Performing Department
(N/A)
Non Technical Summary
Food security is one of the most important global challenges of our age and insect pests continue to be a problem in agricultural systems, contributing to between 36%-42% losses in some staple crop systems. Reducing crop damage by pest insects is one strategy to increase food production and security. Toxins from the insect pathogen Bacillus thuringiensis (Bt) are widely used in biological control. Bt toxins, which are specifically toxic to insects and not mammals, are applied (sprayed) on crops and produced by transgenic crops to control insect pests in agriculture. However, resistance to these toxins in some populations of insect pests is a problem, making Bt toxins less effective. One way to combat resistance to Bt toxins is to use a variety of toxins with different modes of action, including the development of novel toxins. Recently we have discovered that insect-parasitic nematodes, which are considered "beneficial nematodes" since they kill insect pests, produce and release insecticidal proteins into their hosts. We have found that they have oral toxicity to some insect pests and have identified several promising candidates and have established a pipeline to characterize others. We propose to develop our promising candidates and assess their efficacy in transgenic plants and foliar application as well as characterize additional nematode-derived insecticidal proteins and test the feasibility of using these toxins to control insect pests in agricultural systems. The results of this research could lead to the development of new natural compounds useful for killing insect pests. This proposal addresses a program area priority for pests and beneficial species in agricultural production systems by developing strategies to mitigate resistance to pesticides and/or toxins.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
0%
Goals / Objectives
To maximize the use of resources and accelerate the characterization of S. carpocapsae, S. feltiae, and S. hermaphroditum insecticidal proteins, we will perform the proposed research using adult Drosophila melanogaster, larval Manduca sexta, larval Plutella xylostella, larval Spodoptera exigua, and whitefly adults. The fruit fly is a model system that has well-established assays and will allow us to use minimal amounts of protein to identify active fractions and proteins. We will follow-up by performing the same experiments in pest insects. Using insects from several different orders will help us determine the potential breadth of toxicity across insects.The specific objectives of our research are:1. Insecticidal activity-guided purification of candidate toxins from S. carpocapsae, S. feltiae, and S. hermaphroditum ESPs and assessment of candidate toxins by oral application against several insect species (Spodoptera exigua, Plutella, whiteflies, Drosophila melanogaster, and Manduca sexta).2. Develop transgenic tomato plants expressing CLP and evaluate the resistance of these plants against insect pests (Spodoptera exigua, whiteflies, and Manduca sexta)3. Elucidate the mode of action through which CLP has insecticidal activity and define molecular targets for CLP and other candidate toxins identified through activity-guided fractionation.
Project Methods
1A. Identification of insecticidal protein(s) within S. carpocapsae ESPs: For this objective we will collect ESPs from tens of millions of activated S. carpocapsae IJs for insecticidal activity guided fractionation to narrow down the insecticidal candidate proteins and ultimately purify individual toxins. To eliminate possible contamination of the ESPs by toxins from symbiotic bacteria (Xenorhabdus nematophila is the symbiont of S. carpocapsae), we will use axenic (bacteria-free) IJs for activation and ESP collection. We have established a method to obtain large numbers of axenic IJs by feeding nematodes with a mutant strain of X. nematophila that supports nematode growth but is defective in colonizing IJs. We have shown that ESPs from the activated axenic S. carpocapsae are as toxic as the ESPs from symbiotic IJs (7). A total of 8 mg of crude ESPs will be collected in PBS and verified for toxicity as described as previously. Briefly, adult fruit flies will be injected with 20ng of the crude ESPs to verify that they are toxic. The collection will then be buffer exchanged into the mobile phase buffer (20 mM Tris-HCl, pH 8.0) in preparation for anion exchange chromatography using FPLC system. We are employing a Bio-Rad NGC quest-10 FPLC System (P-920 pump, UPC-900 monitor, Frac-920 fraction collector, UNICORN 5.31 workstation software) and a Mono Q 1.66 mL strong anion exchange chromatography column to bind and retain negatively charged proteins. The initial flow-through (detected by UV) will be collected into 1 mL fractions and the retained proteins will be eluted with the elution buffer (20 mM Tris- HCl, pH 8.0, 1 M NaCl) at a rate of 1 mL/min and a gradient of 0 to 100% elution buffer (0 to 1 M NaCl) for 20 column volumes. All the elutions associated with detected UV (280nm) absorbance peaks will be collected into 1 mL fractions those associated with identifiable peaks and across the range of the chromatogram will be selected for toxicity testing. We will then test the activity of the fractions by injecting them into Drosophila melanogaster adults and Manduca sexta larvae. D. melanogaster is small and a model insect while M. sexta is larger and a model pest. We will run SDS-PAGE gels for fractions to determine how many proteins are in each fraction and potentially perform mass spectrometry on the fractions. The most toxic fractions will be further purified using size exclusion chromatography column (Superdex-75 pg 100/600 Ge healthcare). As described above, we will test all the elutions for toxicity with identifiable UV (280nm) peaks against D. melanogaster. The toxic fraction(s) will be subjected to mass spectrometry to identify insecticidal protein(s). As described above we have preliminary data using insecticidal activity guided purification and mass spectrometry with S. carpocapsae ESPs, where we have identified the candidate insecticidal protein (CLP). By preparing a matched set of samples using crude ESPs and a single toxic fraction from that crude ESP sample, using fold enrichment and the statistical significance of the peptide-spectrum match, we were able to find new candidate toxins from ESPs. The method used to identify insecticidal proteins from S. carpocapsae ESPs will also be done with S. hermaphroditum and S. feltiae ESPs. When we do perform mass spectrometry on the ESPs, we will use both the nematode proteome and the symbiotic bacterial proteome as library queries, so that bacterial contamination can be ruled out. If assessment by enrichment and the significance of the spectral match fails to yield a short list of candidate toxins, further fractionation of active fractions using another fractionation technique (i.e. hydrophobic interaction or differential precipitation) may be needed to reduce the number of candidate insecticidal toxins. We would then test the secondary fractions for activity and perform mass spectrometry on active secondary fractions.1B. Characterization of purified insecticidal protein(s): We will generate recombinant versions of candidate insecticidal proteins and test their activity by injection into D. melanogaster adults and M. sexta larvae. We have successfully expressed one such protein chymotrypsin like protease (CLP) recombinantly in E. coli, and tested its activity against D. melanogaster adults, Bemesia tabaci, Plutella xylostella, Manduca sexta and Spodoptera frugiperda. We found CLP is toxic to all the insect and kill them at nanogram quantities except Manduca sexta larvae which has shown significant weight loss when fed orally. Similarly, we will express other purified insecticidal proteins in E. coli and test their efficacy against all the above-mentioned insects by injecting or by oral feeding in a dose-response curve to determine the LD50. If we only find several weak toxins, we will mix them in isomolar quantities and test combined toxicity. With the toxicity tests, we expect to distinguish the possibilities that (1) a few individual proteins are potent toxins; (2) individual proteins are weak toxins but have combinatorial or synergistic effects. We will also want to know what cells or organs in the host are targeted by the toxin proteins we identify.1C. Develop oral feeding method for purified nematode toxins to kill insects: We will feed recombinantly expressed proteins to D. melanogaster adults and larvae, as well as M. sexta larvae, S. frugiperda larvae, P. xylostella larvae and B. tabaci adults to determine the feasibility of using the nematode-derived insecticidal toxins in insect pest control. For feeding toxins to D. melanogaster adults we have used the capillary feeder (CAFE?) assay. This assay allows for the intake of a liquid food source to be measured, which will allow us to determine the LD50 of the toxin proteins for adult flies. A liquid food source is presented to the flies in 25μl microcapillaries with 5μl marks. The food base is a 3 M sucrose solution with food coloring, this will be combined with concentrated amounts of individual recombinant toxins. We will perform a dose-response experiment to determine whether any amount of the candidate toxins or fractions are toxic to adult flies, starting with 100ng/μl. We have observed that 100ng/ml of recombinant CLP was toxic to D. melanogaster. We expect much lower amounts of individual toxins or fractions will have toxic activity, but we will start at a relatively high dose. To determine the toxic effects of individual proteins on fly larvae, the eclosion assay will be used. First instar larvae are reared on medium containing the toxin of interest and the survival and development of the larvae are measured over time. The base food is brown sugar, cornmeal, yeast food, and different concentrations of toxin(s) can be added to evaluate their toxicity when eaten by the larvae. A dose-response experiment using different quantities of proteins will be used, starting with 10μg/mg of food.Oral bioassays for toxicity in Manduca sexta larvae, Spodoptera frugiperda larvae and Plutella xylostella larvae will be performed as previously described: 100μl of toxin preparations, at a known concentration, will be applied to 1cm3 disks of artificial diet. This will be allowed to soak into the food block and dry for approximately 20 minutes under laminar flow. First-instar neonates will be placed on each block before incubation at 25C for 7 days. The percent mortality of the larvae and the weight of surviving larvae will be recorded. Oral toxicity will be assessed as the relative weight gain of animals on the treated diet in comparison to control animals fed on buffer-treated food blocks.For Bemisia tabaci parafilm sandwich method will be used as described. In this method 100 µl (100ng/µl) protein will be mixed with 900 µl artificial liquid diet and sandwiched in between 2 stretched layers of parafilm. The whiteflies will be released in perforated specimen tubes and oral toxicity will be assessed for 7 days.