Differing requirements for actin and myosin by plant viruses for sustained intercellular movement - PubMed (original) (raw)

Differing requirements for actin and myosin by plant viruses for sustained intercellular movement

Phillip A Harries et al. Proc Natl Acad Sci U S A. 2009.

Abstract

The actin cytoskeleton has been implicated in the intra- and intercellular movement of a growing number of plant and animal viruses. However, the range of viruses influenced by actin for movement and the mechanism of this transport are poorly understood. Here we determine the importance of microfilaments and myosins for the sustained intercellular movement of a group of RNA-based plant viruses. We demonstrate that the intercellular movement of viruses from different genera [tobacco mosaic virus (TMV), potato virus X (PVX), tomato bushy stunt virus (TBSV)], is inhibited by disruption of microfilaments. Surprisingly, turnip vein-clearing virus (TVCV), a virus from the same genus as TMV, did not require intact microfilaments for normal spread. To investigate the molecular basis for this difference we compared the subcellular location of GFP fusions to the 126-kDa protein and the homologous 125-kDa protein from TMV and TVCV, respectively. The 126-kDa protein formed numerous large cytoplasmic inclusions associated with microfilaments, whereas the 125-kDa protein formed few small possible inclusions, none associated with microfilaments. The dependence of TMV, PVX, and TBSV on intact microfilaments for intercellular movement led us to investigate the role of myosin motors in this process. Virus-induced gene silencing of the Nicotiana benthamiana myosin XI-2 gene, but not three other myosins, inhibited only TMV movement. These results indicate that RNA viruses have evolved differently in their requirements for microfilaments and the associated myosin motors, in a manner not correlated with predicted phylogeny.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

The effect of LatB on virus spread. (A) Representative images showing GFP lesions formed upon infection with the indicated viruses either in the absence (-) or presence (+) of 5 μM LatB. All images were taken at 6 dpi. (Scale bar, 1 mm.) (B–E) Lesion areas were quantified to determine the effect of LatB on the cell-to-cell movement of (B) TMV, (C) PVX, (D) TBSV, and (E) TVCV at 2, 4, and 6 dpi. N. benthamiana leaf tissue was infiltrated with either the actin inhibitor LatB (circles) or a DMSO buffer control (squares). Bars represent standard errors for 15 lesions per treatment.

Fig. 2.

Fig. 2.

The TVCV 125-kDa protein does not form numerous large inclusions like the TMV 126-kDa protein. Representative images showing TMV 126-kDa protein (A) and TVCV 125-kDa protein (B) GFP fusions expressed in N. benthamiana leaf epidermal cells 3 days following agrobacterium infiltration. The positions of TMV 126-kDa protein bodies (A) and potential TVCV 125-kDa protein bodies (B) are indicated with arrows. Red fluorescent bodies are chloroplasts. (Scale bar, 25 μm.)

Fig. 3.

Fig. 3.

VIGS of individual myosin genes. Quantitative RT-PCR was used to determine the relative expression ratio of target genes (myosin VIII-1, myosin VIII-2, myosin XI-2, and myosin XI-F) in lines treated with the indicated TRV silencing constructs versus a TRV control not expressing a myosin fragment. Elongation factor 1α served as an internal loading control for each sample. Expression analysis was performed on extracts from systemic leaves at 18 dpi with TRV constructs. Bars represent means ± standard errors for three replicates per treatment. Analysis of variance followed by an lsd calculation was used to determine significant differences between treatments. Different letters above the bars indicate significant differences (P = 0.05). The experiment was repeated at least once for each TRV silencing construct.

Fig. 4.

Fig. 4.

TMV utilizes a distinct myosin for virus spread in N. benthamiana. GFP lesion areas reflect the cell-to-cell movement of (A) TMV, (B) TVCV, (C) PVX, and (D) TBSV in N. benthamiana leaves silenced for individual myosin genes (VIII-1, VIII-2, XI-2, XI-F) via TRV VIGS. Plants inoculated with wild-type TRV (WT TRV) or buffer (Mock) were controls. The area of GFP fluorescent lesions (mm2) in inoculated leaves was determined at 3 dpi for tissues carrying a systemic infection with the TRV VIGS vector (approximately 18 dpi). Bars represent means ± standard errors for three replicates per treatment. Analysis of variance followed by an lsd calculation was used to determine significant differences between treatments. Different letters above the bars indicate significant differences (P = 0.05). The experiment was repeated at least once for each virus challenge.

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