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Movement Protein of Cucumber Mosaic Virus Associates with Apoplastic Ascorbate Oxidase

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ABSTRACT

Plant viral movement proteins facilitate virion movement mainly through interaction with a number of factors from the host. We report the association of a cell wall localized ascorbate oxidase (CsAO4) from Cucumis sativus with the movement protein (MP) of Cucumber mosaic virus (CMV). This was identified first in a yeast two-hybrid screen and validated by in vivo pull down and bimolecular fluorescence complementation (BiFC) assays. The BiFC assay showed localization of the bimolecular complexes of these proteins around the cell wall periphery as punctate spots. The expression of CsAO4 was induced during the initial infection period (up to 72 h) in CMV infected Nicotiana benthamiana plants. To functionally validate its role in viral spread, we analyzed the virus accumulation in CsAO4 overexpressing Arabidopsis thaliana and transiently silenced N. benthamiana plants (through a Tobacco rattle virus vector). Overexpression had no evident effect on virus accumulation in upper non-inoculated leaves of transgenic lines in comparison to WT plants at 7 days post inoculation (dpi). However, knockdown resulted in reduced CMV accumulation in systemic (non-inoculated) leaves of NbΔAO-pTRV2 silenced plants as compared to TRV inoculated control plants at 5 dpi (up to 1.3 fold difference). In addition, functional validation supported the importance of AO in plant development. These findings suggest that AO and viral MP interaction helps in early viral movement; however, it had no major effect on viral accumulation after 7 dpi. This study suggests that initial induction of expression of AO on virus infection and its association with viral MP helps both towards targeting of the MP to the apoplast and disrupting formation of functional AO dimers for spread of virus to nearby cells, reducing the redox defense of the plant during initial stages of infection.

No MeSH data available.


In vivo pull down assay showing interaction between MP and CsAO4.Protein complexes were immuno-precipitated using anti-HA antibody (raised in rabbit) and analyzed by SDS-PAGE. Proteins were transferred to PVDF membranes and probed with anti-c-Myc antibody. Abbreviations: MP-nYFP (complete MP in pSPYNE173 vector), CsAO4-cYFP (complete AO in pSPYCE (MR) vector), nYFP (empty pSPYNE173 vector) and cYFP (empty pSPYCE (MR) vector).
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pone.0163320.g004: In vivo pull down assay showing interaction between MP and CsAO4.Protein complexes were immuno-precipitated using anti-HA antibody (raised in rabbit) and analyzed by SDS-PAGE. Proteins were transferred to PVDF membranes and probed with anti-c-Myc antibody. Abbreviations: MP-nYFP (complete MP in pSPYNE173 vector), CsAO4-cYFP (complete AO in pSPYCE (MR) vector), nYFP (empty pSPYNE173 vector) and cYFP (empty pSPYCE (MR) vector).

Mentions: For the in vivo pull down assay, protein was extracted from leaves transiently expressing CMV MP-nYFP and CsAO4-cYFP in BiFC vectors. Proteins were extracted using a low ionic strength buffer suitable for extraction of cell wall proteins [48], as MP and AO were reported to be localized around the cell wall (PD/apoplast). Therefore, the low ionic strength buffer worked well in protein isolation and subsequent detection by epitope specific antibodies (Anti-c-Myc/HA) in western blotting. Protein complexes were captured using Anti-HA tag antibodies (for CsAO4 fusion) and precipitated using protein G magnetic beads. Subsequent detection was done with c-myc antibodies (for CMV MP fusion) which lead to the detection of bands corresponding to the size of the MP-nYFP fusion on the membrane (Fig 4). The results of this assay supported interaction between CsAO4 and MP in plant cells.


Movement Protein of Cucumber Mosaic Virus Associates with Apoplastic Ascorbate Oxidase
In vivo pull down assay showing interaction between MP and CsAO4.Protein complexes were immuno-precipitated using anti-HA antibody (raised in rabbit) and analyzed by SDS-PAGE. Proteins were transferred to PVDF membranes and probed with anti-c-Myc antibody. Abbreviations: MP-nYFP (complete MP in pSPYNE173 vector), CsAO4-cYFP (complete AO in pSPYCE (MR) vector), nYFP (empty pSPYNE173 vector) and cYFP (empty pSPYCE (MR) vector).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5036820&req=5

pone.0163320.g004: In vivo pull down assay showing interaction between MP and CsAO4.Protein complexes were immuno-precipitated using anti-HA antibody (raised in rabbit) and analyzed by SDS-PAGE. Proteins were transferred to PVDF membranes and probed with anti-c-Myc antibody. Abbreviations: MP-nYFP (complete MP in pSPYNE173 vector), CsAO4-cYFP (complete AO in pSPYCE (MR) vector), nYFP (empty pSPYNE173 vector) and cYFP (empty pSPYCE (MR) vector).
Mentions: For the in vivo pull down assay, protein was extracted from leaves transiently expressing CMV MP-nYFP and CsAO4-cYFP in BiFC vectors. Proteins were extracted using a low ionic strength buffer suitable for extraction of cell wall proteins [48], as MP and AO were reported to be localized around the cell wall (PD/apoplast). Therefore, the low ionic strength buffer worked well in protein isolation and subsequent detection by epitope specific antibodies (Anti-c-Myc/HA) in western blotting. Protein complexes were captured using Anti-HA tag antibodies (for CsAO4 fusion) and precipitated using protein G magnetic beads. Subsequent detection was done with c-myc antibodies (for CMV MP fusion) which lead to the detection of bands corresponding to the size of the MP-nYFP fusion on the membrane (Fig 4). The results of this assay supported interaction between CsAO4 and MP in plant cells.

View Article: PubMed Central - PubMed

ABSTRACT

Plant viral movement proteins facilitate virion movement mainly through interaction with a number of factors from the host. We report the association of a cell wall localized ascorbate oxidase (CsAO4) from Cucumis sativus with the movement protein (MP) of Cucumber mosaic virus (CMV). This was identified first in a yeast two-hybrid screen and validated by in vivo pull down and bimolecular fluorescence complementation (BiFC) assays. The BiFC assay showed localization of the bimolecular complexes of these proteins around the cell wall periphery as punctate spots. The expression of CsAO4 was induced during the initial infection period (up to 72 h) in CMV infected Nicotiana benthamiana plants. To functionally validate its role in viral spread, we analyzed the virus accumulation in CsAO4 overexpressing Arabidopsis thaliana and transiently silenced N. benthamiana plants (through a Tobacco rattle virus vector). Overexpression had no evident effect on virus accumulation in upper non-inoculated leaves of transgenic lines in comparison to WT plants at 7 days post inoculation (dpi). However, knockdown resulted in reduced CMV accumulation in systemic (non-inoculated) leaves of NbΔAO-pTRV2 silenced plants as compared to TRV inoculated control plants at 5 dpi (up to 1.3 fold difference). In addition, functional validation supported the importance of AO in plant development. These findings suggest that AO and viral MP interaction helps in early viral movement; however, it had no major effect on viral accumulation after 7 dpi. This study suggests that initial induction of expression of AO on virus infection and its association with viral MP helps both towards targeting of the MP to the apoplast and disrupting formation of functional AO dimers for spread of virus to nearby cells, reducing the redox defense of the plant during initial stages of infection.

No MeSH data available.