Contribution of Host Intracellular Transport Machineries to Intercellular Movement of Turnip Mosaic Virus

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.

6K2-induced vesicles can move cell to cell during turnip mosaic virus infection

“…We showed previously that GFP-HDEL does not move between cells during TuMV infection (Agbeci et al, 2013). Because both gene cassettes are delivered into the same cells, agroinfiltrated cells are characterized by concomitant green and red fluorescence and any intercellular movement of the mCherry fusion would be characterized by the presence of red-only fluorescence.…”

Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection

“…To modify pVPH-GFP for the inclusion of an independent gene expression cassette for coexpression of an mCherry recombinant protein fused with an ER retention signal, the mCherry-HDEL fragment was amplified from the vector pCambiaTuMV/6K2::mCherry//GFP-HDEL (35). The resulting fragment was inserted between the 35S promoter and the Nos terminator of the binary plant expression vector pCaMterX (36).…”

Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming

“…Although rapid movement of protein bodies induced by plant viruses has generally been linked to viral cell-to-cell movement (10,57,58), our results provide another explanation for rapid intracellular movement: the proximity of viral protein bodies to the ER causes passive movements with ER streaming. In other words, rapid movements with ER streaming can reflect spatial and functional relationships between these protein bodies and the ER.…”