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Objective 1. Develop improved knowledge of insecticidal bacterial endotoxin molecular biology, including synthesis, inclusion assembly, and mode of action. Objective 2. Develop new insecticidal proteins and peptides for use in bacterial insecticides and transgenic plants that control chewing and sucking insect pests. Objective 3. Characterize genes in Coelomomyces fungi involved in recognizing and manipulating mosquito larvae for use as mosquito control agents. My project supports the UCR Experiment Station Mission by combining basic and applied research to develop new types of bacterial insecticides and insecticidal proteins and peptides to control important mosquito vectors of disease and agricultural pests, in the latter case, particularly lepidopteran pests of vegetable and field crops. Our basic research focuses on the genetic mechanisms underlying gene expression in insect pathogenic bacteria and viruses, whereas the applied research focuses on the practical use of the insecticidal agents we develop for use in integrated vector and pest management programs. <P>In the renewal of this project, over the next five years we will also be using genomic techniques to identify new targets in the midgut epithelium of insects for which we will construct and screen novel proteins in peptides for insecticidal activity based on new leads developed over the previous project period. This research will also involve the new strategies based on these leads that could well lead to new types of environmentally safe peptide and protein insecticides, as well as novel packing systems. For example, we have recently identified short peptide sequences (5 - 6 amino acids) in different bacterial proteins that target these to a fibrous matrix that contains several different insecticidal proteins in the bacterium, Bacillus thuringiensis subsp. israelensis. Preliminary experiments indicate that placing these short peptides into insecticidal proteins that do not bear them target them to the fibrous matrix. This presents the possibility of constructing a wide range of new types of complex protein insecticides, basically a nanotechnology approach. If we are successful, this should generate patents as well as additional extramural funding. <P>We also plan to initiate research on fungi of the genus Coelomomyces in collaboration with faculty members in our department (Anand Ray) and Plant Pathology and Microbiology (Jason Stajich) aimed at identifying proteins and potentially smaller molecules that enable members of this fungal group to recognized and differentiate the larval stages of different species of mosquitoes. If successful, the research will enable us to identify specific fungal receptors that may enable us to target bacteria to specific nuisance and vector mosquitoes. The era of genomics has opened a wide variety of avenues for producing basic knowledge that will lead to new, effective, and highly selective protein and peptide insecticides.
Non-Technical Summary:<br/>
Over the past sixty years, the control of insect pests of agriculture and vectors responsible for the transmission of the pathogens that cause diseases of humans such as malaria, filariasis, and the viral encephalopathologies has been based primarily on the use of synthetic chemical insecticides. While there is no question that these insecticides have been of great benefit to humankind, there is general recognition that they have also been detrimental for the environment, particularly for non-target organisms. Moreover, extensive and heavy use of chemical insecticides has resulted in high levels of resistance to these in many insect pest and vector populations. As a result, there have been significant efforts over the past several decades to develop pest management programs that employ a combination of more environmentally friendly tactics to suppress insect pests and vectors of disease. These include the use of biological control agents, microbial insecticides, pheromones, newer narrow-spectrum insecticides, modified cultural practices, and insect-resistant plant varieties. An especially important breakthrough occurred in the mid-1990?s with the commercial development of insect-resistant transgenic plants, which were developed though the use of recombinant DNA technology. In addition, recombinant DNA technology has been used to create new types of insecticides based on the highly specific proteins of various pathogens of insects. While still controversial, emerging scientific evidence indicates that the use of this new generation of insect control agents will be much better for the environment, humans, and other animals than synthetic chemical insecticides, and will eventually be widely accepted by the scientific community and public. Thus, the objectives of this new project are to develop new types of bacterial insecticides and insect-resistant transgenic plants based on insecticidal proteins and peptides. Effective development of these new technologies will require considerable advances in our knowledge of insect molecular biology, especially our knowledge of the midgut epithelium, as well as a better understanding of the mode of action of various insecticidal proteins. This new knowledge will serve as the basis for developing new types of bacterial insecticides and transgenic plants that will be constructed through the use of recombinant DNA technology.
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Approach:<br/>
To study the molecular biology of insecticidal protein synthesis in bacteria, we will clone and sequence the regions upstream and downstream of the genes that encode these sequences. Regions of at least 1 kbp upstream and downstream will be cloned. Analysis of these sequences should reveal regulatory elements such as promoters, enhancers, transcription regulators, and mRNA stabilizing sequences. These can then be manipulated by recombinant DNA technology and site-directed mutagenesis to improve synthesis. These techniques have been highly successful in improving our understanding of endotoxin synthesis over the past five years. In addition to these techniques, we are now using, and will continue to use RT-PCR for transcript analyses of endotoxin synthesis combined with electron microscopy to study the timing of the assembly of individual toxins and formation of the glycoprotein matrix that binds these inclusions together to form the insecticidal parasporal body of B. thuringiensis subsp. israelensis. We will also determine and annotate the genome of this bacterium with and without pBtoxis, the plasmid that encodes all of the endotoxin proteins of this species, as well as the enzymes, which synthesize the glycoprotein matrix that holds the endotoxins together to maximize toxicity and delay the evolution of mosquito resistance to this bacterial species. With respect to toxin mode of action, we will continue to use fluorescent dyes to label proteins and define their interactions with other membrane proteins and the insect microvillar membrane. Lastly, we will continue using mass spectroscopy methods that we have used previously to identify peptides and proteins (enzymes and structural proteins) involved in formation of the complex parasporal body of B. thuringiensis subsp. israelensis, the structure responsible for the insecticidal/mosquitocidal activity of this species. In addition to these studies, we are currently collaborating with researchers at Purdue University and Baylor University, who are evaluating insecticidal proteins we identified over the past decade for the potential use in transgenic plants. The collaboration with both universities involves determination of whether the Cry11Aa protein we cloned and characterized can be used in wheat to control the larvae of the Hessian Fly (Purdue) or in the nectar of plants (Baylor) that adult mosquitoes feed on shortly before taking a blood meal. The Hessian Fly is related to mosquitoes (i.e., it is in the dipteran suborder Nematocera, the same suborder to which mosquitoes belong), and in early tests appears to be sensitive to Cry11 proteins. To initiate the research on Coelomomyces fungi parasites of mosquitoes, we are currently in the process of establishing in vivo cultures of C. punctatus in A. gambiae and C. vernalis. We will sequence and annotate its genome using standard sequencing and proteomic techniques as we have done with our insecticidal bacterial endotoxin studies.
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Progress:<br/>
2012/01 TO 2012/12<br/>
OUTPUTS: Strains of Bacillus thuringiensis such as B. thuringiensis subsp. israelensis (ONR-60A) and B. thuringiensis subsp. morrisoni (PG-14), which highly toxic for mosquito larvae, produce a complex parasporal body consisting of several protein endotoxins synthesized during sporulation that form an aggregate of crystalline inclusions bound together by a multilamellar fibrous matrix. Most studies of these strains focus on the endotoxins. Although it is known that parasporal body structural integrity is important to achieving high mosquitocidal toxicity, virtually nothing is known about the matrix that binds the toxin inclusions together. A proteomic analysis of this matrix identified proteins that mediate assembly and stability of the parasporal body. In addition to fragments of their known major toxins, namely Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa, peptides with 100% identity to regions of Bt152, a protein coded for by pBtoxis of B. thuringiensis subsp. israelensis, the plasmid that encodes all endotoxins of this subspecies were obtained. The cry19A operon of B. thuringiensis subsp. jegathesan encodes two proteins, mosquitocidal Cry19A (ORF1; 75 kDa) and an ORF2 (60 kDa) of unknown function. Expression of the cry19A operon in an acrystalliferous strain of B. thuringiensis (4Q7) yielded one small crystal per cell, whereas no crystals were produced when cry19A or orf2 was expressed alone. To determine the function of the ORF2 protein, different combinations of Cry19A, ORF2, and the N- or C-terminal half of Cry1C were synthesized in strain 4Q7.
<br/>PARTICIPANTS: Dennis Bideshi, Hyun-Woo Park, and Margaret Wirth all hold PhD degrees and are part of a highly trained team of very competent scientist that drive the research forward. In addition to these individuals, the genes that we have cloned, which encode highly insecticidal proteins, are now part of collaborations with scientists at Purdue University in a project aimed at control of the Hessian Fly, a serious pest of wheat, and a second project at Baylor University aimed at engineering plants in Africa so that they secret these proteins in nectar in an attempt to kill anopheline mosquitoes that transmit malaria.
<br/>TARGET AUDIENCES: I and other members of my group presented a total of seven papers at meetings of professional societies during the past year. In addition, I gave talks to professional training groups responsible for mosquito control in California to update our research progress on developing new types of environmentally compatible mosquito control agents.
<br/>PROJECT MODIFICATIONS: Not relevant to this project.
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IMPACT: Mutation of this protein using recombinant DNA technology destabilized the parasporal body matrix, and concomitantly, inclusion aggregation. Fluorescence microscopy further demonstrated that Bt152 localized to the parasporal body in both strains, but was absent in other structural or soluble components of the cell, including the endospore and cytoplasm. Ligand blots showed this Bt152 bound to the purified multilamellar fibrous matrix. Together the data show that Bt152 is essential for stability and parasproal larvicidal toxicity of these strains (CA). In a related project, stable crystalline inclusions of these fusion proteins similar in shape to those in the strain harboring the wild-type operon were observed in sporulating cells. Comparative analysis showed that ORF2 shares considerable amino acid sequence identity with the C-terminal region of large Cry proteins. Together, these results suggested that ORF2 assisted in synthesis and crystallization of Cry19A by functioning like the C-terminal domain characteristic of Cry protein in the 130-kDa mass range. To determine whether overexpression of the cry19A operon stabilized its shape and increased Cry19A yield, it was expressed under the control of the strong chimeric cyt1A-p/STAB-SD promoter. In contrast expression achieved with the native promoter, overexpression yielded uniform bipyramidal crystals that were 4-fold larger on average than the wild-type crystal. In bioassays using the 4th instars of Cx quinquefasciatus, the strain producing the larger Cry19A crystal showed moderate larvicidal activity that was 4-fold (95% lethal concentration [LC95] = 1.9 ?g/ml) more toxic than the activity produced in the strain harboring the wild-type operon (LC95 = 8.2 ?g/ml) (CA). This basic research on the molecular biology of parasporal body formation in the mosquitocidal bacteria has identified proteins enabling the use of recombinant DNA technology to improve bacterial strains used in commercial formulations of bacterial mosquito larvicides by as much as ten-fold. Furthermore, laboratory studies on the potential evolution of resistance in anopheline and culicine mosquito species has identified novel combinations of mosquitocidal proteins such as Cyt1A and Cry11B that can be used to delay and overcome the evolution of resistance.