Arabidopsis synaptotagmin SYTA regulates endocytosis and virus movement protein cell-to-cell transport

Abstract

Synaptotagmins are calcium sensors that regulate synaptic vesicle exo/endocytosis. Thought to be exclusive to animals, they have recently been characterized in plants. We show that Arabidopsis synaptotagmin SYTA regulates endosome recycling and movement protein (MP)-mediated trafficking of plant virus genomes through plasmodesmata. SYTA localizes to endosomes in plant cells and directly binds the distinct Cabbage leaf curl virus (CaLCuV) and Tobacco mosaic virus (TMV) cell-to-cell movement proteins. In a SYTA knockdown line, CaLCuV systemic infection is delayed, and cell-to-cell spread of TMV and CaLCuV movement proteins is inhibited. A dominant-negative SYTA mutant causes depletion of plasma membrane-derived endosomes, produces large intracellular vesicles attached to plasma membrane, and inhibits cell-to-cell trafficking of TMV and CaLCuV movement proteins, when tested in an Agrobacterium-based leaf expression assay. Our studies show that SYTA regulates endocytosis, and suggest that distinct virus movement proteins transport their cargos to plasmodesmata for cell-tocell spread via an endocytic recycling pathway. S ynaptotagmins (Syts) are a large family of Ca 2+ /lipid binding proteins widely studied in animals due to their role in neurotransmitter release. They are also found in Drosophila and Caenorhabditis elegans and were recently described in plants (1, 2). Syts have a conserved domain structure: a short uncleaved N-terminal signal peptide that overlaps a transmembrane (TM) domain, followed by a cytosolic variable region and two C-terminal C 2 domains, C 2 A and C 2 B. Whereas C 2 A and C 2 B each bind phospholipids in a Ca 2+ -dependent manner, fold independently and act synergistically, C 2 B is essential for activity (1). SytI, the best studied Syt, is proposed to act as a Ca 2+ sensor to regulate rapid and synchronous synaptic vesicle exocytosis (1). Whether it regulates SNARE complex formation in a temporal and spatial manner, or is itself fusogenic, is unclear. Studies in PC12 cells, and of mouse and Drosophila sytI mutants, suggest that the SNARE complex VAMP1/SNAP25/syntaxin-1 targets the synaptic vesicle to the plasma membrane to create a metastable fusion intermediate. SytI on the vesicle membrane, and perhaps a distinct partner Syt on the plasma membrane, would then interact with phospholipids and the SNARE complex to accelerate SNARE-mediated fusion pore dilation. Liposome studies suggest a direct fusogenic role for SytI, in which shallow insertion of the C 2 region into target membranes induces curvature to destabilize the lipid bilayer and form the fusion pore opening (1, 3). Studies in mice, Drosophila, and C. elegans show that SytI also regulates the kinetics of endocytosis at nerve terminals, apparently in a clathrin-mediated manner (1, 4).Plant virus movement proteins (MPs) mediate the transport of progeny genomes across the cell wall for local and systemic infection. Despite diverse strategies for cell-to-cell movement, two common features have emerged: movement proteins alter plasmodes...

The Arabidopsis Synaptotagmin1 Is Enriched in Endoplasmic Reticulum-Plasma Membrane Contact Sites and Confers Cellular Resistance to Mechanical Stresses

“…Moreover, cell biological and biochemical analyses have reported SYT1 being at the ER, PM, and plasmodesmata (Schapire et al, 2008;Lewis and Lazarowitz, 2010;Yamazaki et al, 2010). To unequivocally establish the SYT1 subcellular localization, we developed new polyclonal anti-SYT1 244-541 antibodies (hereafter, anti-SYT1), and demonstrated their specificity by the absence of signal in the null syt1-2 mutant (hereafter, syt1) via western blot and immunolocalization (Supplemental Fig.…”

“…In these reports, SYT1 was proposed to act as a Ca 2+ -dependent regulator of membrane fusion, in analogy to the classical animal SYTs that mediate Ca 2+ -triggered vesicle fusion during neurotransmission (Carr and Munson, 2007). Another report highlighted a role for SYT1 in viral spreading from cell to cell (Lewis and Lazarowitz, 2010). The recent characterization of mammalian E-Syts in stress tolerance (Herdman et al, 2014) and the phylogenetic relationships of tricalbins and E-Syts with SYT1 (Craxton, 2010;Yamazaki et al, 2010) led us to hypothesize that SYT1 could be a functional ortholog of E-Syts and tricalbins in plants.…”

“…In this article, we characterize the Arabidopsis (Arabidopsis thaliana) type I anchor Synaptotagmin1 (SYT1/ SYTA/NTMC2T1.1 [hereafter SYT1]; Craxton, 2010;Yamazaki et al, 2010;Lewis and Lazarowitz, 2010) as an important component required to withstand mechanical stress in plant cells. SYT1 belongs to a five-member family in Arabidopsis and shares a common modular structure with different members of the mammalian extended synaptotagmins (E-Syts) and yeast (Saccharomyces cerevisiae) tricalbins families of organelle tethers (Manford et al, 2012;Fig.…”

A Membrane Microdomain-Associated Protein, Arabidopsis Flot1, Is Involved in a Clathrin-Independent Endocytic Pathway and Is Required for Seedling Development

“…Endocytosis and vesicle recycling play crucial roles in plant developmental processes, including polar growth of root hair cells (Dhonukshe et al, 2006), cell-to-cell communication (Lewis and Lazarowitz, 2010), establishment of root cell polarity, and other auxin-dependent processes (Kleine-Vehn et al, 2008). As these studies have focused mainly on clathrin-dependent endocytosis, little information is currently available on the role of the non-clathrin-mediated pathway in seedling development.…”

Plasma Membrane Localization of Solanum tuberosum Remorin from Group 1, Homolog 3 Is Mediated by Conformational Changes in a Novel C-Terminal Anchor and Required for the Restriction of Potato Virus X Movement

“…The recent report of the role of Arabidopsis (Arabidopsis thaliana) synaptotagmin1 in the control of Begomovirus and Tobamovirus cell-to-cell movement (Lewis and Lazarowitz, 2010) sheds new light on the involvement of host membrane proteins in virus movement. Arabidopsis synaptotagmin1 resides in the cortical endoplasmic reticulum, endosomes, DIMs at the PM, and plasmodesmata (PDs; Schapire et al, 2008;Minami et al, 2009;Lewis and Lazarowitz, 2010;Yamazaki et al, 2010), and regulates endocytosis and PM repair processes (Schapire et al, 2008;Yamazaki et al, 2008;Lewis and Lazarowitz, 2010). These studies support a link between DIMs and PDs, and provide evidence for a role of DIM proteins in cell-to-cell communication via PDs.…”