Construction Of An Infectious Clone Of Chinese Wheat Mosaic Virus And Functional Analysis Of The Viral Movement Protein

Chinese wheat mosaic virus (CWMV) is a new member of the genus Furovirus, family Virgaviridae, which causes severe damage to winter wheat in eastern Shandong province in co-infections with Wheat yellow mosaic virus (WYMV, genus Bymovirus). The virus is transmitted by the plasmodiophoraceous’fungus’Polymyxa graminis Led., which is widely distributed in the soil and has durable resting spores that cannot easily be controlled by chemicals. Current wheat varieties appear to be susceptible to CWMV and there is an urgent need to discover and introduce disease resistance. A study of the pathogenesis of CWMV not only provides theoretical guidance for disease resistance breeding, but also enriches our knowledge of the molecular biology of plant viruses. This study has two main parts:Firstly, full-length infectious cDNA clones of the CWMV genomic components were constructed based on the published sequences. Secondly, the function of the37K protein encoded by CWMV RNA1was investigated.Primers were designed on the basis of published sequences (accession numbers AJ271838and AJ271839) to amplify fragments of CWMV RNA1and RNA2, respectively. Fragments were cloned into pGEM-T Easy vectors and sequencing confirmed that they were correct and without a frame shift. Then, the fragments of RNA1and RNA2were ligated to construct full-length cDNA clones, respectively. T7RNA polymerase was used for in vitro transcription after linearization. In vitro transcripts were used to inoculate tobacco and wheat at17℃, but no target protein was detected by Western blot analysis. In vitro transcripts were able to replicate and pack virions when transfected to protoplasts of Arabidopsis thaliana. This result indicates that replication of in vitro transcripts perhaps requires a low temperature (17℃), but the system lacks stability and needs further refinement.The37K gene of CWMV RNA1was expressed in Escherichia coli strain BL21(DE3) using the glutathione S-tranferase gene fusion vector pGEX-6P-1. The expressed protein was purified from the inclusion body, and a polyclonal antibody against37K was made in rabbit. This polyclonal antibody can be used to detect the relevant protein in CWMV-infected wheat leaves and after expression in tobacco. A Trans-complementation assay of the movement of PVX mutant (35S:PVX (P25fs)-GFP) was used to investigate the involvement of the CWMV37K gene in cell-to-cell movement. An agrobacterium strain harboring35S:PVX (P25fs)-GFP (+pl9) was diluted10,000fold and mixed (1:1) with agrobacterium harboring35S:CP,35S:19K CRP,35S:37K or35S:P25. The mixtures were co-infiltrated into leaves of N. benthamiana and GFP fluorescence was observed using confocal laser-scanning microscopy at5dpi. Only CWMV37K and P25were able to complement the cell-to-cell movement of movement-defective PVX. Other constructs appeared to be restricted to a single cell. When the plasmids containing35S:37K and35S:eGFP were co-agroinfiltrated into N. benthamiana leaves, eGFP spread between cells showing that37K enlarges the SEL. eGFP was then fused separately to the N-and C-termini of37K to construct the fusion plasmids37K-eGFP and eGFP-37K. These fusion proteins were able to move to the neighboring cells at high dilution (10,000fold). When fusion constructs were introduced to tobacco epidermal cells infiltrated with agrobacterium, GFP accumulated at cell walls in the form of punctuate spots, which may present plamodesmata (PD), and was also distributed in some irregular granule-like structures within cells. The location of the punctuate spots was confirmed by co-infiltrating separately with the PD markers TMV30K and PDLPla. Plasmolysis experiments showed that green punctuate spots were still attached to the cell wall with PDLP1a, while granule-like structures moved along the plasma membrane. These results confirmed that37K was associated with the plasma membrane and the cell wall. Using chemical drugs (BFA and LatB) and protein inhibitor (Sar1[H74L], Ⅷ-1and XI-F) treatments suggested that37K may utilize the ER-to-Golgi secretory pathway and the actomyosin motility system for its intracellular transport and targeting to PD.CWMV37K is intracellular membrane protein including two transmembrane domains predicted by the TMpred program. A series of mutations were designed based on these transmembrane domains. Mutants in which the N-terminus or C-terminus was deleted, or where either of the transmembrane domains (TM1or TM2) was disrupted or deleted could neither target to PD nor complement the cell-to-cell movement of movement-defective PVX. Expressed protein was not targeted to ER if both transmembrane domains were deleted. A mutant, dC1, which included both transmembrane domains was able to spread to neighbouring cells and form granule-like structures, and also had the ability to enlarge PD SEL. These results indicate that TMl and TM2have the ability to target ER although they could not together complete intercellular movement and target to PD, which further demonstrates that the N-terminus of37K, containing the two domains TM1and TM2, has a significant role for the function of the protein.PME cloned from N. benthamiana leaves interacted with CWMV37K, in both BiFC (in vivo) and YTHS (in vitro) assays; the in vivo interaction occurred at the cell wall. Further experiments using BiFC to investigate the interaction between PME and some37K mutants showed that only mutant dN2(N-terminus deletion expressing145-328aa) interacted with PME, showing that the C-terminal region of37K plays a key role for its interaction with PME.