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.
Age synchronized fourth instar larvae of Bt toxin susceptible tobacco budworm strain YDK and resistant CXC, KCB, and YHD2 strains were exposed to sub-lethal doses of Cry 1Ac and/or Cry2Ab. Control groups of insects were either mock treated or fed on non-toxic Cry3Aa. Midgut tissues of the larvae were harvested at 30, 120, 480, and 720 min after exposure to toxins. Total RNA samples from the midgut tissues collected at various time points were used to hybridize glass-slide microarrays developed from the tobacco budworm transcriptome. Bioinformatic analyses were performed to identify genes differentially expressed in susceptible and resistant strains. Results from microarray analyses are being validated using quantitative real-time polymerase chain reaction (qRT-PCR) studies.