Cotton Resistance-related Genes High-throughput Screened By VIGS Technology Responsive To Verticillium Dahliae

Cotton Verticillium wilt(CVW), the most severe disease in cotton,which is a soilborn vascular spreading fungus disease caused byVerticillium dahliae. CVW causes dramatic yield losses worldwide,known as "cancer of cotton". Breeding resistance varieties should bethe most economical approach on CVM’s prevention and control.However, the resistance germplasm shortage and the obscuremolecular mechanism block this procedure, and result in difficultdefending disease. Thus, now a lot of studies still focus on uncoveringthe molecular mechanism of cotton resistance to verticillium wilt,and try to improve cotton resistance to CVW. Because of thelimitation of research materials and the lack of research techniques,it is difficult to explore the molecular mechanism of cotton resistanceto verticillium wilt at the genetic level. Virus-induced genesilencing(VIGS), based on loss-of-function concept to research genefunction in plant. VIGS provides a new opportunity to reveal genes’ functions which participate in the cotton-Verticillium dahliaeinteraction.Verticillium wilt resistant uplant cotton(Gossypium hirsutum)strain cv. Zhongzhimian KV3was used in this study, which wasbreeded by the Institute of Plant Protection, Chinese Academy ofAgricultural Sciences. On the basis of the results of suppressionsubtractive hybridization and gene chip, we selected106genes whichpossibly involved in cotton resistant to verticillium wilt. Then, wereveal the function of this gene through VIGS technology. Cotton leafcrumple virus (CLCrV) was used as VIGS vector. Sixty-two targetgene-silencing vectors were constructed, and classified into six types.Agrobacterium-mediated VIGS experiment was used to silencecorresponding genes, dipping-root method was used to inoculate V.dahliae V991, then detected the expression levels of these genes.Analyzing CVW resistance related genes can facilitate the discoveryof the resistance molecular mechanism, and help to breed resistancevarieties. The main results were showed as follows:1. CLCrV-induced silence system were used to silence CHLI andCLA1. Plants showed obvious phenotypic changes (yellowing andphotoblenching) after gene silencing. In the target-gene silencedplant, the expression of target gene mRNA were reduced significantly.There has no obvious difference between the inoculated V. dahliae V991wild type plants and the VIGS with empty-vector plants. Thisreflects the CLCrV-induced silence system can silencing target genein Zhongzhimian KV3, which could be used to study the function oftarget gene;2. Utilizing VIGS technology, high-throughput screening related geneswhich resistance verticillium wilt in cotton. After three rounds ofrepeat screening, eight related genes were selected in cotton. Sixgenes are regulated resistance cotton verticillium wilt positively,Gh39(bZIP), Gh24(GRAS), Gh58(WRKY), Gh55(EIN2), Gh114(XTH),Gh97(Drigent like protein), respectively. Two negative regulator incotton to against verticillium wilt, Gh15(bZIP), Gh31(NAC5);3. We choose three genes for function annotation and expressioncharacteristics analysis. The Gh55(EIN2) is a positive regulatoryfactor in ethylene signaling pathways. After silenced this gene,inoculated V. dahliae plants’ symptoms increased significantly. Thisindicate that this gene is a positive regulator of CVW. Silenced Gh97(Dirigent like protein) plants inoculated V. Dahliae, the plants’symptoms also increased significantly, cluster analysis showed thatthe gene belongs to DIRb/d family, preferentially expressed in cottonroots, these results suggest that Gh97may mediated cell lignificationin roots, so as to block the invasion and reproduction of fungalpathogen V. dahliae, enhanced resistance of cotton to verticillium wilt. Gh114is xyloglucan endotransglucosylase/hydrolase (XTH),After inoculated V. Dahliae XTH-silenced plants, the plants were moresensitive to V. Dahliae, this indicated that GhXTH involved inresistance to verticillium wilt by changing the structure of plant cellwall in cotton.