Location: Subtropical Plant Pathology Research2013 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: 1. Characterize ecology, biology, epidemiology, molecular genetics, and vector and host (crop and weed) interactions of domestic, exotic, newly emerging, and re-emerging pathogens; 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 Tomato spotted wilt virus (TSWV) surface protein and Bacillius Thuringensis (Bt) toxin that can be used to effectively and specifically control both the spread of virus and thrips, which are pests in their own right. This work is based on our published observation that a cloned and expressed protein derived from the surface of TSWV will attach to thrips gut cells and block the acquisition and transmission of virus. We call this protein GN-S (a soluble form of TSWV glycoprotein) and think of it as a surrogate for the ligand on the virus that interacts with a putative receptor on the insect gut involved in virus endocytosis. Metaphorically, GN-S is the key and the receptor is the lock…when GN-S is in the “lock”…the virus cannot enter. If we replace the normal Bt ligand (which accounts for its insect specificity) with our cloned viral ligand it should now bind to thrips guts (instead of its original target insect) and be toxic to thrips. This is consistent with the normal mode of action wherein Bt is brought in close proximity to the insect gut wall cells and induces pore formation by virtue of this proximity. Work is progressing as planned. We are motivated by a couple of recent results. In collaboration with Kansas State University, we have shown that transgenic plants expressing cloned GN-S fused to GFP can: (1) locate to appropriate cellular sites for thrips acquisition, (2) block acquisition and transmission of TSWV in a highly statistically relevant way when both are present in the same plant. This is good news because these plants will be useful to determine thresholds for control of insect transmission in the field or greenhouse and for the Bt work. Moreover, it suggests that fusions of GN-S and Bt can be produced at biologically relevant levels and delivered to thrips in a natural plant setting. We have also expressed GN-S using an E. coli expression system that is much faster, produces more protein and is less expensive than eukaryotic expression systems. We have not done this previously because the dogma suggested that glycosylation (which does not occur in bacterial cells) is necessary for binding to the putative receptor. In experiments in which E. coli produced GN-S is used as a probe to detect thrips proteins that are binding partners (likely components of the relevant receptor) the results with either the glycosylated GN-S or the non-glycosylated E. coli are the same. This encourages us to move forward making GN-S-Bt fusions using the E. coli expression system. This will make it possible to feed versions of the fusion proteins directly to thrips to assay for toxicity and to other insects (e.g. non-target species) to evaluate their specificity. This may also portend the development of a bacterial expression system using plant flora or the bacteria known to exist in the thrips gut. Future work will involve both bacterially expressed fusions and testing existing transgenic plants constitutively expressing fusions in thrips bioassays for toxicity and specificity of action. Presentation made at NC-APS meeting in June 2013 on “Inhibition of vector transmission of Tomato spotted wilt virus by transgenic tomato plants that express the TSWV glycoprotein, GN” by graduate student and cooperators on project. A refereed manuscript is in preparation on this same topic. We hosted an undergraduate student second semester of last year who worked on this project. This involved bench experiments designed in collaboration with other ARS Scientists, writing a research report about his work, and the preparation and presenting of a poster at an all College Science Fair. He earned two research credits towards his degree as a result.