U.S. patent application number 10/011200 was filed with the patent office on 2003-09-25 for methods for the identification of inhibitors of homocitrate synthase as antibiotics.
Invention is credited to Adachi, Kiichi, Darveaux, Blaise, DeZwaan, Todd M., Frank, Sheryl, Hamer, Lisbeth, Heiniger, Ryan, Lo, Sze-Chung, Mahanty, Sanjoy K., Montenegro-Chamorro, Maria Victoria, Pan, Huaqin, Shuster, Jeffrey, Skalchunes, Amy, Tanzer, Matthew M., Tarpey, Rex W..
Application Number | 20030180829 10/011200 |
Document ID | / |
Family ID | 28038593 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030180829 |
Kind Code |
A1 |
Shuster, Jeffrey ; et
al. |
September 25, 2003 |
METHODS FOR THE IDENTIFICATION OF INHIBITORS OF HOMOCITRATE
SYNTHASE AS ANTIBIOTICS
Abstract
The present inventors have discovered that homocitrate synthase
is essential for fungal pathogenicity. Specifically, the inhibition
of homocitrate synthase gene expression in fungi results in no
signs of successful infection or lesions. Thus, homocitrate
synthase can be used as a target for the identification of
antibiotics, preferably antifungals. Accordingly, the present
invention provides methods for the identification of compounds that
inhibit homocitrate synthase expression or activity. The methods of
the invention are useful for the identification of antibiotics,
preferably antifungals.
Inventors: |
Shuster, Jeffrey; (Chapel
Hill, NC) ; Tanzer, Matthew M.; (Durham, NC) ;
Hamer, Lisbeth; (Durham, NC) ; Adachi, Kiichi;
(Durham, NC) ; DeZwaan, Todd M.; (Apex, NC)
; Lo, Sze-Chung; (Durham, NC) ;
Montenegro-Chamorro, Maria Victoria; (Morrisville, NC)
; Darveaux, Blaise; (Hillsborough, NC) ; Frank,
Sheryl; (Durham, NC) ; Heiniger, Ryan;
(Raleigh, NC) ; Mahanty, Sanjoy K.; (Chapel Hill,
NC) ; Pan, Huaqin; (Apex, NC) ; Skalchunes,
Amy; (Raleigh, NC) ; Tarpey, Rex W.; (Apex,
NC) |
Correspondence
Address: |
PARADIGM GENETICS, INC
108 ALEXANDER DRIVE
P O BOX 14528
RTP
NC
27709-4528
US
|
Family ID: |
28038593 |
Appl. No.: |
10/011200 |
Filed: |
November 9, 2001 |
Current U.S.
Class: |
435/32 ;
435/4 |
Current CPC
Class: |
C12N 9/88 20130101; C12N
9/0008 20130101; C12Y 102/01031 20130101; C12Q 1/18 20130101; C12Q
1/26 20130101; C12Q 1/527 20130101 |
Class at
Publication: |
435/32 ;
435/4 |
International
Class: |
C12Q 001/18; C12Q
001/00 |
Claims
What is claimed is:
1. A method for identifying a test compound as a candidate for an
antibiotic, comprising: a) contacting a homocitrate synthase
polypeptide with said test compound; and b) detecting the presence
or absence of binding between said test compound and said
homocitrate synthase polypeptide; wherein binding indicates that
said test compound is a candidate for an antibiotic.
2. The method of claim 1, wherein said homocitrate synthase
polypeptide is a fungal homocitrate synthase polypeptide.
3. The method of claim 1, wherein said homocitrate synthase
polypeptide is a Magnaporthe homocitrate synthase polypeptide.
4. The method of claim 1, wherein said homocitrate synthase
polypeptide is SEQ ID NO: 3.
5. A method for determining whether a compound identified as an
antibiotic candidate by the method of claim 1 has antifungal
activity, further comprising: contacting a fungus or fungal cells
with said antibiotic candidate and detecting the decrease in
growth, viability, or pathogenicity of said fungus or fungal
cells.
6. A method for identifying a test compound as a candidate for an
antibiotic, comprising: a) contacting said test compound with at
least one polypeptide selected from the group consisting of: a
polypeptide having at least ten consecutive amino acids of a fungal
homocitrate synthase, a polypeptide having at least 50% sequence
identity with a fungal homocitrate synthase, and a polypeptide
having at least 10% of the activity thereof, and b) detecting the
presence and/or absence of binding between said test compound and
said polypeptide; wherein binding indicates that said test compound
is a candidate for an antibiotic.
7. A method for determining whether a compound identified as an
antibiotic candidate by the method of claim 6 has antifungal
activity, further comprising: contacting a fungus or fungal cells
with said antibiotic candidate and detecting a decrease in growth,
viability, or pathogenicity of said fungus or fungal cells.
8. A method for identifying a test compound as a candidate for an
antibiotic, comprising: a) contacting acetyl-CoA and H.sub.2O and
2-oxoglutarate with a homocitrate synthase; b) contacting
acetyl-CoA and H.sub.2O and 2-oxoglutarate with a homocitrate
synthase and said test compound; and c) determining the change in
concentration for at least one of the following:
2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate, acetyl-CoA,
CoA, and/or H.sub.2O; wherein a change in concentration for any of
the above substances between steps (a) and (b) indicates that said
test compound is a candidate for an antibiotic.
9. The method of claim 8, wherein said homocitrate synthase is a
fungal homocitrate synthase.
10. The method of claim 8, wherein said homocitrate synthase is a
Magnaporthe homocitrate synthase.
11. The method of claim 8, wherein said homocitrate synthase is SEQ
ID NO: 3.
12. A method for determining whether a compound identified as an
antibiotic candidate by the method of claim 8 has antifungal
activity, further comprising: contacting a fungus or fungal cells
with said antibiotic candidate and detecting a decrease in growth,
viability, or pathogenicity of said fungus or fungal cells.
13. A method for identifying a test compound as a candidate for an
antibiotic, comprising: a) contacting
2-hydroxybutane-1,2,4-tricarboxylat- e and CoA with a homocitrate
synthase; b) contacting 2-hydroxybutane-1,2,4-tricarboxylate and
CoA with a homocitrate synthase and said test compound; and c)
determining the change in concentration for at least one of the
following: 2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate,
acetyl-CoA, CoA, and/or H.sub.2O; wherein a change in concentration
for any of the above substances between steps (a) and (b) indicates
that said test compound is a candidate for an antibiotic.
14. The method of claim 13, wherein said homocitrate synthase is a
fungal homocitrate synthase.
15. The method of claim 13, wherein said homocitrate synthase is a
Magnaporthe homocitrate synthase.
16. The method of claim 13, wherein said homocitrate synthase is
SEQ ID NO: 3.
17. A method for determining whether a compound identified as an
antibiotic candidate by the method of claim 13 has antifungal
activity, further comprising: contacting a fungus or fungal cells
with said antibiotic candidate and detecting a decrease in growth,
viability, or pathogenicity of said fungus or fungal cells.
18. A method for identifying a test compound as a candidate for an
antibiotic, comprising: a) contacting acetyl-CoA and H.sub.2O and
2-oxoglutarate with a polypeptide selected from the group
consisting of: a polypeptide having at least 50% sequence identity
with a homocitrate synthase, a polypeptide having at least 50%
sequence identity with a homocitrate synthase and having at least
10% of the activity thereof, and a polypeptide comprising at least
100 consecutive amino acids of a homocitrate synthase b) contacting
acetyl-CoA and H.sub.2O and 2-oxoglutarate with said polypeptide
and said test compound; and c) determining the change in
concentration for at least one of the following:
2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate, acetyl-CoA,
CoA, and/or H.sub.2O; wherein a change in concentration for any of
the above substances between steps (a) and (b) indicates that said
test compound is a candidate for an antibiotic.
19. A method for identifying a test compound as a candidate for an
antibiotic, comprising: a) contacting
2-hydroxybutane-1,2,4-tricarboxylat- e and CoA with a polypeptide
selected from the group consisting of: a polypeptide having at
least 50% sequence identity with a homocitrate synthase, a
polypeptide having at least 50% sequence identity with a
homocitrate synthase and at least 10% of the activity thereof, and
a polypeptide comprising at least 100 consecutive amino acids of a
homocitrate synthase b) contacting
2-hydroxybutane-1,2,4-tricarboxylate and CoA, with said polypeptide
and said test compound; and c) determining the change in
concentration for at least one of the following:
2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate, acetyl-CoA,
CoA, and/or H.sub.2O; wherein a change in concentration for any of
the above substances between steps (a) and (b) indicates that said
test compound is a candidate for an antibiotic.
20. A method for identifying a test compound as a candidate for an
antibiotic, comprising: a) measuring the expression of a
homocitrate synthase in a cell, cells, tissue, or an organism in
the absence of said compound; b) contacting said cell, cells,
tissue, or organism with said test compound and measuring the
expression of said homocitrate synthase in said fungus or fungal
cell; c) comparing the expression of homocitrate synthase in steps
(a) and (b); wherein a lower expression in the presence of said
test compound indicates that said compound is a candidate for an
antibiotic.
21. The method of claim 20 wherein said a cell, cells, tissue, or
organism is, or is derived from a fungus.
22. The method of claim 20 wherein said cell, cells, tissue, or
organism is, or is derived from a Magnaporthe fungus or fungal
cell.
23. The method of claim 20, wherein said homocitrate synthase is
SEQ ID NO: 3.
24. The method of claim 20, wherein the expression of homocitrate
synthase is measured by detecting HCS1 mRNA.
25. The method of claim 20, wherein the expression of homocitrate
synthase is measured by detecting homocitrate synthase
polypeptide.
26. A method for identifying a test compound as a candidate for an
antibiotic, comprising: a) providing cells having one form of a
homocitrate synthase gene, and providing comparison cells having a
different form of a homocitrate synthase gene, b) contacting said
cells and said comparison cells with a test compound and
determining the growth of said cells and comparison cells in the
presence of the test compound; wherein a difference in growth
between said cells and said comparison cells in the presence of
said compound indicates that said compound is a candidate for an
antibiotic.
27. The method of claim 26 wherein the cells are fungal cells.
28. The method of claim 26 wherein the cells are Magnaporthe
cells.
29. The method of claim 26 wherein said form and said comparison
form of the homocitrate synthase are fungal homocitrate
synthases.
30. The method of claim 26, wherein at least one form is a
Magnaporthe homocitrate synthase.
31. The method of claim 26 wherein said form and said comparison
form of the homocitrate synthase are non-fungal homocitrate
synthases.
32. The method of claim 26 wherein one form of the homocitrate
synthase is a fungal homocitrate synthase, and the other form is a
non-fungal homocitrate synthase.
33. A method for identifying a test compound as a candidate for an
antibiotic, comprising: d) providing cells having one form of a
gene in the lysine biochemical and/or genetic pathway and providing
comparison cells having a different form of said gene. e)
contacting said cells and comparison cells with a said test
compound, f) determining the growth of said cells and comparison
cells in the presence of said test compound; wherein a difference
in growth between said cells and said comparison cells in the
presence of said compound indicates that said compound is a
candidate for an antibiotic.
34. The method of claim 33 wherein the cells are fungal cells.
35. The method of claim 33 wherein the cells are Magnaporthe
cells.
36. The method of claim 33 wherein said form and said comparison
form of the lysine biosynthesis gene are fungal lysine biosynthesis
genes.
37. The method of claim 33, wherein at least one form is a
Magnaporthe lysine biosynthesis gene.
38. The method of claim 33 wherein said form and said comparison
form of the lysine biosynthesis genes are non-fungal lysine
biosynthesis genes.
39. The method of claim 33 wherein one form of the lysine
biosynthesis gene is a fungal lysine biosynthesis gene, and the
other form is a non-fungal lysine biosynthesis gene.
40. A method for determining whether a test compound identified as
an antibiotic candidate by the method of claim 33 has antifungal
activity, further comprising: contacting a fungus or fungal cells
with said antibiotic candidate and detecting a decrease in growth,
viability, or pathogenicity of said fungus or fungal cells.
41. A method for identifying a test compound as a candidate for an
antibiotic, comprising: (a) providing paired growth media;
comprising a first medium and a second medium, wherein said second
medium contains a higher level of lysine than said first medium;
(b) contacting an organism with said test compound; (c) inoculating
said first and second media with said organism; and (d) determining
the growth of said organism; wherein a difference in growth of the
organism between said first and second media indicates that said
test compound is a candidate for an antibiotic.
42. The method of claim 41, wherein said organism is a fungus.
43. The method of claim 41, wherein said organism is
Magnaporthe.
44. An isolated polynucleotide comprising a nucleotide sequence
that encodes a polypeptide of SEQ ID NO: 3.
45. The polynucleotide of claim 44 comprising the nucleotide
sequence of SEQ ID NO: 1.
46. An expression cassette comprising the polynucleotide of claim
45.
47. The isolated polynucleotide of claim 44 comprising a nucleotide
sequence of at least 50 to at least 95% sequence identity to SEQ ID
NO: 1.
48. A polypeptide consisting essentially of the amino acid sequence
of SEQ ID NO: 3.
49. A polypeptide comprising the amino acid sequence of SEQ ID NO:
3.
Description
FIELD OF THE INVENTION
[0001] The invention relates generally to methods for the
identification of antibiotics, preferably antifungals that affect
the biosynthesis of lysine. This application is co-pending with our
application entitled "Methods for the Identification of Inhibitors
of .alpha.-Aminoadipate Reductase as Antibiotics".
BACKGROUND OF THE INVENTION
[0002] Filamentous fungi are the causal agents responsible for many
serious pathogenic infections of plants and animals. Since fungi
are eukaryotes, and thus more similar to their host organisms than,
for example bacteria, the treatment of infections by fungi poses
special risks and challenges not encountered with other types of
infections. One such fungus is Magnaporthe grisea, the fungus that
causes rice blast disease. It is an organism that poses a
significant threat to food supplies worldwide. Other examples of
plant pathogens of economic importance include the pathogens in the
genera Agaricus, Alternaria, Anisogramma, Anthracoidea, Antrodia,
Apiognomonia, Apiosporina, Armillaria, Ascochyta, Aspergillus,
Bipolaris, Bjerkandera, Botryosphaeria, Botrytis, Ceratobasidium,
Ceratocystis, Cercospora, Cercosporidium, Cerotelium, Cerrena,
Chondrostereum, Chryphonectria, Chrysomyxa, Cladosporium,
Claviceps, Cochliobolus, Coleosporium, Colletotrichium,
Colletotrichum, Corticium, Corynespora, Cronartium, Cryphonectria,
Cryptosphaeria, Cyathus, Cymadothea, Cytospora, Daedaleopsis,
Diaporthe, Didymella, Diplocarpon, Diplodia, Discohainesia,
Discula, Dothistroma, Drechslera, Echinodontium, Elsinoe,
Endocronartium, Endothia, Entyloma, Epichloe, Erysiphe,
Exobasidium, Exserohilum, Fomes, Fomitopsis, Fusarium,
Gaeumannomyces, Ganoderma, Gibberella, Gloeocercospora,
Gloeophyllum, Gloeoporus, Glomerella, Gnomoniella, Guignardia,
Gymnosporangium, Helminthosporium, Herpotrichia, Heterobasidion,
Hirschioporus, Hypodermella, Inonotus, Irpex, Kabatiella, Kabatina,
Laetiporus, Laetisaria, Lasiodiplodia, Laxitextum, Leptographium,
Leptosphaeria, Leptosphaerulina, Leucytospora, Linospora,
Lophodermella, Lophodermium, Macrophomina, Magnaporthe, Marssonina,
Melampsora, Melampsorella, Meria, Microdochium, Microsphaera,
Monilinia, Monochaetia, Morchella, Mycosphaerella, Myrothecium,
Nectria, Nigrospora, Ophiosphaerella, Ophiostoma, Penicillium,
Perenniporia, Peridermium, Pestalotia, Phaeocryptopus, Phaeolus,
Phakopsora, Phellinus, Phialophora, Phoma, Phomopsis, Phragmidium,
Phyllachora, Phyllactinia, Phyllosticta, Phymatotrichopsis,
Pleospora, Podosphaera, Pseudopeziza, Pseudoseptoria, Puccinia,
Pucciniastrum, Pyricularia, Rhabdocline, Rhizoctonia, Rhizopus,
Rhizosphaera, Rhynchosporium, Rhytisma, Schizophyllum, Schizopora,
Scirrhia, Sclerotinia, Sclerotium, Scytinostroma, Septoria,
Setosphaera, Sirococcus, Spaerotheca, Sphaeropsis, Sphaerotheca,
Sporisorium, Stagonospora, Stemphylium, Stenocarpella, Stereum,
Taphrina, Thielaviopsis, Tilletia, Trametes, Tranzschelia,
Trichoderma, Tubakia, Typhula, Uncinula, Urocystis, Uromyces,
Ustilago, Valsa, Venturia, Verticillium, Xylaria, and others.
Related organisms in the classification, oomycetes, that include
the genera Albugo, Aphanomyces, Bremia, Peronospora, Phytophthora,
Plasmodiophora, Plasmopara, Pseudoperonospora, Pythium,
Sclerophthora, and others are also significant plant pathogens and
are sometimes classified along with the true fungi. Human diseases
that are caused by filamentous fungi include life-threatening lung
and disseminated diseases, often a result of infections by
Aspergillus fumigatus. Other fungal diseases in animals are caused
by fungi in the genera, Fusarium, Blastomyces, Microsporum,
Trichophyton, Epidermophyton, Candida, Histoplamsa, Pneumocystis,
Cryptococcus, other Aspergilli, and others. The control of fungal
diseases in plants and animals is usually mediated by chemicals
that inhibit the growth, proliferation, and/or pathogenicity of the
fungal organisms. To date, there are less than twenty known
modes-of-action for plant protection fungicides and human
antifungal compounds.
[0003] A pathogenic organism has been defined as an organism that
causes, or is capable of causing disease. Pathogenic organisms
propagate on or in tissues and may obtain nutrients and other
essential materials from their hosts. A substantial amount of work
concerning filamentous fungal pathogens has been performed with the
human pathogen, Aspergillus fumigatus. Shibuya et al. (Shibuya, K.,
M. Takaoka, et. al. (1999) Microb Pathog 27: 123-31 (PMID:
10455003)) have shown that the deletion of either of two suspected
pathogenicity related genes encoding an alkaline protease or a
hydrophobin (rodlet) respectively, did not reduce mortality of mice
infected with these mutant strains. Smith et al. (Smith, J. M., C.
M. Tang, et al. (1994) Infect Immun 62: 5247-54 (PMID: 7960101))
showed similar results with alkaline protease and the ribotoxin
restrictocin; Aspergillus fumigatus strains mutated for either of
these genes were fully pathogenic to mice. Reichard et al.
(Reichard, U., M. Monod, et al. (1997) J Med Vet Mycol 35: 189-96
(PMID: 9229335)) showed that deletion of the suspected
pathogenicity gene encoding, aspergillopepsin (PEP) in Aspergillus
fumigatus, had no effect on mortality in a guinea pig model system,
and Aufauvre-Brown et al (Aufauvre-Brown, A., E. Mellado, et al.
(1997) Fungal Genet Biol 21: 141-52 (PMID: 9073488)) showed no
effects of a chitin synthase mutation on pathogenicity. However,
not all experiments produced negative results. Ergosterol is an
important membrane component found in fungal organisms. Pathogenic
fungi that lack key enzymes in this biochemical pathway might be
expected to be non-pathogenic since neither the plant nor animal
hosts contain this particular sterol. Many antifungal compounds
that affect this biochemical pathway have been described (Onishi,
J. C. and A. A. Patchett (1990 a, b, c, d, and e) U.S. Pat. Nos.
4,920,109; 4,920,111; 4,920,112; 4,920,113; and 4,921,844, Merck
& Co. Inc. (Rahway N.J.)) and (Hewitt, H. G. (1998) Fungicides
in Crop Protection Cambridge, University Press). D'Enfert et al.
(D'Enfert, C., M. Diaquin, et al. (1996) Infect Immun 64: 4401-5
(PMID: 8926121)) showed that an Aspergillus fumigatus strain
mutated in an orotidine 5'-phosphate decarboxylase gene was
entirely non-pathogenic in mice, and Brown et al. (Brown, J. S., A.
Aufauvre-Brown, et al. (2000) Mol Microbiol 36:1371-80 (PMID:
10931287)) observed a non-pathogenic result when genes involved in
the synthesis of para-aminobenzoic acid were mutated. Some specific
target genes have been described as having utility for the
screening of inhibitors of plant pathogenic fungi. Bacot et al.
(Bacot, K. O., D. B. Jordan, et al. (2000) U.S. Pat. No. 6,074,830,
E. I. du Pont de Nemours & Company (Wilmington Del.)) describe
the use of 3,4-dihydroxy-2-butanone 4-phosphate synthase, and Davis
et al. (Davis, G. E., G. D. Gustafson, et al. (1999) U.S. Pat. No.
5,976,848, Dow AgroSciences LLC (Indianapolis Ind.)) describe the
use of dihydroorotate dehydrogenase for potential screening
purposes.
[0004] There are also a number of papers that report less clear
results, showing neither full pathogenicity nor non-pathogenicity
of mutants. Hensel et al. (Hensel, M., H. N. Arst, Jr., et al.
(1998) Mol Gen Genet 258: 553-7 (PMID: 9669338)) showed only
moderate effects of the deletion of the are A transcriptional
activator on the pathogenicity of Aspergillus fumigatus. Tang et
al. (Tang, C. M., J. M. Smith, et al. (1994) Infect Immun 62:
5255-60 (PMID: 7960102)) using the related fungus, Aspergillus
nidulans, observed that a mutation in para-aminobenzoic acid
synthesis prevented mortality in mice, while a mutation in lysine
biosynthesis had no significant effect on the mortality of the
infected mice.
[0005] Therefore, it is not currently possible to determine which
specific growth materials may be readily obtained by a pathogen
from its host, and which materials may not. Surprising, especially
in light of the results showing that a lysine biosynthesis mutation
in the filamentous fungus, Aspergillus nidulans, had no significant
effect on the pathogenicity in a mouse model system (Tang, C. M.,
J. M. Smith, et al. (1994) Infect Immun 62: 5255-60 (PMID:
7960102)), we have found that Magnaporthe grisea that cannot
synthesize their own lysine are entirely non-pathogenic on their
host organism. To date there do not appear to be any publications
demonstrating an anti-pathogenic effect of the knock-out,
over-expression, antisense expression, or inhibition of the genes
or gene products involved in lysine biosynthesis in filamentous
fungi. Thus, it has not been shown that the de novo biosynthesis of
lysine is essential for fungal pathogenicity. Our co-pending
application entitled "Methods for the Identification of Inhibitors
of .alpha.-Aminoadipate Reductase as Antibiotics" shows that the
disruption of lysine biosynthesis as the result of a disruption of
the gene encoding the enzyme activity,
alpha-aminoadipate-semialdehyde dehydrogenase, also results in a
non-pathogenic phenotype for M. grisea. Thus, it would be desirable
to determine the utility of the enzymes involved in lysine
biosynthesis for evaluating antibiotic compounds, especially
fungicides. If a fungal biochemical pathway or specific gene
product in that pathway is shown to be required for fungal
pathogenicity, various formats of in vitro and in vivo screening
assays may be put in place to discover classes of chemical
compounds that react with the validated target gene, gene product,
or biochemical pathway, and are thus candidates for antifungal,
biocide, and biostatic materials.
SUMMARY OF THE INVENTION
[0006] Surprisingly, the present inventors have discovered that in
vivo disruption of the gene encoding homocitrate synthase in
Magnaporthe grisea prevents or inhibits the pathogenicity of the
fungus. Thus, the present inventors have discovered that
homocitrate synthase is essential for normal rice blast
pathogenicity, and can be used as a target for the identification
of antibiotics, preferably fungicides. Accordingly, the present
invention provides methods for the identification of compounds that
inhibit homocitrate synthase expression or activity. The methods of
the invention are useful for the identification of antibiotics,
preferably fungicides.
BRIEF DESCRIPTION OF THE FIGURES
[0007] FIG. 1 shows the reaction performed by the homocitrate
synthase (HCS1) reaction. The Substrates/Products are Acetyl
CoA+H.sub.2O+2-oxoglutarate and the Products/Substrates are
2-hydroxybutane-1,2,4-tricarboxylate+CoA. The function of the
homocitrate synthase enzyme is the interconversion of Acetyl CoA,
2-oxoglutarate, and H.sub.2O to
2-hydroxybutane-1,2,4-tricarboxylate and CoA. This reaction is part
of the lysine biosynthesis pathway.
[0008] FIG. 2 shows a digital image showing the effect of HCS1 gene
disruption on Magnaporthe grisea pathogenicity using whole plant
infection assays. Rice variety C039 was inoculated with wild-type
and the transposon insertion strains, KO1-1 and KO1-2. Leaf
segments were imaged at five days post-inoculation.
[0009] FIG. 3. Verification of Gene Function by Analysis of
Nutritional Requirements. Wild-type and transposon insertion
strains, KO1-1 and KO1-2, were grown in (A) minimal media and (B)
minimal media with the addition of L-lysine, respectively. The
x-axis shows time in days and the y-axis shows turbidity measured
at 490 nanometers and 750 nanometers. The symbols represent
wildtype (--.diamond-solid.--), transposon strain KO1-1
(--.box-solid.--), and transposon strain KO1-2
(--.tangle-solidup.--).
DETAILED DESCRIPTION OF THE INVENTION
[0010] Unless otherwise indicated, the following terms are intended
to have the following meanings in interpreting the present
invention.
[0011] The term "active against" in the context of compounds,
agents, or compositions having antibiotic activity indicates that
the compound exerts an effect on a particular target or targets
which is deleterious to the in vitro and/or in vivo growth of an
organism having that target or targets. In particular, a compound
active against a gene exerts an action on a target which affects an
expression product of that gene. This does not necessarily mean
that the compound acts directly on the expression product of the
gene, but instead indicates that the compound affects the
expression product in a deleterious manner. Thus, the direct target
of the compound may be, for example, at an upstream component which
reduces transcription from the gene, resulting in a lower level of
expression. Likewise, the compound may affect the level of
translation of a polypeptide expression product, or may act on a
downstream component of a biochemical pathway in which the
expression product of the gene has a major biological role.
Consequently, such a compound can be said to be active against the
gene, against the gene product, or against the related component
either upstream or downstream of that gene or expression product.
While the term "active against" encompasses a broad range of
potential activities, it also implies some degree of specificity of
target. Therefore, for example, a general protease is not "active
against" a particular gene which produces a polypeptide product. In
contrast, a compound which inhibits a particular enzyme is active
against that enzyme and against the gene which codes for that
enzyme.
[0012] As used herein, the term "allele" refers to any of the
alternative forms of a gene that may occur at a given locus.
[0013] The term "antibiotic" refers to any substance or compound
that when contacted with a living cell, organism, virus, or other
entity capable of replication, results in a reduction of growth,
viability, or pathogenicity of that entity.
[0014] The term "binding" refers to a non-covalent or a covalent
interaction, preferably non-covalent, that holds two molecules
together. For example, two such molecules could be an enzyme and an
inhibitor of that enzyme. Non-covalent interactions include
hydrogen bonding, ionic interactions among charged groups, van der
Waals interactions and hydrophobic interactions among nonpolar
groups. One or more of these interactions can mediate the binding
of two molecules to each other.
[0015] The term "biochemical pathway" or "pathway" refers to a
connected series of biochemical reactions normally occurring in a
cell, or more broadly a cellular event such as cellular division or
DNA replication. Typically, the steps in such a biochemical pathway
act in a coordinated fashion to produce a specific product or
products or to produce some other particular biochemical action.
Such a biochemical pathway requires the expression product of a
gene if the absence of that expression product either directly or
indirectly prevents the completion of one or more steps in that
pathway, thereby preventing or significantly reducing the
production of one or more normal products or effects of that
pathway. Thus, an agent specifically inhibits such a biochemical
pathway requiring the expression product of a particular gene if
the presence of the agent stops or substantially reduces the
completion of the series of steps in that pathway. Such an agent,
may, but does not necessarily, act directly on the expression
product of that particular gene.
[0016] As used herein, the term "cDNA" means complementary
deoxyribonucleic acid.
[0017] As used herein, the term "CoA" means coenzyme A.
[0018] As used herein, the term "conditional lethal" refers to a
mutation permitting growth and/or survival only under special
growth or environmental conditions.
[0019] As used herein, the term "cosmid" refers to a hybrid vector,
used in gene cloning, that includes a cos site (from the lambda
bacteriophage). It also contains drug resistance marker genes and
other plasmid genes. Cosmids are especially suitable for cloning
large genes or multigene fragments.
[0020] As used herein, the term "dominant allele" refers to a
dominant mutant allele in which a discernable mutant phenotype can
be detected when this mutation is present in an organism that also
contains a wild type (non-mutant), recessive allele, or other
dominant allele.
[0021] As used herein, the term "DNA" means deoxyribonucleic
acid.
[0022] As used herein, the term "ELISA" means enzyme-linked
immunosorbent assay. "Fungi" (singular: fungus) refers to whole
fungi, fungal organs and tissues (e.g., asci, hyphae, pseudohyphae,
rhizoid, sclerotia, sterigmata, spores, sporodochia, sporangia,
synnemata, conidia, ascostroma, cleistothecia, mycelia, perithecia,
basidia and the like), spores, fungal cells and the progeny
thereof. Fungi are a group of organisms (about 50,000 known
species), including, but not limited to, mushrooms, mildews,
moulds, yeasts, etc., comprising the kingdom Fungi. They can either
exist as single cells or make up a multicellular body called a
mycelium, which consists of filaments known as hyphae. Most fungal
cells are multinucleate and have cell walls, composed chiefly of
chitin. Fungi exist primarily in damp situations on land and,
because of the absence of chlorophyll and thus the inability to
manufacture their own food by photosynthesis, are either parasites
on other organisms or saprotrophs feeding on dead organic matter.
The principal criteria used in classification are the nature of the
spores produced and the presence or absence of cross walls within
the hyphae. Fungi are distributed worldwide in terrestrial,
freshwater, and marine habitats. Some live in the soil. Many
pathogenic fungi cause disease in animals and man or in plants,
while some saprotrophs are destructive to timber, textiles, and
other materials. Some fungi form associations with other organisms,
most notably with algae to form lichens.
[0023] As used herein, the term "fungicide", "antifungal", or
"antimycotic" refers to an antibiotic substance or compound that
kills or suppresses the growth, viability, or pathogenicity of at
least one fungus, fungal cell, fungal tissue or spore.
[0024] In the context of this disclosure, "gene" should be
understood to refer to a unit of heredity. Each gene is composed of
a linear chain of deoxyribonucleotides which can be referred to by
the sequence of nucleotides forming the chain. Thus, "sequence" is
used to indicate both the ordered listing of the nucleotides which
form the chain, and the chain, itself, which has that sequence of
nucleotides. ("Sequence" is used in the similar way in referring to
RNA chains, linear chains made of ribonucleotides.) The gene may
include regulatory and control sequences, sequences which can be
transcribed into an RNA molecule, and may contain sequences with
unknown function. The majority of the RNA transcription products
are messenger RNAs (mRNAs), which include sequences which are
translated into polypeptides and may include sequences which are
not translated. It should be recognized that small differences in
nucleotide sequence for the same gene can exist between different
fungal strains, or even within a particular fungal strain, without
altering the identity of the gene.
[0025] As used in this disclosure, the terms "growth" or "cell
growth" of an organism refers to an increase in mass, density, or
number of cells of said organism. Some common methods for the
measurement of growth include the determination of the optical
density of a cell suspension, the counting of the number of cells
in a fixed volume, the counting of the number of cells by
measurement of cell division, the measurement of cellular mass or
cellular volume, and the like.
[0026] As used in this disclosure, the term "growth conditional
phenotype" indicates that a fungal strain having such a phenotype
exhibits a significantly greater difference in growth rates in
response to a change in one or more of the culture parameters than
an otherwise similar strain not having a growth conditional
phenotype. Typically, a growth conditional phenotype is described
with respect to a single growth culture parameter, such as
temperature. Thus, a temperature (or heat-sensitive) mutant (i.e.,
a fungal strain having a heat-sensitive phenotype) exhibits
significantly different growth, and preferably no growth, under
non-permissive temperature conditions as compared to growth under
permissive conditions. In addition, such mutants preferably also
show intermediate growth rates at intermediate, or semi-permissive,
temperatures. Similar responses also result from the appropriate
growth changes for other types of growth conditional
phenotypes.
[0027] As used herein, the term "H.sub.2O" means water.
[0028] As used herein, the term "HCS1 " means a gene encoding
homocitrate synthase activity, referring to an enzyme that
catalyses the interconversion of acetyl-CoA, H.sub.2O, and
2-oxoglutarate with 2-hydroxybutane-1,2,4-tricarboxylate and
CoA.
[0029] As used herein, the term "heterologous HCS1 gene" means a
gene, not derived from Magnaporthe grisea, and having: at least 50%
sequence identity, preferably 60%, 70%, 80%, 90%, 95%, 99% sequence
identity and each integer unit of sequence identity from 50-100% in
ascending order to SEQ ID NO: 1 or SEQ ID NO: 2; or at least 10% of
the activity of a Magnaporthe grisea homocitrate synthase,
preferably 25%, 50%, 75%, 90%, 95%, 99% and each integer unit of
activity from 10-100% in ascending order.
[0030] As used herein, the term "homocitrate synthase (EC 4.1.3.21)
or homocitrate synthase polypeptide" is synonymous with "the HCS1
gene product" and refers to an enzyme that catalyses the
interconversion of acetyl-CoA, H.sub.2O, and 2-oxoglutarate with
2-hydroxybutane-1,2,4-trica- rboxylate and CoA.
[0031] As used herein, the term "His-Tag" refers to an encoded
polypeptide consisting of multiple consecutive histidine amino
acids.
[0032] As used herein, the term "HPLC" means high pressure liquid
chromatography.
[0033] As used herein, the terms "hph", "hygromycin B
phosphotransferase", and "hygromycin resistance gene" refer to the
E. coli hygromycin phosphotransferase gene or gene product.
[0034] As used herein, the term "hygromycin B" refers to an
aminoglycosidic antibiotic, used for selection and maintenance of
eukaryotic cells containing the E. coli hygromycin resistance
gene.
[0035] "Hypersensitive" refers to a phenotype in which cells are
more sensitive to antibiotic compounds than are wild-type cells of
similar or identical genetic background.
[0036] "Hyposensitive" refers to a phenotype in which cells are
less sensitive to antibiotic compounds than are wild-type cells of
similar or identical genetic background.
[0037] As used herein, the term "imperfect state" refers to a
classification of a fungal organism having no demonstrable sexual
life stage.
[0038] The term "inhibitor", as used herein, refers to a chemical
substance that inactivates the enzymatic activity of homocitrate
synthase or substantially reduces the level of enzymatic activity,
wherein "substantially" means a reduction at least as great as the
standard deviation for a measurement, preferably a reduction by
50%, more preferably a reduction of at least one magnitude, i.e. to
10%. The inhibitor may function by interacting directly with the
enzyme, a cofactor of the enzyme, the substrate of the enzyme, or
any combination thereof.
[0039] A polynucleotide may be "introduced" into a fungal cell by
any means known to those of skill in the art, including
transfection, transformation or transduction, transposable element,
electroporation, particle bombardment, infection and the like. The
introduced polynucleotide may be maintained in the cell stably if
it is incorporated into a non-chromosomal autonomous replicon or
integrated into the fungal chromosome. Alternatively, the
introduced polynucleotide may be present on an extra-chromosomal
non-replicating vector and be transiently expressed or transiently
active.
[0040] As used herein, the term "knockout" or "gene disruption"
refers to the creation of organisms carrying a null mutation (a
mutation in which there is no active gene product), a partial null
mutation or mutations, or an alteration or alterations in gene
regulation by interrupting a DNA sequence through insertion of a
foreign piece of DNA. Usually the foreign DNA encodes a selectable
marker.
[0041] As used herein, the term "LB agar" means Luria's Broth
agar.
[0042] The term "method of screening" means that the method is
suitable, and is typically used, for testing for a particular
property or effect in a large number of compounds. Typically, more
than one compound is tested simultaneously (as in a 96-well
microtiter plate), and preferably significant portions of the
procedure can be automated. "method of screening" also refers to
determining a set of different properties or effects of one
compound simultaneously.
[0043] As used herein, the term "mRNA" means messenger ribonucleic
acid.
[0044] As used herein, the term "mutant form" of a gene refers to a
gene which has been altered, either naturally or artificially,
changing the base sequence of the gene. The change in the base
sequence may be of several different types, including changes of
one or more bases for different bases, deletions, and/or
insertions, such as by a transposon. By contrast, a normal form of
a gene (wild type) is a form commonly found in natural populations
of an organism. Commonly a single form of a gene will predominate
in natural populations. In general, such a gene is suitable as a
normal form of a gene, however, other forms which provide similar
functional characteristics may also be used as a normal gene. In
particular, a normal form of a gene does not confer a growth
conditional phenotype on the strain having that gene, while a
mutant form of a gene suitable for use in these methods does
provide such a growth conditional phenotype.
[0045] As used herein, the term "Ni" refers to nickel.
[0046] As used herein, the term "Ni-NTA" refers to nickel
sepharose.
[0047] As used herein, the term "one form" of a gene is synonymous
with the term "gene", and a "different form" of a gene refers to a
gene that has greater than 49% sequence identity and less than 100%
sequence identity with said first form.
[0048] As used herein, the term "pathogenicity" refers to a
capability of causing disease. The term is applied to parasitic
microorganisms in relation to their hosts.
[0049] As used herein, the term "PCR" means polymerase chain
reaction.
[0050] The "percent (%) sequence identity" between two
polynucleotide or two polypeptide sequences is determined according
to the either the BLAST program (Basic Local Alignment Search Tool;
(Altschul, S.F., W. Gish, et al. (1990) J Mol Biol 215: 403-10
(PMID: 2231712)) at the National Center for Biotechnology or using
Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J
Mol Biol 147:195-7 (PMID: 7265238)) as incorporated into
GeneMatcher Plus.TM.. It is understood that for the purposes of
determining sequence identity when comparing a DNA sequence to an
RNA sequence, a thymine nucleotide is equivalent to a uracil
nucleotide.
[0051] By "polypeptide" is meant a chain of at least two amino
acids joined by peptide bonds. The chain may be linear, branched,
circular or combinations thereof. Preferably, polypeptides are from
about 10 to about 1000 amino acids in length, more preferably 10-50
amino acids in length. The polypeptides may contain amino acid
analogs and other modifications, including, but not limited to
glycosylated or phosphorylated residues.
[0052] As used herein, the term "proliferation" is synonymous to
the term "growth".
[0053] As used herein, the term "reverse transcriptase-PCR" means
reverse transcription-polymerase chain reaction.
[0054] As used herein, the term "RNA" means ribonucleic acid.
[0055] As used herein, "semi-permissive conditions" are conditions
in which the relevant culture parameter for a particular growth
conditional phenotype is intermediate between permissive conditions
and non-permissive conditions. Consequently, in semi-permissive
conditions an organism having a growth conditional phenotype will
exhibit growth rates intermediate between those shown in permissive
conditions and non-permissive conditions. In general, such
intermediate growth rate may be due to a mutant cellular component
which is partially functional under semi-permissive conditions,
essentially fully functional under permissive conditions, and is
non-functional or has very low function under non-permissive
conditions, where the level of function of that component is
related to the growth rate of the organism. An intermediate growth
rate may also be a result of a nutrient substance or substances
that are present in amounts not sufficient for optimal growth rates
to be achieved.
[0056] "Sensitivity phenotype" refers to a phenotype that exhibits
either hypersensitivity or hyposensitivity.
[0057] The term "specific binding" refers to an interaction between
homocitrate synthase and a molecule or compound, wherein the
interaction is dependent upon the primary amino acid sequence
and/or the conformation of homocitrate synthase.
[0058] As used herein, the term "TLC" means thin layer
chromatography.
[0059] "Transform", as used herein, refers to the introduction of a
polynucleotide (single or double stranded DNA, RNA, or a
combination thereof) into a living cell by any means.
Transformation may be accomplished by a variety of methods,
including, but not limited to, electroporation, polyethylene glycol
mediated uptake, particle bombardment, agrotransformation, and the
like. This process may result in transient or stable expression of
the transformed polynucleotide. By "stably transformed" is meant
that the sequence of interest is integrated into a replicon in the
cell, such as a chromosome or episome. Transformed cells encompass
not only the end product of a transformation process, but also the
progeny thereof which retain the polynucleotide of interest.
[0060] For the purposes of the invention, "transgenic" refers to
any cell, spore, tissue or part, that contains all or part of at
least one recombinant polynucleotide. In many cases, all or part of
the recombinant polynucleotide is stably integrated into a
chromosome or stable extra-chromosomal element, so that it is
passed on to successive generations.
[0061] As used herein, the term "transposase" refers to an enzyme
that catalyzes transposition. Preferred transposons are described
in WO 00/55346, PCT/JUS00/07317, and U.S. 09/658,859. As used
herein, the term "transposition" refers to a complex genetic
rearrangement process involving the movement or copying of a
polynucleotide (transposon) from one location and insertion into
another, often within or between a genome or genomes, or DNA
constructs such as plasmids, bacmids, and cosmids.
[0062] As used herein, the term "transposon" (also known as a
"transposable element", "transposable genetic element", "mobile
element", or "jumping gene") refers to a mobile DNA element such as
those, for example, described in WO 00/55346, PCT/US00/07317, and
U.S. 09/658,859. Transposons can disrupt gene expression or cause
deletions and inversions, and hence affect both the genotype and
phenotype of the organisms concerned. The mobility of transposable
elements has long been used in genetic manipulation, to introduce
genes or other information into the genome of certain model
systems.
[0063] As used herein, the term "Tween 20" means sorbitan
mono-9-octadecenoate poly(oxy-1,1-ethanediyl).
[0064] As used in this disclosure, the term "viability" of an
organism refers to the ability of an organism to demonstrate growth
under conditions appropriate for said organism, or to demonstrate
an active cellular function. Some examples of active cellular
functions include respiration as measured by gas evolution,
secretion of proteins and/or other compounds, dye exclusion,
mobility, dye oxidation, dye reduction, pigment production, changes
in medium acidity, and the like.
[0065] The present inventors have discovered that disruption of the
HCS1 gene and/or gene product inhibits the pathogenicity of
Magnaporthe grisea. Thus, the inventors are the first to
demonstrate that homocitrate synthase is a target for antibiotics,
preferably antifungals.
[0066] Accordingly, the invention provides methods for identifying
compounds that inhibit HCS1 gene expression or biological activity
of its gene product(s). Such methods include ligand binding assays,
assays for enzyme activity, cell-based assays, and assays for HCS1
gene expression. Any compound that is a ligand for homocitrate
synthase may have antibiotic activity. For the purposes of the
invention, "ligand" refers to a molecule that will bind to a site
on a polypeptide. The compounds identified by the methods of the
invention are useful as antibiotics.
[0067] Thus, in one embodiment, the invention provides a method for
identifying a test compound as a candidate for an antibiotic,
comprising:
[0068] a) contacting a homocitrate synthase polypeptide with said
test compound; and
[0069] b) detecting the presence or absence of binding between said
test compound and said homocitrate synthase polypeptide;
[0070] wherein binding indicates that said test compound is a
candidate for an antibiotic.
[0071] The homocitrate synthase protein may have the amino acid
sequence of a naturally occurring homocitrate synthase found in a
fungus, animal, plant, or microorganism, or may have an amino acid
sequence derived from a naturally occurring sequence. Preferably
the homocitrate synthase is a fungal homocitrate synthase. The cDNA
(SEQ ID NO: 1) encoding the homocitrate synthase protein, the
genomic DNA (SEQ ID NO: 2) encoding the protein, and the
polypeptide (SEQ ID NO: 3) can be found herein.
[0072] By "fungal homocitrate synthase" is meant an enzyme that can
be found in at least one fungus, and which catalyzes the
interconversion of acetyl-CoA and H.sub.2O and 2-oxoglutarate with
2-hydroxybutane-1,2,4-tri- carboxylate and CoA. The homocitrate
synthase may be from any of the fungi, including ascomycota,
zygomycota, basidiomycota, chytridiomycota, and lichens.
[0073] In one embodiment, the homocitrate synthase is a Magnaporthe
homocitrate synthase. Magnaporthe species include, but are not
limited to, Magnaporthe rhizophila, Magnaporthe salvinii,
Magnaporthe grisea and Magnaporthepoae and the imperfect states of
Magnaporthe in the genus Pyricularia. Preferably, the Magnaporthe
homocitrate synthase is from Magnaporthe grisea.
[0074] In various embodiments, the homocitrate synthase can be from
Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis
cinerea), White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma
adspersum), Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago
maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora
zeae-maydis), Honey Fungus (Armillaria gallica), Root rot
(Armillaria luteobubalina), Shoestring Rot (Armillaria ostoyae),
Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting
Fungus (Monilinia fructigena), Apple-rotting Fungus (Penicillium
expansum), Clubroot Disease (Plasmodiophora brassicae), Potato
Blight (Phytophthora infestans), Root pathogen (Heterobasidion
annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm
Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculatus),
Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia
circinata), Southern Corn Blight (Cochliobolus heterostrophus),
Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus
stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab
Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmorum),
Potato Black Scurf (Rhizoctonia solani), Wheat Black Stem Rust
(Puccinia graminis), White mold (Sclerotinia sclerotiorum), and the
like.
[0075] Fragments of a homocitrate synthase polypeptide may be used
in the methods of the invention, preferably if the fragments
include an intact or nearly intact epitope that occurs on the
biologically active wildtype homocitrate synthase. The fragments
comprise at least 10 consecutive amino acids of a homocitrate
synthase. Preferably, the fragment comprises at least 15, 20, 25,
30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,
170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,
300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,
430 or at least 440 consecutive amino acids residues of a
homocitrate synthase. In one embodiment, the fragment is from a
Magnaporthe homocitrate synthase. Preferably, the fragment contains
an amino acid sequence conserved among fungal homocitrate
synthases.
[0076] Polypeptides having at least 50% sequence identity with a
fungal homocitrate synthase are also useful in the methods of the
invention. Preferably, the sequence identity is at least 60%, more
preferably the sequence identity is at least 70%, most preferably
the sequence identity is at least 80% or 90 or 95 or 99%, or any
integer from 60-100% sequence identity in ascending order.
[0077] In addition, it is preferred that the polypeptide has at
least 10% of the activity of a fungal homocitrate synthase. More
preferably, the polypeptide has at least 25%, at least 50%, at
least 75% or at least 90% of the activity of a fungal homocitrate
synthase. Most preferably, the polypeptide has at least 10%, at
least 25%, at least 50%, at least 75% or at least 90% of the
activity of the M. grisea homocitrate synthase protein.
[0078] Thus, in another embodiment, the invention provides a method
for identifying a test compound as a candidate for a fungicide,
comprising:
[0079] a) contacting said test compound with at least one
polypeptide selected from the group consisting of: a polypeptide
having at least ten consecutive amino acids of a fungal homocitrate
synthase, a polypeptide having at least 50% sequence identity with
a fungal homocitrate synthase, and a polypeptide having at least
10% of the activity thereof; and
[0080] b) detecting the presence and/or absence of binding between
said test compound and said polypeptide;
[0081] wherein binding indicates that said test compound is a
candidate for an antibiotic.
[0082] Any technique for detecting the binding of a ligand to its
target may be used in the methods of the invention. For example,
the ligand and target are combined in a buffer. Many methods for
detecting the binding of a ligand to its target are known in the
art, and include, but are not limited to the detection of an
immobilized ligand-target complex or the detection of a change in
the properties of a target when it is bound to a ligand. For
example, in one embodiment, an array of immobilized candidate
ligands is provided. The immobilized ligands are contacted with a
homocitrate synthase protein or a fragment or variant thereof, the
unbound protein is removed and the bound homocitrate synthase is
detected. In a preferred embodiment, bound homocitrate synthase is
detected using a labeled binding partner, such as a labeled
antibody. In a variation of this assay, homocitrate synthase is
labeled prior to contacting the immobilized candidate ligands.
Preferred labels include fluorescent or radioactive moieties.
Preferred detection methods include fluorescence correlation
spectroscopy (FCS) and FCS-related confocal nanofluorimetric
methods.
[0083] Once a compound is identified as a candidate for an
antibiotic, it can be tested for the ability to inhibit homocitrate
synthase enzymatic activity. The compounds can be tested using
either in vitro or cell based assays. Alternatively, a compound can
be tested by applying it directly to a fungus or fungal cell, or
expressing it therein, and monitoring the fungus or fungal cell for
changes or decreases in growth, development, viability,
pathogenicity, or alterations in gene expression. Thus, in one
embodiment, the invention provides a method for determining whether
a compound identified as an antibiotic candidate by an above method
has antifungal activity, further comprising: contacting a fungus or
fungal cells with said antifungal candidate and detecting a
decrease in the growth, viability, or pathogenicity of said fungus
or fungal cells.
[0084] By decrease in growth, is meant that the antifungal
candidate causes at least a 10% decrease in the growth of the
fungus or fungal cells, as compared to the growth of the fungus or
fungal cells in the absence of the antifungal candidate. By a
decrease in viability is meant that at least 20% of the fungal
cells, or portion of the fungus contacted with the antifungal
candidate are nonviable. Preferably, the growth or viability will
be decreased by at least 40%. More preferably, the growth or
viability will be decreased by at least 50%, 75% or at least 90% or
more. Methods for measuring fungal growth and cell viability are
known to those skilled in the art. By decrease in pathogenicity, is
meant that the antifungal candidate causes at least a 10% decrease
in the disease caused by contact of the fungal pathogen with its
host, as compared to the disease caused in the absence of the
antifungal candidate. Preferably, the disease will be decreased by
at least 40%. More preferably, the disease will be decreased by at
least 50%, 75% or at least 90% or more. Methods for measuring
fungal disease are well known to those skilled in the art, and
include such metrics as lesion formation, lesion size, sporulation,
respiratory failure, and/or death.
[0085] The ability of a compound to inhibit homocitrate synthase
activity can be detected using in vitro enzymatic assays in which
the disappearance of a substrate or the appearance of a product is
directly or indirectly detected. Homocitrate synthase catalyzes the
irreversible or reversible reaction acetyl-CoA and H.sub.2O and
2-oxoglutarate=2-hydroxybutane-1,2,4-tricarboxylate and CoA (see
FIG. 1). Methods for detection of
2-hydroxybutane-1,2,4-tricarboxylate, CoA, acetyl-CoA, and/or
2-oxoglutarate, include spectrophotometry, mass spectroscopy, thin
layer chromatography (TLC) and reverse phase HPLC.
[0086] Thus, the invention provides a method for identifying a test
compound as a candidate for an antibiotic, comprising either:
[0087] a) contacting acetyl-CoA and H.sub.2O and 2-oxoglutarate
with a homocitrate synthase;
[0088] b) contacting acetyl-CoA and H.sub.2O and 2-oxoglutarate
with homocitrate synthase and said test compound; and
[0089] c) determining the change in concentration for at least one
of the following: 2-hydroxybutane-1,2,4-tricarboxylate,
2-oxoglutarate, acetyl-CoA, CoA, and/or H.sub.2O.
[0090] wherein a change in concentration for any of the above
substances indicates that said test compound is a candidate for an
antibiotic. or,
[0091] a) contacting 2-hydroxybutane-1,2,4-tricarboxylate and CoA
with a homocitrate synthase;
[0092] b) contacting 2-hydroxybutane-1,2,4-tricarboxylate and CoA
with a homocitrate synthase and said test compound; and
[0093] c) determining the change in concentration for at least one
of the following: 2-hydroxybutane-1,2,4-tricarboxylate,
2-oxoglutarate, acetyl-CoA, CoA, and/or H.sub.2O.
[0094] wherein a change in concentration for any of the above
substances indicates that said test compound is a candidate for an
antibiotic.
[0095] Enzymatically active fragments of a fungal homocitrate
synthase are also useful in the methods of the invention. For
example, a polypeptide comprising at least 100 consecutive amino
acid residues of a fungal homocitrate synthase may be used in the
methods of the invention. In addition, a polypeptide having at
least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% sequence
identity with a fungal homocitrate synthase may be used in the
methods of the invention. Most preferably, the polypeptide has at
least 50% sequence identity with a fungal homocitrate synthase and
at least 10%, 25%, 75% or at least 90% of the activity thereof.
[0096] Thus, the invention provides a method for identifying a test
compound as a candidate for a fungicide, comprising:
[0097] a) contacting acetyl-CoA and H.sub.2O and 2-oxoglutarate
with a polypeptide selected from the group consisting of: a
polypeptide having at least 50% sequence identity with a
homocitrate synthase, a polypeptide having at least 50% sequence
identity with a homocitrate synthase and having at least 10% of the
activity thereof, and a polypeptide comprising at least 100
consecutive amino acids of a homocitrate synthase
[0098] b) contacting acetyl-CoA and H.sub.2O and 2-oxoglutarate
with said polypeptide and said test compound; and
[0099] c) determining the change in concentration for at least one
of the following: 2-hydroxybutane-1,2,4-tricarboxylate,
2-oxoglutarate, acetyl-CoA, CoA, and/or H.sub.2O.
[0100] wherein a change in concentration for any of the above
substances indicates that said test compound is a candidate for an
antibiotic. or,
[0101] a) contacting 2-hydroxybutane-1,2,4-tricarboxylate and CoA
with a polypeptide selected from the group consisting of: a
polypeptide having at least 50% sequence identity with a
homocitrate synthase, a polypeptide having at least 50% sequence
identity with a homocitrate synthase and at least 10% of the
activity thereof, and a polypeptide comprising at least 100
consecutive amino acids of a homocitrate synthase
[0102] b) contacting 2-hydroxybutane-1,2,4-tricarboxylate and CoA,
with said polypeptide and said test compound; and
[0103] c) determining the change in concentration for at least one
of the following, 2-hydroxybutane-1,2,4-tricarboxylate,
2-oxoglutarate, acetyl-CoA, CoA, and/or H.sub.2O.
[0104] wherein a change in concentration for any of the above
substances indicates that said test compound is a candidate for an
antibiotic.
[0105] For the in vitro enzymatic assays, homocitrate synthase
protein and derivatives thereof may be purified from a fungus or
may be recombinantly produced in and purified from an archael,
bacterial, fungal, or other eukaryotic cell culture. Preferably
these proteins are produced using an E. coli, yeast, or filamentous
fungal expression system. Methods for the purification of
homocitrate synthase may be described in Jaklitsch and Kubicek
(Jaklitsch, W. M. and C. P. Kubicek (1990) Biochem J 269: 247-53
(PMID: 2115771)). Other methods for the purification of homocitrate
synthase proteins and polypeptides are known to those skilled in
the art.
[0106] As an alternative to in vitro assays, the invention also
provides cell based assays. In one embodiment, the invention
provides a method for identifying a test compound as a candidate
for a antibiotic, comprising:
[0107] a) measuring the expression of a homocitrate synthase in a
cell, cells, tissue, or an organism in the absence of said
compound;
[0108] b) contacting said cell, cells, tissue, or organism with
said test compound and measuring the expression of said homocitrate
synthase in said cell, cells, tissue, or organism;
[0109] c) comparing the expression of homocitrate synthase in steps
(a) and (b);
[0110] wherein a lower expression in the presence of said test
compound indicates that said compound is a candidate for an
antibiotic.
[0111] Expression of homocitrate synthase can be measured by
detecting the HCS1 primary transcript or mRNA, homocitrate synthase
polypeptide, or homocitrate synthase enzymatic activity. Methods
for detecting the expression of RNA and proteins are known to those
skilled in the art. See, for example, Current Protocols in
Molecular Biology Ausubel et al., eds., Greene Publishing and
Wiley-Interscience, New York, 1995. The method of detection is not
critical to the invention. Methods for detecting HCS1 RNA include,
but are not limited to amplification assays such as quantitative
reverse transcriptase-PCR, and/or hybridization assays such as
Northern analysis, dot blots, slot blots, in-situ hybridization,
transcriptional fusions using a HCS1 promoter fused to a reporter
gene, DNA assays, and microarray assays.
[0112] Methods for detecting protein expression include, but are
not limited to, immunodetection methods such as Western blots,
ELISA assays, polyacrylamide gel electrophoresis, mass
spectroscopy, and enzymatic assays. Also, any reporter gene system
may be used to detect HCS1 protein expression. For detection using
gene reporter systems, a polynucleotide encoding a reporter protein
is fused in frame with HCS1, so as to produce a chimeric
polypeptide. Methods for using reporter systems are known to those
skilled in the art.
[0113] Chemicals, compounds or compositions identified by the above
methods as modulators, preferably inhibitors, of HCS1 expression or
activity can then be used to control fungal growth. Diseases such
as rusts, mildews, and blights spread rapidly once established.
Fungicides are thus routinely applied to growing and stored crops
as a preventive measure, generally as foliar sprays or seed
dressings. For example, compounds that inhibit fungal growth can be
applied to a fungus or expressed in a fungus, in order to prevent
fungal growth. Thus, the invention provides a method for inhibiting
fungal growth, comprising contacting a fungus with a compound
identified by the methods of the invention as having antifungal
activity.
[0114] Antifungals and antifungal inhibitor candidates identified
by the methods of the invention can be used to control the growth
of undesired fungi, including ascomycota, zygomycota,
basidiomycota, chytridiomycota, and lichens.
[0115] Examples of undesired fungi include, but are not limited to
Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis
cinerea), White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma
adspersum), Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago
maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora
zeae-maydis), Honey Fungus (Armillaria gallica), Root rot
(Armillaria luteobubalina), Shoestring Rot (Armillaria ostoyae),
Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting
Fungus (Monilinia fructigena), Apple-rotting Fungus (Penicillium
expansum), Clubroot Disease (Plasmodiophora brassicae), Potato
Blight (Phytophthora infestans), Root pathogen (Heterobasidion
annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm
Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculatus),
Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia
circinata), Southern Corn Blight (Cochliobolus heterostrophus),
Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus
stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab
Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmorum),
Potato Black Scurf (Rhizoctonia solani), Wheat Black Stem Rust
(Puccinia graminis), White mold (Sclerotinia sclerotiorum),
diseases of animals such as infections of lungs, blood, brain,
skin, scalp, nails or other tissues (Aspergillus fumigatus
Aspergillus sp. Fusraium sp., Trichophyton sp., Epidermophyton sp.,
and Microsporum sp., and the like).
[0116] Also provided is a method of screening for an antibiotic by
determining whether a test compound is active against the gene
identified (SEQ ID NO: 1 or SEQ ID NO: 2), its gene product (SEQ ID
NO: 3), or the biochemical pathway or pathways it functions on.
[0117] In one particular embodiment, the method is performed by
providing an organism having a first form of the gene corresponding
to either SEQ ID NO: 1 or SEQ ID NO: 2, either a normal form, a
mutant form, a homologue, or a heterologous HCS1 gene that performs
a similar function as HCS1. The first form of HCS1 may or may not
confer a growth conditional phenotype, i.e., a lysine requiring
phenotype, and/or a hypersensitivity or hyposensitivity phenotype
on the organism having that altered form. In one particular
embodiment a mutant form contains a transposon insertion. A
comparison organism having a second form of an HCS1, different from
the first form of the gene is also provided, and the two organisms
are separately contacted with a test compound. The growth of the
two organisms in the presence of the test compound is then
compared.
[0118] Thus, in one embodiment, the invention provides a method for
identifying a test compound as a candidate for an antibiotic,
comprising:
[0119] a) providing cells having one form of a homocitrate synthase
gene, and providing comparison cells having a different form of a
homocitrate synthase gene,
[0120] b) contacting said cells and said comparison cells with a
test compound and determining the growth of said cells and
comparison cells in the presence of the test compound,
[0121] wherein a difference in growth between said cells and said
comparison cells in the presence of said test compound indicates
that said test compound is a candidate for an antibiotic.
[0122] It is recognized in the art that the optional determination
of the growth of said first organism and said comparison second
organism in the absence of any test compounds may be performed to
control for any inherent differences in growth as a result of the
different genes. It is also recognized that any combination of two
different forms of an HCS1 gene, including normal genes, mutant
genes, homologues, and functional homologues may be used in this
method. Growth and/or proliferation of an organism is measured by
methods well known in the art such as optical density measurements,
and the like. In a preferred embodiment the organism is Magnaporthe
grisea.
[0123] Conditional lethal mutants may identify particular
biochemical and/or genetic pathways given that at least one
identified target gene is present in that pathway. Knowledge of
these pathways allows for the screening of test compounds as
candidates for antibiotics as inhibitors of the substrates,
products and enzymes of the pathway. Pathways known in the art may
be found at the Kyoto Encyclopedia of Genes and Genomes and in
standard biochemistry texts (Lehninger, A., D. Nelson, et al.
(1993) Principles of Biochemistry. New York, Worth Publishers).
[0124] Thus, in one embodiment, the invention provides a method for
screening for test compounds acting against the biochemical and/or
genetic pathway or pathways in which HCS1 functions,
comprising:
[0125] a) providing cells having one form of a gene in the lysine
biochemical and/or genetic pathway and providing comparison cells
having a different form of said gene.
[0126] b) contacting said cells and comparison cells with a said
test compound,
[0127] c) determining the growth of said cells and comparison cells
in the presence of said test compound.
[0128] wherein a difference in growth between said cells and said
comparison cells in the presence of said compound indicates that
said compound is a candidate for an antibiotic.
[0129] The use of multi-well plates for screening is a format that
readily accommodates multiple different assays to characterize
various compounds, concentrations of compounds, and fungal strains
in varying combinations and formats. Certain testing parameters for
the screening method can significantly affect the identification of
growth inhibitors, and thus can be manipulated to optimize
screening efficiency and/or reliability. Notable among these
factors are variable sensitivities of different mutants, increasing
hypersensitivity with increasingly less permissive conditions, an
apparent increase in hypersensitivity with increasing compound
concentration, and other factors known to those in the art.
[0130] Conditional lethal mutants may identify particular
biochemical and/or genetic pathways given that at least one
identified target gene is present in that pathway. Knowledge of
these pathways allows for the screening of test compounds as
candidates for antibiotics. Pathways known in the art may be found
at the Kyoto Encyclopedia of Genes and Genomes and in standard
biochemistry texts (Lehninger, A., D. Nelson, et al. (1993)
Principles of Biochemistry. New York, Worth Publishers). Thus, in
one embodiment, the invention provides a method for screening for
test compounds acting against the biochemical and/or genetic
pathway or pathways in which HCS1 functions, comprising:
[0131] (a) providing paired growth media; comprising a first medium
and a second medium, wherein said second medium contains a higher
level of lysine than said first medium;
[0132] (b) contacting an organism with said test compound;
[0133] (c) inoculating said first and second media with said
organism; and
[0134] (d) determining the growth of said organism;
[0135] wherein a difference in growth of the organism between said
first and second media indicates that said test compound is a
candidate for an antibiotic.
[0136] It is recognized in the art that the optional determination
of the growth of said organism in the paired media in the absence
of any test compounds may be performed to control for any inherent
differences in growth as a result of the different media. Growth
and/or proliferation of an organism is measured by methods well
known in the art such as optical density measurements, and the
like. In a preferred embodiment, the organism is Magnaporthe
grisea.
EXPERIMENTAL
EXAMPLE 1
Construction of Plasmids with a Transposon Containing a Selectable
Marker
[0137] Construction of Sif transposon: Sif was constructed using
the GPS3 vector from the GPS-M mutagenesis system from New England
Biolabs, Inc. (Beverly, Mass.) as a backbone. This system is based
on the bacterial transposon Tn7. The following manipulations were
done to GPS3 according to Sambrook et al. (1989) Molecular Cloning,
a Laboratory Manual, Cold Spring Harbor Laboratory Press. The
kanamycin resistance gene (npt) contained between the Tn7 arms was
removed by EcoRV digestion. The bacterial hygromycin B
phosphotransferase (hph) gene (Gritz and Davies (1983) Gene 25:
179-88 (PMID: 6319235)) under control of the Aspergillus nidulans
trpC promoter and terminator (Mullaney et al. (1985) Mol Gen Genet
199: 37-45 (PMID: 3158796)) was cloned by a HpaI/EcoRV blunt
ligation into the Tn7 arms of the GPS3 vector yielding pSif1.
Excision of the ampicillin resistance gene (bla) from pSif1 was
achieved by cutting pSif1 with XmnI and Bg1I followed by a T4 DNA
polymerase treatment to remove the 3' overhangs left by the Bg1I
digestion and religation of the plasmid to yield pSif. Top 10F'
electrocompetent E. coli cells (Invitrogen) were transformed with
ligation mixture according to manufacturer's recommendations.
Transformants containing the Sif transposon were selected on LB
agar (Sambrook et al. (1989) Molecular Cloning, a Laboratory
Manual, Cold Spring Harbor Laboratory Press.) containing 50 ug/ml
of hygromycin B (Sigma Chem. Co., St. Louis, Mo.).
EXAMPLE 2
Construction of a Cosmid Library Containing Fungal Genes and a
Selectable Marker
[0138] Cosmid libraries were constructed in the pcosKA5 vector
(Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID:
11296265)) as described in Sambrook et al. (1989) Molecular
Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press.
Cosmid libraries were quality checked by pulsed-field gel
electrophoresis, restriction digestion analysis, and PCR
identification of single genes.
EXAMPLE 3
Construction of Cosmids with Transposon Inserted into Fungal
Genes
[0139] Sif Transposition into a Cosmid: Transposition of Sif into
the cosmid framework was carried out as described by the GPS-M
mutagenesis system (New England Biolabs, Inc.). Briefly, 2 ul of
the 10.times.GPS buffer, 70 ng of supercoiled pSIF, 8-12 .mu.g of
target cosmid DNA were mixed and taken to a final volume of 20 ul
with water. 1 ul of transposase (TnsABC) was added to the reaction
and incubated for 10 minutes at 37.degree. C. to allow the assembly
reaction to happen. After the assembly reaction 1 ul of start
solution was added to the tube, mixed well and incubated for 1 hour
at 37.degree. C. followed by heat inactivation of the proteins at
75.degree. C. for 10 min. Destruction of the remaining untransposed
pSif was done by PISceI digestion at 37.degree. C. for 2 hours
followed by 10 min incubation at 75.degree. C. to inactivate the
proteins. Transformation of Top 10F' electrocompetent cells
(Invitrogen) was done according to manufacturers recommendations.
Sif-containing cosmid transformants were selected by growth on LB
agar plates containing 50 ug/ml of hygromycin B (Sigma Chem. Co.)
and 100 ug/ml of Ampicillin (Sigma Chem. Co.).
EXAMPLE 4
High Throughput Preparation and Verification of Insertion of
Transposon into Fungal Genes
[0140] E. coli strains containing cosmids with transposon
insertions were picked to 96 well growth blocks (Beckman Co.)
containing 1.5 ml of TB (Terrific Broth, Sambrook et al. (1989)
Molecular Cloning, a Laboratory Manual, Cold Spring Harbor
Laboratory Press) supplemented with 50 ug/ml of ampicillin. Blocks
were incubated with shaking at 37 C overnight. E. coli cells were
pelleted by centrifugation and cosmids were isolated by a modified
alkaline lysis method (Marra et al. (1997) Genome Res 7: 1072-84
(PMID: 9371743)). DNA quality was checked by electrophoresis on
agarose gels. Cosmids were sequenced using primers from the ends of
each transposon and commercial dideoxy sequencing kits (Big Dye
Terminators, Perkin Elmer Co.). Sequencing reactions were analyzed
on an ABI377 DNA sequencer (Perkin Elmer Co.).
[0141] DNA sequences adjacent to the site of the insertion were
collected and used to search DNA and protein databases using the
BLAST algorithms (Altschul et al. (1997) Nucleic Acids Res 25:
3389-3402 (PMID: 9254694)). A single insertion of SIF into the
Magnaporthe grisea HCS1 gene was chosen for further analysis. This
construct was designated cpgmra0023008h04 and it contains the SIF
transposon between amino acids 334 and 335 relative to the
Penicillium chrysogenum homologue (total length--474 amino acids,
GENBANK: PCAJ3630 accession number AJ223630).
EXAMPLE 5
Preparation of Cosmid DNA and Transformation of the Fungus
Magnaporthe grisea
[0142] Cosmid DNA from the HCS1 transposon tagged cosmid clone was
prepared using QIAGEN Plasmid Maxi Kit (QIAGEN), and digested by
PI-PspI (New England Biolabs, Inc.). Fungal electro-transformation
was performed essentially as described (Wu et al. (1997) MPMI 10:
700-708). Briefly, M. grisea strain Guy 11 was grown in complete
liquid media (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID:
8312740)) shaking at 120 rpm for 3 days at 25.degree. C. in the
dark. Mycelia was harvested and washed with sterile H.sub.2O and
digested with 4 mg/ml beta-glucanase (InterSpex) for 4-6 hours to
generate protoplasts. Protoplasts were collected by centrifugation
and resuspended in 20% sucrose at the concentration of
2.times.10.sup.8 protoplasts/ml. 50 ul protoplast suspension was
mixed with 10-20 ug of the cosmid DNA and pulsed using Gene Pulser
II (BioRad) set with the following parameters: resistance 200 ohm,
capacitance 25 uF, voltage 0.6 kV. Transformed protoplasts were
regenerated in complete agar media (CM, Talbot et al. (1993) Plant
Cell 5: 1575-1590 (PMID: 8312740)) with the addition of 20% sucrose
for one day, then overlayed with CM agar media containing
hygromycin B (250 ug/ml) to select transformants. Transformants
were screened for homologous recombination events in the target
gene by PCR (Hamer et al. (2001) Proc Natl Acad Sci USA 98: 5110-15
(PMID: 11296265)). Two independent strains were identified and are
hereby referred to as KO1-1 and KO1-2, respectively.
EXAMPLE 6
Effect of Transposon Insertion on Magnaporthe Pathogenicity
[0143] The target fungal strains, KO1-1 and KO1-2, obtained in
Example 5 and the wild type strain, Guy11, were subjected to a
pathogenicity assay to observe infection over a 1-week period. Rice
infection assays were performed using Indian rice cultivar CO39
essentially as described in Valent et al. ((1991) Genetics 127:
87-101 (PMID: 2016048)). All three strains were grown for spore
production on complete agar media. Spores were harvested and the
concentration of spores adjusted for whole plant inoculations.
Two-week-old seedlings of cultivar C039 were sprayed with 12 ml of
conidial suspension (5.times.10.sup.4 conidia per ml in 0.01%
Tween-20 (Polyoxyethylensorbitan monolaureate) solution). The
inoculated plants were incubated in a dew chamber at 27.degree. C.
in the dark for 36 hours, and transferred to a growth chamber
(27.degree. C. 12 hours/21.degree. C. 12 hours 70% humidity) for an
additional 5.5 days. Leaf samples were taken at 3, 5, and 7 days
post-inoculation and examined for signs of successful infection
(i.e. lesions). FIG. 2 shows the effects of HCS1 gene disruption on
Magnaporthe infection at five days post-inoculation.
EXAMPLE 7
Verification of Gene Function by Analysis of Nutritional
Requirements
[0144] The fungal strains, KO1-1 and KO1-2, containing the HCS1
disrupted gene obtained in Example 5 were analyzed for their
nutritional requirement for lysine using the PM5 phenotype
microarray from Biolog, Inc. (Hayward, Calif.). The PM5 plate tests
for the auxotrophic requirement for 94 different metabolites. The
inoculating fluid consists of 0.05% Phytagel, 0.03% Pluronic F68,
1% glucose, 23.5 mM NaNO.sub.3, 6.7 mM KCl, 3.5 mM
Na.sub.2SO.sub.4, 11 mM KH.sub.2PO.sub.4, 0.01%
p-iodonitrotetrazolium violet, 0.1 mM MgCl.sub.2, 1.0 mM CaCl.sub.2
and trace elements. Final concentrations of trace elements are: 7.6
.mu.M ZnCl.sub.2, 2.5 .mu.M MnCl.sub.2.4H.sub.2O, 1.8 .mu.M
FeCl.sub.2.4H.sub.2O, 0.71 .mu.M COCl.sub.2.6H2O, 0.64 .mu.M
CuCl.sub.2.2H.sub.2O, 0.62 .mu.M Na.sub.2MoO.sub.4, 18 .mu.M
H.sub.3BO.sub.3. pH adjusted to 6.0 with NaOH. Spores for each
strain were harvested into the inoculating fluid. The spore
concentrations were adjusted to 2.times.10.sup.5 spores/ml. 100
.mu.l of spore suspension were deposited into each well of the
microtiter plates. The plates were incubated at 25.degree. C. for 7
days. Optical density (OD) measurements at 490 nm and 750 nm were
taken daily. The OD.sub.490 measures the extent of tetrazolium dye
reduction and the level of growth, and OD.sub.750 measures growth
only. Turbidity=OD.sub.490+OD.sub.750. Data confirming the
annotated gene function is presented as a graph of Turbidity vs.
Time showing both the mutant fungi and the wild-type control in the
absence (FIG. 3A) and presence (FIG. 3B) of L-lysine.
EXAMPLE 8
Cloning and Expression Strategies, Extraction and Purification of
the Homocitrate Synthase Protein
[0145] The following protocol may be employed to obtain the
purified the homocitrate synthase protein.
[0146] Cloning and Expression Strategies:
[0147] A HCS1 cDNA gene can be cloned into E. coli (pET
vectors-Novagen),
[0148] Baculovirus (Pharmingen) and Yeast (Invitrogen) expression
vectors containing His/fusion protein tags. Evaluate the expression
of recombinant protein by SDS-PAGE and Western blot analysis.
[0149] Extraction:
[0150] Extract recombinant protein from 250 ml cell pellet in 3 ml
of extraction buffer By sonicating 6 times, with 6 sec pulses at
4.degree. C. Centrifuge extract at 15000.times.g for 10 min and
collect supernatant. Assess biological activity of the recombinant
protein by activity assay.
[0151] Purification:
[0152] Purify recombinant protein by Ni-NTA affinity chromatography
(Qiagen).
[0153] Purification protocol: perform all steps at 4.degree.
C.:
[0154] Use 3 ml Ni-beads (Qiagen)
[0155] Equilibrate column with the buffer
[0156] Load protein extract
[0157] Wash with the equilibration buffer
[0158] Elute bound protein with 0.5 M imidazole
EXAMPLE 9
Assays for Testing Binding of Test Compounds to Homocitrate
Synthase
[0159] The following protocol may be employed to identify test
compounds that bind to the homocitrate synthase protein.
[0160] Purified full length homocitrate synthase polypeptide with a
His/fusion protein tag (Example 8) is bound to a HisGrab Nickel
Coated Plate (Pierce, Rockford, Ill.) following manufacturer's
instructions.
[0161] Buffer conditions are optimized (e.g. ionic strength or pH,
Jaklitsch, W. M. and C. P. Kubicek (1990) Biochem J 269: 247-53
(PMID: 2115771)) for binding of radiolabeled
(acetyl-1-.sup.14C)-coenzyme A (Sigma-Aldrich Co.) to the bound
HCS1.
[0162] Screening of test compounds is performed by adding test
compound and (acetyl-1-.sup.14C)-coenzyme A (Sigma-Aldrich Co.) to
the wells of the HisGrab.TM. plate containing bound HCS1.
[0163] The wells are washed to remove excess labeled ligand and
scintillation fluid (Scintiverse.RTM., Fisher Scientific) is added
to each well.
[0164] The plates are read in a microplate scintillation
counter.
[0165] Candidate compounds are identified as wells with lower
radioactivity as compared to control wells with no test compound
added.
[0166] Additionally, a purified polypeptide comprising 10-50 amino
acids from the M. grisea homocitrate synthase is screened in the
same way. A polypeptide comprising 10-50 amino acids is generated
by subcloning a portion of the HCS1 gene into a protein expression
vector that adds a His-Tag when expressed (see Example 8).
Oligonucleotide primers are designed to amplify a portion of the
HCS1 gene using the polymerase chain reaction amplification method.
The DNA fragment encoding a polypeptide of 10-50 amino acids is
cloned into an expression vector, expressed in a host organism and
purified as described in Example 8 above.
[0167] Test compounds that bind HCS1 are further tested for
antibiotic activity. M. grisea is grown as described for spore
production on oatmeal agar media (Talbot et al. (1993) Plant Cell
5: 1575-1590 (PMID: 8312740)). Spores are harvested into minimal
media (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID:
8312740)) to a concentration of 2.times.10.sup.5 spores/ml and the
culture is divided. The test compound is added to one culture to a
final concentration of 20-100 .mu.g/ml. Solvent only is added to
the second culture. The plates are incubated at 25.degree. C. for
seven days and optical density measurements at 590 nm are taken
daily. The growth curves of the solvent control sample and the test
compound sample are compared. A test compound is an antibiotic
candidate if the growth of the culture containing the test compound
is less than the growth of the control culture.
EXAMPLE 10
Assays for Testing Inhibitors or Candidates for Inhibition of
Homocitrate Synthase Activity
[0168] The enzymatic activity of homocitrate synthase is determined
in the presence and absence of candidate compounds in a suitable
reaction mixture, such as described by Gray and Bhattacharjee
(Gray, G S and Bhattacharjee, J K (1976) Can J Microbiol 22: 1664-7
(PMID: 10066)), or Jaklitsch, W. M. and C. P. Kubicek (1990)
Biochem J 269: 247-53 (PMID: 2115771). Candidate compounds are
identified when a decrease in products or a lack of decrease in
substrates is detected with the reaction proceeding in either
direction.
[0169] Additionally, the enzymatic activity of a polypeptide
comprising 10-50 amino acids from the M. grisea homocitrate
synthase is determined in the presence and absence of candidate
compounds in a suitable reaction mixture, such as described by Gray
and Bhattacharjee (Gray, G S and Bhattacharjee, J K (1976) Can J
Microbiol 22: 1664-7 (PMID: 10066)), or Jaklitsch, W. M. and C. P.
Kubicek (1990) Biochem J 269: 247-53 (PMID: 2115771). A polypeptide
comprising 10-50 amino acids is generated by subcloning a portion
of the HCS1 gene into a protein expression vector that adds a
His-Tag when expressed (see Example 8). Oligonucleotide primers are
designed to amplify a portion of the HCS1 gene using polymerase
chain reaction amplification method. The DNA fragment encoding a
polypeptide of 10-50 amino acids is cloned into an expression
vector, expressed and purified as described in Example 8 above.
[0170] Test compounds identified as inhibitors of HCS1 activity are
further tested for antibiotic activity. Magnaporthe grisea fungal
cells are grown under standard fungal growth conditions that are
well known and described in the art. M. grisea is grown as
described for spore production on oatmeal agar media (Talbot et al.
(1993) Plant Cell 5: 1575-1590 (PMID: 8312740)). Spores are
harvested into minimal media (Talbot et al. (1993) Plant Cell 5:
1575-1590 (PMID: 8312740)) to a concentration of 2.times.10.sup.5
spores/ml and the culture is divided. The test compound is added to
one culture to a final concentration of 20-100 .mu.g/ml. Solvent
only is added to the second culture. The plates are incubated at
25.degree. C. for seven days and optical density measurements at
590 nm are taken daily. The growth curves of the solvent control
sample and the test compound sample are compared. A test compound
is an antibiotic candidate if the growth of the culture containing
the test compound is less than the growth of the control
culture.
EXAMPLE 11
Assays for Testing Compounds or Candidates for Compounds That Alter
the Expression of the Homocitrate Synthase Gene
[0171] Magnaporthe grisea fungal cells are grown under standard
fungal growth conditions that are well known and described in the
art. Wild-type M. grisea spores are harvested from cultures grown
on complete agar or oatmeal agar media after growth for 10-13 days
in the light at 25.degree. C. using a moistened cotton swab. The
concentration of spores is determined using a hemacytometer and
spore suspensions are prepared in a minimal growth medium to a
concentration of 2.times.10.sup.5 spores per ml. 25 ml cultures are
prepared to which test compounds will be added at various
concentrations. A culture with no test compound present is included
as a control. The cultures are incubated at 25.degree. C. for 3
days after which test compound or solvent only control is added.
The cultures are incubated an additional 18 hours. Fungal mycelia
is harvested by filtration through Miracloth (CalBiochem.RTM., La
Jolla, Calif.), washed with water and frozen in liquid nitrogen.
Total RNA is extracted with TRIZOL.RTM. Reagent using the methods
provided by the manufacturer (Life Technologies, Rockville, Md.).
Expression is analyzed by Northern analysis of the RNA samples as
described (Sambrook et al. (1989) Molecular Cloning, a Laboratory
Manual, Cold Spring Harbor Laboratory Press) using a radiolabeled
fragment of the HCS1 gene as a probe. Test compounds resulting in a
reduced level of HCS1 mRNA relative to the untreated control sample
are identified as candidate antibiotic compounds.
EXAMPLE 12
In Vivo Cell Based Assay Screening Protocol with a Fungal Strain
Containing a Mutant Form of Homocitrate Synthase with No
Activity
[0172] Magnaporthe grisea fungal cells containing a mutant form of
the HCS1 gene which abolishes enzyme activity, such as a gene
containing a transposon insertion (see Examples 4 and 5), are grown
under standard fungal growth conditions that are well known and
described in the art. Magnaporthe grisea spores are harvested from
cultures grown on complete agar medium containing 4 mM L-lysine
(Sigma-Aldrich Co.) after growth for 10-13 days in the light at
25.degree. C. using a moistened cotton swab. The concentration of
spores is determined using a hemacytometer and spore suspensions
are prepared in a minimal growth medium containing 100 .mu.M
L-lysine to a concentration of 2.times.10.sup.5 spores per ml.
Approximately 4.times.10.sup.4 spores are added to each well of
96-well plates to which a test compound is added (at varying
concentrations). The total volume in each well is 200 .mu.l. Wells
with no test compound present (growth control), and wells without
cells are included as controls (negative control). The plates are
incubated at 25.degree. C. for seven days and optical density
measurements at 590 nm are taken daily. Wild type cells are
screened under the same conditions. The effect of each compound on
the mutant and wild-type fungal strains is measured against the
growth control and the percent of inhibition is calculated as the
OD.sub.590 (fungal strain plus test compound)/OD.sub.590 (growth
control).times.100. The percent of growth inhibition as a result of
a test compound on a fungal strain and that on the wild type cells
are compared. Compounds that show differential growth inhibition
between the mutant and the wild type are identified as potential
antifungal compounds. Similar protocols may be found in Kirsch and
DiDomenico ((1994) Biotechnology 26:177-221 (PMID: 7749303)).
EXAMPLE 13
In Vivo Cell Based Assay Screening Protocol with a Fungal Strain
Containing a Mutant Form of Homocitrate Synthase with Reduced
Activity
[0173] Magnaporthe grisea fungal cells containing a mutant form of
the HCS1 gene, such as a promoter truncation that reduces
expression, are grown under standard fungal growth conditions that
are well known and described in the art. A promoter truncation is
made by deleting a portion of the promoter upstream of the
transcription start site using standard molecular biology
techniques that are well known and described in the art (Sambrook
et al. (1989) Molecular Cloning, a Laboratory Manual, Cold Spring
Harbor Laboratory Press). Magnaporthe grisea spores are harvested
from cultures grown on complete agar medium containing 4 mM
L-lysine (Sigma-Aldrich Co.) after growth for 10-13 days in the
light at 25.degree. C. using a moistened cotton swab. The
concentration of spores is determined using a hemacytometer and
spore suspensions are prepared in a minimal growth medium to a
concentration of 2.times.10.sup.5 spores per ml. Approximately
4.times.10.sup.4 spores are added to each well of 96-well plates to
which a test compound is added (at varying concentrations). The
total volume in each well is 200%1. Wells with no test compound
present (growth control), and wells without cells are included as
controls (negative control). The plates are incubated at 25.degree.
C. for seven days and optical density measurements at 590 nm are
taken daily. Wild type cells are screened under the same
conditions. The effect of each compound on the mutant and wild-type
fungal strains is measured against the growth control and the
percent of inhibition is calculated as the OD.sub.590 (fungal
strain plus test compound)/OD.sub.590 (growth control).times.100.
The percent of growth inhibition as a result of a test compound on
a fungal strain and that on the wild-type cells are compared.
Compounds that show differential growth inhibition between the
mutant and the wild type are identified as potential antifungal
compounds. Similar protocols may be found in Kirsch and DiDomenico
((1994) Biotechnology 26: 177-221 (PMID: 7749303)).
EXAMPLE 14
In Vivo Cell Based Assay Screening Protocol with a Fungal Strain
Containing a Mutant Form of a Lysine Biosynthetic Gene with No
Activity
[0174] Magnaporthe grisea fungal cells containing a mutant form of
a gene in the lysine biosynthetic pathway (e.g.
L-Aminoadipate-semialdehyde dehydrogenase (E.C. 1.2.1.31)) are
grown under standard fungal growth conditions that are well known
and described in the art. Magnaporthe grisea spores are harvested
from cultures grown on complete agar medium containing 4 mM
L-lysine (Sigma-Aldrich Co.) after growth for 10-13 days in the
light at 25.degree. C. using a moistened cotton swab. The
concentration of spores is determined using a hemacytometer and
spore suspensions are prepared in a minimal growth medium
containing 100 .mu.M L-lysine to a concentration of
2.times.10.sup.5 spores per ml. Approximately 4.times.10.sup.4
spores or cells are harvested and added to each well of 96-well
plates to which growth media is added in addition to an amount of
test compound (at varying concentrations). The total volume in each
well is 200 .mu.l. Wells with no test compound present, and wells
without cells are included as controls. The plates are incubated at
25.degree. C. for seven days and optical density measurements at
590 nm are taken daily. Wild type cells are screened under the same
conditions. The effect of each compound on the mutant and wild-type
fungal strains is measured against the growth control and the
percent of inhibition is calculated as the OD.sub.590 (fungal
strain plus test compound)/OD.sub.590 (growth control).times.100.
The percent of growth inhibition as a result of a test compound on
a fungal strain and that on the wild type cells are compared.
Compounds that show differential growth inhibition between the
mutant and the wild-type are identified as potential antifungal
compounds. Similar protocols may be found in Kirsch and DiDomenico
((1994) Biotechnology 26: 177-221 (PMID: 7749303)).
EXAMPLE 15
In Vivo Cell Based Assay Screening Protocol with a Fungal Strain
Containing a Mutant Form of a Lysine Biosynthetic Gene with Reduced
Activity
[0175] Magnaporthe grisea fungal cells containing a mutant form of
a gene in the lysine biosynthetic pathway (e.g.
L-Aminoadipate-semialdehyde dehydrogenase (E.C. 1.2.1.31)), such as
a promoter truncation that reduces expression, are grown under
standard fungal growth conditions that are well known and described
in the art. A promoter truncation is made by deleting a portion of
the promoter upstream of the transcription start site using
standard molecular biology techniques that are well known and
described in the art (Sambrook et al (1989) Molecular Cloning, a
Laboratory Manual, Cold Spring Harbor Laboratory Press).
Magnaporthe grisea fungal cells containing a mutant form of are
grown under standard fungal growth conditions that are well known
and described in the art. Magnaporthe grisea spores are harvested
from cultures grown on complete agar medium containing 4 mM
L-lysine (Sigma-Aldrich Co.) after growth for 10-13 days in the
light at 25.degree. C. using a moistened cotton swab. The
concentration of spores is determined using a hemacytometer and
spore suspensions are prepared in a minimal growth medium to a
concentration of 2.times.10.sup.5 spores per ml. Approximately
4.times.10.sup.4 spores or cells are harvested and added to each
well of 96-well plates to which growth media is added in addition
to an amount of test compound (at varying concentrations). The
total volume in each well is 200 .mu.l. Wells with no test compound
present, and wells without cells are included as controls. The
plates are incubated at 25.degree. C. for seven days and optical
density measurements at 590 nm are taken daily. Wild type cells are
screened under the same conditions. The effect of each compound on
the mutant and wild-type fungal strains is measured against the
growth control and the percent of inhibition is calculated as the
OD.sub.590 (fungal strain plus test compound)/OD.sub.590 (growth
control).times.100. The percent of growth inhibition as a result of
a test compound on a fungal strain and that on the wild type cells
are compared. Compounds that show differential growth inhibition
between the mutant and the wild type are identified as potential
antifungal compounds. Similar protocols may be found in Kirsch and
DiDomenico ((1994) Biotechnology 26:177-221 (PMID: 7749303)).
EXAMPLE 16
In Vivo Cell Based Assay Screening Protocol with a Fungal Strain
Containing a Fungal HCS1 and a Second Fungal Strain Containing a
Heterologous HCS1 Gene
[0176] Wild-type Magnaporthe grisea fungal cells and M. grisea
fungal cells lacking a functional HCS1 gene and containing a HCS1
gene from Thermus aquaticus (Genbank accession 087198, 56% sequence
identity) are grown under standard fungal growth conditions that
are well known and described in the art. A M. grisea strain
carrying a heterologous HCS1 gene is made as follows:
[0177] A M. grisea strain is made with a nonfunctional HCS1 gene,
such as one containing a transposon insertion in the native gene
(see Examples 4 and 5).
[0178] A construct containing a heterologous HCS1 gene is made by
cloning the HCS1 gene from Thermus aquaticus into a fungal
expression vector containing a trpC promoter and terminator (e.g.
pCB1003, Carroll et al. (1994) Fungal Gen News Lett 41: 22) using
standard molecular biology techniques that are well known and
described in the art (Sambrook et al. (1989) Molecular Cloning, a
Laboratory Manual, Cold Spring Harbor Laboratory Press).
[0179] The said construct is used to transform the M. grisea strain
lacking a functional HCS1 gene (see Example 5). Transformants are
selected on minimal agar medium lacking L-lysine. Only
transformants carrying a functional HCS1 gene will grow.
[0180] Wild-type strains of Magnaporthe grisea and strains
containing a heterologous form of HCS1 are grown under standard
fungal growth conditions that are well known and described in the
art. Magnaporthe grisea spores are harvested from cultures grown on
complete agar medium after growth for 10-13 days in the light at
25.degree. C. using a moistened cotton swab. The concentration of
spores is determined using a hemacytometer and spore suspensions
are prepared in a minimal growth medium to a concentration of
2.times.10.sup.5 spores per ml. Approximately 4.times.10.sup.4
spores or cells are harvested and added to each well of 96-well
plates to which growth media is added in addition to an amount of
test compound (at varying concentrations). The total volume in each
well is 200 .mu.l. Wells with no test compound present, and wells
without cells are included as controls. The plates are incubated at
25.degree. C. for seven days and optical density measurements at
590 nm are taken daily. The effect of each compound on the
wild-type and heterologous fungal strains is measured against the
growth control and the percent of inhibition is calculated as the
OD.sub.590 (fungal strain plus test compound)/OD.sub.590 (growth
control).times.100. The percent of growth inhibition as a result of
a test compound on the wild-type and heterologous fungal strains
are compared. Compounds that show differential growth inhibition
between the wild-type and heterologous strains are identified as
potential antifungal compounds with specificity to the native or
heterologous HCS1 gene products. Similar protocols may be found in
Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID:
7749303)).
EXAMPLE 17
Pathway Specific In Vivo Assay Screening Protocol
[0181] Magnaporthe grisea fungal cells are grown under standard
fungal growth conditions that are well known and described in the
art. Wild-type M. grisea spores are harvested from cultures grown
on oatmeal agar media after growth for 10-13 days in the light at
25.degree. C. using a moistened cotton swab. The concentration of
spores is determined using a hemocytometer and spore suspensions
are prepared in a minimal growth medium and a minimal growth medium
containing 4 mM L-lysine (Sigma-Aldrich Co.) to a concentration of
2.times.10.sup.5 spores per ml. The minimal growth media contains
carbon, nitrogen, phosphate, and sulfate sources, and magnesium,
calcium, and trace elements (for example, see inoculating fluid in
Example 7). Spore suspensions are added to each well of a 96-well
microtiter plate (approximately 4.times.10.sup.4 spores/well). For
each well containing a spore suspension in minimal media, an
additional well is present containing a spore suspension in minimal
medium containing 4 mM L-lysine. Test compounds are added to wells
containing spores in minimal media and minimal media containing
L-lysine. The total volume in each well is 200 .mu.l. Both minimal
media and L-lysine containing media wells with no test compound are
provided as controls. The plates are incubated at 25.degree. C. for
seven days and optical density measurements at 590 nm are taken
daily. A compound is identified as a candidate for an antibiotic
acting against the lysine biosynthetic pathway when the observed
growth in the well containing minimal media is less than the
observed growth in the well containing L-lysine as a result of the
addition of the test compound. Similar protocols may be found in
Kirsch and DiDomenico ((1994) Biotechnology 26: 177-221 (PMID:
7749303)).
[0182] While the foregoing describes certain embodiments of the
invention, it will be understood by those skilled in the art that
variations and modifications may be made and still fall within the
scope of the invention. The foregoing examples are intended to
exemplify various specific embodiments of the invention and do not
limit its scope in any manner.
Sequence CWU 1
1
3 1 1458 DNA Magnaporthe grisea 1 atgtgcccat cctgcgagcc tgagcaagcc
gctgcctcca atggcaatgc gaacggcaat 60 ggcgcctcca atggcaatgg
aaaccacgac ggaatgactg gtattgagac tcgccaagca 120 caaaacgcac
gctaccagcc atcacggaat ccctaccagc ccgtcggtga ctttttgtcc 180
aacgtgaaca acttcaagat cattgagagc accctgcgag agggcgagca gttcgccaat
240 gccttcttcg acacggccaa gaagattgag atcgccaagg cgctggacga
ctttggcgtc 300 gactacatcg agctcaccag cccggctgcc tcggagcagt
ccaggcttga ctgcgctgcc 360 atctgcaagc tgggactcaa ggccaagatc
ctcacccaca tcaggtgcca catggacgac 420 gcgcgcatcg ccgtcgagac
cggtgttgac ggcgtcgaca ttgtcatcgg cacctcttcg 480 ttcctcatgg
agcactcgca cggcaaggac atgacctaca tcaccaacac ggccattgag 540
gtcatcaact ttgtcaagag caagggcatc gaggtccgct tctcatccga ggactcgttc
600 cgcagcaacc tggttgacct gctgagcatc tactcgaccg tcgacaagat
tggtgtcaac 660 cgtgtcggta ttgctgatac cgtcggttgc gcctcgcccc
gccaggtcta cgacctggtc 720 aagaccctgc gtggtgttgt ctcttgtgac
attgagacac acttccacaa cgacactggc 780 tgtgccatct caaatgcttt
ttgcgctttg gaggccggtg ctacccacat cgacacgtgt 840 gtcctgggta
tcggcgagcg taacggaatt acccctcttg gaggtctgat ggctcgcatg 900
attgtcggct ccaaggacta cgttctgagc aagtacaagc tgcacaagct caaggacatt
960 gaggagcttg ttgccgacgc cgttcaggtc aacattcctt tcaataacta
catcactggt 1020 ttctgtgctt tcacccacaa ggccggtatc catgccaagg
ctattctcaa gaacccctca 1080 acatatgaga ttattgaccc gactctgttc
ggcatcactc gctatgtcca cttcgccagc 1140 agattgacgg gatggaacgc
aatcaagagc agagcatcgc agctcaacat tgagatgacg 1200 gatgagcagt
gcaaggagtg cactgccaag atcaagctgt tggctgacat taggccgatc 1260
gctatcgacg acgccgactc catcattcac gcattccacc gcagcatcaa ctcgggccag
1320 cctattcagt ctctcggaag cctgctcccc aacatgacgg aggaggagaa
ggccgccctg 1380 gcagatgtag agcgccgtga gtcgaacgat gccgagcaac
cggcggccaa gagggccaag 1440 gtcgaggctg ttgcatga 1458 2 2346 DNA
Magnaporthe grisea exon (130)..(384) 2 caaagctgga gctccaccgc
ggtggcggcc gctctagaac tagtggatcc cccgggctgc 60 aggaattcgg
cacgagccaa gtcccagcct cccattgagc tttactcaca acaatcccaa 120
accaccaaa atg tgc cca tcc tgc gag cct gag caa gcc gct gcc tcc aat
171 Met Cys Pro Ser Cys Glu Pro Glu Gln Ala Ala Ala Ser Asn 1 5 10
ggc aat gcg aac ggc aat ggc gcc tcc aat ggc aat gga aac cac gac 219
Gly Asn Ala Asn Gly Asn Gly Ala Ser Asn Gly Asn Gly Asn His Asp 15
20 25 30 gga atg act ggt att gag act cgc caa gca caa aac gca cgc
tac cag 267 Gly Met Thr Gly Ile Glu Thr Arg Gln Ala Gln Asn Ala Arg
Tyr Gln 35 40 45 cca tca cgg aat ccc tac cag ccc gtc ggt gac ttt
ttg tcc aac gtg 315 Pro Ser Arg Asn Pro Tyr Gln Pro Val Gly Asp Phe
Leu Ser Asn Val 50 55 60 aac aac ttc aag atc att gag agc acc ctg
cga gag ggc gag cag ttc 363 Asn Asn Phe Lys Ile Ile Glu Ser Thr Leu
Arg Glu Gly Glu Gln Phe 65 70 75 gcc aat gcc ttc ttc gac acg
gtgagtcaag ccacatcgca agcaaatact 414 Ala Asn Ala Phe Phe Asp Thr 80
85 tgctcctcac aacggccgca agcctgggct actttggtag ctcggcggtg
tttttgctgt 474 cgatgtgtcc tggcggcatc ccggcgcaaa aacagacctc
atagactgac tcatgctttt 534 tttaacctcc gcgcag gcc aag aag att gag atc
gcc aag gcg ctg gac gac 586 Ala Lys Lys Ile Glu Ile Ala Lys Ala Leu
Asp Asp 90 95 ttt ggc gtc gac tac atc gag ctc acc agc ccg gct gcc
tcg gag cag 634 Phe Gly Val Asp Tyr Ile Glu Leu Thr Ser Pro Ala Ala
Ser Glu Gln 100 105 110 tcc agg ctt gac tgc gct gcc atc tgc aag ctg
gga ctc aag gcc aag 682 Ser Arg Leu Asp Cys Ala Ala Ile Cys Lys Leu
Gly Leu Lys Ala Lys 115 120 125 atc ctc acc cac atc agg tgc cac atg
gac gac gcg cgc atc gcc gtc 730 Ile Leu Thr His Ile Arg Cys His Met
Asp Asp Ala Arg Ile Ala Val 130 135 140 145 gag acc ggt gtt gac ggc
gtc gac att gtc atc ggc acc tct tcg ttc 778 Glu Thr Gly Val Asp Gly
Val Asp Ile Val Ile Gly Thr Ser Ser Phe 150 155 160 ctc atg gag cac
tcg cac ggc aag gac atg acc tac atc acc aac acg 826 Leu Met Glu His
Ser His Gly Lys Asp Met Thr Tyr Ile Thr Asn Thr 165 170 175 gcc att
gag gtc atc aac ttt gtc aag agc aag ggc atc gag gtc cgc 874 Ala Ile
Glu Val Ile Asn Phe Val Lys Ser Lys Gly Ile Glu Val Arg 180 185 190
ttc tca tcc gag gac tcg ttc cgc agc aac ctg gtt gac ctg ctg agc 922
Phe Ser Ser Glu Asp Ser Phe Arg Ser Asn Leu Val Asp Leu Leu Ser 195
200 205 atc tac tcg acc gtc gac aag att ggt gtc aac cgt gtc ggt att
gct 970 Ile Tyr Ser Thr Val Asp Lys Ile Gly Val Asn Arg Val Gly Ile
Ala 210 215 220 225 gat acc gtc ggt tgc gcc tcg ccc cgc cag gtc tac
gac ctg gtc aag 1018 Asp Thr Val Gly Cys Ala Ser Pro Arg Gln Val
Tyr Asp Leu Val Lys 230 235 240 acc ctg cgt ggt gtt gtc tct tg
gtgagccaca ggtctgatga atcttgtgct 1071 Thr Leu Arg Gly Val Val Ser
Cys 245 gcttggtgct gatgctaaca gttcgatag t gac att gag aca cac ttc
cac aac 1125 Asp Ile Glu Thr His Phe His Asn 250 255 gac act ggc
tgt gcc atc tca aat gct ttt tgc gct ttg gag gcc ggt 1173 Asp Thr
Gly Cys Ala Ile Ser Asn Ala Phe Cys Ala Leu Glu Ala Gly 260 265 270
gct acc cac atc gac acg tgt gtc ctg ggt atc ggc gag cgt aac gga
1221 Ala Thr His Ile Asp Thr Cys Val Leu Gly Ile Gly Glu Arg Asn
Gly 275 280 285 att acc cct ctt gga ggt ctg atg gct cgc atg att gtc
ggc tcc aag 1269 Ile Thr Pro Leu Gly Gly Leu Met Ala Arg Met Ile
Val Gly Ser Lys 290 295 300 305 gac tac gtt ctg agc aag tac aag ctg
cac aag ctc aag gac att gag 1317 Asp Tyr Val Leu Ser Lys Tyr Lys
Leu His Lys Leu Lys Asp Ile Glu 310 315 320 gag ctt gtt gcc gac gcc
gtt cag gtc aac at gtaagttttg ccatcccagt 1369 Glu Leu Val Ala Asp
Ala Val Gln Val Asn Ile 325 330 gcagttttca ttctgggtag gattgctaac
attttgtctc tgtag t cct ttc aat 1424 Pro Phe Asn 335 aac tac atc act
ggt ttc tgt gct ttc acc cac aa gtatgttccg 1469 Asn Tyr Ile Thr Gly
Phe Cys Ala Phe Thr His Lys 340 345 tcacacactg gtatctacta
ttgattcaaa actaactcgt tgctatag g gcc ggt 1524 Ala Gly atc cat gcc
aag gct att ctc aag aac ccc tca aca tat gag att att 1572 Ile His
Ala Lys Ala Ile Leu Lys Asn Pro Ser Thr Tyr Glu Ile Ile 350 355 360
365 gtatgttttt gatctgttca cgcactgtgc cagcatgggt atgatgagcc
gaaaatacta 1632 acccttgatt aatcag gac ccg act ctg ttc ggc atc act
cgc tat gtc cac 1684 Asp Pro Thr Leu Phe Gly Ile Thr Arg Tyr Val
His 370 375 ttc gcc agc aga ttg acg gga tgg aac gca atc aag agc aga
gca tcg 1732 Phe Ala Ser Arg Leu Thr Gly Trp Asn Ala Ile Lys Ser
Arg Ala Ser 380 385 390 cag ctc aac att gag atg acg gat gag cag tgc
aag gag tgc act gcc 1780 Gln Leu Asn Ile Glu Met Thr Asp Glu Gln
Cys Lys Glu Cys Thr Ala 395 400 405 aag atc aag ctg ttg gct gac att
agg ccg atc gct atc gac gac gcc 1828 Lys Ile Lys Leu Leu Ala Asp
Ile Arg Pro Ile Ala Ile Asp Asp Ala 410 415 420 425 gac tcc atc att
cac gca ttc cac cgc agc atc aac tcg ggc cag cct 1876 Asp Ser Ile
Ile His Ala Phe His Arg Ser Ile Asn Ser Gly Gln Pro 430 435 440 att
cag tat ctc gga agc ctg ctc ccc aac atg acg gag gag gag aag 1924
Ile Gln Tyr Leu Gly Ser Leu Leu Pro Asn Met Thr Glu Glu Glu Lys 445
450 455 gcc gcc ctg gca gat gta gag cgc cgt gag tcg aac gat gcc gag
caa 1972 Ala Ala Leu Ala Asp Val Glu Arg Arg Glu Ser Asn Asp Ala
Glu Gln 460 465 470 ccg gcg gcc aag agg gcc aag gtc gag gct gtt gca
t gagcacaacg 2019 Pro Ala Ala Lys Arg Ala Lys Val Glu Ala Val Ala
475 480 485 gaatttttga gcattgtcga agcgtgagcg agtcacatat atattgttga
cgcagaattt 2079 tggtggtcaa agggaagtac agaaaggcct tgggctttga
ttttcctaac cccaaagcgt 2139 tgacattttt attatgtctt cttctgtctg
catacgaagt caaaaaagga aggagaaagg 2199 aaaagtatcg tcaggatggg
atggtttacg gatctacatt ggtaccggag ctattcaagg 2259 atagattgtg
tttgctttga tttgccccca tggatgagtt ggggcctttt gctgatttgc 2319
tatatgttgc tataccattg aatgaaa 2346 3 449 PRT Magnaporthe grisea 3
Met Cys Pro Ser Cys Glu Pro Glu Gln Ala Ala Ala Ser Asn Gly Asn 1 5
10 15 Ala Asn Gly Asn Gly Ala Ser Asn Gly Asn Gly Asn His Asp Gly
Met 20 25 30 Thr Gly Ile Glu Thr Arg Gln Ala Gln Asn Ala Arg Tyr
Gln Pro Ser 35 40 45 Arg Asn Pro Tyr Gln Pro Val Gly Asp Phe Leu
Ser Asn Val Asn Asn 50 55 60 Phe Lys Ile Ile Glu Ser Thr Leu Arg
Glu Gly Glu Gln Phe Ala Asn 65 70 75 80 Ala Phe Phe Asp Thr Ala Lys
Lys Ile Glu Ile Ala Lys Ala Leu Asp 85 90 95 Asp Phe Gly Val Asp
Tyr Ile Glu Leu Thr Ser Pro Ala Ala Ser Glu 100 105 110 Gln Ser Arg
Leu Asp Cys Ala Ala Ile Cys Lys Leu Gly Leu Lys Ala 115 120 125 Lys
Ile Leu Thr His Ile Arg Cys His Met Asp Asp Ala Arg Ile Ala 130 135
140 Val Glu Thr Gly Val Asp Gly Val Asp Ile Val Ile Gly Thr Ser Ser
145 150 155 160 Phe Leu Met Glu His Ser His Gly Lys Asp Met Thr Tyr
Ile Thr Asn 165 170 175 Thr Ala Ile Glu Val Ile Asn Phe Val Lys Ser
Lys Gly Ile Glu Val 180 185 190 Arg Phe Ser Ser Glu Asp Ser Phe Arg
Ser Asn Leu Val Asp Leu Leu 195 200 205 Ser Ile Tyr Ser Thr Val Asp
Lys Ile Gly Val Asn Arg Val Gly Ile 210 215 220 Ala Asp Thr Val Gly
Cys Ala Ser Pro Arg Gln Val Tyr Asp Leu Val 225 230 235 240 Lys Thr
Leu Arg Gly Val Val Ser Cys Asp Ile Glu Thr His Phe His 245 250 255
Asn Asp Thr Gly Cys Ala Ile Ser Asn Ala Phe Cys Ala Leu Glu Ala 260
265 270 Gly Ala Thr His Ile Asp Thr Cys Val Leu Gly Ile Gly Glu Arg
Asn 275 280 285 Gly Ile Thr Pro Leu Gly Gly Leu Met Ala Arg Met Ile
Val Gly Ser 290 295 300 Lys Asp Tyr Val Leu Ser Lys Tyr Lys Leu His
Lys Leu Lys Asp Ile 305 310 315 320 Glu Glu Leu Val Ala Asp Ala Val
Gln Val Asn Ile Pro Phe Asn Asn 325 330 335 Tyr Ile Thr Gly Phe Cys
Ala Phe Thr His Lys Ala Gly Ile His Ala 340 345 350 Lys Ala Ile Leu
Lys Asn Pro Ser Thr Tyr Glu Ile Ile Asp Pro Thr 355 360 365 Leu Phe
Gly Ile Thr Arg Tyr Val His Phe Ala Ser Arg Leu Thr Gly 370 375 380
Trp Asn Ala Ile Lys Ser Arg Ala Ser Gln Leu Asn Ile Glu Met Thr 385
390 395 400 Asp Glu Gln Cys Lys Glu Cys Thr Ala Lys Ile Lys Leu Leu
Ala Asp 405 410 415 Ile Arg Pro Ile Ala Ile Asp Asp Ala Asp Ser Ile
Ile His Ala Phe 420 425 430 His Arg Ser Ile Asn Ser Gly Gln Pro Ile
Gln Ser Leu Gly Ser Leu 435 440 445 Leu
* * * * *