1a.Objectives (from AD-416)
The overall goal of this project is to investigate the risk of development of insect resistance to second generation transgenic crops expressing pyramided Bacillus thuringiensis toxins (Bt). Our work will be focused on characterizing mechanisms that result in crossresistance to Cry1Ac and Cry2Aa or Cry1Fa toxins. Both Cry2Ab and Cry1Fa toxins are expressed with Cry1Ac in second generation transgenic Bt cotton and corn targeted to control heliothine species. The proposed research will also test the possibility of enhanced survivorship of Cry1Ac-resistant insects when exposed to Cry2Ab toxin.
1b.Approach (from AD-416)
As part of our preliminary work we have been able to develop a microarray containing 14,584 expressed sequence tags (EST) from H. virescens (see preliminary data). This array included 144 gut-specific ESTs obtained from a subtraction library. We have expanded the microarray by adding another 124 ESTs from a gut cDNA library. The first goal of aim 1 is to analyze expression of gut genes in susceptible and Cry1Ac-Cry2Ab resistant H. virescens larvae. We propose using a microarray in a high throughput analysis using mRNA labeled with CyDyes. To focus our analyses on proteins directly involved in resistance to both Cry1Ac and Cry2A toxins, we plan to compare susceptible (COW or ARS) with selected strains that are resistant to both toxins (CXC and KCBhyb for COW and EMS for ARS) and a strain that is only resistant to Cry1Ac (YHD2-B).
Once the specific set of genes with altered expression is identified, we propose using quantitative real-time PCR (qRT-PCR) to confirm the differential expression levels in each strain. We expect this genomic approach to successfully identify genes whose products have a relevant role in the Cry intoxication process and whose alteration leads to resistance. These genes would be optimal candidates for the development of highly sensitive DNA-based resistance monitoring strategies.
Microarray design was improved to new manufacturer standards by selecting annotated set of expressed sequence tags to design triplicate probes and by randomly selecting 50 sequences to design 15 replicate probes each for use as internal normalizations controls. Fourth instar larvae of susceptible strain YDK and resistant strains CXC, YHD2, and KCB of Heliothis (H.) virescens were exposed to Bt toxins Cry3Aa, Cry1Ac, Cry2Ab, and the commercial biological insecticide Dipel by allowing to feed on 1ppm toxin containing diet. The insects were dissected and gut tissues were frozen on dry ice and stored in -80 Celsius freezer until used. Labeled complementary RNA was prepared from RNA extracted from gut tissues for hybridization with 24 microarrays. Expression data identified differential modulation of groups of genes involved in cell structure, stress, and development. A membrane bound from of alkaline phosphatase we previously identified as a binding target for Cry protein showed reduced levels of expression after exposure to Cry toxins. Reduced levels of messenger RNA for this protein were also observed in Bt resistant strains of H. virescens compared to susceptible strains. In addition, we have identified storage proteins that are differentially expressed in the gut tissues in response to intoxication. Relative expression levels of membrane bound alkaline phosphatase was identified as a biomarker to identify resistance to transgenic Bt cotton. The project was monitored through emails, telephone calls, and site visits.