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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 interferes with V2 aggregation in TYLCV-infected tomato. Western blot analysis of V2, distributed in linear 10-50% sucrose gradients from detached leaves of infected tomato (49 dpi) untreated (DMSO control) or leaves treated with oryzalin, latB, MG132, oryzalin+ MG132 and latB+ MG132. Gradients were divided into 10 fractions, 1 (top) to 10 (bottom) and aliquots were subjected to SDS-PAGE. The experiment was repeated three times with similar results.
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f8: Inhibition of the cytoskeleton and the 26S proteasome interferes with V2 aggregation in TYLCV-infected tomato. Western blot analysis of V2, distributed in linear 10-50% sucrose gradients from detached leaves of infected tomato (49 dpi) untreated (DMSO control) or leaves treated with oryzalin, latB, MG132, oryzalin+ MG132 and latB+ MG132. Gradients were divided into 10 fractions, 1 (top) to 10 (bottom) and aliquots were subjected to SDS-PAGE. The experiment was repeated three times with similar results.

Mentions: To determine whether the proteasome and cytoskeleton inhibitors have similar effects on V2 aggregation during virus infection, leaves of TYLCV-infected tomato plants were treated with oryzalin, latB, and MG132, separately or in combination (oryzalin + MG132 and latB + MG132). Proteins were extracted after 4 days and separated by ultracentrifugation in sucrose gradients, and the viral V2 was immuno-detected on western blots. In samples from tissues with disrupted cytoskeleton, V2 was observed in fractions reflecting aggregates of smaller sizes than in untreated leaves (Fig. 8). Treatment with oryzalin led to a shift of V2 aggregates from mostly mid-sized (fractions 8, 9) to small aggregates (fractions 6, 7). Treatment with latB led to V2 appearance in fractions containing protein complexes (fractions 3-5) and to its disappearance from mid-sized aggregates (fractions 7-9). Following 26S proteasome inhibition by MG132, most of the V2 was associated with large aggregates (fraction 10) (Fig. 8). The combined treatment of 26S proteasome and cytoskeleton inhibitors diminished the effect of MG132, causing the re-appearance of V2 in small/mid-sized aggregates. Such V2 patterns resembled V2 distribution in sucrose gradients of untreated leaf tissue (Fig. 8). These results demonstrated the involvement of the cytoskeleton and the 26S proteasome in the development of V2 aggregation and its turnover in plants naturally infected with TYLCV.


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 interferes with V2 aggregation in TYLCV-infected tomato. Western blot analysis of V2, distributed in linear 10-50% sucrose gradients from detached leaves of infected tomato (49 dpi) untreated (DMSO control) or leaves treated with oryzalin, latB, MG132, oryzalin+ MG132 and latB+ MG132. Gradients were divided into 10 fractions, 1 (top) to 10 (bottom) and aliquots were subjected to SDS-PAGE. The experiment was repeated three times with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Inhibition of the cytoskeleton and the 26S proteasome interferes with V2 aggregation in TYLCV-infected tomato. Western blot analysis of V2, distributed in linear 10-50% sucrose gradients from detached leaves of infected tomato (49 dpi) untreated (DMSO control) or leaves treated with oryzalin, latB, MG132, oryzalin+ MG132 and latB+ MG132. Gradients were divided into 10 fractions, 1 (top) to 10 (bottom) and aliquots were subjected to SDS-PAGE. The experiment was repeated three times with similar results.
Mentions: To determine whether the proteasome and cytoskeleton inhibitors have similar effects on V2 aggregation during virus infection, leaves of TYLCV-infected tomato plants were treated with oryzalin, latB, and MG132, separately or in combination (oryzalin + MG132 and latB + MG132). Proteins were extracted after 4 days and separated by ultracentrifugation in sucrose gradients, and the viral V2 was immuno-detected on western blots. In samples from tissues with disrupted cytoskeleton, V2 was observed in fractions reflecting aggregates of smaller sizes than in untreated leaves (Fig. 8). Treatment with oryzalin led to a shift of V2 aggregates from mostly mid-sized (fractions 8, 9) to small aggregates (fractions 6, 7). Treatment with latB led to V2 appearance in fractions containing protein complexes (fractions 3-5) and to its disappearance from mid-sized aggregates (fractions 7-9). Following 26S proteasome inhibition by MG132, most of the V2 was associated with large aggregates (fraction 10) (Fig. 8). The combined treatment of 26S proteasome and cytoskeleton inhibitors diminished the effect of MG132, causing the re-appearance of V2 in small/mid-sized aggregates. Such V2 patterns resembled V2 distribution in sucrose gradients of untreated leaf tissue (Fig. 8). These results demonstrated the involvement of the cytoskeleton and the 26S proteasome in the development of V2 aggregation and its turnover in plants naturally infected with TYLCV.

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