Journal: Nature Communications

Article Title: Magnaporthe oryzae MoPh1 perceives ER stress and promotes adaptive responses via a plasma membrane-to-vacuole pathway

doi: 10.1038/s41467-026-70610-0

Figure Lengend Snippet: A Dynamics of ER morphological changes during ER stress (2.5 mM DTT). Visualization of the relative localization between ER (marked with MoSec61-GFP) and PM (dyed with FM4-64). Asterisks and dashed lines indicate sites of close contact between the ER and the PM. “T1” to “T5” represent distinct ER morphological states and spatial distributions under varying ER stress conditions, with corresponding quantitative analysis presented on the right. Scale bar, 5 μm. n = 5 biologically independent samples were examined. Error bars were derived from measurements of five hyphae observed under the microscope. B Co-localization of MoTcb1-GFP with the ER marker MoSec61-CFP and the PM dye FM4-64 under ER stress (induced by 2.5 mM DTT for 2 h). (i) Pseudo-colored fluorescence intensity of MoSec61-CFP. (ii) Pseudo-colored fluorescence intensity of MoTcb1-GFP. White arrows indicate the accumulation of MoTcb1-GFP adjacent to the MoSec61-CFP-labeled ER. The dashed white lines in the merged panel denote the trajectories for line-scan analysis, which are used to generate the corresponding fluorescence intensity profiles. Scale bar, 5 μm. The experiment was performed in triplicate and yielded comparable results. C The BiFC analysis for the interaction of MoPh1-MoTcb1, with the autophagosome marked by RFP-MoAtg8. MoPh1- N YFP (N-terminal of yellow fluorescent protein) and MoTcb1- C YFP (C-terminal of yellow fluorescent protein) constructs are co-transformed into the wild-type Guy11 strain and the fluorescence is detected in aerial hyphae under ER stress (2.5 mM DTT for 1 or 5 h), CM condition was used as a non-stress control. Scale bar, 5 μm. The experiment was performed in triplicate and yielded comparable results. D The in vivo co-IP assay confirms the interaction between MoPh1 and MoTcb1. Short-term ER stress is made by 2.5 mM DTT for 1 h, long-term ER stress is made by 2.5 mM DTT for 5 h. The experiment was performed in triplicate and yielded comparable results. E The localization observation of MoPh1 during the hyphae stage under sustained ER stress (DTT treatment, 2.5 mM DTT, 5 h) in both the wild-type Guy11 and the Δ MoTcb1 mutant. CM condition is used as the non-stress control. CMAC is used as a vacuole dye. Scale bar, 5 μm. The experiment was performed in triplicate and yielded comparable results. F The BiFC analysis for the interaction of MoPh1 and MoDbf2. MoPh1- N YFP and MoDbf2- C YFP constructs are co-transformed into the wild-type Guy11 strain and the fluorescence is detected in aerial hyphae under ER stress (2.5 mM DTT for 1 h), CM condition is used as a non-stress control. Scale bar, 5 μm. The experiment was performed in triplicate and yielded comparable results. G Phos-tag gels analysis for MoPh1 phosphorylation by MoDbf2 during the hyphal stage under ER stress (2.5 mM DTT for 1 h). The experiment was performed in triplicate and yielded comparable results. H Localization of MoPh1-GFP under different conditions in both wild-type Guy11 and Δ Modbf2 mutant. “SA” stands for the constitutively inactivated mutations of MoPh1 phosphorylation sites and “SD” stands for the constitutively activated mutations of MoPh1 phosphorylation sites. ER stress is induced by a treatment of 2.5 mM DTT for 5 h. CMAC is used as a vacuole dye. Scale bar, 5 μm. The experiment was performed in triplicate and yielded comparable results.

Article Snippet: Droplets (35 μl) of conidial suspension (5 × 10 4 spores/ml) were placed on a microscope cover glass (Fisher Scientific, 16938) under humid conditions at 28 °C in darkness, and the samples were microscopically observed at intervals .

Techniques: Derivative Assay, Microscopy, Marker, Fluorescence, Labeling, Construct, Transformation Assay, Control, In Vivo, Co-Immunoprecipitation Assay, Mutagenesis, Phospho-proteomics