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Abstract
The contribution of different host cell transport systems in the intercellular movement of turnip mosaic virus (TuMV) was investigated. To discriminate between primary infections and secondary infections associated with the virus intercellular movement, a gene cassette expressing GFP-HDEL was inserted adjacent to a TuMV infectious cassette expressing 6K2:mCherry, both within the T-DNA borders of the binary vector pCambia. In this system, both gene cassettes were delivered to the same cell by a single binary vector and primary infection foci emitted green and red fluorescence while secondarily infected cells emitted only red fluorescence. Intercellular movement was measured at 72 hours post infiltration and was estimated to proceed at an average rate of one cell being infected every three hours over an observation period of 17 hours. To determine if the secretory pathway were important for TuMV intercellular movement, chemical and protein inhibitors that blocked both early and late secretory pathways were used. Treatment with Brefeldin A or Concanamycin A or expression of ARF1 or RAB-E1d dominant negative mutants, all of which inhibit pre- or post-Golgi transport, reduced intercellular movement by the virus. These treatments, however, did not inhibit virus replication in primary infected cells. Pharmacological interference assays using Tyrphostin A23 or Wortmannin showed that endocytosis was not important for TuMV intercellular movement. Lack of co-localization by endocytosed FM4-64 and Ara7 (AtRabF2b) with TuMV-induced 6K2-tagged vesicles further supported this conclusion. Microfilament depolymerizing drugs and silencing expression of myosin XI-2 gene, but not myosin VIII genes, also inhibited TuMV intercellular movement. Expression of dominant negative myosin mutants confirmed the role played by myosin XI-2 as well as by myosin XI-K in TuMV intercellular movement. Using this dual gene cassette expression system and transport inhibitors, components of the secretory and actomyosin machinery were shown to be important for TuMV intercellular spread.
Highlights
Plant viruses move from the initially infected cell to neighboring cells during local spread and over long distances through vascular tissues to establish a systemic infection in the plant
By using a dual cassette of genes encoding fluorescent proteins that can differentiate between primary infected cells and cells infected after intercellular transport, we provide evidence that turnip mosaic virus (TuMV) needs a functional secretory pathway where pre- and post-Golgi trafficking and the actomyosin network are important for its movement
In order to discriminate initially infected cells from later infected cells in live tissue, we introduced within the T-DNA borders of a binary vector a gene cassette expressing the endoplasmic reticulum (ER)-localized GFP-HDEL adjacent to a TuMV infectious genome cassette expressing 6K2:mCherry (Fig. 1A)
Summary
Plant viruses move from the initially infected cell to neighboring cells during local spread and over long distances through vascular tissues to establish a systemic infection in the plant. Transport of viruses between cells first involves the intracellular movement of the viral RNA from the site of replication to plasmodesmata (PDs) and its delivery into neighboring cells through PDs. PDs are tunnels in the cell wall that connect the cytoplasm, the endoplasmic reticulum (ER) and the plasma membrane between adjoining cells (reviewed in [1]). Intracellular movement likely involves a membrane-associated viral RNA-host and viral protein complex, but the exact configuration of the viral entity that enters the neighboring cells has not yet been determined (reviewed in [2,4]). In the case of tobacco mosaic virus (TMV), the viral RNA appears to spread between cells as membrane MP-associated viral replication complexes (VRCs) [5]. For members of the comovirus and caulimovirus genera, viral particles transit through MP-induced tubules that go through PDs for their delivery into non-infected cells [6,7,8,9,10]
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