2012 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.
To understand how and where in the seed the transgene is expressing an antifungal peptide that may inhibit Aspergillus flavus growth in the plant, we have transformed nuclei or chloroplasts of tobacco plants with a special tagged version of the antifungal peptide D4E1. This tag, called hemagglutinin (HA), makes it possible to visualize where this transgenic peptide is expressed in plant cells and how they act on invading fungi. We have also developed and optimized several procedures for localizing this peptide using Transmission Electron Microscopy. We used a bioassay of 26 different cotton cultivars to identify which cultivars or species exhibited enhanced resistance to Aspergillus flavus infection. Based on our first assay, we have chosen candidate cultivars and species for further study. These studies are ongoing. The mechanism of action for the lytic (ability to break open cells) peptide d4E1 is unknown. To determine the mechanism of action of the peptide, we are performing a series of assays using the dye SYTOX green. SYTOX green can not penetrate intact cells, and its uptake is a measure of membrane integrity. When SYTOX green binds nucleic acids, the fluorescence emission increases by more than 500-fold. We are in the process of developing a microtiter plate (a plate with many small wells for multiple assays) assay to determine the effect d4E has on membranes. In this assay, Aspergillus flavus spores are incubated with varying concentrations of d4E in the presence of SYTOX green and the emission values quantified. These studies are currently in progress. An extension of these studies that will be performed this summer is to perform microscopic analysis of germinating Aspergillus flavus spores in the presence of different concentrations of d4E followed by staining the dyes with calcafluor white and SYTOX green. This will enable us to visualize SYTOX green that has permeated the Aspergillus flavus cell membranes which should be elevated relative to the peptide dose, as well as to determine if there are any effects on the morphology of the cells at sub-lethal concentrations of the peptide.