Extended Data Fig. 6: Functional analysis of mutant DspE proteins in Xenopus oocytes and tobacco.
From: Bacterial pathogens deliver water- and solute-permeable channels to plant cells
a, Effect of mutations on DspE-induced currents in oocytes. Mean ± s.e.m. (n = 5 oocytes) current values at different test pulses from oocytes expressing wild-type or mutant DspE proteins at 15 h after injection with 0.01 or 0.1 ng cRNA. Note: At 0.01 cRNA injection, all mutant DspE proteins did not induce currents. Next, 10-fold increase in mutant dspE cRNA (i.e., 0.1 ng) was injected into oocytes, revealing current induction by DspEK1399E/K1401E, suggesting the K1399E/K1401E double mutations only partially affect ion conductance, consistent with results in panels b and c. b, Effect of mutations on DspE-induced baseline swelling in oocytes. Plots represent mean ± s.e.m. (n = 6 oocytes) and individual values of increased oocyte volume in relation to its initial volume 15 h after 1 ng cRNA injection. c, Water-soaking assay in tobacco leaves. 1 × 108 CFU/mL of Agrobacterium tumefaciens GV3101 containing pER8 empty vector, pER-dspE, pER-dspE mutants were syringe-inoculated into Nicotiana benthamiana leaves (infiltration areas circled) and kept at 22 °C for 24 h before leaves were painted with 90 µM estradiol to induce DspE expression. Water-soaking symptom (dark-coloured appearance) was assessed at 8 h and 24 h after estradiol induction, showing a complete loss of water-soaking induction by DspEβ-barrel and DspEL1776E/L1777E/L1778E, but only delayed water-soaking by DspEK1399E/K1401E. Experiments were independently performed two times. Two- (a) or one- (b) way ANOVA values and exact P-values for all comparisons are detailed in the Source Data files. See Extended Data Figs. 4b and d for immunoblotting of DspE and DspE mutant expression.
