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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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Dominant negative repression of specific myosins inhibits TMV-GFP cell-to-cell spread.A, example of an N. benthamiana leaf with TMV-GFP infection sites at 4 dpi in the presence of RFP in one half of the leaf and of inhibitory myosin VIII-1 tails in the other half. B, areas of viral infection sites are reduced in N. benthamiana half-leaves transiently expressing myosin VIII-1, VIII-2, VIII-B, XI-2 or XI-K tails as compared to half-leaves expressing RFP. Transient expression of myosin XI-F tails has no effect on the area of TMV-GFP viral infection sites. Boxplots depict the 25th to 75th percentile of measured areas of viral infection sites. Error bars indicate the range of the 5th and 95th percentile of measured areas of viral infection sites. The horizontal bar in each boxplot indicates the median value. Circles above the graphs represent outliers. n represents the number of viral infection sites measured on three half-leaves. Mean area of viral infection sites is given in red. (***) represents significant differences as determined by unbalanced ANOVA (p<0.001). Data are from three independent experiments. C, immunoblot analysis using HA- (top panel) antibodies revealed the expression of the HA-tagged myosin tails. Bands corresponding to class XI (≈100 kDa) and class VIII (≈40 kDa) myosin tails are marked by asterisks. Coomassie blue staining (bottom panel) is shown as loading control. All leaves were inoculated with equal volumes of TMV-GFP in-vitro transcription reaction mix and agro-infiltrated for myosin tail/RFP expression two days later. Analyses were conducted at 4 dpi.
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ppat-1004448-g001: Dominant negative repression of specific myosins inhibits TMV-GFP cell-to-cell spread.A, example of an N. benthamiana leaf with TMV-GFP infection sites at 4 dpi in the presence of RFP in one half of the leaf and of inhibitory myosin VIII-1 tails in the other half. B, areas of viral infection sites are reduced in N. benthamiana half-leaves transiently expressing myosin VIII-1, VIII-2, VIII-B, XI-2 or XI-K tails as compared to half-leaves expressing RFP. Transient expression of myosin XI-F tails has no effect on the area of TMV-GFP viral infection sites. Boxplots depict the 25th to 75th percentile of measured areas of viral infection sites. Error bars indicate the range of the 5th and 95th percentile of measured areas of viral infection sites. The horizontal bar in each boxplot indicates the median value. Circles above the graphs represent outliers. n represents the number of viral infection sites measured on three half-leaves. Mean area of viral infection sites is given in red. (***) represents significant differences as determined by unbalanced ANOVA (p<0.001). Data are from three independent experiments. C, immunoblot analysis using HA- (top panel) antibodies revealed the expression of the HA-tagged myosin tails. Bands corresponding to class XI (≈100 kDa) and class VIII (≈40 kDa) myosin tails are marked by asterisks. Coomassie blue staining (bottom panel) is shown as loading control. All leaves were inoculated with equal volumes of TMV-GFP in-vitro transcription reaction mix and agro-infiltrated for myosin tail/RFP expression two days later. Analyses were conducted at 4 dpi.

Mentions: To identify myosin motors involved in TMV movement, N. benthamiana plants were inoculated with TMV-GFP, an engineered TMV variant expressing GFP in place of the CP [41]. At two days post inoculation (dpi), the inoculated leaves where agro-infiltrated for expression of a specific N. benthamiana myosin tail in one half of the leaf blade and for expression of free RFP as control in the other half (Figure 1A). At 4 dpi, the sizes of fluorescent infection sites were measured and compared between the different treatments. Figure 1B shows that the mean area of TMV-GFP infection sites in the half leaves expressing myosin tails VIII-1, VIII-2, or VIII-B was reduced by ∼50% as compared to the corresponding control half leaves. The sizes of the TMV-GFP infection sites were also reduced upon overexpression of myosin XI-K and XI-2 tails, of which the former had a stronger effect than the latter (Figure 1B). In contrast, overexpression of the myosin XI-F tail did not exert a statistically significant change in the size of virus infection sites. The expression of myosin tails in infected leaves at 4 dpi was validated by immunoblot analysis that clearly showed that the observed effects on TMV spread were dependent on each myosin's identity rather than on differential expression levels (Figure 1C). Using the same assays, similar reductions in the sizes of viral infection sites upon transient expression of myosin tails were observed using TMV-GFP-JL24 [42], a TMV variant that expresses both the GFP and CP.


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)

Dominant negative repression of specific myosins inhibits TMV-GFP cell-to-cell spread.A, example of an N. benthamiana leaf with TMV-GFP infection sites at 4 dpi in the presence of RFP in one half of the leaf and of inhibitory myosin VIII-1 tails in the other half. B, areas of viral infection sites are reduced in N. benthamiana half-leaves transiently expressing myosin VIII-1, VIII-2, VIII-B, XI-2 or XI-K tails as compared to half-leaves expressing RFP. Transient expression of myosin XI-F tails has no effect on the area of TMV-GFP viral infection sites. Boxplots depict the 25th to 75th percentile of measured areas of viral infection sites. Error bars indicate the range of the 5th and 95th percentile of measured areas of viral infection sites. The horizontal bar in each boxplot indicates the median value. Circles above the graphs represent outliers. n represents the number of viral infection sites measured on three half-leaves. Mean area of viral infection sites is given in red. (***) represents significant differences as determined by unbalanced ANOVA (p<0.001). Data are from three independent experiments. C, immunoblot analysis using HA- (top panel) antibodies revealed the expression of the HA-tagged myosin tails. Bands corresponding to class XI (≈100 kDa) and class VIII (≈40 kDa) myosin tails are marked by asterisks. Coomassie blue staining (bottom panel) is shown as loading control. All leaves were inoculated with equal volumes of TMV-GFP in-vitro transcription reaction mix and agro-infiltrated for myosin tail/RFP expression two days later. Analyses were conducted at 4 dpi.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004448-g001: Dominant negative repression of specific myosins inhibits TMV-GFP cell-to-cell spread.A, example of an N. benthamiana leaf with TMV-GFP infection sites at 4 dpi in the presence of RFP in one half of the leaf and of inhibitory myosin VIII-1 tails in the other half. B, areas of viral infection sites are reduced in N. benthamiana half-leaves transiently expressing myosin VIII-1, VIII-2, VIII-B, XI-2 or XI-K tails as compared to half-leaves expressing RFP. Transient expression of myosin XI-F tails has no effect on the area of TMV-GFP viral infection sites. Boxplots depict the 25th to 75th percentile of measured areas of viral infection sites. Error bars indicate the range of the 5th and 95th percentile of measured areas of viral infection sites. The horizontal bar in each boxplot indicates the median value. Circles above the graphs represent outliers. n represents the number of viral infection sites measured on three half-leaves. Mean area of viral infection sites is given in red. (***) represents significant differences as determined by unbalanced ANOVA (p<0.001). Data are from three independent experiments. C, immunoblot analysis using HA- (top panel) antibodies revealed the expression of the HA-tagged myosin tails. Bands corresponding to class XI (≈100 kDa) and class VIII (≈40 kDa) myosin tails are marked by asterisks. Coomassie blue staining (bottom panel) is shown as loading control. All leaves were inoculated with equal volumes of TMV-GFP in-vitro transcription reaction mix and agro-infiltrated for myosin tail/RFP expression two days later. Analyses were conducted at 4 dpi.
Mentions: To identify myosin motors involved in TMV movement, N. benthamiana plants were inoculated with TMV-GFP, an engineered TMV variant expressing GFP in place of the CP [41]. At two days post inoculation (dpi), the inoculated leaves where agro-infiltrated for expression of a specific N. benthamiana myosin tail in one half of the leaf blade and for expression of free RFP as control in the other half (Figure 1A). At 4 dpi, the sizes of fluorescent infection sites were measured and compared between the different treatments. Figure 1B shows that the mean area of TMV-GFP infection sites in the half leaves expressing myosin tails VIII-1, VIII-2, or VIII-B was reduced by ∼50% as compared to the corresponding control half leaves. The sizes of the TMV-GFP infection sites were also reduced upon overexpression of myosin XI-K and XI-2 tails, of which the former had a stronger effect than the latter (Figure 1B). In contrast, overexpression of the myosin XI-F tail did not exert a statistically significant change in the size of virus infection sites. The expression of myosin tails in infected leaves at 4 dpi was validated by immunoblot analysis that clearly showed that the observed effects on TMV spread were dependent on each myosin's identity rather than on differential expression levels (Figure 1C). Using the same assays, similar reductions in the sizes of viral infection sites upon transient expression of myosin tails were observed using TMV-GFP-JL24 [42], a TMV variant that expresses both the GFP and CP.

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