Fig. 4: SYP72 directly binds to the mechanosensitive channel MSL8 and is required for its plasma membrane localization. | Nature Communications

Fig. 4: SYP72 directly binds to the mechanosensitive channel MSL8 and is required for its plasma membrane localization.

From: SYP72 interacts with the mechanosensitive channel MSL8 to protect pollen from hypoosmotic shock during hydration

Fig. 4

a Dual-membrane yeast two-hybrid assays with SYP72 and MSL8. b Co-immunoprecipitation (Co-IP) assays of GFP-tagged SYP72 and Myc-tagged MSL8. Arabidopsis flower buds expressing GFP-SYP72/MSL8-6×Myc or GFP/MSL8-6×Myc in pollen grains were used for Co-IP assay. c Pull-down assays between MBP-tagged SYP72 and Myc-tagged MSL8. d, e SYP72 strongly colocalizes with MSL8 in pollen. d Representative intracellular localization images of GFP-SYP72 and MSL8-RFP in pollen (n = 15 biologically independent pollen grains). Scale bars, 5 µm. e Profile of relative fluorescence signal intensity for GFP-SYP72 (green line) and MSL8-RFP (red line) along the dashed line indicated in d. f–h MSL8 mRNA levels (f) and MSL8 protein levels (g, h) are not altered in the syp72 mutant compared to those of WT plants. f RT-qPCR was performed to determine relative mRNA levels of MSL8 in WT and syp72-1. mRNA levels of MSL8 in the WT were set to 1. Data represent the mean ± SD (n = 3). g Western blot was used to determine protein levels of MSL8 in WT and syp72-1. Myc antibody was used to probe MSL8-6×Myc in WT and syp72-1 plants. Plant actin antibody was used as the control. This experiment was repeated 3 times with similar results. h Relative MSL8-RFP protein level was quantified according to the average RFP fluorescence intensity of pollen grains, with the level from WT plants set to 100. (n = 12 biologically independent pollen grains for WT and 20 for syp72-1). Box plots indicate the 25th, median, and 75th percentiles. The whiskers indicate the minimum and maximum values. i The percentages of pollen grains showing MSL8-RFP plasma membrane signals in WT and syp72-1 plants. Data represent the mean ± SD from three independent experiments (n = 180 pollen grains). j The syp72 mutation mislocalizes MSL8 in pollen (n = 16 biologically independent pollen grains). Scale bars, 5 µm. k Profile of relative fluorescence signal intensity for MSL8-RFP (red line) along the dashed lines (indicated in j) drawn across pollen from WT and syp72-1 plants. cw cell wall, pm plasma membrane. (two-tailed Student’s t test; ns, no significant difference, P > 0.05; ****P < 0.0001). Yeast two-hybrid, Co-IP, pull-down, and RT-qPCR experiments were performed independently three times with similar results.

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