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

Lei Xiong; Zhaoxia Liu; Jingge Li; Shuyuan Yao; Zeyun Li; Xuanhao Chen; Lingling Shen; Zhen Zhang; Yongbin Li; Qing Hou; Yuhang Zhang; Minsheng You; Zhiguang Yuchi; Shijun You

doi:10.3390/toxins15040273

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

Toxins (Basel). 2023 Apr 6;15(4):273. doi: 10.3390/toxins15040273.

Authors

Lei Xiong^{1

2

3}, Zhaoxia Liu^{1

2

3

4}, Jingge Li^{1

2

3}, Shuyuan Yao^{1

2

3}, Zeyun Li^{1

2

3}, Xuanhao Chen^{1

2

3}, Lingling Shen^{1

2

3}, Zhen Zhang^{1

2

3}, Yongbin Li^{1

2

3}, Qing Hou^{1

2

3}, Yuhang Zhang^{1

2

3}, Minsheng You^{1

2

3}, Zhiguang Yuchi⁵, Shijun You^{1

2

3}

Affiliations

¹ State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
² Ministerial and Provincial Joint Innovation Centre for Safety Production of Cross-Strait Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
³ Joint International Research Laboratory of Ecological Pest Control, Ministry of Education, Fuzhou 350002, China.
⁴ College of Oceanology and Food Science, Quanzhou Normal University, Quanzhou 362000, China.
⁵ School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Bacillus thuringiensis Toxins / metabolism
Bacillus thuringiensis* / genetics
Bacillus thuringiensis* / metabolism
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Bacterial Proteins / pharmacology
CRISPR-Cas Systems
Endotoxins / genetics
Endotoxins / metabolism
Endotoxins / pharmacology
Hemolysin Proteins / genetics
Hemolysin Proteins / metabolism
Hemolysin Proteins / pharmacology
Insect Proteins / metabolism
Insecticide Resistance / genetics
Larva
Moths*
Multidrug Resistance-Associated Protein 2

Substances

Bacillus thuringiensis Toxins
Endotoxins
Multidrug Resistance-Associated Protein 2
Hemolysin Proteins
Bacterial Proteins
Insect Proteins

Grants and funding

This research was funded by the central government-guided local science and technology development projects (2022L3087), National Natural Science Foundation of China (No. 31972271), the Fujian Natural Science Fund for Distinguished Young Scholars (2022J06013), the Outstanding Young Scientific Research Talents Program of Fujian Agriculture and Forestry University (xjq201905).