The ER-Membrane Transport System Is Critical for Intercellular Trafficking of the NSm Movement Protein and Tomato Spotted Wilt Tospovirus

Zhike Feng,Min Zhu,Min Xu,Maria J Garcia-Murria,Wenyang Zhao,Ying Huang,Ismael Mingarro,Yong Liu,Fan Xue,Xiaorong Tao,Xiaojiao Chen,Lei Jiang

doi:10.1371/journal.ppat.1005443

Abstract

Plant 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.

Highlights

Plasmodesma-mediated macromolecular trafficking plays important roles in plant growth and development [1,2,3] and in plant–pathogen interactions [4,5,6]
In this study, using in vitro and in vivo systems for characterizing membrane proteins, we identified that the TSWV NSm movement protein was physically associated with the endoplasmic reticulum (ER) membrane
Taking advantage of TSWV NSm being tightly associated with ER membrane and trafficked between cells through plasmodesmata, we demonstrated here by robust biochemical, cellullar and genetic evidence that the ER membrane transport system of plants serves as an important route for intercellular trafficking of the NSm movement protein and TSWV

Summary

Introduction

Plasmodesma-mediated macromolecular trafficking plays important roles in plant growth and development [1,2,3] and in plant–pathogen interactions [4,5,6]. Besides the long-established cell-to-cell transport of small molecules via plasmodesmata, macromolecules such as proteins and RNAs have been shown in the last two decades to traffic between cells through plasmodesmata (PD) Such macromolecular trafficking is crucial for viral infection [4,5,6], plant defense [8,9], and developmental regulation [1,2,3]. Plant viruses need to move within and between cells to establish systemic infection To accomplish this task, the plant virus encodes a movement protein (MP) to facilitate intracellular trafficking of the viral genomes from the replication site to PD and to assist the spread of the viral replication complexes or viral particles between plant cells through PD [5,6,10,11,12,13]. The endocytic pathway has been shown to influence the movement of three viruses [41,42,43]

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