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.
Highlights
To establish successful infection in a susceptible host, viruses initially move from cell to cell followed by systemic movement through phloem sieve elements
Identification of plant host factors interacting with Cucumber mosaic virus (CMV)-movement protein (MP)
Considering that, in vivo generated cucumber expression library in pGADT7 was screened with MP as bait having co-transformation efficiency of approximately 2.2×105 cfu/μg
Summary
To establish successful infection in a susceptible host, viruses initially move from cell to cell followed by systemic movement through phloem sieve elements. Viral movement proteins (MPs) play a dynamic role in the spread of viruses by managing their transport through symplasmic. MPs modulate PD structure by increasing the size exclusion limit (SEL) through severing actin filaments or degradation of callose around PD, either directly or by employing host proteins to facilitate movement [4,5,6,7,8]. The virus is well known as one of the rapidly evolving pathogens with the largest host range (infecting 1200 plant species from 100 families). Functional validation of the gene revealed its role in virus infection and in plant development. This study adds to the information on host factors playing important roles during viral spread at the early stages of infection
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