Mechanism Of GhSDH1-1 Positively Regulating Cotton Resistance To Verticillium Wilt

Verticillium wilt caused by the soil-borne fungus of Verticillium dahliae Kleb.is one of the most harmful diseases of cotton.The complex mechanism underlying cotton resistance to Verticillium wilt remains uncharacterized.Identifying an endogenous resistance gene may be helpful to control this disease.Previous studies have shown that succinate dehydrogenase?SDH?as a mitochondrial marker enzyme can catalyze succinate to fumarate,and it is involved in reactive oxygen species?ROS?-induced stress signaling pathway that is likely to be triggered by salicylic acid?SA?.Metabolomics and gene differential expression analysis in Gossypium hirsutum inoculated with Verticillium dahliae,we noticed that GhSDH1-1 gene may play an important role in the resistance of cotton to V.dahlia.Then we report GhSDH1-1 gene and its functional analysis in relation to the resistance of cotton to V.dahliae.Using virus-induced gene silencing?VIGS?and gene overexpression methods,the function of GhSDH1-1 gene was verified in cotton and Arabidopsis thaliana,and its defense response mechanism and related signaling pathways were further analyzed by biological and cytological methods.The results are as follows:We compared the SDH protein sequence of Arabidopsis thaliana with the SDH protein sequence of Gossypium hirsutum and identified 19 genes.To further study the functions of these genes,qRT-PCR analysis was performed to detect the expression pattern of SDH gene in cotton roots.The results showed that the expression level of Gh SDH1-1 increased significantly after being infected by Verticillium dahliae,and reached a peak at 6 and 12 hours after infection,it was 2.27 and 2.18 times that of the control group.SA can also induce the up-regulation of GhSDH1-1 expression.This indicated that GhSDH1-1 may play an important role in the defense response of Verticillium dahliae to cotton.Using VIGS technology to successfully silence GhSDH1-1 in cotton.Inoculation experiments showed that GhSDH1-1-silenced cotton were more susceptible to Verticillium dahliae than the controls.The leaves of silenced cotton showed the typical symptoms of verticillium wilt,yellowing,wilt,shedding,and even dead;while the control plants had significantly fewer yellow and wilt leaves,without severe shedding and dead plants.At 20 and 25 days after inoculation,the disease index of the TRV::GhSDH1-1plant was 27.2 and 40.4,which were significantly higher than the controls of 18.7 and23.0.After the gene was overexpressed in Arabidopsis thaliana,inoculation experiments showed that the transgenic lines had significantly increased resistance to Verticillium dahliae.These experiments indicated that GhSDH1-1 positively regulated cotton resistance to Verticillium wilt.Analysis of the silenced cotton plants revealed that the expression of TRV::Gh SDH1-1resistance-related genes POD,PPO,PAL,HIN,JAZ,NPR1,PR1,and PR3 decreased significantly.And TRV::GhSDH1-1 plants had more dead cells in the leaves,fewer active oxygen bursts,more brown changes in stem vascular bundles,more pathogens colonize,the content of H₂O₂ and NO reduced significantly,and the content of SA and fumaric acid was also significantly reduced.It showed that the defense response of TRV::GhSDH1-1plants to Verticillium wilt was obviously weakened.Subcellular localization test of GhSDH1-1 in N.benthamiana leaves,through constructing a fusion expression vector,and co-localizing with the mitochondrial marker gene Mt-rk,confirming that the gene is localized to mitochondria.After treating different cotton varieties with succinic acid and fumaric acid,investigate changes in disease resistance.It was found that after treatment with succinic acid,the disease resistance of Zhongzhimian 2 and Jimian 11 increased,and Yumian 21 had no significant change.After treatment with fumaric acid,the disease resistance of Zhongzhimian 2 and Yumian 21 decreased,and there was no significant change in Jimian 11.It was speculated that disease resistance may be related to enzyme activity of SDH.