Construction Of Chinese Wheat Mosaic Virus (CWMV) Infectious Clones And The Interaction Of Cwmv With Host HSP70S

At the end of the last century,a new virus transmited by Polymyxa graminis,was characterized on winter wheat plants in Shandong Province,China,and designed Chinese wheat Mosaic virus(CWMV).The virus causes wheat mosaic disease,leading to serous losses of wheat yields in China.In recent years,significant progress has made in its biology and molecular biology,however,its reverse genetics and the interaction between the virus and host plants remains largely unknown.Heat shock protein 70(HSP70)is recognized as among the most conserved molecular chaperones in all living organisms and play important roles in many cellular process.HSP70s also are known to be key host factors involved in some viral infection.In this study,we first focused on the construction of CWMV cDNA clones and its infectivity on plants and then analyzed the interaction relationship between CWMV and HSP70s.The full-length cDNA clones of Chinese wheat mosaic virus(CWMV)RNA1 and RNA2 were first constructed under the control of T7 promoter.A mixture of in vitro transcripts was mechanically inoculated into the young leaves of wheat plants.Its infectivity was characterized by Northern blot,Western blot,and electron microscopy.The full-length cDNAs were further cloned into a binary vector under the control of 35S promoter.An efficient and reliable agroinfiltration method was then developed in N.benthamiana,providing a basis for reverse genetic assays of CWMV.Based on the infectious clones,we further investigated the relationship between CWMV and HSP70s.The results of qRT-PCR and western blots showed that HSP70s were richer in CWMV-infected plants,indicating that CWMV infection could up-regulate the expression of host HSP70s.When the expression of HSP70s was inhibited by Quercetin treatment or Virus-induced gene silencing(VIGS),the accumulation of CWMV RNAs in N.Benthamian was severely reduced;when HSP70s were over-expressed,the accumulation of CWMV RNAs in N.Benthamian was significantly enhanced,indicating that HSP70s were associated with the accumulation of CWMV RNAs.By yeast two-hybird(YTH)assays and BiFC,we validated that HSP70s could interact with CWMV Replicase,and the region aa 167-333 was identified to be crucial for its interaction with HSP70.In plant cells,the interaction between the viral replicase and HSP70 was observed to form a large number of irregular fluorescent granules,which appeared to be like virus inclusion body(VIB).The subcellular fractionation assays showed that the viral replicase could recruit host HSP70 to cellular membrane,suggestion that HSP70 might be involved in the formation of viral replication complex and thus promote the replication of the CWMV.This is the first host factor identified to be involved in furoviral infection,which extends the list and functional scope of HSP70 chaperones.