Jiong Chen

A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family

Summary.u2002A universal primer (Sprimer: 5′-GGX AAY AAY AGY GGX CAZ CC-3′, Xu2009=u2009A, G, C or T; Yu2009=u2009T or C; Zu2009=u2009A or G), designed from the consensus sequences that code for the conserved sequence GNNSGQP in the NIb region of members of the family Potyviridae, was used to amplify by RT-PCR the 3′-terminal genome regions from infected plant samples representing 21 different viruses in the family. Sequencing of some of the fragments (c. 1.7u2009kb) showed that the type strain (ATTC PV-107) of Oat necrotic mottle virus is not a distinct species in the genus Rymovirus, but is synonymous with Brome streak mosaic virus (genus Tritimovirus) and that Celery mosaic virus is a distinct member of the genus Potyvirus not closely related to any other sequenced species. Potyviruses infecting crops in China were also investigated, showing that viruses on cowpea and maize in Hangzhou, Zhejiang province were respectively Bean common mosaic virus and Sugarcane mosaic virus and that one on garlic in Nanjing, Jiangsu province was Onion yellow dwarf virus. Fragments were also sequenced from Chinese isolates of Lettuce mosaic virus and Soybean mosaic virus (from Hangzhou), Turnip mosaic virus (2 different isolates from Zhejiang province) and RNA1 of Wheat yellow mosaic virus (from Rongcheng, Shandong province).

Molecular characterisation of a complex mixture of viruses in garlic with mosaic symptoms in China

Summary.u2002Degenerate primers were used to detect and amplify cDNA of viruses of the genera Carlavirus, Allexivirus and Potyvirus from garlic plants with mosaic symptoms growing in Zhejiang province, China. Plants contained a complex mixture of viruses and strains. Three distinct stains of Garlic latent virus were detected; the most frequent one was completely sequenced and partial sequences were obtained for the other two. The complete sequence (8363u2009nt) was 76.4% identical to a Korean isolate. Two allexiviruses were detected and completely sequenced. One (8319u2009nt) was identified as Garlic virus X and comparisons showed that a published Korean isolate (which had 90.2% identical nucleotides) had an N-terminal deletion in the serine-rich ORF4. The other isolate (8451u2009nt), tentatively named Garlic virus E, appeared to be a new member of the genus. Phylogenetic analyses of the different viral proteins and distinctive conserved sequence motifs within the genus are discussed. This is the first report of allexiviruses from China. Using potyvirus primers, three distinct isolates of Onion yellow dwarf virus and one of Leek yellow stripe virus were detected and the 3′-terminal sequences of their genomes were determined. In a coat protein phylogenetic analysis, the new isolates were most closely related to other published isolates from Japan and China.

A potyvirus p1 protein interacts with the Rieske Fe/S protein of its host

ABSTRACT Yeast two-hybrid (Y2H) screens were used to test for interactions between the P1 protein of Soybean mosaic virus Pinellia isolate (SMV-P) and a cDNA expression library of its host, the aroid Pinellia ternata. Of the 13 independent interacting clones identified, ten were identical and had an open reading frame predicted to encode a 23.7-kDa protein closely related to the cytochrome b6/f complex Rieske Fe/S genes of plants. The interaction between SMV-P-P1 and the mature Rieske Fe/S protein (without transit peptide) of the host was confirmed by in vitro co-immunoprecipitation of the two proteins. Y2H assays using different parts of the two proteins showed that only the N-terminal part (amino acids 1-82) of SMV-P P1 was responsible for the interaction with the Rieske Fe/S protein and that amino acids 1-33 interacted only with the transit peptide, while amino acids 34-82 could interact with the entire Rieske Fe/S protein. SMV-P P1 also interacted moderately with the Rieske Fe/S protein of its other hosts, soybean and Zantedeschia aethiopica, but weakly with that of the non-host Arabidopsis thaliana. The P1-Rieske Fe/S protein interactions are likely to be involved in symptom development, and the very variable N-terminus of P1 may play an important role in host adaptation.

Molecular characterisation of an isolate of Dasheen mosaic virus from Zantedeschia aethiopica in China and comparisons in the genus Potyvirus.

Summary.u2002The complete nucleotide sequence of an isolate of Dasheen mosaic virus from Zantedeschia aethiopica in Zhejiang Province, China, was determined. The 9991 nucleotide genome was typical of the genus Potyvirus and phylogenetic analysis showed it to be a member of the Bean common mosaic virus subgroup. The 3′-terminal sequence, including the coat protein region, was determined for three further isolates from China and Japan. Variations in the length and composition of the N-terminus of the coat protein were not related to geographic origin or plant host. An analysis of all potyvirus cleavage sites revealed patterns related to phylogenetic groupings.

Characterisation of potyviruses from sugarcane and maize in China

Summaryu2002Sugarcane or maize leaves with mosaic virus symptoms were collected from 13 sites in China. Sequence data showed that all 8 samples from maize contained Sugarcane mosaic virus (SCMV); complete sequences were determined from 2 samples and partial sequences (the CI coding region and the 3′-part of the genome) from the others. The 5 sugarcane samples all contained a virus tentatively described as Sorghum mosaic virus (SrMV) and in three of them SCMV was also detected; 2 SrMV sequences and the 3 SCMV ones were completely determined and partial SrMV sequences were obtained from the remaining 3 samples. The features of the complete sequences of SCMV and SrMV are described for the first time. Sequence comparisons and phylogenetic analysis showed three distinct groups of Chinese SCMV sequences (sugarcane isolates from Zhejiang province, a maize isolate from Guangdong and maize isolates from other provinces). The SrMV sequences were similar to one another (>u200993% identical nucleotides); they resembled published sequences in the coat protein but were less similar in the 3′-UTR. The complete sequences of SCMV and SrMV had about 70% nucleotides identical to one another and to Maize dwarf mosaic virus (MDMV). MDMV was not detected in any of the samples.

Protein-protein interactions in two potyviruses using the yeast two-hybrid system.

Interactions between all ten mature proteins of the potyviruses Soybean mosaic virus (Pinellia ternata isolate) and Shallot yellow stripe virus were investigated using yeast two-hybrid (Y2H) assays. Consistently strong self-interactions were found between the pairs of HC-Pro, VPg, NIa-Pro, NIb and CP in both viruses. Apart from the NIb, such interactions have been previously reported for some other potyviruses. The 6K1/NIa-Pro combination gave a consistently moderate to strong interaction in both directions for both viruses. This interaction occurred even when the 6K1 of SMV-P was truncated to eliminate the C-terminal motif that acts as a recognition site for cleavage by the NIa-Pro. Many other interactions occurred only in one direction or only for one of the two viruses. When taken together with other published reports, the data suggest that interactions detected by Y2H should be regarded as only preliminary indications.

Characterisation of some carla- and potyviruses from bulb crops in China

Summary.u2002Conserved carla- and potyvirus primers were used in RT-PCR to amplify virus fragments from garlic and other bulb crops in China and the fragments were subsequently sequenced and compared in phylogenetic analyses. Garlic plants from Henan, Hubei, Jiangsu, Shangdong and Yunnan provinces all contained at least one isolate each of Garlic latent virus (genus Carlavirus), Onion yellow dwarf virus (OYDV, genus Potyvirus) and Leek yellow stripe virus (LYSV, genus Potyvirus). The complete sequence of a Zhejiang isolate of LYSV was also determined, providing the first complete sequence of this virus. The genome was 10142 nucleotides long excluding the poly(A) tail and had the typical features of the genus Potyvirus, although some of the amino acids surrounding the polyprotein cleavage sites were unusual. Shallot yellow stripe virus (SYSV) was amplified from bunching onion (Allium fistulosum var. caespitosum) in Zhejiang province, providing the first record of SYSV in China. Lily mottle virus was amplified from dragon-teeth lily (Lilium brownii var. viridulum).

Bean common mosaic virus isolates causing different symptoms in asparagus bean in China differ greatly in the 5′-parts of their genomes

Summaryu2002Potyvirus isolates from asparagus bean (Vigna sesquipedalis) plants in Zhejiang province, China, caused either rugose and vein banding mosaic symptoms (isolate R) or severe yellowing (isolate Y) in this host, but were otherwise similar in host range. Both isolates were completely sequenced and shown to be isolates of Bean common mosaic virus (BCMV). The complete sequences were 9992 (R) or 10062 (Y) nucleotides long and shared 91.7% identical nucleotides (93.2% identical amino acids) in their genomes and were more distantly related to the BCMV-Peanut stripe virus sequence (PStV). The isolates were much less similar to one another in the 5′-UTR and the N-terminal region of the P1 protein. In the P1, isolate Y was closer to PStV (76.1% identical amino acids) than to isolate R (64.8%). Phylogenetic analyses of the coat protein region showed that the new isolates grouped with other isolates from Vigna spp., forming the blackeye cowpea mosaic strain subgroup of BCMV with 94–98% nucleotides (96–99% amino acids) identical to one another and about 90% identity to other BCMV isolates. Other significant subgroupings amongst published BCMV isolates were detected.

Sequence analysis of a soilborne wheat mosaic virus isolate from Italy shows that it is the same virus asEuropean wheat mosaic virus andSoilborne rye mosaic virus

The complete sequence of the two RNAs of a furovirus isolate from durum wheat in Italy was determined. Sequence comparisons and phylogenetic analysis were done to compare the Italian virus withSoilborne wheat mosaic virus (SBWMV) from the USA and with furovirus sequences recently published asEuropean wheat mosaic virus (EWMV), from wheat in France, andSoilborne rye mosaic virus (SBRMV), from rye and wheat in Germany. Over the entire genome, the Italian isolate RNA1 and RNA2 had respectively 97.5% and 98.6% nucleotide identity with EWMV, 95.5% and 85.8% with SBRMV-G and 70.6% and 64.5% with SBWMV. The Italian isolate was therefore clearly distinct from SBWMV The European isolates all appear to belong to the same virus and the nameSoilborne cereal mosaic virus may resolve earlier ambiguities.

Molecular analysis of barley yellow mosaic virus isolates from China

The complete sequences of both RNAs of an isolate of barley yellow mosaic virus from Yancheng, Jiangsu province, China, were determined. The sequences resembled those of an isolate from Japan (96.8% identical nucleotides for RNA1; 95.7% for RNA2) more closely than one from Germany (93.9 and 91.0%, respectively). The greatest differences between the Chinese and Japanese isolates were in the 5-UTRs of RNAs 1 and 2 (88.9 and 91.6% identical nucleotides, respectively) and there were also some other regions of difference in P1 (RNA2) and P3, CI, NIa and the 5 end of the coat protein (CP) (RNA1). Molecular differences between isolates from ten sites widely distributed in Eastern China were studied by sequencing RNA regions coding for the CP (RNA1) and the N-terminus of the P2 protein (RNA2). The P2 fragment was more variable than the CP, and phylogenetic analysis of both regions showed that Asian and European isolates formed distinct clusters. Differences between isolates were also revealed by single-strand conformation polymorphism of reverse transcription-polymerase chain reaction products, spanning the full lengths of both RNA1 and RNA2. However, molecular variations between isolates could not be linked to earlier results showing differences in cultivar response.