Fig. 4: MHZ1 physically interacts with and inhibited by ethylene receptors.
From: Histidine kinase MHZ1/OsHK1 interacts with ethylene receptors to regulate root growth in rice
a mhz1 suppressed the ethylene hypersensitivity of Osetr2 (Dongjin) and Osers2 (Dongjin) loss-of-function mutants. (Left) Seedlings were treated with air or 1 ppm ethylene. (Right) Data are means ± SD, n > 30. **P < 0.01; Student’s t-test. DJ, Dongjin; Nip, Nipponbare. Bars indicate 10 mm. b Ethylene response of Osers2d/MHZ1-OE lines. Etiolated seedlings were treated with air or 10 ppm ethylene. Bars indicate 10 mm. c Root length quantification of each line in b. Total proteins of each line were immunoblotted for MHZ1-FLAG with anti-FLAG antibody. Data are means ± SD, n > 30. **P < 0.01; Student’s t-test. d Schematic structures of MHZ1 and OsERS2 and their truncated versions. e Split-ubiquitin Y2H assay for interaction of MHZ1 and OsERS2. Combination of pTSU2-APP and pNubG-Fe65 (provided in the kit) was used as a positive control. f Pull-down of MBP-MHZ1 with GST-OsERS2ΔTM in E. coli. g Co-IP assays for interaction of MHZ1 and OsERS2. The constructs were cotransformed into rice protoplasts. Total proteins were immunoprecipitated with GFP-Trap and immunoblotted with anti-GFP, anti-FLAG, and anti-UGPase antibodies. h OsERS2 facilitates the ER membrane localization of MHZ1. MHZ1-GFP and OsERS2-mCherry proteins were transiently expressed in tobacco leaf epidermal cells. Bars indicate 20 μm. i Co-IP assays for interaction domain mapping of MHZ1 and OsERS2. The constructs were cotransformed into rice protoplasts. Total proteins were immunoprecipitated with GFP-Trap and immunoblotted with anti-FLAG, anti-GFP antibodies. j OsERS2 inhibits MHZ1 kinase activity. GST was added as a control. Values at the bottom indicate relative phosphorylation levels of GST-MHZ1. k OsERS2 inhibited phosphate groups relayed to OsAHP1. Values at the bottom indicate relative phosphorylation levels of GST-AHP1. l GAF domain of OsERS2 inhibits MHZ1 kinase activity. GST was added as a control. Values at the bottom indicate relative phosphorylation levels of GST-MHZ1. m Histidine phosphorylation of MHZ1 is suppressed by OsERS2 and Osers2d. Vectors carrying OsERS2-myc and Osers2d-myc were transformed into protoplasts of MHZ1OE 4-4. Total protein was immunoprecipitated with anti-FLAG affinity gel and immunoblotted with anti-FLAG or anti-1-p-His (Millipore, MABS1330) antibodies. Values at the bottom indicate relative phosphorylation levels of MHZ1. Protoplasts of ProMHZ1:MHZ1(H375Q) transgenic line transformed with the control vector were used as negative control. Source data are provided as a Source Data file.
