Figure 2: Detection of P3N-PIPO and P3N-ALT products by Agrobacterium-mediated transient expression in N. benthamiana. | Scientific Reports

Figure 2: Detection of P3N-PIPO and P3N-ALT products by Agrobacterium-mediated transient expression in N. benthamiana.

From: Truncated yet functional viral protein produced via RNA polymerase slippage implies underestimated coding capacity of RNA viruses

Figure 2

(a) Schematic diagrams of the plasmids used. The original or mutated G2A6 motif is indicated above each diagram with mutated nucleotides in lower case red letters. The P3 (green box with vertical stripes), pipo (blue box with horizontal stripes) and alt (magenta box) ORFs from Cl30 and RB strains of ClYVV, and CS strain of BYMV (CS), and the FLAG-tag (yellow box with dots) are marked. These modified P3 cistrons were inserted in a binary vector between a DEX-inducible promoter (prom) and a poly(A) addition signal (pAs). The binary vectors with P3(PIPO:FLAG–1) express FLAG-tagged P3N-PIPO from the –1 reading frame, and the vectors with P3(ALT:FLAG+1) express FLAG-tagged P3N-ALT from the +1 reading frame. These constructs coincidently express proteins without the FLAG-tag from other frames (shaded). Their expected amino acid sequences are presented in Supplementary Fig. 2. P3N-PIPO:FLAGmk and P3N-ALT:FLAGmk RNAs are designed to express FLAG-tagged proteins in-frame, respectively. (b) Western blotting using anti-FLAG antibody to detect FLAG-tagged P3N-PIPO as –1 reading frame products after translation of P3(PIPO:FLAG–1) RNA (lanes 1–3) and as in-frame products after translation of P3N-PIPO:FLAGmk RNA (lanes 5–7). FLAG-tagged P3N-ALT was detected as +1 reading frame products after translation of P3(ALT:FLAG+1) RNA (lanes 9–11) and in-frame products after translation of P3N-ALT:FLAGmk RNA (lanes 13–15). In the control (Ctrl, lanes 4, 8, 12 and 16), protein samples prepared from leaves that expressed yellow fluorescence by agroinfiltration with the vector carrying the YFP gene were analysed as negative controls. Samples in lanes RB, Cl30 and CS were prepared from leaves expressing the P3 derivatives of RB, Cl30 and BYMV-CS, respectively. Open and closed arrowheads indicate the positions of P3N-PIPO:FLAG and P3N-ALT:FLAG, respectively. The asterisks denote background signals derived from non-specific cross-reaction of the anti-FLAG antibody. Positions of molecular mass markers (kDa) are indicated on the right side of each panel. The analysis was repeated four times, and typical images are shown.

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