Cloning And Functional Analysis Of Verticillium Wilt Resistance Gene GaVdr1 From Asiatic Cotton Gossypium Arboretum L.

Verticillium wilt, caused by the vascular fungus Verticillium dahliae Kleb., is one of the most serious diseases during cotton growth in China. Once the fungus infects the cotton, it results in cotton buds and cotton boll abscission, even fall into a polished rod or death while the morbidity is serious. The resistance varieties mainly exist in wild germplasm resources of Asiatic cotton and Island cotton, due to the backcross selection and hybridization of barriers, it is difficult to use directly. The first reported Verticillium wilt resistance gene Vel was cloned from tomato (Lycopersicon esculentum). According to homologous cloning, there have been cloned SlVelgene from diploid tomato, StVe gene from wild tomato, mVel gene from Mentha and Gbvel from island cotton. In this study, we cloned a Verticillium wilt resistance gene GaVdrl from Asiatic cotton Gossypium arboretum L., which encodes a cellular receptor-like protein. The Verticillium wilt-resistant function of GaVdrl was verified by Virus induced gene silencing (VIGS) in Nanlingxiaozi and its over-expression in Nicotiana benthamiana. The expression of pathogen related genes, H2O2 production, callose deposition and cell death in transformants and wild type plants were analyzed after the inoculation with V991, BP2 and JR22.The Verticillium wilt-resistant characters of Asiatic cotton cv. Nanlingxiaozi (G. arboretum) was identified firstly and the susceptible upland cotton cv. Simian 2 (G. hirsutum.) was set as control. The results showed the diseases index of Nanlingxiaozi was only 15.6 and 30.6 respectively after inoculation with V991 and BP2 at 24days, while the disease index of Simian 2 was 73.2 and 71.7.Based on a cotton EST in Genbank, a lkb fragment was amplified from the upland cotton cv. Changkang, and its 5’and 3’unknown flanking sequence was isolated by genomewalking. Primers were designed according to the assembled sequence, then they were used to amplify the full sequence of GaVdrl gene from Asiatic cotton. GaVdrl has 9 LRR conserved domains and a trans-membrane domain in the C-terminal by protein structure prediction online. The amino acid sequence similarity of GaVdrl comparison with Vel and Ve2 is 47% and 44.9% respectively. The Real-time RT-PCR confirmed GaVdr1 was up-regulated by the V991, BP2 and JR22 inoculation.The VIGS was conducted in the Asiatic cotton and the expression level of GaVdrl was decreased about 85%. The silenced cotton plants showed more sensitive to V. dahliae. The disease index of silenced plants was 44.6,59.3 and 43.2 respectively while the control plants was 27.2,36.4 and 22.7 respectively at 30 days after inoculation with V991, BP2 and JR22.The over expression vector of GaVdrl was transformed to Nicotiana benthamiana, the transformed plants showed more resistant to V. dahlias. The incidence of disease was 21.2%,18.3%,12.9% in transgenic line 3 and 25%,22.5%,13.8% in transgenic line 22 while the wild type plants was 58.3%,54.5%,55.9% respectively after inoculation with V991, BP2 and JR22 at 24 days. We have also found the GaVdrl overexpression enhanced the resistance to P. nicotianae. The lesion area of transformed plants was 2.38cm2 while that of the WT plants was 7.28cm2 after P. nicotianae infection. The expression pattern of some pathogen related genes was varied in the transformed and wild type plants were found faster and higher in the transgenic plants than the wild type. The peak expression of PRb2-SA, ERF1-ET and LOX-JA was appeared at 24 hours after inoculation with V991 and at 72 hours by the infection of BP2 and JR22. The accumulation of callose, superoxide and cell deaths were observed much more in the transgnic plants after inoculation with V. dahliae. The results showed that GaVdrl owned the resistant function to V. dahlia and P. nicotianae, which might accomplished by H2O2 producing, callose deposition or cell death.