Fig. 5: The role of ATG5 and ATG7 in mediating autophagic degradation of ADF7 and Profilin2 via the NBR1 receptor.

a Western blotting detection of ADF and Profilin levels in Arabidopsis WT, atg5-1 and atg7-2 pollen grains by using anti-ADF and anti-Profilin antibodies. Western blotting was performed in three independent experimental replicates, yielding consistent results. Source data of blotting are provided in Source data file. b Yeast two-hybrid analysis of protein interactions between NBR1 and ATG8d, ADF7, Profilin2. Positive interactions were determined by the appearance of yeast colonies on quadruple dropout agar medium SD/–Leu/–Trp/–His/–Ade (right column). A total of three independent experimental replicates were conducted for each sample, consistently yielding similar results. c Luciferase complementation assay to investigate protein interactions between NBR1 and ATG8d, ADF7, Profilin2. Positive interactions were determined by the appearance of fluorescent colors. Three independent experimental replicates were conducted for each sample, and similar results were consistently obtained. d Immunoprecipitation assay for detection of protein interactions between NBR1 and ADF7, and Profilin2. Total proteins were extracted from transgenic Arabidopsis expressing pUBQ::GFP, pADF7::GFP-ADF7 and pProfilin2::GFP-Profilin2. Immunoprecipitation was conducted by using GFP-trap beads and determined by western blotting with indicated specific antibodies. Immunoprecipitation assays were performed in three independent experimental replicates, yielding consistent results. Source data of blotting are provided in Source data file. e Schematic illustration of different domains of the full-length Arabidopsis NBR1 and its six different types of truncations. They were subsequently used to identify the functional domain(s) for its interaction with ADF7 and Profilin2. f Yeast two hybrid analysis of the interaction between NBR1∆UBA, NBR1∆PB1∆ZZ∆FW, NBR1∆ZZ∆FW∆UBA, NBR1∆ZZ, NBR1∆FW and NBR1∆ZZ∆FW with ADF7 and Profilin2, respectively. Yeast cells transformed with various plasmids were cultured on synthetic complete medium to exam the protein interactions. Positive interactions were determined by the appearance of yeast colonies on quadruple dropout agar medium SD/–Leu/–Trp/–His/–Ade (right column). A total of three independent experimental replicates were conducted for each sample, consistently yielding similar results. g Luciferase complementation assays for determination of the interaction between NBR1∆UBA, NBR1∆PB1∆ZZ∆FW, NBR1∆ZZ∆FW∆UBA, NBR1∆ZZ, NBR1∆FW and NBR1∆ZZ∆FW with ADF7 and Profilin2, respectively. Three independent experimental replicates were conducted for each sample, and similar results were consistently obtained. Positive interactions were determined by the detection of fluorescent colors.