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The Tomato yellow leaf curl virus V2 protein forms aggregates depending on the cytoskeleton integrity and binds viral genomic DNA.

Moshe A, Belausov E, Niehl A, Heinlein M, Czosnek H, Gorovits R - Sci Rep (2015)

Bottom Line: Similar to CP, V2 localized to cytoplasmic aggregates of increasing size and as infection progressed was also found in nuclei, where it co-localized with CP.V2 was associated with viral genomic DNA molecules, suggesting that V2 functions as a DNA shuttling protein.We propose that the cytoskeleton-dependent formation and growth of V2 aggregates play an important role during TYLCV infection, and that microtubules and actin filaments are important for the delivery of V2 to the 26S proteasome.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Sciences and Genetics in Agriculture and the Otto Warburg Minerva Center for Agricultural Biotechnology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.

ABSTRACT
The spread of Tomato yellow leaf curl virus (TYLCV) was accompanied by the formation of coat protein (CP) aggregates of increasing size in the cytoplasm and nucleus of infected tomato (Solanum lycopersicum) cells. In order to better understand the TYLCV-host interaction, we investigated the properties and the subcellular accumulation pattern of the non-structural viral protein V2. CP and V2 are the only sense-oriented genes on the virus circular single-stranded DNA genome. Similar to CP, V2 localized to cytoplasmic aggregates of increasing size and as infection progressed was also found in nuclei, where it co-localized with CP. V2 was associated with viral genomic DNA molecules, suggesting that V2 functions as a DNA shuttling protein. The formation and the 26S proteasome-mediated degradation of V2 aggregates were dependent on the integrity of the actin and microtubule cytoskeleton. We propose that the cytoskeleton-dependent formation and growth of V2 aggregates play an important role during TYLCV infection, and that microtubules and actin filaments are important for the delivery of V2 to the 26S proteasome.

No MeSH data available.


Related in: MedlinePlus

Inhibition of the cytoskeleton and the 26S proteasome leads to changes in the size and number of V2:GFP aggregates inN. benthamiana. a: sizes of aggregates. Images obtained as described for figs. 5,7 were analyzed with ImageJ. Bars and numbers indicate the mean sizes of aggregates as measured for leaf areas of 50 mm2 in 5 independent experiments for each treatment. b: average number of aggregates per cell. Displayed are the mean values +/− standard error of counts in leaf areas of 50 mm2 in 5 independent experiments for each treatment. Statistical significance between treatments and control were determined by one-way ANOVA (* = P<0.05).
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f6: Inhibition of the cytoskeleton and the 26S proteasome leads to changes in the size and number of V2:GFP aggregates inN. benthamiana. a: sizes of aggregates. Images obtained as described for figs. 5,7 were analyzed with ImageJ. Bars and numbers indicate the mean sizes of aggregates as measured for leaf areas of 50 mm2 in 5 independent experiments for each treatment. b: average number of aggregates per cell. Displayed are the mean values +/− standard error of counts in leaf areas of 50 mm2 in 5 independent experiments for each treatment. Statistical significance between treatments and control were determined by one-way ANOVA (* = P<0.05).

Mentions: Aggregation of viral proteins often depends on interactions with proteins involved in intracellular movement. Since the cytoskeleton plays an important role in the intracellular movement of viral proteins (reviewed in 3, 10), we tested whether V2:GFP aggregation was dependent on the integrity of microtubules and actin filaments. The potential involvement of microtubules in V2 aggregation was investigated by labeling microtubules with the microtubule marker MAP4:RFP (microtubule binding domain of MAP4 fused to RFP27. Co-expression of V2:GFP (green color) and MAP4:RFP (red color) revealed that some V2 aggregates localized in the vicinity of microtubules (yellow color) (Fig. 4b). The involvement of microtubules in V2 aggregation was further studied using oryzalin, a herbicide known to depolymerize microtubules30. In N. benthamiana leaves infiltrated with V2:GFP and treated with oryzalin, indeed the microtubules were depolymerized as shown by the appearance of the red fluorescence associated with MAP4:RFP (Fig. 5a). While V2:GFP aggregates occurred in the vicinity of microtubules in untreated tissues, in oryzalin-treated leaves V2:GFP appeared as smaller aggregates and fewer co-localized with MAP4:RFP (Fig. 6a, to be compared with Fig. 4b). The observation of a large number of cells showed that in oryzalin-treated cells, the number of aggregates diminished by about 80% and their average size decreased by about 2.5 folds (Figs. 5b,6).


The Tomato yellow leaf curl virus V2 protein forms aggregates depending on the cytoskeleton integrity and binds viral genomic DNA.

Moshe A, Belausov E, Niehl A, Heinlein M, Czosnek H, Gorovits R - Sci Rep (2015)

Inhibition of the cytoskeleton and the 26S proteasome leads to changes in the size and number of V2:GFP aggregates inN. benthamiana. a: sizes of aggregates. Images obtained as described for figs. 5,7 were analyzed with ImageJ. Bars and numbers indicate the mean sizes of aggregates as measured for leaf areas of 50 mm2 in 5 independent experiments for each treatment. b: average number of aggregates per cell. Displayed are the mean values +/− standard error of counts in leaf areas of 50 mm2 in 5 independent experiments for each treatment. Statistical significance between treatments and control were determined by one-way ANOVA (* = P<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4419519&req=5

f6: Inhibition of the cytoskeleton and the 26S proteasome leads to changes in the size and number of V2:GFP aggregates inN. benthamiana. a: sizes of aggregates. Images obtained as described for figs. 5,7 were analyzed with ImageJ. Bars and numbers indicate the mean sizes of aggregates as measured for leaf areas of 50 mm2 in 5 independent experiments for each treatment. b: average number of aggregates per cell. Displayed are the mean values +/− standard error of counts in leaf areas of 50 mm2 in 5 independent experiments for each treatment. Statistical significance between treatments and control were determined by one-way ANOVA (* = P<0.05).
Mentions: Aggregation of viral proteins often depends on interactions with proteins involved in intracellular movement. Since the cytoskeleton plays an important role in the intracellular movement of viral proteins (reviewed in 3, 10), we tested whether V2:GFP aggregation was dependent on the integrity of microtubules and actin filaments. The potential involvement of microtubules in V2 aggregation was investigated by labeling microtubules with the microtubule marker MAP4:RFP (microtubule binding domain of MAP4 fused to RFP27. Co-expression of V2:GFP (green color) and MAP4:RFP (red color) revealed that some V2 aggregates localized in the vicinity of microtubules (yellow color) (Fig. 4b). The involvement of microtubules in V2 aggregation was further studied using oryzalin, a herbicide known to depolymerize microtubules30. In N. benthamiana leaves infiltrated with V2:GFP and treated with oryzalin, indeed the microtubules were depolymerized as shown by the appearance of the red fluorescence associated with MAP4:RFP (Fig. 5a). While V2:GFP aggregates occurred in the vicinity of microtubules in untreated tissues, in oryzalin-treated leaves V2:GFP appeared as smaller aggregates and fewer co-localized with MAP4:RFP (Fig. 6a, to be compared with Fig. 4b). The observation of a large number of cells showed that in oryzalin-treated cells, the number of aggregates diminished by about 80% and their average size decreased by about 2.5 folds (Figs. 5b,6).

Bottom Line: Similar to CP, V2 localized to cytoplasmic aggregates of increasing size and as infection progressed was also found in nuclei, where it co-localized with CP.V2 was associated with viral genomic DNA molecules, suggesting that V2 functions as a DNA shuttling protein.We propose that the cytoskeleton-dependent formation and growth of V2 aggregates play an important role during TYLCV infection, and that microtubules and actin filaments are important for the delivery of V2 to the 26S proteasome.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Sciences and Genetics in Agriculture and the Otto Warburg Minerva Center for Agricultural Biotechnology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.

ABSTRACT
The spread of Tomato yellow leaf curl virus (TYLCV) was accompanied by the formation of coat protein (CP) aggregates of increasing size in the cytoplasm and nucleus of infected tomato (Solanum lycopersicum) cells. In order to better understand the TYLCV-host interaction, we investigated the properties and the subcellular accumulation pattern of the non-structural viral protein V2. CP and V2 are the only sense-oriented genes on the virus circular single-stranded DNA genome. Similar to CP, V2 localized to cytoplasmic aggregates of increasing size and as infection progressed was also found in nuclei, where it co-localized with CP. V2 was associated with viral genomic DNA molecules, suggesting that V2 functions as a DNA shuttling protein. The formation and the 26S proteasome-mediated degradation of V2 aggregates were dependent on the integrity of the actin and microtubule cytoskeleton. We propose that the cytoskeleton-dependent formation and growth of V2 aggregates play an important role during TYLCV infection, and that microtubules and actin filaments are important for the delivery of V2 to the 26S proteasome.

No MeSH data available.


Related in: MedlinePlus