ANK, a Host Cytoplasmic Receptor for the Tobacco mosaic virus Cell-to-Cell Movement Protein, Facilitates Intercellular Transport through Plasmodesmata

Abstract

Plasmodesma (PD) is a channel structure that spans the cell wall and provides symplastic connection between adjacent cells. Various macromolecules are known to be transported through PD in a highly regulated manner, and plant viruses utilize their movement proteins (MPs) to gate the PD to spread cell-to-cell. The mechanism by which MP modifies PD to enable intercelluar traffic remains obscure, due to the lack of knowledge about the host factors that mediate the process. Here, we describe the functional interaction between Tobacco mosaic virus (TMV) MP and a plant factor, an ankyrin repeat containing protein (ANK), during the viral cell-to-cell movement. We utilized a reverse genetics approach to gain insight into the possible involvement of ANK in viral movement. To this end, ANK overexpressor and suppressor lines were generated, and the movement of MP was tested. MP movement was facilitated in the ANK-overexpressing plants, and reduced in the ANK-suppressing plants, demonstrating that ANK is a host factor that facilitates MP cell-to-cell movement. Also, the TMV local infection was largely delayed in the ANK-suppressing lines, while enhanced in the ANK-overexpressing lines, showing that ANK is crucially involved in the infection process. Importantly, MP interacted with ANK at PD. Finally, simultaneous expression of MP and ANK markedly decreased the PD levels of callose, β-1,3-glucan, which is known to act as a molecular sphincter for PD. Thus, the MP-ANK interaction results in the downregulation of callose and increased cell-to-cell movement of the viral protein. These findings suggest that ANK represents a host cellular receptor exploited by MP to aid viral movement by gating PD through relaxation of their callose sphincters.

DEVELOPMENTALLY REGULATED PLASMA MEMBRANE PROTEIN of Nicotiana benthamiana Contributes to Potyvirus Movement and Transports to Plasmodesmata via the Early Secretory Pathway and the Actomyosin System

“…Plasmodesmata (PD) are plasma membranemediated channels in cell walls that control the intercellular trafficking of micromolecules and macromolecules, including plant viruses (Boevink and Oparka, 2005;Lucas et al, 2009). Plant viruses encode movement proteins (MPs) that can regulate the size exclusion limit (SEL) of PD and mediate virus trafficking between cells (Lucas, 2006;Raffaele et al, 2009;Amari et al, 2010;Ueki et al, 2010). Based on the functions of MPs during virus movement, the viral MPs are divided into three major groups.…”

“…Viruses recruit host factors for their movement in plants Raffaele et al, 2009;Amari et al, 2010;Ueki et al, 2010). Compared with the progresses on viral MP characterization, identifications of MP-interacting host proteins are much behind Oparka, 2004;Raffaele et al, 2009;Amari et al, 2010).…”

“…To date, about 20 host proteins have been identified to interact with specific viral MPs (Pallas and García, 2011). For example, the pectin methylesterase interacted with TMV MP, increased the SEL of PD, and facilitated TMV movement between cells ; an ankyrin repeatcontaining protein (ANK) interacted with TMV MP at PD, down-regulated callose formation, and aided viral movement (Ueki et al, 2010); the Arabidopsis (Arabidopsis thaliana) PLASMODESMATA-LOCALIZED PRO-TEIN1 (AtPDLP1) was reported to interact with GFLV MP and mediate tubule assembly during GFLV cell-tocell movement in plants (Amari et al, 2010(Amari et al, , 2011. TuMV P3N-PIPO was shown to interact with AtPCaP1, a plasma membrane cation-binding protein of Arabidopsis, and colocalize with this host protein at the PD.…”

The Rubisco Small Subunit Is Involved in Tobamovirus Movement and Tm-22-Mediated Extreme Resistance

“…These TMV MPbinding host factors include cell wall-associated proteins such as pectin methylesterase (Chen et al, 2000), calreticulin (Meshi et al, 1989), ANK1 (Ueki et al, 2010), and the cellular DnaJ-like protein MPIP1 (Shimizu et al, 2009). Many cytoskeletal components such as actin filaments (McLean et al, 1995), microtubules (Heinlein et al, 1995), and the microtubuleassociated proteins MPB2C (Kragler et al, 2003) and EB1a (Brandner et al, 2008) also interact with TMV MP.…”

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins <em>in vitro</em>

“…Strong YFP signal was reconstructed when MP and ANK, fused to C-terminal and N-terminal halves of YFP, respectively, were coexpressed in tobacco epidermis following microbombardment of their encoding genes. This BiFC signal accumulated in puncta at the cell periphery, which are diagnostic of plasmodesmata 15 . (B) ANK-MP interaction in vitro as detected by protein membrane overlay assay.…”