Molecular Mechanism Of GhPR5K In Regulation The Disease Resistance Of Cotton To Verticillium Dahliae

Verticillium wilt is an important disease in cotton production in China and even the world.Unclear of the disease resistance mechanism of host is the bottleneck problem for its effective control.In this paper,a large number of differential proteins are obtained based on the analysis of the phosphorylation of the root proteins in resistant and susceptible cotton before and after the induction of V.dahliae.One of the receptor-like protein kinase GhPR5 K showed significant differences.The Virus Induced Gene Silencing(VIGS)technology was used to verify its function of resistance to V.dahliae.Its mechanism of defense was analyzed and interacting protein was screened.The main results were as follows:1.1716 phosphorylated proteins were detected by analyzing the modified protein quantitative of Zhongzhimian 2(a resistant variety)and Jimian 11(a susceptible variety)infected by V.dahliae Vd080,of which 639 proteins were significantly different,mainly involved in plant hormone signal transduction pathway,MAPK signaling pathway,AMPK signaling pathway,plant pathogen interaction and mTOR signaling pathways.Among them,Gh_D04G1868 had the highest difference multiple between the treatment and the control in Zhongzhimian 2,which reached 12.69.While there was no obvious difference in Jimian 11.Homology comparison found that the similarity between Gh_D04G1868 and the pathogen-associated protein kinase AtPR5 K of arabidopsis was 84.7%,so it was named GhPR5 K.Further analysis showed the expression of GhPR5 K in Zhongzhimian 2 was greatly improved after infected by Vd080.It was preliminarily concluded that GhPR5 K involved in the defense response to V.dahliae in cotton.2.The total length of GhPR5 K gene was obtained by gene cloning.It located in Dt_chr12 and its CDS include 1887 bp and encode 628 amino acids.The protein sequence analysis showed that there was a transmembrane region at the 252-274 base area,a signal peptide at the N-terminal and a serine/threonine concentration area at the C-terminal.3.The gene was successfully silenced in cotton by using VIGS technology.Inoculation experiment showed that the leaves of the TRV:GhPR5K plants were yellowing,wilting,even falling off and dying,which indicated that they were seriously damaged by V.dahliae.However the leaves of control plants showed light yellowing and wilting,without severe shedding and dead plants.The disease index(DI)of TRV:GhPR5K were 19.5 and 54.2 respectively which were significantly higher than that of control plants(10.3 and 25.2).This results showed that GhPR5 K regulated the resistance of cotton to V.dahliae positively.After that GhPR5 K overexpressed tobacco plants were obtained on selective medium.4.Defense reaction and related genes of TRV:00 and TRV:GhPR5K were analyzed after infected by Vd080.The results showed that,compared with TRV:00,in leaves of TRV:GhPR5K plants,the active oxygen bursts decreased and dead cells increased,and lignification degree in stem weakened significantly.More V.dahliae colonized in the stem of TRV:GhPR5K plant and the browning degree of vascular was also significantly increased.The qRT-PCR showed that the PR genes were significantly decreased in TRV:GhPR5K compared with TRV:00 with 4CL,CHI,PAL,POD,CAD,and C4H1 decreased by 70.95%,43.57%,55.33%,43.47%,78.09% and 71.89% respectively.It showed that the defense response to V.dahliae in TRV:GhPR5K plants was significantly weakened.5.The interaction protein DNA J of GhPR5 K in cotton was screened out based on yeast-two hybrid experiment.Bioinformatics analysis showed that there was a 22 amino acid signal peptide at the N-terminal,a transmembrane structure and a protein kinase domain at C-terminal which maybe the main domain interaction with GhPR5 K protein.The above studies partly explicated the molecular mechanism of GhPR5 K to defense V.dahliae.The GhPR5 K overexpression tobacco and its interacion protein still need to be further detected,verificated and analysed.