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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

Immunostaining of V2 in cross sections of midribs of infected tomato leaves and of stems at 28 and 49 dpi. a: cross sections of midribs of infected tomato leaf and stem; nuclei are DAPI stained (blue); V2 is stained with Cy3-labeled antibody (red); V2 localized in nuclei appears as pink (arrows). Bar: 100 μm. b: co-localization of CP and V2 in infected (49 dpi) leaves by triple-stain immunolocalization. 1. Cy-3 labeled V2 (red), Cy5-labeled CP (green) and DAPI-stained nuclei (blue). 2. Cy-3 labeled V2 and DAPI-stained nuclei. 3. Cy-3 labeled V2 and Cy5-labeled CP. 4. Cy5-labeled CP and DAPI-stained nuclei. V2 with CP in nuclei is indicated with arrows. Bar: 100 μm. c: cross sections of midribs immunostained with anti-V2 antibody were analyzed with ImageJ. Displayed are the values of counts in 10 leaf areas of 50 mm2 and 10 stem areas of 70 mm2 in 5 individual experiments for 28 dpi and 48 dpi. 1: size of aggregates. Bars and numbers indicate the mean sizes of aggregates. 2: number of aggregates. Bars: standard error.
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f2: Immunostaining of V2 in cross sections of midribs of infected tomato leaves and of stems at 28 and 49 dpi. a: cross sections of midribs of infected tomato leaf and stem; nuclei are DAPI stained (blue); V2 is stained with Cy3-labeled antibody (red); V2 localized in nuclei appears as pink (arrows). Bar: 100 μm. b: co-localization of CP and V2 in infected (49 dpi) leaves by triple-stain immunolocalization. 1. Cy-3 labeled V2 (red), Cy5-labeled CP (green) and DAPI-stained nuclei (blue). 2. Cy-3 labeled V2 and DAPI-stained nuclei. 3. Cy-3 labeled V2 and Cy5-labeled CP. 4. Cy5-labeled CP and DAPI-stained nuclei. V2 with CP in nuclei is indicated with arrows. Bar: 100 μm. c: cross sections of midribs immunostained with anti-V2 antibody were analyzed with ImageJ. Displayed are the values of counts in 10 leaf areas of 50 mm2 and 10 stem areas of 70 mm2 in 5 individual experiments for 28 dpi and 48 dpi. 1: size of aggregates. Bars and numbers indicate the mean sizes of aggregates. 2: number of aggregates. Bars: standard error.

Mentions: These results were confirmed by in situ immuno-fluorescent visualization of V2 in leaf and stem sections of symptomatic tomato plants at 28 and 49 dpi. The first appearance of V2 aggregates was detected at 28 dpi. At later stages (49 dpi), the abundance of V2 aggregates increased (Fig. 2a), some of the V2 aggregates were associated with the nucleus (Fig. 2a). Quantification of V2 aggregates (Fig. 2c) indicated that between 28 and 49 dpi, the number of aggregates per area doubled and that their size increased by about 50%. Double in situ immuno-detection of V2 and CP revealed that V2 and CP occasionally co-localized in large nuclear inclusions (Fig. 2b, shown for leaf). The low abundance of V2 in the nuclei correlates with the low amounts of V2 in infected tomato tissues.


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)

Immunostaining of V2 in cross sections of midribs of infected tomato leaves and of stems at 28 and 49 dpi. a: cross sections of midribs of infected tomato leaf and stem; nuclei are DAPI stained (blue); V2 is stained with Cy3-labeled antibody (red); V2 localized in nuclei appears as pink (arrows). Bar: 100 μm. b: co-localization of CP and V2 in infected (49 dpi) leaves by triple-stain immunolocalization. 1. Cy-3 labeled V2 (red), Cy5-labeled CP (green) and DAPI-stained nuclei (blue). 2. Cy-3 labeled V2 and DAPI-stained nuclei. 3. Cy-3 labeled V2 and Cy5-labeled CP. 4. Cy5-labeled CP and DAPI-stained nuclei. V2 with CP in nuclei is indicated with arrows. Bar: 100 μm. c: cross sections of midribs immunostained with anti-V2 antibody were analyzed with ImageJ. Displayed are the values of counts in 10 leaf areas of 50 mm2 and 10 stem areas of 70 mm2 in 5 individual experiments for 28 dpi and 48 dpi. 1: size of aggregates. Bars and numbers indicate the mean sizes of aggregates. 2: number of aggregates. Bars: standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Immunostaining of V2 in cross sections of midribs of infected tomato leaves and of stems at 28 and 49 dpi. a: cross sections of midribs of infected tomato leaf and stem; nuclei are DAPI stained (blue); V2 is stained with Cy3-labeled antibody (red); V2 localized in nuclei appears as pink (arrows). Bar: 100 μm. b: co-localization of CP and V2 in infected (49 dpi) leaves by triple-stain immunolocalization. 1. Cy-3 labeled V2 (red), Cy5-labeled CP (green) and DAPI-stained nuclei (blue). 2. Cy-3 labeled V2 and DAPI-stained nuclei. 3. Cy-3 labeled V2 and Cy5-labeled CP. 4. Cy5-labeled CP and DAPI-stained nuclei. V2 with CP in nuclei is indicated with arrows. Bar: 100 μm. c: cross sections of midribs immunostained with anti-V2 antibody were analyzed with ImageJ. Displayed are the values of counts in 10 leaf areas of 50 mm2 and 10 stem areas of 70 mm2 in 5 individual experiments for 28 dpi and 48 dpi. 1: size of aggregates. Bars and numbers indicate the mean sizes of aggregates. 2: number of aggregates. Bars: standard error.
Mentions: These results were confirmed by in situ immuno-fluorescent visualization of V2 in leaf and stem sections of symptomatic tomato plants at 28 and 49 dpi. The first appearance of V2 aggregates was detected at 28 dpi. At later stages (49 dpi), the abundance of V2 aggregates increased (Fig. 2a), some of the V2 aggregates were associated with the nucleus (Fig. 2a). Quantification of V2 aggregates (Fig. 2c) indicated that between 28 and 49 dpi, the number of aggregates per area doubled and that their size increased by about 50%. Double in situ immuno-detection of V2 and CP revealed that V2 and CP occasionally co-localized in large nuclear inclusions (Fig. 2b, shown for leaf). The low abundance of V2 in the nuclei correlates with the low amounts of V2 in infected tomato tissues.

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