Limits...
Movement Protein of Cucumber Mosaic Virus Associates with Apoplastic Ascorbate Oxidase

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.


In planta interaction between CsAO4 and CMV MP by BiFC.The lower surface of N. benthamiana leaves was observed under the confocal microscope for fluorescence from YFP: A) and B) CsAO4-cYFP and MP-nYFP. C) and D) MP-nYFP and cYFP. E) and F) CsAO4-cYFP and nYFP. YFP reconstitution observed in A and B showed punctate sites around the cell wall periphery. Confocal images were merged with bright field images. Fluorescence was detected 48 h post agroinfiltration. Scale bars are shown in the figures.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5036820&req=5

pone.0163320.g003: In planta interaction between CsAO4 and CMV MP by BiFC.The lower surface of N. benthamiana leaves was observed under the confocal microscope for fluorescence from YFP: A) and B) CsAO4-cYFP and MP-nYFP. C) and D) MP-nYFP and cYFP. E) and F) CsAO4-cYFP and nYFP. YFP reconstitution observed in A and B showed punctate sites around the cell wall periphery. Confocal images were merged with bright field images. Fluorescence was detected 48 h post agroinfiltration. Scale bars are shown in the figures.

Mentions: In-vivo protein-protein interaction between CMV-MP and CsAO4 was confirmed by BiFC assay in N. benthamiana. CsAO4 was fused with the C-terminal half of YFP (CsAO4-cYFP); and MP was fused with the N-terminal half of YFP (MP-nYFP). Proteins were infiltrated along with the silencing suppressor p19 and lower epidermal cells were observed for reconstituted YFP signals. Fluorescence was detected only in presence of CsAO4-cYFP and MP-nYFP (Fig 3A and 3B). Fluorescence was observed around the cell wall periphery as punctate spots. No distinct fluorescent signals were found except some background fluorescence in control experiments, when expressing MP-nYFP/cYFP (Fig 3C and 3D) and CsAO4-cYFP/nYFP only (Fig 3E and 3F).


Movement Protein of Cucumber Mosaic Virus Associates with Apoplastic Ascorbate Oxidase
In planta interaction between CsAO4 and CMV MP by BiFC.The lower surface of N. benthamiana leaves was observed under the confocal microscope for fluorescence from YFP: A) and B) CsAO4-cYFP and MP-nYFP. C) and D) MP-nYFP and cYFP. E) and F) CsAO4-cYFP and nYFP. YFP reconstitution observed in A and B showed punctate sites around the cell wall periphery. Confocal images were merged with bright field images. Fluorescence was detected 48 h post agroinfiltration. Scale bars are shown in the figures.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0163320.g003: In planta interaction between CsAO4 and CMV MP by BiFC.The lower surface of N. benthamiana leaves was observed under the confocal microscope for fluorescence from YFP: A) and B) CsAO4-cYFP and MP-nYFP. C) and D) MP-nYFP and cYFP. E) and F) CsAO4-cYFP and nYFP. YFP reconstitution observed in A and B showed punctate sites around the cell wall periphery. Confocal images were merged with bright field images. Fluorescence was detected 48 h post agroinfiltration. Scale bars are shown in the figures.
Mentions: In-vivo protein-protein interaction between CMV-MP and CsAO4 was confirmed by BiFC assay in N. benthamiana. CsAO4 was fused with the C-terminal half of YFP (CsAO4-cYFP); and MP was fused with the N-terminal half of YFP (MP-nYFP). Proteins were infiltrated along with the silencing suppressor p19 and lower epidermal cells were observed for reconstituted YFP signals. Fluorescence was detected only in presence of CsAO4-cYFP and MP-nYFP (Fig 3A and 3B). Fluorescence was observed around the cell wall periphery as punctate spots. No distinct fluorescent signals were found except some background fluorescence in control experiments, when expressing MP-nYFP/cYFP (Fig 3C and 3D) and CsAO4-cYFP/nYFP only (Fig 3E and 3F).

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.