Regulation Of Host Gene Expression By VsiRNA-20 Derived From Chinese Wheat Mosaic Virus (CWMV)

In China,a soil-borne virus causing a disease of winter wheat and leading to serous yield losses has been identified to be a new species,Chinese wheat mosaic virus(CWMV),within genus Furovirus.The virus is transmitted by Polymyxa graminis,an obligate parasite of graminaceous roots.Since its discovery in Shandong Province in 1990s,more progress has been made in understanding its genomic structure and functions.In our previous studies,numerous virus-derived small interference RNAs(vsiRNAs)derived from CWMV,were characterized from infected wheat plants by deep sequencing,in which CWMV vsiRNAs of 21-22nt in length are predominant and display a strong bias for the 5'-terminal nucleotide,suggesting that CWMV vsiRNAs might be potentially loaded into diverse AGO-containing RISCs to disturb the gene expressions of host plants.In this study,the regulation functions of CWMV vsiRNAs were further investigated using a series of molecular assays.Quantitative polymerase chain reaction(qPCR)assays showed that the 20 vsiRNAs,including vsiRNA-1~5,-7~13,and-15~22,were highly expressed in CWMV-infected wheat plants,in which 6 vsiRNAs,including vsiRNA-1,-3,-5,-12,-13,and-20,could be significantly detectable in Northern blotting assays.Using CleaveLand computer program,three targets were predicted from our previous degradome data.In qPCR assays,accumulation of vsiRNA-20 was rapidly enhanced after inoculation,reached to peak at20-25 days post inoculation(dpi),and then was stabilized at higher expression level in infected plants while its putative target,a Triticum aestivam vacuolar H~+-pyrophosphatase(TaVP),was down-regulated after infection and maintained at lower expression level at15-30 dpi,indicating that there was a negative relationship between vsiRNA-20 and TaVP.The full-length cDNA of TaVP was cloned from healthy plants of a wheat cultivar,Yannong 22,and sequence analysis showed that the TaVP contained a conserved ppi binding site and shared 75.19-89.29%homologies with those vacuolar H~+-pyrophosphatase genes in NCBI database.Phylogenetic tree suggested that the TaVP was close to its homologs from Oryza sativa.RNA ligase mediated rapid amplification of cDNA 5'-end(RLM-RACE)assays showed that TaVP gene was cleaved within its 3'-untranslated terminal region(3'-UTR)in infected wheat plants,suggesting that vsiRNA-20 could regulate the cleavage of TaVP mRNA.To validate the relationship,both binary vectors containing artificial vsiRNA-20 and green fluorescence protein(GFP)fused with the3'-UTR of TaVP were constructed and co-infiltrated into Nicotiana benthamiana leaves.The results showed that vsiRNA-20 could specifically down-regulate the expression of TaVP gene.Further assays showed that virus-induced gene silencing of NbVP and TaVP could increase the accumulation of CWMV RNAs and lead to more severe symptoms in both Nicotiana benthamiana and wheat plants.Taken together,the vsiRNA derived from CWMV could be involved in symptom development and viral pathogenicity by down-regulating the expression of its target at the posttranscriptional level.This study provided new insight into the interactions between CWMV and its host plant.