Location: Subtropical Plant Pathology Research2012 Annual Report
1a. Objectives (from AD-416):
The objective of this cooperative research is to develop a specific and safe Bacillus thuringiensis toxin (Bt) for management of thrips.
1b. Approach (from AD-416):
Previous work from our laboratory has demonstrated that a protein ligand on the surface of Tomato spotted wilt virus binds to a receptor in the gut of thrips to facilitate cell entry and replication. Since we know that the specificity of Bt toxin (that is why they only kill a small subset of insects) depends on targeting Bt to an insect gut receptor by means of a specific ligand as well, we are using standard molecular techniques to re-engineer several versions of Bt by replacing their native ligand with that from the virus. The rationale being that the fusion protein thus created will bind to thrips guts (and only thrips guts because the virus is not transmitted by any other insect) and create a specific (no harm to beneficial insects) benign (years of use of Bt toxins have shown them to be safe) and effective pesticide.
3. Progress Report:
This research relates to inhouse project objectives 2. Develop/refine rapid, sensitive reliable detection/sampling methods for pathogens, and 3. Develop or improve comprehensive integrated disease management strategies. Our goal is to produce a fusion protein between a soluble, truncated form of a Tomato spotted wilt virus surface protein (Gn-S) and Bacillus thuringiensis (Bt) toxin. Our expectation is that this fusion will re-target Bt to thrips gut receptors and create a control strategy for these insects. Thrips are pests in their own right and serve as vectors for Tospoviruses. In the last grant period, we re-engineered Gn-S to include several smaller forms of the original molecule to produce more efficient expression systems. Again this year, funds were used to support an undergraduate research project involving expression in and recovery from plants of prototype fusions using transient Agrobacterium infiltration techniques. This project provided additional data to develop systems that will deliver these molecules to insects in a realistic feeding array. The student again wrote the findings as a report that will be used by new students who will contribute to the project. We also used funds for supplies for a senior postdoctoral researcher whose salary is now funded from other sources. In this grant period, University of Wisconsin has successfully expressed, solubilized (with significant difficulty) and purified GnS from E. coli. This is an improvement due to the production very large amounts of protein (30 mg from 1 liter culture). Importantly, they have also expressed, recovered and purified the ligand that is secreted into the insect cell culture media using a more facile plasmid system (compared to the tedious Bauclovirus expression used originally). We know that insect expressed protein is active in binding and if E. coli expressed protein, which is not glycosylated, is active we will use both of these systems to test fusions and mutants for biological activity (gut binding and virus transmission inhibition). We have learned that stable transformation (as compared to transient Agrobacterium expression) is a far better way to deliver the ligand and or fusions thereof to insects as judged by superior expression and blocking of virus transmission when thrips are fed on stably transformed plants expressing GnS-GFP fusions. Expression levels were higher, uniform and the subcellular location was shown to be in regions that will insure acquisition by insects during feeding visits to plants. Accordingly, during collaboration with Kansas State University we have started transforming Arabidopsis plants with Gn-S-Bt fusions to test their efficacy against thrips. We believe this is a good approach because these plants are comparatively easy to transform, can be produced relatively rapidly, are good feeding hosts for thrips and likely will express high levels of protein. We will increase our efforts in this area by collaborating with New Mexico State University who will also produce transformed plants using additional fusion constructs and Bt species.