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

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.

Figure 1. Sampling locations for pink bollworm field populations in India.

We screened DNA of 425 pink bollworm collected from all seven sites for cadherin resistance alleles r1, r2, and r3 (triangles). We sequenced cadherin cDNA and gDNA of 11 larvae from three sites: Akola (AMH), Anand (AGJ), and Khandwa (KMP) (circles) and conducted bioassays with 130 larvae from two sites: AMH and AGJ (squares). Based on cadherin DNA sequences (circles) and bioassay data (squares) from this study, red indicates evidence of resistance for AGJ and KMP; blue indicates evidence of susceptibility for AMH. Resistance was reported previously from four districts of Gujarat including Rajkot –.

Figure 2. Predicted cadherin proteins in pink bollworm from three populations in India.

We isolated and sequenced full-length PgCad1 cDNA clones from 11 individuals: three from Akola, Maharashtra (AMH-1 to AMH-3), three from Anand, Gujarat (AGJ-1 to AGJ-3), and five from Khandwa, Madhya Pradesh (KMP-4 to KMP-8). Predicted proteins are shown for cDNA of the PgCad1 susceptible (s) allele and 19 isoforms (r5A, r5B, etc.) of mutant alleles r5–r12. The amino-terminal membrane signal sequence (S), cadherin repeats (1–11), membrane-proximal region (MPR), transmembrane region (T), and cytoplasmic domain (C) are shown for the s allele. Red triangles indicate mutations predicted to cause loss of at least 33 amino acids (see Table 1). Truncated structures indicate proteins predicted from cDNA with premature stop codons. Gray indicates missing regions of proteins caused by deletions. The 3-bp deletion (corresponding to bp 72–74 in the s allele) that occurred in one sequence from AMH-3 and four sequences from KMP-8 as well as in two sequences from AGJ-1 and one sequence from KMP-7 is not shown.

Figure 3. Cadherin mRNA transcripts of a susceptible allele and three severely disrupted alleles found in three resistant pink bollworm larvae from Anand, Gujarat (AGJ).

Exons are numbered (1–34). Sequences are shown for exons missing from transcripts. Blue boxes show insertions, green boxes show deletions, and stars show premature stop codons. The six transcript isoforms shown are r5A-r7B (GenBank accession KJ480757-KJ480762).

Figure 4. Cadherin mRNA transcripts from five severely disrupted alleles found in four pink bollworm larvae collected on Bt cotton in Khandwa, Madhya Pradesh (KMP).

Transcript isoforms of alleles r8–r12 from individuals KMP-4, KMP-5, KMP-6, and KMP-7. Exons are numbered. Sequences are shown for exons missing from transcripts. Blue boxes show insertions, green boxes show deletions, black boxes show substitutions, and stars show premature stop codons. The 13 transcript isoforms shown are r8A-r12D (GenBank accession KJ480763-KJ480775).