Progress 02/15/10 to 02/14/14
Outputs
Target Audience: The information generated from this research was presented to the scientific community at professional meetings. Graduate and undergraduate student participated in the work, gaining laboratory and technical experience. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Chenxing Esther Niu worked on this project a part of her PhD research. Her training included fungal molecular biology and bioinformatics analysis of large data sets of fungal expression obtained from next generation sequencing technology. Brooklyn Biese, Jamie Moore, and Andy Delks were three undergraduate students that work on the project this year. The received training in mycotoxin analysis (HPLC), mycological, and molecular biology techniques. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Our research results have provided new knowledge about the biology of Fusarium verticillioides and its ability to produce the mycotoxin fumonisin. The pathogen produces the mycotoxin during infection of maize seeds, and the gene FST1 is required. We began this project with the hypothesis that Fst1 is an environmental sensor. During the course of the project we discovered that a functional FST1 also is required for pathogenicity, macroconidia production and resistance to reactive oxygen. This knowledge provides evidence that Fst1 influences the expression of many genes, especially in secondary metabolism, host defense response and fungal development. Through peer-reviewed publications of our results we have provided knowledge about the function of FST1 and about the influence of hexose kinases on fumonisin production and pathogenicity. Three PhD graduate students were trained, one has completed and two are forthcoming. Two technicians and a number of undergraduates were trained and contributed to the work. Finally, numerous presentations describing the results were presented both nationally and internationally. Objective 1. To determine the biochemical function of the gene FST1 and its role in the regulation of fumonisin biosynthesis during colonization of maize kernels, to characterize two hexose kinases and determine their involvement in the regulation of fumonisin biosynthesis. We have hypothesized that the central loop and C-terminus structures are involved in the functionality of Fst1. Constructs of FST1 containing deletions three amino acids) in the coding regions of the central loop and C-terminus were transformed into a strain lacking a functional FST1. The resulting transformant strains were tested to determine the impact on the complementation of three defects associated with the FST1-deletion mutant, fumonisin production, macrospore production and sensitivity to reactive oxygen (hydrogen peroxide). All the constructs failed to complement fumonisin and reactive oxygen sensitivity, suggesting that the structure of both regions of the protein is critical for function. One of the two deletions in the central loop was able to complement the macrospore defect, where as all the other deletions failed to complement. This result suggests that the various phenotypic effects of FST1 can be separated relative to protein structure. Objective 2. To profile the extracellular protein produced during pathogenesis of maize kernel This objective was previously altered away from the secreted protein analysis to gene expression analysis. We have hypothesized that Fst1 function impacts expression of secretome genes during colonization of kernels. We made a comparison of the transcriptome of the wild type (WT) at 4 and 6 days after inoculation (DPI) of maize kernels with the Δfst1 mutant at 6DPI. Growth of Δfst1 at 6DPI is the same as the WT at 4DPI. Pairwise comparisons of genes expressed by WT and Δfst1 indicated a total of 1826 significant differentially expressed genes among three sets of comparisons (p<0.05, >2 fold change). The lowest number of differentially expressed genes was found in the comparison of WT-4 to 6DPI. The highest number of genes was identified in the comparison of WT-4 to Δfst1-6DPI. The products of genes identified as significant were categorized by BLAST analysis and placed into one of nine functional categories or a group designated “unknown” The result indicates that different subsets of genes are upregulated during kernel infection. Expression of 33% of the 1412-secretome genes was not detected in Δfst1-6DPI. Of these, 36% were expressed in the WT at both 4 and 6 DPI, which indicates a major affect of FST1 on the secretome. Our current goal is to complete the analysis of the secretome data and obtain validation of the results by quantitative PCR.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Niu, C., Payne, G., Woloshuk, C. 2013. Effect of mutations to FST1 in Fusarium verticillioides on functionality and the regulation of gene expression.. Phytopathology 103(Suppl. 2):104.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Shu, X., Livingston, D., Franks, R. G. and Payne, G. A. 2013. Histology and transcriptional changes of maize seed infected by Aspergillus flavus and Fusarium verticillioides. Phytopathology 103(Suppl. 2):133.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Woloshuk: Pathogenesis and Fumonisin Biosynthesis in Fusarium verticilliodes. Gordon Research Conference. Stonehill College, Easton, MA. June 2013. (Oral presentation)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Shu, X., Livingston, D., Franks, R. G. and Payne, G. A. 2013. Interaction between Maize Seed and Its Fungal Pathogens Aspergillus flavus and Fusarium verticillioides. 2013 Gordon Research Seminar. Easton, MA. June 15-16, 2013. (Oral presentation)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Shu, X., Livingston, D., Franks, R. G. and Payne, G. A. 2013. Functional Genomic Tools to Analyze Pathogenesis of Aspergillus flavus and Fusarium verticillioides in Developing Maize Seeds. 2013 Gordon Research Seminar. Easton, MA. June 15-16, 2013. (Poster presentation)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Shu, X. and Payne, G. A. Host Response of Developing Maize Kernels During Infection by Aspergillus flavus and Fusarium verticillioides. Nov. 2013. Sigma XI Student Research Conference. Research Triangle Park, NC. (Poster presentation)
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Progress 02/15/12 to 02/14/13
Outputs
OUTPUTS: Activities: Our goal has been to provide a better understanding of the molecular mechanisms that regulate metabolism during the colonization phase of pathogenesis of Fusarium verticillioides, especially as they relate to kernel infection and fumonisin biosynthesis. We have three specific aims: 1) Determine the biochemical function of FST1 and its role in the regulation of fumonisin biosynthesis during colonization of maize kernels. 2) Characterize HXK1 and HXK2 and determine their involvement in the regulation of fumonisin biosynthesis. 3) Profile the extracellular protein produced during pathogenesis of maize kernel. The focus of the research over the 2012 period was on completing Aim 3. Our original approach was to use proteomics methodology. Due to a loss of a key collaborator from NCSU and unsatisfactory result from the proteomics facility at Purdue University, we modified our approach in 2012, opting to examine the expression of genes that encode putative secreted proteins. Ears from field-grown maize were inoculated with the wild type and mutant Δfst1, and kernels were collected at various time-points after inoculation. Total RNA was isolated from kernel collected at 6 day, a time point where visible difference can be seen in the amount of disease caused by the two strains. Also the amount of fungal tissue is sufficient for expression analysis. High throughput sequencing of the RNA (RNAseq) was done and we analyzed the expression of the 1412 genes predicted to encode extracellular proteins. Dissemination: Woloshuk presented a lecture entitled "Biosynthesis and regulation of mycotoxins: the increasing impact of genomics" at the International Symposium on Mycotoxin and Phycotoxins in Rotterdam, The Netherlands November 2012. Graduate student Chenxing Nui presented a poster entitled "Function of FST1 in Fusarium verticillioides during colonization of maize kernels" at the Botany and Plant Pathology Poster Session in Lafayette, IN November 2012. PARTICIPANTS: Chenxing Nui, a PhD graduate student at Purdue University, worked under the supervision of Charles Woloshuk. Greg O'Brian of North Carolina Sate University has worked under the supervision of Gary Payne. Brooklyn Biese, an undergraduate student in Animal Science at Purdue, worked as an assistant on the project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: The original approach was to use proteomics methodology in Objective 3. Due to a loss of a key collaborator from NCSU and unsatisfactory result from the proteomics facility at Purdue University, we modified our approach. We are examining the expression of genes that encode putative secreted proteins. This change in the approach has caused a delay in progress and resulted in our request for a no-cost extension of the project.
Impacts
The research conducted was a major learning effort for a PhD graduate student (Chenxing Nui). New knowledge was discovered about the response of F. verticillioides to the maize kernel environment. A comparison of the transcriptome of the wild type and the fst1 mutant six days after inoculation of maize kernels revealed that expression that over 2000 genes in the mutant were significantly (P<0.05) different than wild type. Of the genes that are predicted to encode secreted proteins, expression of over 250 were significantly altered (≥ 2 fold) in the mutant strain. Among these genes was the reduced expression of an extracellular peroxidase, likely involve in resistance to reactive oxygen.
Publications
- Woloshuk, C.P. and Shim, W-B. 2013. Aflatoxins, Fumonisins, and Trichothecenes: A Convergence of Knowledge. FEMS Microbiology Reviews. 37:94-109.
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Progress 02/15/11 to 02/14/12
Outputs
OUTPUTS: Poster: Functional characterization of Fst1 from Fusarium verticillioides.
Kim, H. and Woloshuk, C. P. Annual Meeting of the American Phytopathological Society, Charlotte, NC Poster: Fst1, a gene involved in pathogenicity and fumonisin production by Fusarium verticillioides, confers resistance to reactive oxygen species. Aparna Natarajan, A., Payne, G. A., and Woloshuk, C. P. 2011. Genetics of Maize Disease Workshop 2011, Raleigh, NC. Raleigh NC. Training: Hun Kim received his PhD degree from Purdue University. Aparna Natarajan is receiving research training in a post-MS scientist position at NCSU. Presentation: Understanding the pathogen response to the maize kernel environment. Woloshuk at seminar at Purdue 10/26/2011. Presentation: The function of Fst1 in Fusarium verticillioides is linked to both fumonisins biosynthesis and pathogenesis of maize kernels. Woloshuk at USDA/NIFA Microbial Biology and Microbial Functional Genomics Programs Awardee Meeting 7/26/2011. PARTICIPANTS: Charles Woloshuk Gary Payne Hun Kim Aparna Natarajan Burt Bluhm J. E. Smith J. B. Ridenour Pui Kar Loh TARGET AUDIENCES: Maize Pathologists. PROJECT MODIFICATIONS: Delays have occurred because students graduated and it took time to find replacements.
Impacts
We demonstrated that Fst1 belongs to a class of proteins that are structurally similar to sugar transporter but may have a regulatory function. The most important discovery was a new phenotype associated with fst1-disruption mutant. This mutant is sensitive to active oxygen. We have hypothesized that this sensitivity is responsible for the decreased virulence of the mutant. Our studies of the hexose kinase gene Hxk1 linked gene function to fructose uptake, pathogenicity, fumonisin biosynthesis, and trehalose biosynthesis.
Publications
- Kim, H., Smith, J. E., Ridenour, J. B., Woloshuk, C. P., and Bluhm, H. B. 2011. HXK1 regulates carbon catabolism, sporulation, fumonisin B1 production, and pathogenesis in Fusarium verticillioides. Microbiology 157:2658-2669.
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Progress 02/15/10 to 02/14/11
Outputs
OUTPUTS: Activities: Over the past year, we continued studies on gene regulation of fumonisin biosynthesis. We also continued our analysis of extracellular proteins produced by F. verticillioides during colonization of maize kernels. Events: Results from our work were presented at two conference: Poster: Functional characterization of FST1 from Fusarium verticillioides.
Kim, H. and Woloshuk, C. P. Annual Meeting of the American Phytopathological Society, Charlotte, NC. Phytopathology 100:S62. Poster: Fst1, a gene involved in pathogenicity and fumonisin production by Fusarium verticillioides, confers resistance to reactive oxygen species. Aparna Natarajan, A., Payne, G. A., and Woloshuk, C. P. 2011. Genetics of Maize Disease Workshop 2011, Raleigh, NC. Raleigh NC. Products: Hun Kim was granted Ph.D. from Purdue University (Major Advisor: Charles Woloshuk, Department of Botany and Plant Pathology) The following manuscript has been submitted for publication: Kim, H., Smith, J. E., Ridenour, J. B., Woloshuk, C. P., and Bluhm, H. B. HXK1 regulates carbon catabolism, sporulation, fumonisin B1 production, and pathogenesis in Fusarium verticillioides. Microbiology (Accepted with revisions). PARTICIPANTS: Charles Woloshuk, PI; Hun Kim, Graduate Student; Aparna Natarajan, Graduate Student; TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We demonstrated that Fst1 belongs to a class of proteins that are structurally similar to sugar transporter but may have a regulatory function. We identified a new phenotype related to the function of the gene Fst1. Mutants lacking a functional Fst1 are more susceptible to reactive oxygen than the wild-type strain. Our studies of the hexose kinase gene Hxk1 discovered links between the function of the gene and fructose uptake, pathogenicity, fumonisin biosynthesis, and trehalose biosynthesis.
Publications
- Kim, H. and Woloshuk, C. P. 2010. Functional characterization of fst1 in Fusarium verticillioides during colonization of maize kernels. Mol. Plant Microbiol. Interact. 24:18-24.
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