2011 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. 3.Progress Report Introduction of the new antifungal genes into chloroplast, the green pigment organelles in plant cells, is being studied as a means to reduce Aspergillus flavus invasion and subsequent aflatoxin contamination of cottonseed. Successful production of the antifungal protein/peptide will depend in large part on the ability of the promoter sequence of the gene, which serves as trigger mechanism, 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. This work addresses objective 3 of the project plan to identify potential antifungal genes and optimize their expression in transgenic crops such as cotton. We have identified two promoters called psbA and rrn16 that direct high levels of expression (more than 200 fold then that of the reference gene) in at least one sample. Other promoters have been identified as likely candidates, if preferential expression in specific tissues is required. The goal of this study has been met and the research published. We have also developed transgenic tobacco plants that express an antifungal gene D4E1 tagger with another master gene (called HA g hemoglutenin) in either nuclei or chloroplasts. These plants are currently being studied using microscopy and a protein tracking procedure called immunolocalization to determine the sites and levels of expression of the antifungal peptide. We are in the final stages of developing and optimizing protocols that are appropriate for visualization of the introduced proteins in tobacco leaf tissues. In another study, we are growing 34 cotton cultivars in the greenhouse to generate sufficient seeds for a kernel screening assay (KSA). At this point, we have sufficient seeds (>30) for a preliminary assay of 26 of these cotton varieties (namely, 2 Gossypium (G.) barbadense, 5 G. arboreum and 19 G. hirsutum). The KSA will monitor the progression and growth of Aspergillus flavus on these seeds using a green fluorescent protein (GFP)-expressing strain of Aspergillus flavus. Research progress was monitored through teleconferencing, frequent email communications and reports.