Molecular Mechanisms Of Resistance To Bacillus Thuringiensis Cry1Ac Toxin In Diamondback Moth, Plutella Xylostella(L.)

Bacillus thuringiensis(Bt), a gram-positive entomopathogen, is the most widely used microbial insecticide due to its highly specific activity and environmental safety. Insecticidal crystal protein(ICP) genes derived from Bt during its sporulation are widely used to be expressed in many transgenic crops(Bt crops) to control insect pests in the fields. However, massive and improper use of Bt biopesticide and large-scale plant Bt crops have led to field-evolved resistance in several pests. Insufficient understanding of insect resistance mechanisms to Bt seriously restricts its resistance management and the further exploitation and application of its novel insecticidal proteins, moreover, it also seriously threatens the future sustainable utilization of Bt transgenic crops. Therefore, given the economic and environmental importance of Bt biopesticides, unraveling insect molecular resistance mechanisms to Bt are of great importance for delaying insect field resistance evolution and sustainable utilization of Bt. The diamondback moth, Plutella xylostella(L.), is a global notorious pest of cruciferous crops that can rapidly evolve resistance to different kinds of insecticides and cause US $4-5 billion in management costs annually. Furthermore, P. xylostella is the first pest that has been reported to be developed field-evolved resistance to Bt sprays. Thus, P. xylostella has been an excellent model to study the complex molecular resistance mechanisms to Bt, especially when its whole genome sequencing has been completed and released recently. Although recent studies have found that resistance to Bt Cry1 Ac toxin in P. xylostella was genetically mapped to a mutation of an ATP-binding cassette(ABC) transporter gene(Px ABCC2) in the Bt R-1 resistance locus, comprehensive understanding of the molecular resistance mechanisms to Bt Cry1 Ac toxin in P. xylsotella still remains to be further explored. In this study, we used several Bt-susceptible and Bt Cry1 Ac toxin/Btk formulation-resistant P. xylsotella strains as study objects to deeply study the molecular resistance mechanisms to Bt Cry1 Ac toxin via a series of techniques and methods of insect toxicology, insect physiology, molecular biology, biochemistry, molecular genetics and bioinformatics. Taken together, our findings demonstrate that Bt Cry1 Ac resistance of P. xylostella is independent of the midgut cadherin gene(Px CAD) and ABC transporter gene(Px ABCH1), whereas it is tightly associated with differential expression of a midgut membrane-bound alkaline phosphatase gene(Pxm ALP) and an ABC transporter gene(Px ABCG1) outside Bt R-1 resistance locus and a suite of ABC transporter genes(Px ABCC1-3) inside Bt R-1 resistance locus, which is all trans-regulated by a constitutively transcriptionally-activated upstream gene(Px MAP4K4) in the mitogen-activated protein kinase(MAPK) signaling pathway outside Bt R-1 resistance locus. The results of this study are of great theoretical and practical significance for the development of novel Bt crops, the detection and integrated management of insect resistance to Bt crops or Bt biopesticides in the field and the further exploitation and application of novel Bt insecticidal proteins.The main research contents and conclusions are as follows:1. Rearing of P. xylsotella in the laboratory, resistance selection and bioassays Take advantage of good insect rearing conditions and the unique cabbage-radish seedling method, we developed a series of mature and standard artificial mass-rearing techniques of P. xylsotella in the laboratory, meanwhile, we also developed the standard Bt resistance selection and bioassay methods of P. xylsotella larvae. The bioassay result indicated that all the four Bt resistant strains have developed high-level resistance to Bt Cry1 Ac toxin/Btk formulation, which provided good tested P. xylsotella larvae for subsequent funtional experiments.2. Binding analysis between Bt Cry1 Ac toxin and midgut BBMV proteins of P. xylostella The binding between trypsin-activited Bt Cry1 Ac toxin and the midgut BBMV proteins of P. xylostella was semiquantitatively analyzed by Western Blot, and the result showed that reduced Cry1 Ac toxin binding to midgut BBMV proteins was a common phenotype observed in all resistant strains compared to the Bt susceptible reference strain, suggesting midgut receptor alterations(gene mutations or expression alterations) as the major Bt resistance mechanism in these P. xylostella resistant strains.3. Studies on relationship between midgut CAD gene and Bt resistance of P. xylostella The full-length c DNA sequences of the midgut Px CAD gene were PCR cloned and compared among all the Bt-susceptible and-resistant P. xylostella strains, and we didn’t find any contant mutations in this gene in all the resistant P. xylostella strains. Subsequently, we utilize the real-time quantitative PCR(q PCR) indicated that Px CAD transcript levels in the midgut did not significantly differ among all the susceptible and resistant P. xylostella strains. RNA interference(RNAi)-mediated suppression of cadherin gene expression did not affect larval susceptibility to Cry1 Ac toxin. Furthermore, genetic linkage assays confirmed that the cadherin gene is not linked to Cry1 Ac resistance trait in P. xylostella. Therefore, our findings demonstrate that the Cry1 Ac resistance of P. xylostella is independent of this midgut Px CAD gene.4. Studies on relationship between midgut ALP gene and Bt resistance of P. xylostella Detection of enzymatic activity of the alkaline phosphatase(ALP) and aminopeptidase N(APN) in the brush border membrane vesicles(BBMV) showed that ALP enzymatic activity dramatically reduced in BBMV from larvae of all resistant strains compared to the Bt susceptible strain, while APN activity did not differ, suggesting midgut ALP gene might be associated with Bt resistance in P. xylostella. Then, rapid amplificathion of c DNA ends(RACE) was used to clone the full-length c DNA sequence of the midgut membrane-bound alkaline phosphatase gene(Pxm ALP). Sequence comparison analysis showed that the Pxm ALP gene didn’t contain any constant mutation sites in all the resistant strains. q PCR results indicated that Pxm ALP transcript levels in the midgut significantly reduced in all the Bt resistant P. xylostella strains compared to the Bt susceptible strain. Enzyme linked immunosorbent assays(ELISA), immunolocalization and cytotoxicity assays confirmed that recombinant expressed Pxm ALP proteins in Sf9 cells can serve as the funtional receptor of Bt Cry1 Ac toxin. RNAi-mediated suppression of Pxm ALP gene expression significantly reduced larval susceptibility to Cry1 Ac toxin. Furthermore, genetic linkage assays confirmed that the reduced Pxm ALP gene expression is tightly linked to Cry1Ac resistance trait in P. xylostella. Therefore, our findings demonstrate that the Cry1 Ac resistance of P. xylostella is closely related to reduced expression of the midgut Pxm ALP gene.5. Studies on relationship between midgut ABCC1-5 gene and Bt resistance of P. xylostella We sucessfully assembled the approximately 3.15 Mb chromosome region representing Bt R-1 resistance locus assisted by linkage mapping data, genomic data of Bombyx mori and P. xylostella and the genetic synteny between P. xylostella and B. mori. Bioinformatics analysis found that the Bt R-1 resistance locus contains five ABC transport genes(Px ABCC1-5). The full-length c DNA sequences of the midgut Px ABCC1-5 genes were PCR cloned and compared among all the Bt-susceptible and-resistant P. xylostella strains, and we didn’t find any contant mutations in these genes in all the resistant P. xylostella strains. Subsequently, q PCR results indicated that Px ABCC2 and Px ABCC3 transcript levels in the midgut significantly reduced in the resistant P. xylostella strains compared to the Bt susceptible strain, while Px ABCC1 transcript levels in the midgut significantly increased in all the resistant P. xylostella strains compared to the Bt susceptible strain. RNAi-mediated suppression of Px ABCC2 and Px ABCC3 gene expression dramatically decreased larval susceptibility to Cry1 Ac toxin, and combinatorial suppression of Pxm ALP, Px ABCC2 and Px ABCC3 gene expression can further dramatically decreased larval susceptibility to Cry1 Ac toxin. More importantly, genetic linkage assays revealed that the Cry1 Ac resistance trait was tightly linked to down-regulation of Px ABCC2 and Px ABCC3 genes, whereas it was not linked to up-regulation of Px ABCC1 gene. Therefore, our findings demonstrate that the Cry1 Ac resistance of P. xylostella is closely related to reduced expression of the midgut Px ABCC2 and Px ABCC3 genes.6. Studies on relationship between midgut MAP4K4 gene and Bt resistance of P. xylostella Bioinformatics analysis also found that the Bt R-1 resistance locus contains three genes involved in the MAPK signaling pathway, among them, the upstream Px MAP4K4 gene in the MAPK signaling pathway locates extremely close to the three Px ABCC1-3 genes. The full-length c DNA sequences of the midgut Px MAP4K4 gene were PCR cloned and compared among all the Bt-susceptible and-resistant P. xylostella strains, and we didn’t find any contant mutations in the gene in all the resistant P. xylostella strains. Subsequently, q PCR results indicated that Px ABCC2 and Px ABCC3 transcript levels in the midgut increased in all the resistant P. xylostella strains compared to the Bt susceptible strain. RNAi-mediated suppression of Px MAP4K4 gene expression led to a transient increased Pxm ALP, Px ABCC2 and Px ABCC3 gene expression and decreased Px ABCC1 gene expression thereby restoring the susceptibility in resistant larvae. Therefore, our findings demonstrate that the Cry1 Ac resistance of P. xylostella is closely related to reduced expression of the midgut Pxm ALP and Px ABCC1-3 genes trans-regulated by the constitutively transcriptionally-activated upstream gene(Px MAP4K4) in the MAPK signaling pathway.7. Studies on relationship between midgut ABCG1 gene and Bt resistance of P. xylostella After re-analysis of our previous midgut transcriptome and RNA-Seq data, we found the expression level of a novel ABC transporter gene(Px ABCG1 or Pxwhite) significantly reduced in Bt-resistant P. xylostella strains. Subsequently, the full-length c DNA sequences of the midgut Px ABCG1 gene were PCR cloned and compared among all the Bt-susceptible and-resistant P. xylostella strains, and we didn’t find any contant mutations in this gene in all the resistant P. xylostella strains. q PCR results indicated that Px ABCG1 transcript levels in the midgut significantly reduced in all the resistant P. xylostella strains compared to the Bt susceptible strain. RNAi-mediated suppression of Px ABCG1 gene expression dramatically decreased larval susceptibility to Cry1 Ac toxin. Furthermore, genetic linkage assays confirmed that the reduced Px ABCG1 gene expression is tightly linked to the Cry1 Ac resistance trait in P. xylostella. Therefore, our findings demonstrate that Cry1 Ac resistance of P. xylostella is closely related to reduced expression of the midgut Px ABCG1 gene.8. Studies on relationship between midgut ABCH1 gene and Bt resistance of P. xylostella After re-analysis of our previous midgut transcriptome and RNA-Seq data, we also found the expression level of another novel ABC transporter gene(Px ABCH1) altered among Bt-susceptible and-resistant P. xylostella strains. Subsequently, the full-length c DNA sequences of the midgut Px ABCH1 gene were PCR cloned and compared among all the Bt-susceptible and-resistant P. xylostella strains, and we didn’t find any contant mutations in this gene in all the resistant P. xylostella strains. q PCR results indicated that Px ABCH1 transcript levels in the midgut did not significantly differ among all the susceptible and resistant P. xylostella strains. Sublethal RNAi-mediated suppression of Px ABCH1 gene expression using a relative low dose of ds RNA did not affect larval susceptibility to Cry1 Ac toxin. Therefore, the Cry1 Ac resistance of P. xylostella is independent of this Px ABCH1 gene. However, silencing of Px ABCH1 by a relatively high dose of ds RNA dramatically reduced its expression and resulted in larval and pupal lethal phenotypes in both Bt-susceptible and-resistant P. xylostella strains, which suggested that Px ABCH1 gene can be used as an excellent target for RNAi-based insect pest control and resistance management.In conclusion, these findings identify a series of key Bt Cry1 Ac resistance genes and reveal a novel trans-regulatory signaling mechanism responsible for modulating the expression of these pivotal genes in P. xylostella. Thus, this study provides the first comprehensive mechanistic description responsible for the field-evolved Bt Cry1 Ac resistance in P. xylostella.