Molecular Variability Of Four Potyviruses And The Effect Of HC-Pro Structure On Suppressing RNA Silencing

The molecular variability of four potyviruses, Potato virus Y (PVY), Tobacco vein banding mosaic virus (TVBMV), Turnip mosaic virus (TuMV) and Zucchini yellow mosaic virus (ZYMV), which are prevalent and cause great economical losses to crop production in China, was analyzed with phylogenetic methods. The structure and function relationship of helper component proteinase (HC-Pro), the multi-functional protein and first identified silencing suppressor, was also studied with site-directed mutagenesis. The main results read as follows:Forty-four isolates of PVY infecting tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) were collected from five provinces in China (Shandong, Henan, Anhui, Gansu and Heilongjiang province) and characterized in this study. Analysis of the 3′genomic sequences of 44 PVY isolates and the helper component proteinase (HC-Pro) encoding sequences in 25 of these isolates revealed high genetic variability of PVY population and incidence of N, O, N:O and NTN strains in Chinese tobacco with N:O strain as the most prevalent one. Genetic diversity analysis confirmed that coat protein (CP) encoding sequences of PVY were under positive selection, while HC-Pro encoding sequences were under negative selection. Further analysis showed that host and geographical origin play roles in the evolution of PVY CP encoding sequences.Red flesh of radish was proved to be a viral disease, and the causal agent was identified to be TuMV with evidences at biological, serological and molecular levels. The CP gene of TuMV was further cloned into expression vector pET22b(+), transferred into E. coli BL21(DE3) and expressed as a 38.0 kD fusion protein when induced with IPTG. The result of SDS-PAGE and western blotting assay showed that CP gene had been correctly expressed. TuMV population was highly varied within or between lineages. Forty-four TuMV isolates from East China were obtained from 2004 to 2007. The 3′-terminal genomic sequences (including partial NIb, complete CP and 3′-UTR) of these 44 TuMV isolates characterized are 1125 or 1126 nucleotides (nts) long excluding the poly(A) tail. They all contain one partial open reading frame of 912 nts, encoding 304 amino acids, followed by a stop codon and a non-translated region of 209-210 nts. Results of phylogenetic analyses showed that TuMV isolates from China and East Asia clustered into three groups: basal-BR, Asian-BR and world-B. It's the first time that TuMV isolates in basal-BR group were occurred in China and the population expanded to 14 isolates. Using the recombinant analysis software, RDP and PhylPro1.00, there were potential recombinant site within the CP and 3′-UTR of the WFLB-04, BJ1-06 and BJ2-06 isolates. The ratios of non-synonymous and synonymous substitutions and results of amino acid alignment provided evidence that the amino acid of CP encoding region were conserved,and TuMV populations of East Asia constrained with purifying or negative selection.TVBMV is of increasing importance in tobacco production in Chinese mainland. The 3′-terminal genomic sequences (1624 nucleotides) of 12 TVBMV isolates from China were determined and compared to the sequences of only four TVBMV isolates available in databanks. The results revealed that TVBMV consists of several phylogenetically distinguishable strains that show a degree of correlation with the geographical origin. Two isolates from Yunnan province had a unique putative NIb/CP proteolytic cleavage site of Q/N that is uncommon for potyviruses, whereas other TVBMV isolates had the more typical Q/G amino acid sequence at the proteolytic site. The CP gene of YND isolate was cloned into expression vector pET22b(+) and transferred into E. coli BL21 (DE3). The result of SDS-PAGE showed TVBMV CP gene was expressed as a 35.0 kDa fusion protein to high level when induced with IPTG. Western blotting and dot-immunobinding assay (DIBA) results showed that the acquired antiserum could specifically react with both TVBMV CP expressed in E. coli and extracts from diseased plants.ZYMV is one of the major factors that causes great economical losses to cucurbits. Four viruses, ZYMV, WMV, TMV and CMV were detected from one mix-infected pumpkin(Cucurbita moschata)plants in Liaocheng, Shandong Province, using RT-PCR method. The phylogenetic trees constructed with the complete nucleotide sequence of the CP genes indicated that the 42 ZYMV isolates analyzed could be clustered into 6 genotypes and showed close relationship with geographical origin. The isolate we obtained and other 11 isolates (including Con, Cal, Flo) belonged to genotypeⅢ. Chinese ZYMV isolates showed highest diversity and formed specific branches.To confirm whether the motifs that play roles in aphid transmission and virus synergism are involved in RNA silencing suppression, we designed three pairs of primers for site-directed mutagenesis. Mutations were introduced to amino acids at position 7, 12, 250 and 251, where Phe7 and Asn12 were deleted, Ile250 and Gly251 were changed to Asp and Glu respectively. The results showed that all of these three mutations of HC-Pro result in the loss of suppressing RNA silencing activity. The results would provide some insight for studying structure and function of HC-Pro, which will surely better our understanding on the pathogenesis of potyviruses.