Fig 1.
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.
Fig 2.
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).
Fig 3.
In planta interaction between CsAO4 and CMV MP by BiFC.
The lower surface of N. benthamiana leaves was observed under the confocal microscope for fluorescence from YFP: A) and B) CsAO4-cYFP and MP-nYFP. C) and D) MP-nYFP and cYFP. E) and F) CsAO4-cYFP and nYFP. YFP reconstitution observed in A and B showed punctate sites around the cell wall periphery. Confocal images were merged with bright field images. Fluorescence was detected 48 h post agroinfiltration. Scale bars are shown in the figures.
Fig 4.
In vivo pull down assay showing interaction between MP and CsAO4.
Protein complexes were immuno-precipitated using anti-HA antibody (raised in rabbit) and analyzed by SDS-PAGE. Proteins were transferred to PVDF membranes and probed with anti-c-Myc antibody. Abbreviations: MP-nYFP (complete MP in pSPYNE173 vector), CsAO4-cYFP (complete AO in pSPYCE (MR) vector), nYFP (empty pSPYNE173 vector) and cYFP (empty pSPYCE (MR) vector).
Fig 5.
Relative AO expression levels in C. sativus cv. Summer Green at different time points during CMV infection.
Buffer inoculated plants served as control samples for each time interval. Relative expression levels were determined at various time points (6, 12, 24, 36, 48, 72, 96, 144 and 168 h) in comparison to mock inoculated plants at the same time points. The cucumber Actin gene was used to normalize all data and error bars illustrated the standard deviation about the mean for three independent biological replicates. Relative expression was plotted as 2-ΔΔCT (fold change) values. h- hours post inoculation.
Fig 6.
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.
Fig 7.
Effect of NbAO4 (VIGS) silencing on N. benthamiana two weeks post agro-infiltration.
A) Mock control plant: developed no symptoms. B) Empty TRV vector: mosaic symptoms. C) Partial N. benthamiana AO (NbΔAO-pTRV2): caused downward leaf curling at earlier stages and then severe leaf distortion affecting leaf lamina and developing chlorosis, as indicated by arrows. D) PDS-pTRV2 control: newly emerging leaves showed photo bleaching effect.
Fig 8.
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.