Analysis of the Effect of Plutella xylostella Polycalin and ABCC2 Transporter on Cry1Ac Susceptibility by CRISPR/Cas9-Mediated Knockout

Abstract

Many insects, including the Plutella xylostella (L.), have developed varying degrees of resistance to many insecticides, including Bacillus thuringiensis (Bt) toxins, the bioinsecticides derived from Bt. The polycalin protein is one of the potential receptors for Bt toxins, and previous studies have confirmed that the Cry1Ac toxin can bind to the polycalin protein of P. xylostella, but whether polycalin is associated with the resistance of Bt toxins remains controversial. In this study, we compared the midgut of larvae from Cry1Ac-susceptible and -resistant strains, and found that the expression of the Pxpolycalin gene was largely reduced in the midgut of the resistant strains. Moreover, the spatial and temporal expression patterns of Pxpolycalin showed that it was mainly expressed in the larval stage and midgut tissue. However, genetic linkage experiments showed that the Pxpolycalin gene and its transcript level were not linked to Cry1Ac resistance, whereas both the PxABCC2 gene and its transcript levels were linked to Cry1Ac resistance. The larvae fed on a diet containing the Cry1Ac toxin showed no significant change in the expression of the Pxpolycalin gene in a short term. Furthermore, the knockout of polycalin and ATP-binding cassette transporter subfamily C2 (ABCC2) genes separately by CRISPR/Cas9 technology resulted in resistance to decreased susceptibility to Cry1Ac toxin. Our results provide new insights into the potential role of polycalin and ABCC2 proteins in Cry1Ac resistance and the mechanism underlying the resistance of insects to Bt toxins.

Keywords: ABCC2; Bacillus thuringiensis; CRISPR/Cas9; polycalin.

Figure 1

Structure of Pxpolycalin gene in P. xylostella. (A) Genomic structure of the Pxpolycalin gene in P. xylostella. Green boxes indicate the exons, and the spaces between the two boxes indicate the introns. The figure is drawn to scale, and the corresponding scale bar is shown. The dotted boxes above exons 22, 35, and 49 are fixed site mutations between G88 and Cry1S1000 strains. (B) NCBI conserved domain database (CDD)-based annotation of the Pxpolycalin gene sequence.

Figure 2

R_Po mutation of Pxpolycalin from the Cry1S1000 strain. (A) The difference in the genotype of Pxpolycalin between the G88 and Cry1S1000 strains based on PCR and sequencing. S_PoS_Po is the sequencing result of the G88 strain, R_PoR_Po is the sequencing result of the Cry1S1000 strain, and S_PoR_Po is the sequencing result of hybrid F₁ between G88 and Cry1S1000 strains. (B) Alignment of gDNA sequences of the S_Po allele and the R_Po allele. Sequences highlighted in orange are exons 26 and 27.

Figure 3

Neighbor-joining tree based on the amino acid sequences of Pxpolycalin in P. xylostella and other insects. The scale bar indicates the phylogenetic distance (bootstrap = 1000). The polycalin protein of P. xylostella is represented by a red asterisk.

Figure 4

Relative polycalin transcription detected by qRT-PCR in G88 susceptible and Cry1S1000 resistant larvae of P. xylostella. Asterisks (*) indicate significant difference for p < 0.05. The data were evaluated by Student’s t test using SPSS v.26.0.

Figure 5

Relative expression level of Pxpolycalin in different developmental stages and different tissues of P. xylostella. (A,B) Relative expression level of Pxpolycalin in G88 strain; (C,D) Relative expression level of Pxpolycalin in Cry1S1000 strain. E: egg; L1: 1st instar larvae; L2: 2nd instar larvae; L3: 3rd instar larvae; 4th instar larvae; PP: prepupae; P: pupae; AF: female adults; AM: male adults. MG: midgut; IN: integument; HD: head; MT: malpighian tubules; SG: silk gland. Expression level was calculated according to the value of the egg (E) or midgut (MG), which was given an arbitrary value of 1. Data was represented with three biological replicates and each replication was repeated three times. The bars were shown as mean ± SD. Different letters above the bars indicate significant differences in different development stages or different tissues. Statistically significant differences were analyzed with one-way ANOVA (Tukey’s test for multiple comparisons, p < 0.05).

Figure 6

Effect of LC₁₀ of Cry1Ac on the relative expression of PxABCC2 (A) and Pxpolycalin (B) in the 3rd instar P. xylostella of the G88 strain. The results are shown as the mean ± SD. Asterisks above error bars represent significant differences (**, p < 0.01; ***, p < 0.001) using Student’s t-test.

Figure 7

Mutagenesis of Pxpolycalin induced by CRISPR/Cas9. Partial sequences from the G88 and homozygous Pxpolycalin mutant showing the indels at the target sequence (polycalin-sgRNA) in exon 2 of Pxpolycalin.

Figure 8

Relative mortality of different types of mutant homozygous strains after Cry1Ac toxin treatment. G88 indicates the Cry1Ac-susceptible strain. PoKO21KI16 indicates the homozygous strain for the Pxpolycalin mutation. A3KO28 indicates the homozygous strain for the PxABCC2 mutation. The bars were shown as mean ± SD. Different letters above the bars indicate significant differences in different types of mutant homozygous strains. Statistically significant differences were analyzed with one-way ANOVA (Tukey’s test for multiple comparisons, p < 0.05).

Figure 9

Diagram showing the genetic linkage analysis strategy of Pxpolycalin with Cry1Ac resistance. A female G88 (susceptible) was crossed with a male Cry1S1000 (resistant) to produce F1 progeny. Next, the hybrid F1 was backcrossed with Cry1S1000. Half of the backcross family a and b were treated with 0.5 μg/mL Cry1Ac toxin, and the other half together with the surviving larvae/adults from the treated group were sampled directly.

Figure 10

The strategy of homozygous mutant line construction. The unmated adult of the G₀ generation was hybridized with the G88 strain, and the gDNA of the adult G₀ was extracted after oviposit. Then, the mutant was detected by PCR sequencing, and the offspring of the same mutation type was selected to obtain homozygous mutant line.