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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.

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Overexpression of CsAO4 in A. thaliana; Analysis of transgene integration and effect of overexpression on CMV infection.A) Diagrammatic representation of CsAO4 constructs in pCAMBIA1302 vector as GFP fusion product. CaMV 35S promoter: Cauliflower mosaic virus 35S promoter, GFP: Green fluorescent protein, Hyg: Hygromycin, LB: left border, RB: right border. B) Validation of CsAO4 transgene expression in Arabidopsis transgenic lines and WT by RT-PCR. Arabidopsis actin (AR Actin) was used as an internal control, HPPD (4-Hydroxy phenyl pyruvate dioxygenase) is a single copy gene in Arabidopsis and CsAO4 is the cucumber Ascorbate oxidase 4. C) Relative CMV CP accumulation in CsAO4 overexpressing Arabidopsis lines and WT plants. Relative CP accumulation in infected transgenic plants was calculated relative to CMV infected WT plants at 3 dpi. The 18S RNA 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; dpi—days post inoculation.
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pone.0163320.g006: Overexpression of CsAO4 in A. thaliana; Analysis of transgene integration and effect of overexpression on CMV infection.A) Diagrammatic representation of CsAO4 constructs in pCAMBIA1302 vector as GFP fusion product. CaMV 35S promoter: Cauliflower mosaic virus 35S promoter, GFP: Green fluorescent protein, Hyg: Hygromycin, LB: left border, RB: right border. B) Validation of CsAO4 transgene expression in Arabidopsis transgenic lines and WT by RT-PCR. Arabidopsis actin (AR Actin) was used as an internal control, HPPD (4-Hydroxy phenyl pyruvate dioxygenase) is a single copy gene in Arabidopsis and CsAO4 is the cucumber Ascorbate oxidase 4. C) Relative CMV CP accumulation in CsAO4 overexpressing Arabidopsis lines and WT plants. Relative CP accumulation in infected transgenic plants was calculated relative to CMV infected WT plants at 3 dpi. The 18S RNA 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; dpi—days post inoculation.

Mentions: To investigate the effect of CsAO4 overexpression, transgenic constitutively-expressing Arabidopsis lines were generated using CsAO4-pCAMBIA1302 (Fig 6). These lines did not exhibit any phenotypic variation in comparison to WT except for early flowering (S5 Fig). Two T3-generation transgenic lines (T1 and T2) as well as WT plants were checked for viral CP RNA accumulation at 3, 5 and 8 dpi. Transgenic lines showed only mild increase in CP RNA accumulation in comparison to wild type control plants at 3 and 5 dpi. CP RNA accumulation reached almost similar levels at 8 dpi, and no significant difference in expression was seen at this stage (Fig 6C). These results showed that overexpression of AO did not affect virus accumulation in arabidopsis significantly during early infection. However, anthocyanin pigmentation was observed on leaves of infected transgenic plants and not on infected WT plants (S6 Fig).


Movement Protein of Cucumber Mosaic Virus Associates with Apoplastic Ascorbate Oxidase
Overexpression of CsAO4 in A. thaliana; Analysis of transgene integration and effect of overexpression on CMV infection.A) Diagrammatic representation of CsAO4 constructs in pCAMBIA1302 vector as GFP fusion product. CaMV 35S promoter: Cauliflower mosaic virus 35S promoter, GFP: Green fluorescent protein, Hyg: Hygromycin, LB: left border, RB: right border. B) Validation of CsAO4 transgene expression in Arabidopsis transgenic lines and WT by RT-PCR. Arabidopsis actin (AR Actin) was used as an internal control, HPPD (4-Hydroxy phenyl pyruvate dioxygenase) is a single copy gene in Arabidopsis and CsAO4 is the cucumber Ascorbate oxidase 4. C) Relative CMV CP accumulation in CsAO4 overexpressing Arabidopsis lines and WT plants. Relative CP accumulation in infected transgenic plants was calculated relative to CMV infected WT plants at 3 dpi. The 18S RNA 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; dpi—days post inoculation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036820&req=5

pone.0163320.g006: Overexpression of CsAO4 in A. thaliana; Analysis of transgene integration and effect of overexpression on CMV infection.A) Diagrammatic representation of CsAO4 constructs in pCAMBIA1302 vector as GFP fusion product. CaMV 35S promoter: Cauliflower mosaic virus 35S promoter, GFP: Green fluorescent protein, Hyg: Hygromycin, LB: left border, RB: right border. B) Validation of CsAO4 transgene expression in Arabidopsis transgenic lines and WT by RT-PCR. Arabidopsis actin (AR Actin) was used as an internal control, HPPD (4-Hydroxy phenyl pyruvate dioxygenase) is a single copy gene in Arabidopsis and CsAO4 is the cucumber Ascorbate oxidase 4. C) Relative CMV CP accumulation in CsAO4 overexpressing Arabidopsis lines and WT plants. Relative CP accumulation in infected transgenic plants was calculated relative to CMV infected WT plants at 3 dpi. The 18S RNA 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; dpi—days post inoculation.
Mentions: To investigate the effect of CsAO4 overexpression, transgenic constitutively-expressing Arabidopsis lines were generated using CsAO4-pCAMBIA1302 (Fig 6). These lines did not exhibit any phenotypic variation in comparison to WT except for early flowering (S5 Fig). Two T3-generation transgenic lines (T1 and T2) as well as WT plants were checked for viral CP RNA accumulation at 3, 5 and 8 dpi. Transgenic lines showed only mild increase in CP RNA accumulation in comparison to wild type control plants at 3 and 5 dpi. CP RNA accumulation reached almost similar levels at 8 dpi, and no significant difference in expression was seen at this stage (Fig 6C). These results showed that overexpression of AO did not affect virus accumulation in arabidopsis significantly during early infection. However, anthocyanin pigmentation was observed on leaves of infected transgenic plants and not on infected WT plants (S6 Fig).

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