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.
Greenhouse-grown immature cotton bolls were inoculated with a toxigenic strain of Aspergillus flavus. Samples of pericarp (outer skin of cotton bolls) and immature seeds from infected locules and adjacent locules were collected at different time points and total ribonucleic acid (RNA) was isolated from the tissues. An anneal control primer (ACP) technique was used to study the genes that are differentially expressed in pericarp and seeds in response to the fungal infection. A total of 32 differentially expressing gene products or transcripts were isolated and sequenced. Functional analysis of the sequences indicated that the genes are involved in different stress response pathways. Semiquantitative analysis of eight genes, in different tissues of the cotton bolls during the fungal infection process to define their function, has been completed. Genes identified in this study will lead to future development of sequence based functional markers (known as single-nucleotide polymorphism [SNP] or EST-derived Simple Sequence Repeats [ESSR]) for use in crop breeding to obtain a cotton variety resistant to aflatoxin contamination. Research progress was monitored through teleconferencing, visits to collaborator’s lab, and scientific discussions at professional society meetings.