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Characterization of the X4 protein of Tomato ringspot virus and analysis of its variability among virus isolates Jafarpour, Bita


The genome of Tomato ringspot virus (nepovirus) encodes a unique X4 protein of unknown function. X4 is absent from the genome of many other nepoviruses and does not have significant sequence homology with other proteins in available databases. I have studied the possible function(s) of the ToRSV-X4 protein. I used three ToRSV isolates, Rasp2, Rasp1 and PYB. Sequence analysis of the X4 region from these isolates showed a surprising level of sequence variability. The central region of the X4 protein contains two series of amino acid sequence tandem repeats. The number of these repeats varies among ToRSV isolates. N. benthamiana plants inoculated with the ToRSV-Rasp1 recover from infection at temperatures equal to or above 27°C. At 21°C, recovery does not occur and plants eventually die. In ToRSV-PYB1 infection, plants recover from infection at all temperature tested. Since RNA silencing is a ubiquitous plant defence response and was observed in nepovirus infected plants, I used agroinfiltration assays to test the effect of the X4 protein on the induction of RNA silencing directed at a green fluorescent protein reporter gene (GFP). Co-expression of X4-Rasp1 or X4-Rasp2 proteins with GFP in N. benthamiana plants transiently enhanced the expression of GFP. To detect the ToRSV X4 protein in virus infected plants, I used polyclonal antibodies raised against the C-terminal region of the X4-Rasp2 protein. This antibody detected a 60 kDa protein in ToRSV-Rasp1 infected plants. The cross-reactivity of this X 4 (64-65) antibody with the CP suggested that the 60 kDa protein corresponds to the CP. However, X4(3743-44) and X4 (3741-42) antibodies derived from the X4 sequence, did not detect this 60 kDa protein. The predicted 82 kDa X4 full-length protein was not detected by any of the anti-X4 antibodies. This suggests that X4 may be an unstable protein. In conclusion, the variability of the X4 protein among ToRSV isolates combined with different symptomatology and the preliminary evidence that X4 may act as a suppressor of silencing in plants, suggest that X4 may be a multifunctional protein that is involved in protein–protein interaction, host specificity, symptomatology and/or interaction with host defence responses.

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