The Establishment And Application Of Cotton Leaf Crumple Virus-induced Gene Silencing System

Cotton is one of the most important economic crops worldwide. Functional analysis of genes involved in disease resistance and tolerence to abiotic and biotic stresses will be useful to improve the quality and yield of cotton fibers. Due to its long growth cycle, large genome size and recalcitrance to stable transformation, traditional methods for the analysis of gene function in cotton plants through T-DNA insertion mutagenesis or chemical-induced mutagenesis are difficult and labour intensive. Hence, development of a convenient and high-throughput reverse genetics system for gene function analysis in cotton is becoming urgent.Here, we report a cotton leaf crumple virus (CLCrV)-based vector and its application in gene function analysis through virus-induced gene silencing (VIGS) and overexpression of microRNAs (miRNAs), small tandem target mimic (STTM) and artificial miRNA (amiRNA) in cotton via an Agrobacterium-mediated infiltration approach. Using this system, we were able to efficiently silence two endogenous genes, magnesium chelatase subunit/(CHLI) and elongation factor-1α(EF-1α), in Gossypium species and the Bacillus thuringiensis cryl A gene in transgenic cotton. Furthermore, our results show that this vector can be used to ectopically express endogenous miR156in G. hirsutum, causing a reduction in miR156-targeted RNA transcripts resulting in the development of abnormal leaf phenotypes. Ectopic expression of miR165/166STTM with this vector led to downward curling and crumpled leaves, and a significant increase in the miR165/166target mRNAs. This versatile system is easy to use and can provide more uniform and persistent gene silencing in multiple cotton cultivars, thereby providing a powerful approach for gene discovery in cotton.We further used the CLCrV VIGS vector to study gene functions in cotton plants. Previous studies showed that LysM-containing kinases (LYK) are receptor proteins that play important roles in plant innate immunity against fungi infection. To decipher the role of cotton LYK in resistance against Verticillium wilt, five cotton LYK genes, designated as Gh-LYK1to Gh-LYK5, were obtained by bioinformatic analysis. Using quantitative reverse transcription PCR, we found that expression of Gh-LYK1and Gh-LYK2were up-regulated in resistant cotton cultivar (c.v.3503) during Verticillium dahliae infection. Silencing of Gh-LYK1or Gh-LYK2in c.v.3503induced more sensitive symptoms to V. Dahliae, indicating that these two genes are important for resistance to Verticillium wilt. Using chitin binding assay, we showed that both Gh-LYK1and Gh-LYK2ectodomain was able to bind chitin. However, unlike Gh-LYK1, the kinase domain of Gh-LYK2is inactive in the kinase assay. Moreover, sequence analysis showed that transmembrane domain(TM) was deficient in Gh-LYK2isolated from two susceptible cultivar c.v.Zn905and c.v.Junl.. Silencing of Gh-LYK2in c.v.Zn905has no effect on the resistance to Verticillium wilt, suggesting that the truncated Gh-LYK2is non-functional in chitin-triggered immunity. Subcellular localization of Gh-LYK2showed that the truncated Gh-LYK2localized at plasma membrane. We conclude that part of the truncated Gh-LYK2might insert into the phospholipid bilayer and inhibit the recognition of chitin by LysM domains. Further research will be carried out to elucidate the role of TM domain in the recognition of chitin.