FIGURE 4.

RSV infection activates the bZIP17/28 branch of the unfolded protein response (UPR). (a) Confocal images of RSV‐infected Nicotiana benthamiana leaves expressing Myc‐eGFP‐NbbZIP17/28A/28B. Myc‐eGFP‐NbbZIP17/28A/28B were expressed in RSV‐infected N. benthamiana leaves at 12 days postinoculation (dpi), and images were taken 48 h later. RFP‐H2B was expressed as nucleus marker. The magnified areas are indicated by blue boxes. Bars, 20 μm. (b, c) The reverse transcription quantitative PCR (RT‐qPCR) analyses of the UPR‐related genes when expressing NSvc2 (b) or NSvc4 (c). NSvc2, NSvc4 or empty control was expressed in N. benthamiana leaves through agroinfiltration. The total RNA of the leaf samples was extracted for RT‐qPCR analyses at 60 h postinfiltration (hpi). NbActin served as an internal reference in relative quantification. The values represent the means of the expression levels ± SD relative to the empty vector expressed leaves (n = 3 biological replicates). The values were analysed by analysis of variance followed by Dunnett's test, and asterisks denote significant differences between NSvc2 or NSvc4 and empty vector‐expressing leaves (two‐sided, *p < 0.05, **p < 0.01). (d) Confocal images of N. benthamiana leaves co‐expressing Myc‐eGFP‐NbbZIP17/28A/28B with NSvc2 or NSvc4. Myc‐eGFP‐NbbZIP17/28A/28B were co‐expressed with NSvc2, NSvc4 or empty vector control through agroinfiltration. The images were taken at 48 hpi. (e) Western blot analyses of N. benthamiana leaves co‐expressing Myc‐eGFP‐NbbZIP17/28A/28B with NSvc2, NSvc4 or empty vector control. The leaf samples in (d) were harvested and total protein was extracted for western blot analyses. Actin was used as a loading control. Molecular mass markers are indicated on the left