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Title: Alternative splicing and highly variable cadherin transcripts associated with field-evolved resistance of pink bollworm to Bt cotton in India

Author
item Fabrick, Jeffrey
item PONNURAJ, JEYAKUMAR - National Institute Fo Plant Health Management
item SINGH, AMAR - National Centre For Integrated Pest Management
item TANWAR, RAJ - National Centre For Integrated Pest Management
item UNNITHAN, GOPLALAN - University Of Arizona
item YELICH, ALEX - University Of Arizona
item LI, XIANCHUN - University Of Arizona
item CARRIERE, YVES - University Of Arizona
item TABASHNIK, BRUCE - University Of Arizona

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/25/2014
Publication Date: 5/19/2014
Publication URL: http://handle.nal.usda.gov/10113/58810
Citation: Fabrick, J.A., Ponnuraj, J., Singh, A., Tanwar, R.K., Unnithan, G., Yelich, A.J., Li, X., Carriere, Y., Tabashnik, B.E. 2014. Alternative splicing and highly variable cadherin transcripts associated with field-evolved resistance of pink bollworm to Bt cotton in India. PLoS One. 9(5):e97900.

Interpretive Summary: Genetically modified or transgenic crops producing insecticidal toxins of the bacterium, Bacillus thuringiensis (Bt), are key management tools for many insect pests. These Bt toxins kill many major insect pests while having little impact on beneficial insects or other plants or animals. Unfortunately, the development of resistance to Bt by insect pests threatens to diminish the long term benefits of Bt transgenic crops. To date, five pests have exhibited resistance to Bt crops in the field, including the pink bollworm to Bt cotton in India. Whereas alterations to a class of “cell adhesion proteins” (cadherins) that bind the Bt toxin are important for resistance to Bt in the laboratory, the underlying genetic basis for resistance occuring in the field is unknown. An ARS scientist at Maricopa, AZ and collaborators found completely new mutations to cadherin genes in Bt resistant pink bollworm collected from the field in India. Furthermore, many of these mutations were generated through a previously undocumented mechanism of altering the cadherin gene, called “alternative splicing”. These findings represent the first example of alternative splicing associated with Bt resistance that developed in the field. Because a variety of altered cadherin proteins was found in only a few field-collected pink bollworm, current molecular methods that rely on detection of specific cadherin mutations to monitor resistance may not be effective.

Technical Abstract: Evolution of resistance by insect pests can reduce the benefits of insecticidal proteins from Bacillus thuringiensis (Bt) that are used extensively in sprays and transgenic crops. Despite considerable knowledge of the genes conferring insect resistance to Bt toxins in laboratory-selected strains and in field populations exposed to Bt sprays, understanding of the genetic basis of field-evolved resistance to Bt crops remains limited. In particular, previous work has not identified the genes conferring resistance in any cases where field-evolved resistance has reduced the efficacy of a Bt crop. Here we report that mutations in a gene encoding a cadherin protein that binds Bt toxin Cry1Ac are associated with field-evolved resistance of pink bollworm (Pectinophora gossypiella) in India to Cry1Ac produced by transgenic cotton. We conducted laboratory bioassays that confirmed previously reported resistance to Cry1Ac in pink bollworm from the state of Gujarat, where Bt cotton producing Cry1Ac has been grown extensively. Analysis of DNA from 436 pink bollworm from seven populations in India detected none of the four cadherin resistance alleles previously reported to be linked with resistance to Cry1Ac in laboratory-selected strains of pink bollworm from Arizona. However, DNA sequencing of pink bollworm derived from resistant and susceptible field populations in India revealed eight novel, severely disrupted cadherin alleles associated with resistance to Cry1Ac. For these eight alleles, analysis of complementary DNA (cDNA) revealed a total of 19 transcript isoforms, each containing a premature stop codon, a deletion of at least 99 base pairs, or both. Seven of the eight disrupted alleles each produced two or more different transcript isoforms, which implicates alternative splicing of messenger RNA (mRNA). This represents the first example of alternative splicing associated with field-evolved resistance that reduced the efficacy of a Bt crop.