccgtacgac 360ctcgtcatcg tcggctggac cttcgaccac
gcacacaacc gggcgcagct tgaggccatg 420tggcacaacg ccgtggtgag gctcaagccc
agcactggcc gcctgctcgt cgtccgcaac 480ggcgatccgc gcagccctgc cgtcaccggc
ggccgctacg gcatccgcta cgccggccat 540gtacccatcc ccggcgggtt ccggttccgc
gatcagatga ttcgttgggg aggcggcggc 600caacagcagg ggacaaaacc cgaccagttc
gagatcctcg ccgactacga gaccaccgcc 660ctcgaggtca tgtactccgg ctcgcacgag
atgtaccatc agttcggcct taccgacatc 720cgcacccagc cctatgaaga gacggctgcc
gtccgagccg acccggcctt ttgggcccag 780ttcctcgaga acccgtgtct ggccgtcgtc
acggcccgga agatgggcaa ggtggaatag 840401987DNAEpichloe festucae
40atgtcgaaca tgagagctac gattgcgggg ggtgcgcggt ggcaagaggt agcagcagac
60tgccaacagc atcgcgatgc gactgttacc aaaatccacc ctcccattcc agatacacaa
120gcactggaga gccttttcgc aagcggtgat ccgcgcgacg tctcgagcat tccaactctc
180gtcctcagcg agggggaact tgccatcacg tcggccaatg tcgaggatct tgttcccagg
240ctggccagcg gagagtggag cgccagcacg gttttgaagg ccttcttgcg cagggcggcg
300ctagcccagc ggctcgtgaa ttgcgtgaca gagatgcttt cagaaacggc cttgaagaga
360gcagcagaac tggatgagca ccttgcggtc cacgggaagc ccattggccc actccacggc
420gtccccatca gcgtcaaaga gcacatcgct atgaaaggat tggacgtcaa tggtggctac
480gtatccgagg ttgggcgcgt tgctgaggaa gatgcgctga tcttgaacat cctccgggat
540gcgggcgcca tcttctacgt taggacaaca gagccacagt catcaatgca cctggagacg
600agtagcagtc tctatgggtg agtcatacgc ggcaatttgg cgtctacttg acaaacaatc
660gactaaccat tggacagaga gactgtgaac ccctttaaca ccaccctcac atcgggcggg
720tcttcgggcg gagagggcgc catcatcgcc atgcgaggct cggtgctagg ggtcggcagt
780gacatcgggg gaagcatccg ctcaccagcg cattgtaacg gcatcttcgg gttcaagcct
840actaccggac gtctacccac cctcggatgg ttcgcgctca tggtgggctc tgaggctatc
900cacgcaacta ctggtcccct ctctaccagc atagaaggtc ttcggctgtt taccaaaaca
960ctgttggacg cgaagccatg gctccaggac cccagcctga cacccatgga gtggagagat
1020atgtccacgg cctttgccgg gcgaagactg aaggtcgcgg tgatgtggga cgatggcgtc
1080gttaagccgc atccgcccgt cacgcgggct ttgaaaagcg tggtggaaga tttgaagaag
1140agcgagaaga ttgaggtggt ggactggtga ggactattct aggggaatca cccctttttt
1200cccctccccc cttgtcattc catcgtcggc ccgcaagtct ttgcttctcg tcggtctggc
1260gcccccaaaa caacgagcgg gcaacatcaa gtcagactaa cggctgatat tcttgtagga
1320aaccatggaa acacgacctt gcctggtcca tcattgtaag ctgctccaca aagtcacctt
1380tcaccttccc gactaaggca taagtgatta ggcaggcttg tacttttgcg acggcggtgc
1440tcagctaaac gctgcgtttg aagcagccaa ggaaccgctg cggccgcttt cacactggat
1500tttgaaggag aacccccacg tcaagcatca ctccatagcc tcactgtgga gtgcgtgcgc
1560cgagagagac gcataccgtc tcaaatatgc cgagctctgg aacgacactg caaagggggg
1620gggcgggcca gtggacgtga tcctgtgccc ggccgggcca ggagcggcgc ccaaactcaa
1680tacttcgcga tattggggtt atacggcaca gtggaaccta ctggactatc cggccgtcgt
1740gttccccacg ggtgatatcg tcagcgtcga gaaggatggc gcggcgggcg agcagggagg
1800cggcgacccg gccagtggtg ccgacctgga caattggagt ctatggacag agcatggggc
1860tgaagggtac agcaatgcgc ccctcgcttt gcagctcgta gctcgcagat gtgacgacga
1920aaagctcctg cacgctttag agatggtaat gaaggaggct ggactagcca cggagctcgt
1980tggatga
1987411818DNAEpichloe festucae 41atggatctga ctcaattcaa aacagcgggc
atcgtttggc cgacggttgc tgccatggcc 60atatcctata tcctgctgtc gagctttctc
tcttggtaca ggctacggca catccccggc 120ccgttcctgg cctcgatctc aagtctttgg
aatgttctaa acatcgtgac tgggcgcacg 180tcgcctgtgc tcgagaaact gccaggaaag
tacggcccga tggtgcgaac cggccccaac 240tacgttctta cagatgatgc cgaaattttg
cgtcacgtta atggcgctcg cagcacatac 300ccccgtaatg ggtgtaagtc tgtccatatc
acatgtcttt tgaaatgtag ggagactcag 360agactcactc acactgtggc ttccagggta
tgaaggcttc aaggtcgatg aacacgacca 420tatggggtcc catatcgaca cgtcggtaca
tgacgccatc aaaagcaagg tgattggggg 480gtacaacggc aaggatggga tagacctcga
gggggccatc ggatcgcagg tcaagaccct 540ggtcagtgag atccggcgcc gtcacctagg
gcaacctgtc gacttctctc gtctgatgcg 600tcagatggcg ctcgacgcca tcaccgccgt
agcttttggc gaggccctcg ggttcctgac 660ggccgaagac ggagacgtgt tcggctatgt
cagcgccgtt gacaagatgc tgacctacct 720gacacttgcc agcgacctgc ccatagtgcg
cagctttgtc cgttcacgcc gcatggcgcc 780ggcggtgcgt tgcgtcctgg cctatactgg
catcggccgc atgctcaacc atacacgccg 840ggtggtggcg gagcgctacg cggccgacga
ccccgggaag ggcgacatga cggcctcatt 900cattcgcaag gggctcacgc agatcgagtg
cgagggcgag agccacctgc agctcatcgc 960cggcgccgac actgccgtca cggtgctgcg
ctccacgctg ctgtacatca tgacgacgcc 1020gcgcgtgtac acgcggctca aggccgagat
caaggccgcg gtggatgccg gcgaggtggt 1080cgaggtcatc accatggccc aggcccaggg
gctgccgtat ctgcaggctg tcgtgctcga 1140gggcttccgc atgcgcccgg ccgtcgtgta
cgggcacttc aagtcggtgc cggccggcgg 1200cgatacgctg ccgaatggtg tacgcctgcc
tgccggcact gccatcgccc ccaactacat 1260agcactgacc cggcgaaccg acgtctatgg
cgctgatgtc gatttgttcc ggcccgagcg 1320tttcctcgac gccgagccgg ccaagcgcca
cgagatggag cgcgccatgg acctgaattt 1380cgggcttggc cgctggcagt gcgctggcag
gaacattgct ctcatggaaa tgaataaggt 1440tttcttcgag gtcggtggat gtgcatctcc
gctcttttgc ttcggttctt ttttcctgcc 1500attgccctcg ccctcctttg ctatcctgac
gcaggcgagg tatgagacga gatgagagac 1560tgattcaatg cgcagttatt acgccacttc
gacctccaga tcgtgtatcc aggcaaagca 1620tgggatgaat acacgtaagg ccttctgaac
cctttttttt tttttttttt tttccccacc 1680tttcgcgcat atgcgtctcg ggtggcgtga
gcagcatgcc atggatattg gagtgctaac 1740caggttactt cttacctctg caggggcgtg
gtatattcgc agcataacat gtgggtacaa 1800atcacggaga gctcgtga
1818422055DNAEpichloe uncinata
42agaacaagtt ggcgattttc caacttggtt actcacccaa gccgtcactc tgttcatatc
60attactcgcc ttccagtgtc ttgcccacct tgtttcaata ttgtcgtcga gcaaaatgac
120agtagatacg attacttcga cttctaacgg gaaccaagat gttccaaagg agttcttccc
180aaaagaattc gaaactcagc ttctccatgt tgggtaggtt atcgcgcctc atatgacggt
240cctctatccc agaactaatc agtttgcgat tcaagccggt tcccggacat tttaggcagt
300tgcgcggtgc ctgtatacag ttcggcagta agatcaagcc accgcctcct cgaaacccac
360caactatgcc acatgtactg aattcttttc cctttttgcg catcttgata ggcctttgag
420ttcaacagcg ttgcccacgg tgcgcgtctt ctaaacttga cgcagttcgg caacatctac
480agccgcttca ccaatgtttg tctctttctc cttctccctc atgactgctt ttttaacgag
540gacacaagct aataaaacaa aaccatcctt caacagccca ccgtcaatgt attgcaaaat
600cgactggccg ggctggaagg aggcgtcgct gcttgtgccg tcgcatccgg ctctgcggcg
660gtagtcgtga cggtaatggc cctcgcaggc gttggcgaca acttcgtctc atcctttcac
720gttcatgctg gcactttcca ccagttcgag agtttagcca agcagatggg catcgagtgc
780cgctttgtga agtctcgaga ccctgcagac tttgcggcgg ccatcgacga caagaccaag
840ttcgtctggc ttgagaccat cagcaaccct ggcaacgtaa tactagacct tgaggcagtc
900tcgatggtct gccacaccaa gggcattcct ttgattgtta gtatcccaat gaaaactgtc
960cgtcccatag ggggggttgg ggctaaaatt cggggggatg tggttcccat ccaagatttt
1020agtgcgataa cacctttggc tgtgccgggt acttttgtcg tcccatcaac cacggcgtcg
1080atatcgtcgt tcactcggcc accaagtgga tcggcggcca cggcactacg gtaggcggtg
1140tcatcgtcga cggcggtacc tttgactggg gccagcaccc ggatcggttc ccccagttcc
1200atgatccacg gacgcgactc tgggaacgct tttcccgtcg ggcgtttgct gtccgctgcc
1260agtttgagat cctgcgcgat acggggagca ccctcagcgc tcctgcggcc cagcagctgc
1320tggttggcct cgaatccctt gccgtgcgct gcgagcgcca cgcgcagaat gcggccaaga
1380ttgccgactg gctgcgtgag catcccctcg tggcctgggt cagctatgtt ggtcacccga
1440accaccccga tcaccaggga gcgctcaagt acctcaagcg aggctttggc tcggtcatct
1500gctttggtct acgggggggt ttcgaagcag gtgccctgtt ctgcgatgcg ttgaagatgg
1560tcatcaccac taccaagtgc gtaattaaac tagccccttc ttctcccgag agagcatcgt
1620tctgccattc taacctcgct ttgcagcctg ggggatgcca agaccctgat cctccatccc
1680gcctcgacta ctcatgagca cttcagttcc gagcatcgag ctgaggctgg cgtcacagat
1740gatatgatca ggctgtctgt gggtattgag cagatcaagg atatcaaggc cgacttcgag
1800caggcctttg agcaagtgct tcggggtaag aagagtcttc gtaagccttg catcggaaag
1860attctcttgc aggatgagat caatgaagac ttatttggac cttcagcttg tcgtacgtaa
1920ataggggtca ttgcgaggca tggtaaatgt tctaccagaa gtggcatgga agtattttcc
1980aatagacctg taattcacgg ttgctacgtt ttttcatact taccatgcgg atttattttg
2040ataatatttc tttaa
2055431875DNAEpichloe uncinata 43atgacagtag atacgattac ttcgacttct
aacgggaacc aagatgtccc aaaggaattc 60cttccaattg aattcgaaac tcagcttctt
catcttgggt aggttatcgc atctcatatg 120acggtcctct attccagaac taatcagttt
gcgattcaag ccgattcccg gacattttag 180gcagttgcgc agtgcctgta tacagttcgg
cagtaagatc aagccaccgc ctcctcgaaa 240cctaccaact atgcacatgt actgaattgt
tttccttttt gcgcattctt aataggcctt 300tgagttcaac agcgttgccc acggtgcgcg
tcttctaaac ttgacgcagt tcggcaacat 360ctacagccgc ttcaccaatg tttgtctctt
tctccttctc ccgcatgact gcttttccct 420gacgaggata caagctaata aaacacaacc
atccttcaac agcccaccgt caatgtattg 480caaaatcgac tggccgggct ggaaggaggc
gtcgctgctt gtggcgtcgc atccggctct 540gcggcggtag tcgtgacggt aatggccctc
acaggcgttg gcgacaactt cgtctcatcc 600tttcacgttc atgctggcac tttccaccag
ttcgacagtt tagccaagca gatgggcatc 660gagtgccgct ttgtgaagtc tcgagaccct
gcagactttg cggcggccat cgacgacaag 720accaagtttg tctggcttga gaccatcagc
aaccctggca acgtaatact agaccttgag 780gcagtctcga cggtctgcca caccaagggc
attcctttga ttgttagtat cccaatgaat 840actgtccatc ccataggggg agttggggct
aaaattaggg gggatggggt ttccatccgg 900gggattttag tgcgataaca cctttggctg
tgccgggtac ttttgtcgtc ccatcgacca 960cggtgtcgat atcgtcgttc actcggccac
caagtggatc ggcggccacg gcactacggt 1020aggcggtatc atcgtcgacg gcggtacctt
tgactggggc cagcacccgg atcgctttcc 1080ccagttccat gatccacgga cgcgactctg
ggaacgcttt tcccgtcggg cgtttgctgt 1140ccgctgccag tttgagatcc tgcgcgatac
ggggagcacc ctcagcgccc ctgcggccca 1200gcagctgctg gtaggcctcg aatcgcttgc
cgtgcgctgc gagcgccacg cgcagaatgc 1260ggccaagatt gccgactggc tgcgcgagta
tcccctcgtg gcctgggtca gctatgttgg 1320tcacccgaac caccccgatc accagggagc
gctcaagtac ctcaagcgag gctttggctc 1380ggtcatctgc ttcggtctac gggggggttt
cgaagcaggt gccctgttct gcgatgcgtt 1440gaagatggtc atcaccacta ccaagtgcgt
aattaaacta gccccttctt ctcctgcgag 1500aacatcgttc tgccattcta acctcgcttt
gcagcctggg ggatgccaag accctaatcc 1560tccatcccgc ctcgactact catgagcact
tcagttccga gcatcgagct gaggctggcg 1620tcacagatga tatgattagg ctgtctgtgg
gtattgagca gatcaaggat atcaaggccg 1680acttcgagca ggcctttaag caagtgcttc
ggggtaaaaa gagtcttcgt aagccttgca 1740tcggaaagat tctcatgcag gatgagatca
atgaagactt atttggacct tcggcttgtc 1800gtacgtaaat aggggtcatt gcgaggcatg
gtaagttttc ctaatagacc cgtaattcac 1860ggttgttact ttttt
1875441854DNAEpichloe uncinata
44ttgccattgt attactccag aacacgtatc ttgcacaaac tccaatctca gcaatggcca
60ccgtcgtacg agaagcattt gagaaccatg tcaagctggt cgagagtcgg aactcgcccg
120gccatgtgct tgcttcttct gaggcctcct tctttgttgc cgacttgaac gacattgttc
180gtaagtgggc ggcgtggaag aaagctctcc cggatgtcac cccttttttt ggtacgtcct
240caattccccc catttcatgt ctcactaagc tgggaagtcc cgtccgaggg tccgaatcaa
300ttgacttgga ggcagccgtg aaaagcagct acgatcgacg gctgatccag actctggcca
360cctgtggagc cggatttgac tgtgcctcgg tggaggagat tgagttgatc ctctccttgg
420gcattggcgc agaacgaatc gtcttcactc atccgtgcaa gcccgtctcc tccctcgggc
480tttgccgcaa gcttgggatc acgctcatca cttttgacaa cgaatgcgag ctccgtaagc
540tccaccatca ctatcccgag gctcagaccg tcctccgcgt cttcgccgac gacccaacca
600atgccgatcc tttgggtacc aagtttggcg ccgcgcggga ggacattgat ggactcgtgc
660gtctggtcaa ggagttgaac atgaagctgg ccggcgccag ctttcatgca ggtgcgtctc
720gctctcggta cctcggccgg tgaaatgcat gcagctcgct aacggcggca attccacgcc
780ctgttctagc ccctagcgtc gctgtcgacg cagctgcata cgtgcggggc atccgggacg
840cagccgaggt cttcgcgcgg gcccgacggg tcgggttgaa ccctacggtg ctggatattg
900gcggcggcta cacagactcg acgtttcaac agattgcagg ggcggtcagg ccggccatcg
960ccgagtgctt caagtcgcaa gtggtcgagg gacgccttcg catccttgcg gagccgggga
1020ctctcttctc ctgcagcccg ttctatctag cagtcaaggt tgtcgcgcgg aggaggaacg
1080ccgctgcgtt tgggaatgag ccagccacgc gtctctacat caacgacggc atctacagca
1140acttcatgat gcgtttcatc gtcaacatga ccttctcgcc cgtggccgtc atccggaagg
1200gggtgtggta cgatcagacg gagcaaacga tgcgccgcga ggcgtgctct ctttggggcc
1260ggagctgtga ctccaacgac tgcatcaaca gggattgccg gctcgatccg gaagtggggg
1320tcggggactg gcttgtcttc aaagacatgg gtggtgagcg tctcaacttt tccccccttt
1380tccttttcgt ttttgggtcc ctgtggagaa tagcatatgt aggagctaac catgcatggt
1440gggcagccta cacaacggta tgtaacacca ccttcaatgg cttcaccagt tccaatcaca
1500caatctacat tgaacccacc caagtcgaca aagcccagtc gacctttgaa cagttggagt
1560tggccatctg agtgccagtt cggggagacc cacacgcggc acgtgccgtc ccgggttccc
1620gggcgtggct gcatgacgct agacgcgcta gtctagtacc tactccgttc cgtactgtcc
1680ttgcagcagt ccgtagtcac aacagatggc ttggatcaat tgatgcacac tccctgatta
1740gcttgtttga cacattccat ttggcttcgt gcacatcatg atacaaccag tacatgtttc
1800ctccagtcct tttgtacatg gccacgccga gctcttgtaa gtacctcgtc gagc
1854451555DNAEpichloe uncinata 45ttgccattgt tttacttcag aacacgtatc
ttgcacaatt ttcagtcgca gcaatgacga 60cagtcgtacg agaagcattt gagaaccatg
tcaagctggt cgagagtcgg aactcgcccg 120gccatgtgct tgcttcttct gaggcctcct
tctttgttgc cgacttgaac gacgtcgttc 180gtaagtgggc ggcgtggaag gaagctctcc
cagatgtcac cccttttttt ggtacgtctt 240caattccccc ccatttcatg tctcactaag
ctgggaagtc ccgtccgagg gtccgaatca 300attgacttgg aggcagccgt gaaaagcagc
tatgatcgac ggctgatcca gactctggcc 360acctgtggag ccggatttga ctgtgcctcg
acggaggaga ttgagttgat cctgtccttg 420ggcattgggg cagaacgaat catcttcact
catccgtgca agcccgtctc ctccctgggg 480ctgtgccgca agcttgggat cacgctcatc
acttttgaca acgaatgtga gcttcgtaag 540ctccaccatc actatcccga ggctcagacc
gtgctccgag tcttcgccga cgatccaacc 600aatgccgatc ccttgggtac caagtttggc
gccgcgcggg acgactttga tggactcgtc 660cgtctggtta aggagttgaa catgcagctg
gccggcgcca gctttcatgc aggtgcgtct 720cgctctcggt atcttggccg gtaaaatgca
tgcatacagc tcgctaacgg cggcaattcc 780acaccctgtt ccactgttcc agcccccagc
gtcgctgtcg atgcagctgc atacgtacgg 840ggcatccggg acacagccga ggtcttcgcg
cgggcccgac aggtggggct gaaccctacg 900gtgctggata tcggcggcgg ctacacggac
tcgacgtttc aacagattgc aggggcggtc 960aggccggcga ttgccgagtg cttcaagtcg
gaagtgggcg agggacgcct gcgcatcctt 1020gcggagccgg ggactctctt ctcctgcagc
ccgttctatc tagcagtcaa ggttgtcgcg 1080cggagggtga acgccactgc gtttgggcat
gagccagcca cgcgtctcta catcaacgac 1140ggcatctaca gcaacttcat gatgcgtttc
atcgtcaaca tgaccttctc gcccgcggcc 1200gtcatccggg agggtgtgtg gcacgatcag
gcggatcata cgatgcgcgg cgaggcgtgc 1260tctctttggg gccggagctg cgactccaac
gactgcatca acagggattg ccggctcggt 1320ccggaagtga gggtcgggga ctggcttgtc
ttcaaagaca tggggggtga gcgtttcccc 1380ttttccccct gtggagaata gcatacatgt
attagcatag gagctaacca tgcatggtgt 1440ggcagcctac acaacggtat gcaacaccac
cttcaatggc ttcaccagct ccaatcacac 1500aatctacctg gaacctggaa cccacccaag
tcgacgaagc ccagtcgacc tttga 1555461820DNAEpichloe uncinata
46ttctgcatat gattatttaa ttcttcttca tttcactaag atgacattga ccaatttgga
60cgtaatcgtc gttggcgctg gtttttcagg tattctcgct gtctacaggt gagagtctac
120gttgccatcc aagacacgaa tagagtagag aggtaggact gaactatgac tacagactac
180gaaagctcgg atttcgagtc caagggtttg agcgccagga acgtcttgga ggtgtctggc
240gcgagaacgc ttatccggga gcagcagtcg acagcctgtt tcccttctac cagttctatg
300atgcggagct tctccaagat tgggaatggg tagagcaatt tcccacccgt gcagagatgc
360tgagatattt tgaccacgtg gacaagcgat gggaaatctc tgctagtttt gagtttggcg
420tttcggtttc cgcggcccgg tactcagaaa ctacccagag atggaccgtc tctttagaag
480atggcagaag agccgaggcg cgatggttca ttccagctgt agggttttcg tccgttctca
540acatccccag gattccagga atgtctcgat tccgcggtcc catctaccac accgcaaaat
600ggcctcatga cgctgtcagt atgcgcggta agagggtggc tgtcattgga acggggccaa
660gcggagttca gatcatccaa tctgtgggta agatagccaa ggccatgacg atattccagc
720agtctccatg cctcactcta cgcaaatacg gcagcccgag ccagacggca acggcacttt
780gcatgagacc cgacgaccac agagaagccc tacgacttgg actgcagact tcaaacggtt
840tcggctacgt gccccgtgac caggacacgt tggatgtccc aatagaggag cgaaaccatt
900tttatcaaca acgctatctg gcgggaggct gggctttctg gatggctggg ttccgggatc
960tgtgccagaa catccaagcc aaccgggatg cgtatgattt ctgggctcgg cggacgcgag
1020ctagaatcag cgatgtggcc aaacgggagc tcctggtgcc tcaaattcca tcttttgcct
1080ttggtatcaa gcgaccctgt ttggaagagg atctttacga ggttatggac caaccccatg
1140tgaaggttat tgacatcagc aaccagcaaa tcgagttaat tacagagaca ggtatccgcg
1200ttcatgggca gacagttgaa tgcgaagcca taattcttgc caccgggttt ggcgacgagg
1260caagcgggct caggagtctt catatcagag gccggaatgg catccgttta gaagatgcct
1320ggtccgatgg tgtcgagtcg catctcggaa tggccattca ttcattcccc aacatggtca
1380tcctctatgg accccagtgc cctacacttc tggtcaactc ccccgcggtc atcaccgttc
1440aggtagagtg gttgtgcgaa atcatcgcaa ggtgccaaca ggcgggcatt tgtcaactcg
1500aggcgacttc caaatcccac tgccagtggg agaggaagat gagcctactt tgggacaaga
1560cactctacca tacacatgca cgcaaaagca agaaaacagc tgaggctaac aaggaagaga
1620aaacttggta ggttgcacaa gggctcttgt cgctttgttc tgtggactgc tctaacaaac
1680tgatcagggt tggggggttg attctgtatc gtcgggagct ggaaaactgt ctggccaaca
1740acctggaggg gtttcaagcg tggcatgtag aggagacggg tcttctgtga agccactttc
1800tgtcgtatat cgagtcgata
1820471776DNAEpichloe uncinata 47ttcttcattt cactaagatg acattgacca
atttggacgc aatcgtcgtt ggcgctggtt 60tttcaggtat tctcgctgtc tacaggtgag
tctacgttgc catccaagac acgaatagag 120tagagaggta ggactgaact atgactacag
actgcgaaag ctcggatttc gagtccaagg 180gttcgagcgc caggaacgtc ttggaggtgt
ctggcgcgag aacgcttatc cgggagcagc 240agtcgacagc ctatttccct tctaccagtt
ctatgatgca gagcttctcc aagattggga 300atggggagag caatttccca cccgtgcaga
gatgctcaga tattttgacc acgtggacaa 360gcgatgggaa atctctgcta gttttgagtt
tggtgtttcg gtttccgcgg cgaggtactc 420agaaactacc cagagatgga ccgtcacttt
agaagatggc agaagagccg aggcgcgatg 480gttcattcca gctgtagggt tttcgtcggt
tctcaatatc cctaggattc caggaatgtc 540tcgattccgc ggtgccatct accacaccgc
aaaatggcct catgacgctg tcagtatgcg 600cggtaagaga gtggctgtca ttggaacggg
gccaagcgga gttcagatca tccaatctgt 660gggtaagata gccaaggcca tgacgatatt
ccagcagtct ccatgcctca ctctacgcaa 720gtacggcagc ccgaaccaga cggcaacggc
actttgcatg agacccgacg accacagaga 780agccctacga cttggactgc agacttcgaa
cggtttcggc tacgtgcccc gtgaccagga 840cacgttggat gtcccaatag aggagcgaaa
ccatttttat caacaacgct atctggcggg 900aggctgggct ttctggatgg ctgggttccg
ggatctgtgc cagaacatcc aagccaaccg 960ggatgcgtat gatttctggg ctcggcggac
acgagctaga atcggcgatg tgaccaaacg 1020ggagctcctg gtgcctcaga ttccatcttt
tgcctttggc atcaagcgac cctgtttgga 1080agaggatctt tacgagatca tggaccaacc
acatgtgaag attattgaca tcagcaacca 1140gcaaatcgag ttgattacag agacaagtat
tcgcgttcat gggcagacag ttgaatgcga 1200agcgatcatt tttgccaccg ggtttggtga
cgaggcaagc gggctcagga gtcttcatat 1260cagaggccgg aatggcatcc gtttagaaga
tgcctggtcc gatggtgtcg agtcgcatct 1320cggaatggcc atccattcat tccctaacat
gtttttcctc tatggacccc agtgtcctac 1380acttctggtc aactcccccg cggtcatcac
tgttcaggta gagtggttgt gcgaaatcat 1440ctcaaagtgc caacaggcgg gcatttgtca
actcgaggcg acttccaagt cccactgcca 1500gtgggagaag aagatgagcc tactttggga
caagacactc taccatacac atgcacgcaa 1560aagcaagaaa acagctgagg ctaacaagga
agagaaaact tggtaggttg cacaaaggct 1620cttgtcgctt tgtactgtgg actgctctaa
caaaccggtc agggttgggg ggttgattct 1680gtatcgtcgg gagctggaaa actgtctggc
caacaacctg gaggggtttc aagcatggta 1740tgtagaggag acggctcttc tgtgaagcca
ctttct 177648843DNAEpichloe uncinata
48gtcagtgaca ccaaacccat tcagcccacc cacaaaatct caattcgact gtattttcta
60acgatgctcg atgaaagccc aatgaggaag ggtgattctg tgagcaacga ccaaagcaac
120ccggagagca atgcatctgt ttcaatccat cagcagaatc aaatcatcac ctgtgtctcc
180ccaggcccag tctgccccaa tgcgatagaa atcaagcgtg acattgtgat cgtgagacta
240cggcccgtcg aaagttgtcc aggctatcgc ttctttcgcc gagtctttga gacactagag
300aaatggcagc tgcaagtcga catgttctca accagtctgg ggcgaataac cttggcgctg
360ggggccgcag cgctgcaagc cggcattagt gactcatgca gtgccagaaa tgacatgatg
420agccgggatc tcatgcacgg catgcagaag ctactcccgg acgatcacat agagctcttt
480cctcacatgg ccatcatctc ggtcgttgga cacccgagcc gacgaatggc cggccacatc
540ttcgccacca tggatgccaa tgatattccc acggtcatga tttcgcacgg tatggcctca
600atcctttccc gtttccatag caaatcatcg tcccattctt ctgaccagca tgttcagacg
660ccgccaggct cggtatagcc tgtgccatct cggagcagta cactgctaaa gccttgtgcg
720tcttcgagca atgcttattc cggtactcct tgacacattg aaccgttatc tcagtctctt
780catcaaaact cttctcgttg cagaattaga tagattagat gcacaagaca ctcgttgtgg
840gct
84349944DNAEpichloe uncinata 49acggaacgtc agtaacacca aacccatcca
gcccacccac aaaatctcaa ttgaactgta 60ttttctaacg atgcttgatg aaagcccgat
gcggaagggt aattctgtga gcaacgacca 120aggcaaccca gagagcaatg catctgtttc
aatccaccag cagaatcaga tcatcacctg 180tgcctcccca ggtccagtct gccccaatgc
cataggcatc aagcgtgaca ttgtggtcgt 240gagactacgg cccgtcaaaa gttgtccaga
ctatcgcttc tttcgccgag tctttgagac 300actagagaaa tggcagctgc aagtcgacat
gttctcaacc agtctggggc gaataacctt 360ggcgctgggg gccgcagcgc tgcaagccgg
cattggtgac tcatgcagtg ccagaaatga 420catgatgagc cgggatctca tgcacggcat
gcagaagctg ctcccggacg atcacatatt 480agagctcttt cctcacatgg ccatcatctc
ggtcgttgga cacccgagcc gacgaattgc 540tggccacatc ttcgccacca tggatgccaa
tgatattctc acggtcatga tttcgcacgg 600tatggcctca atcctttccc gtttccatag
aaggtcatcg tcccactctt ctgaccagca 660tgttcagacg ccgccaggct cggtatagcc
tgtgtcatct cggagcagca cactgctaaa 720gctttgggcg tcttcgagca atgcttattc
cggtactcct tgacacattg aacagttatc 780tcagtctctt catcaaaacg cttctcattg
cagaatcata ttagatgcac aagacactcc 840ttgtgggcta tggttttttt ggccgcgata
gacagctaaa actaaattct cattgtagct 900acaagcagct ttatcacttc ccgtgatact
gacttttctt tggc 944501517DNAEpichloe uncinata
50atgacagtga acagcaagcg tatcccattc ggcaagccta tgctggaggc tttctgcatg
60gatccggaat acaccaacct caattcttgt cagttgcccg tcacacgatg tgcccgtttc
120aatttccatg ttgactgtat ctgaactaac aagacgtggt cgtagcatct tgtgggtcat
180ggcccaaggt ggtgagcaag cagatcagag attactggtc cctgctggag gcccagcccg
240acttgttctc cgagttttcc caaggcttgg tgctgcagga ggcacgtatc ggcctcgctc
300atctagttca tgccgccgtc tcggagtgcg tcctcgtctc caacgtcact actggtatct
360tcaccgtcct ttacaaccag gcatttgagg aacgggacgt cgtggtgact ctatcaacta
420cttacggtgc catcgaccat ggcatcactt ccttggccga gactcggccc ttcaagacac
480gtagggtgga gtttgagctc cccacgacgg gccaaaagat tgtgtcccag tttgagactg
540ccatggccca aatcagggct gacggtctac gcccgcgcct agcaattcta gagacgatag
600tgagcatccc tgctgttagg atgccgttcg aagacttgct gcgcgtatgc cagaaggaag
660gcatcatgac gttggtcgac ggggcgcata gcgtgggcca gttcgaggtc aatctccagg
720agctgcagcc tgacttcttc gtctctgatt gccacaagta tgcagatcca acgtcacaca
780cgcccttaat acggcggttc catgctgata ctctacgcat tgatcttact aggtggttat
840ttgttcctcg accttgtgct gtcctatacg ttgccgagcg caaccagcac atgatgcgct
900ccgtcatccc gacctctttc ggatttattc ccaagaatgg caactctcga cttcccctgt
960ggtcgcagat ggtcagtgcc agcgaaacgg cgtcttcgtt tgagacactg ttcgcctaca
1020ctgccacgag cgattacatg ccccatctgt gcatcccggc cgccctccgc ttcaggcgag
1080acgtctgtgg tggcgaggcg gcaatttacg agtacataaa gtggctcgcg aaagagggtg
1140gtgacaagat cgccgatatt cttcagacag aggtcttgga ggagcctggt cttggggccg
1200gggtagatgg ccagatgaga aactgcggca tcgtgacagt gcgacttccc ttggccattg
1260ccacgggccc gtccactgcc ccagctcatg tgccgggcgg cgctctgacg gagaaagagg
1320tcggcccggc agttcgttac ttgacaaagg ctctggcgga aagatacaag acctggatac
1380ccatcatcga ttaccgcgga tggatatggg ctagactctg tgcgcaagta tacttggagg
1440tcagtgattt tgagatggcc ggcaattctc tcaaggtaat atgcgaagag atactcaaca
1500gggagatggg acaatag
1517511558DNAEpichloe uncinata 51ccttgtcaag atgacagtga atagcaagcg
tatcccattt ggcaagccta tgctggaggc 60tttctgcatg gatccggaat acaccaacct
caattcttgt cagttgctca ttacacgatg 120tgcccgtttc agtttccatg tgaaatgtat
ctgaactaac aagacgtggt cgtagcatct 180tgtgggtcat ggcccaaggt ggtgagcaag
cagatcagag attactggtc cctgctggag 240gcccagcccg acttgttctc cgagttttac
caaggcttgg tgctgcagga ggcacgtctc 300ggccttgctc gtctcgttca cgccgccgtc
tcggagtgcg tcctcgtctc caacgtcact 360actggcatct tcaccgtcct ttacaaccag
gaatttgagg aacgagacgt tgtggtgact 420ctatcaacta cttacggtgc catcgaccat
ggcataactt ccttggccga gactcgatcc 480ttcaaaacac gtagggtaga gtttgagctc
cccacgactg gcgaaaagat tgtgtctcag 540tttgagacta caatagctca aatcagggct
aagggtctac gcccgcgcct agcaattcta 600gagacgatag tgagcatccc tgctgttagg
atgccgtttg aggacttgct gcgcgtatgc 660cagaaggaat gcatcatgac attggtcgac
ggggcgcaca gcgtgggcca gtttgaggtc 720aatctccagg agctgcaccc tgacttcttc
gtctctgatt gccacaagta tgcggatcca 780acgtctctcc acgcccttga tacggtggtg
ccatgctgat cctctgcgca ttcactttac 840taggtggcta tttgttcctc gaccttgtgc
tttcttatac gttgccgagc gcaaccagca 900catgatgcgc tccgccatcc cgacctcttt
cggatttatt cccaagaatg gcaactctca 960acttccccta tggtcgcaga tggtcagtgc
aaacggaaca gcgtcttcat tcgagacact 1020gtttgcctac acagccacga gcgataacat
gccccatctg tgcatcccga ccgccctccg 1080cttcaggcga gacgtctgtg gtggcgaggc
ggcaatttac gagtacatca agtggctcgc 1140taaagagggt ggtgacaagg tcgccgagat
acttcagaca gaggtcttgg aggagcctgg 1200tctcggggcc ggggcggatg gccagatgag
agactgcggc atcgtgacag tgcgacttcc 1260cttggccatt gccacgggcc cgtccactgc
cccagctcat gtgccgggcg gcgctctgac 1320ggagaaagag gtcggcccgg cagttcgtta
cttgacaaag gctctggcgg atagatacaa 1380gacctggata cccatcgccg attgccgcgg
atggatatgg gctagactct gtgcgcaagt 1440atacttggag gtcagtgatt ttgagatggc
cggcaatgct ctcaaggtaa tatgcgaaga 1500gatactcagc agggagatgg gacaagagat
ttcggactca tataggtggc acgactaa 155852852DNAEpichloe uncinata
52tcatgaccgc tgcttcttcc cctcacccag gcgtctctgc agaggacatc gaattctacc
60aagccaacgg atatcttcgc ctgccccaag aggctcacgg cctgttcgac gacttggcaa
120agttgcaggc atgggtggca gaaatctccc agtggggcct ggaaacgggg aaatggcgac
180attactacga gacgacgaat ggcaagcatc ttctctgggg gacggagaag ctcatggaat
240accacgcgcc catgcgagac ctgattgctg gcgaggcacc tctcacactg ctcaagtcgc
300tgacgggcaa agacatggtg gtcttcaagg acgagatagg gtggaaactc ccaggcggga
360agggggcggt ccctcacctc gaccggcccg cgtactccat gtttgccccc gagttcatcg
420agatcatgat cgccgtcgat gcccatacgg tcgagaatgg ttgtttacag tttgtaccag
480gctctcacaa ggaggcagtc ccgatttcgg ccgacggccg cattgcatcg gcgtggctag
540agggcaagga gttcatcccc atggtcctcg atcccggcga cgtcttgatc ttcaacgaga
600gcatggccca tcggttggat cctaacaaga cggaccaaag acgtgcagct gtctttggca
660cctaccactt tgaccggtcc cagcccgacc tgcgggacaa attctacgcc caccggctca
720tccacagccc cccagaaaac ggtaaggctt ttccttggcc agatgatgtt tgcatgtttg
780gaggccaatg ctaacatgat gcgtgaccaa tctcacgtag cctgggttga aacagtggaa
840gcgcagactt ga
85253917DNAEpichloe uncinata 53aaagaacagt ccaacctacg agacaatcac
ggtgaagccc tctgtttcct gtaatccatc 60atgaccgctg cttcttcccc tcacccaggc
gtctctgcag aggacatcga attctaccaa 120gccaacggat atcttcgcct gccccaagag
gctcacggtc tgttcgacga cttggcaaag 180ctgcaggtat gggtggcaga aatctcccag
tggggcctgg aaacgggaaa atggcgacat 240tattacgaga caacgaatgg caagcatctt
ctctggggga cggagaaact aatggaatac 300cacgcgccca tgcaagactt gatttctggc
gaggcacctc tcgcactgct caagtcgctg 360acgggcaaag acatggtggt cttcaaggac
gagatagggt ggaaactccc aggcgggaag 420ggggcggttc ctcacctcga ccggcccgcg
tactccatgt ttgcccccga gttcatcgag 480atcatgatcg ccgtagatgc ccatacggtc
gagaatggtt gtttgcagtt tgtgccaggc 540tctcacaagg aagcagcccc gatctcggcc
gacggccgca ttgcatcggc gtggctagag 600ggcaaggagt tcatccccat ggtcctcgac
ccgggcgacg tcttgatctt caacgagagc 660atggcccatc ggttggagcc taacaagacg
gaccaaagac gtgcagccgt ctttggcacc 720taccactttg acctgtccca gcccgacctg
cgggataaat tctacgccca ccggctcatc 780cacagccctc cggaaaacgg taaggctttt
ccatgaaaag atgatgtttg catgtttgga 840gaccaatgct aacatgatac gtgaccaatc
ttacgtagcc tgggttgaaa aagtgggagc 900gcagacttga caagaac
917541372DNAEpichloe uncinata
54atatcacgat tcctcactct ttccgaggca ctaatcccaa attcaacatc aagattctat
60ctgttcaact cccaagatga cggtaacaaa caagcctgtt aagcctgcta atgtgccagt
120gatggacttt gaggcaatcc atgccagtgt cgggaatgag cgcaagaaat acttgcgaca
180gctcgacgag gcatggagcc atcacggagc catctacgtt attaaccaca gtattggaac
240taggacgctc gaggaagcat tcgcctgggt aagtagctgg cctggttact caagaatggg
300ctggcattcc ccatgcaagt taggatcgct aattgatctt gtgtgccttt tgaaatgcag
360tgcaagaagt tttttgacct gcctctggcg gtcaagaact cggttcacat cccacctgac
420gtatcaaagc atttccaggg ctggacgggc acaggcgagg ccatctcctc gcagggcgtc
480tgggatcccg acgagatcga gaggctccgc aaggagacgc cgacggagct caaagaggcc
540atggagctgc aggacccttg cggaacgtac cccccgggcg cccccgatct aaacttggtg
600gagcagcatc tcccgggctt tctcgacttc ttgaagaagt ggttcgcggc ctgctacaag
660caatctctcc agaacatgcg gctcgtgtgc gaaatcctcg ggatggagga tctggattac
720attggaaaaa agtttgagcc gcgccacatg agcacccact caacttggaa ctacttcctg
780gggcagcccg tttcacagct ggccagcgga tccgcgaacc ggctcaacgc gcatacggac
840tactgccagt tcaccatgct cttccaggac atggtcgggg ggcttgagct gcacgactac
900gaggaagaca tttaccgacc tgtgcctccg atcaaggggg ctatgattgt tcaagttggg
960gacctgcttg agaagcagac caacggcaga tggcggagcg ccctccaccg cgtcacggcg
1020ccgagccggt acatgtacga aggaagtgcc ggtgacgatg acgagctggt gcagcgctac
1080tcgctcgtct tcttcggaca cttgaatctg gacgagatga tcgaacctct gcctggctgc
1140gagaagcaag gaaagtggag cacgctcgag tggaaggacc ggatgacggc agggcagtgg
1200ctggcccgcc gagttgctct tgagtatgag cgcaagacag cagcaacggt catgtaggag
1260gcgacggagg cctgaggctt gtagtagtga agtgatttgc actgaaacaa cgaactagtc
1320cgtacagtct gctacctagt attcaatcat gagcacttat gccagtcggt ca
1372551376DNAEpichloe uncinata 55acaaatcaca attcttcact ctttccgaga
cgctaatccc aaattcagca tcaagatttt 60atctgtctaa ctccaaagat gacggtaaca
aacaagcctg ttgagcctgc taatgtgcca 120gtgatggact ttgaggcaat ccatgccagt
gtcgggaatg agcgtaagga atacttgcga 180cagcttgacg aggcgtggag ccatcacgga
gccgtctatg ttattaatca cagtattggc 240actgagacgc tcgaggaagc attcgtctgg
gtaagtagct ggcctggtta ctcaagaatg 300ggctggcatt ccccatacaa gttaggagcg
ctaattgatc ccgtgtgcct ttgaaatgca 360gtgcaagaag ttttttgacc tgcctctggc
ggtcaagaac tcggttcaca tcccacctga 420cgtatcaaag catttccagg gctggacggg
cacaggtgag gccatctcct cgcagggcgt 480ctgggacccc gacgagatcg agaggctccg
caaggagatg ccgacggagc tcaaagaggc 540catggagctg caggaccctt gcggaacgta
ccccccgggc aacccagatc taaacttggt 600ggagcagcat ctcccgggct atctcgactt
cttgaagaag tggttcgcgg cctgctacaa 660gcaatctctc cagaacatgc gcctcgtgtg
cgaaatcctc gggatggagg atttggatta 720cattggaaaa aagtttgagc cgcgccacat
gagcacccat tcaacctgga actatttcct 780ggggcagccc gtttcacagc tggccagcgg
atcctcgaac cggctcaacg cgcatacgga 840ctactgccag ttcaccatgc tcttccagga
catggtcggg gggcttgagc tgcacgacta 900cgaggaagac atttacagac ctgtgcctcc
gatcaagggg gctatgattg ttcaagttgg 960ggacctgctt gagaagcaga ccaacggcag
atggcggagc gccctccacc gcgtgacggc 1020gccgagccgg tacatgtacg gaggaagtcc
cggtggcgat gacgagctgg tgcagcgcta 1080ctcgctcgtc ttcttcggac acttgaatct
ggacgagatg atcaaacctc tgcctggctg 1140cgagaagcca gggaagtgga gcacgctcga
gtggaaggat ctgatgacgg cagggcagtg 1200gctggcccgc cgagttgctc ttgagtatga
gcgcaagaca gcagcaacgg tcatgtagga 1260ggggacggag gcctgaggct tatagcagtg
aagtgatttg caccgaaaca atgaactagt 1320ccgtacagtc tgctgctacc tagtattcaa
tcatgagcac ttctgccagt cggtca 1376561735DNAEpichloe uncinata
56ggtgctggtt tgaaacttac ttcttacatt gcaaggtttt cgtcgtcaat atccaatagc
60cctcactgct ttactctccc agactatccc cgctgttctt aacaggcaga gcagccatgg
120ccagcccagg gaatcatgcc attgtcgctc ccggatggac cagatcagaa gacggctctc
180tcacacggcc cctcgacttg gtggagaact ggctgcttgc ccgtatccaa cgcgccaaca
240cgcctcctgg tcgtgaagcc gagggtctca cctacaagct caaattacga ctgccccaag
300acattgatga ccccatcccc tacctccgcc gcgcttggct tgtttttcga tatgtccagc
360cgctcatagg cgcaatatat ccgccctatt cagaacgaga tgagacaggg cggtacctgg
420ttacggttcc cctcatggat cctgaggagt ggctgcggct aagtttccac gtaaaccaag
480ggacccaggc tgttttcagg gacgtggacg atgccggaaa gatctttcaa cctcgtccaa
540cggcaatggc ctactggttt cctccttcgt cgaccttgat tattcgcagc actcacctcc
600gatttgacgc tgtgggaata tacaaggcga caaacacctt catgctcggt ctcgagtcag
660tcttccgtct cggcctcgac gccaacctag attgctacac taccgatgtc aaacaaccgt
720ccctcccgcc cggcatcgac tatattctgg ggttcccgcc gcaggagaca cccgtcccac
780accgcgtgga gcgtgccgtc gatgagctga tgcggcattg gcatcatggt ttgtacagct
840tgtctctccc cgtgcgtgag gggtccgagg acgccgcgcc tgccaacacc caacacctgg
900tgactttgtt cgacgagccg acgctcgagg ccatcgtggc gggttgcaag aagctgggcg
960tgagcgtctc ggccgccgtc cacgcgagta ttgtccgcgt ctgggcctca ttccctcaac
1020aacagcacac cggagcgcgc aacatgctca ttcccctcgt cgcgaacctg cgtccacttc
1080tggaccccaa gtgggttgtt ccggactacg cactcggcct ctgtatcttt gtcgtgccgt
1140tctgtctcac gggtggcttc gaggacctca cgcaacgtct gggtgctgtt tactcgcgag
1200atctgtcggc gctgccctca gacccggcag gcgaccctgt gagctttctc gagctgctgc
1260cgctgtatga aagccgggag gccgctttcc ttggctcctt gcccgttgcc ggctgtccgc
1320ccttccgggt acccaacctc agtagcctcg gcgtgttgga gagatatcta gcgcgcgcgt
1380acgggaaaaa gggggcacaa gcccctgttt gtgagatcga agacgtagcc ctcgtcaatg
1440caacgactga tccaaccatc gagttccagc tatttacctt ccgcggcact atgcggctgt
1500acttgtacta caatgatgca tactatacgg aagactttct ggctcccgtc atggagatgg
1560tgcgcgacag tcttctccag gagctaggac tcggtggaag ttagtcttca gaagcactag
1620atccgaagga atcttgaacg ttgttgtcac tagagtggtc gatcagctgc taaagaaaag
1680tcagtatcac gggaagtact aaagctgctt gtaactacaa tgaggtttta gtttt
1735571629DNAEpichloe uncinata 57ctctcccaga ctatccccgc cgttcttaac
aggcagagca gccatggtca gcgcagggaa 60tcatgccatt gtcgctctcg gatggaccac
atcagaagac ggcactctca cacgccccct 120cgacttggtg gagaactggc tgcttgcccg
tatccaacgc gccaacacgc ctcctggtcg 180tgaagccgag ggcctcacct acaagctcaa
attacgactg ccccaagaca ttgatgaccc 240catcccctac ctccgccgcg cttggcttgt
ttttcgatac gtccagccgc tcataggcgc 300aatctatccg ccctattcag agcgagatga
gacagggcgg tacctggtta cggttccccc 360aatggatcct gaggagtggc tgcggctaag
tttccacgta aaccaaggga gccaggccgt 420tttcagggac gtggacgatg ccggaatgat
ctttcgacct cgtccaacgg caatggccta 480ctggtttcct ccgtcgtcga ccttggttat
tcgcagcact cacctccgat ttgacgctgt 540gggactatac aaggcgacaa acaccttcat
gctcggtctt gagtcagtct tccgtctcgg 600cctcgacgcc aacctagatt gctacactac
cgacgtcaaa caaccgtccc tcccgcccgg 660catcgactat attctggggt tcccgccgca
ggagacaccc gtctcacacc gggtggggtg 720tgccgtcgat gagctgatgc ggcattggca
tcatggtttg tacagcttgt ctctccctgt 780gcgtgagggg tccgaggacg ccgcgcctgc
caacacccaa cacatggtga ccttgttcga 840cgagccgacg cttgaggcaa tcgtggcggg
ttgcaaggag ctgggcgtga gcgtctcggc 900cgccgtgcac gcgagtattg tccgcgtctg
ggcctcattc cctcaacagc agcacaccgg 960agcgcgcaac atgctcattc ccctcgtcgc
gaacctgcgt ccacttctgg accccaaatg 1020ggtggttccg gactacgcac ttagcctctg
tatctttgtc gtgccgttct gtctcacggg 1080tggcttcgag gacctcacgc aacgtctggg
tgctgtttac tcgcgagatc tgtcggcgct 1140gccctcagac tcggcaggcg accctgtgag
ttttctcgag ttgctgccgc tgtatgacag 1200ccaggaggcc gctttccttg gctccttgcc
cgttgccggc tgtccgccct tccgggtacc 1260gaacctcagc agcctcggcg tgttggagag
atacctagcg cgtgcgtacg ggcaaaaggg 1320ggcacaagct cctgtttgtg agatcgaaga
cgtagccctc gtcaatgcaa cgactgatcc 1380aaccatcgag ttccagctat ttaccttccg
cggcactatg cggctgtact tgtactacaa 1440tgatgcttac tatacggaag actttctggc
ttccgtcatg gagatggtgc gcgaaagtct 1500tctccaggag ttgggactcg atggaagtga
gtcttcagaa ggactagatc cgaaggaggc 1560ttgaacgtcg ttgtcatgtc actagagtgg
tcggtcagct gccaaagaaa agtcagtatc 1620acgggaagt
162958840DNAEpichloe uncinata
58atgacggtga acagcagcgt gaagcaggag tacgacgccc aggccgccat ctacgatggc
60tacatggacc ggcccagcgg cgtgatcgag cggcagctct tcacggcagc actcggcaac
120tgcacagggc tgacggtgct cgacctgggc ggcgggacgg ggctcaaggc gcgcgaggcg
180gccgacgcag gggcatcggc cgtcgacgtg atcgacctgt cgcccgagat gatgcgggtg
240ggtcgagatg ccgagcaggc gggccctcgc cgcggaaaag acattctccg gtggtacgag
300ggcgacgtga ctcgcgccga cctcgtcgag acgctgccgg gactgcgcgg gccgtacgac
360ctcgtcatcg tcggctggac cttcgaccac gcgcacgacc gggcgcagct tgaggccatg
420tggcacaacg ccgtggtgag gctcaagctc ggcactggcc gcctgctcgt cgtccgcaac
480ggcgatccgc gcagccctgc cgtcaccggc ggccgctacg gcatccgcta cgccgaccat
540gtacccatcc ccggcgggtt ccggttccgc gatcagatga ttcgttgggg aggcggcggc
600caaaagcagg ggaaaaaccc cgaccagttc gagatcctcg ccgactacga gtgcaccgcc
660ctcgaggtca tgtactccgg ctcgcacgag atgtaccatc agttcggcct taccgacatc
720cgcgtccagc cctatgaaga gacggctgcc gtccgagccg acccggcctt ttgggcccag
780ttcctcgaga acccgtgtct ggccgtcgtc acggcccgga agatgggcat ggtggaatag
840592005DNAEpichloe uncinata 59atgtcgaaca taagagctac gattgcgggg
ggtgcgcggt ggcaagaggt agcagcagac 60tgccaacagc atcgcgatgc gactattgcc
aaaatccacc ctcccattcc agatacacaa 120gcactcgaga gccttttcgc aggcggtgat
ccgcgcgacg tcttgagcat tcccactctc 180gtcctcacga agggggaact tgccatcacg
tcggccaatg tcgaggatct tgttcccagg 240ctggctagcg gagagtggag cgccagcacg
gttttgaagg ccttcttgcg cagggcggcg 300ctagcccagc ggctcgtgaa ttgcgtgaca
gagatgcttt cagagacggc cttgaagaga 360gcagcagaac tggatgaaca ccttgcggtc
cacgggaaac ccattggccc actccacggc 420gtccctatca gcgtcaaaga gcacatagct
atgaaaggat tggacgttaa tggtggctac 480gtatccgagg ttgggcgcgt cgctgaggaa
gatgcgctga ttctaggcat cctccgggat 540gcgggcgcca tcttctacgt caggacaaca
gagccacagt catcaatgca cctggagacg 600agtagcagtc tctatgggta agtcgcacgc
ggcaatttgg cgcctgcttg accaacaatc 660gactgaccat tggacagaga gactgtgaac
ccctttaaca ccaccctcac ttcgggcggg 720tcttcgggcg gagagggcgc catcatcgcc
atgcgaggct cggtgctagg ggtcggcagt 780gacatcgggg gaagcatccg ctcaccggcg
cattgcaacg gcatcttcgg gttcaagcct 840actgccggac gtctacccac cctcggatgg
ttcgcgctca tggtgggctc tgaggctatc 900cacgcaacta ctggtcccct ctctaccagc
atagaaggtc tttggctgtt caccaaaaca 960ctgttggacg cgaagccatg gctccaggac
cccagcctga cacccatgga gtggagagat 1020atgtccacgg cctttgccgg gcgaagactg
aaggtcgcgg tgatgtggga tgatggcgtc 1080gttaagccgc atccgcccgt cacgcgggct
ttgaaaagcg tggttgaagc tttgaagaag 1140agcgagaaga ttgaggtggt ggactggtaa
ggactatact atgggaataa cccccttttc 1200ccccttcccc cttgccattc catcgtcggc
cctcaagtct ttgcttctcg tcggctggtg 1260ccccccaaaa caacgaccgg gcaacatcaa
gtcagactaa cgcctgacat tcttgtagga 1320aaccatggaa acacgacctt gcctggtcca
tcattgtaag ctgctccaca aactcacctt 1380gcaccttccc gactaaggca taagtgatca
ggcaggcttg tacttttgcg acggcggtgc 1440tcagctaaac gctgcgtttg aagcagccaa
ggaaccgctg cggccgcttt cacactggat 1500tttgaaggag aacccccacg tcaagcacca
ctccatagcc tcgctgtgga gtgcgtgcgc 1560cgagagagac gcataccgtc tcaaatatgc
cgagctctgg aacgacactg caaagggggg 1620gggcgggcca gtggacgtga tcctgtgccc
ggccgggcca ggagcggcgc ccaagctcaa 1680tacttcgcga tattggggtt atacggcaca
gtggaaccta ctggactatc ccgccgtcgt 1740gttccccacc ggtgatatcg tcagcgtcga
gaaggatggg gcagcaggcg agcagggagg 1800cggcgacccg gccagtggtg ccgacctgga
caattggagt ctatggacag agcatggggc 1860tgaagggtac agcaatgcgc ccctcacttt
gcagctcgta gctcgcagat atgacgacga 1920aaagctcctg cacgctttgg agatggtgat
gaaggaggct ggactaccca cggagctcgt 1980cggaagaagc cggggcccgg cgtga
2005601990DNAEpichloe uncinata
60cttgatggat ctgacccagt tcaacacagc gggcatcgtt tggccgacgg ttgctgccat
60cgccatctcc tatatcctgc tgtcgagctt tctctcttgg tataggctac ggcacatccc
120cggccctttc ttggcctcga tctcaagtct ttggaatgtt ctaaacatcg tgactgggcg
180cacgtcgcca gtgctcgaga aactgccagg aaagtacggc ccgctggtgc gaaccggccc
240caactacgtt ctcacagatg atgccgaaat tttacgtcac gtcaatggcg ctcgcagcac
300atacccccgt aatgggtgta agtctgtcca tatcacatgt cttttgaaat gtagggagac
360tcagagaccc actcacactg tggcttccag ggtatgaagg cttcaaggtc gatgaacacg
420accatatggg gtcccatatc gacacgtcgg tacatgacgc catcaaaagc aaggtgattg
480gggggtacaa cggcaaggat gggatagacc tcgagggggc catcggatcg caggtcaaga
540ccctggtcag tgagatccgg cgccgtcacc ttgggcaacc tgtcgacttc tctcgtctga
600tgcgtcagat ggcgctcgac gccatcaccg ccgtagcctt tggcgaggcc cttgggttcc
660tgacggccga agacggagac gtgttcggct acgtcagcgc cgttgacaag atgctgacct
720acctgacact tgccagcgac ctgcccgtag tgcgcagcgt tgtccggtca cgccgcatgg
780cgccggcggt gcgttgcgtc ctggcctata ctggcattgg ccgcatgctc aaccatacac
840gccgggtggt ggcggagcgc tacgcggccg acgaccccgg gaagggcgac atgacggcct
900cattcatccg caaggggctc acgcagatcg agtgcgaggg cgagagccac ctgcagctca
960tcgccggcgc cgacactgcc gtcacggtgc tgcgctccac gctgctgtac atcatgacga
1020cgccgcgcgt gtacacgcgg ctcaaggccg agatcaaggc cgcggtggat gccggcgagg
1080tggtcgaggt catcaccatg gcccaggccc agaggctgcc gtatctgcag gctgtcgtgc
1140tcgagggctt ccgcatgcgc ccggccgtcg tgtacgggca cttcaagtcg gtgccggccg
1200gcggcgatac gctgccgaat ggtgtacgcc tgcctgcagg caccgccatc gcccccaact
1260acatagcact gacccggcgc gccgacgtct atggcgccga tgtcgatttg tttcggcccg
1320agcgtttcct cgacgccgag ccggccaagc gccacgagat ggagcgcgcc atggacctga
1380acttcgggct tggccgctgg cagtgcgctg gcaggaacat tgctctcatg gagatgaaca
1440aggttttctt cgaggtcggt ggatgtgcat ctccgctctt tgcttttgtt tcttttcctg
1500ctattactct cgccctcctt tgctatcctg acgcgggcga ggtatgagac gagatgagag
1560actgattcaa tgcgcagtta ttacgccact tcgacctcca gatcttgtat ccgggcaaag
1620catgggatga atacacgtaa ggccttctga aacctttttt aatacctttc gcgcataggc
1680gtctcgggtg gcgtgagcag cgtgccatgg atattggagt gctaaccagg ttacctctta
1740cctctgcagg ggcgtggtat attcgcagca taacatgtgg gtacaaatca ccgagagctc
1800gtgagagagc gcaaaggtga gtatagtgta cagggataca catggcaggg gtggctacga
1860acgtccatga ccacgtacga ggcggtaccg atgggcggga aaggacacga ctgagacggc
1920tggagagaac gatgaagatg gggtaaggaa ttatagcaat cagaataacg atcgtgtttg
1980tgcgcgtcgg
199061446PRTPenicillium expansum 61Met Thr Val Thr Ala Glu Ile Glu Ala
Ser Pro Ser Arg Asp Pro Arg1 5 10
15Ala Phe Glu Thr Leu Leu Leu His Gly Gly Arg Phe Pro Asp Val
Leu 20 25 30Gly Ser Cys Ala
Val Pro Val Tyr Asn Ser Ala Pro Thr Val Asn Val 35
40 45Leu Gln Asn Arg Ile Ala Leu Leu Glu Gly Gly Val
Ala Ala Cys Ala 50 55 60Val Ser Ser
Gly Ser Ala Ala Val Ala Leu Thr Met Met Ala Leu Ala65 70
75 80Gly Thr Gly Asp Asn Phe Val Ser
Ser Phe His Val His Gly Gly Thr 85 90
95Phe His Gln Phe Ser Val Leu Ala Lys Gln Met Gly Ile Glu
Cys Arg 100 105 110Phe Ile Lys
Ser Glu Asp Pro Gln Ala Tyr Ala Asp Ala Val Asp Glu 115
120 125Arg Thr Lys Phe Val Trp Ile Glu Thr Ile Ser
Asn Pro Gly Asn Val 130 135 140Ile Pro
Asp Phe Glu Glu Leu Ala Lys Val Ala Arg Glu Asn Gly Leu145
150 155 160Pro Leu Ile Cys Asp Asn Thr
Phe Gly Cys Ala Gly Tyr Phe Cys Arg 165
170 175Pro Ile Asp Tyr Gly Val Asp Ile Val Val His Ser
Ala Thr Lys Trp 180 185 190Ile
Gly Gly His Gly Thr Thr Ile Gly Gly Leu Ile Val Asp Gly Gly 195
200 205Thr Phe Asp Trp Gly Arg His Arg Asp
Arg Phe Pro Gln Phe His Ala 210 215
220Thr Asp Thr Arg Leu Trp Glu Lys Phe Gly Arg Arg Ala Phe Ala Met225
230 235 240Arg Cys Gln Phe
Glu Ile Leu Arg Asp Thr Gly Ser Thr Leu Ser Ala 245
250 255Ser Ser Ala Gln Gln Leu Leu Ile Gly Leu
Glu Ser Leu Ala Val Arg 260 265
270Cys Gln Arg His Ala Glu Asn Thr Gln Ala Leu Ala Asp Trp Leu Cys
275 280 285Gly Asn Ser Asn Ser Gln Lys
Arg Gln Val Ser Trp Val Asn Tyr Ile 290 295
300Gly His Pro Asp His Pro His His Ala Leu Ala Lys Arg Tyr Leu
Arg305 310 315 320Arg Gly
Phe Gly Ser Val Phe Thr Phe Gly Ile Thr Gly Gly Val Ala
325 330 335Ala Ser Ala Arg Phe Cys Asp
Ala Leu Lys Leu Val Ile Ile Thr Thr 340 345
350Asn Leu Gly Asp Ala Lys Thr Leu Val Val His Pro Ala Ser
Thr Thr 355 360 365His Glu His Phe
Thr Pro Glu Asp Arg Lys Ala Cys Gly Val Thr Asp 370
375 380Glu Met Ile Arg Leu Ser Val Gly Ile Glu Gln Ile
Asp Asp Leu Gln385 390 395
400Ala Asp Phe Ala Gln Ala Phe Ala Gln Leu His Ile Pro Cys Ala Pro
405 410 415Gln Pro Pro Glu Leu
Ala Leu Leu Glu Glu Ala Gln Glu Glu Val Val 420
425 430Ala Ala Leu Tyr Asn Tyr Pro Pro Gly Ile Phe Met
Gly Asn 435 440
44562409PRTPenicillium expansum 62Met Ile Glu Pro Gln Thr Ile Lys Gly Ala
Ile Glu Arg Gln Ile Gln1 5 10
15Gln Asn Asn Ala Gln Lys Thr Ser His Leu Ser Glu Ala Ser Phe Phe
20 25 30Val Ala Asp Leu Ser Glu
Val Ile Thr Lys Tyr Asn Leu Trp Gln Lys 35 40
45Thr Leu Pro Gly Val Thr Pro Phe Phe Glu Ala Asn Ile Gln
Pro Gln 50 55 60Thr Ala Val Lys Ser
Asn Asn Asp Arg Gln Leu Leu Gln Thr Leu Ser65 70
75 80Gln Cys Gly Ala Gly Phe Asp Cys Ala Ser
Ala Glu Glu Ile Glu Leu 85 90
95Val Leu Ser Leu Gly Val Pro Ala Lys Arg Ile Ile Tyr Thr His Pro
100 105 110Cys Lys Pro Ile Ser
Ser Ile Glu Phe Cys Arg Arg Ala Ser Ile Glu 115
120 125Leu Ile Thr Phe Asp Asn Val Glu Glu Leu Gln Lys
Met Lys Asp His 130 135 140Tyr Pro Glu
Ala Arg Leu Met Leu Arg Val Phe Ala Asp Asp His Thr145
150 155 160Gly Val Asp Pro Leu Gly Ser
Lys Phe Gly Val Ala Thr Gln Asp Ala 165
170 175Pro Ser Leu Leu Thr Thr Ile Lys Ala Leu Gln Leu
Asn Phe Glu Gly 180 185 190Val
Ser Phe His Val Ala Pro Thr Asn Ala Asp Pro Ala Gly Tyr Val 195
200 205Arg Ala Ile Gln Asp Ala Ala Lys Val
Phe Lys Asp Ala Arg Gly Leu 210 215
220Gly Leu Gln Pro Asn Thr Leu Asp Ile Gly Gly Gly Tyr Thr Asp Glu225
230 235 240Thr Phe Pro His
Ile Ala Ala Glu Ala Lys Arg Thr Leu Asp Glu Cys 245
250 255Phe Gly Asn Gly Ser Ile Ile Pro Arg Pro
Gln Leu Ile Ala Glu Pro 260 265
270Gly Thr Leu Leu Ser Cys Ser Ser Phe His Leu Ala Val Gln Val Ile
275 280 285Ala Arg Arg Thr Asn Ala Thr
Gly Phe Gly Gly Glu Ala Pro Thr Arg 290 295
300Leu Tyr Ile Asn Asp Gly Ile Tyr Ser Asn Phe Met Met Arg Phe
Ile305 310 315 320Val Thr
Ser Ser Phe Val Pro Val Ala Val Ile Arg Asp Gly Lys Trp
325 330 335His Asp Glu Asn Ala Gln Gly
Thr Leu Glu Cys Ser Val Trp Gly Arg 340 345
350Ser Cys Asp Gln Asn Asp Cys Ile Asn Ser Arg Cys Met Phe
Ser Gln 355 360 365Glu Val Arg Thr
Gly Asp Trp Leu Val Phe Lys Asp Met Gly Ala Tyr 370
375 380Thr Thr Val Cys Ser Thr Thr Phe Asn Gly Phe Thr
Ser Pro Asn His385 390 395
400Val Ile Tyr Leu Asp Ala Pro Leu Asn
40563531PRTPenicillium expansum 63Met Thr Arg Asp Val Leu Asp Val Ala Ile
Val Gly Ala Gly Phe Ser1 5 10
15Gly Ile Leu Ala Leu His Arg Leu Arg Gln Leu Gly Leu Arg Val Arg
20 25 30Gly Phe Glu Arg Lys Ala
Ser Leu Gly Gly Val Trp Arg Glu Asn Ala 35 40
45Tyr Pro Gly Ala Ala Val Asp Ser Pro Phe Pro Phe Tyr Gln
Phe Tyr 50 55 60Asp Ala Gly Leu Leu
Gln Asp Trp Gln Trp Arg Glu Glu Tyr Pro Ser65 70
75 80Arg Ala Glu Met Leu Arg Tyr Phe Glu Gln
Val Asp Arg Glu Trp Ser 85 90
95Ile Ser Glu Gly Phe Glu Phe Gly Ala His Ile Thr Gly Ala Arg Phe
100 105 110Ser Ala Glu Ser Gln
Gln Trp Ala Ile Ser Leu Ala Asp Gly Arg Glu 115
120 125Ile Tyr Ala Gln Trp Phe Ile Pro Ala Val Gly Phe
Asn Ser Val Val 130 135 140Asn Met Pro
Gln Ile Pro Gly Leu Asp Arg Phe Gln Gly Gln Val Tyr145
150 155 160His Thr Ala Gln Trp Pro His
Asp Ala Val Ser Met Glu Asn Lys Asn 165
170 175Val Ala Ile Ile Gly Thr Gly Pro Ser Gly Val Gln
Ile Ile Gln Ser 180 185 190Val
Gly Glu Val Ala Lys Ser Leu Thr Ile Tyr Gln Gln Thr Pro Phe 195
200 205Leu Thr Leu Pro Lys Tyr Gly Asn Arg
Pro Pro Lys Leu Ala Gly Ser 210 215
220Asp Leu Leu Glu Met Gly Val Glu Ala Phe Asp Ala Ala Phe Gln Arg225
230 235 240Gly Leu Gln Ser
Phe Ser Gly Phe Asp Tyr Thr Met Arg Asp Gln Asp 245
250 255Thr Leu Ser Ala Ser Thr Thr Glu Arg Leu
Glu Phe Tyr Gln Lys Arg 260 265
270Ile Arg Glu Gly Gly Trp Ala Phe Trp Met Gly Gly Phe Arg Asp Leu
275 280 285Asn Tyr Asp Ala Arg Ala Asn
Arg Asp Thr Tyr Asp Phe Trp Ala Glu 290 295
300Asn Val Arg Pro Arg Leu Gln Asp Ala Met Lys Arg Asp Leu Leu
Val305 310 315 320Pro His
Gln Pro Gly Ser Pro Phe Gly Val Lys Arg Pro Cys Leu Glu
325 330 335Asp Arg Leu Tyr Glu Met Ile
Asp Arg His His Val Asp Met Ile Asp 340 345
350Val Ser Gln Arg Pro Ile Gln Ala Ile Thr Ile Gly Gly Ile
Gln Ala 355 360 365His Asp Glu Ile
Arg Ser Phe Asp Val Ile Ile Met Ala Thr Gly Phe 370
375 380Gly Asp Asp Ala Ser Gly Leu Lys Gln Leu Ser Ile
His Gly Arg Asp385 390 395
400Gly Val Ser Leu Ala Glu Met Trp Ser Asp Asp Ile His Ser Phe Leu
405 410 415Gly Met Ala Val His
Asn Phe Pro Asn Met Leu Tyr Leu Tyr Gly Pro 420
425 430Gln Cys Pro Ser Leu Leu Val Ser Ser Pro Ala Val
Ile His Val Gln 435 440 445Val Glu
Trp Ile Cys Gln Ala Leu Met Cys Phe Arg Lys Ala Gly Val 450
455 460Val Gln Val Glu Ser Thr Ala Glu Ser Gln Lys
Leu Trp Arg Glu Lys465 470 475
480Ile Asp Arg Leu Trp Ser Lys Ser Leu Tyr Cys Arg Pro Gly Ala Lys
485 490 495Asn Lys Gly Ala
Thr Trp Ile Gly Gly Leu Val Glu Tyr Gln Lys Glu 500
505 510Leu Cys Lys Cys Leu Asp Glu Gly Phe Pro Gly
Phe Asp Leu Thr Phe 515 520 525Ala
Gln Asn 53064474PRTPenicillium expansum 64Met Pro Ala Leu Pro Leu Ser
Glu Asn Glu Gly Trp Lys Arg Pro Thr1 5 10
15Thr Pro Phe Gly Lys Pro Met Leu Lys His Phe Cys Met
Asn Pro Glu 20 25 30Tyr Arg
Asn Leu Asn Ala Pro Ser Cys Gly Ser Trp Pro Lys Thr Val 35
40 45Arg Asp Gln Trp Arg Arg Tyr Leu Asp Asp
Leu Glu Ala Gln Pro Asp 50 55 60Tyr
Phe Ser Glu Val Lys Gln Gly Pro Val Ile Gln Glu Ala Arg Arg65
70 75 80Glu Val Ala Gln Leu Leu
His Ala Arg Val Ser Glu Cys Val Phe Ile 85
90 95Ser Asn Ala Thr Thr Gly Ile Tyr Thr Val Leu His
Asn Ile Pro Phe 100 105 110Asp
Lys Asp Asp Val Ile Ile Thr Phe Ser Thr Thr Tyr Gly Ala Ile 115
120 125Asp Asn Ala Ile Ala Ser Met Ala Glu
Thr Gln Pro Phe Gln Thr Arg 130 135
140Lys Val Thr Val Asp Leu Pro Met Arg Gly Glu Asp Ile Val Ala Arg145
150 155 160Phe Glu Gly Met
Val Ala Gln Ile Lys Ala Glu Gly Leu His Pro Arg 165
170 175Leu Ala Val Leu Glu Thr Ile Val Ser Ile
Pro Ala Ile Arg Met Pro 180 185
190Phe Glu Ser Leu Val Gln Ala Cys Gln Arg Glu Gly Val Leu Ser Leu
195 200 205Val Asp Gly Ala His Ser Ile
Gly Gln Phe Ser Leu Asn Leu Glu Val 210 215
220Leu Gln Pro Asp Phe Phe Ile Met Asp Cys His Lys Trp Leu Phe
Val225 230 235 240Pro Arg
Pro Cys Ala Ala Leu Tyr Val Pro Glu Arg Asn Gln His Tyr
245 250 255Ile Arg Ser Thr Ile Pro Pro
Ser Phe Gly Phe Ile Pro Arg Asp Gly 260 265
270Lys Pro Ala Leu Pro Leu Trp Ser Lys Gln Ser Gly Gly Gly
Ser Ser 275 280 285Gly Ser Thr Ala
Thr Asp Phe Glu Thr Ile Phe Ala Tyr Val Ala Thr 290
295 300Ser Asp Asn Met Pro His Met Cys Ile Pro Thr Ala
Leu Lys Phe Arg305 310 315
320Arg Glu Val Cys Gly Gly Glu Glu Ala Ile Tyr Gln Tyr Leu Arg Val
325 330 335Leu Ala Lys Glu Gly
Gly Asp Arg Val Ala Ala Ile Leu Gly Thr Glu 340
345 350Val Leu Asp Glu Lys Pro Ala Gly Glu Tyr Lys Ser
Gln Arg Thr Pro 355 360 365Ser Glu
Met Arg Asp Cys Gly Ile Ala Thr Val Arg Leu Pro Leu Ala 370
375 380Val Ser Ser Ser Leu Lys Pro Pro Pro His Ser
Gly Thr Pro Tyr Ser385 390 395
400Pro Leu Ser Asp Glu Glu Val Gly Pro Ala Val His Tyr Leu Ser Met
405 410 415Thr Leu Ala Glu
Thr His Lys Thr Trp Leu Pro Leu Ile Asp His Gly 420
425 430Gly Tyr Ile Trp Val Arg Leu Cys Ala Gln Ile
Tyr Leu Asp Thr Ser 435 440 445Asp
Phe Glu Trp Ile Gly Asn Val Leu Lys Glu Ile Cys Glu Thr Ile 450
455 460Gly Lys Lys Gly His Val Ile Ser Lys
His465 47065262PRTPenicillium expansum 65Met Pro Ser Leu
Pro Ser Leu Ala His Asp Pro Ala Pro Lys Leu Pro1 5
10 15Thr Leu Thr Pro Lys Gln Ile Gln His Tyr
Gln Glu Lys Gly Tyr Leu 20 25
30Ile Leu Pro Gln Ala Gln His His Leu Phe Pro Ser Leu Gln Ser Phe
35 40 45Lys Ser Trp Ile His Glu Val Ser
Val Trp Pro Ser Pro Pro Asp Pro 50 55
60Thr Lys Pro Glu Ser Tyr Arg Leu Tyr Tyr Glu Pro Ser Pro Val Pro65
70 75 80Gly Glu Asp Pro Leu
Leu Phe Asp Thr Glu Arg Val Ser Glu Ser His 85
90 95Gln Pro Leu Ala Asn Ile Ile Thr Gly Pro Ala
Ala Ile Ser Leu Leu 100 105
110His Gln Leu Thr Gly Gln Gln Met Leu Leu Phe Lys Asp Glu Val Ala
115 120 125Trp Lys Leu Pro Gly Gly Arg
Gly Ala Ile Pro His Ile Asp Leu Pro 130 135
140Ala Tyr Gly Asp Phe Ala Pro Glu Phe Val Glu Ile Met Ile Ala
Val145 150 155 160Asp Ala
His Thr Ala Glu Asn Gly Cys Met Glu Val Val Asp Gly Ser
165 170 175His Arg Glu Glu Val Pro Phe
Gly Glu Gly Gly Arg Ile Val Gly Asp 180 185
190Trp Val Gln Lys Leu Glu Gly Gln Gly Arg Glu Phe Val Pro
Val Val 195 200 205Leu Glu Ala Gly
Asp Ile Leu Ile Phe Gly Glu Lys Leu Ala His Arg 210
215 220Leu Gly Pro Asn Lys Thr Asp Gln Arg Arg Ala Ala
Val Phe Ala Thr225 230 235
240Tyr His Phe Asp Leu Glu Lys Pro Asp Leu Arg Asp Glu Phe Phe Ala
245 250 255His Arg Leu Val Phe
Asp 26066361PRTPenicillium expansum 66Met Val Gly Ile Glu Ile
Val Glu Pro Ala Asp Val Pro Val Ile Asp1 5
10 15Leu Asn Ala Leu Ser Ser Pro Ser Pro Ala Glu Arg
Arg Val Ala Leu 20 25 30Ala
Gln Leu Asp Glu Ala Tyr Arg Thr Tyr Gly Ala Ile Trp Leu Val 35
40 45Asn His Ser Ile Gly Val Asp Leu Val
Glu Glu Ala Leu Ala Trp Arg 50 55
60Phe Phe Gln Leu Pro Arg Glu Gln Lys Gln Thr Val Ser Met Pro Thr65
70 75 80Lys Asn Ala Ser Glu
Arg Ile Glu Gly Trp Ser Asp Val Gly Ala Ser 85
90 95Ile Ser Ser Gln Gly Val Trp Asp Pro Asn Glu
Leu Glu Lys Ile Arg 100 105
110Ala Ala Ser Ser Ile Glu Leu Lys Glu Val Leu Asp Ser Leu Asp Pro
115 120 125Thr Ser Glu Ala Ala Gln Asp
Ser Glu Arg Leu Arg Lys Leu Asp Gln 130 135
140Met Leu Pro Gly Phe Pro Ala Phe Ile Glu Arg Trp Trp Asp Ala
Cys145 150 155 160Phe Lys
Gln Gln Thr Glu Leu Met Arg Cys Leu Cys Glu Ile Leu Gly
165 170 175Ile Ala Asp Thr Asp Phe Ile
Val Lys Gln Gln Gln Thr Pro Arg His 180 185
190Gly Ser Thr His Leu Thr Trp Asn Tyr Phe Leu Gly Met Pro
Leu Ser 195 200 205Pro Leu Ser Ser
Gly Ser Ala Asn Arg Leu Asn Pro His Thr Asp Tyr 210
215 220Gly Gln Leu Thr Leu Leu Phe Gln Asp Met Gln Gly
Gly Leu Glu Ile225 230 235
240Leu Asp Pro Val Ala Gly Ile Tyr Arg Pro Val Pro Pro Leu Lys Gly
245 250 255Ala Met Ile Ile Gln
Val Gly Asp Ile Leu Glu Lys Gln Ser Asn Gly 260
265 270Arg Trp Arg Ser Pro Leu His Arg Val Thr Ala Pro
Asn His Leu Met 275 280 285Tyr Gly
Gly Asn Pro Gly Glu Arg Ser Asp Gln Gln Glu Asp Ala Leu 290
295 300Val Ser Arg Cys Ser Ile Val Phe Leu Cys Tyr
Pro Gly Tyr Glu Thr305 310 315
320Val Ile Glu His Leu Pro Gly Cys Glu Lys Lys Gly Asn Trp Lys Thr
325 330 335Leu Glu Trp Glu
Gly Asn Met Thr Ala Gly Glu Trp Ile Lys Arg Arg 340
345 350Ala Ala Leu Glu Tyr Glu Arg Pro Glu
355 36067554PRTPenicillium expansum 67Met Ala Val Ala Ser
Gln Gln Lys Leu Ser Trp Gln Glu Ser Ala Arg1 5
10 15Gln Val Gln Ala Ala Arg Asp Arg Ser Ile Glu
Asp Val Asp Ser Ala 20 25
30Ile Ala Ala Leu Pro Ala Thr Tyr Thr Gly Arg Val Ile Asp Phe Pro
35 40 45Arg Lys His Leu Ser Gln Thr Glu
Ile Ala Ile Thr Glu Ser Ser Ala 50 55
60Glu Thr Leu Val Ala Ser Leu Ala Thr Gly Lys Leu Thr Ala Thr Ala65
70 75 80Val Ala Asn Ala Phe
Leu Arg Arg Ala Ala Ile Ala Gln Lys Leu Thr 85
90 95Asn Cys Ile Tyr Glu Leu Leu Pro Asp Arg Ala
Ile Ala Arg Ala Lys 100 105
110Glu Leu Asp Asp Tyr Leu Ala Lys His Gly Lys Pro Ser Gly Pro Leu
115 120 125His Gly Leu Pro Ile Ser Ile
Lys Gly His Ile Gly Leu Lys Gly Arg 130 135
140Asp Leu Ser Ala Gly Phe Val Ala Trp Leu Asp Arg Glu Ser Pro
Asp145 150 155 160Asp Ala
Asn Ile Val Lys Ile Leu Leu Asp Ala Gly Ala Val Val Tyr
165 170 175Ala Arg Thr Thr Glu Pro Gln
Gly Leu Met Ala Leu Glu Thr Cys Ser 180 185
190Asn Ile Thr Gly Ile Thr Thr Asn Pro His Asn Thr Ala Leu
Thr Pro 195 200 205Gly Gly Ser Ser
Gly Gly Glu Ser Ala Leu Gln Ala Leu His Gly Ser 210
215 220Pro Leu Gly Ile Gly Ser Asp Ile Gly Gly Ser Ile
Arg Ser Pro Ala225 230 235
240Ala Asn Cys Gly Leu Tyr Gly Leu Lys Pro Ser Thr Gly Arg Leu Pro
245 250 255Leu Ile Gly Cys Ala
Ser Tyr Val Leu Gly Cys Glu Thr Ile Val Gly 260
265 270Thr Leu Gly Pro Ile Ser Pro Thr Phe Gly Gly Ile
Glu Leu Phe Leu 275 280 285Lys Thr
Ile Ile Glu Ser Lys Pro Trp Val Lys Asp Ser Met Met Leu 290
295 300Pro Ile Pro Trp Arg Asp Gln Glu Lys His Ile
His Leu Asp Asn Lys305 310 315
320Lys Leu Thr Val Gly Val Met Trp Thr Asp Asp Val Val Thr Pro Ala
325 330 335Pro Ala Val Thr
Arg Ala Leu Lys Glu Val Val Gly Arg Leu Lys Leu 340
345 350Val Asp Ser Val Glu Val Ile Glu Trp Lys Ala
Tyr Gln Gln Lys Glu 355 360 365Ala
Leu Glu Ile Leu Thr Arg Leu Tyr Ala Pro Asp Gly Gly Lys Ala 370
375 380Phe Ala Gly His Leu Glu Ala Ser Gly Glu
Pro Phe Thr Pro Leu Thr385 390 395
400Ala Trp Ser Leu Arg Asp Ala Pro Gly Ile Glu Glu Leu Ser Gln
Gln 405 410 415Gly Leu Trp
Asp Trp Thr Gly Lys Arg Glu Met Phe Arg Tyr Ala Tyr 420
425 430Leu Gln Gln Trp Asn Asn Val Ala Pro Glu
Met Asp Val Ile Leu Cys 435 440
445Pro Ala Phe Pro Thr Pro Ala Pro Leu His Phe Thr Ser Arg Tyr Trp 450
455 460Gly Tyr Thr Ser Leu Phe Asn Leu
Leu Asp Tyr Pro Ala Leu Val Phe465 470
475 480Pro Val Thr Lys Val Asp Pro Asp Arg Asp Ala Lys
His Thr Thr Tyr 485 490
495Thr Pro Lys Asn Glu Phe Asp Ser Trp Ala Tyr Glu His Tyr Asp Ser
500 505 510Val Lys Gln Lys Asp Ala
Pro Val Ser Leu Gln Leu Val Ser Lys Lys 515 520
525Leu Glu Glu Glu Lys Leu Leu Gln Ala Phe Lys Glu Ile Gln
Glu Arg 530 535 540Ile Gly Leu Pro Phe
Val Asn Cys Leu Ala545 550681802DNAPenicillium expansum
68atgacagtga ctgctgaaat tgaggcttct cctagtcggg atccgagagc gtttgagacg
60ctgcttctcc atggagggtg ggtttgattg aaggagagct agaggaatat cagtaatgct
120aagtgattta ttgtggcgca ggagatttcc cgacgtgttg ggcagttgtg ctgtacccgt
180ttacaattcc gcggtagggt attcaattca agttcatgca gtctcaagcc cttctctaac
240tctcccttcg tgtcttttgg ggtcgctatc taggctttcc aattcaacag taccgagcat
300ggtgcgcgat tgttgaatgt tacacaactc ggacacatct atagtcggtt cacaaatgtc
360tgtctctatc actttctaga tcctttcgga tcttatatgc tggtagccac tgacactttg
420tttctagccc acggtaaatg ttcttcagaa ccgcatcgct ctcctggaag gaggagtagc
480cgcttgtgcg gtctcgtcgg ggtctgcggc agtggcgttg acgatgatgg cactggcggg
540cacgggggac aatttcgttt cctcgttcca tgtccacggg ggcactttcc accaatttag
600cgtcttggct aaacagatgg gcatcgagtg tcggtttatc aagtccgagg atccgcaggc
660ctatgccgac gccgtggatg agcgcaccaa gtttgtatgg atcgaaacca tcagcaatcc
720cggtaatgtg atcccggact tcgaagagtt ggccaaggtg gcacgggaaa acggactacc
780actgattgta agactactga ttctttgctt cttgattttg tttaccggat taggctcttc
840ccatgactca gtactcgtgg aaatctaaca atctggcagt gcgacaacac cttcggctgc
900gccggatatt tctgccgacc tatcgattat ggggtggata ttgtggttca ctcagccacc
960aaatggattg gcgggcacgg cacgaccatc gggggtctca tcgtggacgg cggcaccttc
1020gattggggcc ggcaccgcga ccggttcccc cagttccacg cgaccgacac gcgcctatgg
1080gagaagttcg gccggcgggc gttcgcgatg cggtgccagt ttgagatctt gcgggacacg
1140gggagcacac tcagcgcgag ctcggcgcag cagctgctga tcgggctcga gtcgctggcc
1200gtgcgatgtc aacgccacgc ggaaaatacg caggcgctgg ctgattggtt gtgcggcaac
1260agcaacagcc aaaagcgcca ggtcagctgg gtcaactata tcggccatcc ggaccatcca
1320catcatgccc ttgctaagcg gtatctgcgg cgggggttcg ggtccgtgtt tacatttggc
1380atcacgggcg gtgttgcggc cagcgcgagg ttctgcgatg cattgaaatt ggtgattatt
1440acgacgaagt acgtataccc tctctgaggg gatgatactg gcagagtaaa gccgaacaac
1500actaacagct acatccaagc ctcggagatg ctaagaccct tgtcgttcat cctgccagca
1560ccacacacga gcatttcacg ccagaggatc gcaaggcctg cggggtgacg gacgagatga
1620ttcggttgtc ggtggggatt gagcagatcg acgatctgca agccgatttc gcccaggctt
1680ttgctcagct ccacattccg tgtgccccac agccgcccga gttggctctg ctggaggagg
1740cccaggaaga agttgtggct gcgctatata attacccacc agggattttt atgggcaatt
1800ga
1802691425DNAPenicillium expansum 69atgattgaac cccaaacgat caaaggagcg
atcgagcgac aaattcagca aaataatgcc 60caaaagacca gtcatctatc cgaggcttcc
ttcttcgtcg ctgacctgag tgaagtcatt 120accaaatata acctctggca aaagactctc
ccaggggtga caccattttt cggtaagagg 180tgtgatgatg aagtttccaa atcagcaaaa
caaccataaa acaaccggga gaggctaaca 240tccagcccca aacagctgtc aagagcaaca
atgatcgaca gttactccag acgctttccc 300agtgcggcgc cggcttcgac tgcgcctcag
ccgaggaaat tgaattggtt ctctctctcg 360gggtgccggc gaagcgcatc atctacacgc
acccctgcaa gcccatcagt tccatcgagt 420tctgtcgccg ggccagcatt gagctcataa
cctttgacaa tgtcgaggag ctccagaaga 480tgaaagacca ctatccggag gcccggctga
tgctgcgagt cttcgccgac gaccacacgg 540gcgtcgatcc cctgggcagc aagttcggcg
tcgccaccca agatgctccc agccttctca 600cgactatcaa agcactgcaa ttgaactttg
agggagtgag cttccatgtt ggttagttct 660ctcccttccc tcatggagcg atcccgaaaa
aaacggactg acaattcttg tttggatttt 720ccagccccga ccaacgccga cccagccggc
tacgtgcgcg caattcaaga cgcagccaaa 780gtattcaaag atgcacgggg cctggggctg
cagcctaaca ccctcgatat cggcggcggg 840tacacggacg aaacattccc gcacatcgcc
gccgaagcaa agcggacgct ggacgagtgt 900ttcggcaacg ggtcgatcat cccacgaccg
caacttatcg ccgagcccgg gacactgctc 960tcgtgcagtt ccttccatct agcggtgcag
gtcatcgcgc gccgcacgaa tgcgacgggt 1020tttggcggcg aggcgcctac ccgcctctac
atcaatgacg gcatttacag taacttcatg 1080atgcgcttca tcgtcacctc gagctttgta
cccgtggcag ttatccgcga tggcaagtgg 1140cacgatgaga acgctcaggg tactttggag
tgctcggtct ggggccgctc ctgtgaccag 1200aacgactgca tcaatagccg gtgtatgttc
agtcaggagg tcaggacggg cgattggttg 1260gttttcaagg atatgggagg tctgtttgcc
ccccttcccg gctaatatgc gatagttttg 1320ctaacaatca tgacggctgc agcgtacact
accgtctgtt caaccacctt taacggattc 1380actagtccca accacgtcat ttacctggac
gctccgttga attag 1425701727DNAPenicillium expansum
70atgactcgcg acgttttaga cgtggccatt gtcggagccg gattttcagg catcttggct
60ctccataggt aagtaagcta gttgacgttc gtttcaggtc cgaatcctag agaccgaacg
120ctcacgaatc aattagacta cggcagttag ggcttcgagt tcgaggcttc gagcggaaag
180cgtcgctggg gggggtgtgg cgagaaaatg cctatcccgg ggccgcggtc gatagtccat
240ttccattcta ccagttctac gatgctgggt tactccagga ctggcagtgg cgcgaagaat
300accccagtcg agccgagatg ttacgatact tcgagcaagt cgacagagaa tggagcatct
360cggaaggctt cgaattcggg gcccatatca cgggagcccg tttctcggcg gaatcccagc
420agtgggcaat cagtctcgct gatgggcgtg agatctatgc gcagtggttc atccccgcgg
480tgggcttcaa ctcagtggtc aatatgcccc aaatccccgg cctggatcgg ttccagggcc
540aggtctatca cacagcccag tggccgcacg atgccgtctc gatggagaac aaaaacgtgg
600ccatcattgg taccggaccc agtggcgtgc aaattatcca gagcgtgggc gaggtggcca
660aaagcctaac gatctaccag cagaccccct ttttgacact ccccaaatac ggcaaccggc
720caccaaagct ggcggggtct gacctgctag aaatgggcgt ggaagccttt gatgccgcgt
780ttcagcgcgg cctgcagtcg ttcagcggat tcgactatac gatgcgcgat caggacacct
840tatctgcctc gaccacagag aggttggagt tctatcagaa gcgaatccgc gaaggcggct
900gggcattctg gatgggaggc ttccgtgacc tcaactacga tgcccgcgcc aaccgggaca
960cctatgactt ctgggcagag aacgtccgac cccgtctgca ggatgcgatg aaacgtgatc
1020tgctagtgcc acaccagccc ggctcaccct ttggggtcaa acggccctgt ctggaggaca
1080ggctgtatga gatgatcgat agacaccacg tggacatgat cgatgtcagc cagcgaccca
1140tccaggccat taccattggc ggtatccagg cccacgatga gatccggtcg tttgacgtga
1200tcatcatggc gacgggattc ggggacgatg cgagtggctt gaagcagctg tcgattcacg
1260gtcgcgatgg tgtctcgcta gcggagatgt ggtccgacga tatccactcc ttcctcggca
1320tggctgtgca taatttcccc aacatgctat atctttacgg accgcagtgt ccttccctgc
1380tagtcagctc tcccgccgtc attcatgtcc aagtcgaatg gatctgtcag gcgctcatgt
1440gtttccgaaa agctggggtg gtccaggtcg agtcaacggc tgagtcacaa aagttatggc
1500gcgagaagat cgaccggctt tggtcaaaat ctttgtattg ccggccgggc gcgaagaaca
1560agggagcaac atggtacgtt atctctctac agatatagta gcttggcacg tttgtctctg
1620acttaaggcc taataggatt ggagggttgg ttgaatacca gaaggagttg tgcaagtgcc
1680tagatgaagg gttcccaggg tttgatctga catttgcaca gaattaa
1727711528DNAPenicillium expansum 71atgcctgcac tccctctctc tgagaatgag
ggctggaagc gcccgaccac cccctttgga 60aagccgatgc tgaagcattt ctgcatgaac
cccgagtatc gaaatctcaa tgcgcgtagg 120taagcttcaa ccgaatatga ggtctcgggg
gactaatacg caccagcatc ctgtggttcc 180tggccgaaga ccgttcgcga tcagtggagg
cgttacctcg acgatctcga ggcccagcct 240gactacttct ccgaggtgaa acaaggccct
gtgattcaag aggcacggcg ggaggttgcc 300caactacttc atgcccgcgt ctccgagtgt
gtgttcatct ccaacgccac cacggggata 360tacaccgtcc tccataacat tccttttgac
aaggacgacg tgatcatcac cttctcgacc 420acgtacggcg ccatcgataa tgccatcgcc
tcgatggcgg agacccagcc cttccagaca 480cgcaaggtga ccgttgacct gcctatgcgc
ggagaggata tcgtcgcccg gtttgagggc 540atggtagctc agatcaaggc ggaggggttg
catcctaggc tggccgtcct cgagaccatc 600gttagcattc ccgccatacg gatgcccttc
gaatccctcg tgcaagcctg ccagagggag 660ggggttttga gtctggtgga tggtgcgcat
agtatcggcc aattctccct gaacctcgag 720gtgctgcagc cggacttttt tattatggac
tgccacaagt atgaacagaa aactcaattg 780attcttgaat gtttctaaca cagaaaatag
atggctgttc gtgcctcgtc cctgcgctgc 840actgtatgtc cccgagcgca atcagcacta
tattcggagc accatcccgc catcgttcgg 900cttcatcccg agggatggca agcccgctct
gcccctctgg tctaagcagt ccggcggtgg 960tagtagtggc tccacggcaa cggatttcga
gactattttt gcctatgtgg cgactagcga 1020caatatgccg catatgtgca tccccacagc
cctcaaattc cgtcgtgagg tttgcggcgg 1080tgaggaggca atttatcagt atctgcgcgt
tctagcaaag gaaggcgggg atagggtggc 1140cgctattctg ggcacggagg tgcttgacga
gaaaccagcc ggagaataca agagtcaacg 1200gacgccgagc gagatgagag attgtggcat
tgcaacagtg cgcttgcccc tggccgtatc 1260gtcatccctt aagcctccac cccacagcgg
cacaccgtat tccccacttt cggacgagga 1320agtgggaccc gcagttcatt atctgtccat
gaccttagcc gaaactcaca aaacatggct 1380tccactcatt gaccacggag gatacatctg
ggtgagattg tgtgcacaga tatatctaga 1440tacctcggac tttgagtgga ttggcaatgt
gcttaaggag atatgtgaga caattgggaa 1500gaaggggcat gtcatttcta aacactag
152872955DNAPenicillium expansum
72atgccttctc tgccatcact tgcccatgat ccggccccta aactcccaac tttaaccccc
60aagcaaatcc aacactacca agaaaaaggc tacctcatcc ttccccaagc acaacaccat
120ctcttcccct ctctgcagtc cttcaaatcc tggatccatg aagtgtccgt ctggccatca
180cccccagacc ccacgaagcc cgaatcttac cgcctgtact acgaaccctc ccctgtacca
240ggggaggacc cactcctctt cgacacagag cgcgtatccg agtcccacca gccactagct
300aatatcatca cgggccccgc agccatttcc ctcctccacc aattgaccgg ccagcaaatg
360ctgctcttca aagacgaagt tgcctggaag ctacccggtg ggcgtggcgc gatcccgcac
420attgatctcc cggcatacgg ggacttcgca ccggagtttg tcgagatcat gatcgcggtg
480gatgctcaca cagccgagaa tgggtgtatg gaggttgtag atgggagtca tcgagaggaa
540gttccgtttg gggagggggg caggattgtt ggggattggg ttcagaagtt ggagggacag
600gggagggaat ttgtgccggt tgttttggag gctggtatgt atttcttctg gggatttggt
660tgaggagatg ggctgggctg actatgtgcg tacgtacgta ctataggaga tatactcatc
720ttcggggaga agctggcaca tcggttgggg ccgaataaga cggaccagcg gcgcgcggcg
780gtgttcgcaa cttatcattt tgatttggag aagccggatc tgagggacga gttctttgcg
840cacaggcttg tggtgtgtcc accggggatt ggtaggttat ctatttcctt tcattgtgga
900aataataagg gcatagagga tgtcgctaac atttcgtcta tcttagtttg attaa
955731214DNAPenicillium expansum 73atggtaggaa ttgagatcgt ggaaccggcc
gacgtgcccg tgatcgatct aaatgccctc 60tcctcccctt cccctgcgga acgccgagtt
gccctggcgc agctggatga ggcctaccga 120acctacggtg ccatttggct agtcaaccat
agcattggcg tggacctggt tgaggaggct 180ttagcatggg ttagtagaga tcttttcctc
ttcccccttt cctagaaagt tcccagacaa 240gatagcagca gatggggaaa aaggacccct
gaaagtttga gtgaaacaaa gatatctaat 300agctgctata gtgtcagcgc ttcttccaac
tgccgcgcga gcagaaacag acggtctcta 360tgccgaccaa gaatgcctcc gagcggatcg
aagggtggtc ggacgtcggg gcaagcatct 420catctcaggg ggtgtgggat ccgaacgagc
tggagaagat ccgtgcggcc agctcgatcg 480agctgaagga ggtgctcgac agcctagacc
cgacctcgga ggcggcgcag gactcggaac 540gactgcgcaa gctggaccag atgctgcccg
gattcccggc attcatcgaa cggtggtggg 600acgcttgctt taagcagcag accgaactga
tgcgctgcct gtgcgagatc cttggcatcg 660ccgacaccga tttcattgtc aagcagcagc
agacgccccg ccacggctcc actcatctga 720cctggaatta cttcttgggg atgccgctct
cgcctctgag cagcggcagc gccaaccgcc 780tcaacccgca cactgactat gggcagttga
ccctgctctt ccaggatatg cagggcgggt 840tggagatcct tgaccccgtc gctggcatct
atcgtcctgt cccgcctctg aagggcgcga 900tgatcatcca ggtaggcgat atcctggaaa
agcagtcgaa tggtcggtgg aggagtccgt 960tgcaccgcgt gacagcgccg aatcatctca
tgtatggcgg gaaccctggg gaaagatctg 1020accagcagga ggatgccttg gtgtcacggt
gctccatcgt gtttttgtgt tatcccggct 1080atgagacggt gattgaacat cttcctggat
gtgaaaagaa aggaaactgg aagaccttgg 1140aatgggaggg taacatgacg gcgggggagt
ggatcaagcg ccgggccgct ctggagtatg 1200aacggcccga gtga
1214741973DNAPenicillium expansum
74atggctgtgg cgtctcaaca gaagctttct tggcaagagt cggcccgtca ggtccaggca
60gcgcgcgatc gttcgataga ggacgttgac tcagccatcg ctgctcttcc tgctacatat
120actggcaggg tgatcgattt ccccagaaag cacctttcgc agaccgagat cgctattaca
180gagagctccg ctgagacctt ggtggcgtcc cttgcaacgg gtaaactaac tgcgaccgcg
240gtagcaaacg catttcttcg gcgggctgcg atcgctcaga agttggtgag taaagctgga
300ggctgtgccc ggacaatgcc ttgaattttc aattctaaca atcaatcgtc ttattagacc
360aactgtatct atgagcttct tcctgaccgt gccatcgccc gtgccaagga gcttgatgat
420tatcttgcga agcatggaaa gccctctggt ccgcttcacg gtttaccaat tagcatcaaa
480ggacatatag gcctcaaggg acgagacttg tctgctggat tcgtcgcttg gctcgaccgg
540gagagcccag acgatgcaaa tattgtcaag attctgttgg atgctggtgc ggttgtttat
600gcaaggacaa ccgagccgca gggactggtt agcagatcta gctttcctac agtgttgata
660tgtggctgat atcttgattt cgcggtagat ggctcttgaa acttgcagca atatcactgg
720catcaccacg aacccacata acaccgctct cacaccaggc ggttcatctg gcggagagtc
780agctttacaa gcccttcatg gcagccctct aggaattggt tcagacatcg gtatattgcc
840agattctctg cattaatttc ctaattgtta ataacagatc caggtggtag tattcgttct
900cccgccgcca attgtggcct ttacggccta aagccatcaa ccggtcgact gccgctaatc
960ggatgcgcgt cctatgtact cggatgtgag acaattgtgg gcacacttgg gcctatttcg
1020ccaacatttg gcggtattga acttttcctg aagacgatca tcgaatcaaa gccttgggtc
1080aaagattcaa tgatgcttcc aatcccgtgg agagaccaag agaaacacat tcacctggat
1140aacaagaagc taaccgtcgg tgtaatgtgg accgacgatg tcgttactcc cgcaccggca
1200gtgacaaggg cattgaagga ggtggttggg cgtcttaaat tggtggatag tgttgaggtt
1260attgaatgga aggcatacca acagaaagag gcactcgaga ttcttgtaag aggtctatat
1320cgcttcaata atcaatgtga gctgaactaa ccaatgaacg tatatagact aggctttatg
1380caccagatgg cggaaaggca tttgccgggc atctcgaggc ctctggtgag ccatttacgc
1440cactcacggc ttggagcctt cgggatgccc ctggcatcga agaacttagc cagcagggct
1500tatgggactg gaccggaaaa cgagagatgt tccgctacgc ctatttacaa cgttagtaca
1560gcaataatcc atgatcgttc ctgattctaa tccgaattcg gtccaatgca gagtggaaca
1620atgttgcgcc cgaaatggat gttatactct gccctgcatt ccccacaccc gcccctcttc
1680actttacttc gaggtactgg gggtatactt ctttattcaa tcttctcgat tacccggctc
1740ttgtgttccc ggttaccaag gtggatcccg atagggatgc caagcacacc acttacaccc
1800cgaagaacga attcgacagc tgggcatacg agcattatga ctccgtaaaa cagaaggatg
1860cgcccgtttc tttacagcta gtttccaaga agttggagga agaaaagtta cttcaagcat
1920tcaaagagat ccaggagaga atcggcctgc cgtttgtgaa ttgtctggct tga
1973
User Contributions:
Comment about this patent or add new information about this topic: