Progress 09/01/08 to 08/31/13
Outputs
Target Audience: Scientists at USDA labs, universities, and national labs. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two postdocs and two grad students received training. How have the results been disseminated to communities of interest? In peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
All of the goals were accomplished, except that secreted proteins that induce seedling death could not be purified..
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Paper, J. M., J. S. Scott-Craig, D. Cavalier, A. Faik, R. E. Wiemels, M. S. Borrusch, M. Bongers, and J.D. Walton (2013) ?-Fucosidases with different substrate specificities from two species of Fusarium. Appl. Microbiol. Biotechnol. 97:5371-5380.
Walton, J. D., H. Luo, and H. Hallen-Adams (2012) Ribosomally encoded cyclic peptide toxins from mushrooms. Meth Enzymol 516, part B (D.A. Hopwood, ed.), pp. 63-77.
Wight, W.D., R. Labuda, J.D. Walton (2013) Conservation of the genes for HC-toxin biosynthesis in Alternaria jesenskae. BMC Microbiol. 13:165.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Scientific findings wre reported at meetings and in peer-reviewed journals. PARTICIPANTS: Jonathan Walton, PI Wanessa Wight, graduate student Janet Paper, graduate student John Scott-Craig, postdoctoral research associate Melissa Borrusch, technician TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Two fucosidases were characterized from two plant pathogenic fungi.
Publications
- Luo, H., H. E. Hallen-Adams, J. S. Scott-Craig, and J. D. Walton (2012) Ribosomal biosynthesis of alpha-amanitin in Galerina marginata. Fung. Genet. Biol. 49:123-129. Arnison, P. G., M. J. Bibb, G. Bierbaum, A. A. Bowers, G. Bulaj, J. A. ...J. D. Walton, et al (2012) Ribosomally synthesized and post-translationally modified peptide natural products: Overview and recommendations for a universal nomenclature. Nat Prod Rep, in press.
- Paper, J. M., J. S. Scott-Craig, D. Cavalier, A. Faik, R. E. Wiemels, M. S. Borrusch, M. Bongers, and J.D. Walton. 2012 alpha-Fucosidases with different substrate specificities from two species of Fusarium. Appl. Microbiol. Biotechnol., in press
- Banerjee, G., S. Car, T. Liu, D. L. Williams, S. Lopez Meza, J. D. Walton, and D. B. Hodge (2012) Scale-up and integration of alkaline hydrogen peroxide pretreatment, enzymatic hydrolysis, and ethanolic fermentation. Biotechnol. Bioengineer. 109:922-931.
- Walton, J. D., H. Luo, and H. Hallen-Adams (2012) Ribosomally encoded cyclic peptide toxins from mushrooms. Meth Enzymol 516, part B (D.A. Hopwood, ed.), pp. 63-77.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Scientific findings were reported at scientific meetings and in peer-reviewed publications. PARTICIPANTS: Janet Paper, graduate student and postdoctoral researcher Wanessa Wight, graduate student John Scott-Craig, postdoctoral researcher associate TARGET AUDIENCES: The scientific community of enzymologists and plant pathologists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Two fucosidases were characterized from the plant pathogens Fusarium oxysporum and Fusarium graminearum. One enzyme uses the model compound p-NP-fucoside as substrate but not native pea xyloglucan, whereas the other works on the native substrate but not the model substrate. The two fucosidases group into two branches of the GH29 family of glycosyl hydrolases.
Publications
- Luo, H., H.E. Hallen-Adams, J.S. Scott-Craig, and J.D. Walton (2010) Co-localization of amanitin and a candidate toxin-processing prolyl oligopeptidase in Amanita basidiocarps. Eukaryotic Cell 9: 1891-1900. Paper et al., Two fucosidases from species of Fusarium, in preparation.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: We discovered and began analysis of a gene cluster in Alternaria jesenskae that is responsible for biosynthesis of the cyclic tetrapeptide HC-toxin. This toxin was previously shown by us to be an essential virulence determinant for the pathogenic fungus Cochliobolus carbonum on its host, maize. It is remarkable that an unrelated fungus make the same complex secondary metabolite. We used 454 sequencing technology to survey the genome of A. jesenskae. We found clear orthologs of HTS1, TOXD, TOXE, TOXC, and TOXA, all of which are involved in HC-toxin biosynthesis in C. carbonum. Although the genes are strongly similar in sequence, the organization of the genes in different in C. carbonum and A. jesenskae. We also continued analysis of putative virulence factors in the wheat pathogen Fusarium graminearum. We found a gene that when mutated results in a less virulent fungus. We also cloned and are expressing a novel alpha-fucosidase gene from F. graminearum. We continued our studies on the biosynthesis of the fatal toxins of Amanita mushrooms. We showed co-localization of the toxins and the biosynthetic enzymes in mushrooms. PARTICIPANTS: Jonathan Walton, PI. Oversaw all aspects of research. Wanessa Wight, graduate student. Performed the experiments that led to discovery and analysis of the HC-toxin gene cluster in Alternaria jesenskae. Hong Luo, postdoctoral research associate. Performed all of the toxin and enzyme localization studies in Amanita basidiocarps using confocal immunomicroscopy. Heather Hallen, postdoctoral research associate. Analyzed genes involved in Amanita toxin biosynthesis. John Scott-Craig, senior postdoctoral research associate. Participated in all aspects of the above projects. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The studies on HC-toxin biosynthesis by A. jesenskae are relevant to an understanding of how virulence traits evolve in pathogenic fungi. The studies on F. graminearum address the critical virulence traits of this major pathogen of wheat and barley, with the ultimate goal of developing new control strategies.
Publications
- Luo, H., H.E. Hallen-Adams, J.S. Scott-Craig, and J.D. Walton (2010) Co-localization of amanitin and a candidate toxin-processing prolyl oligopeptidase in Amanita basidiocarps. Eukaryotic Cell 9:1891-1900.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Four publications in peer-reviewed journals. Six talks at national/international meetings, universities, and research institutes. One talk at an undergraduate institution (Swarthmore College). PARTICIPANTS: Jonathan Walton, PI. Oversaw all aspects of research. Wanessa Wight, graduate student. First author on depudecin gene cluster paper. Performed all experiments none performed by collaborators at Virginia Tech (Chris Lawrence and K. Kim). Hong Luo, postdoctoral associate. Purified and cloned gene for prolyl oligopeptidase that processes the Amanita toxins. Suba Nagendran, postdoctoral associate. Analyzed the secretome of Trichoderma and the genome of Laccaria bicolor. Heather Hallen, postdoctoral associate. Identified the genes for Amanita toxin genes. John Scott-Craig, postdoctoral associate. Assisted with cloning of Amanita toxin genes. Harry Van Erp, graduate student. Analyzed the regulation of the CSL gene family in maize. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Discovered and analyzed the gene cluster in the plant pathogenic fungus Alternaria brassicicola that makes the histone deacetylase inhibitor depudecin. Showed that the lethal peptide toxins of Amanita mushrooms are processed by a serine protease in the prolyl oligopeptidase family. Analyzed the secreted proteins of the industrial fungus Trichoderma reesei. Used this information to show that the ectomycorrhizal (symbiotic) mushroom Laccaria bicolor is deficient in enzymes that can degrade plant cell walls and trigger plant defense responses. Analyzed the cellulose-synthase like (CSL) genes of maize and rice and showed that they are regulated by light and auxin in maize.
Publications
- Nagendran, S., H. Hallen-Adams, J. M. Paper, N. Aslam, and J. D. Walton (2009) Reduced genomic potential for secreted plant cell-wall-degrading enzymes in the ectomycorrhizal fungus Amanita bisporigera, based on the secretome of Trichoderma reesei. Fung. Genet. Biol. 46:427-435.
- Luo, H., H.E. Hallen-Adams, and J.D. Walton (2009) Processing of the phalloidin proprotein by prolyl oligopeptidase from the mushroom Conocybe albipes. J. Biol. Chem. 284:18070-18077.
- Wight, W., K.-H. Kim, C.B. Lawrence, and J.D. Walton (2009) Biosynthesis and role in virulence of the histone deacetylase inhibitor depudecin from Alternaria brassicicola. Mol. Plant-Microbe Interact. 22:1258-1267.
- Van Erp, H., and J.D. Walton (2009) Regulation of the cellulose synthase-like gene family by light in the maize mesocotyl. Planta 229:885-897.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Identified gene cluster for biosynthesis of the fungal toxin depudecin, from the plant pathogenic fungus Alternaria brassicicola. Analyzed gene structure of gene cluster by genomic and cDNA sequencing. Identified multiple genes in the cluster required for depudecin production by targeted disruption. Identified secreted Fusarium proteins that induce cell death/hypersensitivity in model test plants. PARTICIPANTS: Jonathan Walton, PI Janet Paper, graduate student Wanessa Wight, graduate student TARGET AUDIENCES: Scientists working on natural products biosynthesis. Scientists interested in new cancer control strategies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Generated novel information on the biosynthesis of an unusual epoxide-containing histone deacetylase inhibitor (depudecin).
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Research results were disseminated to the public through peer-reviewed publications, talks to High School teachers, and through presentations at national and international scientific conferences.
PARTICIPANTS: Jonathan Walton Janet Paper Wanessa Wight John Scott-Craig Collaborators: H. Corby Kistler, USDA, Univ. Minnesota
TARGET AUDIENCES: Other scientists working on basic mechanisms of fungal virulence.
Impacts
In 2007 we continued our work on the proteomics of Fusarium graminearum, a major pathogen of wheat and barley in the US. We established that >120 fungal proteins are secreted during the infection of wheat heads. These are now being tested by targeted gene disruption for a role in pathogenicity. Furthermore, we are testing the secreted proteins for recognition by plants in triggering plant defense responses in both wheat and in non-host plants. These studies should elucidate the biochemical mechanisms by which Fusarium invades crop plants, and by which wheat resists invasion by Fusarium and other pathogens. We also worked on the biosynthetic genes for depudecin in the cabbage pathogen Alternaria brassicicola. Depudecin is an inhibitor of histone deacetylase. We had earlier shown that HC-toxin, an HDAC inhibitor made by Cochliobolus carbonum, a pathogen of maize, is an essential virulence determinant. Our goal is to test the role of depudecin in virulence by A.
brassicicola and to elucidate the biosynthetic pathway of depudecin.
Publications
- Cuomo, C.A., U. Guldener J.R. Xu, F. Trail, B.G. Turgeon, A. Di Pietro, J.D. Walton, L.J. Ma, S.E. Baker, M. Rep, G. Adam, J. Antoniw, Baldwin T, Calvo S, Chang YL, Decaprio D, Gale LR, Gnerre S, Goswami RS, Hammond-Kosack K, Harris LJ, Hilburn K, Kennell JC, S. Kroken, J.K. Magnuson, G. Mannhaupt, E. Mauceli, H.W. Mewes, R. Mitterbauer, G. Muehlbauer, M. Munsterkotter, D. Nelson, K. O'Donnell, T. Ouellet, W. Qi, H. Quesneville, M.I. Roncero, K.Y. Seong, I.V. Tetko, M. Urban, C. Waalwijk, T.J. Ward, J. Yao, B.W. Birren, H.C. Kistler (2007) The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science 317:1400-1402.
- Paper, J.M., J.S. Scott-Craig, N.D. Adhikari, C.A.Cuomo, J.D. Walton (2007) Comparative proteomics of extracellular proteins in vitro and in planta from the pathogenic fungus Fusarium graminearum. Proteomics 7:3171-3183
- Hallen, H.E., H. Luo, J.S. Scott-Craig, J.D. Walton. (2007) A gene family encoding the major toxins of lethal Amanita mushrooms. Proc. Natl. Acad. Sci. 104:19097-19101.
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Progress 01/01/06 to 12/31/06
Outputs
Research in 2006 focused on our continued analysis of genes and enzymes that enable plant pathogenic fungi to parasitize crop plants (maize and wheat). We characterized two genes that regulate the transcription of xylanases and other cell wall degrading enzymes such as cellulase. Disruption of both copies of XLNR in the maize pathogen Cochliobolus carbonum caused the fungus to be unable to grow on xylose, but growth on other substrates was normal. The double mutant was still fully pathogenic, indicating that the genes regulated by XLNR are not critical to virulence. We also studied the extracellular proteins of Fusarium graminearum, cause of head blight of wheat and ear rot of maize. High-throughput proteomics identified 120 proteins that F. graminearum secretes during pathogenesis. Some of these proteins encode degradative enzymes (e.g., xylanase), whereas others have unknown function or function as small elicitors of plant defense responses. Yet others are
housekeeping proteins that might be recognized as allergens by plants.
Impacts
Plant diseases cost farmers billions of dollars every year. In addition, the possibility of massive epidemics of crops could cause severe enonomic disruption of the food supply. This research will contribute to a better understanding of how plant pathogens attack plants, and thus to the development of new control strategies.
Publications
- Walton, J.D. (2006) Molecules of interest: HC-toxin. Phytochemistry 67:1406-1413.
- Walton, J.D., K. Ohtani, D. Baidyaroy, N.J. Tonukari, K.F. Pedley, and J.S. Scott-Craig (2006) General and specific virulence factors in fungal pathogens. In: In: F. Sanchez, S. Quinto, I.M. Lopez-Lara, and O. Geiger eds., Biology of Plant-Microbe Interactions, Vol. 5. Intl. Soc. Mol. Plant-Microbe Interact., St. Paul, MN, pp. 586-596.
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Progress 01/01/05 to 12/31/05
Outputs
Research in 2005 focused on our continued analysis of genes and enzymes that enable plant pathogenic fungi to parasitize crop plants (maize and wheat). We characterized two genes that regulate the transcription of xylanases and other cell wall degrading enzymes such as cellulase. Disruption of both copies of XLNR in the maize pathogen Cochliobolus carbonum caused the fungus to be unable to grow on xylose, but growth on other substrates was normal. The double mutant was still fully pathogenic, indicating that the genes regulated by XLNR are not critical to virulence. We also studied the extracellular proteins of Fusarium graminearum, cause of head blight of wheat and ear rot of maize. High-throughput proteomics identified 63 proteins that F. graminearum secretes during pathogenesis. Some of these proteins encode degradative enzymes (e.g., xylanase), whereas others have unknown function or function as small elicitors of plant defense responses.
Impacts
Plant diseases cost farmers billions of dollars every year. In addition, the possibility of massive epidemics of crops could cause severe enonomic disruption of the food supply. This research will contribute to a better understanding of how plant pathogens attack plants, and thus to the development of new control strategies.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
In 2004 we continued our search for the genes and enzymes that enable plant pathogenic fungi to attack plants. We characterized two genes that regulate the transcription of xylanases and other cell wall degrading enzymes such as cellulase. Disruption of both copies of XLNR in the maize pathogen Cochliobolus carbonum caused the fungus to be unable to grow on xylose, but growth on other substrates was normal. The double mutant was still fully pathogenic, indicating that the genes regulated by XLNR are not critical to virulence. C. carbonum also makes a cyclic tetrapeptide host-selective toxin called HC-toxin. HC-toxin is an inhibitor of histone deacetylase (HDAC). In 2004 we established that HDACs are inhibited during infection in a host-selective fashion. Using newly developed maize gene arrays, we have identified >80 genes that are induced by fungal attack and suppressed by the presence of HC-toxin, as predicted by our current working model of the role of HDACs in
infection. These genes will be evaluated for a role in resistance to C. carbonum and other fungi.
Impacts
Plant diseases cost farmers billions of dollars every year. In addition, the possibility of massive epidemics of crops could cause severe enonomic disruption of the food supply. This research will contribute to a better understanding of how plant pathogens attack plants, and thus to the development of new control strategies.
Publications
- Walton, J.D., D.G. Panaccione, and H. Hallen (2004) Peptide synthesis without ribosomes. In: Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine. J. Tkacz and L. Lange, eds., Kluwer Academic, New York, pp. 127-162.
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Progress 01/01/03 to 12/31/03
Outputs
We are working to elucide the mechanisms by which filamentous fungi attack crop plants. Some pathogenic fungi produce low-molecular compounds called host-selective toxins. The work in our laboratory has helped to establish the importance of these compounds in pathogenicity. The fungus Cochliobolus carbonum produces HC-toxin, a toxin that it needs to parasitize maize. We have been studying the biosynthesis of this compound with the ultimate goal of understanding how new pathogenic fungi evolve in nature. In the past year we have been characterizing two genes involved in the HC-toxin biosynthetic pathway, called TOXE and TOXG. The role of these genes has been studied genetically and biochemically. We have established that TOXG encodes an alanine racemase that converts L-alanine to D-alanine, which is a component of HC-toxin. Our work has shown that TOXE is a pathway-specific transcription factor that controls the expression of all of the other genes necessary for
HC-toxin biosynthesis. HC-toxin is a potent inhibitor of histone deacetylase (HDAC), a key enzyme that links chromatin structure and gene expression. We are trying to understand how C. carbonum protects itself against its own toxin. The answer to this question could have importance for understanding how C. carbonum attacks maize, for developing maize lines with novel resistance to C. carbonum, and would also contribute to our understanding of the general biology of HDAC enzymes. We have discovered that the HDACs of C. carbonum are uniquely resistant to HC-toxin, whereas all other known HDACs (e.g., from mammals, yeast, other fungi, and Drosophila) are sensitive to HC-toxin. In the past year we have isolated three HDAC genes from C. carbonum, characterized its HDACs biochemically, and established that C. carbonum makes a factor that can protect sensitive HDACs against the inhibitory effects of HC-toxin. We postulate that when C. carbonum makes HC-toxin it simultaneously makes a factor
that protects its own enzymes from inhibition. This work is being done in collaboration with Peter Loidl and collaborators at the University of Innsbruck, Austria. All pathogenic fungi secrete enzymes that can degrade the cell walls of their host plants. Despite a widespread assumption that these enzymes are important in pathogenesis (for penetration of the host, ramification, and/or acquisition of nutrients), direct evidence for their importance has been hard to obtain. We have been systematically studying the cell wall degrading enzymes made by C. carbonum, which includes cellulases, xylanases, pectinases, and proteases. We have isolated a gene, ccSNF1, that when mutated causes decreased expression of most or all of the known cell wall degrading enzymes. Significantly, the SNF1 mutant has sharply decreased pathogenicity due to a decreased ability to penetrate the maize leaf epidermis. Previous work had indicated that penetration by C. carbonum is enzymatic and not by mechanical
force. These results indicate that cell wall degrading enzymes are important in pathogenicity of maize by C. carbonum.
Impacts
Plant diseases cost farmers billions of dollars every year. In addition, the possibility of massive epidemics of crops could cause severe enonomic disruption of the food supply. This research will contribute to a better understanding of how plant pathogens attack plants, and thus to the development of new control strategies.
Publications
- Hazen, S.P., R.M. Hawley, G.L. Davis, B. Henrissat, and J.D. Walton (2003) Quantitative trait loci and comparative genomics of cereal cell wall composition. Plant Physiol. 132:263-271. Walton, J.D., D.G. Panaccione, and H. Hallen (2003) Peptide synthesis without ribosomes. In: Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine. J. Tkacz, ed., Kluwer Academic, in press. Tonukari, N.J., J.S. Scott-Craig, and J.D. Walton (2003) Isolation of the carbon catabolite repressor (CREA) gene from the plant-pathogenic fungus Cochliobolus carbonum. DNA Sequence 14:103-107.
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Progress 01/01/02 to 12/31/02
Outputs
We are working to elucidate the mechanisms by which filamentous fungi attack crop plants. Some pathogenic fungi produce low-molecular compounds called host-selective toxins. The work in our laboratory has helped to establish the importance of these compounds in pathogenicity. The fungus Cochliobolus carbonum produces HC-toxin, a toxin that it needs to parasitize maize. We have been studying the biosynthesis of this compound with the ultimate goal of understanding how new races of pathogenic fungi evolve. In the past year we have characterized a gene involved in the HC-toxin biosynthetic pathway called TOXE. We have shown that TOXE is a pathway-specific transcription factor that controls the expression of all of the other genes necessary for HC-toxin biosynthesis. HC-toxin production is controlled by a Mendelian locus called TOX2. TOX2 is composed of multiple genes involved in biosynthesis, regulation, and secretion of HC-toxin. Unlike other known gene clusters, most
of the genes are present in multiple functional copies. Only simultaneous mutation of all copies of any particular TOX2 gene gives a toxin-minus phenotype. We extended our mapping of TOX2 to show that it contains, at a minimum, fifteen genes extending over more than 600 kb of contiguous DNA. HC-toxin is a potent inhibitor of histone deacetylase (HDAC), a key enzyme that links chromatin structure and gene expression. We are trying to understand how C. carbonum protects itself against its own toxin. The answer to this question could have importance for understanding how C. carbonum attacks maize, for developing maize lines with novel resistance to C. carbonum, and would also contribute to our understanding of the general biology of HDAC enzymes. We have discovered that the HDAC activity of C. carbonum is uniquely resistant to HC-toxin, whereas all other known HDACs (e.g., from mammals, yeast, other fungi, and Drosophila) are sensitive. In the past year we have isolated four HDAC genes
from C. carbonum, biochemically characterized the HDACs, and established that C. carbonum makes a factor that can cross-protect sensitive HDACs against the inhibitory effects of HC-toxin. We postulate that when C. carbonum makes HC-toxin it simultaneously makes a factor that protects its own enzymes from inhibition. This work is being done in collaboration with Peter Loidl and collaborators at the University of Innsbruck, Austria. We also discovered that one of the C. carbonum HDAC genes, HDC1, is essential for virulence. Cytological studies indicate that the defect in virulence is at the stage of penetration of the maize leaf. The HDC1 mutant fungus cannot fully express its panoply of cell wall degrading enzymes (cellulase, xylanase, pectinase, etc.) that it needs to digest plant cell walls. Apparently, these enzymes have a critical role in the penetration process.
Impacts
Filamentous fungi are the most important pathogens of crop plants. We are working to understand the molecular mechanisms by which fungi attack plants. Specific areas of interest are host-selective toxins, which are small pathogen-produced molecules that interfere with the host plant's metabolism; and extracellular enzymes that degrade the plant cell wall. Our results indicate that both toxins and enzymes are important virulence factors for at least some plant pathogenic fungi.
Publications
- Ahn, J.-H., Y.-Q. Cheng, and J.D. Walton (2002) An extended physical map of the TOX2 locus of Cochliobolus carbonum required for biosynthesis of HC-toxin. Fung. Genet. Biol. 35:31-38.
- Baidyaroy, D., G. Brosch, S. Graessle, P. Trojer, and J.D. Walton (2002) Characterization of inhibitor-resistant histone deacetylase activity in plant pathogenic fungi. Eukaryotic Cell 1:538-547.
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Progress 01/01/01 to 12/31/01
Outputs
We are working to elucidate the mechanisms by which filamentous fungi attack crop plants. Some pathogenic fungi produce low-molecular compounds called host-selective toxins. The work in our laboratory has helped to establish the importance of these compounds in pathogenicity. The fungus Cochliobolus carbonum produces HC-toxin, a toxin that it needs to parasitize maize. We have been studying the biosynthesis of this compound with the ultimate goal of understanding how new races of pathogenic fungi evolve. In the past year we have characterized a gene involved in the HC-toxin biosynthetic pathway called TOXE. We have shown that TOXE is a pathway-specific transcription factor that controls the expression of all of the other genes necessary for HC-toxin biosynthesis. HC-toxin production is controlled by a Mendelian locus called TOX2. TOX2 is composed of multiple genes involved in biosynthesis, regulation, and secretion of HC-toxin. Unlike other known gene clusters, most
of the genes are present in multiple functional copies. Only simultaneous mutation of all copies of any particular TOX2 gene gives a toxin-minus phenotype. We extended our mapping of TOX2 to show that it contains, at a minimum, fifteen genes extending over more than 600 kb of contiguous DNA. HC-toxin is a potent inhibitor of histone deacetylase (HDAC), a key enzyme that links chromatin structure and gene expression. We are trying to understand how C. carbonum protects itself against its own toxin. The answer to this question could have importance for understanding how C. carbonum attacks maize, for developing maize lines with novel resistance to C. carbonum, and would also contribute to our understanding of the general biology of HDAC enzymes. We have discovered that the HDAC activity of C. carbonum is uniquely resistant to HC-toxin, whereas all other known HDACs (e.g., from mammals, yeast, other fungi, and Drosophila) are sensitive. In the past year we have isolated four HDAC genes
from C. carbonum, biochemically characterized the HDACs, and established that C. carbonum makes a factor that can cross-protect sensitive HDACs against the inhibitory effects of HC-toxin. We postulate that when C. carbonum makes HC-toxin it simultaneously makes a factor that protects its own enzymes from inhibition. This work is being done in collaboration with Peter Loidl and collaborators at the University of Innsbruck, Austria. We also discovered that one of the C. carbonum HDAC genes, HDC1, is essential for virulence. Cytological studies indicate that the defect in virulence is at the stage of penetration of the maize leaf. The HDC1 mutant fungus cannot fully express its panoply of cell wall degrading enzymes (cellulase, xylanase, pectinase, etc.) that it needs to digest plant cell walls. Apparently, these enzymes have a critical role in the penetration process.
Impacts
Filamentous fungi are the most important pathogens of crop plants. We are working to understand the molecular mechanisms by which fungi attack plants. Specific areas of interest are host-selective toxins, which are small pathogen-produced molecules that interfere with the host plant's metabolism; and extracellular enzymes that degrade the plant cell wall. Our results indicate that both toxins and enzymes are important virulence factors for at least some plant pathogenic fungi.
Publications
- Baidyaroy, D., Brosch, G., Ahn, J.H., Graessle, S., Wegener, S., Tonukari, N.J., Caballero, O., Loidl, P., Walton, J.D. 2001. A gene related to yeast HOS2 histone deacetylase affects extracellular depolymerase expression and virulence in a plant pathogenic fungus. Plant Cell 13:1609-1624.
- Kim, H., Ahn, J.H., Gorlach, J.M., Caprari, C., Scott-Craig, J.S., Walton, J.D. 2001. Mutational analysis of two beta-glucanase genes, EXG2 and MLG2, from the plant pathogenic fungus Cochliobolus carbonum. Mol. Plant-Microbe Interact. 14:1436-1443.
- Ahn, J.-H., Sposato, P., Kim, S.I., Walton, J.D. 2001. Molecular cloning and characterization of cel2 from the fungus Cochliobolus carbonum. Biosci. Biotech. Biochem. 65:1406-1411.
- Ahn, J.H., Cheng, Y.Q., Walton, J.D. 2001. An extended physical map of the TOX2 locus of Cochliobolus carbonum required for biosynthesis of HC-toxin. Fung. Genet. Biol., in press.
- Brosch, G., Dangl, M., Graessle, S., Loidl, A., Trojer, A., Brandtner, E.M., Mair, K., Walton, J.D., Baidyaroy, D., Loidl, P. 2001. An inhibitor-resistant histone deacetylase in the plant pathogenic fungus Cochliobolus carbonum. Biochemistry 40:12855-12863.
- Pedley, K.F., Walton, J.D. 2001. Regulation of cyclic peptide biosynthesis in a plant pathogenic fungus by a novel transcription factor. Proc. Natl. Acad. Sci. U.S.A. 98:14174-14179.
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Progress 01/01/00 to 12/31/00
Outputs
We are working to elucide the mechanisms by which filamentous fungi attack crop plants. Some pathogenic fungi produce low-molecular compounds called host-selective toxins. The work in our laboratory has helped to establish the importance of these compounds in pathogenicity. The fungus Cochliobolus carbonum produces HC-toxin, a toxin that it needs to parasitize maize. We have been studying the biosynthesis of this compound with the ultimate goal of understanding how new pathogenic fungi evolve in nature. In the past year we have been characterizing two genes involved in the HC-toxin biosynthetic pathway, called TOXE and TOXG. The role of these genes has been studied genetically and biochemically. We have established that TOXG encodes an alanine racemase that converts L-alanine to D-alanine, which is a component of HC-toxin. Our work has shown that TOXE is a pathway-specific transcription factor that controls the expression of all of the other genes necessary for
HC-toxin biosynthesis. HC-toxin is a potent inhibitor of histone deacetylase (HDAC), a key enzyme that links chromatin structure and gene expression. We are trying to understand how C. carbonum protects itself against its own toxin. The answer to this question could have importance for understanding how C. carbonum attacks maize, for developing maize lines with novel resistance to C. carbonum, and would also contribute to our understanding of the general biology of HDAC enzymes. We have discovered that the HDACs of C. carbonum are uniquely resistant to HC-toxin, whereas all other known HDACs (e.g., from mammals, yeast, other fungi, and Drosophila) are sensitive to HC-toxin. In the past year we have isolated three HDAC genes from C. carbonum, characterized its HDACs biochemically, and established that C. carbonum makes a factor that can protect sensitive HDACs against the inhibitory effects of HC-toxin. We postulate that when C. carbonum makes HC-toxin it simultaneously makes a factor
that protects its own enzymes from inhibition. This work is being done in collaboration with Peter Loidl and collaborators at the University of Innsbruck, Austria. All pathogenic fungi secrete enzymes that can degrade the cell walls of their host plants. Despite a widespread assumption that these enzymes are important in pathogenesis (for penetration of the host, ramification, and/or acquisition of nutrients), direct evidence for their importance has been hard to obtain. We have been systematically studying the cell wall degrading enzymes made by C. carbonum, which includes cellulases, xylanases, pectinases, and proteases. We have isolated a gene, ccSNF1, that when mutated causes decreased expression of most or all of the known cell wall degrading enzymes. Significantly, the SNF1 mutant has sharply decreased pathogenicity due to a decreased ability to penetrate the maize leaf epidermis. Previous work had indicated that penetration by C. carbonum is enzymatic and not by mechanical
force. These results indicate that cell wall degrading enzymes are important in pathogenicity of maize by C. carbonum.
Impacts
Filamentous fungi are the most important pathogens of crop plants. We are working to understand the molecular mechanisms by which fungi attack plants. Specific areas of interest are host-selective toxins, which are small pathogen-produced molecules that interfere with the host plant's metabolism; and extracellular enzymes that degrade the plant cell wall. Our results indicate that both toxins and enzymes are important virulence factors for at least some plant pathogenic fungi.
Publications
- Pitkin, J.W., A. Nikolskaya, J.-H. Ahn, and J.D. Walton. 2000. Reduced virulence caused by meiotic instability of the TOX2 chromosome of the maize pathogen Cochliobolus carbonum. Mol. Plant-Microbe Interact. 13:80-87.
- Cheng, Y.-Q., and J.D. Walton. 2000. A eukaryotic alanine racemase involved in cyclic peptide biosynthesis. J. Biol. Chem. 275:4906-5004.
- Tonukari, N.J., J.S. Scott-Craig, and J.D. Walton. 2000. The Cochliobolus carbonum SNF1 gene is required for cell wall-degrading enzyme expression and virulence on maize. Plant Cell 12:237-248.
- Lechner, T., A. Lusser, A. Pipal, G. Brosch, A. Loidl, M. Goralik-Schramel, R. Sendra, S. Wegener, J. D. Walton, and P. Loidl. 2000. RPD3-type histone deacetylases in maize embryos. Biochemistry 39:1683-1692.
- Graessle, S., M. Dangl, H. Haas, K. Mair, P. Trojer, E.-M. Brandtner, J.D. Walton, P. Loidl, and G. Brosch. 2000. Characterization of two putative histone deacetylase genes from Aspergillus nidulans. Biochim. Biophys. Acta 93405:1-7.
- Walton, J.D., J.S. Scott-Craig, Y.-Q. Cheng, N.J. Tonukari, and K. Pedley. 2000. Fungal virulence factors: some things are and some things aren't. In: P.J.G.M. deWit, T. Bisseling, and W.J. Stiekema, eds., Biology of Plant-Microbe Interactions, Vol. 2. Intl. Soc. Mol. Plant-Microbe Interact., St. Paul, MN, pp. 175-179.
- Walton, J.D. 2000. Horizontal gene transfer and the origin of secondary metabolite gene clusters in fungi: an hypothesis. Fung. Genet. Biol. 30:167-171.
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Progress 01/01/99 to 12/31/99
Outputs
The research under this grant studies various aspects of virulence and resistance in the interaction between plant pathogenic fungi and their hosts. Plant pathogenic fungi make many secreted enzymes that can degrade plant cell walls. We are systematically studying the role of these enzymes in pathogenesis by targeted gene disruption. We isolated a homolog of the yeast gene SNF1 from the pathogenic fungus Chochlibolus carbonum. When disrupted, the mutant can no longer grow on complex media such as cellulose or corn cell walls, but can still grow on glucose. The expression of many secreted enzymes is severely down-regulated. The mutant is greatly impaired in virulence. These results present positive evidence that secreted enzymes are important in virulence, probably for the process of penetration of leaf surfaces. This knowledge could be used to develop new control strategies for plant pathogenic fungi. We have also continued work on the biosynthesis of HC-toxin, a
cyclic peptide toxin used by C. carbonum to infect corn. We discovered two new genes, TOXF and TOXG, that are involved in HC-toxin biosynthesis. TOXF encodes a branched chain aminotransferase and probably aminates a precursor of the epoxide amino acid of HC-toxin. We demonstrated that TOXG encodes an alanine racemase, an enzyme previously thought to be restricted to bacteria. The alanine racemase makes D-alanine from L-alanine. The genes involved in HC-toxin biosynthesis have a very complex genomic arrangement. Furthermore, the genes are very unstable. We documented this instability during meiosis: about 5% of all progeny had major deletions in the chromosome containing the genes involved in toxin production. Apparently, this trait is undergoing rapid evolutionary change.
Impacts
Plant pathogenic fungi cause severe crop losses every year in the United States. A better understanding of how fungi attack plants, and how plants respond to fungi, will allow the eventual development of novel strategies to control diseases, thus saving growers money and enhancing the quality of our food. This project addresses the mechanisms by which fungi attack plants. In particular, we are studying the role in disease of fungal enzymes that degrade the plant cell wall and of fungal toxins that compromise the host's metabolism.
Publications
- Cheng, Y.-Q., Le, L.D., Walton, J.D., and Bishop, K.D. 1999. 13C labelling indicates that the epoxide-containing amino acid of HC-toxin is biosynthesized by head-to-tail condensation of acetate. J. Nat. Prod. 62:143-145.
- Wegener, S., Ransom, R.F., and Walton, J.D. 1999. A unique eukaryotic beta-xylosidase gene from the phytopathogenic fungus Cochliobolus carbonum. Microbiology 145:1089-1095.
- Cehng, Y.-Q., Ahn, J.-H., and Walton, J.D. 1999. A putative branched-chain-amino-acid transaminase gene required for HC-toxin biosynthesis and pathogenicity in Cochliobolus carbonum. Microbiology 145:3539-3546.
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Progress 01/01/98 to 12/31/98
Outputs
We have continued to make progress on the elucidation of the mechanisms by which filamentous fungi attack crop plants. In 1998 we discovered two new genes involved in biosynthesis of the host-selective toxin HC-toxin, which the pathogen Cochliobolus carbonum uses to parasitize maize. The two genes encode enzymes, branched-chain amino acid transferase (TOXF) and alanine racemase (TOXG). Gene disruption experiments indicate that both TOXF and TOXG are required for biosynthesis of HC-toxin and for pathogenicity. We also reported on the discovery of a gene that regulates HC-toxin production called TOXE. A long-standing question has been how C. carbonum protects itself against its own toxin. Some mechanism of self-protection must exist because, like all eukaryotic organisms, C. carbonum has histone deacetylase, which we earlier showed is the site of action of HC-toxin. In 1998 we discovered that the HDA of C. carbonum is resistant to HC-toxin; all other known HDAs are
sensitive (mammals, yeast, other fungi, Drosophila, etc.). We are currently exploring the molecular basis of this resistance. In collaboration with Peter Loidl of the University of Innsbruck we have cloned two HDA genes from C. carbonum and are testing whether they encode the resistant HDA activity. All pathogenic fungi make enzymes that can degrade the cell walls of their host plants. Despite a widespread assumption that these enzymes are important in pathogenesis (for penetration of the host, ramification, and/or acquisition of nutrients), evidence for or against this hypothesis has been scarce. We have been systematically studying the enzymes made by C. carbonum, including cellulases, xylanases, pectinases, and proteases. However, none of our mutant strains have altered pathogenicity, indicating that these enzymes are, in fact, not required for disease. We are now taking a more global approach to the question of the role of these enzymes in disease by identifying genes that
regulate the production of these enzymes. In 1998 we cloned a gene, SNF1, that in yeast regulates production of invertase and other secreted hydrolases. We have disrupted this gene in C. carbonum and are testing the ability of the snf1 mutant to grow on complex polysaccharides such as corn cell walls, and to pathogenize maize.
Impacts
(N/A)
Publications
- Itoh, Y., Kiyohara, R., Kawamoto, Y., Kodama, M., Otani, H., Walton, J.D., Kohmoto, K. 1998. A catalytic domain of a cyclic peptide synthetase that is specific for the apple pathotype of Alternaria alternata and its possible involvement in host-specific AM-toxin production. In: Kohmoto, K., Yoder, O.C., eds., Molecular Genetics of Host-specific Toxins in Plant Disease, Kluwer Academic, Dordrecht, pp. 53-61.
- Scott-Craig, J.S., Apel-Birkhold, P.C., Gorlach, J.M., Nikolskaya, A., Pitkin, J.W., Ransom, R.F., Sposato, P., Ahn, J.-H., Tonukari, N.J., Wegener, S., Walton, J.D. 1998. Cell wall degrading enzymes in HST-producing fungal pathogens. In: Kohmoto, K., Yoder, O.C., eds., Molecular Genetics of Host-specific Toxins in Plant Disease, Kluwer Academic, Dordrecht, pp. 245-252.
- Cheng, Y.-Q., Le, L.D., Walton, J.D., Bishop, K.D. 1998. 13C labelling indicates that the epoxide-containing amino acid of HC-toxin is biosynthesized by head-to-tail condensation of acetate. J. Nat. Prod., in press.
- Ahn, J.-H., Walton, J.D. 1998. Regulation of cyclic peptide biosynthesis and pathogenicity in Cochliobolus carbonum by TOXE, a gene encoding a novel protein with a bZIP basic DNA binding motif and four ankyrin repeats. Mol. Gen. Genet. 260:462-469.
- Nikolskaya, A., Pitkin, J.W., Schaeffer, H.J., Walton, J.D. 1999. EXG1p, a novel beta-1,3-glucanase from the fungus Cochliobolus carbonum, contains a repeated motif present in other proteins that interact with polysaccharides. Biochim. Biophys. Acta, in press
- Gorlach, J.M., Van Der Knaap, E., Walton J.D. 1998. Cloning and targeted disruption of MLG1, a gene encoding two of three extracelluar mixed-linked glucanases of Cochliobolus carbonum. Applied and Environmental Microbiology 64:385-391.
- Scott-Craig, J.S., Poduje, L., Casida, J.E., Walton, J.D. 1998. Herbicide safener binding protein of maize: purification, and cloning and expression of an encoding cDNA. Plant Physiology 116:1083-1089.
- Scott-Craig, J.S., Cheng, Y.-Q., Cervone, F., DeLorenzo, G., Pitkin, J.W., Walton, J.D. 1998. Targeted mutants of Cochliobolus carbonum lacking the two major extracellular polygalacturonases. Applied and Environmental Microbioliology 64:1497-1503.
- Walton, J.D., Ahn, J.-H., Pitkin, J.W., Cheng, Y., Nikolskaya, A.N., Ransom, R., S. Wegener. 1998. Enzymology, molecular genetics, and regulation of biosynthesis of the host-selective toxin HC-toxin. In: Kohmoto, K., Yoder, O.C., eds., Molecular Genetics of Host-specific Toxins in Plant Disease, Kluwer Academic, Dordrecht, pp. 25-34.
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