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INVESTIGATOR: Meihls, L.; Meihls, L. N.; Wang, P.

PERFORMING INSTITUTION:
BOYCE THOMPSON INSTITUTE
TOWER ROAD
ITHACA, NEW YORK 14853

MOLECULAR MECHANISMS OF RESISTANCE TO BACILLUS THURINGIENSIS CRY3BB1 TOXIN IN DIABROTICA VIRGIFERA VIRGIFERA (WESTERN CORN ROOTWORM)

NON-TECHNICAL SUMMARY: WCR is one of the most significant pests of corn in the United States. Its range includes most of the north central and north eastern United States, parts of southern Canada, northern Mexico, and an expanding region in Europe. Larvae feed on roots of corn causing yield loss. Traditionally, WCR in continuous corn cultivation has been controlled by the application of soil insecticides for larval control, or aerial application of insecticides for adult control to reduce the number of eggs laid. However, WCR is extremely adaptive and populations have become resistant to a number of insecticides as well as crop rotation. Transgenic corn constitutively producing Bt protoxins was approved for WCR management beginning in 2003. Since then, utilization of rootworm-resistant transgenic corn has increased dramatically, with ~20% of the current US corn crop expressing Bt toxins for rootworm control. Adoption of a single control strategy over a wide geographic area creates significant selection pressure that can lead to resistance development. Given WCR's proven ability to adapt to control measures and the significant selection pressure applied by widespread adoption of transgenic corn, WCR resistance to transgenic corn in the field is a real possibility. The goal of my research is to identify the mechanisms by which populations of WCR have become resistant to a Bt toxin (Cry3Bb1) and to identify genes contributing to resistance in WCR. Initial experiments will examine whether the insect immune system contributes to resistance to Cry3Bb1. Immune function can be inferred through an increase in melanization, a mechanism for dealing with foreign bodies. Increases in melanization were found to contribute to resistance to Bt in other insects. In addition, I will determine if Cry3Bb1 is processed in a similar manner in both resistant and susceptible insects. A reduction in the processing of the toxin in the insect midgut will decrease toxicity. This will be accomplished by examining protease activity in the midgut. Finally, binding of the toxin to receptors in the insect midgut will be examined. Mutations resulting in reduced binding have been shown to contribute to Bt resistance in other insects. Differences identified between resistant and susceptible colonies will be correlated with gene expression differences as identified by Illumina sequencing. Gene expression data and associated sequences will be combined with the WCR genome to identify genes contributing to resistance. WCR is one of the most significant pests of corn, thus maintaining the efficacy of available control measures is crucial for corn production in the United States. In addition, a number of promising biofuel crops may be impacted by WCR. One potential biofuel crop, Miscanthus, is a host for WCR. Populations of WCR performed equally well on Miscanthus and corn and recognized both plants a potential egg laying sites. Insect herbivory is one factor (besides abiotic factors) which limits America's agricultural potential. My results translate directly into ways to mitigate herbivory by a significant pest, leading to increased success of corn both as a food crop and a biofuel source.

OBJECTIVES: The western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte), one of the most significant insect pests of corn (Zea mays L.) in the United States, can decrease yield by as much as 45%. Since 2003, when transgenic corn plants expressing toxins derived from Bacillus thuringiensis Berliner (Bt) that are effective against WCR larvae became commercially available, there has been increasing evidence that this pest will develop resistance in the field. During my Ph.D. research at the University of Missouri-Columbia, I selected and characterized WCR laboratory colonies with elevated resistance to corn expressing the commercially relevant Cry3Bb1 Bt toxin. Now, as a postdoctoral fellow, I propose to bring this research to a new level by studying the molecular biology and genetics of Cry3Bb1 resistance in WCR. The goal of my research is to discover mechanisms responsible for WCR resistance to Bt toxin. At the Boyce Thompson Institute I will be working together with Georg Jander, who has expertise in the area of gene expression studies, genetic mapping, and insect behavior assays. My project will also involve collaboration with Ping Wang at the New York State Agricultural Experiment Station in Geneva, New York, who has extensive experience studying the physiology and biochemistry of Bt toxin resistance in Lepidoptera. Specific objectives and associated main hypotheses that I will address in the course of my research are: 1. Investigate possible Cry3Bb1 resistance mechanisms in WCR Hypotheses: 1.1 Immune responses differ between resistant and susceptible colonies. 1.2 Cry3Bb1is differentially processed by WCR proteases. 1.3 There is altered binding to the brush border membrane. 2. Identify gene expression differences between resistant and susceptible insects Hypotheses: 2.1 Gene expression changes underlie Bt toxin resistance. 2.2 Single nucleotide polymorphisms show linkage to resistance traits.