2009 Annual Report 1a.Objectives (from AD-416) Clone gene from Tex6 coding for a chitinase that is inhibitory to growth of A. flavus. Produce recombinant chitinase in a bacterial expression system to confirm that the corresponding gene encodes the inhibitory activity. Use the recombinant chitinase to determine the organ specificity and abundance of the protein in corn. Develop a specific probe from the sequence of the gene that can be used by researchers working on resistance to aflatoxin contamination for mapping resistance in corn populations. 1b.Approach (from AD-416) A partial amino acid sequence for the purified chitinase will be used to design a series of nested degenerate primers to amplify a DNA fragment unique to the Tex6 chitinase. Positive clones will be characterized by sequencing. The cloned gene will be over-expressed in bacteria, purified, and tested for activity. The organ specificity and abundance of the protein in corn will be determined by co-migration of extracted protein with the recombinant protein. To develop a specific probe from the sequence of the gene that can be used to follow resistance in corn to aflatoxin accumulation, we will design highly specific primer pairs of at least 20 bp each to target the unique sequences of the coding region and 5' and 3' untranslated regions of the gene. The primer pairs will be used to amplify the chitinase gene sequence with PCR, and the parents of several mapping populations will be screened with these primers to identify amplification product length polymorphism that can be mapped. In a new initiative, A. flavus microarrays containing the 5,200 expressed gene sequences will be analyzed for identification of specific genes involved in plant-fungal interactions, as well as those expressed under conditions conducive to aflatoxin production in the fungus. 3.Progress Report The overall goal of the research at North Carolina State University (NCSU) is to identify compounds that inhibit growth or aflatoxin production by Aspergillus flavus and develop genetic markers that can be used for selecting maize genotypes with resistance to aflatoxin accumulation. We are currently characterizing maize kernel compounds that perturb aflatoxin biosynthesis and Aspergillus flavus development. Production of pathogenesis-related (PR) proteins in response to pathogen infection is a hallmark of plant disease resistance. We isolated and characterized a protein fraction from kernels of the resistant maize line Tex6 that inhibits growth and aflatoxin production by A. flavus in vitro. Two protein bands with inhibitory activity were identified as chitinase A (ChitA) and zeamatin. We used techniques, such as bioinformatics and analysis of gene expression, to investigate other maize genes that may be involved in seed defense responses. These maize genes grouped into four major genetic groups based on the relative origin of species (phylogeny) similar to those described in rice. Genes corresponding to Thaumatin-like proteins (TLP are a Family of Plant Peptides) from maize generally followed their previously described phylogeny in rice. Ribonucleic acid (RNA) profiling of infected kernels in the field showed that RNA transcripts (gene messages) for a subset of the seed defense response protein families are induced during infection. In related research to identify target sites for inhibition of aflatoxin biosynthesis, we identified a small gene, called hypE, situated between the genes aflM (ver-1) and aflN (verA) in the aflatoxin gene cluster (a specific part of the fungal chromosome). A hypE deletion mutant of A. flavus produced less aflatoxin B1 and B2 than control strains and accumulated an unknown metabolite, which we assigned as a tentative HypE substrate (HESUB). Aflatoxin biosynthesis could be restored in this hypE mutant by replacement of the mutanted gene with a functional gene back into the fungus. Restored aflatoxin biosynthesis was associated with loss of HESUB accumulation. These data suggest that the hypE gene product is involved in formation of aflatoxins, although its precise biochemical function remains unclear. Research progress was monitored through teleconferencing, emails, and reports.