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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.

APPROACH: The goal of this 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. Briefly, hemolymph will be collected from WCR larvae via a capillary tube and the degree of melanization (observed as an increase in optical density) recorded over time. If sufficient hemolymph cannot be obtained using a capillary tube, the entire larvae will be ground up and used to measure melanization. In addition, I will determine if Cry3Bb1 is processed in a similar manner in both resistant and susceptible insects. The 77 kDa Cry3Bb1 protoxin must be proteolytically cleaved to become active against first instar larvae (the protoxin is not effective against later larval instars). 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. Toxin processing will be investigated both in vivo, by detecting the protein in whole larvae extracts using SDS-PAGE, and in vitro, by collecting midgut extracts from larvae to determine timing of toxin processing. 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. The processed Cry3Bb1 binds to the brush border membrane in larval midguts. The brush border membrane can be isolated as brush border membrane vesicles (BBMV). Using BBMV, total protein content will be quantified by Bradford assays and later separated by SDS-PAGE. To visualize tissues in the midgut where Cry3Bb1 binds, anti-Cry3Bb1 antibodies will be exposed to fixed larval midguts. Afterwards, fluorescent secondary antibodies can be used for visualization. Differences identified between resistant and susceptible colonies will be correlated with gene expression differences as identified by Illumina sequencing. To do this, cDNA libraries created from resistant and susceptible colonies will be sequenced and gene expression data then combined with the WCR genome to identify genes contributing to resistance.

PROGRESS: 2011/09 TO 2013/08
Target Audience: Available data has been shared with both industry and academic rootworm researchers. Changes/Problems: Objective 1.1: Western corn rootworm has a unique hemolymph defense strategy which makes traditional measures of immune response such as phenoloxidase activty or lysozyme activity difficult (1).As an alternative, the gene expression data obtained from the RNAseq analysis allows us to examine immune response-related genes (2). Objective 1.3: There was not enough time to investigate this aspect. 1. J. G. Lundgren, T. Haye, S. Toepfer, U. Kuhlmann, Biocontrol Sci Techn 19, 871 (2009). 2. D. Freitak, C. W. Wheat, D. G. Heckel, H. Vogel, BMC Biol 5, (Dec, 2007). What opportunities for training and professional development has the project provided? This project has allowed me to expand my training in bioinformatics. I attended two courses toprepare me to analyze next generation sequencing data: 2012 Cold Spring Harbor Computational & Comparative Genomics Course, Instruction on handling next generation sequencing data, covering everything from checking sequencing read quality to de novo assembly of transcriptomes. 2012 BTI Plant Bioinformatics Course, hosted by the BTI bioinformatics lab. Introduction to bioinformatics web based tools, databases, and SQL. In addition to training, this project allowed me to attend both the USDA NIFA meeting and the 2012 Entomological Society of America annual meeting (the major entomology meeting in the US). This allowed me to maintain my professional contacts and investigate potentialnew collaborations. How have the results been disseminated to communities of interest? Results of RNAseq analysis (objective 2) are still pending. Results of objectives 1 were scheduled to be presented at the Entomological Society of America annual meeting in November 2013. However, due toUSDA travel restrictions, I was not allowed to attend. What do you plan to do during the next reporting period to accomplish the goals? Data from theRNAseqexperiment will be available soon. This data will be analyzed, published, and presented at the 2014 annual Entomological Society of America meeting.

IMPACT: 2011/09 TO 2013/08
What was accomplished under these goals? 1.2 Cry3Bb1is differentially processed by WCR proteases. Western blot analysis revealed quantitative and qualitative differences in the processing of Cry3Bb1 in resistant vs. susceptible larvae. These data will be used in conjunction with RNAseq data tocompareprotease gene sequences and protease gene expression differences between resistant and susceptible larvae. 2.1 Gene expression changes underlie Bt toxin resistance. 2.2 Single nucleotide polymorphisms show linkage to resistance traits. RNAseq data are pending. These objectives will be addressed when the data become available.

PUBLICATIONS (not previously reported): 2011/09 TO 2013/08
1. Type: Journal Articles Status: Published Year Published: 2013 Citation: Meihls LN, et al. (2013) Natural variation in maize aphid resistance is associated with 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside methyltransferase activity. Plant Cell 25(6):2341-2355.
2. Type: Journal Articles Status: Published Year Published: 2013 Citation: Mijares V, Meihls LN, Jander G, & Tzin V (2013) Near-isogenic lines for measuring phenotypic effects of DIMBOA-Glc methyltransferase activity in maize. Plant Signal Behav 8(10).

PROGRESS: 2011/09/01 TO 2012/08/31
OUTPUTS: Activities Initial experiments to optimize insect melanization assay's have been conducted. Initial western blot experiments have been conducted to verify antibody activity and optimize the procedure. During this first year, I also mentored one undergraduate student for a three month period. This involved introducing the student to proper experimental design, data collection, and data analysis/interpretation. Events During the past year, I attended one conference where I presented results of my data. I also attended two workshops: the Boyce Thompson Institute Plant Bioinformatics Course and the Cold Spring Harbor Computational and Comparative Genomics course. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

IMPACT: 2011/09/01 TO 2012/08/31
Much of my research deals with proprietary materials which must be provided by Monsanto. After a long delay in obtaining the required material transfer agreements, the necessary materials finally arrived in August. Due to this delay, many of the proposed experiments are just now beginning. Despite initial difficulties, the year was very productive in other areas. Prior to my receiving the USDA NIFA grant, I was attempting to identify genes for resistance to Rhopalosiphum maidis, the corn leaf aphid. Using a corn mapping population, I was able to identify a gene involved in corn secondary metabolite pathway which affects aphid reproduction. To identify genes for resistance to corn leaf aphid, I utilized the publicly available maize nested association mapping population (NAM). This mapping population was derived by crossing 25 diverse inbred lines to a reference parent, B73. B73 was chosen because it was the first sequenced corn line. I began by screening the maize parental lines to determine if a phenotypic difference (measured by aphid reproduction) was detectable between the lines. Significant differences in aphid reproduction were seen in the parental lines, so I began screening the recombinant inbred lines (RIL's) of parental lines which showed susceptibility to corn leaf aphid. Using a publically available program, WinQTL cartographer, several quantitative trait loci (QTL) associated with corn leaf aphid reproduction were discovered. One QTL, located on chromosome one, was consistent across several RIL's screened. This interval included several genes of interest, most notably there were three O-methyltransferase genes identified. This was exciting, as the major secondary metabolite pathway in maize, the benzoxazinoid pathway, was known to involve several O-methyltransferases and is the primary defense against insect herbivores in maize. Analysis of the RIL's for individual components of the benzoxazinoid pathway identify a significant QTL associate with HDMBOA, which overlaps with the QTL identified for corn leaf aphid. Towards the end of the benzoxazinoid pathway, DIMBOA-glucoside can either be converted to HDMBOA-glucoside via an O-methyltransferase or be converted to DIMBOA via a β-glucosidase. Analysis of the parental lines for DIMBOA-glucoside and HDMBOA-glucoside content indicate that those parental lines which had high aphid reproduction also had high levels of HDMBOA-glucoside. As more data are collected, it is increasingly certain that the O-methyltransferases identified in this interval are the methyltransferases responsible for the conversion of DIMBOA-glucoside to HDMBOA-glucoside.

PUBLICATIONS: 2011/09/01 TO 2012/08/31
1. Meihls LN, Kaur H, Jander G (2012) Natural Variation in Maize Defense Against Insect Herbivores. Cold Spring Harb Symp Quant Biol. pp. Accessed December 6, 2012. DOI 2010.1101/sqb.2012.2077.014662.
2. Meihls LN, Higdon ML, Ellersieck MR, Gassmann AJ, Hibbard BE (2012)Greenhouse-selected resistance to Cry3Bb1-producing corn in three western corn rootworm populations. PloS ONE In Press.
3. Meihls LN, Higdon ML, Ellersieck MR, Hibbard BE (2011) Selection for resistance to mCry3A-expressing transgenic corn in western corn rootworm. J Econ Entomol 104: 1045-1054.