Progress 09/15/05 to 09/14/09
Outputs
OUTPUTS: Results and discoveries were disseminated to both the scientific community and the general public. 1. A paper authored by a graduate student won the best student paper award from the British Society for Plant Pathology. http://www.bspp.org.uk/society/bestpaper.htm. 2. Popular press articles and news releases: a. "Defining gene's role may lead to prevention of dangerous corn toxin", Purdue University March 24, 2008. b. "Defining gene's role may lead to prevention of dangerous corn toxin". CSA News [May 2008 vol 53 issue 5]. c. "Fight disease with DNA". The Furrow [September-October 2008 issue]. 3. Two fungal strains generated from our work were shared with Dr. V. Chaturvedi at the University of Albany School of Public Health, Albany, NY to be used in studies investigating human corneal infections. 4. Presentations include: a. Woloshuk 2006. Understanding the response of the Fusarium ear rot pathogen to the maize kernel environment. 2008 Specialty Products Conference. St. Francisville, IL.Woloshuk, C. P. 2006. b. Woloshuk. 2006. Amylopectin in maize kernels induces fumonisin production by Fusarium verticillioides during colonization. European Fusarium Seminar. Wageningen, The Netherlands. c. Shim 2008. Regulation of fumonisin biosynthesis in Fusarium verticillioides. 2008 Texas AgriLife Conference. College Station, TX d. Shim 2009. Unraveling the mechanism of fungal pathogenesis in field crops by molecular genetics and genomics approaches. Biosciences eastern and central Africa (BecA)-ILRI Hub, International Livestock Research Institute, Nairobi, Kenya. e. Shim. 2009. Unraveling the mechanism of fungal pathogenesis in field crops by molecular genetics and genomics approaches. University of Nairobi, Kenya. f. Woloshuk 2009. Aspects of habitat important to Fusarium verticillioides during pathogenesis of maize kernels. American Phytopathological Society annual meeting. Portland, OR. 5. Posters presented: a. Kim, H. and Woloshuk, C.P. 2007. AREA, nitrogen regulatory gene, influences fumonisin B1 production of Fusarium verticillioides. APS North Central Division Meeting. b. Kim, H. and Woloshuk, C.P. 2007. AREA in Fusarium verticillioides affects growth and fumonisin biosynthesis in colonized maize kernels. 24th Fungal Genetics Conference. c. Kim, H. and Woloshuk, C.P. 2008. Two putative hexose kinase genes, HXK1 and HXK2, are involved in FB1 biosynthesis of Fusarium verticillioides. APS annual meeting. d. Kim, H. and Woloshuk, C.P. AREA, nitrogen regulatory gene, influences fumonisin B1 production of Fusarium verticillioides. APS annual meeting. e. Mukherjee, M., and W. B. Shim. 2008. Functional characterization of heterotrimeric G protein regulators in Fusarium verticillioides. APS annual meeting. f. Kim, J.-E. and W. B. Shim. 2009. FDL1, a putative cytochrome P450, is involved in macroconidiation, conidia germination, and fumonisin B1 production in Fusarium verticillioides. APS annual meeting. g. Shin, J.-H. and W. B. Shim. 2009. Characterization of PPR1 and PPR2, genes encoding regulatory subunits of protein phosphatase 2A, in Fusarium verticillioides. APS annual meeting. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Analysis of a Zfr1-disruption indicated that the gene might regulate sugar uptake. The ZFR1 deletion strain grew approximately 2.5 fold less than wild type on endosperm tissue and a variety of other carbon sources. The mutant displayed higher expression of genes involved in starch saccharification than wild type. By microarray analysis revealed that expression of 6 putative sugar transporters was greater on endosperm tissue than germ tissue, and expression of at least 3 genes was reduced by disruption of ZFR1. Results indicated that disruption of FST1 had no effect on growth but decreased fumonisin (FB1) production. We discovered that disruption of AREA, a regulator of nitrogen metabolism, the fungus grew poorly on maize kernels, but grew normally with the addition of ammonium phosphate. The mutant under all condition did not produce FB1. This study indicated that the blister kernel environment is repressive to genes regulated by AREA, including the FB1 biosynthetic genes. The repression is likely due to the free amino acids in these kernels. The results also indicated that because the kernels lack sufficient carbohydrates, the pathogen utilizes the amino acids as carbon and secrete ammonia into the kernel environment. Such metabolic activity causes the pH of kernel environment to increase resulting in additional repression of FB1. We investigated genes identified in the microarray study as well as genes known to affect secondary metabolism in other fungi. These genes were targeted for gene knockout, and subsequently analyzed for FB1 biosynthesis and other phenotypic abnormalities. The first two genes we targeted for mutation affected fungal conidiation and hyphal morphology but not FB1 production. Another candidate gene CPP1, which encodes a putative protein phosphatase 2A, played a key role in FB1 regulation. Gene disruption mutant showed elevated FB1 level when grown on corn kernels. In addition to CPP1, we characterized GAP1, encoding a glycolipid anchored surface protein, which was identified as a differentially expressed gene in F. verticillioides. GAP1 null mutants GAM126 and GAG8 exhibited restricted growth with more aerial hyphae compared to their wild-type progenitor. GAM126 and GAG8 strains also produced significantly fewer conidia and produced comparatively densely branched hyphae. Results indicated that the GAP1 deletion altered the cell wall carbohydrate composition/ deposition process. The deletion of GAP1 did not affect FB1 production. Mutation of GBP1, encoding a monomeric G protein, increased FB1 production. The gene may function as a negative regulator of FB1 production in F. verticillioides. GBB1 encodes a putative beta subunit of a heterotrimeric G protein. A GBB1 deletion mutant (BM83) showed no significant differences in radial growth and mycelial mass but produced significantly less FB1 than the wild type. Expression results suggested that GBB1 is directly involved in FB1 regulation. Stalk rot virulence, as measured by mean lesion length and by area, was not significantly different, suggesting that GBB1 does not regulate virulence in F. verticillioides.
Publications
- Kim, H., and Woloshuk, C. P. 2008. Role of AREA, a regulator of nitrogen metabolism, during colonization of maize kernels and fumonisin biosynthesis in Fusarium verticillioides. Fungal Genet. Biol. 45:957-953.
- Yoon-E Choi and Won-Bo Shim. 2008. Functional characterization of Fusarium verticillioides CPP1, a gene encoding a putative protein phosphatase 2A catalytic subunit. Microbiology 154: 326-336.
- Uma Shankar Sagaram, Brian D. Shaw, and Won-Bo Shim. 2007. Fusarium verticillioides GAP1, a gene encoding a putative glycolipid-anchored surface protein, participates in conidiation and cell wall structure but not virulence. Microbiology 153:2850-2861.
- Xiquan Gao, Won-Bo Shim, Cornelia Gobel, Susan Kunze, Ivo Feussner, Robert Meeley, and Michael Kolomiets. 2007. Disruption of a maize 9-lipoxygenase results in increased resistance to fungal pathogens and reduced levels of contamination with mycotoxin fumonisin. Molecular Plant-Microbe Interactions 20: 922-933.
- Uma Shankar Sagaram, Robert A. E. Butchko, and Won-Bo Shim. 2006. GBP1, a putative monomeric G-protein, is negatively associated with fumonisin B1 production in Fusarium verticillioides. Molecular Plant Pathology 7: 381-389.
- Uma Shankar Sagaram and Won-Bo Shim. 2007. Fusarium verticillioides GBB1, a gene encoding heterotrimeric G protein subunit, is associated with fumonisin B1 biosynthesis and hyphal development but not with fungal virulence. Molecular Plant Pathology 8: 375-384.
- Yoon-E Choi and Won-Bo Shim. 2008. Identification of genes associated with fumonisin biosynthesis in Fusarium verticillioides via proteomics and quantitative real-time PCR. Journal of Microbiology and Biotechnology 18: 648-657.
- Hye-Young Yu, Jeong-Ah Seo, Kap-Hoon Han, Won-Bo Shim, Sung-Hwan Yun, and Yin-Won Lee. 2008. Functional analyses of heterotrimeric G protein G-alpha and G-beta subunits in Gibberella zeae. Microbiology 154: 392-401.
- Bluhm, B. H., Kim, H., Butchko, R. A. E., and Woloshuk, C. P. 2008. Involvement of ZFR1 of Fusarium verticillioides in kernel colonization and the regulation of FST1, a putative sugar transporter gene required for fumonisin biosynthesis on maize kernels. Molecular Plant Pathology 9:203-211.
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Progress 09/15/07 to 09/14/08
Outputs
OUTPUTS: Activities: Over the past year, we continued studies on gene regulation of fumonisin biosynthesis. We characterized several new genes that impact toxin production and pathogen development. Events: Results from our work were present at several conferences including: Presentation: Regulation of fumonisin biosynthesis in Fusarium verticillioides. 2008 Texas AgriLife Conference. January 2008, College Station, TX Presentation: Altered expression of polyketide biosynthetic gene clusters in fumonisin-deficient mutants of Fusarium verticillioides. Gordon conference on Cellular & Molecular Fungal Biology, July 2008, Holderness, NH. Presentation: Understanding the response of the Fusarium ear rot pathogen to the maize kernel environment. 2008 Specialty Products Conference. June 26, 2008, St. Francisville, IL. Poster: AREA, nitrogen regulatory gene, influences fumonisin B1 production in Fusarium verticillioides. American Phytopathological Society Meeting, Minneapolis, MN July 2008. Poster: Two putative hexose kinase genes, HXK1 and HXK2, are involved in FB1 biosynthesis in Fusarium verticillioides. American Phytopathological Society Meeting, Minneapolis, MN July 2008. Poster: Functional characterization of heterotrimeric G protein regulators in Fusarium verticillioides. 2008 American Phytopathological Society Meeting, Minneapolis, MN July 2008 Products: Mr. Yoon-E Choi was granted Ph.D. from Texas A&M University (Major Advisor Won-Bo Shim, Department of Plant Pathology and Microbiology) Dissemination: Several popular press articles and news releases were produced including: Purdue University news article "Defining gene's role may lead to prevention of dangerous corn toxin" March 24, 2008; the cover picture and story, "Defining gene's role may lead to prevention of dangerous corn toxin" in the CSA News [May 2008 vol 53 issue 5]; and "Fight disease with DNA" in The Furrow [September-October 2008]. PARTICIPANTS: Several graduate students and undergraduates worked on this project. They received training in fungal biology, plant pathology, and molecular biology. TARGET AUDIENCES: Information generated from the research was developed into peer reviewed articles, presentations at professional meetings and presented to agricultural stakeholders, namely corn germplasm producers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results from our research has provided new knowledge about genes that are important to the corn pathogen Fusarium verticillioides. We have documented gene expression patterns during growth on corn kernels, and discovered genes involved in pathogen development as well as genes that appear to function in the perception of environmental cues that are responsible for initiating mycotoxin metabolism. In addition, we have investigated differential expression of proteins during fumonisin induced and suppressed conditions in F. verticillioides. We have also characterized key signaling genes, i.e., heterotrimeric G proteis and protein phophatases, to better understand their role in mycotoxin biosynthesis.
Publications
- Bluhm, B. H., Kim, H., Butchko, R. A. E., and Woloshuk, C. P. 2008. Involvement of ZFR1 of Fusarium verticillioides in kernel colonization and the regulation of FST1, a putative sugar transporter gene required for fumonisin biosynthesis on maize kernels. Molecular Plant Pathology 9:203-211.
- Yoon-E Choi and Won-Bo Shim. 2008. Identification of genes associated with fumonisin biosynthesis in Fusarium verticillioides via proteomics and quantitative real-time PCR. Journal of Microbiology and Biotechnology 18: 648-657.
- Hye-Young Yu, Jeong-Ah Seo, Kap-Hoon Han, Won-Bo Shim, Sung-Hwan Yun, and Yin-Won Lee. 2008. Functional analyses of heterotrimeric G protein G and Gβ subunits in Gibberella zeae. Microbiology 154: 392-401.
- Kim, H., and Woloshuk, C. P. 2008. Role of AREA, a regulator of nitrogen metabolism, during colonization of maize kernels and fumonisin biosynthesis in Fusarium verticillioides. Fungal Genet. Biol. 45:957-953.
- Yoon-E Choi and Won-Bo Shim. 2008. Functional characterization of Fusarium verticillioides CPP1, a gene encoding a putative protein phosphatase 2A catalytic subunit. Microbiology 154: 326-336.
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Progress 09/15/06 to 09/14/07
Outputs
OUTPUTS: Our overall goal is to discover metabolic targets in Fusarium verticillioides that yield management strategies to lower the risk of fumonisin contamination in food and feed. To achieve our goal we have investigated the genes that affect fumonisin biosynthesis. GBB1, a gene encoding a putative beta subunit of a heterotrimeric G protein, was disrupted and the effects on fumonisin biosynthesis and virulence were evaluated. A GBB1 deletion mutant (??83) showed no significant differences in radial growth and mycelial mass but produced significantly less FB1 than its wild-type progenitor. Reduced expression of the key FB1 biosynthetic genes, FUM1 and FUM8, in ??83 provides further evidence that GBB1 is involved in FB1 regulation. Stalk rot virulence, as measured by mean lesion length and by area, was not significantly different in ??83 compared to the wild type, suggesting that GBB1 does not regulate virulence in F. verticillioides. Complementation of ??83 with GBB1 restored FB1
production and hyphal growth to wild-type strain. We completed molecular characterization of GAP1, a gene encoding a protein that belongs to a glycolipid anchored surface (GAS) protein family. GAP1 null mutants GAM126 and GAG8 exhibited restricted growth with more aerial hyphae compared to their wild-type progenitor on solid media. When grown in suspended conditions, GAM126 and GAG8 strains produced significantly fewer conidia and produced comparatively densely branched hyphae. Concanavalin A staining indicated that the GAP1 deletion altered the cell wall carbohydrate composition/deposition process. Significantly, contrary to our hypothesis, deletion of GAP1 did not affect FB1 production level. The ZFR1 deletion strain grew approximately 2.5 fold less than wild type on endosperm tissue and a variety of other carbon sources including glucose and amylopectin. However, the zfr1 strain displayed higher ?-amylase activity and expression of genes involved in starch saccharification than
wild type, thus indicating that the reduced growth of the zfr1 strain was not due to inhibition of amylolytic enzymes. In the wild-type strain, expression of six genes encoding putative sugar transporters was significantly greater on endosperm tissue relative to germ tissue, and expression of at least 3 of the 6 genes was negatively affected by disruption of ZFR1. Intriguingly, disruption of FST1 had no effect on growth, kernel colonization, or kernel pH but decreased FB1 production by approximately 82% on maize kernels. An AREA-deletion mutant of Fusarium verticilliodes grew poorly on mature maize kernels, but grew similar to wild type (WT) with the addition of ammonium phosphate. Fumonisin B1 (FB1) was not produced by areA under any condition or by the WT with added ammonium phosphate. Constitutive expression of AREA (strain AREA-CE) rescued the growth and FB1 defects in areA. Growth of WT, areA, and AREA-CE on blister stage kernels was similar. After seven days of growth, none of
the strains produced FB1 and the pH of the kernel tissue was 8.0. Addition of amylopectin to the blister kernels resulted in a pH near 6.6 and FB1 production by WT and AREA-CE.
TARGET AUDIENCES: Plant pathologists
Impacts
Our results to date demonstrate that heterotrimeric G protein ? subunit plays an important role in regulation of FB1 biosynthesis and hyphal growth, but not virulence in F. verticillioides. However, contrary to our hypothesis, GAP1 is not directly associated with pathogenicity or regulation of FB1, but with growth, development and conidiation in F. verticillioides. Significantly, maize-fungal cross-talk research may provide new avenues for developing strategies to control mycotoxin contamination in field. The results support the hypothesis that FB1 biosynthesis is regulated by AREA. Also the failure to produce FB1 in blister kernels is due to high pH conditions generated because of an unfavorable carbon/nitrogen environment. Based on these findings, we hypothesize that ZFR1 controls FB1 biosynthesis by regulating genes involved in the perception or uptake of carbohydrates.
Publications
- Uma Shankar Sagaram, Brian D. Shaw, and Won-Bo Shim. 2007. Fusarium verticillioides GAP1, a gene encoding a putative glycolipid-anchored surface protein, participates in conidiation and cell wall structure but not virulence. Microbiology 153:2850-2861.
- Xiquan Gao, Won-Bo Shim, Cornelia Gobel, Susan Kunze, Ivo Feussner, Robert Meeley, and Michael Kolomiets. 2007. Disruption of a maize 9-lipoxygenase results in increased resistance to fungal pathogens and reduced levels of contamination with mycotoxin fumonisin. Molecular Plant-Microbe Interactions 20: 922-933.
- Uma Shankar Sagaram and Won-Bo Shim. 2007. Fusarium verticillioides GBB1, a gene encoding heterotrimeric G protein ? subunit, is associated with fumonisin B1 biosynthesis and hyphal development but not with fungal virulence. Molecular Plant Pathology 8: 375-384.
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Progress 09/15/05 to 09/14/06
Outputs
We found that F. verticillioides grew equally well on all stages of the developing kernel. Fumonisin B1 (FB1) was not produced in the most immature kernels. We have hypothesized that this lack of fumonisin production results from either nitrogen repression due to high levels of free amino acids or to a lack of an inducer such as starch. Microarray analysis of FUM gene expression indicated between 16 to 64-fold increase in gene expression in endosperm versus germ tissue. Disruption of the ?-amylase gene of F. verticillioides results in low levels of FB1 biosynthesis on endosperm fractions. Analyses of FB1 production in maize kernel mutants and in defined liquid media indicate that amylopectin, a component of starch, induces FB1 biosynthesis. To better understand the results we obtain with the immature corn kernels, we disrupted AreA in F. verticillioides. Homologues of AreA in other fungi are known to be global regulators of nitrogen metabolism, but also effect the
transcription of many other genes, including genes involved in secondary metabolism. The AreA-disruption mutant can grow on media containing ammonium salts or amino acids, but not on media containing nitrate or protein. The mutant also grows poorly on mature corn kernels. However the mutant does grow well on immature (blister) kernels. Disruption of Zfr1 in F. verticillioides results in a loss of FB1 production on colonized corn kernels. Our original hypothesis was that Zfr1 is a regulatory of fumonisin gene expression. Although we have not disproved this hypothesis, further analysis of the Zfr1-disruption mutant indicated that the strain might be impaired in sugar uptake. We found that the mutant grows poorly on kernel endosperm but growth on germ tissue is much greater than the wild-type fungus. Amylase production is not impaired in the mutant. By microarray analysis of RNA isolated from the wild-type strain grown on germ and endosperm tissues we found that expression of two of the
21 sugar transporter genes are up in the endosperm grown fungus. Analysis of RNA isolated from the Zfr1 mutant indicated that expression of the two transporter genes are greatly reduced. We have made progress to functionally characterize a subset of genes that are expressed during fumonisin biosynthesis. To date, we have generated four gene-disruption F. verticillioides mutants. Mutation of two of these genes affected fungal conidiation and hyphal morphology but not fumonisin production. These results are not unexpected because fungal development and FB1 production were closely linked phenotypes during the gene selection process. We found that disruption of a third gene (GBP1), a gene encoding a monomeric G protein, actually increased fumonisin production. The gene appears to function as a negative regulator of fumonisin production in F. verticillioides. We are currently investigating the fourth gene disrupted (CPP1), a putative protein phosphatase 2A, which appears to have a role in
fumonisin regulation.
Impacts
Our results provide a better understanding of how the metabolites in a developing corn kernel influences growth and fumonisin production in F. verticillioides when it colonizes each kernel stage. We have also determined discovered the fumonisin-inducing activity of amylopectin. This work suggests that starch chemical structure and metabolism may be a target for control strategies.
Publications
- Burton Bluhm and Charles P. Woloshuk 2005. Amylopectin induces fumonisin B1 production by Fusarium verticillioides during colonization of maize kernels. Molecular Plant-Microbial Interactions 12:1333-1339.
- Uma Shankar Sagaram, Robert A. E. Butchko, Won-Bo Shim. 2006. GBP1, a putative monomeric G-protein, is negatively associated with fumonisin B1 production in Fusarium verticillioides. Molecular Plant Pathology 7: 381-389.
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