Fig. 3: MHZ9 is localized in P-body and interacts with OsEIN2.

a Subcellular localization of MHZ9. GFP-MHZ9 was transiently expressed in tobacco leaves by Agrobacterium infiltration and treated with or without 10 μL/L of ethylene for 16 h at 2 days post infiltration. GFP was used as the control. Scale bars, 20 µm. b The co-localization tests of MHZ9, OsEIN5 (P-body marker), OsEIN2-C, and OsDCP2 (P-body marker). The co-localization assays of MHZ9 with OsEIN5 (the first row), MHZ9 with OsEIN2-C (the second row), OsEIN2-C with OsEIN5 (the third row), the three proteins (OsEIN5, MHZ9, and OsEIN2-C), and MHZ9 with OsDCP2 were performed by using Agrobacterium-mediated transient expression in tobacco leaves followed by treatment of 10 μL/L of ethylene for 16 h at 2 d post infiltration. Scale bars are 5 µm. c Diagrams of full-length and truncated versions of MHZ9 used in subcellular localization and interaction domain mapping studies. d The co-localization study of truncation (upper panel) and deletion (lower panel) variants of MHZ9 with OsEIN5 in mhz9 protoplasts. Scale bars, 2 µm. e Yeast-two-hybrid assay for interaction between MHZ9 and OsEIN2-C. Full-length and truncated versions of MHZ9 used are the same as in (c). The region for MHZ9-PRP4 is 72–233 aa and the region for MHZ9-GYF-C is 502–1530 aa. f BiFC assays for interaction between different truncated versions of MHZ9 and OsEIN2-C. Scale bar, 10 µm. Interaction analysis of full-length of MHZ9 with OsEIN2-C (g), and different MHZ9 domains with OsEIN2-C (h) by co-IP assays. The total proteins were immunoprecipitated with anti-Myc affinity gel (g) or GFP-Trap (h), GFP-Myc (g) or GFP (h) was used as the negative control. The estimated molecular weight of each protein in (right panel of h) was indicated in parentheses. The band corresponding to each of the truncated proteins is marked by a triangle, and the upper bands with higher molecular weights than expected may arise from protein modifications. Each experiment was repeated at least three times with similar results. Source data are provided as a Source Data file.