Class VIII myosins are required for plasmodesmatal localization of a closterovirus Hsp70 homolog

Abstract

The Hsp70 homolog (Hsp70h) of Beet yellows virus (BYV) functions in virion assembly and cell-to-cell movement and is autonomously targeted to plasmodesmata in association with the actomyosin motility system (A. I. Prokhnevsky, V. V. Peremyslov, and V. V. Dolja, J. Virol. 79:14421-14428, 2005). Myosins are a diverse category of molecular motors that possess a motor domain and a tail domain involved in cargo binding. Plants have two classes of myosins, VIII and XI, whose specific functions are poorly understood. We used dominant negative inhibition to identify myosins required for Hsp70h localization to plasmodesmata. Six full-length myosin cDNAs from the BYV host plant Nicotiana benthamiana were sequenced and shown to encode apparent orthologs of the Arabidopsis thaliana myosins VIII-1, VIII-2, VIII-B, XI-2, XI-F, and XI-K. We found that the ectopic expression of the tail domains of each of the class VIII, but not the class XI, myosins inhibited the plasmodesmatal localization of Hsp70h. In contrast, the overexpression of the motor domains or the entire molecules of the class VIII myosins did not affect Hsp70h targeting. Further mapping revealed that the minimal cargo-binding part of the myosin VIII tails was both essential and sufficient for the inhibition of the proper Hsp70h localization. Interestingly, plasmodesmatal localization of the Tobacco mosaic virus movement protein and Arabidopsis protein RGP2 was not affected by myosin VIII tail overexpression. Collectively, our data implicate class VIII myosins in protein delivery to plasmodesmata and suggest that more than one mechanism of such delivery exist in plants.

FIG. 1.

Diagram showing the domain architecture of the N. benthamiana myosins characterized in this work. Color-coded dotted lines correspond to the truncated myosin variants used for overexpression. The exact nucleotide sequences of these variants are presented in Table 1. DIL-motif, dilute motif.

FIG. 2.

Subcellular localization of BYV Hsp70h-GFP coexpressed with the N. benthamiana myosin tails. (A) Coexpression of Hsp70h-GFP with an empty vector control (no myosin) (top row) or the tails of myosins XI-2, XI-F, and XI-K. (B) Coexpression of Hsp70h-GFP with A. thaliana (At) Hsc70 (control) (top row) or the tails of myosins VIII-1, VIII-2, and VIII-B. The scale bars in the left columns in both panels A and B represent 50 μm. The images in the right columns in panels A and B are close-ups of the areas in white boxes in the corresponding left panels; scale bars represent 10 μm. (C) Immunoblot analyses of the coexpressed proteins using HA-specific antibody to detect HA-tagged myosin tails and Hsc70 (Hsc; no, empty vector control) (top panel) or GFP-specific antibody to detect Hsp70h-GFP (middle panel). Positions of selected molecular weight markers are shown on the left. (Bottom panel) Coomassie-stained Rubisco subunit.

FIG. 3.

The overexpression of the myosin VIII-B tail does not induce observable changes in the architecture of actin microfilaments in the epidermal cells of N. benthamiana. The actin cytoskeleton was visualized using confocal laser scanning microscopy and the yellow fluorescent protein-tagged actin-binding domain 2 of the A. thaliana fimbrin as a reporter (36). (A) Actin microfilaments in a cell overexpressing myosin VIII-B tails. (B) Actin microfilaments in the cells transformed with an empty vector (control). The scale bars in the left panels represent 50 μm. The right panels are close-ups of the areas in white boxes in the corresponding left panels; scale bars represent 10 μm.

FIG. 4.

Mapping the minimal myosin VIII domain required for dominant negative inhibition of Hsp70h-GFP localization to plasmodesmata. (A) Coexpression of Hsp70h-GFP with the full-size myosin VIII-1, its motor domain alone, or the motor domain with the coiled-coil (CC) motif. (B) Coexpression of Hsp70h-GFP with the truncated myosin VIII-1 variants as indicated on the panels. The scale bars in the left columns in both panels A and B represent 50 μm. The images in the right columns in panels A and B are close-ups of the areas in white boxes in the corresponding left panels; scale bars represent 10 μm. (C) Immunoblot analysis of the HA-tagged myosin VIII-1 derivatives using HA-specific antibody. F, full-size myosin VIII-1; MC, motor domain and coiled-coil motif; M, motor domain only. (D) Similar analysis of the HA-tagged myosin VIII-1 tail (IQC, IQ motifs to the C terminus) and its truncated variants, the coiled-coil motif to the C terminus (CCC) and the GTD only. The maps and limits of the myosin variants are also shown in Fig. 1 and Table 1. Positions of selected molecular weight markers are shown on the right.

FIG. 5.

Mapping the minimal myosin VIII domain required for dominant negative inhibition of Hsp70h-GFP localization to plasmodesmata. (A) Coexpression of Hsp70h-GFP with the full-size myosin VIII-2, its motor domain, or the motor domain with the coiled-coil motif. (B) Coexpression of Hsp70h-GFP with the truncated myosin VIII-2 variants as indicated on the panels. The scale bars in the left columns represent 50 μm. The images in the right columns are close-ups of the areas in white boxes in the corresponding left panels; scale bars represent 10 μm. Abbreviations are the same as those in Fig. 4.

FIG. 6.

The coexpression of the myosin VIII-2 tails does not affect the plasmodesmatal localization of TMV MP-GFP (A and B) or A. thaliana RGP2-GFP (C and D) but does abolish the plasmodesmatal localization of Hsp70h-mRFP (B and D). (C) GFP-tagged TMV-MV-p30 coexpression with both mRFP-tagged Hsp70h and the myosin VIII tail. (D) GFP-tagged A. thaliana RGP2 coexpression with both mRFP-tagged Hsp70h and the myosin VIII tail. The scale bars in the left images in panels A to D represent 50 μm. The right images in panels A to D are close-ups of the areas in white boxes in the corresponding left panels; scale bars represent 10 μm.