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. 2017 Nov 13;176(1):649–662. doi: 10.1104/pp.17.01198

Figure 3.

Figure 3.

TuMV infection induces large aggregate-like structures containing NBR1, ATG8, and ubiquitin. A, RT-qPCR analysis of ATG8a and NBR1 transcript levels in TuMV infected wild-type plants at 10 DAI compared to noninfected plants. Values represent mean ± sd (n = 3) relative to noninfected control and were normalized to UBQ9 and PP2a. Statistical significance (**P < 0.01) was revealed by Student’s t test. B, Immunoblot analysis of ATG8a and NBR1 protein levels in TuMV-infected and control plants at 10 DAI. Two biological replicates were analyzed and accumulation of the cell cycle protein CDC2 served as reference. C, Immunoblot analysis of NBR1 and ubiquitin (Ub) in noninfected (-) and infected (+) wild-type, atg5, atg7, and nbr1 plants. Ponceau S staining (PS) of the membrane was used as loading control. D, Localization of stably coexpressed NBR1-RFP and GFP-ATG8a in TuMV-infected transgenic Col-0 plants at 14 DAI. The images are Z-stack projections and scale bar = 20 μm. E, Localization of GFP-ubiquitin and NBR1-RFP in stably expressing transgenic Col-0 plants. Images represent single confocal plains. Scale bar = 20 μm. F, The number of GFP-ATG8a puncta was quantified from TuMV-infected and healthy Col-0 that stably expressed GFP-ATG8a at 14 DAI. Puncta were calculated from single plains of individual Z-stacks (n = 7 per condition) using ImageJ. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles and data points are plotted as white circles. G, Immunoblot analysis of NBR1 accumulation and GFP-ATG8a processing in stably expressing Col-0 plants in the absence (control) or presence of TuMV, using anti-NBR1 and anti-GFP antibodies. PS of the membrane served as loading control. The non-GFP tagged TuMV-2 isolate was used in D, E, F and G. H, Association of RFP-tagged NBR1 mutants K11A, W661A I664A, and ΔUBA2 in comparison to NBR1 wild type with virus factories (arrow) upon coexpression with TuMV-GFP in N. benthamiana. Scale bar = 10 μm. N indicates nucleus. Images represent single confocal planes.