2012 Annual Report 1a.Objectives (from AD-416): (1) Identification and isolation of genes/promoters for Aspergillus (A.) flavus resistance in corn/cotton; (2) Express the candidate genes isolated in transgenic crops (tobacco, cotton, corn) by over/under expression; and (3) Evaluate the genes for tolerance to abiotic and biotic stress, for example, in response to A. flavus infection. Transgenic plants developed with these genes will be tested for their efficacy against A. flavus and other microbial pathogens. Genes identified in this study will lead to future development of sequence based functional marker (such as single-nucleotide polymorphism (SNP), EST-derived Simple Sequence Repeats (ESSR)) for use in marker-assisted breeding. 1b.Approach (from AD-416): Identification of novel genes/promoters through differential gene expression analysis in response to Aspergillus (A.) flavus infection such as messenger ribonucleic acid (mRNA) differential display/ subtractive suppression hybridization to complement proteomics/genomic research. Heterologous or native genes/promoters identified through this research or from research on other plant species, such as Louisiana native Smooth Cordgrass (marsh adapted, salt-tolerant), will be employed in transgenic systems to evaluate their efficacy for tolerance to biotic and abiotic stress. Smooth Cordgrass has been shown to have high sequence similarity with corn. Promising gene constructs in transgenic tobacco model system will be advanced for use in transgenic cotton or corn. Finally, transgenic plants will be evaluated for resistance to A. flavus under laboratory, greenhouse and field conditions. 3.Progress Report: Aflatoxin contamination of food and feed crops is often exacerbated when plants undergo physiological stress due to environmental factors such as heat, drought, and salinity. Several salt tolerant grass species (called halophytes) regulate salt transport across membranes in their cells and organelles by using a key enzyme called vacuolar ATPase. We isolated a v-ATPase gene (SaVHAc1) from Spartina alterniflora or smooth cordgrass, a plant native to Louisiana coastal salt marshes and expressed it in transgenic tobacco and rice. The transgenic plants showed salt tolerance, and other physiological adaptations such as reduced stomatal density and closure of stomata to overcome stress. This study demonstrated that halophytes are not only useful to understand gene regulation mechanism for their natural adaptation to salinity, but also could be effectively used as donors for improving tolerance to salinity and other stress factors in several cultivated crops. This research finding will be useful to other crop breeders and biotechnologists in developing new germplasm and crop varieties resistant to environmental stress due to salinity and drought. In another study, we have isolated 44 genes that showed altered expression in response to Aspergillus (A.) flavus infection in pericarp (outer skin of cotton bolls) and cotton seed. Four genes had significant match to different functional groups of cotton-specific genes suggesting that the fungus induced a complex response in the cotton plant. Quantitative expression analysis of eight genes representing transcription factors, kinase, and stress responsive genes, revealed their tissue and time-dependent response to the fungal infection. The upregulated genes would have potential use in development of A. flavus resistance in cottonseed through genetic engineering and/or molecular marker-assisted breeding.