2010 Annual Report
1a.Objectives (from AD-416)
Identify cotton plastid promoters that demonstrate high expression levels in both green and non-green plant tissues for use in development of cotton plastid transformation vectors. Determine levels of expression of reporter genes in both green (leaf and outer boll) and non-green (cottonseed and root) cotton tissues under control of select, engineered plastid promoters. Generate cotton plastid transformation vectors that place antifungal genes and selectable marker genes under control of selected cotton plastid promoters and transform cotton. Test transformed tissues for expression of antifungal genes and selectable marker genes under both light and dark growth conditions. Perform in planta bioassays for antifungal activity in transplastomic cotton plants.
1b.Approach (from AD-416)
Total RNA isolated from developing cotton plants and cottonseed will be hybridized with PCR-generated probes for selected cotton plastid genes using standard Northern hybridization technology. The promoters from those genes that demonstrate high levels of expression in green and/or non-green cotton tissues based on Northern hybridization results will be cloned and characterized using standard molecular biological methods. Promoter fragments of select plastid genes will be fused to reporter genes (GUS, GFP, etc.) and transformed into tobacco and cotton plastids in order to identify the minimal functional promoter sequences. While tobacco plastid transformation protocols have been developed, the same cannot be said of cotton, and protocols will have to be optimized for this plant. Once an efficient cotton plastid transformation system has been developed, cotton plastid will be transformed with transformation vectors in which reporter, antifungal, and selectable marker genes are placed under control of selected cotton plastid gene promoters. Transplastomic cotton plants will be analyzed for expression and production of reporter, antifungal, and selectable marker genes by standard molecular biological techniques (PCR, Northern and Western blotting). In planta antifungal bioassays will be performed to determine levels of resistance to A. flavus, as well as other cotton fungal pathogens.
Transformation of the chloroplast (a compartment within a plant cell that contains chlorophyll and deoxyribonucleic acid (DNA)) genome of cotton with antifungal genes is being studied as a means to reduce Aspergillus (A.) flavus invasion and subsequent aflatoxin contamination of cottonseed. Aflatoxin is a toxic and carcinogenic compound produced by A. flavus during growth on some plants. Successful production of the antifungal protein/peptide will depend in large part on the ability of the promoter structure controlling expression of the antifungal gene to generate high levels of gene expression in the chloroplast genomes present in cells of both green and non-green tissues. Based on northern hybridization (a method to measure how much a gene is being expressed) studies using 20 different plastid DNA probes and total ribonucleic acid (RNA) extracted from cotton tissues grown in the light or dark, 6 promoters were selected for real time quantitative polymerase chain reaction (PCR) gene expression analysis (qPCR). Two gene promoters directed levels of expression more than 200 fold higher than that of the RNA polymerase gene that was used as a control: the psbA and rrn16 gene promoters. Since rrn16 is expressed in all tissues at higher levels than psbA (at least ~10 fold higher than that of psbA in all tissues except light-grown cotyledons and leaves), its promoter is our best candidate for generation of a plastid transformation vector for high level expression in a wide variety of tissues.
Both nuclear- and chloroplast-transformed transgenic tobacco plants have been developed that express an antifungal peptide capable of disrupting the cell membrane of the fungus. These plants are currently being studied using microscopic techniques to determine the sites and levels of expression of the antifungal peptide. We have completed a preliminary study of tobacco tissues to determine which wavelengths of light are appropriate for these microscopic studies and will be using a commercially available antibody to directly visualize the peptide. These studies should be completed by the end of August 2010.
Thirty-one cotton cultivars are currently being grown in the greenhouse to generate sufficient seeds for a cottonseed screening assay to determine if any of the seed of these cultivars demonstrate increased resistance to infection by the fungus. Once sufficient seed is obtained from these and any additional varieties that we obtain as well as ancestral strains, we will infect the cottonseed with a strain of Aspergillus flavus that produces a fluorescent protein that will allow us to quantitatively determine its growth in the cottonseed screening assay.
Development of transgenic cottons expressing antifungal genes will be used to control aflatoxin contamination in cottonseed. Research progress is monitored by phone, email, and site visits.