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Myosins VIII and XI play distinct roles in reproduction and transport of tobacco mosaic virus.

Amari K, Di Donato M, Dolja VV, Heinlein M - PLoS Pathog. (2014)

Bottom Line: The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation.The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane.Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.

View Article: PubMed Central - PubMed

Affiliation: Zürich-Basel Plant Science Center, Botany, Department of Environmental Sciences, University of Basel, Basel, Switzerland.

ABSTRACT
Viruses are obligatory parasites that depend on host cellular factors for their replication as well as for their local and systemic movement to establish infection. Although myosin motors are thought to contribute to plant virus infection, their exact roles in the specific infection steps have not been addressed. Here we investigated the replication, cell-to-cell and systemic spread of Tobacco mosaic virus (TMV) using dominant negative inhibition of myosin activity. We found that interference with the functions of three class VIII myosins and two class XI myosins significantly reduced the local and long-distance transport of the virus. We further determined that the inactivation of myosins XI-2 and XI-K affected the structure and dynamic behavior of the ER leading to aggregation of the viral movement protein (MP) and to a delay in the MP accumulation in plasmodesmata (PD). The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation. The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane. These results suggest that class XI myosins contribute to the viral propagation and intracellular trafficking, whereas myosins VIII are specifically required for the MP targeting to and virus movement through the PD. Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.

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Inhibition of myosin XI-2 and XI-K reduces the efficiency by which MP is targeted to PD.A-C, Kyomographs showing examples of fluorescence recovery during the time of the FRAP experiments (50 min, from the left to the right) after bleaching of several PD (arrows) at 1 dpa. A, FRAP experiment in the presence of RFP. MP:GFP fluorescence at photobleached PD recovers to 70% during 50 minutes after bleaching (lower panel). B, FRAP experiment in the presence of myosin XI-2 tails. MP:GFP fluorescence at PD recovers only to 30% during 50 minutes (lower panel). C, FRAP experiment in the presence of myosin XI-K tails. Like in the presence of XI-2, MP:GFP fluorescence at PD recovers only to 30% during 50 minutes (lower panel). Fat arrows at the left side of the kymographs indicate the location of photobleached PD. Arrows on the top of the kymographs indicate the bleaching time point.
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ppat-1004448-g006: Inhibition of myosin XI-2 and XI-K reduces the efficiency by which MP is targeted to PD.A-C, Kyomographs showing examples of fluorescence recovery during the time of the FRAP experiments (50 min, from the left to the right) after bleaching of several PD (arrows) at 1 dpa. A, FRAP experiment in the presence of RFP. MP:GFP fluorescence at photobleached PD recovers to 70% during 50 minutes after bleaching (lower panel). B, FRAP experiment in the presence of myosin XI-2 tails. MP:GFP fluorescence at PD recovers only to 30% during 50 minutes (lower panel). C, FRAP experiment in the presence of myosin XI-K tails. Like in the presence of XI-2, MP:GFP fluorescence at PD recovers only to 30% during 50 minutes (lower panel). Fat arrows at the left side of the kymographs indicate the location of photobleached PD. Arrows on the top of the kymographs indicate the bleaching time point.

Mentions: Although the inactivation of myosins XI-2 and XI-K had no significant effect on the PD localization of the MP, the obvious effect on the structure and dynamic behavior of the ER could affect the dynamics and efficiency of MP targeting to PD. To examine this possibility, we performed Fluorescence Recovery After Photobleaching (FRAP) assays. Upon bleaching the MP:GFP fluorescence at PD, the rate of the MP:GFP fluorescence recovery over time reflects the efficiency with which MP:GFP is delivered to PD. Figure 6 shows the results of FRAP analysis in the format of kymographs providing both the visual representation of PD fluorescence recovery over time (upper panels) and a quantitative assessment in the form of diagrams (lower panels). As expected, overexpression of RFP did not affect the recovery of MP:GFP fluorescence at PD upon bleaching: the fluorescence levels recovered to 70% of the pre-bleach level within 50 minutes (Figure 6A). In contrast, the recovery of MP:GFP fluorescence at PD was severely reduced to less than 30% upon expression of myosin XI-2 or XI-K tails (Figure 6B and C, respectively). Thus, the effects of myosin XI-2 or XI-K inhibition on the organization and dynamic behavior of the ER, as well as on the distribution of MP along the membrane correlate with a reduced efficiency of the MP in targeting to the PD and with significant inhibition of virus transport between cells.


Myosins VIII and XI play distinct roles in reproduction and transport of tobacco mosaic virus.

Amari K, Di Donato M, Dolja VV, Heinlein M - PLoS Pathog. (2014)

Inhibition of myosin XI-2 and XI-K reduces the efficiency by which MP is targeted to PD.A-C, Kyomographs showing examples of fluorescence recovery during the time of the FRAP experiments (50 min, from the left to the right) after bleaching of several PD (arrows) at 1 dpa. A, FRAP experiment in the presence of RFP. MP:GFP fluorescence at photobleached PD recovers to 70% during 50 minutes after bleaching (lower panel). B, FRAP experiment in the presence of myosin XI-2 tails. MP:GFP fluorescence at PD recovers only to 30% during 50 minutes (lower panel). C, FRAP experiment in the presence of myosin XI-K tails. Like in the presence of XI-2, MP:GFP fluorescence at PD recovers only to 30% during 50 minutes (lower panel). Fat arrows at the left side of the kymographs indicate the location of photobleached PD. Arrows on the top of the kymographs indicate the bleaching time point.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199776&req=5

ppat-1004448-g006: Inhibition of myosin XI-2 and XI-K reduces the efficiency by which MP is targeted to PD.A-C, Kyomographs showing examples of fluorescence recovery during the time of the FRAP experiments (50 min, from the left to the right) after bleaching of several PD (arrows) at 1 dpa. A, FRAP experiment in the presence of RFP. MP:GFP fluorescence at photobleached PD recovers to 70% during 50 minutes after bleaching (lower panel). B, FRAP experiment in the presence of myosin XI-2 tails. MP:GFP fluorescence at PD recovers only to 30% during 50 minutes (lower panel). C, FRAP experiment in the presence of myosin XI-K tails. Like in the presence of XI-2, MP:GFP fluorescence at PD recovers only to 30% during 50 minutes (lower panel). Fat arrows at the left side of the kymographs indicate the location of photobleached PD. Arrows on the top of the kymographs indicate the bleaching time point.
Mentions: Although the inactivation of myosins XI-2 and XI-K had no significant effect on the PD localization of the MP, the obvious effect on the structure and dynamic behavior of the ER could affect the dynamics and efficiency of MP targeting to PD. To examine this possibility, we performed Fluorescence Recovery After Photobleaching (FRAP) assays. Upon bleaching the MP:GFP fluorescence at PD, the rate of the MP:GFP fluorescence recovery over time reflects the efficiency with which MP:GFP is delivered to PD. Figure 6 shows the results of FRAP analysis in the format of kymographs providing both the visual representation of PD fluorescence recovery over time (upper panels) and a quantitative assessment in the form of diagrams (lower panels). As expected, overexpression of RFP did not affect the recovery of MP:GFP fluorescence at PD upon bleaching: the fluorescence levels recovered to 70% of the pre-bleach level within 50 minutes (Figure 6A). In contrast, the recovery of MP:GFP fluorescence at PD was severely reduced to less than 30% upon expression of myosin XI-2 or XI-K tails (Figure 6B and C, respectively). Thus, the effects of myosin XI-2 or XI-K inhibition on the organization and dynamic behavior of the ER, as well as on the distribution of MP along the membrane correlate with a reduced efficiency of the MP in targeting to the PD and with significant inhibition of virus transport between cells.

Bottom Line: The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation.The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane.Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.

View Article: PubMed Central - PubMed

Affiliation: Zürich-Basel Plant Science Center, Botany, Department of Environmental Sciences, University of Basel, Basel, Switzerland.

ABSTRACT
Viruses are obligatory parasites that depend on host cellular factors for their replication as well as for their local and systemic movement to establish infection. Although myosin motors are thought to contribute to plant virus infection, their exact roles in the specific infection steps have not been addressed. Here we investigated the replication, cell-to-cell and systemic spread of Tobacco mosaic virus (TMV) using dominant negative inhibition of myosin activity. We found that interference with the functions of three class VIII myosins and two class XI myosins significantly reduced the local and long-distance transport of the virus. We further determined that the inactivation of myosins XI-2 and XI-K affected the structure and dynamic behavior of the ER leading to aggregation of the viral movement protein (MP) and to a delay in the MP accumulation in plasmodesmata (PD). The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation. The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane. These results suggest that class XI myosins contribute to the viral propagation and intracellular trafficking, whereas myosins VIII are specifically required for the MP targeting to and virus movement through the PD. Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.

Show MeSH
Related in: MedlinePlus