Location: Food and Feed Safety Research2013 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:
To improve the host plant resistance to the toxin-producing fungus, Aspergillus (A.) flavus, it is essential to understand ongoing molecular events during the infection process. In this regard, we have previously reported on the identification of 44 A. flavus-responsive genes in the outer wall of cotton bolls (pericarp) and seed tissues, using a small-scale approach through a novel gene fishing technique that is target-specific. Out of these 44 genes we are in the process of identifying candidate genes for overexpression in cotton bolls and seeds to measure their impact in resistance against A. flavus 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. Recently, efforts were made to profile the entire gene pool of cotton. Immature cotton bolls were inoculated with both non-toxin producing (Aspergillus flavus36) and toxin producing (Aspergillus flavus13) strains of A. flavus. Immature seed and pericarp tissues were harvested at different time points (0, 6, 24, 48, 72 hours post inoculation) from inoculated and adjacent segments of cotton bolls (locules). Total ribonucleic acid (RNA) was isolated and equal aliquot of RNA was pooled over different time points independently for seed and pericarp. The RNA samples were normalized for equal representation of all genes by removing abundant house-keeping genes (genes required for the maintenance of basic cellular functions in all types of cells) before proceeding for profiling using next generation sequencing (NGS) technology. Gene libraries for each sample were first created and were processed for sequencing by chemically joining to specific adapters (a special technique). Processed libraries were sequenced at the deoxyribonucleic acid facility of the Iowa State University in a Hi-Seq 2000 genome analyzer. The average counts of fragments derived from genes per sample were about 139 million, each with a size of 100 nucleotides long. Bioinformatic analysis (using computer tools to generate useful biological knowledge) of these sequences is currently being pursued.