Screening Of Virulence Effectors Of Ralstonia Solanacearum UW551 Based On Transient Expression System In Tobacco

Bacterial wilt caused by Ralstonia solanacearum is an important bacterial disease.The T3 SS is essential for the pathogenicity of Ralstonia solanacearum in host plants.Ralstonia solanacearum possesses complex type III effector(T3E)repertoires of effectors(T3Es)that are translocated inside the host cells to cause disease.However,only a minor proportion of these effectors have been assigned a function,the molecular mechanism of its pathogenesis is still poorly understood.In order to further quickly approach the virulence functional studies of Ralstonia solanacearum effector proteins,our research used transient expression system screening of effector proteins in tobacco,we found that some of effector proteins can induce cell death in tobacco and preliminary identification of plant immune pathway mediated,and some can interfere with the plant ETI and PTI immune pathway.This study will lay a foundation for the further exploration of the virulence function of Ralstonia solanacearum effector proteins,and pave the way for further exploring the resistant gene against Ralstonia solanacearum.The results are as follows:1.Root inoculation and leaf inoculation were used on N.benthamiana and 3 cultivars of tobacco(D101,Yunyan 87,K326)and 4 species of wild type tobacco(N.langsdorffii,N.repanda,N.alata,N.goodspeedii)by Ralstonia solanacearum UW551.The results showed that R.solanacearum UW551 cannot lead to all tobacco species above wilt,but can induce the reaction of cell death on tobacco leaves.Tobacco leaf did not produced cell death reaction while inoculated with the T3 SS hrpB mutant of UW551.These results indicated that the cell death induced by UW551 is mediated by T3 SS.2.47 effectors were cloned from Ralstonia solanacearum UW551,and the transient expression vectors of the 42 effector genes were constructed successfully by using potato virus dual expression vector pGR106.3.Analyzed of the 42 effector genes by Agrobacterium mediated transient expression system in N.benthamiana and 7 kinds of tobacco,the effector of Rip5 could induce cell death in all test plant leaf tissue;Rip6 could induce cell death in N.benthamiana and 4 species of wild type tobacco;In addition,Rip25,Rip26,Rip32,Rip 40,Rip 55,Rip 56,Hyp9 can cause different levels of cell death in N.benthamiana.4.In the research of the immune pathway mediated by Rip5,Rip6,Rip25,Rip26,Rip55,Rip56 in N.benthamiana,respectively silencing NbBAK1,NbNDR1,NbEDS1,NbSGT1,NbWIPK and NbCoi1,that the key genes in plant immune pathways.The cell death induced by Rip26 was found enhanced in NbBAK1 silencing plants,while weaker in NbNDR1 silencing plants.5.The effectors of UW551 on the inhibition of HR reaction mediated by ETI and PTI in N.benthamiana leaves was found.There were 7 effectors,Rip6,Rip13,Rip10,Rip28,Rip30,Rip32,Rip47 and Hyp11 completely inhibited the INF1 induced HR;and 9 effectors,Rip6,Rip13,Rip10,Rip30,Rip31,Rip32,Rip36,Rip53 and Hyp11,could inhibit the excitation of HR by PiCRN2;and only Rip6 was able to partially inhibit HR stimulated by Avr3a/R3 a or Avr4/Cf4.Some of the effectors of Ralstonia solanacearum UW551 play the similar role of inhibition of PCD,and the may inhibit the immune response of PTI and ETI during the pathogen infection.