Intracellular Trafficking Of Nucleocapsid Protein And Intercellular Trafficking Of Movement Protein Of Tomato Spotted Wilt Tospovirus

Multipartite negative-strand RNA plant viruses are important plant viruses for agricultural production, which cause severe diseases to agricultural crops, leading to significant economic loss each year in the world. Meanwhile, because of their unique genomic structure, the viruses are of important research value. From discovery to date,biological processes of the virus life cycle has been largely revealed through the constant efforts; however, the movement mechanisms for such viruses still remain unclear. Tomato spotted wilt tospovirus (TSWV) is a representative member among multipartite negative-strand RNA plant viruses. In the present study, TSWV is used as a model virus to study the mechanisms of intracellular and intercellular movement of multipartite negative-strand RNA plant viruses; nucleocapsid protein and movement protein are selected to launch the projects. Research focused on the following two aspects:1. Intracellular trafficking of nucleocapsid protein of Tomato spotted wilt tospovirusA number of viral proteins from plant viruses, other than movement proteins, have been shown to traffic intracellularly along actin filaments and to be involved in viral infection. However, there has been no evidence that a viral capsid protein may traffic within a cell by utilizing the actin/endoplasmic reticulum (ER) network. We used Tomato spotted wilt tospovirus (TSWV) as a model virus to study the cell biological properties of a nucleocapsid (N) protein. We found that TSWV N protein was capable of forming highly motile cytoplasmic inclusions that moved along the ER and actin network. The disruption of actin filaments by latrunculin B, an actin-depolymerizing agent, almost stopped the intracellular movement of N inclusions, whereas treatment with a microtubule-depolymerizing reagent, oryzalin, did not. The overexpression of a myosin XI-K tail, functioning in a dominant-negative manner, completely halted the movement of N inclusions. Latrunculin B treatment strongly inhibited the formation of TSWV local lesions in Nicotiana tabacum cv Samsun NN and delayed systemic infection in N.benthamiana. Collectively, our findings provide the first evidence that the capsid protein of a plant virus has the novel property of intracellular trafficking along the actin/ER system.2. Contribution of plant ER membrane transport system to the intercellular spread of TSWV and its movement protein NSmPlant viruses move through plasmodesmata to infect new cells. The plant endoplasmic reticulum (ER) is interconnected among cells via the ER desmotubule in the plasmodesma across the cell wall,forming a continuous ER network throughout the entire plant. This ER continuity is unique to plants and has been postulated to serve as a platform for the intercellular trafficking of macromolecules. In the present study, the contribution of the plant ER membrane transport system to the intercellular trafficking of the NSm movement protein and Tomato spotted wilt tospovirus (TSWV) is investigated. We showed that TSWV NSm is physically associated with the ER membrane in Nicotiana benthamiana plants. An NSm-GFP fusion protein transiently expressed in single leaf cells was trafficked into neighboring cells. Mutations in NSm that impaired its association with the ER or caused its mis-localization to other subcellular sites inhibited cell-to-cell trafficking.Pharmacological disruption of the ER network severely inhibited NSm-GFP trafficking but not GFP diffusion. In the Arabidopsis thaliana mutant rhd3 with an impaired ER network,NSm-GFP trafficking was significantly reduced, whereas GFP diffusion was not affected.We also showed that the ER-to-Golgi secretion pathway and the cytoskeleton transport systems were not involved in the intercellular trafficking of TSWV NSm. Importantly,TSWV cell-to-cell spread was delayed in the ER-defective rhd3 mutant,and this reduced viral infection was not due to reduced replication. On the basis of robust biochemical,cellular and genetic analysis, we established that the ER membrane transport system serves as an important direct route for intercellular trafficking of NSm and TSWV.