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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.

No MeSH data available.


GAL4 Yeast two hybrid assay.Yeast transformants spotted on selection medium (SD/-LTHA/+4mM AT) showing interaction of full length AO and its domains with MP domains along with positive (SV40 Large T antigen and p53) and negative (MP-pGBKT7 and empty pGADT7 vector) controls. Abbreviations used in the figure: MPc (C-terminal region of MP); FAO (Full length CsAO4); MPn (N-terminal region of MP); AO-N (N-terminal, cupredoxin domain 1 region of CsAO4); AO-M (Middle region, cupredoxin domain 2 region of CsAO4) and AO-C (C-terminal, cupredoxin domain 3 region of CsAO4).
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pone.0163320.g002: GAL4 Yeast two hybrid assay.Yeast transformants spotted on selection medium (SD/-LTHA/+4mM AT) showing interaction of full length AO and its domains with MP domains along with positive (SV40 Large T antigen and p53) and negative (MP-pGBKT7 and empty pGADT7 vector) controls. Abbreviations used in the figure: MPc (C-terminal region of MP); FAO (Full length CsAO4); MPn (N-terminal region of MP); AO-N (N-terminal, cupredoxin domain 1 region of CsAO4); AO-M (Middle region, cupredoxin domain 2 region of CsAO4) and AO-C (C-terminal, cupredoxin domain 3 region of CsAO4).

Mentions: Interactors from the cDNA library screening covered the 3’ region of cupredoxin domain1 (AO-N) and complete domain 2 (AO-M) of CsAO4, and showed growth on selection plates (SD/-LTHA/+4mM AT). However, full length CsAO4-pGADT7 did not show growth on co-transformation with MP-pGBKT7. The larger size or aggregate formation may prevent proper folding of the protein, restricting their interaction. Therefore, the interaction was dissected out at the region/domain level. The MP was divided randomly (Fig 1A) into N-terminal (MPn-pGBKT7) and C-terminal regions (MPc-pGBKT7), while CsAO4 was divided into different regions for the cupredoxin domains (AO-N-pGADT7, AO-M-pGADT7 and AO-C-pGADT7) (Fig 1B). MPn-pGBKT7 consisted of 180 aa (1–180); MPc-pGBKT7 of 179 aa (101–279) and both constructs covered the common region of 80 aa (Fig 1A). During this screening, we found that co-transformants MP-pGBKT7/AO-M-pGADT7, MP-pGBKT7/AO-C-pGADT7, MPn-pGBKT7/AO-N-pGADT7, MPc-pGBKT7/AO-M-pGADT7, and MPc-pGBKT7/CsAO4-pGADT7 showed growth on -LTHA (4mM AT) (Fig 2) and was also positive during β-galactosidase assay. The dilution assay was also performed for yeast transformants to determine the strength of interactions (S3 Fig).


Movement Protein of Cucumber Mosaic Virus Associates with Apoplastic Ascorbate Oxidase
GAL4 Yeast two hybrid assay.Yeast transformants spotted on selection medium (SD/-LTHA/+4mM AT) showing interaction of full length AO and its domains with MP domains along with positive (SV40 Large T antigen and p53) and negative (MP-pGBKT7 and empty pGADT7 vector) controls. Abbreviations used in the figure: MPc (C-terminal region of MP); FAO (Full length CsAO4); MPn (N-terminal region of MP); AO-N (N-terminal, cupredoxin domain 1 region of CsAO4); AO-M (Middle region, cupredoxin domain 2 region of CsAO4) and AO-C (C-terminal, cupredoxin domain 3 region of CsAO4).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036820&req=5

pone.0163320.g002: GAL4 Yeast two hybrid assay.Yeast transformants spotted on selection medium (SD/-LTHA/+4mM AT) showing interaction of full length AO and its domains with MP domains along with positive (SV40 Large T antigen and p53) and negative (MP-pGBKT7 and empty pGADT7 vector) controls. Abbreviations used in the figure: MPc (C-terminal region of MP); FAO (Full length CsAO4); MPn (N-terminal region of MP); AO-N (N-terminal, cupredoxin domain 1 region of CsAO4); AO-M (Middle region, cupredoxin domain 2 region of CsAO4) and AO-C (C-terminal, cupredoxin domain 3 region of CsAO4).
Mentions: Interactors from the cDNA library screening covered the 3’ region of cupredoxin domain1 (AO-N) and complete domain 2 (AO-M) of CsAO4, and showed growth on selection plates (SD/-LTHA/+4mM AT). However, full length CsAO4-pGADT7 did not show growth on co-transformation with MP-pGBKT7. The larger size or aggregate formation may prevent proper folding of the protein, restricting their interaction. Therefore, the interaction was dissected out at the region/domain level. The MP was divided randomly (Fig 1A) into N-terminal (MPn-pGBKT7) and C-terminal regions (MPc-pGBKT7), while CsAO4 was divided into different regions for the cupredoxin domains (AO-N-pGADT7, AO-M-pGADT7 and AO-C-pGADT7) (Fig 1B). MPn-pGBKT7 consisted of 180 aa (1–180); MPc-pGBKT7 of 179 aa (101–279) and both constructs covered the common region of 80 aa (Fig 1A). During this screening, we found that co-transformants MP-pGBKT7/AO-M-pGADT7, MP-pGBKT7/AO-C-pGADT7, MPn-pGBKT7/AO-N-pGADT7, MPc-pGBKT7/AO-M-pGADT7, and MPc-pGBKT7/CsAO4-pGADT7 showed growth on -LTHA (4mM AT) (Fig 2) and was also positive during β-galactosidase assay. The dilution assay was also performed for yeast transformants to determine the strength of interactions (S3 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.