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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.


Construct preparation for yeast two hybrid.A) Diagram of CMV MP showing cysteine and histidine (Cys-His) rich region (126–194 aa); and RNA binding regions (174–233 aa). N-terminal region (MPn: 1–180 aa); C-terminal region (MPc: 101–279 aa) of MP; and complete MP (1–279 aa) used to prepare constructs in the pGBKT7 vector for yeast two-hybrid screening. B) Diagram of CsAO4 describing the location of the signal peptide, and of domains selected from three regions: Full AO (FAO: 1-578aa); N-terminal AO region (AO-N: 44-161aa); C-terminal AO region (AO-C: 170-336aa); middle AO region (AO-M: 453-561aa); and the signal peptide (1–33 aa) highlighted in grey color.
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pone.0163320.g001: Construct preparation for yeast two hybrid.A) Diagram of CMV MP showing cysteine and histidine (Cys-His) rich region (126–194 aa); and RNA binding regions (174–233 aa). N-terminal region (MPn: 1–180 aa); C-terminal region (MPc: 101–279 aa) of MP; and complete MP (1–279 aa) used to prepare constructs in the pGBKT7 vector for yeast two-hybrid screening. B) Diagram of CsAO4 describing the location of the signal peptide, and of domains selected from three regions: Full AO (FAO: 1-578aa); N-terminal AO region (AO-N: 44-161aa); C-terminal AO region (AO-C: 170-336aa); middle AO region (AO-M: 453-561aa); and the signal peptide (1–33 aa) highlighted in grey color.

Mentions: Based on the cDNA library clones sequence information, full length AO was amplified from C. sativus using high fidelity Advantage 2 Polymerase mix (Clontech, TakaraBio, Japan). Percent identity index of AO from different plants (the nucleotide sequences were taken from the NCBI database) was carried out using Bioedit version 7.1.9 software [44]. Cucumber AO (CsAO4) was cloned in the pGADT7 vector at restriction sites EcoRI and SacI (CsAO4-pGADT7). Further, to identify the region responsible for interaction between two proteins, constructs were prepared from different regions of the MP region (N terminal: MPn-pGBKT7 and C terminal: MPc-pGBKT7); and AO (N-terminal: AO-N-pGADT7, Middle: AO-M-pGADT7 and C-terminal: AO-C-pGADT7) (Fig 1). Interaction between transformants was checked by co-transformation into AH109. The transformants were first selected on SD/-LT and then transferred to SD/-LTHA (4mM AT) plates. To check the specificity of interaction between CsAO4 and MP, CsAO4-pGADT7 was also transformed with other CMV proteins as baits: the RdRp region (2a), the 2b protein, the methyl transferase and the helicase regions (1a) (amplified from cDNA clones of CMV-SG genomic RNA). Empty bait vector and lamin C were used as negative controls.


Movement Protein of Cucumber Mosaic Virus Associates with Apoplastic Ascorbate Oxidase
Construct preparation for yeast two hybrid.A) Diagram of CMV MP showing cysteine and histidine (Cys-His) rich region (126–194 aa); and RNA binding regions (174–233 aa). N-terminal region (MPn: 1–180 aa); C-terminal region (MPc: 101–279 aa) of MP; and complete MP (1–279 aa) used to prepare constructs in the pGBKT7 vector for yeast two-hybrid screening. B) Diagram of CsAO4 describing the location of the signal peptide, and of domains selected from three regions: Full AO (FAO: 1-578aa); N-terminal AO region (AO-N: 44-161aa); C-terminal AO region (AO-C: 170-336aa); middle AO region (AO-M: 453-561aa); and the signal peptide (1–33 aa) highlighted in grey color.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0163320.g001: Construct preparation for yeast two hybrid.A) Diagram of CMV MP showing cysteine and histidine (Cys-His) rich region (126–194 aa); and RNA binding regions (174–233 aa). N-terminal region (MPn: 1–180 aa); C-terminal region (MPc: 101–279 aa) of MP; and complete MP (1–279 aa) used to prepare constructs in the pGBKT7 vector for yeast two-hybrid screening. B) Diagram of CsAO4 describing the location of the signal peptide, and of domains selected from three regions: Full AO (FAO: 1-578aa); N-terminal AO region (AO-N: 44-161aa); C-terminal AO region (AO-C: 170-336aa); middle AO region (AO-M: 453-561aa); and the signal peptide (1–33 aa) highlighted in grey color.
Mentions: Based on the cDNA library clones sequence information, full length AO was amplified from C. sativus using high fidelity Advantage 2 Polymerase mix (Clontech, TakaraBio, Japan). Percent identity index of AO from different plants (the nucleotide sequences were taken from the NCBI database) was carried out using Bioedit version 7.1.9 software [44]. Cucumber AO (CsAO4) was cloned in the pGADT7 vector at restriction sites EcoRI and SacI (CsAO4-pGADT7). Further, to identify the region responsible for interaction between two proteins, constructs were prepared from different regions of the MP region (N terminal: MPn-pGBKT7 and C terminal: MPc-pGBKT7); and AO (N-terminal: AO-N-pGADT7, Middle: AO-M-pGADT7 and C-terminal: AO-C-pGADT7) (Fig 1). Interaction between transformants was checked by co-transformation into AH109. The transformants were first selected on SD/-LT and then transferred to SD/-LTHA (4mM AT) plates. To check the specificity of interaction between CsAO4 and MP, CsAO4-pGADT7 was also transformed with other CMV proteins as baits: the RdRp region (2a), the 2b protein, the methyl transferase and the helicase regions (1a) (amplified from cDNA clones of CMV-SG genomic RNA). Empty bait vector and lamin C were used as negative controls.

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.