Fig. 6

MoCsn5 inhibits autophagy by promoting the K48-ubiquitination of MoAtg6. A The interaction between MoCsn5 and MoAtg6 was detected by yeast two-hybrid assays. pGADT7-T and pGBKT7-53 were used as positive controls. pGADT7-T and MoCsn5-BD and pGBKT7 and MoAtg6-AD served as two pairs of negative controls. B The interaction between MoCsn5 and MoAtg14 was detected by yeast two-hybrid assays. pGADT7-T and pGBKT7-53 were used as positive controls, and pGADT7-T and MoCsn5-BD and pGBKT7 and MoAtg14-AD were used as two pairs of negative controls. C Pulldown assays to detect the interaction between MoCsn5 and MoAtg14 in vitro. GST-MoAtg14 and His-MoCn5, empty GST, and His-MoCsn5 were incubated sequentially with glutathione agarose gel beads with GST labels for 2 h. The final eluents were detected by western blot assays. D The relationship between MoCsn5 and MoAtg14 was examined by in vivo coimmunoprecipitation assays. The MoCsn5-Flag bands were detected following MoAtg14-GFP immunoprecipitation. E The relationship between MoCsn5 and MoAtg6 was examined by in vivo coimmunoprecipitation assays. The MoCsn5-Flag bands were detected following MoAtg6-GFP immunoprecipitation. F Interaction intensity of MoAtg6 and MoAtg14 in 70-15 and ΔMocsn5. MoCsn5 knockout was performed in 70-15 strain expressing MoAtg6-GFP and MoAtg14-Flag to ensure consistent MoAtg6 and MoAtg14 contents. The strains were lysed and immunoprecipitated with anti-GFP beads and then subjected to immunoblotting with anti-GFP and anti-Flag antibodies. The formula MoAtg14-Flag/MoAtg6-GFP was used to calculate the interaction intensity of MoAtg6 and MoAtg14 in the different strains. G Interaction intensity of MoAtg6 and MoAtg14 in 70-15 and ΔMocsn5. MoCsn5 knockout was performed in 70-15 strain expressing MoAtg6-GFP and MoAtg14-Flag to ensure consistent MoAtg6 and MoAtg14 contents. The strains were lysed and immunoprecipitated with anti-Flag beads and then subjected to immunoblotting with anti-GFP and anti-Flag antibodies. The formula MoAtg6-GFP/MoAtg14-Flag was used to calculate the interaction intensity of MoAtg6 and MoAtg14 in the different strains. H Ubiquitination levels of MoAtg14 in 70-15 and ΔMocsn5. Gene knockout of MoCSN5 was performed in 70-15 expressing MoAtg14-Flag to ensure consistent transcript levels of MoATG14. The strain was immunoprecipitated with anti-Flag beads after lysis, and a western blot analysis was then performed with an anti-Flag antibody and an anti-ubiquitin antibody. The formula ubiquitin/MoAtg14-Flag was used to calculate the ubiquitination levels of MoAtg14-Flag. I Deletion of MoCSN5 inhibited the ubiquitination and K48-ubiquitination of MoAtg6. Gene knockout of MoCSN5 was performed in 70-15 expressing MoAtg6-GFP to ensure consistent transcript levels of MoATG6. The strain was immunoprecipitated with anti-GFP beads after lysis, and a western blot analysis was then performed with anti-GFP antibody, anti-ubiquitin antibody, and anti-K48 antibody. The ubiquitination and K48-ubiquitination levels of MoAtg6-GFP were calculated as ubiquitin/MoAtg6-GFP and K48-ubiquitin/MoAtg6-GFP, respectively. J Ubiquitination levels and K48-ubiquitination levels of MoAtg6 in 70-15, ΔMocsn5 and Mocsn5-C. K The protein levels of MoAtg6 in the 70-15, ΔMocsn5 and Mocsn5-C strains were measured with an anti-Beclin1 antibody. The strains were cultured in liquid CM at 25 ℃ for 36 h. The protein content of actin was used as a control. L Transcript levels of MoATG6 in 70-15 and ΔMocsn5. The error bars represent the standard deviations