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. 2024 Jul 16;15:5987. doi: 10.1038/s41467-024-50290-4

Fig. 5. MHZ3 is required for the interaction between the ethylene receptors and OsCTR2.

Fig. 5

a, b Co-IP assays indicate that MHZ3 mutation disrupted the interaction between OsCTR2 and OsETR2 (a) or OsERS2 (b). Transgenic rice seedlings stably expressing OsETR2-Myc/WT, OsETR2-Myc/mhz3, OsERS2-Myc/WT, and OsERS2-Myc/mhz3 were grown in the dark for 2 days. Total proteins were immunoprecipitated with anti-c-Myc affinity gel and immunoblotted with anti-c-Myc, anti-OsCTR2, anti-MHZ3, and anti-BiP antibodies. WT and mhz3 were used as negative controls. c Membrane association of OsCTR2 in WT and mhz3-1. Equal amounts of total protein (T), soluble protein (S), and microsomal membranes (M) were immunoblotted for OsCTR2, BiP (ER membrane marker), and UGPase (cytoplasm marker). d, e Luciferase complementation imaging assays demonstrate that MHZ3 enhances the interaction between OsCTR2 and OsETR2 (d) or OsERS2 (e). An equal amount of the Agrobacteria harboring Cluc-OsETR2 or Cluc-OsERS2 expressing vectors plus OsCTR2-Nluc vector was co-infiltrated into N. benthamiana leaves. The Agrobacteria harboring GFP or MHZ3-GFP vectors were introduced to compare the effects. The relative luminescence values represent the means ± SD, with n = 20 (d) and n = 19 (e) biologically independent samples (**P < 0.01, two-tailed Student’s t-test; compared with GFP). Immunoblot analysis showed similar expression levels of Cluc-OsETR2, Cluc-OsERS2, and OsCTR2-Nluc proteins in each experimental group. f, g MHZ3 enhances interactions of OsETR2 (f) and OsERS2 (g) with OsCTR2 in semi-in vitro pull-down (PD) assays. Equal amounts of OsETR2-Myc and OsERS2-Myc protein purified from OsETR2-Myc/mhz3 and OsERS2-Myc/mhz3 plants are added to protein homogenates from WT, mhz3, and MHZ3-OE#22. Total homogenate proteins were immunoprecipitated with anti-c-Myc affinity gel and immunoblotted with anti-c-Myc, anti-OsCTR2, anti-MHZ3, and anti-BiP antibodies. Three independent experiments were repeated with similar results. Uncropped blots and source data are in the Source Data file.