Verticillium Dahliae Infection Inspection And Transcriptomic And Phosphoproteomic Analysis In Cotton

Cotton Verticillium wilt is one of the most serious diseases in cotton,however,there is no effective chemical control method until now.Breeding resistant varieties is a most economic and effective way to control the cotton Verticillium wilt.Accurate identification of Verticillium wilt is crucial for genetic studies and selection of Verticillium wilt resistance.The widely-used dipping root inoculation method has been based on making wound on cotton roots to facilitate the ability of pathogens to invade cotton vascular system.The disease resistance identification are greatly influenced by wounding degree,inducing condition,enviroment,and operating process,and thus the resistance identification results are usually poor in consistency and reproducibility.Furtermore,the disease identification can not be conducted until the Verticillium wilt symptoms appear by using these classic methods,so it can not be used to inspect the early process of the pathogen invasion and expansion.The molecular mechanism of Verticillium wilt resistance in cotton is not clear,Together with few germplasms have been found to resist Verticillium wilt in upland cotton,little progress has been made in Verticillium wilt resistant breeding.In the present study,a susceptible upland cotton cultivar TM-1 and a resistant sea island cultivar Hai 7124 were used as plant materials.We transferred the GFP gene into Verticillium dahliae to generate Vd991-GFP-2 to study the pathogenesis of cotton Verticillium wilt by the method of pathogen quantitatively inoculation in a sterile medium.Moreover,we identified the differently expressed genes and the changes of protein phosphorylation in the root cells of TM-1 upon infection with Verticillium dahliae,which provided new clues to identify the key genes and protein phosphorylation(loci)resistance to Verticillium wilt.The results are as follows:1.The Verticillium wilt-resistant island cotton cv Hai7124 and susceptible upland cotton cv TM-1 were infected with Vd991-GFP-2 by the method of pathogen quantitatively inoculation in a sterile medium.Through fluorescence microscope,we found that the intensity of GFP fluorescence was much lower in Hai7124 than that of TM-1.Especially at 6 days post infection(dpi),GFP fluorescence was strongly detected in the root,stem and leaf of TM-1,however,it only can be detected in the root of Hai7124,and the disease index of Hai7124 was significantly lower than TM-1.The results indicated that Hai7124 has the ability to against the invasion and expansion of Verticillium dahliae.2.We used the RNA-seq methed to research the possible mechanisms of the Cotton-Verticillium wilt pathogen interactions by camparing infected and unifected TM-1.935 differentially expressed genes were identified,including 651 up-regulated genes and 284 down-regulated genes.The infection of Verticillium dahliae could induce the accumulation of lignin and phenylalanine metabolism as well as the up-regulation of WRKY,MYB,cytochrome P450 and E3 ubiquitin ligases which may be play an important role in the resistance process.These results provide candidate genes for further research on the molecular mechanism of cotton resistance to Verticillium wilt.3.The optimization method was established for extraction and enrichment of phosphorylated proteins in cotton.We have compared two different protein extraction and digestion methods and three methods of phosphopeptide enrichment,furthermore,discussed the different factors impacting on the quantity and specificity of phosphopeptide enrichment,including the starting amount of total protein and acidic modifiers used in affinity chromatography.The results showed that the most phosphopeptides were indentified by the method of Filter-Aided Sample Preparation(FASP)and phosphopeptides enrichment by using TiO2.When in the incubation procedure,the addition of lactic acid can improve the efficiency and specificity of phosphopeptide recovery from TiO2.In a certain range,the number of phosphopeptides is proportional to the amount of protein.4.Based on the above phosphorylation method and Label-free quantitative method,a comparative posphoprotein analysis was carried out between the infected and unifected TM-1 seedlings at 1dpi.262 different phosphoproteins were detected.These proteins are mainly involved in endocytosis,glycolysis/gluconeogenesis,starch and sucrose metabolism,carbon metabolism and biosynthesis of secondary metabolites.In addition,Abscisic acid signal transduction and ubiquitin-mediated protein degradation may play important roles in the resistance to Verticillium dahliae in TM-1.Our results provide clues and candidate proteins for further research on mechanism of cotton resistance to Verticillium wilt.