This Small Business Innovation Research Phase I project proposes to enhance the delivery of a novel class of small insecticidal peptides. Insect damage to crops threatens global food security and as a result farmers grow millions of acres of crops with insecticides expressed as transgenes, and protect millions more acres with spray-on pesticides. The inevitable evolution of insect resistance to insecticides occurs despite rotating mode-of-action chemicals and proper transgenic plant refuge stewardship. Research into safe new insecticides is needed now to protect our future crop production. This project proposes to develop an enhanced delivery of a new class of insecticidal peptides from the Blue Mountains funnel-web spider. Individual peptides have been identified and characterized from a venom screen. Their mode of action is insect-specific and interferes with various ion channels in the central nervous system (CNS). Toxicity occurs when insect pests ingest the peptide and it passes through the gut wall, into the hemolymph and into the CNS. Vestaron Corporation is developing spray-on pesticide products from these peptides are looking to enhance their activity further by aiding in their delivery to the CNS. This proposed research involves combining Vestaron's peptides to one of three classes of proteins that will aid in crossing the gut wall. The tests will be done in vitro by spraying the purified proteins onto leaf disks and running insect mortality assays and screening insect hemolymph for elevated levels of the insecticidal peptide. It is anticipated that one or many of the combinations will generate a technology that enhances delivery of the peptide and results in increased efficacy. Completion of this Phase I project will allow a focused transition to in planta assays in Phase II where the spray-on technology will be manufactured as a transgene and the proof of concept will generate a research partnership within the agriculture industry to develop a new plant incorporated protectant (PIP). The improvements from Phase I will also be captured by Vestaron's own spray-on product pipeline resulting in this improvement being available to both the PIP and spray-on markets in the future. OBJECTIVES: The goal of this grant is to enhance the delivery of a novel class of Vestaron's small insecticidal peptides. There are five objectives to accomplish this goal. The first objective is the DNA synthesis of yeast expression cassettes of three combinations of proteins that we hypothesize will aid in the transit of the insecticidal peptide to its site of action. The second objective is then to create and screen populations of yeast strains transformed with these cassettes using small scale fermentation to identify those individuals with the highest level of expression. The third objective is to do larger scale fermentation and recovery of adequate levels of the proteins for insect bioassays. The forth objective is to quantify and qualify the peptide concentrations and their insecticidal activity. The final objective is to compare the proteins for their increase in insecticidal activity and correlate that to the localization of the protein within the insect. The expected output is a candidate sequence that enhances the delivery of the insecticidal peptide and increases it potency. The proof of concept will be incorporated into a phase II application with the ultimate goal of transferring the technology to transgenic plants and generating a proof of concept for eventual commercialization to protect global food production. APPROACH: Each of the five objectives in this grant will require individual methods to generate material, and evaluate results. This first objective, the DNA synthesis of a yeast expression cassette, will be contracted out. The company employed to construct the DNA will also sub-clone the cassette into the yeast transformation vector which will be transformed into yeast through electroporation and selection for individual transgenic colonies in house. The second objective involves picking individual yeast transformants and conducting milliliter scale fermentation in a 48 deepwell plate. Each strain's production will be evaluated and quantified with an iELISA using an antibody that binds to the peptide. The third objective will build from the previous screen by conducting liter scale fermentations with the highest expressing strains. Proteins will be partially purified from the beer through tangential flow filtration, and turned into a powder using spray drying with a Buchi dryer. The fourth objective will quantify and quality the peptide in the powders using an iELISA and a housefly injection bioassay. The iELISA, conducted first, will establish the amount of peptide recovered across the powders and allow for establishing dosage for downstream dipteran and lepidopteran testing. The housefly injection bioassay will be compared to a purified yeast-expressed version of Vestaron's insecticidal peptide as a control to establish that the yeast expression cassette design is not interfering with the insecticidal nature of the peptides. The fifth objective has two stages. Solutions of equivalent peptide concentration from the powders from objective three, and the purified yeast-expressed version from objective four, will be sprayed onto organic lettuce leaf disks. The first test will be to infest those disks with small instar Spodoptera exigua (Beet Armyworm) and Trichoplsia ni (Cabbage looper) to look rate oral insecticidal activity. Enhanced activity should result in increased mortality compared to moderately lethal doses of the control. The disks with the enhanced insecticide version will be feed to larger larva stage S. exigua and T. ni and where hemolymph samples will be taken to confirm that changes in insecticidal activity correlate with the increased efficacy of delivery of the peptide into the hemolymph. From start to finish this project: creates the DNA, screens for the best strain, generates sufficient material, validates the material and then compares it to the standard to identify a more efficient delivery of the insecticidal peptide
