Preliminary Identification Of The Host Factors Associated With Tomato Spotted Wilt Virus In N.benthamiana

Tomato spotted wilt virus(TSWV)is a typical member of the plant-infecting tospovirus genus of the Bunyaviridae family.TSWV belongs to negative strand RNA virus which contains three single strand RNA.The main insect transmission mediator of tomato spotted wilt virus is western flower thrips(Frankliniella occidentalis).Thrips have mixed food habits,more than 500 species of host plants are currently known,including important cash crops such as pepper,eggplant and tomato.With the continued migratory flight of western flower thrips,its host species will continue to increasing.TSWV have a wide host range and can infect as many as 1,000 families of more than 1,000 monocotyledonous and dicotyledonous plants,including important cash crops such as tomatoes,peppers,tobacco,and flowers,causing serious economic losses in worldwide.TSWV is list as one of the world's top ten most dangerous plant viruses,due to its' great harm.Therefore,the study of the TSWV have great significance.The diseased systemic leaf was collected by expanding propagate TSWV on the model plant Nicotiana benthamiana.After grinded in the liquid nitrogen,the virus particles were purified by sucrose density gradient centrifugation,then polyacrylamide gel electrophoresis(SDS-PAGE),and did mass spectrometry analysis after cutting gal,determined that high purity virus particles were obtained.The results of mass spectrometry showed that the virus particles extracts contained viral replication protein RdRP,glycoprotein Gn/Gc,non-structural protein NSs,and nucleocapsid protein N.Vrus extracts also contained many different plant proteins,such as tubulin,heat shock protein(HSP70/80),calnexin,clathrin,vacuolar-ATPase.The vacuolar membrane proton pump(vacuolar-ATPase)is particularly abundant in viral particle extracts.This protein is a multi-subunit membrane protein complex which is present on plant cell and organelle membrane.The latest research show that vacuolar-ATPase may participate in the spread of virus in host cells by maintaining the endosomal acidic environment.The mass spectrometry analysis results of the virus extracts show that the V-ATPase A subunit abundance is the highest in all component.Due to the importance role of V-ATPase protein of the TSWV replication and movement pattern in plants,polyclonal antibodies of V-ATPase-A should prepared for further study.After four-times immunoreaction in New Zealand white rabbit,we got rabbit anti-V-ATPase-A with a titer of 1:10,000.Western blot analysis demonstrated that the polyclonal antibody can specifically recognize the V-ATPase-A protein and has a good effect.