Location: Subtropical Plant Pathology Research2011 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 project is related to inhouse objective 3: Develop or improve comprehensive integrated disease management strategies. Our goal to produce a fusion protein between a Tomato spotted wilt virus surface protein and Bacillius Thuringensis toxin that will kill its thrips vector is progressing as planned. In this grant period we have re-engineered the viral protein to include several smaller forms of the original molecule to produce a more efficient expression system. Several of these are now constructed and we are testing them for binding to the mid-gut receptor. 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 important data for use going forward to develop systems to deliver these molecules to insects in a realistic feeding array. The student wrote this up as a report that will be used by new students to move the work forward. We also used funds for supplies for a senior postdoctoral researcher who worked pro bono for several months and has now been hired full time on other funds. Post Doc is currently expressing the modified viral ligand using a Baculovirus system currently being optimized for production of the new ligands and Bt fusions that will be subsequently test fed to insects. In preliminary experiments we have shown that fusions between full length Bt toxin (Cry3A) and our viral ligand remain toxic to Colorado potato beetles. This confirms that the fusion construct does not inactivate the toxic moiety of the molecule. We are now creating fusions between the toxic Bt domain and omitting the beetle binding domain to test for thrips lethality and specificity. This work was reported at the Entomological Society of America Annual Meeting in 2010, San Diego, Ca. at a symposium Emerging Pests and Research Approaches in Vegetable Pest Management. Developing a strategy to control thrips with Bt toxin. Progress was monitored via through direct involvement in lab and field activities, research meetings, telephone calls and email communications.