Date Published: October 3, 2013
Publisher: Public Library of Science
Author(s): Maxime Agbeci, Romain Grangeon, Richard S. Nelson, Huanquan Zheng, Jean-François Laliberté, Christophe Ritzenthaler.
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.
Plant viruses move from the initially infected cell to neighboring cells during local spread and then 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 then 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 ). The size exclusion limit (SEL) of PD is normally too small to allow passive transport of large molecular complexes, but plant viruses encode movement proteins (MPs) that increase the SEL of PDs to allow passage of the viral RNA (reviewed in , ). 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 , ). In the case of tobacco mosaic virus (TMV), the viral RNA appears to spread between cells as membrane MP-associated viral replication complexes (VRCs) . 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 –.
Studies on intercellular movement have shown that plant viruses may use different trafficking pathways to move from one cell to another (reviewed in , ). In this study, by discriminating infiltrated and primary-infected cells from cells infected following intercellular virus movement, we were able to evaluate the contribution of the secretory pathway and the cytoskeleton for TuMV intercellular movement.