Fig. 4.

APUM5 repressed the CMV 3′ UTR reporter at the translational level and inhibited CMV replication in protoplast. (A) Schematic diagrams of GFP-fused reporter constructs. Endochitinase 3′ UTR was used as a negative control reporter. CMV mutant 3′ UTR reporter was generated by mutating the Pumilio binding core motif of the wild-type CMV 3′ UTR. (B) GFP-fused reporter constructs with endochitinase 3′ UTR or CMV 3′ UTR were transformed into protoplasts of Col-0 and 35S-APUM5 transgenic plants by the polyethylene glycol-mediated transformation method. Next, the GFP reporter signal was detected by LSM 700 confocal microscope (Carl Zeiss). (C) Western blot analysis was performed with 5 μg of total protoplast protein. Luciferase (LUC) was used as an internal control. Rubisco protein was used as an equal loading control. Total cellular mRNA was extracted and GFP mRNA levels were determined by RT-PCR. Actin7 mRNA level was monitored for equal loading control. (D) GFP signal intensities were quantified by LSM 700 ZEN software (Carl Zeiss) and the ImageJ program. Significant difference (Student t test; *P < 0.05, ***P < 0.001) is indicated by asterisks. Quantification of Western blot analysis was performed by Multi Gauge V3.0 (Fujifilm). Significant difference (Student t test; *P < 0.05) is indicated by an asterisk. (E) A GFP-fused CMV mutant 3′ UTR reporter construct was transformed into protoplasts of Col-0 and 35S-APUM5 transgenic plants. Next, the GFP reporter signal was detected by confocal microscopy. (F) Protein blot analysis of the GFP-fused CMV mutant 3′ UTR reporter was performed with 5 μg of total protoplast protein. LUC was used as an internal control. Rubisco protein was used as an equal loading control. Total cellular mRNA was extracted, and GFP mRNA levels were determined by RT-PCR. (G) GFP signal intensities were quantified by LSM 700 ZEN software and the ImageJ program. Quantification of protein blots was performed by Multi Gauge V3.0 software. (H) Wild-type and mutant-type CMV RNA 1, 2, and 3 in vitro transcripts were cotransformed into Col-0 and 35S-APUM5 transgenic protoplasts. After 24 h, total RNAs were extracted and RNA blot analysis was performed. For quantification of CMV replication in Col-0 and 35S-APUM5 transgenic protoplasts, signal intensities of RNA blots were quantified using the ImageJ program. Significant difference (Student t test; *P < 0.05) is indicated by an asterisk.