| APPROACH:
The common theme in this proposal is locating specific DNA regions associated with virulence or toxin production that can be used for reliable identification of pathogenic individuals within a population. Three fungi have been chosen because they are important plant pathogens, they represent fungi having different tropic interactions with their hosts, and they occupy different ecological niches. Pathogenicity genes will be identified in Magnaporthe grisea based on differential gene expression, comparison with non-pathogens at the genus level, genes involved in secondary metabolism and recovery of genes from pathogenicity mutants. Bottom-up approaches will be used to examine the evolutionary processes that have given rise to the specific organization and function of genes in the aflatoxin gene pathway. To do this, we will identify nucleotide sequence variation within coding and noncoding portions of the aflatoxin gene cluster in population samples of A. flavus or A.
parasiticus. Comparative genomics and gene expression profiling will be used to obtain a better understanding of the evolution of secondary metabolism in these fungi, its regulatory elements and its linkage to fungal development. Finally, we propose to examine the quinic acid gene cluster in Rhizoctonia solani to better understand how the down regulation of the shikimic acid pathway associated with the dsRNAs contributes to virulence in R. solani.
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CRIS NUMBER: 0203696
SUBFILE: CRIS
PROJECT NUMBER: NC09002
SPONSOR AGENCY: CSREES
PROJECT TYPE: SPECIAL GRANT
PROJECT STATUS: TERMINATED
MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Jul 1, 2005
TERMINATION DATE: Jun 30, 2007
GRANT PROGRAM: CROP PATHOGENS, NC
GRANT PROGRAM AREA: Special Research Grant
CLASSIFICATION
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| 212 | 1310 | 1040 | 4.2 | 80% |
| 712 | 1510 | 1070 | 4.1 | 20% |
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CLASSIFICATION HEADINGS
KA212 - Pathogens and Nematodes Affecting Plants
KA712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins
S1310 - Potato
S1510 - Corn
F1040 - Molecular biology
F1070 - Ecology
G4.2 - Reduce Number and Severity of Pest and Disease Outbreaks
G4.1 - Reduce Incidence of Foodborne Illnesses and Contaminants
RESEARCH EFFORT CATEGORIES
| BASIC |
50% |
| APPLIED |
25% |
| DEVELOPMENTAL |
25% |
KEYWORDS: evolution; pathogenicity; fungus physiology; genes; fungus genetics; gene action; biosecurity; genomes; food contamination; potatoes; corn; magnaporthe grisea; fungus diseases (plants); aspergillus flavus; aflatoxin; gene analysis; rhizoctonia solani; toxins; dna; virulence; mutants; metabolic pathways; protein dna interactions; ds rna
PROGRESS: Jul 1, 2005 TO Jun 30, 2007
Our ability to detect and counter threats of bio-terrorism to our food security is limited. In order to develop rapid and reliable methods to detect pathogens, it is necessary to identify genes required for pathogenicity and toxin production, characterize their regulation, and understand their evolution. The fungi being examined limit the production of potato, maize, rice, and wheat. Magnaporthe grisea is widely recognized as a leading model for studying numerous aspects of host-pathogen interactions. In addition to known pathogenicity genes, numerous potential pathogenicity genes are being identified based on differential gene expression, comparison with non-pathogens at the genus level, genes involved in secondary metabolism and recovery of genes from pathogenicity mutants. In addition, studies have continued to evaluate the extent and organization of repetitive DNA (mainly transposable elements) in the genome of Magnaporthe grisea. Aspergillus flavus is pathogenic
on plants and animals and also produces the carcinogen, aflatoxin. Aspergillus oryzae is a closely related non-pathogenic species that does not produce aflatoxin. The recently available whole genome sequences of Aspergillus flavus and A. oryzae presented an opportunity to carefully examine two fungi with different ecologies, but very similar morphology. The goal of the comparison is to identify genes required for pathogenicity and aflatoxin production in A. flavus. The synthesis of aflatoxin is currently being used as a model to better understand gene function and evolution. Population level variation in the aflatoxin gene cluster has allowed for the reconstruction of their evolutionary history of mutation, recombination, and selection. The 3.57 kb M2 double-stranded RNA (dsRNA) found in the soil fungus R. solani has been shown to influence its disease causing activity and may play a role in the utilization of the carbon source quinic acid which is needed for virulence. Experiments
were conducted to determine whether quinic acid can be used as a sole carbon source by R. solani and 12 additional species of soil fungi. An understanding of the transmission of the M2 dsRNA between isolates of Rhizoctonia solani is important for examining its effect on the disease causing activity of the fungus. Nine donor isolates of R. solani AG-3 containing the M2 dsRNA were paired on potato dextrose agar with each of three different recipient isolates where the M2 dsRNA was absent. Reverse-transcription PCR (RT-PCR) was used to detect horizontal transmission of the M2 dsRNA via hyphal anastomosis from donor to recipient isolates by examining hyphal explants taken 3-cm from the hyphal interaction zone. PCR-RFLP genetic-based markers of two nuclear loci and one mitochondrial locus were used to confirm identity and transmission between donor and recipient isolates of R. solani AG-3.
IMPACT: 2005-07-01 TO 2007-06-30
The avirulence gene AVR-pita in Magnaporthe grisea was found to be interrupted by the element MAGGY, suggesting that insertion and recombination of mobile elements can have a profound impact on the evolution of pathogenicity. Active transposition during reproduction cycles may provide a mechanism for rapid evolution and expansion of host range. In experiments to examine the ability of fungi to utilize quinic acid, all fungi examined, except for Rhizopus oryzae and Trichoderma hamatum, exhibited significantly greater growth in Vogel's minimal medium when amended with 1% quinic acid. The frequency of transmission observed between 72 pairings of the eight donor and three recipient isolates was approximately 4% of the total pairings and differences in the phenotype of the recipient isolates after acquisition of the M2 dsRNA via horizontal transmission were observed. To our knowledge, this represents the first demonstration of transmission of dsRNA between genetically
different individuals of R. solani confirmed with nuclear and mitochondrial markers. These results suggest that transmission can occur between somatically incompatible isolates of R. solani AG-3, but that maintenance of the dsRNA in the recipient isolates was not stable following repeated subculturing on nutrient medium. A comparison of Aspergillus flavus and A. oryzae at the genomic level showed that these two fungi are very similar. The estimated genome size of 36.8 Mb for A. flavus is similar to that for A. oryzae (36.7). Further, these two fungi have a similar number of nonribosomal peptide synthases and polyketide synthases, the two major multifunctional enzymes involved in secondary metabolism. One striking difference between the two fungi is a translocation in A. flavus between chromosomes 2 and 6. In addition, these fungi have approximately 300 genes unique to each species. Functional analysis of the genes unique to A. flavus may reveal genes important for pathogenicity and
aflatoxin production. We have developed two new software tools, SNAP Combine and Map, to facilitate our comparative analyses of molecular genetic variation in the aflatoxin gene clusters of A. flavus and A. parasiticus. These analyses will lead to a better understanding of the evolutionary forces that influence aflatoxigenicity in natural populations of these species, allowing us fine-tune biocontrol strategies. The primary goal of this research is to develop an internet accessible genomic DNA database which contains additional information on pathogenicity, toxin production, geographic distribution and other biologically relevant data. These data will be invaluable for understanding the molecular and evolutionary basis of how fungi cause plant disease. This research will contribute significantly towards the development of methods that can be deployed in a state-of-the art diagnostic laboratories to reduce the threat to our food safety.
PUBLICATION INFORMATION: 2005-07-01 TO 2007-06-30
Aylor, D. L., Price, E.W. and I. Carbone. 2006. SNAP: Combine and Map modules for multilocus population genetic analysis. Bioinformatics 22: 1399-1401.
PROJECT CONTACT INFORMATION
| NAME: |
Payne., G. A. |
| PHONE: |
919-515-6994 |
| FAX: |
919-513-0024 |
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