Fig. 8
Mechanism of GRP regulating the expression of MST1 and MST2. A Growth states of TgGluSensor::CON and TgGluSensor::OE-Tggrp on MM + straw, grown at 28 °C for 4 days. B Fluorescence images of TgGluSensor::CON and TgGluSensor::OE-Tggrp. The newly grown hyphae of TgGluSensor::CON and TgGluSensor::OE-Tggrp were gently scraped by glass, FY and FC were observed by a CLSM. Note that FY and FC were same in TgGluSensor::CON and FY was brighter than FC in TgGluSensor::OE-Tggrp. YFP and CFP were excited at 500 nm and 430 nm and received at 525 nm and 470 nm respectively; hyphae length in visual field was 60 µm. C Relative fluorescence intensity of hyphae calculated by software (ZEN3.4). D The intracellular glucose content of TgGluSensor::CON and TgGluSensor::OE-Tggrp, grown for 4 days at 28 °C. E The median FY/FC value of TgGluSensor::CON and TgGluSensor::OE-Tggrp. F1 GSH content of treatments (T1, T2, T3, and T4). F2 GSH content of OE-Tgatps and OE-TgcysK. G Western blot verified that GSH or DTT could affect the glutathionylation level of GRP; (g1) 1:500 anti-GSH antibody was used in Western blot; GSH (0.1 mmol) or DTT (0.1 mmol) was added to MM + straw, “ + ” means added, “−” means not added. With same Input, the hybridization signal intensity of anti-GSH antibody was increased in GSH added treatment (+ GSH, − DTT), and decreased in DTT added treatment (− GSH, + DTT). g2 The glutathionylation level of GRP in different treatments. H The effect of different glutathionylation levels of GRP on MST1/2 expression level, see Additional file 1: Fig. S6E for original image. (h1) 1:1000 anti-6 × His antibody was used in Western blot; GSH (0.1 mmol) or DTT (0.1 mmol) was added to MM + straw; the hybridization signal intensity of anti-6 × His antibody was increased in GSH added treatment (+ GSH, − DTT), and decreased in DTT added treatment (− GSH, + DTT). (h2) The expression level of MST1/2 in different treatment. Bars represent mean ± SEM, with n = 3 biological repeats; red dots resemble values from individual experiments. Student’s t-testing was conducted in (D, E), *significant difference to TgGluSensor::CON at two-tailed P = 0.015 (D, TgGluSensor::OE-Tggrp); *significant difference to TgGluSensor::CON at two-tailed P = 0.031 (E, TgGluSensor::OE-Tggrp). ANOVA was conducted in (F1), Tukey's HSD test was used for post hoc comparisons, and the letters “a”, “b”, and “c” were used for significance exhibition. Inorganic sulfide has a significant effect on the intracellular GSH content (P < 0.05 level). Student’s t-testing was conducted in (F2), *significant difference to WT at two-tailed P = 0.012 (F2, OE-Tgatps), 0.012 (F2, OE-TgcysK)