Insecticidal crystal toxins derived from the soil bacterium Bacillus thuringiensis (Bt) are widely used as biopesticide sprays or expressed in transgenic crops to control insect pests. However, large-scale use of Bt has led to field-evolved resistance in several lepidopteran pests. Resistance to Bt Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.), was previously mapped to a multigenic resistance locus (BtR-1). Here, we assembled the 3.15 Mb BtR-1 locus and found high-level resistance to Cry1Ac and Bt biopesticide in four independent P. xylostella strains were all associated with differential expression of a midgut membrane-bound alkaline phosphatase (ALP) outside this locus and a suite of ATP-binding cassette transporter subfamily C (ABCC) genes inside this locus. The interplay between these resistance genes is controlled by a previously uncharacterized trans-regulatory mechanism via the mitogen-activated protein kinase (MAPK) signaling pathway. Molecular, biochemical, and functional analyses have established ALP as a functional Cry1Ac receptor. Phenotypic association experiments revealed that the recessive Cry1Ac resistance was tightly linked to down-regulation of ALP, ABCC2 and ABCC3, whereas it was not linked to up-regulation of ABCC1. Silencing of ABCC2 and ABCC3 in susceptible larvae reduced their susceptibility to Cry1Ac but did not affect the expression of ALP, whereas suppression of MAP4K4, a constitutively transcriptionally-activated MAPK upstream gene within the BtR-1 locus, led to a transient recovery of gene expression thereby restoring the susceptibility in resistant larvae. These results highlight a crucial role for ALP and ABCC genes in field-evolved resistance to Cry1Ac and reveal a novel trans-regulatory signaling mechanism responsible for modulating the expression of these pivotal genes in P. xylostella.

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Published in PLOS Genetics, v. 11, no. 4, article e1005124, p. 1-32.

© 2015 Guo et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

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This work was supported by the National Natural Science Foundation of China (30871659; 30471159; 31201532), the 863 Program (2012AA101502), the Agricultural Science and Technology Innovation Program, the Beijing Key Laboratory for Pest Control and Sustainable Cultivation of Vegetables, and the Biotechnology Risk Assessment Grant Program competitive grants Nos. 2010-33522-21700 and 2014-33522-22215 from the USDA National Institute of Food and Agriculture. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

journal.pgen.1005124.s001.TIF (1165 kB)
S1 Fig. Nucleotide and deduced amino acid sequence of PxmALP gene.

journal.pgen.1005124.s002.TIF (3555 kB)
S2 Fig. Phylogenetic relationship of alkaline phosphatase genes in five insect orders.

journal.pgen.1005124.s003.TIF (2014 kB)
S3 Fig. Detection of heterologous expression of PxmALP in Sf9 cells and Cry1Ac binding to the recombinant protein.

journal.pgen.1005124.s004.TIF (450 kB)
S4 Fig. Determination of dsRNA concentration and time post-injection for optimum PxmALP silencing by RNAi.

journal.pgen.1005124.s005.TIF (1091 kB)
S5 Fig. Sequence variations in the PxABCC1, PxABCC2 and PxABCC3 cDNA regions within exons 4–11 among susceptible and resistant strains of P. xylostella.

journal.pgen.1005124.s006.TIF (1066 kB)
S6 Fig. Detection of cDNA isoforms in two domains of PxABCC1 (A), PxABCC2 (B) and PxABCC3 (C) genes in susceptible and resistant P. xylostella larvae.

journal.pgen.1005124.s007.TIF (1088 kB)
S7 Fig. Genomic structure and phylogenetic tree of ABCC1, ABCC2 and ABCC3 genes.

journal.pgen.1005124.s008.TIF (682 kB)
S8 Fig. Experimental design for analysis of linkage between resistance to Cry1Ac and differential expression of PxmALP and three PxABCC genes in the NIL-R strain of P. xylostella.

journal.pgen.1005124.s009.TIF (2202 kB)
S9 Fig. Analysis of linkage between PxABCC1, PxABCC2 and PxABCC3 isoforms with resistance to Cry1Ac in the NIL-R strain of P. xylostella.

journal.pgen.1005124.s010.TIF (1402 kB)
S10 Fig. The location of three PxABCC and PxMAP4K4 genes in the P. xylostella genome.

journal.pgen.1005124.s011.TIF (5750 kB)
S11 Fig. Amino acid sequence alignment of representative MAP4K4 homologs.

journal.pgen.1005124.s012.TIF (365 kB)
S12 Fig. Detection of Cry1Ac toxin-induced expression of PxMAP4K4 gene in the susceptible and resistant P. xylostella strains.

journal.pgen.1005124.s013.DOC (43 kB)
S1 Table. Susceptibility to Cry1Ac toxin or a B. thuringiensis var. kurstaki (Btk) formulation in larvae from five strains of Plutella xylostella.

journal.pgen.1005124.s014.DOC (74 kB)
S2 Table. List of primers used for PxmALP study.

journal.pgen.1005124.s015.DOC (123 kB)
S3 Table. Unigenes with high identity to PxmALP, five different PxABCC, and PxMAP4K4 genes identified in an RNA-seq experiment comparing gene expression in the DBM1Ac-S (MM), DBM1Ac-R (MK), and GZ-R (GK) strains of P. xylostella.

journal.pgen.1005124.s016.XLS (87 kB)
S4 Table. Genes in the BtR-1 resistance locus of P. xylostella.

journal.pgen.1005124.s017.DOC (53 kB)
S5 Table. List of primers used for PxABCC1 study.

journal.pgen.1005124.s018.DOC (67 kB)
S6 Table. List of primers used for PxABCC2 study.

journal.pgen.1005124.s019.DOC (60 kB)
S7 Table. List of primers used for PxABCC3 study.

journal.pgen.1005124.s020.DOC (50 kB)
S8 Table. List of primers used for PxABCC4 study.

journal.pgen.1005124.s021.DOC (50 kB)
S9 Table. List of primers used for PxABCC5 study.

journal.pgen.1005124.s022.DOC (86 kB)
S10 Table. Genomic structure of three P. xylostella ABCC genes in the BtR-1 locus.

journal.pgen.1005124.s023.DOC (39 kB)
S11 Table. Effect of silencing PxABCC2, PxABCC3 and multigenes on biological parameters of P. xylostella strain DBM1Ac-S.

journal.pgen.1005124.s024.DOC (55 kB)
S12 Table. List of primers used for PxMAP4K4 study.

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