Study Of Cytopathology Of Tomato Spotted Wilt Virus And Tomato Zonate Spot Virus And Functions Of MP

Tospovirus is the causal agent for large economic losses in a wide variety of plant hosts. Since then, increasing number of tospoviruses have been found in many regions of China. Similar to other members of the tospoviruses, tomato zonate spot virus(TZSV)—an isolate of a new tospovirus species,has quasi-spherical, enveloped particles (80～110nm in diameter) and a tripartite single-stranded (ss)RNA genome denoted as large (L), medium (M) and small(S) RNAs. Sequence analysis indicated that the identities of S and M RNA IGR sequences shared by TZSV with other tospoviruses are below37.8and54.6%, respectively. In order to study the large-particle virus intercellular movement mechanism inclouding Tomato spotted wilt virus (TSWV)and TZSV, the cytopathological structure of these viruses was observed, the subcellular localization of movement potein NSms were studied.Use two kinds of method, the conventional chemical method and the ultra-low-temperature fast-frozen method, to processing the virus-infected plants by TSWV and TZSV. The ultrastructure of the host cells is observed using transmission electron microscope. The results showed that the distribution of the virus particles and the cytopathological structure has no significant difference between the two virus. Quasi-spherical particles enclosed in a membranous envelope, typical of tospoviruses, were observed in both of the viruses. Howere, the cellular organelles of leaves infected by TZSV are destoried more serious.For example, the chloroplasts are vacuolar in the leaves infected by the two virus,but the vacuoles are more and larger in the leaves infected by TZSV. There are also some vacuoles can be observed in the mitochondria in the leaves infected by TZSV,but invisible in the leaves infected by TSWV. These may be to some extent explain why TZSV has more serious hazards to crops in the field state.The expression and subcellular location of the NSm protein of TSWV and TZSV were studied using two different methods by both plant cells and insect cells. First, the NSm gene, located on the ambisense M RNA segment of TSWV and TZSV, was cloned into the pCHF3vector which include a GFP gene. Agrobacterium-mediated transient expression from N.benthamiana leaves was used to study the location of NSm in plant cells. Second, in order to test whether plant-specific components were involved in tubule formation, the NSm gene was also expressed in a heterologous expression system, i.e., insect cells. So T. ni (Tn) cells were infected with a recombinant baculovirus expressing the NSm gene. The results showed that NSm-GFP fusion proteins of the both virus diffused in the tobacco epidermal cells and were located at the edge of the cell walls, forming discontinuous green fluorescent spots at the plasmodesmata, which were sometimes present in pairs between two neighboring cells, while GFP proteins expressed alone distributed evenly around the cell wall and in the nucleus. In the entomic Tn cells,TSWV NSm proteins formed a large number of tubular structures extending from the surface.But the localization of TZSV NSm proteins are difference.They can just diffused in the cytoplasm and the surface of the cell nucleus.The NSm gene of TSWV and TZSV were expressed in E. coli strain BL21(DE3) pLys S using pET32a vector, and recombiant proteins were purified with Ni2+-NTA agarose affinity chromatography. The polyclonal antibodies against the NSm of TSWV and TZSV, were produced in rabbits, respectively. Western blot analysis of purifed recombinant NSm protein of TSWV,purified NSm protein was shown to bind specifically to intact the polyclonal antibodies.These polyclonal antibodies were prepared for the next immunocytochemistry observation.