Complete Nucleotide Sequnenc Of Shallot Yellow Stripe Virus Isolated From Shaanxi Onion And Interaction Between P3 And Its Host

A disease was found to be causing rugose and yellow stripe symptoms in welsh onion in Shaanxi province. Using universal primers for the family Potyviridae, the complete nucleotide sequence of this potyvirus was determined. It was 10 427 nucleotides (nt) in length excluding the poly(A) tail. The complete sequence contains a single large predicted ORF and was predicted to encode a polyprotein 3 340 amino acids (aa) long with a calculated molecular weight of 379.7kDa. A BLAST search showed that the most closely related sequence was a Shallot yellow stripe virus, isolate from Zhangqiu, Shandong province (SYSV-ZQ2). The two complete nucleotide sequences have 93.2% identity and their complete polyproteins have 97.5% amino acid identity. The virus isolated from shaanxi onion is therefore clearly a SYSV isolate, and was named SYSV-O.After alignment with the complete sequences of other potyviruses, the characteristic proteolytic cleavage sites of the ten mature potyvirus proteins were identified. The two isolates differed at the NIb-CP cleavage site, that of SYSV-O was Q/V but Q/A in SYSV-ZQ2. As in Onion yellow dwarf virus, the SYSV-O sequence reported here contains a large P3 protein. The function of the potyviral P3 is not well understood.The 6K1 region of SYSV-O was identical to that of SYSV-ZQ2. The NIa-Pro, HC-Pro, P3 and 6K2 regions were relatively conserved, whereas the NIa-VPg region was moderately variable (93.6% aa identity). Nucleotide and amino acid identity amongst all SYSV coat protein sequences ranged from 87.3% to 84.1% and from 89.2% to 98.4% respectively. Phylogenetic analysis of the 3′-terminal nucleotide sequences of SYSV isolates confirmed the existence of four distant groupings, partially related to geographical origin and host plant species: (1) Indonesian shallot isolates; (2) Japanese Welsh onion, rakkyo, bunching onion isolates and an Indonesian Welsh onion one; (3) Chinese Hangzhou bunching onion and Zhangqiu isolates (4) Chinese Hangzhou welsh onion, Zhangqiu welsh onion ZQ1 and Shaanxi onion isolates.A recombinant plasmid was constructed by ligating the P3 gene of SYSV-O into pGBKT7, then transforming into yeast AH109. Western blot analysis revealed that the P3 was expressed as a fusion protein with BD in AH109. The plasmid pGBKT7-(SYSV-O) P3 is not toxic to yeast strain AH109 and is unable to activate transcription of reporter genes by itself, and therefore it can serve as a bait plasmid in the yeast two-hybrid system.The total RNA was isolated from onion with Trizol. The first-strand cDNA was synthesized with SMART technology, and the dscDNA was further synthesized with 5′and 3′PCR primers by long distance PCR. The yeast strain AH109 was transformed with dscDNA, pGADT7-Rec and pGBKT7-(SYSV-O) P3. Putative positive interacting clones were obtained by nutritional selection screening and X-α-gal assay. Nine putative positive clones were obtained and their plasmids were isolated. The AD library inserts were amplified by PCR and the PCR result showed that the inserted fragments were about 700 bp. Finally, these positive clones were confirmed and the sequences were determined. The nine sequences were identical and were submitted to GenBank. They encode 3′terminal region of the ribulose-bisphosphate carboxylase large subunit.