Molecular Characterization Of Potyviruses Infecting Aroid Plants For Medicinal Use In China

Many aroid plants (family Araceae) are traditionally used for medicinal purposes in China. For thousands of years, the plants were simply collected from the wild but recently the requirements for such medicinal materials have greatly increased. Virus diseases have proved to be a particularly severe problem in vegetative cultivation of these plants.Recently, we found Pinellia ternata (Thunb.) Ten.ex Breitenb., in Hangzhou city severely infected with a member of the genus Potyvirus. Sequencing of this virus indicated that it was most closely related to Soybean mosaic virus (SMV). We have proposed that this virus should be regarded as an aroid strain of SMV, and designated SMV-P.To characterize the potyviruses infecting aroid plants for medicinal use in China at the molecular level, RT-PCR was done using degenerate primers specific for members of the Potyviridae to detect and identify potyviruses from several aroid plant species growing at different sites in China. Of the 20 samples of aroid plants from different sites, fragments of three distinct potyviruses were amplified from 7 samples. Of these seven samples, five were infected with SMV-P, one from Sheyang was infected with a mixture of SMV-P and Dasheen mosaic virus (DsMV), and the other one, Typhonium flagelliforme from Nanning, Guangxi, was infected with Konjak mosaic virus (KoMV-T), which was the first report of this species infecting ariod plants in China. The complete nucleotide sequence of a new Hangzhou isolate of SMV-P (SMV-PHZ3) was determined. Comparisons for each of the proteins encoded showed that, in most parts of the genome, SMV-PHZ3 had about 90% aa identity to an earlier SMV-P isolate (SMV-PHZ1) and a soybean isolate (SMV-GHZ), but that the P1 protein region of SMV-PHZ3 was substantially more similar to DsMV than to SMV soybean isolates. Phylogenetic analysis was done for the nucleotide sequence of the 3'-terminal region using the SMV-P isolates and other related potyviruses in the Bean common mosaic virus (BCMV) subgroup. The SMV-P, Watermelon mosaic virus (WMV) and SMV soybean isolates formed three distinct clusters with 100% bootstrap support but the distances between them were similar to those between different strains of BCMV and DsMV.The 3' region (1743 nts) of DsMV-SY1 was sequenced. In phylogenetic analyses, DsMV-SY1, Vanilla mosaic virus (VanMV) and other DsMV sequences grouped together and the differences between them were quite similar to those between different strains of SMV. Serological assays revealed a weak serological relationship between SMV-P and DsMV that can be detected using antiserum prepared to the overexpressed coat proteins.The complete sequence of KoMV-T was determined. Comparison between the amino acid sequences revealed that the P3 gene of KoMV-T was 6 amino acid longer than that of KoMV-F (the konjak isolate), which may be related to the viral pathogenicity. In phylogenetic analyses, the isolates of KoMV grouped with Zantedeschia mosaic virus (ZaMV) and Japanese hornwort mosaic virus (JHMV), although JHMV and ZaMV isolates formed separate subgroups, showing that the evolution of isolates correlated with the host and geographical distribution.No detectable interaction was found between P1 and other viral proteins of SMV-P in yeast two-hybrid tests. Using SMV-P P1 protein as bait, an interacting plant cDNA clone was identified. Sequence analysis revealed that the insertion encoded part of a protein closely related to the cytochrome b6/f complex Riseke Fe/S gene of plants. Using 5'-RACE, the complete sequence of the Riseke Fe/S gene was obtained. The P1-Fe/S interaction was also detected by in vitro co-immunoprecipitation assay. It was confirmed by deletion mutation that the N-terminal part (aa 1-82) of SMV-P P1 interacted with the Rieske Fe/S protein of P. ternata, and that the Rieske Fe/S protein transit peptide was also involved in the interaction. Interactions between DsMV P1 and the Rieske Fe/S protein of its host Zantedeschia aethiopica, and of SMV P1 with that of soybean were also found. Although a P1-GFP fusion protein was expressed in the cytoplasm, and SMV-P interacted with the Rieske Fe/S protein of Arabidopsis thaliana, there was no obvious symptom on transgenic A. thaliana plants. It is possible that the development of symptoms is a complex process involving many viral and host proteins. In sections of virus-infected leaves, there was consistent labelling at the regions of cell cytoplasm and chloroplast using antiserum raised to the GST-PI protein while only a few (or no) scattered gold particles were seen in a control using healthy leaves.Full-length clones of SMV-P under the control of T7 or 35S promoters were constructed. Their infectivity needs to be tested prior to their use for studying gene functions and the interactions between SMV-P and its host.