Preliminary Study On The Virulence Function Of Rip56 And Rip6 Of Ralstonia Solanacearum

Bacterial wilt is a worldwide bacterial disease caused by R.solanacearum,which poses a huge hazard to many crops,including potatoes.R.solanacearum contains many virulence factors,and the type III effector protein is a key factor in its pathogenesis.This experiment intends to explore the potential disease resistance mechanism of potato by studying the function of R.solanacearum effector protein to interfere with plant immunity.In this study,the virulence function of the R.solanacearum Rip56 was identified,and the transgenic lines of Rip56 were obtained by genetic transformation system,and its effect on the innate immune pathway of potato was identified.In addition,this study also studied the effect of the effector protein Rip6 on calcium signaling pathway of potato,revealing the effect of calmodulin on R.solanacearum.This study provides a theoretical basis for the breeding of potato bacterial wilt disease resistance.This study mainly includes the following five aspects:1.Identification of virulence function of effector protein Rip56.Wild type potato Solanum chacoense(C9701)was inoculated with wild-type strain UW551,mutant ?Rip56 and mutant ?hrcv.The results showed that the virulence of mutant ?Rip56 was significantly decreased compared with wild-type UW551,indicating the effector protein Rip56 has a significant virulence effect to the wild type potato Solanum chacoense(C9701).At the same time,the transient expression of the effector protein Rip56 in tobacco leaves can produce a cell death phenotype.At the same time,the key genes of silencing immune pathways NbNDR1,NbSGT1,NbNTF6,NbEDS1,NbRBOH?NbMEK2 ? NbMEK1 cannot interfere with Rip56-mediated cell death phenotype.2.Construction of effector protein Rip56 transgenic lines.The cultivar Solanum tuberosum E3 was successfully transformed by Agrobacterium-mediated potato genetic transformation system.Eight transgenic lines of R.solanacearum effector protein Rip56 were obtained.After positive detection,the transgenic lines Rip56-4,Rip56-6 and Rip56-7 were selected for subsequent immune pathway identification.3.Phenotypic identification of effector protein Rip56 transgenic lines.The results showed that there was no significant difference in plant growth height and growth between wild type and transgenic lines,but the results of disease resistance identification showed that the transgenic lines of effector protein Rip56 were more resistant to R.solanacearum UW551 than control E3,suggesting The effector protein Rip56 activates the disease-resistant pathway of the cultivar Solanum tuberosum E3.4.Identification of PTI immune pathway in Rip56 transgenic lines.Reactive oxygen species experiments showed that there was no significant difference in the burst of ROS between the transgenic lines and the wild type,while the PTI marker gene PTI5 was significantly up-regulated.To further verify the immune response of PTI5 activation,some key genes about upstream and downstream of PTI5 were tested.The results showed that the expression of protein kinase PTO,transcription factor PTI5 and the basic defense gene PR1 was significantly increased,which is similar to the previously reported avirulence gene AvrPTO-activated immune pathway,suggesting that Rip56 may enhance resistance of the potato cultivation Solanum tuberosum E3 by activating this pathway.5.Effector protein Rip6 inhibits potato calcium signaling pathway.The effector protein Rip6 is a virulence protein,and transcriptome data show that Rip6 can significantly down-regulate calmodulin expression.Inoculation experiment results show that compared with the effector mutant ?Rip6,the wild-type strain indeed inhibited the expression of the calmodulin genes StCML5,StCML23,StCML-CAST,StCDPK2 and StCNGC1.At the same time,the overexpression vectors of StCML5,StCML19,StCML23,StCML-CAST and StCDPK2 genes and the Solanum tuberosum E3 transgenic line were constructed.The StCML-CAST transgenic plants were obtained and inoculated.The results showed that the overexpression of StCML-CAST transgene was overexpressed.The transgenic line can significantly increase the resistance of Solanum tuberosum E3 to R.solanacearum UW551.This study preliminarily studied the virulence function of Rip56 and found that Rip56 protein can activate the resistance of Solanum tuberosum E3 to R.solanacearum,and found that the resistance is through protein kinase PTO,transcription factor PTI5 and basic defense gene PR1.In addition,this study also verified the transcriptional regulation of the effector protein Rip6 on calmodulin,and found that overexpression of the calmodulin gene StCML-CAST can improve the resistance of potato to R.solanacearum.Above results laid a good theoretical foundation for genetic improvement of R.solanacearum resistance.