Investigating The Inhibitory Effects Of Ralstonia Solanacearum Type ? Effectors On TGS Mediated By DNA Methylation With Plant Viral Vectors

DNA methylation is an important way to mediate transcriptional gene silencing(TGS)in plants.It has been reported that the infection of some pathogenic bacteria affects the DNA methylation level and status of host plants.As the disease-associated protein that enters directly into host cells,pathogen type III effectors are likely to play a role in this process.However,there is currently no convenient experimental system to study this.In view of this,this article has tried the application of plant virus vector and plant virology experimental system in this aspect.16c-TGS Nicotiana benthamiana contains a GFP driven by the 35 S promoter,but due to methylation of the 35 S promoter,the GFP occurs TGS.Ralstonia solanacearum is one of the most widely distributed and most influential plant pathogenic bacteria in the world.In this paper,R.solanacearum effectors rip-6,rip-21,rip-22,rip-35 and rip72 were constructed on potato virus X(PVX)vectors to obtain PVX-rip-6 and PVX-rip-21,PVX-rip-22,PVX-rip-35 and PVX-rip72.Five recombinant viruses were used to infect 16c-TGS N.benthamiana,respectively.7 days later,the green fluorescence was observed under the portable ultraviolet lamp,and the accumulation level of GFP gene was detected by fluorescence quantitative RT-PCR,and the infected plants were detected by bisulfite sequencing 35 S promoter DNA methylation level.It was found that the 16c-TGS N.benthamiana infected with PVX-rip72 showed green fluorescence on the top leaf,and showed a higher level of GFP accumulation,while the CHH methylation site in the 35 S promoter region was reduced by nearly 30%.As a class of DNA viruses,the infection efficiency of begomovirus is limited by TGS mediated by DNA methylation.Studies have shown that ?C1 encoded by begomovirus satellite molecules promotes viral infection by inhibiting DNA methylation-mediated TGS.In order to further verify the inhibitory activity of rip72 on TGS mediated by DNA methylation,this article replaced Sida yellow mosaic China virus(Si YMCNV)and Cotton leaf curl Multan virus(CLCu Mu V)with rip72,respectively.Two begomovirus satellite molecules,?C1,were inoculated with Si YMCNV and CLCu Mu V with recombinant satellite molecules,respectively,and then observed symptoms and detected the accumulation of virus by Southern-blot at different time points.The results show that rip72 can replace the function of ?C1: plants inoculated with virus and recombinant satellite molecules at the same time as plants inoculated with virus and wild-type satellite molecules at the same time,have more severe symptoms than plants inoculated with Si YMCNV and CLCu Mu V alone,and the virus accumulation higher.As a control,we replaced the ?C1 of Si YMCNV and CLCu Mu V satellite molecules with GFP.The results showed that GFP cannot replace the function of ?C1.It is unclear the specific relationship between Ralstonia solanacearum and the host DNA methylation pathway.Therefore,the role of rip72's TGS inhibitory activity in Ralstonia solanacearum infection requires further study.However,this article initially shows that the existing experimental system in plant virology can be used to screen plant pathogenic effectors that have inhibitory activity against DNA methylation-mediated TGS.