Silencing GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt

Abstract

SUMMARYCotton is an important cash crop worldwide, and is a significant source of fiber, feed, foodstuff, oil and biofuel products. Considerable effort has been expended to increase sustainable yield and quality through molecular breeding and genetic engineering of new cotton cultivars. Given the recent availability of the whole-genome sequence of cotton, it is necessary to develop molecular tools and resources for large-scale analysis of gene functions at the genome-wide level. We have successfully developed an Agrobacterium-mediated virusinduced gene silencing (VIGS) assay in several cotton cultivars with various genetic backgrounds. The genes of interest were potently and readily silenced within 2 weeks after inoculation at the seedling stage. Importantly, we showed that silencing GhNDR1 and GhMKK2 compromised cotton resistance to the infection by Verticillium dahliae, a fungal pathogen causing Verticillium wilt. Furthermore, we developed a cotton protoplast system for transient gene expression to study gene functions by a gain-of-function approach. The viable protoplasts were isolated from green cotyledons, etiolated cotyledons and true leaves, and responded to a wide range of pathogen elicitors and phytohormones. Remarkably, cotton plants possess conserved, but also distinct, MAP kinase activation with Arabidopsis upon bacterial elicitor flagellin perception. Thus, using gene silencing assays, we have shown that GhNDR1 and GhMKK2 are required for Verticillium resistance in cotton, and have developed high throughput loss-of-function and gain-of-function assays for functional genomic studies in cotton.

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

“…However, CLA1 -silenced cotton plants hardly developed into the fibre or seed stage, which is likely due to strong defects on chloroplast development. The successful development of cotton VIGS provides an alternative to readily silence the genes of interest for loss-of-function assays and lays foundation for cotton functional genetics/genomics in the fast approaching post-genome era 8 .…”

Proteomic and Virus-induced Gene Silencing (VIGS) Analyses Reveal That Gossypol, Brassinosteroids, and Jasmonic acid Contribute to the Resistance of Cotton to Verticillium dahliae

“…The constructs were transformed into A. tumefaciens GV3101 by electroporation. TRV vectors were agro-infiltrated as described (31) into the cotyledons of 10-day-old seedlings of G. barbadense cv7124 or G. hirsutum cvYZ-1. The seedlings were then grown at 25°C with a 16 h/8 h light/dark photoperiod cycle in a controlled environment chamber.…”

“…Ve1 is the only R gene isolated using map-based cloning from tomato (Solanum lycopersicum) and has been shown to provide race-specific resistance to race 1 strains of V. dahliae and V. albo-atrum in tomato and Arabidopsis (17,29). Although several genes homologous to Ve1 have been cloned from cotton (30,31), it is unclear whether Ve1-mediated resistance signaling also exists in cotton.…”

A Pectin Methylesterase Inhibitor Enhances Resistance to Verticillium Wilt

“…Verticillium wilt leads to severe cotton yield loss each year and represents a major 156 concern for cotton producers (Gao et al, 2011). However, the physiology of plant 157 resistance against Verticillium,is largely unexplored.…”

“…These vector 682 derivatives were used to transform Agrobacterium tumefaciens GV3101 strain. The 683 cultures and cells were handled as described earlier (Gao et al, 2011). Cultures of 684 bacteria harboring pTRV1 and pTRV2 vectors were mixed in a 1:1 ratio, and the 685 cotyledons of 2-week-old cotton seedlings were infiltrated with a needleless syringe.…”