Identification of Insect Resistance Genes by Cotton CDPK Gene Family Mutation Library

Cotton is an important source of natural fibers, vegetable fats and oils and feed, and makes a significant contribution to global development. Cotton has a long growth cycle and is threatened by a wide range of phytophagous insects during its reproductive period, which seriously affects cotton yield and fiber quality.

Insect pests have always been a key factor restricting the healthy development of the cotton industry. The traditional molecular breeding strategy for insect resistance in cotton is mainly to introduce exogenous Bacillus thuringiensis (Bt) insecticidal protein genes into the cotton genome to enhance the insect resistance of cotton. Although this method temporarily relieved the pressure of insect pests, with the long-term cultivation of Bt cotton, the target pests of Bt protein - cotton bollworm, red bollworm, etc. gradually evolved resistance, and the narrow spectrum of the Bt protein's resistance to aphids, thrips, blind stinkbugs, etc. almost no effect. With the intensification of Bt non-target insect pests, the search for new insect-resistant genes and the cultivation of new broad-spectrum insect-resistant varieties have become a major industrial demand for cotton production nowadays.

On August 12, 2014, Shuangxia Jin's group at Huazhong Agricultural University published a research article entitled “CRISPR/Cas9-mediated generation of a mutant library of cotton CDPK gene family for identifying insect-resistant genes” in Plant Communications. They developed a CRISPR/Cas9-mediated generation system to create a saturated mutant library of the CDPK gene, which they used to screen for endogenous insect resistance genes in cotton and analyze the insect resistance mechanism.

In the past, the group developed tools such as Cas9, Cas12a, Cas12b, CBE, ABE, ABE8e, dCas9-TV, Cas13a, etc., which are capable of precise modifications such as knock-out, knock-in and SNP point mutation in the target locus, and created new germplasm such as cottonseed free of cotton phenol, high oleic acid, herbicide resistance, insect resistance, color cotton, etc., which provides a complete toolbox and technical system for the study of the functional genomes of cotton and the molecular design of breeding.

Calcium-dependent protein kinases (CDPKs) are an important class of serine/threonine protein kinases that are widely distributed in multiple tissues and organelles of plants, and play important roles in several life processes. Among them, CDPKs play an important role in the signal transduction network of plants in response to phytophagous insect attack. Previous studies have demonstrated that multiple CDPKs from different species can modulate plant resistance to phytophagous insects by regulating gene expression or hormone signaling.

Figure 1. Construction a mutant library of the CDPK gene family in upland cotton. (Wang, et al., 2024)

Gene editing technology provides a powerful tool for deciphering gene function, and the development of genome sequencing has effectively promoted the use of gene editing tools. Due to its simple design, CRISPR/Cas9 can target most of the DNA sequence fragments in the entire genome, which enables rapid construction of large-scale knockout mutation libraries for gene function screening. Therefore, high-throughput loss-of-function screening based on the construction of mutation libraries by the CRISPR/Cas9 gene editing system has been rapidly applied to plants. For example, functional gene screens based on CRISPR mutant libraries have been performed for tomato, rice, soybean, and maize, and these studies have amply demonstrated the effectiveness of CRISPR-saturated mutation screening.

The research team first identified 82 GhCPKs in the terrestrial cotton genome through a comprehensive analysis of the GhCPKs gene family, and predicted that this gene family is involved in multiple life processes in cotton, laying the foundation for functional analysis of the GhCPKs family. Next, a saturation mutant library of the GhCPKs gene family was constructed by a mixed-pool library building technique with 246 sgRNAs targeting the 82 GhCPKs and 41 pairs of homologous GhCPKs, and a total of 518 independent T0 mutants were obtained. The target gene coverage reached 86.18%, and the average gene editing rate reached 89.49%. Then, 14 insect-resistant or susceptible mutants were screened by field phenotype identification combined with insect resistance experiments.

Finally, the cotton line cpk33/74 with the most pronounced insect-resistant phenotype (with homozygous genes GhCPK33 and GhCPK74 knocked out at the same time) was selected for further study, and the insect-resistant mechanisms of the screened candidate insect-resistant genes GhCPK33 and GhCPK74 were preliminarily explored.

GhCPK33 and GhCPK74 co-negatively regulated the increase of jasmonic acid content in cotton leaves induced by the oral secretion of Spodoptera litura with the Ca²⁺ in-flow in the chloroplasts and affected the expression of S -adenosylmethionine synthase (SAMS) genes, which enhanced the insect resistance of the mutant cpk33/74.

This study provides an effective strategy for constructing a mutant library of polyploid plant gene families and offers valuable insights into the role of CDPKs in plant-insect interactions. In addition, the large number of mutants provides valuable genetic resources for functional studies of CDPKs and for insect-resistant germplasm in cotton.

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Related Services

• Gene Knockout Services with CRISPR/CAS9 Technology
• Gene Knock-in Services with CRISPR/Cas9 Technology
• Genetic Engineering of Insecticidal Proteins
• Identification of Bt Pesticide Protein Genotypes
• Identification of Insecticide Protein Genes
• Genetic Engineering of Insect Resistance
• Development of Insecticidal Hormones
• Plant Insect Resistance Identification
• Plant Functional Gene Network Bioinformatics Analysis

Reference

1. Wang, F., et al. CRISPR/Cas9-mediated generation of a mutant library of cotton CDPK gene family for identifying insect-resistant genes. Plant Commun. 2024 Aug 12: 101047.