The Molecular Mechanism Of HCPro Viral Suppressor Induce The Expression Of Auxin Biosynthesis Genes

Helper component proteinase(HCPro)is a typical RNA silencing suppressor.The studies have shown that HCPro plays an important role in virus transport,virus aphid transmission and virus particle replication,moreover,HCPro can reduce the accumulation of small interfering RNA(si RNA)and inhibit RNA silencing.Auxin is a key regulator in plant growth and development,viral infection affects host auxin accumulation.However,the molecular mechanism that the virus induces auxin accumulation is not clear.In this study,we used Arabidopsis thaliana as plant material,p BI121-HCPro as gene expression vector,Agrobacterium-mediated leaf infection and vacuum infiltration were used to express HCPro in Arabidopsis.The result of gene expression test showed that we successfully completed HCPro transient expression in Arabidopsis.The YUC gene family regulates auxin biosynthesis,and adjusts auxin content in plants.We detected the expression level of YUC5 gene by semi-quantitative RT-PCR in Arabidopsis expressing HCPro,and the Arabidopsis infected with empty vector was used as control plant.The result showed that the expression level of YUC5 gene was obviously increased in Arabidopsis expressing HCPro,bisulfite sequencing result showed that the DNA methylation level of YUC5 gene promoter repeat sequences was significantly reduced.Moreover,the expression of YUC5 gene clearly increased in DNA methylase deletion mutants drm1/2 and met1,indicating that YUC5 gene is the target gene of the Rd DM pathway,and revealing that Rd DM plays an important role in the process of YUC5 gene in response to virus and the viral suppressor.These results confirmed that HCPro can induce the DNA demethylation of YUC5 gene promoter and activate the gene expression.DR5:GUS transgenic plants have been widely used,the expression of reporter gene GUS can indicate the accumulation of auxin in this plant.We completed HCPro transient expression and GUS staining in DR5:GUS transgenic Arabidopsis.The results had shown that GUS expression was clearly increased in DR5:GUS transgenic Arabidopsis infected with Agrobacterium carrying p35S-HCPro,when compared with DR5:GUS transgenic Arabidopsis infected with empty vector.Moreover,semi-quantitative RT-PCR test result confirmed that the expression level of GUS gene was obviously increased.These results demonstrated that HCPro can induce auxin biosynthesis and accumulation in plants.In this study,we revealed that the viral suppressor HCPro activated the expression of auxin biosynthetic gene by inducing the DNA demethylation of YUC5 promoter,and clarified the molecule mechanism that the viral suppressor HCPro induces the accumulation of auxin in plants.