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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.

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Quantification of NbAO and CMV CP RNA levels in silenced plants.A) Transcript levels of NbAO were assessed by semi-quantitative RT-PCR using complete gene primers. NbActin was used as an internal control and TRV infection was checked by detecting the presence of TRV RNA1. B) Relative CMV CP RNA accumulation in TRV control and NbΔAO-pTRV2 silenced plants. CP RNA accumulation in AO silenced plants was calculated relative to CMV-infected TRV control plants at 3 dpi. The 18SrRNA gene was used to normalize all data and error bars represent the standard deviation about the mean for three independent biological replicates. Relative expression was plotted as Log2 values.
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pone.0163320.g008: Quantification of NbAO and CMV CP RNA levels in silenced plants.A) Transcript levels of NbAO were assessed by semi-quantitative RT-PCR using complete gene primers. NbActin was used as an internal control and TRV infection was checked by detecting the presence of TRV RNA1. B) Relative CMV CP RNA accumulation in TRV control and NbΔAO-pTRV2 silenced plants. CP RNA accumulation in AO silenced plants was calculated relative to CMV-infected TRV control plants at 3 dpi. The 18SrRNA gene was used to normalize all data and error bars represent the standard deviation about the mean for three independent biological replicates. Relative expression was plotted as Log2 values.

Mentions: To understand the effect of AO transient suppression on the viral spread, TRV-VIGS vector was used to suppress the N. benthamiana CsAO4 gene (http://solgenomics.net/), which was 97.6% and 64.3% identical at the nucleotide level to the N. tabacum (D43624) and C. sativus (FR750377) genes, respectively (S2 Fig). Along with NbAO (NbΔAO- pTRV2), CsAO4 (CsFAO-pTRV2 and CsΔAO-pTRV2) constructs were used for transient silencing of AO (S7 Fig). Results showed that low sequence identity can also affect silencing. After 8 days after agro-infiltration, the phenotypic effect of silencing phytoene desaturase started appearing on positive control plants and similarly, symptoms of silencing NbAO appeared on newly emerging leaves of silenced plants. Plants infected by NbΔAO-pTRV2 at first developed necrosis around veins and mosaic appearance in leaves above the inoculated ones, but later, exhibited marked effect on leaf development, like curling of leaves and deformation of leaf lamina (Fig 7). Similarly, plants infected with the CsAO4 silencing constructs (CsFAO-pTRV2 and CsΔAO-pTRV2) also showed symptoms like necrosis around veins in leaves above inoculated ones, and later, leaf deformation along with chlorotic patches (S8 Fig). TRV RNAs were detected in non-inoculated upper leaves of all inoculated plants through RT-PCR, showing its systemic movement in the plant. The expression of NbAO was reduced significantly in NbΔAO-pTRV2 silenced plants in comparison to mock inoculated plants indicating effective silencing (Fig 8). As single copy of the AO gene was reported in tobacco, silencing of the homologous N. benthamiana gene was considered specific. But no major effect was observed on transcript levels of NbAO in CsFAO- and CsΔAO-silenced plants indicating poor efficiency regardless of observed symptoms (Fig 8).


Movement Protein of Cucumber Mosaic Virus Associates with Apoplastic Ascorbate Oxidase
Quantification of NbAO and CMV CP RNA levels in silenced plants.A) Transcript levels of NbAO were assessed by semi-quantitative RT-PCR using complete gene primers. NbActin was used as an internal control and TRV infection was checked by detecting the presence of TRV RNA1. B) Relative CMV CP RNA accumulation in TRV control and NbΔAO-pTRV2 silenced plants. CP RNA accumulation in AO silenced plants was calculated relative to CMV-infected TRV control plants at 3 dpi. The 18SrRNA gene was used to normalize all data and error bars represent the standard deviation about the mean for three independent biological replicates. Relative expression was plotted as Log2 values.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036820&req=5

pone.0163320.g008: Quantification of NbAO and CMV CP RNA levels in silenced plants.A) Transcript levels of NbAO were assessed by semi-quantitative RT-PCR using complete gene primers. NbActin was used as an internal control and TRV infection was checked by detecting the presence of TRV RNA1. B) Relative CMV CP RNA accumulation in TRV control and NbΔAO-pTRV2 silenced plants. CP RNA accumulation in AO silenced plants was calculated relative to CMV-infected TRV control plants at 3 dpi. The 18SrRNA gene was used to normalize all data and error bars represent the standard deviation about the mean for three independent biological replicates. Relative expression was plotted as Log2 values.
Mentions: To understand the effect of AO transient suppression on the viral spread, TRV-VIGS vector was used to suppress the N. benthamiana CsAO4 gene (http://solgenomics.net/), which was 97.6% and 64.3% identical at the nucleotide level to the N. tabacum (D43624) and C. sativus (FR750377) genes, respectively (S2 Fig). Along with NbAO (NbΔAO- pTRV2), CsAO4 (CsFAO-pTRV2 and CsΔAO-pTRV2) constructs were used for transient silencing of AO (S7 Fig). Results showed that low sequence identity can also affect silencing. After 8 days after agro-infiltration, the phenotypic effect of silencing phytoene desaturase started appearing on positive control plants and similarly, symptoms of silencing NbAO appeared on newly emerging leaves of silenced plants. Plants infected by NbΔAO-pTRV2 at first developed necrosis around veins and mosaic appearance in leaves above the inoculated ones, but later, exhibited marked effect on leaf development, like curling of leaves and deformation of leaf lamina (Fig 7). Similarly, plants infected with the CsAO4 silencing constructs (CsFAO-pTRV2 and CsΔAO-pTRV2) also showed symptoms like necrosis around veins in leaves above inoculated ones, and later, leaf deformation along with chlorotic patches (S8 Fig). TRV RNAs were detected in non-inoculated upper leaves of all inoculated plants through RT-PCR, showing its systemic movement in the plant. The expression of NbAO was reduced significantly in NbΔAO-pTRV2 silenced plants in comparison to mock inoculated plants indicating effective silencing (Fig 8). As single copy of the AO gene was reported in tobacco, silencing of the homologous N. benthamiana gene was considered specific. But no major effect was observed on transcript levels of NbAO in CsFAO- and CsΔAO-silenced plants indicating poor efficiency regardless of observed symptoms (Fig 8).

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.


Related in: MedlinePlus