Multi-Toxin Resistance Enables Pink Bollworm Survival on Pyramided Bt Cotton

Transgenic crops producing Bacillus thuringiensis (Bt) proteins kill key insect pests, providing economic and environmental benefits. However, the evolution of pest resistance threatens the continued success of such Bt crops. To delay or counter resistance, transgenic plant "pyramids" producing two or more Bt proteins that kill the same pest have been adopted extensively. Field populations of the pink bollworm (Pectinophora gossypiella) in the United States have remained susceptible to Bt toxins Cry1Ac and Cry2Ab, but field-evolved practical resistance to Bt cotton producing Cry1Ac has occurred widely in India. Here we used two rounds of laboratory selection to achieve 18,000- to 150,000-fold resistance to Cry2Ab in pink bollworm. Inheritance of resistance to Cry2Ab was recessive, autosomal, conferred primarily by one locus, and independent of Cry1Ac resistance. We created a strain with high resistance to both toxins by crossing the Cry2Ab-resistant strain with a Cry1Ac-resistant strain, followed by one selection with Cry2Ab. This multi-toxin resistant strain survived on field-collected Bt cotton bolls producing both toxins. The results here demonstrate the risk of evolution of resistance to pyramided Bt plants, particularly when toxins are deployed sequentially and refuges are scarce, as seen with Bt cotton and pink bollworm in India.Peer reviewed: YesNRC publication: Ye

Efficacy of Bt toxins Cry1AbMod, Cry1Ac and Cry2Ab singly and in combinations against a susceptible strain of pink bollworm (APHIS-S) (see Methods for details).

a<p>All mortality values are adjusted for control mortality.</p>b<p>Observed mortality</p>c<p>Expected mortality for combinations of two or three toxins</p>d<p>Observed mortality - expected mortality; synergism causes positive values and antagonism causes negative values</p>e<p>Probability that the difference between observed and expected mortality occurred by chance based on Fisher’s exact test </p

Efficacy of native Bt toxins Cry2Ab, Cry1Ab, Cry1Ac and genetically modified Bt toxins Cry1AbMod, and Cry1AcMod against a resistant strain (BX-R) and a susceptible strain (APHIS-S) of pink bollworm.

a<p>Slope of the concentration-mortality line with its standard error in parentheses</p>b<p>Concentration killing 50% with 95% fiducial limits in parentheses, in µg toxin per ml diet.</p>c<p>Resistance ratio, the LC₅₀ for a strain divided by the LC₅₀ for APHIS-S for the same toxin.</p>d<p>Survival at 10 µg toxin per ml diet adjusted for control mortality, n = 40 to 120 (mean = 82) larvae for each estimate.</p>e<p>Not available</p>f<p>90% fiducial limits, 95% fiducial limits not available</p

Potency of modified Bt toxins relative to native Bt toxins against resistant and susceptible strains of pink bollworm.

a<p>LC₅₀ of a native toxin divided by the LC₅₀ of a modified toxin (based on data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080496#pone-0080496-t001" target="_blank">Table 1</a>). Potency ratios > 1 indicate the modified toxin was more potent than the native toxin; potency ratios <1 indicate the modified toxin was less potent than the native toxin.</p

Alternative Splicing and Highly Variable Cadherin Transcripts Associated with Field-Evolved Resistance of Pink Bollworm to Bt Cotton in India

<div><p>Evolution of resistance by insect pests can reduce the benefits of insecticidal proteins from <i>Bacillus thuringiensis</i> (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 (<i>Pectinophora gossypiella</i>) 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.</p></div

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

<p>Transcript isoforms of alleles <i>r8</i>–<i>r12</i> 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 <i>r8A</i>-<i>r12D</i> (GenBank accession KJ480763-KJ480775).</p

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

<p>We isolated and sequenced full-length <i>PgCad1</i> 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 <i>PgCad1</i> susceptible (<i>s</i>) allele and 19 isoforms (<i>r5A, r5B,</i> etc.) of mutant alleles <i>r5</i>–<i>r12</i>. 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 <i>s</i> allele. Red triangles indicate mutations predicted to cause loss of at least 33 amino acids (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097900#pone-0097900-t001" target="_blank">Table 1</a>). 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 <i>s</i> 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.</p

Similarity between transposons and the insertion in intron 20 of the <i>r5 PgCad1</i> allele.

a<p>Nucleotide position in the 3,827-bp fragment from pink bollworm cadherin (which includes the 3,120-bp insertion in the <i>r5</i> allele) cloned from AGJ-1 gDNA using primers 20PgCad5 + 81PgCad3 (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097900#pone.0097900.s008" target="_blank">Figure S8</a>).</p>b<p>LYDIA_LTR, long terminal repeat retrotransposon from LYDIA, a gypsy-like endogenous retrovirus from <i>Lymantria dispar</i>; TED, internal part of retrotransposon TED inserted in <i>Autographa californica</i> nuclear polyhedrosis virus; CoeSINE4, coelacanth SINE non-long terminal repeat retrotransposon from <i>Latimeria chalumnae</i>; HaSE3, SINE non-long terminal repeat retrotransposon from <i>Helicoverpa armigera</i>; HATN3_DR, nonautonomous DNA transposon from <i>Danio rerio</i>; Transib-4_DBp, Transib-type DNA transposon from the <i>Drosophila bipectinata</i> genome; ISL2EU-3_HM, autonomous ISL2EU DNA transposon from <i>Hydra magnipapillata</i>.</p>c<p>Nucleotide position in the transposon sequence.</p>d<p>Orientation of the insertion sequence relative to the corresponding sequence in the transposon; comp. indicates complementary.</p>e<p>Similarity between the fragment sequence and the corresponding sequence in the transposon; calculated as the number of exact matches/(alignment length - total length gaps in the fragment sequence - total length of gaps in the transposon sequence + total number of gaps).</p>f<p>Alignment score from BLAST.</p

Nineteen transcript isoforms of eight disrupted cadherin alleles in seven pink bollworm larvae from two populations in India: Anand, Gujarat (AGJ) and Khandwa, Madhya Pradesh (KMP).

a<p>Mutations shown in bold cause premature stop codons.</p>b<p>Region of cadherin protein where major mutations occur (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097900#pone-0097900-g002" target="_blank">Figure 2</a>).</p>c<p>The 478-bp deletion found in <i>r5A</i>, <i>r5B</i> and <i>r5C</i> is caused by insertion of 3,120 bp similar to transposons (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097900#pone-0097900-t002" target="_blank">Table 2</a>), causing the loss of exons 21–24 from gDNA and cDNA.</p>d<p>The 3-bp deletion in <i>r5B</i> and <i>r12B</i> is caused by mis-splicing, occurs at exon-intron splice junction 1, and is found in both <i>r</i> and <i>s PgCad1</i> alleles.</p>e<p>gDNA from AGJ-2 was not available to compare with cDNA, but the absence of exons 8–13 occurs exactly at the exon-intron junctions, suggesting that mis-splicing occurred.</p>f<p><i>r9A</i> includes A-to-G (I) RNA editing at base position 2,289 and results in the introduction of a premature stop codon (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097900#pone-0097900-g004" target="_blank">Fig. 4</a>). The 165-bp deletion causes the loss of exon 32.</p>g<p>The 23-bp deletion corresponds to the final 23 nucleotides of exon 20 in cDNA clone KMP-6_3.</p>h<p>The single base insertion introduces a premature stop codon and truncates the mRNA transcript in CR11.</p>i<p>The 118-bp deletion causes the loss of exon 11 resulting in the introduction of a premature stop codon and truncates the mRNA transcript in CR4.</p>j<p>The 11-bp deletion occurs in the membrane signal sequence of <i>r12C</i> and <i>r12D</i> transcripts resulting in the introduction of a premature stop codon.</p>k<p>The 148-bp deletion causes the loss of exon 5 in mRNA transcript between the membrane signal sequence and CR1.</p>l<p>The 230-bp deletion causes the loss of exons 11–12 in mRNA transcript found in CR4.</p

Sampling locations for pink bollworm field populations in India.

<p>We screened DNA of 425 pink bollworm collected from all seven sites for cadherin resistance alleles <i>r1</i>, <i>r2</i>, and <i>r3</i> (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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097900#pone.0097900-Dhurua1" target="_blank">[29]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097900#pone.0097900-Monsanto1" target="_blank">[30]</a>.</p