Fig. 6: MORF2s are degraded during immune activation and stabilized by SsCm1. | Nature Communications

Fig. 6: MORF2s are degraded during immune activation and stabilized by SsCm1.

From: A fungal effector promotes infection via stabilizing a negative regulatory factor of chloroplast immunity

Fig. 6

a, b SsCm1-RFP expression enhances the fluorescence signal of GmMORF2a-GFP in N. benthamiana. c SsCm1 stabilizes MORF2s to promote protein accumulation. MORF2s-GFP or GmPETE-GFP co-expressed with SsCm1-FLAG or SsVSP25-FLAG in N. benthamiana. d Cell-free assay shows His-SsCm1 inhibits MORF2s degradation. His-SsCm1 or His-SsVSP28 was purified and co-incubated with total protein extracted from N. benthamiana expressing GmMORF2a-GFP or AtMORF2-GFP for indicated times. e SsCm1 stabilizes GmMORF2a from degradation during immune activation. Leaves of N. benthamiana expressing GmMORF2a-GFP (Control) or co-expressed with SsCm1-FLAG were treated 36 h after agroinfiltration. The treatments include infiltration with H2O, 1 µM flg22, 200 µg/mL chitin, or 50 µg/mL SCFE for 30 min, and inoculation with DC3000 (OD600 = 0.0005), 2 × 106/mL spore suspension of B. cinerea, or mycelia of ΔSsCm1-17 for 3 h. f MORF2s were rapidly degraded after SCFE or Flg22 treatments. SsCm1 secreted by S. sclerotiorum inhibits endogenous AtMORF2 (g) and GmMORF2s (h) degradation. Leaves of Col-0 or soybean WT were treated indicated treatment and time. The treatments include infiltrated with H2O, SCFE of UF-1 or ΔSsCm1-17, and inoculation with mycelia of UF-1 or ΔSsCm1-17. i SsCm1 inhibits MORF2s degradation in chloroplasts. Chloroplast proteins from Col-0 or soybean WT were incubated with His-SsCm1, His-SsVSP28, SCFE of UF-1 or ΔSsCm1-17 for 2 h, respectively. Chloroplast proteins were probed with α-Rubisco large subunit (α-RbcL) and served as loading controls. j, k SsCm1 secreted by S. sclerotiorum inhibits GmMORF2a-GFP degradation in chloroplasts of N. benthamiana. For a, j, the solid triangles represent the typical chloroplast co-localization. Scale bar, 25 µm. For b, k, data were analyzed by two-tailed Student’s t test (*p < 0.05 and ****p < 0.0001). Error bars represent SD (n = 4 biological replicates). For c–h, blots stained with CBB served as loading controls. For c–i, numbers below the blot indicate relative abundances of the proteins quantified using ImageJ, the relative abundances when co-expressed with SsVSP25 (c), before incubation (d), H2O treatment (e, f), before treatments (g, h), and incubated with His-SsCm1 (i) are set to baseline of 1, respectively. Source data are provided as a Source Data file.

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