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.


Relative AO expression levels in C. sativus cv. Summer Green at different time points during CMV infection.Buffer inoculated plants served as control samples for each time interval. Relative expression levels were determined at various time points (6, 12, 24, 36, 48, 72, 96, 144 and 168 h) in comparison to mock inoculated plants at the same time points. The cucumber Actin gene was used to normalize all data and error bars illustrated the standard deviation about the mean for three independent biological replicates. Relative expression was plotted as 2-ΔΔCT (fold change) values. h- hours post inoculation.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0163320.g005: Relative AO expression levels in C. sativus cv. Summer Green at different time points during CMV infection.Buffer inoculated plants served as control samples for each time interval. Relative expression levels were determined at various time points (6, 12, 24, 36, 48, 72, 96, 144 and 168 h) in comparison to mock inoculated plants at the same time points. The cucumber Actin gene was used to normalize all data and error bars illustrated the standard deviation about the mean for three independent biological replicates. Relative expression was plotted as 2-ΔΔCT (fold change) values. h- hours post inoculation.

Mentions: To determine the effect of CMV infection on CsAO4 expression, qRT-PCR analysis was carried out. CMV-SG developed visible symptoms in 3–4 days on cucumber leaves. Leaf samples of cucumber were collected at different time points up to 168 h from inoculated and mock treated plants. In melon, AO4 expression was reported to be affected by wounding stress [29]. Considering this, gene expression in infected plants was determined in comparison to mock control plants of the same time points. After infection, AO transcript levels were repressed at early time points (up to 24 h) followed by upregulation of 3.7 fold and reached a maximum of about 7.6 fold at 72 h in comparison to the mock control. Subsequently, there was gradual decline in transcript level but expression remained higher in comparison to mock plants (Fig 5).


Movement Protein of Cucumber Mosaic Virus Associates with Apoplastic Ascorbate Oxidase
Relative AO expression levels in C. sativus cv. Summer Green at different time points during CMV infection.Buffer inoculated plants served as control samples for each time interval. Relative expression levels were determined at various time points (6, 12, 24, 36, 48, 72, 96, 144 and 168 h) in comparison to mock inoculated plants at the same time points. The cucumber Actin gene was used to normalize all data and error bars illustrated the standard deviation about the mean for three independent biological replicates. Relative expression was plotted as 2-ΔΔCT (fold change) values. h- hours post inoculation.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0163320.g005: Relative AO expression levels in C. sativus cv. Summer Green at different time points during CMV infection.Buffer inoculated plants served as control samples for each time interval. Relative expression levels were determined at various time points (6, 12, 24, 36, 48, 72, 96, 144 and 168 h) in comparison to mock inoculated plants at the same time points. The cucumber Actin gene was used to normalize all data and error bars illustrated the standard deviation about the mean for three independent biological replicates. Relative expression was plotted as 2-ΔΔCT (fold change) values. h- hours post inoculation.
Mentions: To determine the effect of CMV infection on CsAO4 expression, qRT-PCR analysis was carried out. CMV-SG developed visible symptoms in 3–4 days on cucumber leaves. Leaf samples of cucumber were collected at different time points up to 168 h from inoculated and mock treated plants. In melon, AO4 expression was reported to be affected by wounding stress [29]. Considering this, gene expression in infected plants was determined in comparison to mock control plants of the same time points. After infection, AO transcript levels were repressed at early time points (up to 24 h) followed by upregulation of 3.7 fold and reached a maximum of about 7.6 fold at 72 h in comparison to the mock control. Subsequently, there was gradual decline in transcript level but expression remained higher in comparison to mock plants (Fig 5).

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.