Molecular Biology Of Shallot Yellow Stripe Virus Isolated From Shaanxi Onion

Virus diseases of Allium are common and seriously affect both quality and yield. Molecular biology techniques are essential to detect and identify the pathogens. In this work, the complete nucleotide sequence of a potyvirus infecting onion in Shaanxi Province was determined (AM267479) using universal primers for members of the family Potyviridae and further specific primers based on the sequence determined. Using these data the interactions between the 10 proteins encoded by the virus were investigated using the yeast two hybrid system (YTHS). For comparison, the same study was also carried out on another potyvirus, an isolate of Soybean mosaic virus from Pinellia ternata (SMV-P). Moreover, the interaction between the P3 protein and the host proteins has been also studied. The complete cDNA of this potyvirus was 10,427 nucleotides in length excluding the poly(A) tail. It contains a single large open reading frame and was predicted to encode a polyprotein of 3,340 amino acids. BLAST showed that the sequence of Shallot yellow stripe virus SYSV-ZQ2 was related to it mostly. They had 93.2% nucleotide identity and 97.5% amino acid identity. Obviously, the virus isolated from onion in Shaanxi is an isolate of SYSV, named SYSV-O, and this provides the first report that SYSV can infect the onion. The proteolytic cleavages sites of the SYSV-O ten mature proteins were the same as those of SYSV-ZQ2 except NIb/CP, which are VSYQ/A and VSYQ/V, respectively. Phylogenetic analysis of the 3′-terminal nucleotide sequences of SYSV isolates confirmed the existence of four distinct groups. One is the isolate from Indonesia shallot, and the other includes the isolates from Japan Welsh onion, rakkyo bunching onion and Indonesian Welsh onion. China Hangzhou bunching onion isolates and Zhangqiu isolates make up the third group. The last group includes Chinese Hangzhou welsh onion isolates, Zhangqiu welsh onion ZQ1 isolate and the new Shaanxi onion isolate. The evolutionary relationship between different isolates was significantly correlated with their geographical origin and their host plant species.YTHS has been an effective and powerful tool for studying potyvirus protein functions, and some interesting interactions between the viral proteins have previously been found. However, it is still not clear what interactions occur amongst all ten of the potyvirus proteins. In these studies we have mapped the complete set of interactions between the proteins of SYSV-O and SMVP. The results showed that: P1 protein can self-interact and interact with P3, 6K1, CI, VPg, NIa-pro, CP; HC-Pro can self-interact and interact with P3, NIa-pro, VPg, CP; P3 can self-interact and interact with all of the other viral proteins except CP; the two 6K proteins can interact with P3, NIa-pro, VPg; CI can self-interact and interact with P1, P3, 6K2, NIa-pro, VPg; VPg can self-interact and interact with all of the other viral proteins except NIa-pro and CP; NIa-pro can self-interact and interact with all of the other viral proteins except VPg; NIb can self-interact and interact with VPg, NIa-pro, CP; CP can self-interact and interact with P1, HC-Pro, CI, VPg, NIa-pro, NIb. The interaction of SMV-P proteins is similar to those of SYSV-O in many aspects, but some differences occurred. Although many of these interactions are identical to those in previous reports, there are some new findings including the 6K interactions with the other viral proteins and the self-interactions of P1, P3 and NIb. Many interactions showed directionality, suggesting that proteins fused in one direction may have more favourable protein folding or exposure of binding sites than those in the other direction. A potentially significant and novel finding in our experiments was that the interaction between 6K1 and NIa-Pro are consistently strong in both directions for both viruses. This activity was retained in a slightly truncated form of the SMV-P protein 6K1. We also detected interaction of three (AD/BD) combinations SMV-HC-Pro/SYSV-NIa-Pro, SYSV-HC-Pro/SMV-NIa-Pro and SYSV-HC-Pro/ SMV-NIb from the 38 combinations.The genomic structure of SYSV-O is similar to that of another potyvirus, Onion yellow dwarf virus (OYDV). Both contain a large P3 protein, whose function is not yet clearly understood. Using SYSV-O P3 as bait, we screened for interacting proteins of onion. The results showed that the Ribulose-1, 5-bisphosphate carboxylase/ oxygenase (RuBisCO) large subunit (rbcL) interacts with P3 of SYSV-O strongly. Such interaction was further confirmed by co-immunoprecipitation assay in vitro. RuBisCO is the key protein of photosynthesis and its carboxylase/oxygenase activity center is located on the rbcL. This implies that the P3 protein is involved in symptom expression through interfering with its host plant's photosynthesis. Studies using deletion mutants revealed that the N-terminal of SYSV-P3 and OYDV-P3 was involved in the interaction with the N-terminal of rbcL (137aa). It is predicted P3 is a trans-membrane protein and the outer-membrane part consists of the interaction domain. The P3/rbcL interaction also occurs in OYDV and SMV-P, and rbcL can interact with eight proteins of SYSV-O (except 6K1 and CP) and seven proteins of SMV-P (except 6K1, CI, CP). These results showed that the interaction between P3 of Potyvirus and rbcL is common, and that many other potyvirus proteins may also be involved in symptom development.