Systemic Necrosis On Solanum Lycopersicum Infected By Tomato Mosaic Virus Related To Movement Protein

Tomato mosaic virus (ToMV) is the type species of the genus Tobamovirus and it is apositive-sense single-stranded RNA virus whose genome length is about6.4kb. ToMV has aremarkably host range and distributed worldwide. It is one of the most important economicallyplant viruses. Up to now, domestic and international research on ToMV focuses on theinteraction of the protein and host plant. In this study, agrobacterium-mediated infectious clonewas constructed and analyzed the mechanism of systematic necrosis on Solanum lycopersicuminfected by ToMV.ToMV-S1induced S. lycopersicum hezuo903to mild mosaic phenotype and systematicnecrosis expressed on host plant infected by ToMV-N5, while the nucleotide sequence andamino acid similarity was as high as98.8%and97.2%respectively。In this study, completegenomic RNA of both ToMV strains was obtained by RT-PCR respectively. cDNAs of themwere cloned into vector pCB301which carrying duplicated35S promoter. Positivedrecombinant plasmids pToMV-N5and pToMV-S1were transformed into AgrobacteriumGV3101and the agrobactium infectious clones of ToMV-S1and ToMV-N5were infiltrated onNicotinia benthamiana. At3post-inflitrated days, all of the host plants infected by ToMV werewilt at the top new leaves and were dead in7days. Brown spots emerged on the top new leavesof S. lycopersicum hezuo903infected by pToMV-N5and gradually lead to plant growth arrest7days, only mild mosaic was observed on the plants which inoculated by infectious clone ofpToMV-S1. The two strains of virus could be detected in the systemic leaves of tamoto byWestern Blot, but the virus accumulation of pToMV-S1was lower than pToMV-N5. Theagrobacterium infectious clones of both two virus strains produced the same phenotypic on thehost plants as that caused by the wild-type virus. It is showed that agrobactium infectious clonesof both ToMV-N5and ToMV-S1were constructed successfully.Exchange both the MP and CP of ToMV-N5and ToMV-S1were performed by digestionand ligation to obtain chimeric virus pToMV-N5MPCPS1and pToMV-S1MPCPN5. Results ofinfiltration on N. benthamiana showed that the two infectious clones have infection activity.pToMV-N5MPCPS1which carrying MP and CP of ToMV-S1showed wild mosaic phenotype onhezuo903, while ToMV-N5showed systemic necrosis. And the phenotype of chimeric viruspToMV-S1MPCPN5which carrying the MP and CP of ToMV-N5aggravated by light mosaic to systemic necrotizing compare to the infectious clone of ToMV-S1. These results indicated thatsystemic necrosis phenotype on tomato infected by ToMV-N5was related to MP and CP.In order to analyze the determinant of which related to the phenotype of systemic necrosison tomato, MP between that of ToMV-N5and ToMV-S1was exchanged by digestion andligation to get the chimeric virus pToMV-N5MPS1and pToMV-S1MPN5. The chimeric viruspToMV-S1MPN5which carrying ToMV-N5MP can cause systemic necrosis on host plants;while chimeric virus pToMV-N5MPS1which carrying ToMV-S1MP just induced mild mosaicsymptoms expression on the seedlings of tomato. Thus, the systemic necrosis phenotype onthe host plant might be related to the movement protein. Northern Blot assay showed that thevirus genome accumulation of ToMV-S1was lower than ToMV-N5. But once its MP wasreplaced by that form ToMV-N5, the virus genomic RNAs accumulation of ToMV-S1MPN5wassignificantly higher than that of ToMV-S1. The result of Western blot was consistent with thoseof Northern blot. The results suggested that the MP of ToMV-N5increased the accumulation ofviral genomic RNAs. Moreover, systemic necrosis on tomato probably related to the higherlevel of ToMV-N5. The homology of amino acid of MPN5and MPS1is up to98.8%, anddifferences only in aa92,170and240. Therefore, in this study we also construct the chimericvirus with a point mutation. Preliminary results showed that the difference of the symptoms ofthe two strains on the host was not determined by a single amino acid of ToMV-N5MP.