Function Analysis Of Cotton NADPH Oxidase GhRbohD Regulating Cotton Resistance To Verticillium Wilt

Verticillium wilt of cotton is a type of vascular disease,mainly caused by the soil fungus Verticillium dahliae.It is one of the most harmful and widely spread diseases in the world’s cotton planting areas,posing a serious threat to the yield and quality of cotton.Therefore,there is an urgent need to study the resistance of cotton to Verticillium wilt,especially to explore the molecular mechanism of cotton resistance to Verticillium wilt from the perspective of phytopathology.In the early stage of this study,we performed post-translational metabonomics and differential expression analysis on cotton plants inoculated with V.dahliae.We found that the expression of GhRbohD gene in cotton has changed significantly,and the expression and phosphorylation level of GhRbohD increased significantly.The phosphorylation level in disease-resistant varieties increased by 1.6 times.So it is speculated that GhRbohD plays an important role in the disease resistance of cotton to V.dahliae.In order to further explore the role of GhRbohD protein in cotton disease resistance,this study used relevant experimental methods such as plant pathology,molecular biology,and plant biotechnology to study its participation in the disease resistance mechanism of cotton Verticillium wilt.The results are as follows:1.The GhRbohD gene was cloned from the cotton variety Zhongzhimian No.2,with a total length of 2793 bp,encoding 930 amino acids,containing 6transmembrane proteins,the N-terminus contains the EF-hand structure,and the C-terminus contains NADPH and FAD binding motifs.2.Real-time quantitative analysis showed that after Zhongzhimian NO.2 was inoculated with V.dahliae(Vd080),the gene expression level increased significantly,and the expression level was the highest at 6 hpi.At the same time,the phosphorylation level at 1 hpi also increased to 1.6 times the original.3.The expression of the gene was effectively silenced in cotton through VIGS technology.After inoculation with Vd080,the plants after the silence treatment showed wilting,dead and even shedding leaves in a large area.About 20 days after inoculation,the disease indexes of TRV::00 and TRV::GhRbohD were 38.03 and59.62,respectively,which were significantly higher than those of the control plants.In addition,the active oxygen burst of TRV::GhRbohD plant leaves was greatly reduced,callus deposition ability was significantly reduced,lignin synthesis in cotton stems was also significantly reduced.The expression of defense genes Gh NOA1,Gh HIN1,Gh CHI,Gh PR3 and other related genes were also down-regulated.The results showed that the defense ability of cotton plants against V.dahliae was significantly reduced after GhRbohD was silenced.4.The morbidity of Rboh D transgenic Arabidopsis after inoculation with V.dahliae was significantly reduced.The amount of hyphae attached to the roots of overexpressing Arabidopsis was also significantly less than that of the wild type,which further proved that GhRbohD can enhance the resistance of plants to V.dahliae.5.Subcellular localization results show that GhRbohD is localized in the cell membrane.In addition,the interaction proteins Gh PBL9 and Gh RPL12 C of GhRbohD were screened out through the yeast two-hybrid experiment.The specific interaction mechanism needs to be further explored.