Functional Analysis Of Verticillium Wilt Resistant Related Gene GbSBT1 And Its Resistance Mechanism Study In Cotton

Verticillium wilt,a vascular disease caused by Verticillium dahliae,leads to great loss of cotton yield in our country every year.V.dahliae can survive for many years as the type of microsclerotia in soil,and parasite a varities of plant as the hosts.Because there is limited resource of resistant genes,it is difficult to isolate the resistant genes and transfer them into commercial cotton varieties.V.dahliae can secrete different effectors to help them invading xylem vessels.In the long-term evolution process,it is pivotal for plants to recognize the pathogens effectively during the defense process.Therefore,the analysis of the mutual recognition between cotton and V.dahliae is important to limit pathogens'infection.In this study,the verticillium wilt resistance related gene GbSBT1 was cloned and identified from island cotton Pima-90.Gene function analysis of virulence related gene Tri15 was also analyzed to understand the recognition process between cotton and V.dahliae.These results could lay a foundation for exploring the cotton resistant mechanism.The main contents and results are as follows:1.GbSBT1 could transduce resistance signal through interacting with prohibitinOur previous study revealed that GbSBT1 could interact with verticillium wilt resistance protein VE in island cotton.Further study revealed that expression of GbSBT1 was upregulated upon induction with V.dahliae V991,jasmonic acid?JA?,and ethylene.GbSBT1 protein is localized in the plasma membrane,and moved into cytoplasm by inoculation with V.dahliae.Knockdown of GbSBT1 in Pima-90through VIGS resulted in decreased resistance of cotton plants to V.dahliae infection.Subtilase1 suppression in the sensitive genotype?Coker-312?failed to facilitate V.dahliae infection.When ectopically expressed in Arabidopsis,overexpressed GbSBT1plants conferred enhanced disease resistance to F.oxysporum and V.dahliae.Meanwhile,expression of defense-related genes in Arabidopsis was increased.GbSBT1 could interact with secreted proteins of V.dahliae during the infection process.Of all the secreted proteins,prohibitin?PHB?could interact with GbSBT1 in vivo through yeast-two-hybridization and bimolecular fluorescence complementation?biFC?analysis.These results indicated that GbSBT1 could sense the verticillium wilt signal?e.g.prohibitin protein?and trigger the resistant response of cotton.2.Significant difference in gene expression mainly related to signal transduction pathway could be detected in different virulent strain of V.dahliaeIn this study,a comparative transcriptome analysis of V991 and V250 under induction of upland cotton was conducted.309 and 480 genes were upregulated in V991 and V250 after induction of upland cotton,respectively.94 genes were upregulated in both V991 and V250.Expression of 578 and 897 genes were downregulated in V991 and V250 after induction of upland cotton,respectively.And181 genes were downregulated in both V991 and V250.The differentially expressed genes?DEGs?in both V991 and V250 were mainly related to the basal metabolism pathway.Conversely,DEGs in V991 or V250 were mainly related with Ferredoxin synthesis,signal transduction?upregulated?,protein processing in endoplasmic reticulum,Sugar-based phosphatidyl inositol?GPI?anchor biosynthesis?downregulated?pathways.These results indicated that significant difference of pathogenic signals in verticillium wilt fungus with different virulence at the initial infection stage could be observed.The inhibition of high virulent signaling pathways could facilitate to restrain disease development.Expression of Tri15 was upregulated significantly upon cotton induction.Tri15contains a zinc finger structure.The growth of V991 was not affected significantly after Tri15 gene knocking out.However,Tri15 deficient mutant could lead to decreased virulence to upland cotton.A certain degree of pathogenicity recovery could be obtained when metabolites extracts of wild type strain and Tri15 deficient stain were co-inoculated on cotton.A component with a molecular mass of 407.295was affected significantly after analyzing the toxin component of wild type stain V991 and Tri15 deficient stain by using Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry technique.The molecular organization of this component is C₂₈H₃₈O₂.Numbers of isomerides could be detected after analyzing the mass spectrometric data.Results indicated that the content of C₂₈H₃₈O₂ could be regulated by Tri15 gene during the infection process of V991.In summary,the interaction of GbSBT1 and Prohibitin was involved in the recognition process between V.dahliae and cotton.The pathogenicity of V.dahliae was affected through regulation the production of V.dahliae metaboliteby Tir15 gene.