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. 2019 Feb 18;15(7):1234–1257. doi: 10.1080/15548627.2019.1580104

Figure 5.

Figure 5.

MoAtg3 acetylation affects autophagy and pathogenicity in M. oryzae. (a and b) Autophagy in Guy11, ∆Moatg3 and ∆Moatg3/MoATG3K262,267R (K262R K267R) mutant strains treated in MM-N (nitrogen starvation minimal medium) for 2 or 5 h were analyzed by western blot analysis for (a) GFP-MoAtg8 cleavage and (b) translocation of GFP-MoAtg8 into vacuoles (n = 100). Bars with asterisks represent significant differences (Duncan’s new multiple range method p < 0.01). (c) Cellular location of APs during infection-related appressorium development in Guy11, ∆Moatg3 and ∆Moatg3/MoATG3K262,267R (K262R K267R) mutant strains. (d) Bar charts showing mean APs numbers present in conidia, germ tube and appressorium at 0, 2 and 4 h after germination. Two transformants of ∆Moatg3 (∆Moatg3 #3 and ∆Moatg3 #13) and ∆Moatg3/MoATG3K262,267R (K262R K267R) (K262R K267R #11 and K262R K267R #16) mutant strains expressing GFP-MoATG8 construct were used for statistical analysis, respectively. Asterisks represent significant differences (p < 0.01). (e and f) Rice spraying assays and detailed observation with statistical analysis for infectious growth in rice sheath cells at 24 hpi. Four milliliters of conidial suspension (5 × 104 spores/ml and 1 × 105 spores/ml) of each strain were used for spraying and injection, respectively. Appressorium penetration sites (n = 100) were observed and invasive hyphae were rated from type 1 to 4. Ap, appressorium; IH, invasive hyphae. Error bars represent SD from 3 independent experiments. (g) Effect of MoAtg3 acetylation on MoAtg3-MoAtg8 interaction. Association of MoAtg3 and MoAtg8 in the wild-type (Guy11) strain, ∆Moatg3 and K262R K267R mutant strains expressing GFP-MoAtg8 and MoAtg3-S was incubated with GFP beads and analyzed by western blot analysis to detect the amount of coimmunoprecipitated MoAtg3 using the S tag antibody. Quantitation of the co-immunoprecipitated MoAtg3 intensity was analyzed by the ODYSSEY infrared imaging system (application software Version 2.1). T, total; S, suspensions; E, elution.