| Beet severe curly top virus(BSCTV)can infect the model plant Arabidopsis thaliana.Studies have shown that BSCTV can interfere with DNA methylation of plant endogenous genes and affect the expression of plant endogenous genes.Auxin plays an important role in the growth and development of plants,and YUCCA(YUC)genes are key auxin biosynthesis genes.Studies have shown that plant virus infection affects auxin accumulation in the host.However,it is not clear whether BSCTV induces YUC genes expression and auxin accumulation,and its related molecular mechanism is also unclear.Since the DR5 promoter contains auxin response elements,the expression of reporter gene GUS in DR5: GUS transgenic plants can indicate the amount of auxin accumulation.DR5: GUS transgenic plants are widely used in the study of auxin accumulation changes.We used GUS chemical tissue staining to detect the expression level of GUS gene in BSCTV-infected DR5: GUS transgenic Arabidopsis thaliana to analyze the changes in auxin accumulation in the host.The results showed that compared with empty vector-infected DR5:GUS plants,the accumulation level of GUS gene in BSCTV-infected DR5: GUS transgenic plants was significantly up-regulated.The results of q PCR further confirmed that BSCTV can induce the expression of GUS gene,indicating that BSCTV can induce the accumulation of auxin in the host.In this study,p Cambia1300-BSCTV was used as the vector to express BSCTV,and the Agrobacterium-mediated infiltration were used to complete the BSCTV virus infection of wild-type Arabidopsis thaliana Columbia(Col-0)ecotype.The expression levels of YUC genes in BSCTV-infected Col-0 were detected by semi-quantitative PCR(sq PCR)and quantitative PCR(q PCR).The results showed that the expression levels of YUC1 and YUC10 genes in BSCTV-infected Col-0 were obviously increased compared with the empty vector-infected Arabidopsis.The promoter regions of the YUC1 and YUC10 genes contain highly methylated repeats.The results of sulfite sequencing demonstrated that DNA methylation levels in the promoter of these genes in BSCTV-infected Col-0 plants were significantly decreased.These results suggest that BSCTV activates the expression of YUC1 and YUC10 genes by inducing DNA demethylation.In the RNA-directed DNA methylation(Rd DM)important element Arabidopsis thaliana mutants ago4 and dcl3,the expression levels of YUC1 and YUC10 genes were significantly up-regulated,confirming that these genes are the target genes of Rd DM pathway,and Rd DM can affect DNA methylation of YUC1 and YUC10 genes.Repressor of Silencing 1(ROS1)can negatively regulate the Rd DM pathway.q PCR was used to compare and analyze the expression of these genes in BSCTV-infected wild-type Arabidopsis Col-0 and BSCTV-infected ros1 mutants.The results showed that the increased expression of YUC1 and YUC10 genes were partly repressed in the BSCTV-infected ros1 plants in comparison to the BSCTV-infected Col-0 plants,suggesting that transcriptional activation of these genes partly dependent on ROS1-mediated DNA demethylation.This study confirmed that BSCTV transcriptionally activates the expression of related genes by inducing DNA demethylation in the promoters of auxin synthesis genes YUC1 and YUC10.Further research found that Rd DM pathway and DNA demethylase ROS1 are involved in regulating the expression of YUC1 and YUC10 genes.This article clarified the molecular mechanism of BSCTV-induced expression of the auxin synthesis genes YUC1 and YUC10,providing research basis for the molecular mechanism of plant virus and host interaction. |
