Functional Analysis Of JA Pathway And Lipid Transfer Protein GhLTPds In Cotton Ssn Lesion Mimic Mutant

Verticillium wilt is one of the main disease affecting cotton production.The analysis of cotton resistance mechanism and the exploitation of resistance genes are the basis for disease resistance molecular breeding.In the laboratory,it has been found that knockdown of SSN,one of the cotton P450 gene,by RNAi leads to systemic immune activation in cotton.The genes involved in JA synthesis and the signaling pathway in transgenic plants are significantly upregulated and induce lesions mimic phenotypes in stem and leaf.Grafting experiments confirmed that the appearance of lesions on stems was induced by some kind of disease-resistance signaling molecules in the roots,which were transmitted upward into the stems.To investigate whether the lesions mimic phenotype depends on the activation of JA signal pathways and whether the transmission of disease-resistance signaling molecules depends on systemic acquired resistance,we carried out relevant research and obtained the following main results:1.The phenotypes of ssn and Gh JAZ2 overexpression line JO39,Gh JAZ2 RNAi line JR13 hybrids and JA pathway gene expression analysis showed that the JA pathway don't play a major role in the formation of lesions.2.Five genes that are homologous to Arabidopsis lipid transfer protein At DIR1 were screened in upland cotton by bioinformatics and named Gh LTPds.According to sequence alignment analysis and conservative motif analysis,the polypeptides encoded by these five genes all have some conserved structural features,including the N-terminal signal peptide region and eight cysteine residues engaged in four disulfide bridges;the proline-rich P××P motifs that are thought to be involved in protein interactions;and the CLC domain related to the formation of hydrophobic cavities.3.Virus-induced gene silencing vectors of Gh LTPd was constructed based on sequence conservation to explore the role of Gh LTPd in interaction between cotton and Verticillium dahliae.The results showed that after knocked down of Gh LTPd,the resistance of cotton to Verticillium dahliae weakened.This shows that systemic acquired resistance(SAR)plays an important role in the interaction between cotton and Verticillium dahliae.4.The relative expression of Gh LTPds in cotton before the lesion appears and after the lesion formation on stems of ssn was analyzed.The results showed that the expression of Gh LTPds was significantly activated after lesion formation,suggesting that Gh LTPd was related to the generation of lesions;gene expression analysis after treatment with the SAR signal substance Az A shows that Az A-mediated SAR responses depend on Gh LTPds in cotton.Co-interfering of Gh LTPds and SSN in upland cotton by VIGS,the results showed that inhibition of Gh LTPd expression can restore the activation of LOXs pathway and constitutive activation of Gh PR1 caused by inhibition of SSN expression,meanwhile reducing the reactive oxygen species accumulation.Taken together,we believe that activation of the JA pathway is not the main cause of lesions generated on ssn mutants;Gh LTPd plays an important role in the generation of lesions and maybe participate in the transmission of resistance signals in ssn mutants.At the same time,inhibiting the expression level of Gh LTPd can weaken the resistance of cotton to Verticillium dahliae,indicating that Gh LTPd may be involved in the response of plants to biotic stress.