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.
A microarray containing 14,834 features was developed using expressed sequence tags (ESTs) obtained from various sequencing projects. The Cry toxin resistant Ethyl Methyl Sulfonate (EMS) strain of Heliothis virescens was used to profile gene expression patterns in response to Cry toxin feeding. Age and size synchronized 4th instar larvae of EMS strain and susceptible ARS strain was exposed to sub-lethal concentrations of Cry1Ac and Cry2Ab toxins with larvae exposed to non-lethal Cry3Aa was used as the controls. Midguts were dissected after exposure to toxins for 30, 120, 420, and 720 minutes and frozen at -80°C until used for RNA extractions. Agilent microarrays were hybridized with control and experimental samples labeled with AlexaFluor-555 and AlexaFluor-647 fluorescent dyes, respectively. Microarrays were scanned, data extracted, and analyzed to identify changes in gene expression patterns with the length of exposure to Cry toxins. Expression patterns of groups of genes implicated in binding to Cry proteins, such as aminopeptidases, alkaline phosphatases, and cadherins, during intoxication process were closely examined. Expression profiles indicated that Cry1Ac and Cry2Ab feeding triggered differential expression of a large number of genes compared to those fed with non-lethal Cry3Aa toxin. This project was monitored by site visit, conference call and e-mail.