Construction And Application Of Chinese Wheat Mosaic Virus-induced Gene Silencing Vectors

Chinese wheat mosaic virus(CWMV)is one of the important pathogens causing soil-borne wheat mosaic disease.Its genome contains two single-strand positive RNAs(RNA1-2).Recently,its infectious full-length cDNA clone has been successfully constructed on basis of previous hard works in our laboratory.Virus-induced gene silencing(VIGS)vectors have become one of the key tools for gene functions in plants.By VIGS vectors,the function of many genes,including transcription factors and photosynthesis-related genes,have been understood.In order to develop the CWMV VIGS vector,we modified the infectious clone with molecular methods and attempted to apply the vector to endogenous gene silencing in plants.The main results were as follow:1.Based on the mutant M5 of infectious CWMV clone,we constructed the?CWMV:00 vector,in which a multiple cloning site(MCS)was inserted at the downstream of the viral coat protein gene.Infectivity analysis showed that?CWMV:00 could retain its ability to infect both N.benthamiana and wheat and cause mosaic or yellow symptoms similar to those of viral infection in plants.Northern blot assays showed that?CWMV:00 could accumulate in leaves of N.benthamiana and wheat,reflecting its excellent systemic infectivity in plants.2.The vectors of?CWMV:NbPDS~30000 and?CWMV:TaPDS~30000 were constructed,which harboured about 300bp fragment of N.benthamiana or wheat phytoene desaturase(PDS)gene within MCS,respectively.Quantitative reverse-transcriptase polymerase chain reaction(qRT-PCR)assays showed that the expression levels of endogenous NbPDS or TaPDS genes reduced by 65-75%when compared with the controls;Stronger photobleaching was observed in both N.benthamiana and wheat plants inoculated with the?CWMV:NbPDS~30000 or?CWMV:TaPDS~30000 vectors.In experiments using fragments of PDS ranging from 500 to 1500 bp,sequencing and quantitative assays suggested that the larger insertion could influence the vector stability and the efficiency of VIGS.3.The vectors,?CWMV:STTMmiR165/166 and?CWMV:STTMmiR3134a,were constructed,which mimic the targets of miR165 or miR3134a,respectively.The vectors were inoculated into N.benthamiana or wheat,respectively.QRT-PCR analysis showed that the relative expression levels of mature miR165/166 and miR3134a were down-regulated while the transcript levels of their target genes were upregulated in the inoculated plants,indicating that the CWMV VIGS vector can be applied to the silencing of endogenous miRNAs.Thus the CWMV VIGS vector could provide an alternative valuable tool for investigation of gene functions in monocots and dicots.4.Cysteine proteases(CysPs)are a family of important proteases that are involved in a wide range of plant physiological processes and viral infection.To further characterize the funtions,a gene encoding cysteine protease(NbCysP)was cloned from N.benthamiana plants and expressed in E.Coli.The recombinant NbCysP fusion protein was purified with affinity chromatography and used for producing polyclonal antibodies by immunizing rabbits.In assays of Western blotting,the multiclonal antibodies could react strongly with recombinant Nb CysP protein in a single band,indicating that the antibodies were specific against the proteinase.Further detection showed that the NbCysP expressed at high level but suppressed by low temperature in the N.benthamiana plants.In yeast two hybrid assays,the interaction between the proteinase NbCysP and CWMV coat protein was discovered.The interaction was also validated by in vitro pull-down assays.These results suggested that NbCysP could play an important role in CWMV infection.