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-to-cell spread via an endocytic recycling pathway.

Fig. 1.

SYTA binds Begomovirus MP and TMV 30K in vitro. (A) Annotated SYTA locus (Upper) and predicted SYTA domain structure (Lower). Shown are 5′- and 3′-UTRs, exons (boxes), nucleotides in genomic locus (Upper) and in full-length cDNA (Lower), and syta-1 (Sail775A08) T-DNA insertion (cDNA nt 1,728). TM, transmembrane domain. (B) SDS/PAGE analysis of ³⁵S-met-labeled MP_CaLCuV or MP_SqLCV (33 kDa), TMV 30K [32–34 kDa doublet (5)], or luciferase (61 kDa) bound (BND) to GST-SYTA^ΔTM or GST in vitro, and 8% of unbound proteins (UN). Small MP_CaLCuV products are from two internal AUG starts.

Fig. 2.

CaLCuV infection is delayed in syta-1. (A) Wild-type (WT) Col-0 (red) and syta-1 line 1 (blue) were inoculated with equal amounts of CaLCuV (Table S3, Trial 2). (B) CaLCuV or mock inoculated (GV3101) WT Col-0 or syta-1 line 1 at 30 dpi. (C) sqRT-PCR of RNA from 4-wk-old syta-1 inflorescences or WT Col-0. Primers detected SYTA transcripts (SYTA) or 3′ end (SYTA^3′’) or 18S RNA (18S, loading control). C, no template control. Leaf extracts from 10-day-old seedlings gave identical results. (D) Immunoblots of extracts from 10-day-old seedlings probed with preimmune (1:2,000) or anti-SYTA (1:5,000) sera. 3X, three times the syta-1 line 1 extract loaded to show truncated 59.1-kDa SYTA^T. Identity of anti-SYTA cross-reacting band is unknown. (E) sqPCR of CaLCuV DNA in symptomatic (symp) and asymptomatic (asymp) leaves of WT Col-0 or syta-1 line 1 at 15 dpi. NSP, CaLCuV DNA; NSI, At-NSI loading control; mock, GV3101; C, no template control. Markers in bp (C and E) or kDa (D).

Fig. 3.

SYTA localizes to endosomes. CLSM projected Z-series of (A–E and N) SYTA-GFP or (F–M) SYTA^ΔC2B-GFP expressed in N. benthamiana protoplasts (A–M) or leaf epidermal cell (N). Cells imaged at 20 h or 40 h posttransfection as labeled. (C–E, 10 min FM4-64) SYTA-GFP localized to endosomes. (K–M, 6 min FM4-64) SYTA^ΔC2B-GFP at plasma membrane (merged yellow-orange). (G–J) SYTA^ΔC2B-GFP colocalizes with ER marker KDEL-CFP. (Scale bars, 8 μm.)

Fig. 4.

SYTA^ΔC2B inhibits the formation of plasma membrane-derived endosomes. (A–E) CLSM images (C and D) or projected Z-series (A, B, and E) of FM4-64 labeled N. benthamiana leaf sectors infiltrated with (A) GV2260, (B) SYTA-Myc, or (C–E) SYTA^ΔC2B-Myc. (F–J) CSLM projected Z-series of N. benthamiana leaf sectors infiltrated with RabF1-GFP and (F) SYTA-myc or (G–J) SYTA^ΔC2B-Myc. H is the cell in G scanned at higher magnification. Scale bars marked.