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

Domain: chemistry
Published: 2026-03-16
Editorial outcome: major_revision

Abstract
During growth and development, cells experience both internal and external stresses, which can exert harmful impacts if they are poorly managed. Endoplasmic reticulum (ER) stress is an internal stress that is induced when protein misfolding or perturbations occur at excess rates, and the conventional response pathways from the ER to the nucleus are activated to address the stress. However, the involvement of the plasma membrane (PM) system in response to this internal stress has been insufficiently investigated. Here, a PM sensor, MoPh1, was observed to perceive stress through ER-PM contact sites and target the autophagosome and vacuole, consequently stimulating the autophagy process and supporting stress relief. The PMS-to-vacuole pathway mediated by MoPh1 is independent of the classical ER-to-nucleus pathway and might be highly important in both fungi and plants, as it plays a crucial role in alleviating ER stress and promoting cellular adaptation for cell survival.

Introduction
The endoplasmic reticulum (ER), the largest organelle in eukaryotic cells, is responsible for lipid and protein biosynthesis, providing locales for modifying and folding nascent integral membranes and secreted proteins. Overload of protein folding or perturbations in cellular metabolism instigate ER stress, which disrupts essential cellular processes1, 2, leading to serious human diseases (such as Alzheimer's disease, Parkinson's syndrome, diabetes, and even cancer) and plant diseases (e.g., rice blast and bacterial diseases)3-11. Therefore, the detection of ER stress is highly important for disease prevention and treatment.

Both the plasma membrane (PM) and organelle membrane, two types of membrane systems, are crucially involved in the perception and transmission of stress induced by both internal and external stimuli12-14. The response to ER stress is initiated by the activation of Ire1, an ER membrane protein, by oligomerization and autophosphorylation, which leads to the induction of the transcriptional activator Hac1 by variable shear stress, followed by the subsequent regulation of the expression of downstream stress-responsive genes through entry into the nucleus15-17. These genes reduce protein misfolding and induce membrane synthesis, upon which ER function is enhanced and ER stress is alleviated. The unfolded protein response (UPR), the stress sensing and response from the ER to the nucleus, which is initiated by Ire1 at the ER membrane, is the most well-characterized response to ER stress. Ire1 is considered one of the most important ER stress sensors present in various species, including mammals, plants, and fungi17-19. However, the critical role of the PM structure, where stress sensing and signal transduction start, in this internal stress response remains uncertain.

PM proteins play crucial roles in the perception of biotic and abiotic stresses across various species, thereby increasing their adaptive capacity20-25. Magnaporthe oryzae, the causative agent of devastating fungal blast disease in rice, requires the perception of rice surface signals by plasma membrane proteins as a prerequisite for the formation of its essential infection structure, the appressorium. Multiple PM proteins in M. oryzae, including MoPth11, MoSho1, MoMsb2, and MoRgs7, are critical for recognizing hydrophobic signals, waxes, cutin, and other surface cues from rice26-29. Upon perceiving diverse signals, these receptors undergo state changes (such as phosphorylation) or modulate their interaction networks, thereby initiating intracellular signal transduction pathways, including the cAMP and MAPK cascades, to relay signals downstream and regulate the expression of genes associated with appressorium formation and consequently modulating pathogenicity. Although several PM proteins that are involved in stress signal perception and pathogenicity have been identified in M. oryzae, only a few of these proteins have been thoroughly characterized in terms of their mechanistic actions, specific signal responses, and comprehensive regulatory networks.

Our previous study demonstrated a regulatory mechanism under ER stress conditions, which is essential for appressorium function and virulence, in which a core autophagy-related protein kinase, MoAtg1, directly targets the MAPK K kinase MoMkk1, phosphorylates it, and activates the cell wall integrity pathway independently of the conserved upstream MAPKKK kinase3. Given the unprecedented nature of this regulatory mechanism wherein a protein substitutes for MAPKKK function under ER stress, it appears that M. oryzae employs regulatory modalities and networks to respond to ER stress, warranting further investigation.

In this study, we further investigated putative key PM proteins involved in the ER stress response through comparative membrane proteomic profiling. Combined with expression analysis and subcellular localization assessment, we identified a pleckstrin homology (PH) domain-containing protein, MoPh1. MoPh1 is localized to the PM under non-stress conditions, whereas upon ER stress, it perceives stress signals through inter-organellar communication mediated by the ER-PM contact site regulatory protein MoTcb1 and is subsequently phosphorylated by the protein kinase MoDbf2, leading to its dissociation from the PM. Phosphorylated MoPh1 then targets autophagosomes and interacts with key autophagy-related proteins, including the core scaffold protein MoAtg11 and the autophagosome vacuole fusion-related protein MoYpt7, to promote the initiation and progression of autophagy, thereby facilitating cellular stress adaptation. Concurrently, MoPh1 enhances the efficiency of phase-separated droplet formation by interacting with Ire1, a key sensor in the canonical ER stress response pathway, thereby amplifying the activity of the classical stress response machinery. Surprisingly, this PM-to-vacuole stress response pathway mediated by MoPh1 does not depend on conventional stress sensors or the classical response pathway; however, it increases the intensity of the classical pathway and appears to be specific to PH domain-containing proteins in M. oryzae. Overall, our study not only identified a PM-localized ER stress sensor, MoPh1, in both microorganisms and plants but also elucidated a distinct response model in which it activates autophagy via a non-canonical PM-to-vacuole pathway to mitigate internal stress, enhance fungal fitness, and promote pathogenicity, providing new perspectives for exploring internal stress perception and response in biological systems.

Results
Endoplasmic reticulum stress perception and response are vitally important for both fungal survival and pathogenicity

We initially examined the changes in key factors involved in the ER stress response in M. oryzae at the hyphal stage following treatment with ER stress stimulators [dithiothreitol (DTT) and tunicamycin (TM)]30. The results revealed that these treatments significantly increased the phosphorylation level of Ire1, the ratio of spliced Hac1, and the expression of downstream target genes involved in the unfolded protein response (UPR) (Figure 1A-1C), indicating the promotion of ER stress accumulation and response by these agents. The changes in ER stress levels at various time points during the formation of the appressorium, a crucial structure of infection by M. oryzae, were subsequently examined. The accumulation of ER stress during the period of appressorium formation showed a fluctuating trend, peaking after 8 h of treatment, followed by a gradual decrease (Figure 1D-1F), indicating that ER stress could be induced not only exogenously but also naturally in a fluctuating manner during the development of its infectious structures. To further investigate whether the dynamics of stress during appressorium formation are associated with fungal pathogenicity, 4-PBA was introduced as an inhibitor of ER stress31, while DTT and TM were used as stimulators to treat fungal conidia at different concentrations to interfere with the accumulation of stress. According to our findings, DTT, TM, and 4-PBA treatments interfered with the germination of fungal conidia, which delayed the germination process and appressoria formation rate and led to lower turgor pressure than that of wild-type Guy11 (Supplementary Figure 1A-1C). Further determination of the pathogenicity of these treated conidia on rice leaves revealed that either excessive stimulation or inhibitory stress led to reduced fungal pathogenicity and disease area (Figure 1G-1H). Similarly, infection assays on rice leaf sheaths also revealed that at 24 h post-inoculation, conidia treated with DTT, TM, or 4-PBA either failed to form infective appressoria or produced only short primary invasive hyphae. By 48 h, only limited invasive hyphae restricted to initially infected cells were observed, whereas the wild-type and solvent (DMSO) control groups presented extensive hyphal expansion into neighboring cells (Figure 1I-1J). These results suggest that the accumulation of ER stress and an appropriate response are required for appressorium formation and pathogenicity in M. oryzae.

In addition to its role in fungal pathogenicity, we were also curious about whether the ER stress response is essential for fungal viability under stress. Thus, the survival rate of fungal conidia under ER stress after the application of two ER stress response inhibitors in the wild-type Guy11 strain, including the dual inhibitor of the kinase and RNase activities of Ire1, GSK285016332, and the sole inhibitor of Ire1 RNase activity, STF-08301033, 34, was determined. The results revealed a significant reduction in the survival rate of the conidia and mycelia caused by an abnormal ER stress response under Ire1 inhibitor treatment (Figure 1K). We also obtained an Ire1 knockout mutant in a specialized KU80 background, which does not impair the developmental processes or pathogenicity of M. oryzae but increases the frequency of targeted gene replacement27, 35, and evaluated its virulence and sensitivity to ER stress. The results revealed that deletion of the MoIRE1-encoding gene led to similar reduction of survival rate, the ratio of spliced Hac1 and pathogenicity of M. oryzae (Figure 1L and Supplementary Figure 2A-2B), suggesting that an appropriate response to ER stress is crucial for not only fungal infection but also fundamental survival. However, we also noted that, as the key sensor of ER stress reported to date in fungi, the deletion of MoIRE1 did not result in a complete loss of survival under stress (Supplementary Figure 2A), indicating that there might be other important sensors and processes involved in the ER stress response.

Identification of the plasma membrane protein MoPh1, which is involved in the ER stress response and fungal survival in a stress environment

As one of the key components in perceiving various stress signals, plasma membrane (PM) sensors play crucial roles in both recognizing and transducing signals. During the perception and response process, these sensors might undergo some protein modifications and localization alterations36. To investigate the putative PM proteins that are critical for the ER stress response, we conducted a quantitative membrane proteomics analysis to identify differentially expressed membrane proteins in both the absence and presence of ER stress (DTT+TM co-treatment). Through further screening and analysis, we focused more on the candidate proteins with PM-associated domains whose gene expression significantly increased but whose membrane protein content decreased (Figure 2A and Supplementary Figure 3, Table S1), which might be more likely to be associated with the ER stress response. Two putative proteins with the lowest p value attracted our attention since they both contain a pleckstrin homology (PH) domain (MGG_03558 and MGG_11259). Further protein localization screening confirmed the localization of MoPh1 (MGG_11259) to the PM in the absence of stress and its significant response to ER stress (Supplementary Figure 4).

To elucidate the role of the PM protein MoPh1 in the stress response, we generated deletion mutants of MoPH1 (Moph1#6, Moph1#8) via homologous recombination and examined their ER stress response. The results revealed that a significant defect in the ER stress response caused by the deletion of the MoPH1 gene occurred under both the DTT and TM treatments (Figure 2B-2D) and during appressorium formation (Supplementary Figure 5A-5B), including the ratio of spliced MoHAC1 and the expression of downstream UPR target genes, but not during the phosphorylation of MoIre1. The deletion of MoPh1 also concurrently increased the susceptibility of the pathogen to ER stress, which caused a marked reduction in its survival rate and inhibition rate of mycelia in the presence of DTT or TM (Figure 2E-2F and Supplementary Figure 6A-6B), indicating the possible role of MoPh1 in regulating the ER stress response. Since deletion of MoPh1 did not affect the phosphorylation, it seems that its function might be independent of the classical ER stress sensor Ire1.

Furthermore, as a pathogenic fungus, we examined the phenotypic effects of MoPH1 deletion on appressorium function and pathogenicity. The loss of MoPh1 significantly reduced the infection and virulence of the pathogen in both rice and barley (Figure 2G and Supplementary Figure 7A-7B), and the appressorium turgor pressure in the mutant strains were markedly lower than that in the wild-type and complemented strains (Moph1/MoPH1) (Figure 2H). Consistent with the pathogenicity results, infection assays on rice leaf sheaths and barley leaf abaxial epidermis revealed that at 24 h post-inoculation, the MoPH1 deletion mutant either failed to penetrate the plant epidermis or produced only a minimal infection peg. In contrast, the wild-type and complemented strains generated extensive invasive hyphae with branching within the infected cells. When the infection period was extended, the MoPh1 deletion mutant still produced only limited invasive hyphae, whereas the wild-type and complemented strains fully colonized the initially infected cells and expanded into adjacent cells (Figure 2I and Supplementary Figure 7C-7D). These findings indicated that MoPh1 was essential for appressorium function and pathogenicity in M. oryzae and that the pathogenicity defect observed in the deletion mutant was likely associated with the impaired appressorium function.

MoPh1 exhibits dynamic changes in its subcellular localization in response to fluctuations in ER stress levels

Further observation of MoPh1 localization under different stress conditions revealed that it was localized to the PM (Figure 3A) under non-stress conditions. Upon short-term ER stress (0.5 and 1 h), a portion of MoPh1 formed punctate aggregates inside the cells. This aggregation process could be inhibited by the endocytosis inhibitor latrunculin B (Supplementary Figure 8), indicating that MoPh1 could enter the cytoplasm via endocytosis under stress conditions. When the ER stress treatment was prolonged (2 and 5 h), the internalized MoPh1 further localized to autophagosomes and vacuoles (Figure 3A and Figure 3C-3D). Notably, unlike artificial ER stress treatment during the hyphal stage, MoPh1 constantly maintained partial PM localization under the natural fluctuations of ER stress during appressorium formation (Figure 3B). However, the proportion of MoPh1 internalized into the cells exhibited similar fluctuating changes in response to variations in stress levels: a gradual increase in the proportion of intracellular MoPh1 co-localized with autophagosomes was recorded within the initial 0-8 h of conidial germination, and subsequently declined (Figure 3E). These results suggest that synchronous variation in MoPh1 localization corresponds to different levels of ER stress. Combined with the findings that MoPH1 gene expression was induced by ER stress and that the loss of MoPh1 severely impaired the stress response and tolerance, we concluded that MoPh1 might serve as a PM sensor capable of sensing and responding to ER stress.

MoPh1 is involved in the ER stress response through regulating the autophagy process

Autophagy is an evolutionarily conserved process that plays a role in degrading and recycling the cytosol and organelles in eukaryotic cells by delivering the materials destined for degradation to lysosomes or vacuoles37, 38. Given that MoPh1 can target autophagosomes and vesicles under ER stress, we wondered whether this stress response initiated from the PM leads to the activation of autophagy to help relieve stress. Thus, we first examined the number of autophagosomes in the wild-type and mutant strains under ER stress using transmission electron microscopy and detected a significant reduction in the number of autophagosomes in the MoPh1 deletion mutant (Figure 4A). The intensity of both selective (marked with MoSec61) and non-selective (marked with MoAtg8) autophagy in M. oryzae under ER stress was examined, and both were found to be significantly induced in the presence of ER stress in the wild-type strain Guy11 and largely influenced in the absence of MoPh1 (Figure 4B-4D), indicating the role of MoPh1 in the regulation of selective and non-selective autophagy triggered by ER stress.

To elucidate the mechanisms involved in the entry of MoPh1 into autophagosomes and its regulatory impact on autophagy, MoPh1 was used as bait, and its interaction patterns under ER stress were screened. Our findings revealed the specific potential MoPh1-interacting proteins involved in several biological processes, including the plasma membrane, autophagy, vesicular transport and protein modification, during this stress (Figure 4E and Table S2). Among the processes related to autophagy, MoPh1 was found to interact directly with MoAtg11, which is a key scaffold protein that plays a role in autophagy, specifically in autophagosome formation under ER stress30, under both DTT treatment at the hyphal stage and appressorium formation stage (Figure 4F and Supplementary Figure 9A-9D). Notably, the deletion of MoPh1 disrupted the localization of MoAtg11 to the autophagosome (Figure 4G-4I and Supplementary Figure 27A) and also led to an aberrant interaction strength between MoAtg11 and another core autophagy regulator, MoAtg9 (Supplementary Figure 10), manifested as the enhancement of MoAtg11-MoAtg9 under ER stress compared with that in the wild type, which resulted in a marked reduction in the characteristic punctate distribution of Atg9 but accumulation in large aggregates (Figure 4J-4L and and Supplementary Figure 27B and Supplementary Figure 11A-11B). Furthermore, the analysis of MoPh1-interacting proteins revealed the interaction of MoPh1 with MoYpt7 (Supplementary Figure 12A-12B), which is responsible for regulating the fusion of autophagosomes with the vacuolar membrane39, and the deletion of MoPH1 affects the aggregation of MoYpt7 in vesicles in the context of ER stress (Figure 4M-4O and Supplementary Figure 27C and Supplementary Figure 13A-13B). Additionally, MoYpt7 serves as a marker of late endosomes during the processes associated with endocytosis40, which corroborates our previous finding that the entry of MoPh1 into the cytoplasm via endocytosis.

Given that the interactions of both MoPh1-MoAtg11 and MoPh1-MoYpt7 are associated with the autophagy process, specifically autophagosome formation and subsequent fusion with vacuoles, we investigated whether these interactions influence each other. To address this, we examined whether the MoPh1-MoYpt7 interaction was affected in the MoATG11 deletion mutant. The results revealed that the MoPh1-MoYpt7 interaction under ER stress was significantly weakened in the absence of Atg11 (Supplementary Figure 14A). Conversely, in the MoYPT7 deletion mutant, the MoPh1-MoAtg11 interaction remained largely unaffected (Supplementary Figure 14B).

MoPh1 percepts ER stress through ER-PM contacting sites and undertakes phosphorylation activation

To investigate how MoPh1, a PM-localized protein, receives signals from the ER, which induce the subsequent initiation of the relevant response, we reanalyzed the data of MoPh1-interacting proteins and identified an interesting component, MoTcb1, which functions in regulating the contact between the ER and the PM41. ER-PM contact sites (EPCSs) are formed that allow the exchange of material and information between these two structures41-44. Here, MoSec61 was used as an ER marker, and FM4-64 was used to label the PM. We observed that under normal conditions without ER stress, the ER predominantly exhibited a regular, continuous tubular network that was relatively independent of the PM (Figure 5A). In contrast, under ER stress conditions, the ER displayed not only a tubular morphology, but also a more dispersed distribution, with a significantly increased proportion of regions closely apposed to the PM, especially under relatively short-term ER stress (0.5 and 1 h) (Figure 5A), indicating that frequent ER-PM contacts during the response to ER stress in M. oryzae and that the ER might transmit signals to the PM through these contact sites. Notably, following prolonged ER stress (2 and 5 h), the proportion of dispersed ER structures in close proximity to the PM gradually decreased, while the regular ring-like ER morphology became increasingly predominant. This dynamic redistribution suggested that the stress-induced ER remodeling and its close association with the PM represent a transient signaling phase rather than a sustained state (Figure 5A). Localization tracking revealed that MoTcb1 localized to the PM with punctate aggregation under normal conditions (Supplementary Figure 15A), whereas the puncta-MoTcb1 on the PM were significantly enriched near the ER-PM contact sites and partially co-localized with them under ER stress (Figure 5B), indicating that MoTcb1 could localize to the ER-PM contact sites during stress conditions. Concurrently, a fraction of MoTcb1 internalized and colocalized with autophagosomes and vacuoles under stress conditions (Supplementary Figure 15A). We then confirmed the interaction between MoTcb1 and MoPh1 and found that this interaction occurred specifically under short-term ER stress, as puncta on the PM closely coincided with the distribution pattern of ER-PM contact sites but did not interact under long-term ER stress or normal conditions (Figure 5C-5D and Supplementary Figure 15B). We further generated a MoTCB1 deletion mutant (Motcb1) and introduced GFP-tagged MoPh1 into this background. The results revealed that MoPh1 failed to dissociate from the PM under ER stress in the Motcb1 mutant (Figure 5E and Supplementary Figure 27D), along with the significant impairment of ER stress-induced autophagy (Figure 4D-4E and Supplementary Figure 16A-16B), confirming the generation of ER stress signals perceived by MoPh1 from EPCSs through MoTcb1.

In addition, most PM receptors undergo modifications before and after they sense signals45-47. Our mass spectrometric analysis of MoPh1 phosphorylation led to the identification of two specific phosphorylation sites (S226 and S230) under ER stress (Supplementary Figure 17A). We then validated the authenticity of these two phosphorylation sites using Phos-tag analysis and found that, constitutively unphosphorylated MoPh1S226, 230A still maintained the localization on the PM even under ER stress, whereas the constitutively activated MoPh1S226, 230D internalized spontaneously without stress treatment (Figure 5H and Supplementary Figure 17B and Supplementary Figure 27E), suggesting that the phosphorylation of MoPh1 was essential for its dissociation from the PM. Notably, among the proteins that specifically interact with MoPh1 under ER stress, we identified a potential PM-localized, mitosis-related protein kinase, MoDbf2 (Supplementary Figure 17C), and verified the specific interaction on the PM under stress conditions through BiFC, Co-IP and Y2H assays (Figure 5F and Supplementary Figure 17D-17F). The validation of phosphorylation was performed both in vivo and in vitro to verify whether MoDbf2 can directly phosphorylate MoPh1 at two specific phosphorylation sites (S226 and S230) (Figure 5G and Supplementary Figure 18A-18B). Consistent with these findings, MoPh1 also failed to dissociate from the PM in the MoDBF2 deletion mutant (Modbf2), which was similar to the constitutively unphosphorylated MoPh1S226,230A (Figure 5H and Supplementary Figure 27E). These results together demonstrated that the specific phosphorylation of MoPh1 by MoDbf2 was also a prerequisite for its dissociation from the PM. We also observed that neither the constitutively inactive nor the constitutively active phosphorylation mimic of MoPh1 could fully restore the pathogenicity defect caused by MoPH1 deletion (Supplementary Figure 18C-18D), indicating that while phosphorylation is essential for MoPh1 function, it must be maintained in a relative equilibrium. Consistent with this, although the constitutively active form of MoPh1 internalized under non-stress conditions, it failed to localize to the vacuole (Figure 5H and Supplementary Figure 27E).

Given that the perception of stress signals at ER-PM contact sites and the subsequent phosphorylation of MoPh1 are both critical for its dissociation from the PM, how is there a relationship between them? We then examined the phosphorylation of MoPh1 in the Motcb1 mutant and found that deletion of MoTcb1 interfered with the phosphorylation of MoPh1 (Supplementary Figure 19A), indicating that the interaction of MoTcb1-MoPh1 and the reception of ER stress signals were prerequisites for MoPh1 phosphorylation. However, the MoTcb1-MoPh1 interaction under ER stress remained unaffected by the loss of MoDbf2 (Supplementary Figure 19B). These findings demonstrated that under ER stress conditions, MoPh1 first receives stress signals at ER-PM contact sites and subsequently utilizes the protein kinase MoDbf2 to phosphorylate itself, thereby enabling its dissociation from the PM.

The PM-to-vacuole ER stress response pathway mediated by MoPh1 is independent of the classical ER-to-nucleus response pathway

Considering that both the classical ER-to-nucleus response pathway mediated by the ER membrane sensor MoIre1 and the PM-to-vacuole response pathway mediated by the PM sensor MoPh1 influence the fungal response to ER stress and survival, we investigated the relationship between these two pathways. We thus impaired the classical response pathway through different Ire1 enzymatic inhibitors and a MoIRE1 deletion mutant and examined whether the MoPh1-mediated response was affected. The results revealed that neither the inhibition of Ire1 enzymatic activity nor the deletion of MoIRE1 affected the interaction between MoTcb1 and MoPh1 under ER stress, the dynamic changes in MoPh1 localization, or the autophagy process (Figure 6A-6B and Supplementary Figure 20A-20B and Supplementary Figure 27F), indicating that the MoPh1-mediated response does not depend on the classical ER stress sensor. However, we previously reported that the deletion of MoPh1 affects the splicing of Hac1 and the response of downstream UPR target genes in the classical ER stress response pathway but not Ire1 phosphorylation (Figure 2B-2D), suggesting that these two pathways function relatively independently but exhibit crosstalk at specific nodes.

We therefore sought to further identify the crosstalk node between these two pathways. A recent study demonstrated that mammalian Ire1 can form efficient Ire1 machinery after oligomerization and autophosphorylation through the formation of phase-separated droplets, which enhances Ire1 splicing efficiency and consequently improves the response capacity to stress48. Interestingly, we found that MoIre1 in M. oryzae could form similar droplets under ER stress, and the number of these droplets was significantly reduced in the MoPh1 deletion mutant (Moph1) (Figure 6E-6G and Supplementary Figure 27G-27H), suggesting the involvement of MoPh1 in manufacturing highly efficient Ire1 machinery, which likely blocked Hac1 splicing and UPR downstream target gene expression in the Moph1 mutant. Furthermore, we observed that MoPh1 could partially co-localize with Mo Ire1 under ER stress conditions and that there was a direct interaction between them (Figure 6H-6J). Notably, the interaction domain was mapped to the intrinsically disordered region of MoIre1, which was related to phase separation, rather than the kinase domain responsible for phosphorylation (Supplementary Figure 21), which corroborated that while MoPh1 did not affect Ire1 phosphorylation, it specifically modulated the downstream splicing function of Ire1 toward Hac1. We also found that MoIre1 was important for fungal pathogenicity that deletion of MoIRE1 led to severely attenuated virulence and a complete loss of conidiation (Figure 6K-6L).

The PM-to-vacuole ER stress response pathway mediated by MoPh1 is relatively conserved in both fungi and plants

A comprehensive search of a total of 19 proteins containing the PH domain, including some previously reported proteins49, in M. oryzae was performed. For localization, we randomly selected 10 proteins from the pool of proteins, and the results revealed a PM-to-vacuole response only for MoPh1 under ER stress conditions (Supplementary Figure 22A-22B), suggesting the distinctiveness of MoPh1 in terms of its sensor function in M. oryzae.

Moreover, to determine whether this MoPh1-mediated stress response pathway is prevalent across different species, bioinformatics analysis of the homologs of MoPh1 and the protein involved in ER-PM regulation (MoTcb1) in the model plant Arabidopsis thaliana was performed. The results revealed two homologs of MoPh1, AtPH1 (AT2G29700.1) and AtPH2 (AT5G05710.1), whereas 26 homologs of MoTcb1 was found (Supplementary Figure 23A-23B). The AlphaFold3 model was used to predict the interactions among these proteins, two of MoTcb1 homologs, AtSYT1 (AT2G20990.3) and AtAGD12 (AT4G21160.1), are likely to interact with MoPh1 homologs (Supplementary Figure S24A). These interactions were further validated via a split-luciferase complementation imaging assay in tobacco under ER stress, in which interactions between AtPH2 and both AtSYT1 and AtAGD12 were observed, whereas AtPH1 could interact with only AtSYT1 (Supplementary Figure 24B). Additionally, AtPH1 and AtPH2 could both translocate from the PM to the cytoplasm in response to ER stress (Supplementary Figure 25A-25B), and their deletion caused a significant reduction in plant tolerance to ER stress (Supplementary Figure 25C-25D). These findings collectively suggest that both the ER stress perception and response pathways mediated by MoPh1 also occur in plants.

Methods
Strains and culture conditions.
M. oryzae strain Guy11 and specialized KU8027, 35 were used as the wild type (WT). All strains were generally cultured on solid complete medium (CM) for 7 days before used and in liquid CM for 2 days at 28℃ before observation or collection3. For vegetative growth, small agar blocks were cut from the edge of 7-day-old cultures and placed onto fresh media, followed by incubation in the dark at 28°C. The radial growth was measured after incubation for 7 days. Mycelia were harvested from the liquid CM with or without additional treatment for DNA, RNA and total protein extractions. SDC (a semi-natural medium) medium was used for conidiation87 with 5 days darkness cultivation following by 3 days of continuous illumination under fluorescent.

Antibodies
Antibodies used: Anti-His antibody (TransGen Biotech, China, Code#HT501, Anti-His Mouse Monoclonal Antibody, Lot#R20602); Anti-GST antibody (TransGen Biotech, China, Code#HT601, Anti-GST Mouse Monoclonal Antibody, Lot#R10427); Anti-FLAG antibody (TransGen Biotech, China, Code#HT201, Anti-DYKDDDDK Mouse Monoclonal Antibody, Lot#Q21129); Anti-GFP antibody (TransGen Biotech, China, Code#HT801, Anti-GFP Mouse Monoclonal Antibody, Lot#Q31105); anti-beta-actin antibody (Sino Biological Inc. Code#100166-MM10, beta-Actin Loading Control Antibody, Mouse MAb); anti-Mouse HRP antibody (Sino Biological Inc. Code#HS201-01, Goat Anti-Mouse IgG (H+L), HRP Conjugate, Lot#R20619) Validation: These antibodies were all used for Western Blot analysis. Anti-His antibody (WB, 1:5000); Anti-GST antibody (WB, 1:5000); Anti-FLAG antibody (WB, 1:5000); Anti-GFP antibody (WB, 1:5000); anti-beta-actin antibody (WB, 1:5000); anti-Mouse HRP antibody (WB, 1:10000).

Target gene deletion and complementation.
The MoPH1 gene, MoDBF2, MoAtg11, MoIRE1, as well as MoTCB1 deletion mutant were generated using a standard one-step gene replacement strategy as described previously70. The complement fragment, which contained the entire gene coding region and its native promoter region, was amplified by PCR with primers using 2× Planta Master Mix (Vazyme Biotech Company, P511-02) and inserted into pYF11 (bleomycin resistance) to restore mutant strains. The vector was constructed through a series of molecular biology procedures. Yeast plasmid extraction was performed using Yeast Plasmid Extraction Kit (Cat# D1160, Bei jing Solarbio Science & Technology Co., Ltd.), and PCR product purification was carried out with SPARKeasy Gel/PCR Purification Kit (Cat# AE0101-C, Shandong Sparkjade Biotechnology Co., Ltd.).

Appressorium formation and appressorium related function test.
To analyze fungal appressorium formation, conidia were harvested from 10-day-old SDC cultures and then filtered with two-layers of Miracloth (EMD Millipore Corporation, 475855-1R) and washed with double-distilled water (ddH O) for 3 times. Droplets (35 μl) of conidial suspension (5 × 104 spores/ml) were placed on microscope cover glass (Fisher Scientific, 16938) under humid conditions at 28°C in darkness and the samples were microscopically observed at intervals28. The percentages of conidia germinating and conidia forming appressoria were observed and counted by microscopic examination of at least 100 conidia for more than three times as described before70. For appressorium related function test, the appressorium turgor was measured using an incipient cytorrhysis (cell collapse) assay with a 1.0 to 4.0 M glycerol solution88. For the treatment of different ER stress activators (DTT or TM) and inhibitors (4-PBA), the reagents in different concentrations (DTT for 2.5 mM or 5 mM, TM for 0.25 g/mL or 2.5 g/mL, 4-PBA for 1 mM or 5 mM) were first mixed with the fungal conidia and allowed to stand for 10 minutes, and followed by subsequent droplet application, spraying, or injection. 4-Phenylbutyric acid (4-PBA) was purchased from MedChemExpress, Monmouth Junction, NJ, USA; Cat# HY-A0281. [DTT (Dithiothreitol), Cat：D8220; tunicamycin (TM), Cat T8480 Beijing Solarbio Science & Technology Co., Ltd.].

Fungal viability test.
Fresh conidial suspensions of the wild-type Guy11, ΔMoph1(#6, #8) and ΔMoph1/MoPH1 were spotted on CM at 28°C for 3 days with or without ER stressors (2.5 mM DTT or 0.25 g/mL TM). Each experiment was repeated four times independently. For the treatment of different ER stress response inhibitors (GSK2850163 and STF-083010), the reagents was mixed with the fungal conidia respectively and allowed to stand for 10 minutes before inoculated onto plates containing ER stress inducer (2.5 mM, 5 mM DTT or 0.25 g/mL, 2.5 g/mL TM ). After 7 days of growth, the colony diameter was measured to calculate the inhibition rate.

Assays of fungal pathogenicity and infection.
Conidial suspensions were applied to plants using two methods. For spray and spot inoculation, a suspension at 5 × 10⁴ spores/mL was used on 14-day-old susceptible rice seedlings (Oryza sativa cv. CO-39) or 7-day-old barley seedlings (Hordeum vulgare cv. Four-arris). For leaf sheath injection, a suspension at 1 × 10⁵ spores/mL was used on 21-day-old susceptible rice seedlings (Oryza sativa cv. CO-39). Rice plants at the cultivation stage were supplied with Coolaber's 1000× Kimura B Nutrient Salts Solution (Beijing Coolaber Science & Technology Co., Ltd.). To assess the effects of disrupted ER homeostasis on spore functionality and pathogenicity, Magnaporthe oryzae conidia were pretreated for 10 minutes with ER stress modulators (1 mM and 5 mM 4-PBA, 2.5 mM and 5 mM DTT or 0.25 g/mL and 2.5 g/mL TM) prior to spray and injection inoculation. For mutant strains impaired in sporulation, pathogenicity was assessed by inoculating mycelial plugs onto detached leaves. Inoculated plant materials will be kept in complete darkness for the first 24 hours at a heliophobe growth chamber and then transferred into another transparent growth chamber following a light dark alternation after each 12 hours. The lesion area was analyzed by ImageJ bundled with 64-bit Java 8 which is used for synthetically evaluating the disease severity. Rice leaf sheaths or barley leaf epidermis were inoculated to observe the development of invasive hyphae. The invasive hyphae were quantified by “invasive-type” scoring assay which divided the hyphae into 5 types according to their severity (AP, appressorium with no invasive hyphae; IH0, appressorium with only one short invasive hyphae; IH1, appressorium containing less than three invasive hyphae with short branches; IH2, more than three invasive hyphae with several branches filled with a plant cell; IH3, numerous invasive hyphae that expand to adjacent cells). More than 50 infection courts were observed used for analysis for each sample.

ER stress related analysis.
The ER stress level was detected according to the phosphorylation level of MoIre1, the ratio of spliced MoHAC1, and the expression of downstream target genes involved in the unfolded protein response (UPR)3. Dithiothreitol (DTT) and tunicamycin (TM) were used as ER stress inducers, 4-PBA was introduced as an inhibitor of ER stress. Also, two ER stress response inhibitors, including the dual inhibitor of the kinase and RNase activities of Ire1, GSK2850163 (GSK2850163 was purchased from MedChemExpress, Monmouth Junction, NJ, USA; Cat# HY-U00459), and the sole inhibitor of Ire1 RNase activity, STF-083010 5 M (STF-083010 was purchased from MedChemExpress Monmouth Junction, NJ, USA; Cat# HY-15845). For MoIre1 phosphorylation, the MoIre1-GFP construct was transformed into both the wild-type Guy11 and Moph1 mutant strains. The proteins extracted from mycelium stage (with or without DTT/TM treatment) or appressorium formation stage (generated on 25 × 75 × 1.0 mm glass coverslips (Fisher Scientific, 12-550-15) after generation for 16 hours) were resolved on 8% SDS-polyacrylamide gels prepared with 50 µM acrylamide-dependent Phos-tag ligand and 100 µM MnCl as described before28, 89, 90. Gel electrophoresis was run at a constant voltage of 80 V for 3–6 h. Before transferring, gels were equilibrated in transfer buffer with 5 mM EDTA for 20 min 2 times and followed with another transfer buffer without EDTA for 20 min. Protein transferred from the Mn2+-phos-tagTM acrylamide (NARD institute Limited company, 18D-01) gel to the PVDF membrane by the semi-dry transfer system (Beijing Hongtao Jiye Technology Development Co., Ltd, TRANS-BLOT TURBO, HT1000TBT) for 15 min and then the membrane was analyzed by western blotting using anti-GFP antibody.
For ratio of spliced Hac1 analysis, different spliced states of MoHAC1 (sMoHAC1 and uMoHAC1) mRNA under different conditions were detected with primer pairs sMoHAC1 (Table S3). The intensity stands for the ratio of sHAC1 to uHAC1 and sHAC1, and the intensity ranging from 0 (blue) to 1 (red).
For the expression of UPR target genes (MoKAR2, MoSIL1, MoDER1, MoSCJ1 and MoERV29), total RNA was extracted from mycelium stage (with or without DTT/TM treatment) or appressorium formation stage and was reverse transcribed into first-strand cDNA through the oligo (dT) primers and HiScript II Q RT SuperMix for qPCR (Vazyme Biotech Company, R233-01). qRT-PCR reactions were performed following previously procedures91. qRT-PCR was performed with three independent pools of tissues in three sets of experimental replicates.

Bimolecular fluorescence complementation (BiFC) assay.
The MoPh1-NYFP fusion constructs were generated by cloning the MoPH1 fragment into pHZ68 and similarly, MoAtg11-CYFP, CYFP-MoYpt7, MoTcb1-CYFP, MoDbf2-CYFP were generated by cloning the MoATG11, MoYPT7, MoTCB1, and MoDBF2 fragment into pHZ65 (BiFC vectors from Dr. J. R. Xu of Purdue University). Pairs of MoPh1-NYFP and MoAtg11-CYFP, MoPh1-NYFP and CYFP-MoYpt7, MoPh1-NYFP and MoTcb1-CYFP, as well as MoPh1-NYFP and MoDbf2-CYFP were co-transformed into protoplasts of Guy11 strain, respectively. Transformants resistant to both hygromycin (Solarbio® Life Sciences, H8080) and zeocin (Thermo Fisher Scientific, R25001) were isolated and confirmed by PCR analysis. RFP-MoAtg8 was used to mark autophagosome. YFP and RFP signals were examined by Zeiss LSM880, 63× oil.

Split luciferase assay.
A luciferase complementation imaging assay was employed to investigate the interaction of proteins. The coding sequences (CDS) of AtPH2 and AtPH1 were ligated to the laboratory-created HA-NLUC-pRI101 vector, while the CDS regions of AtAGD12 and AtSYT1 were ligated to the Myc-CLUC-pRI101 vector, also created in the laboratory. Vector creation incorporates pNL and pCL92. The sequences were verified, and the vectors were subsequently introduced into Agrobacterium tumefaciens GV3101. The leaves of 1-month-old Nicotiana benthamiana were infected with strains containing the vectors. Following infection, the plants were maintained in the dark for 12 hours and then incubated under normal conditions for an additional 72 hours. The infected leaves were injected with 1 mM luciferin and exposed to a low-light cooled CCD apparatus (NightSHADE evo LB 985N). The images were captured using IndiGO software.

Co-immunoprecipitation (co-IP)
Total protein was extracted from lyophilized M. oryzae hyphae or appressoria (generated on 25 × 75 × 1.0 mm glass coverslips (Fisher Scientific, 12-550-15) after generation for 16 hours) according to a previous study55, 93, 94. To confirm the interactions of MoPh1-MoAtg11, MoPh1-MoYpt7, MoPh1-MoTcb1, and MoPh1-MoDbf2 in vivo, different pairs of constructs were prepared and then con-transformed into protoplasts of Guy11 strain. Different constructs con-transformed with empty GFP were used as negative controls. Total proteins were extracted from different positive transformants with or without DTT treatment and incubated with anti-GFP agarose (Chromo Tek, gta-20) at 4°C for 2 to 12 h with gently shaking. Proteins bound to the beads were eluted after a series of washing steps by 1× PBS (diluted from 10× PBS). Elution buffer (200 mM glycine, pH 2.5) and neutralization buffer (1 M Tris base, pH 10.4) were used for the elution process. Input and output proteins were then analyzed by western blot using anti-GFP (mouse, 1:5000; Abmart, 293967) or anti-FLAG (rabbit, 1:5000; Abcam, ab93713). NcmColor (Cat.No.P9005,New Cell & Molecular Biotech was used as the potein marker). The Omni-Easy™ One-Step UV Imaging PAGE Gel Rapid Preparation Kit (Cat No. PG112; Epizyme, Shanghai, China) was used for the preparation of protein gel.

Epifluorescence microscopy and three-dimensional imaging.
Cells of M. oryzae during hyphae, conidia, and appressoria (germinating conidia) expressing fluorescent protein-fused chimera were incubated under appropriate conditions. The constructs including GFP-MoAtg8 (G418 resistance), RFP-MoAtg8 (bleomycin and G418 resistance), MoPh1-GFP (bleomycin resistance), MoPh1-RFP (bleomycin resistance), MoAtg11-GFP (bleomycin resistance), GFP-MoAtg9 (bleomycin resistance), GFP-MoYpt7 (G418 resistance), MoDbf2-GFP (bleomycin resistance) were transformed or co-transformed into different strains including the wild-type Guy11, Moph1 mutant, Tcb1 mutant, as well as Modbf2 mutant. Epifluorescence microscopy was performed by using microscope of Zeiss LSM710, 63 x oil. Lipophilic dye FM4-64 was solved in distilled water to a final concentration 5 μg/ml to observe the plasma membrane and endocytic endosomes as previously described (FM4-64 was purchased from MedChemExpress, Monmouth Junction, NJ, USA; Cat# HY-103466)28. Latrunculin B (LatB) (Cayman, USA) was stocked in DMSO in a concentration of 25 mg/ml and the final used concentration was 0.1 g/ml. The vacuole dye 7-amino-4-chloromethylcoumarin (CMAC) was stocked in DMSO in a concentration of 1 mM and the final used concentration was 5 M (Cell Tracker Blue CMAC was purchased from MedChemExpress, Monmouth Junction, NJ, USA; Cat# HY-D1462). Typically, three-dimensional imaging capture image stacks that consisted of 0.5 mm-spaced sections d. Image processing and figure preparation was performed using Bitplane Imaris for 3D surface rendering, and Adobe Photoshop，Zen and Microsoft Excel (for figure preparation).

Phosphorylation analysis through Phos-tag gel and in vitro phosphorylation analysis.
The MoIre1-GFP, MoPh1-GFP and MoPh1S226, 230A-GFP were transformed into different strains (the wild-type Guy11, Modbf2, and Moph1 mutant). The proteins extracted from mycelium stage (with or without DTT treatment) were resolved on 8% SDS-polyacrylamide gels prepared with 50 µM acrylamide-dependent Phos-tag ligand and 100 µM MnCl as described before28, 89. Gel electrophoresis was run at a constant voltage of 80 V for 3–6 h. Before transferring, gels were equilibrated in transfer buffer with 5 mM EDTA for 20 min 2 times and followed with another transfer buffer without EDTA for 20 min. Protein transferred from the Mn2+-phos-tagTM acrylamide (NARD institute Limited company, 18D-01) gel to the PVDF membrane by the semi-dry transfer system (Beijing Hongtao Jiye Technology Development Co., Ltd, TRANS-BLOT TURBO, HT1000TBT) for 15 min and then the membrane was analyzed by western blotting using anti-GFP antibody. For the in vitro phosphorylation, the fusion proteins GST-MoPh1, GST-MoPh1S226, 230A (S226, 230A) and His-MoDbf2 were expressed in E. coli BL21-CodonPlus (DE3) cells (Sigma, CMC0014) and purified as described before3. Then the purified proteins were used for protein kinase reaction in the presence of 50 μM APTP in a kinase reaction buffer and then dyed with Pro-Q® Diamond Phosphorylation Gel Stain95 as described in our previous studies3.

Mass-spectrometry analysis.
To identify putative interacting patterns of MoPh1, proteins were extracted from strains cultured in ample liquid CM condition with or without ER stress [2.5 mM DTT for both 0.5, 1 hour (short time), 3, 5 hours (long time)], and then purified by Co-IP assay described previously. The elution was detected and then used for MS analysis. To identify phosphorylation sites of targeted proteins, samples were separated on 10% SDS-PAGE. The gel bands corresponding to the targeted protein were excised from the gel, reduced with 10 mM of DTT and alkylated with 55 mM iodoacetamide (Sigma-Aldrich, I1149). Subsequent steps for MS analysis were carried according to previous studies28. To investigate putative PM proteins that are involved in ER stress response, a quantitative membrane proteomics analysis was done under ER stress (DTT+TM co-treatment), nonstress CM condition was used as control. Total membrane of the fungus was extracted from lyophilized M. oryzae with the Minute™ Plasma Membrane Protein Isolation Kit for Plants (Invent Biotechnologies, INC., SM-005-P). Filter-aided sample preparation (FASP digestion) procedure was done as before96. A total of 3 biological replicates were analyzed for the Control and Treatment groups. Protein digestion was performed according to the ：Trypsin (Promega) was added at a 1:50 (w/w) ratio and incubated at 37°C overnight. The peptides were desalted using MCX columns (Omicsolution) and concentrated. The peptide content was estimated by UV light spectral density at 280 nm and for Data Independent Acquisition (DIA) experiments, the indexed retention time calibration peptides were spiked into the sample. The peptides from each sample were analyzed by OrbitrapTM AstralTM mass spectrometer (Thermo Scientific) connected to an Vanquish Neo system liquid chromatography (Thermo Scientific) in the DIA mode. Precursor Ions were scanned at a mass range of 380-980m/z，MS1 resolution was 240,000 at 200 m/z, normalized AGC target: 500%，maximum IT: 5 ms. 299 windows were set for DIA mode in MS2 scanning, isolation window: 2 m/z, HCD collision energy: 25ev, normalized AGC target: 500%, maximum IT: 3 ms. Then, DIA data was analyzed with DIA-NN 1.8.1. The search was performed against the uniprotkb_Pyricularia_oryzae_53206_2024_06_07.fasta database. Main software parameters were set as follows: enzyme is trypsin, max missed cleavages is 1, fixed modification is carbamidomethyl (C), dynamic modification is oxidation(M) and acetyl (Protein N-term). All reported data were based on 99% confidence for protein identification as determined by false discovery rate (FDR) ≤ 1%. Statistical significance was evaluated using Student’s t-test. This experiment was conducted with technical support from Zoonbio Biotechnology.

Southern Blot Hybridization.
Southern blot hybridization was performed to confirm the successful generation of all mutants used in this study. The DNA fragments were amplified using the 2× Hieff Canace® Plus PCR Master Mix (Cat No. 10154ES; Yeasen, Shanghai, China). Genomic DNA was digested with restriction endonucleases and the resulting fragments were separated by agarose gel electrophoresis. Following denaturation, the DNA fragments were transferred in situ from the gel onto a nylon membrane. The membrane was then immobilized by baking or UV crosslinking and hybridized with structurally specific, DIG-labeled probes. Finally, target DNA molecules were detected via an enzyme-based colorimetric reaction.This experiment was conducted with technical support from Shanghai Applied Protein Technology.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study elucidates that in Magnaporthe oryzae, PM-localized sensor MoPh1 detects ER stress through ER-PM contact sites and mediates an autophagic pathway from PM to vacuoles, operating independently of the canonical ER-to-nucleus signaling pathway. This pathway regulates autophagosome formation via phosphorylation modifications, promotes ER stress mitigation, and influences fungal pathogenicity. The innovation resides in the first identification of the autonomous role of PM sensors in ER stress response, transcending the conventional paradigm centered on the ER membrane protein Ire1. Functional validation of plant homologous proteins unveils the cross-species conservation of this pathway, providing novel evidence for the unified stress response mechanisms between fungi and plants. The manuscription is enjoyable to read, however, several concerns need to be raised here.\nFirst, line 122\"the Plasma membrane protein MoPh1 inhibition...\" make me confused, as the function of Ph1 is not enzyme or sth. like, why the author used inhibition?\nAlso, as a very long story here, several results should be carefully arranged, such as the results about the classical ER-to-nucleus response pathway, is there any sensor among this pathway? in my opinion UPR pathway is inherently distinct from endoplasmic reticulum autophagy. Maybe some disussion should be provided to the different role of Ph1-mediated response and Ire-mediated UPR upon various signal upstream.\nSpecific Recommendations:\n1. Fig.4A Localization clarification: Where is Tcb specifically localized in Fig.4A? Please provide explicit labeling.\n2. Quantitative analysis: Fig.1A/D and Fig.2B lack quantitative assessments of phosphorylation results. Statistical analysis (e.g., grayscale quantification) should be supplemented.\n3. Language refinement (Line 91):\nReplace \"leads to\" with \"resulting in\" and replace \"every year\" with \"annually\" for formal tone\n4. Sentence restructuring (Line 142): Original: \"When further examination...\" → Revised: \"Upon further examination under varying levels of ER stress, it was found that MoPh1 localized to the PM in the absence of ER stress.\"\n5. Figure adjustment (Fig.S4B): The statistical chart appears partially obscured during typesetting. Although this does not affect conclusions, layout optimizationis recommended to ensure full data visibility.", "reviewer_2": "Endoplasmic reticulum (ER) stress is an internal stress that is induced when protein folding or perturbations occur at excess rates. In this manuscript, a novel plasma membrane sensor, MoPh1, was observed to perceive stress through ER-PM contact sites. MoPh1 interacts with Atg11 to target the autophagosome, and interacts with Ypt7 to vacuole, Tcb1 helps MoPh1 to detach from PM under ER stress, and MoDbf2 kinase phosphorylates MoPh1 for the detachment. The novel PM-to-vacuole pathway mediated by MoPh1 is independent of the classical ER-to-nucleus pathway. The sight that a plasma membrane localized sensor could perceive ER stress is novel to the field. However, there are some key points to clarify.\nWhat is the definition of a plasma membrane sensor, what kind of signal is sensed by MoPh1 was not mentioned here.\nThe relationship of Ire1 and MoPh1 is not fully clear here. As in Fig 2B and Fig 4J, it seems that deletion of MoPh1 does not interfere with the phosphorylation of Ire1, and block of Ire1 using inhibitors (GSK and STF) does not interfere with the MoPh1 localization. However, in Figure 4L-4N, the number of Ire clusters were largely decreased in the deletion mutant △moph1, suggesting that detailed observation was required to interpretate the relationship of Ire1 and MoPh1. Therefore, a deletion mutant of MoIre1 is necessary to test whether the localization and function of MoPh1 is different.\nThe author tested different aspects of MoPh1, it interacts with Atg11 and Ypt7 for autophagy formation and vacuole localization, and Tcb1 and MoDbf2 kinase for the detachment of PM. However, the relationship of those different interaction was not clear. For example, in the absent of Tcb1 and Dbf2, MoPh1 could not detach from PM to endosomes, then how is the phosphorylation stage of MoPh1 here in the PM, is the phosphorylation took place earlier than the targeting to Tcb1 for detachment?\nAnd some minor points:\nFigure 1, DTT and Mo infection could induce ER stress, including HAC1 slicing and UPR gene expression, these were already reported in Yin et al., 2020, Autophagy, Shedding light on autophagy coordinating with cell wall integrity signaling to govern pathogenicity of Magnaporthe oryzae. So it is not new here.\nThe phosphorylation of Ire1 is increased after DTT or TM treatment and in appressoria, here it is better to calculate the ratio of P-Ire1 versus total Ire1 (P-Ire1 and U-Ire1), which is similar to the intensity of sHAC1 versus total HAC1.\nFigure2, here the 2L and 2M were not mentioned in the text. And the figure order should follow the main text. It is not easy to find the correct figure here.\nLine 175, in the main text, it mentioned that the deletion of MoPh1 diminishes the interaction of Atg11-Atg9, but Figure S6 legends (line 764) says that “the in vivo co-immunoprecipitation (co-IP) assay validated the interaction between Atg11-Atg9 was significantly enhanced in ΔMoph1 mutant under ER stress”. So I got confused about the effect of Atg11-Atg9 interaction with or without MoPh1.", "reviewer_3": "The manuscript submitted by Yin et al. presents an intriguing study on the role of the plasma membrane (PM) system in response to endoplasmic reticulum (ER) stress. The authors have identified a novel PM sensor in Magnaporthe oryzae, MoPh1, which senses stress via ER-PM contact sites and subsequently triggers the autophagy process. This finding is significant as it unveils a PM-to-vacuole pathway that operates independently of the classical ER-to-nucleus stress response pathway.\nHowever, a major revision is required before it is accepted to publish.There are a few aspects that could be further clarified or expanded unpon to strengthen the manuscript, here are some suggestions.\nMajor concerns:\n1.Combining DTT and TM as ER stressors harbors the risk of mechanistic ambiguity and misinterpretation of the data. While both agents activate canonical UPR sensors (e.g., Ire1 phosphorylation and Hac1/XBP1 splicing), their distinct modes of action- DTT disrupts disulfide bond formation by reducing the oxidative folding environment, whereas TM inhibits N-linked glycosylation-create overlapping but mechanistically divergent stress signals. This combinatorial approach risks conflating stressor-specific adaptations with universal UPR responses. Previous evidences have established by single-stressor models (e.g., DTT or TM alone), so a comparative analysis of individual stressors, followed by intersectional validation of overlapping candidates, is more methodologically rigorous and biologically interpretable.\n2.According to the figure legends in Fig1, plants were treated with DTT, TM and 4-PBA before being infected, then should consider what happened in the plant itself after treatment. If it was my misunderstanding, then the authors claim that both ER stressors (DTT, TM) and the ER stress inhibitor 4-PBA suppress M. oryzae infection, but the mechanistic basis for this seemingly paradoxical observation remains insufficiently clarified. If DTT/TM inhibit infection by over-activating UPR, which minght disrupt effector secretion or appressorium function), then 4-PBA, acting as a chemical chaperone should theoretically “rescue” the effect by alleviating ER stress, more details should be provided. Meanwhile, it’s better to clarify whether these treatments disrupt appressorium formation, which is a key virulence determinant.\n3. The results section lacks sufficient detail and specificity, which severely limits the interpretability of the findings. E.g. in lines 134-135, the authors claim that deletion of MoPH1 impairs the ER stress response but based on the data provided, seems more selectively affected Hac1 splicing without markedly affecting Ire1 activation (which consistent with the description in lines 240-242). Addressing how ΔMoph1 impacts the formation of the appressorium is equally important.\n4. The authors propose that ER stress signals are transmitted via MoTcb1 to PM-localized MoPh1, thereby trigger autophagic activation, leading to the vacuolar degradation of MoPh1through Atg8/Atg11-mediated trafficking. However, the model raises a critical paradox: if MoPh1 is progressively degraded in vacuolar under increasing ER stress, how can the PM-vacuole signaling axis be sustained over time? The unidirectional trafficking of MoPh1 (PM → vacuole) without recycling implies a self-limiting pathway. If the degradation of MoPh1 is irreversible, its PM pool would rapidly deplete, thereby terminating downstream autophagy.\n5. As for the MoPh1-MoTcb1 interaction at the PM-ER contact sites, the transient nature of this association is not yet clear. If MoTcb1-PM binding occurs only during early ER stress (when MoPh1 is PM-enriched), but diminishes as MoPh1 internalizes, this suggests a stress-phase-dependent interaction. The authors need to first, define the temporal window of MoPh1-MoTcb1 interaction using FRAP (fluorescence recovery after photobleaching) or time-lapse BiFC (bimolecular fluorescence complementation); also, map the dynamics of Tcb1 localization: Does MoTcb1 accumulate at ER-PM contact sites (marked by VAP/Syx proteins) as stress progresses, or does it distribute alongside MoPh1 in autophagosomes? Super-resolution imaging (STED or SIM) could clarify this. Furthermore, the claim that MoPh1-MoTcb1 binding occurs at ER-PM junctions lacks ultrastructural validation (e.g., TEM with immunogold labeling). Critically, if MoTcb1 itself relocalizes to autophagic compartments under prolonged stress, this could decouple it from MoPh1 trafficking, explaining the loss of PM interaction. Quantitative colocalization analysis of MoTcb1 with ER/PM/autophagosome markers across stress time points is necessary.\n6. I believe it would be more appropriate to rearrange the main text of the manuscript into at least three parts: introduction, results and discussion, which could possess a clearer structure of the manuscript. And the experimental data needs to be reorganized to make it more logical.\nSome more specific comments:\n1.Please confirm the null mutants created in this manuscript with a Southern blot.\n2.The authors have presented data on the changes in protein levels at various nodes. However, a critical aspect that requires improvement is the lack of quantitative analysis for these changes. Specifically, while the authors have analyzed the splicing isoform ratio of Hac1, the phosphorylation status of Ire1 remains unaddressed. It is essential to provide a comprehensive quantitative assessment of all relevant protein modifications and levels. Moreover, it would be better to measure the relative gray value of the bands for the ratio analysis instead of signal intensity showed in Figure1 and Figure 2.\n3.The authors should provide a more detailed description of the materials and methods used in this study, including, when and how to treat the conidia with each inhibitors for fungal viability testing. Also, some of the reagents lack of source information.\n4.Please ensure consistency between the figure legends and the images. Like, Figure S3 (there’re no “P” and “U” on the images ) and Fig. S6 (What’s the meaning of “T”, “S”, “E” in the pictures). Additionally, the figure legends should clearly specify the concentrations of reagents and the time periods employed in each experiment.\n5.The title of Figure S2 needs revision for accuracy. Additionally, the data presented on hyphal localization are unclear and should be improved for better interpretation.\n6.The quality of some micrograph images presented is not suitable for publication. DIC images are essential for assessing hyphal health and the context of fluorescent signal. And some ambiguous arrows and line charts hinder data interpretation. Colocalization can be quantified via Pearson’s correlation coefficient or Manders’ overlap coefficient, and provided methodological details (background correction, thresholding).\n7.The statistical analysis for Figure 2 C, D should be carefully reviewed to ensure accuracy and reliability.\n8.The nomenclature of the MoTcb1 and its mutant in Figure 4 A-D is inappropriate.\n9.Latin scientific names should be italicized. (line 128-129 in supp.)"}, "author_response": "Point-by-point response to reviewers' comments\nReviewer 1:\n1. First, line 122\"the Plasma membrane protein MoPh1 inhibition...\" make me\nconfused, as the function of Ph1 is not enzyme or sth. like, why the author used\ninhibition?\nResponse: Thank you for your comment. Our description here was not precise and we\nhave revised in the revised version to more clearly state that we identified MoPh1 as a\nplasma membrane protein involved in stress response. (Line 186-187 in the tracked\nversion)\n2. Also, as a very long story here, several results should be carefully arranged, such\nas the results about the classical ER-to-nucleus response pathway, is there any sensor\namong this pathway? in my opinion UPR pathway is inherently distinct from\nendoplasmic reticulum autophagy. Maybe some disussion should be provided to the\ndifferent role of Ph1-mediated response and Ire-mediated UPR upon various signal\nupstream.\nResponse: We sincerely thank you for this valuable suggestion. In the revised\nmanuscript, we have expanded our discussion on classical response pathways and\ntheir key sensor proteins in both the Introduction and Discussion sections.\nFurthermore, we have obtained and analyzed a knockout mutant of Ire1, who encoded\nthe most critical classical sensor in Magnaporthe oryzae, providing evidence to\ndelineate the relationship between the classical UPR pathway mediated by Ire1 and\nthe pathway governed by the plasma membrane-localized sensor MoPh1.\nSpecific Recommendations:\n1. Fig.4A Localization clarification: Where is Tcb specifically localized in Fig.4A?\nPlease provide explicit labeling.\nResponse: Thank you for the comment. In the revised version of the manuscript, we\nhave now included a detailed observation of the subcellular localization of MoTcb1 as\nsuggested. Localization tracking revealed that MoTcb1 localized to the PM with\npunctate aggregation under normal conditions, whereas the puncta-MoTcb1 on the\nPM were significantly enriched near the ER-PM contact sites and partially co-\nlocalized with them under ER stress, indicating that MoTcb1 could localize to the ER-\nPM contact sites during stress conditions. Concurrently, a fraction of MoTcb1\ninternalized and colocalized with autophagosomes and vacuoles under stress\nconditions. The relevant content has been described in Lines 328-334 of the revised\nmanuscript according to Figure 5B and Supplementary Figure S15A.\n2. Quantitative analysis: Fig.1A/D and Fig.2B lack quantitative assessments of\nphosphorylation results. Statistical analysis (e.g., grayscale quantification) should be\nsupplemented.\nResponse: Thank you for your helpful suggestions. In the revised manuscript, we have\nnow incorporated grayscale quantification and phosphorylation ratio analysis for all\nMoIre1-related results across the relevant figures (Figure 1A and 1D, Figure 2B,\nSupplementary Figure S5A).\n3. Language refinement (Line 91):Replace \"leads to\" with \"resulting in\" and replace\n\"every year\" with \"annually\" for formal tone\n4. Sentence restructuring (Line 142): Original: \"When further examination...\" →\nRevised: \"Upon further examination under varying levels of ER stress, it was found\nthat MoPh1 localized to the PM in the absence of ER stress.\"\n5. Figure adjustment (Fig.S4B): The statistical chart appears partially obscured\nduring typesetting. Although this does not affect conclusions, layout optimizationis\nrecommended to ensure full data visibility.\nResponse: Thank you for the constructive suggestions. All the points raised above\nhave been addressed and corresponding revisions have been incorporated into the\nrevised version of the manuscript.\nReviewer 2:\n1. What is the definition of a plasma membrane sensor, what kind of signal is sensed\nby MoPh1 was not mentioned here.\nResponse: Thanks for this insightful question. Through examination of nearly 20\nrecently published studies on sensor proteins (referenced behind), we note that while\nno universally rigid definition exists for stress sensors, proposed criteria for plasma\nmembrane (PM)-localized sensors generally include the following:\n(1) Subcellular localization to the PM;\n(2) Induced gene expression under the corresponding stress;\n(3) Post-translational modification or biochemical alteration (e.g., phosphorylation)\nupon stress perception;\n(4) Impaired stress perception or response in knockout mutants.\nBased on these criteria, MoPh1 satisfies the requirements of a potential ER stress\nsensor at the PM: MoPh1 localizes to the PM, especially under no stress conditions;\nthe expression of MoPH1 is significantly upregulated under ER stress; MoPh1\nundergoes specific phosphorylation under ER stress, which is functionally linked to\nits stress responsiveness; the ΔMoph1 mutant exhibits significantly reduced viability\nunder ER stress conditions. Taken together, these lines of evidence support the\nconclusion that MoPh1 functions as a novel PM-localized sensor for ER stress in M.\noryzae.\nSome of the latest relevant reports are as follows:\n①Hall, A. E., Lisci, M., & Rose, M. D. (2021). Differential Requirement for the Cell Wall\nIntegrity Sensor Wsc1p in Diploids Versus Haploids. Journal of fungi (Basel, Switzerland), 7(12),\n1049. https://doi.org/10.3390/jof7121049\n②Wang, Y., Wang, J., Zhang, J., Wang, Y., Wang, Y., Kang, H., Zhao, W., Bai, W., Miao, N., &\nWang, J. (2024). Stiffness sensing via Piezo1 enhances macrophage efferocytosis and promotes\nthe resolution of liver fibrosis. Science advances, 10(23), eadj3289.\nhttps://doi.org/10.1126/sciadv.adj3289\n③Woo, S. H., Lukacs, V., de Nooij, J. C., Zaytseva, D., Criddle, C. R., Francisco, A., Jessell, T.\nM., Wilkinson, K. A., & Patapoutian, A. (2015). Piezo2 is the principal mechanotransduction\nchannel for proprioception. Nature neuroscience, 18(12), 1756–1762.\nhttps://doi.org/10.1038/nn.4162\n④Liu, X., Wang, J., & Sun, L. (2018). Structure of the hyperosmolality-gated calcium-permeable\nchannel OSCA1.2. Nature communications, 9(1), 5060. https://doi.org/10.1038/s41467-018-\n07564-5\n⑤Zheng, W., Rawson, S., Shen, Z., Tamilselvan, E., Smith, H. E., Halford, J., Shen, C., Murthy, S.\nE., Ulbrich, M. H., Sotomayor, M., Fu, T. M., & Holt, J. R. (2023). TMEM63 proteins function as\nmonomeric high-threshold mechanosensitive ion channels. Neuron, 111(20), 3195–3210.e7.\nhttps://doi.org/10.1016/j.neuron.2023.07.006\n⑥Yao, J., Liu, B., & Qin, F. (2011). Modular thermal sensors in temperature-gated transient\nreceptor potential (TRP) channels. Proceedings of the National Academy of Sciences of the United\nStates of America, 108(27), 11109–11114. https://doi.org/10.1073/pnas.1105196108\n⑦Yin, Y., Park, C. G., Zhang, F., G Fedor, J., Feng, S., Suo, Y., Im, W., & Lee, S. Y. (2024).\nMechanisms of sensory adaptation and inhibition of the cold and menthol receptor TRPM8.\nScience advances, 10(31), eadp2211. https://doi.org/10.1126/sciadv.adp2211\n⑧Kimura, T., Tsuchida-Mayama, T., Imai, H., Okajima, K., Ito, K., & Sakai, T. (2020).\nArabidopsis ROOT PHOTOTROPISM2 Is a Light-Dependent Dynamic Modulator of\nPhototropin1. The Plant cell, 32(6), 2004–2019. https://doi.org/10.1105/tpc.19.00926IF: 11.6 Q1\n⑨Solis, A. G., Bielecki, P., Steach, H. R., Sharma, L., Harman, C. C. D., Yun, S., de Zoete, M. R.,\nWarnock, J. N., To, S. D. F., York, A. G., Mack, M., Schwartz, M. A., Dela Cruz, C. S., Palm, N.\nW., Jackson, R., & Flavell, R. A. (2019). Mechanosensation of cyclical force by PIEZO1 is\nessential for innate immunity. Nature, 573(7772), 69–74. https://doi.org/10.1038/s41586-019-\n1485-8\n⑩He, Y., Yu, Y., Wang, X., Qin, Y., Su, C., & Wang, L. (2022). Aschoff's rule on circadian\nrhythms orchestrated by blue light sensor CRY2 and clock component PRR9. Nature\ncommunications, 13(1), 5869. https://doi.org/10.1038/s41467-022-33568-3\n2. The relationship of Ire1 and MoPh1 is not fully clear here. As in Fig 2B and Fig 4J,\nit seems that deletion of MoPh1 does not interfere with the phosphorylation of Ire1,\nand block of Ire1 using inhibitors (GSK and STF) does not interfere with the MoPh1\nlocalization. However, in Figure 4L-4N, the number of Ire clusters were largely\ndecreased in the deletion mutant moph1, suggesting that detailed observation was\nrequired to interpretate the relationship of Ire1 and MoPh1. Therefore, a deletion\nmutant of MoIre1 is necessary to test whether the localization and function of MoPh1\nis different.\nResponse: Thank you for this constructive comment. First, our data found that while\nthe deletion of MoPH1 does not affect the phosphorylation of the ER sensor protein\nMoIre1, it significantly impairs the formation of MoIre1 phase-separated condensates\nunder ER stress. Sequence analysis reveals that the protein kinase domain (residues\n849-1147 aa) of MoIre1 governs its phosphorylation, whereas its intrinsically\ndisordered region (IDR, residues 641-825 aa) primarily regulates phase separation,\nwhich make it reasonable that MoPh1 modulates the phase separation of MoIre1\nwithout altering its phosphorylation status. Second, following the reviewer's\nsuggestion, we examined the potential direct interaction between MoIre1 and MoPh1,\nand found that a specific direct interaction between MoIre1 and MoPh1, and notably,\nthe intrinsically disordered region (IDR) of MoIre1 is essential for this interaction.\nThird, we attempted to generate a MoIRE1 knockout mutant in the M. oryzae KU80\nbackground, a strain widely used as a wild-type control that enhances gene targeting\nefficiency without affecting pathogenic development (reference cited). After\nscreening over 5,000 transformants, we successfully obtained a ΔMoIre1 mutant.\nPhenotypic characterization revealed that the ΔMoIre1 mutant exhibited significantly\nenhanced sensitivity to ER stress, accompanied by strongly impaired splicing of Hac1\nmRNA and reduced expression of downstream UPR target genes. Pathogenicity\nassays further confirmed severely attenuated virulence in the ΔMoIre1 mutant,\ndemonstrating that Ire1 functions as a conserved ER stress sensor essential for both\nstress adaptation and pathogenicity in M. oryzae. Notably, in the ΔMoIre1 mutant, the\nsubcellular localization of MoPh1 under ER stress remained unaffected. Moreover,\nthe stress-induced interactions between MoPh1 and MoTcb1, as well as MoPh1 and\nMoAtg11, together with the phosphorylation status of MoPh1, were all maintained\nnormally in the absence of MoIre1. Based on these evidences collectively, we\nconclude that MoPh1-mediated ER stress perception and downstream signaling are\nindependent of the classical sensor MoIre1. Instead, MoPh1 promotes the formation\nof MoIre1-based phase-separated condensates under ER stress through specific\ninteraction with its intrinsically disordered region (IDR), thereby potentiating the\nclassical UPR pathway. The relevant content has been shown in Figure 1L, Figure 6A\nand 6C and 6H-6K and Supplementary Figure S20B and Figure S21 in the tracked\nversion.\nrelevant reports\n①Liu, W. et al. Multiple plant surface signals are sensed by different mechanisms in the rice blast\nfungus for appressorium formation. PLoS Pathog 7, e1001261 (2011).\n②Villalba, F. et al. Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80\nrequired for non-homologous end joining. Fungal Genet Biol 45, 68-75 (2008).\n3. The author tested different aspects of MoPh1, it interacts with Atg11 and Ypt7 for\nautophagy formation and vacuole localization, and Tcb1 and MoDbf2 kinase for the\ndetachment of PM. However, the relationship of those different interaction was not\nclear. For example, in the absent of Tcb1 and Dbf2, MoPh1 could not detach from PM\nto endosomes, then how is the phosphorylation stage of MoPh1 here in the PM, is the\nphosphorylation took place earlier than the targeting to Tcb1 for detachment?\nResponse: We sincerely thanks for your insightful question and valuable suggestion.\nTo clarify the functional relationships among the proteins that interact with MoPh1 at\ndistinct stages (during stress-induced dissociation from the plasma membrane and\nsubsequent targeting to autophagosomes), we have performed the following additional\nexperiments based on our previous results:\n∙ Measuring the phosphorylation level of MoPh1 in the absence of\nMoTcb1;( Supplementary Figure S19A in the tracked version)\n∙ Assessing the interaction between MoPh1 and MoTcb1 in the absence of MoDbf2;\n( Supplementary Figure S19B in the tracked version)\n∙ Examining the interaction between MoPh1 and MoYpt7 in the ΔMoatg11 mutant;\n( Supplementary Figure S14A in the tracked version)\n∙ Evaluating the interaction between MoPh1 and MoAtg11 in the ΔMoypt7 mutant.\n( Supplementary Figure S14B in the tracked version)\nIntegrating these new data with previous findings, we found that deletion of\nMoDbf2 does not affect the MoTcb1-MoPh1 interaction under ER stress, whereas loss\nof MoTcb1 impairs MoPh1 phosphorylation by MoDbf2, indicating that although\nboth MoTcb1 and MoDbf2 are required for MoPh1 dissociation from the plasma\nmembrane, the MoTcb1-MoPh1 interaction occurs prior to MoDbf2-mediated\nphosphorylation of MoPh1. Moreover, loss of MoAtg11 disrupts the MoPh1-MoYpt7\ninteraction. In contrast, deletion of MoYpt7 has no evident effect on the MoPh1-\nMoAtg11 interaction, suggesting that the MoPh1-MoAtg11 interaction likely precedes\nMoPh1-MoYpt7 binding, and that the former is necessary for the latter. The relevant\ncontent has been described in Lines 298-306 and Lines 375-386 of the revised\nmanuscript\n4. And some minor points:\nFigure 1, DTT and Mo infection could induce ER stress, including HAC1 slicing and\nUPR gene expression, these were already reported in Yin et al., 2020, Autophagy,\nShedding light on autophagy coordinating with cell wall integrity signaling to govern\npathogenicity of Magnaporthe oryzae. So it is not new here.\nThe phosphorylation of Ire1 is increased after DTT or TM treatment and in\nappressoria, here it is better to calculate the ratio of P-Ire1 versus total Ire1 (P-Ire1\nand U-Ire1), which is similar to the intensity of sHAC1 versus total HAC1.\nFigure2, here the 2L and 2M were not mentioned in the text. And the figure order\nshould follow the main text. It is not easy to find the correct figure here.\nLine 175, in the main text, it mentioned that the deletion of MoPh1 diminishes the\ninteraction of Atg11-Atg9, but Figure S6 legends (line 764) says that “the in vivo co-\nimmunoprecipitation (co-IP) assay validated the interaction between Atg11-Atg9\nwas significantly enhanced in ΔMoph1 mutant under ER stress”. So I got confused about\nthe effect of Atg11-Atg9 interaction with or without MoPh1.\nResponse: Thank you for the thorough reading and valuable suggestions regarding the\ndetails of our manuscript. In response, we have carefully revised both the figures and\nthe main text as recommended. The specific modifications are outlined below:\n(1) Although a previous study by Yin et al., 2020, Autophagy has reported some of the\nDTT and Mo infection could induce ER stress in the wild-type M. oryzae including\nHAC1 slicing and UPR gene expression, we still showed these results together with\nother ER stress response phenotypes mainly based on two aspects of considerations:\nFirst, to enable a comprehensive comparison of ER stress responses between the wild-\ntype strain and ΔMoph1 mutant, we present a complete set of ER stress phenotypic\ndata in the wild-type background, including not only the previously reported\nMoHAC1 splicing and UPR target gene expression, but also MoIre1 phosphorylation\nlevel and tunicamycin (TM) treatment results, which were not presented\nbefore. Second, while earlier studies reported general ER stress accumulation during\ninfection, our current analysis provides higher temporal resolution by examining\nstress responses at specific time points during appressorium development. This\napproach more accurately captures the dynamic fluctuations of ER stress and enables\nprecise correlation with the stage-specific subcellular localization of MoPh1,\nthereby strengthening the functional connection between stress dynamics and MoPh1 behavior.\n(2) Following the reviewer's suggestion, we have quantified the phosphorylation\nlevels of Ire1 across relevant figures by calculating its phosphorylation ratios,\nsimilar to the approach used for Hac1 splicing analysis. This normalization provides a\nmore accurate and comparable quantification of MoIre1 phosphorylation dynamics under\ndifferent experimental conditions. (Figure 1A and 1D, Figure 2B, Supplementary\nFigure S5A in the tracked version)\n(3) As suggested by the reviewer, we have repositioned the statistical data on disease\nlesion area and fungal biomass measurement for the ΔMoph1 mutant to directly\nfollow the pathogenicity assay results in the relevant figure. Corresponding\ndescriptions and citations have been updated in the main text to reflect this change.\n(Line 219-223 in the tracked version, Supplementary Figure S7-S7B)\n(4) Our results found that the interaction between MoAtg9 and MoAtg11 is altered in\nthe MoPH1 deletion mutant, showing a significant increase in their binding strength\nunder ER stress (Supplementary Figure S10 in the tracked version). We have\ncorrected the description of this finding in the revised manuscript, as the initial\nversion inaccurately characterized this interaction. It was found that just since this\naberrant enhancement of the MoAtg9-MoAtg11 interaction in the ΔMoph1 mutant\ndisrupts the normal dynamic punctate localization of MoAtg9 under ER stress\nconditions, MoAtg9 predominantly accumulates at autophagosomes instead of\nforming dynamic puncta (Figure 4J-4L in the tracked version).\nReviewer 3 :\nMajor concerns：\n1.Combining DTT and TM as ER stressors harbors the risk of mechanistic ambiguity\nand misinterpretation of the data. While both agents activate canonical UPR sensors\n(e.g., Ire1 phosphorylation and Hac1/XBP1 splicing), their distinct modes of action-\nDTT disrupts disulfide bond formation by reducing the oxidative folding environment,\nwhereas TM inhibits N-linked glycosylation-create overlapping but mechanistically\ndivergent stress signals. This combinatorial approach risks conflating stressor-\nspecific adaptations with universal UPR responses. Previous evidences have\nestablished by single-stressor models (e.g., DTT or TM alone), so a comparative\nanalysis of individual stressors, followed by intersectional validation of overlapping\ncandidates, is more methodologically rigorous and biologically interpretable.\nResponse: Thank you for the valuable suggestion. We agree with the reviewer that\nDTT and TM, while differing in their modes of action, both elicit canonical ER stress\nresponses, leading to similar outcomes such as enhanced Ire1 phosphorylation and\nincreased Hac1 splicing. Although simultaneous application of both stressors may\nintroduce some confounding effects, our primary goal was to identify potential plasma\nmembrane-localized sensors from a broad spectrum of stress-responsive factors. The\ncombined stress approach was intended to increase the likelihood of capturing such\nregulators, and fortunately, we successfully identified MoPh1 as a key PM-localized\nregulatory factor. Considering that re-performing the proteomic analysis under\nindividual stress conditions might introduce substantial variability across biological\nreplicates and the phenotypic validations conducted in subsequent experiments\nincluded separate assessments using both DTT and TM, we will follow the reviewer's\nsuggestion to optimize stress treatment conditions in future screening and validation\nexperiments and retain the original experimental data in this study.\n2.According to the figure legends in Fig1, plants were treated with DTT, TM and 4-\nPBA before being infected, then should consider what happened in the plant itself\nafter treatment. If it was my misunderstanding, then the authors claim that both ER\nstressors (DTT, TM) and the ER stress inhibitor 4-PBA suppress M. oryzae infection,\nbut the mechanistic basis for this seemingly paradoxical observation remains\ninsufficiently clarified. If DTT/TM inhibit infection by over-activating UPR, which\nminght disrupt effector secretion or appressorium function), then 4-PBA, acting as a\nchemical chaperone should theoretically “rescue” the effect by alleviating ER stress,\nmore details should be provided. Meanwhile, it’s better to clarify whether these\ntreatments disrupt appressorium formation, which is a key virulence determinant.\nResponse: Thank you for the valuable comments. We have carefully reviewed the\nfigure legends and agree that the original descriptions were unclear. The ER stress-\ninducing agents (DTT and TM) and inhibitor (4-PBA) were applied directly to\nMagnaporthe oryzae to create conditions of hyper-stimulated or suppressed ER stress\nin the pathogen, not to the rice plants. The conidia were treated for 10 minutes before\nbeing inoculated onto rice leaves. We have revised the relevant figure legends to\naccurately reflect this experimental procedure. (Line 1123-1128 in the tracked\nversion).\nIn addition, following the reviewer's suggestion, we have included new data on\nappressorium formation rates and turgor pressure of M. oryzae under different\ntreatment conditions which are conducted on artificial interfaces without plant-related\nfactors (Supplementary Figure S1A-S1C in the tracked version). These results further\nsupport the importance of ER stress homeostasis during pathogenic development.\nConsistent with the pathogenicity assays, both excessive ER stress induction and\nsuppression significantly reduced appressorium formation and impaired turgor\npressure generation.\n3. The results section lacks sufficient detail and specificity, which severely limits the\ninterpretability of the findings. E.g. in lines 134-135, the authors claim that deletion\nof MoPH1 impairs the ER stress response but based on the data provided, seems more\nselectively affected Hac1 splicing without markedly affecting Ire1 activation (which\nconsistent with the description in lines 240-242). Addressing how ΔMoph1 impacts\nthe formation of the appressorium is equally important.\nResponse: We sincerely thank you for the constructive suggestions. To improve the\nprecision and clarity of the results, we have thoroughly restructured the manuscript\naccording to the standard scientific format, with clear separation among the\nIntroduction, Results, and Discussion sections. Regarding the ER stress response\ndefects in the ΔMoph1 mutant, we have provided a more detailed description: while\nthe phosphorylation level of the key ER stress sensor MoIre1 remains largely\nunchanged in the mutant, both the splicing of MoHAC1 and the expression of\ndownstream UPR target genes are significantly impaired (Line 206-211 in the tracked\nversion). Taking together with the subsequent findings, we conclude that MoPh1\nregulates the activity of the classical ER stress response pathway by modulating the\nformation of MoIre1 phase-separated condensates, which in turn affects the efficiency\nof Hac1 splicing.\nFurthermore, in both the Results and Discussion sections, we have elaborated on\nthe mechanism by which MoPh1 influences appressorium function. During\nappressorium development and host infection, M. oryzae accumulates ER stress and\nthe PM sensor MoPh1 perceives stress signals through its interaction with MoTcb1 at\nER-PM contact sites. Following phosphorylation, MoPh1 dissociates from the PM\nand targets autophagosomes, where it interacts with the autophagy scaffold protein\nMoAtg11 and the late endosomal protein MoYpt7, in order to promote\nautophagosome formation and subsequent fusion with the vacuolar membrane,\nthereby facilitating autophagy to alleviate ER stress. Since autophagy is essential for\nappressorium turgor generation and host invasion, this process represents a key\nmechanism through which MoPh1 regulates pathogenicity. Additionally, given that\nMoPh1 also modulates components of the classical ER stress response pathway, it\nmay further influence appressorium function through these parallel regulatory\nmechanisms.\nTherefore, MoPh1 coordinates appressorium function under stress not only by\ninitiating autophagy to facilitate turgor pressure, but may also through regulating\nappressorium-development genes via the classical stress response pathway.\n4. The authors propose that ER stress signals are transmitted via MoTcb1 to PM-\nlocalized MoPh1, thereby trigger autophagic activation, leading to the vacuolar\ndegradation of MoPh1through Atg8/Atg11-mediated trafficking. However, the model\nraises a critical paradox: if MoPh1 is progressively degraded in vacuolar under\nincreasing ER stress, how can the PM-vacuole signaling axis be sustained over time?\nThe unidirectional trafficking of MoPh1 (PM → vacuole) without recycling implies a\nself-limiting pathway. If the degradation of MoPh1 is irreversible, its PM pool would\nrapidly deplete, thereby terminating downstream autophagy.\nResponse: Thank you for raising this important question regarding the sustainability\nof the MoPh1-mediated PM-to-vacuole pathway. Based on our current data, we\npropose the following model to explain how this process is maintained:\nFirst, the MoPh1-mediated stress perception mechanism, involving ER\nremodeling, proximity to the PM, and signal transmission via MoTcb1, appears to be\ntemporally restricted rather than constitutive. Consistent with this, morphological\nanalyses show that ER structural changes and ER-PM contacts are predominantly\nformed under brief stress, whereas prolonged stress restores the regular ring-like ER\narchitecture (Figure 5A in the tracked version). Similarly, the MoTcb1-MoPh1\ninteraction is only detected under short-term ER stress (Figure 5C-5D in the tracked\nversion). Together, these results indicate that ER-PM proximity and subsequent\nMoTcb1-MoPh1 signaling represent a transient, early stress response.\nSecond, during the natural time course of appressorium development, MoPh1\nexhibits dynamic localization: while it largely dissociates from the PM and\ninternalizes at 8 hours post-induction, it retains partial PM localization at other time\npoints (Figure 3B and 3E in the tracked version). This spatiotemporal distribution\nsuggests that the pathogen maintains a balance between PM-localized and internalized\npools of MoPh1, allowing flexible adaptation to fluctuating stress conditions and\nensuring continuity of the PM-to-vacuole pathway.\nWe also acknowledge the reviewer's intriguing hypothesis that a subpopulation\nof MoPh1 might recycle back to the PM after facilitating autophagy. However,\nwhether such recycling occurs, and through what mechanisms, involving which\nmodifications or interacting partners, remains an open question and represents an\nimportant direction for our future research.\n5. As for the MoPh1-MoTcb1 interaction at the PM-ER contact sites, the transient\nnature of this association is not yet clear. If MoTcb1-PM binding occurs only during\nearly ER stress (when MoPh1 is PM-enriched), but diminishes as MoPh1 internalizes,\nthis suggests a stress-phase-dependent interaction. The authors need to first, define\nthe temporal window of MoPh1-MoTcb1 interaction using FRAP (fluorescence\nrecovery after photobleaching) or time-lapse BiFC (bimolecular fluorescence\ncomplementation); also, map the dynamics of Tcb1 localization: Does MoTcb1\naccumulate at ER-PM contact sites (marked by VAP/Syx proteins) as stress progresses,\nor does it distribute alongside MoPh1 in autophagosomes? Super-resolution imaging\n(STED or SIM) could clarify this. Furthermore, the claim that MoPh1-MoTcb1\nbinding occurs at ER-PM junctions lacks ultrastructural validation (e.g., TEM with\nimmunogold labeling). Critically, if MoTcb1 itself relocalizes to autophagic\ncompartments under prolonged stress, this could decouple it from MoPh1 trafficking,\nexplaining the loss of PM interaction. Quantitative colocalization analysis of MoTcb1\nwith ER/PM/autophagosome markers across stress time points is necessary.\nResponse: Thank you for your valuable suggestion. To clarify the spatial and dynamic\ndetails of the MoPh1-MoTcb1 interaction, we performed co-localization studies using\nthe ER membrane protein MoSec61 as an ER marker, FM4-64 to label the PM, and\nMoAtg8 as a marker for autophagosomes and vacuoles. In the absence of ER stress,\nthe ER exhibited a regular, continuous ring-like structure and remained largely\nseparate from the PM. Under ER stress conditions, however, the ER displayed not\nonly the ring-like form but also a more dispersed distribution, with a significantly\nincreased frequency of close apposition to the PM (Figure 5A in the tracked version).\nThese observations suggest that M. oryzae enhances ER-PM contact during ER stress,\npotentially facilitating inter-organellar signaling. Notably, following prolonged ER\nstress, the proportion of dispersed ER structures in close proximity to the PM\ngradually decreased, while the regular ring-like ER morphology became increasingly\npredominant (Figure 5A in the tracked version). This dynamic redistribution suggests\nthat the stress-induced ER remodeling and its close association with the PM represent\na transient signaling phase rather than a sustained state.\nSubcellular localization analysis of MoTcb1 revealed that under non-stress\nconditions, it resides predominantly at the PM with occasional punctate formations\n(Supplementary Figure S15A in the tracked version). Upon ER stress induction, a\nsubset of MoTcb1 retained punctate PM localization, which was significantly\nenriched at sites where the ER closely apposes the PM and colocalized with known\nER-PM contact site markers (Figure 5B in the tracked version), indicating that\nMoTcb1 is specifically localized to ER-PM contact sites. Concurrently, a fraction of\nMoTcb1 internalized and colocalized with autophagosomes and vacuoles under stress\nconditions (Supplementary Figure S15A in the tracked version).\nHowever, interaction analysis between MoTcb1 and MoPh1 showed that their\nbinding occurred exclusively under short-term ER stress, appearing as puncta at the\nPM that closely matched the distribution of ER-PM contact sites. No interaction was\ndetected in the absence of stress or after prolonged ER stress exposure (Figure 5C-5D\nin the tracked version). These findings indicate that although both proteins can\ninternalize under ER stress and exhibit overlapping intracellular localization, their\ninteraction is restricted to the early phase of stress response when ER-PM proximity is\nenhanced. This is consistent with our observation that prolonged ER stress leads to a\nreduction in dispersed ER structures closely associated with the PM (Figure 5A in the\ntracked version).\nAdditionally, as suggested by the reviewer, we constructed and observed the\nsubcellular localization of the VAP protein (MGG_06183) in M. oryzae. We found\nthat it primarily localizes to ER-associated structures. Although there might be partial\nco-localize with the ER-PM contact sites under ER stress, this signal was difficult to\ncapture clearly and was therefore not included in the manuscript.\n6. I believe it would be more appropriate to rearrange the main text of the manuscript\ninto at least three parts: introduction, results and discussion, which could possess a\nclearer structure of the manuscript. And the experimental data needs to be\nreorganized to make it more logical.\nResponse: Thank you for your constructive suggestions. In response, we have\nthoroughly reorganized the manuscript following the standard structure of\nIntroduction, Results, and Discussion. Additionally, we have optimized the sequence\nin which results are presented to enhance the logical flow and coherence of the study.\nSome more specific comments:\n1.Please confirm the null mutants created in this manuscript with a Southern blot.\nResponse: Thanks for this suggestion. We have now included Southern blot analysis\nfor Moph1, Modbf2, Motcb1, Moatg11, Moypt7, and the newly\ngenerated Moire1 mutant in Supplementary Figure S26 in the tracked version.\n2.The authors have presented data on the changes in protein levels at various nodes.\nHowever, a critical aspect that requires improvement is the lack of quantitative\nanalysis for these changes. Specifically, while the authors have analyzed the splicing\nisoform ratio of Hac1, the phosphorylation status of Ire1 remains unaddressed. It is\nessential to provide a comprehensive quantitative assessment of all relevant protein\nmodifications and levels. Moreover, it would be better to measure the relative gray\nvalue of the bands for the ratio analysis instead of signal intensity showed in Figure1\nand Figure 2.\nResponse: Thank you for this valuable suggestion. To more clearly and quantitatively\npresent the phosphorylation status of different proteins, we have now included ratio\nanalysis for Ire1 phosphorylation, which allows readers to better visualize the extent\nof changes (Figure 1A and 1D, Figure 2B, Supplementary Figure S5A in the tracked\nversion). Regarding the Hac1 splicing data in Figure 1 and Figure 2, we would like to\nclarify that the previous version already displayed the relative ratio of different Hac1\nsplicing isoforms rather than raw band intensities, but the labeling in the figures was\ninaccurate. We have now corrected these labels as suggested in the revised version.\n3.The authors should provide a more detailed description of the materials and\nmethods used in this study, including, when and how to treat the conidia with each\ninhibitors for fungal viability testing. Also, some of the reagents lack of source\ninformation.\nResponse: Thanks for this suggestion. In response, we have expanded the Materials\nand Methods section to include detailed information on the reagents used.\nAdditionally, we have provided more comprehensive descriptions of key experimental\nprocedures, such as the treatment of fungal conidia with inhibitors.\n4.Please ensure consistency between the figure legends and the images. Like, Figure\nS3 (there’re no “P” and “U” on the images ) and Fig. S6 (What’s the meaning of “T”,\n“S”, “E” in the pictures). Additionally, the figure legends should clearly specify the\nconcentrations of reagents and the time periods employed in each experiment.\nResponse: Thank you for this comment. We have now revised the figure legends in\nboth the main and supplementary figures to include detailed information such as\nreagent concentrations and treatment durations for each experiment.\n5.The title of Figure S2 needs revision for accuracy. Additionally, the data presented\non hyphal localization are unclear and should be improved for better interpretation.\nResponse: Thanks a lot for this comment. We have rechecked and revised the title of\nFigure S2 to \"The localization of two PH domain-containing proteins under different\nlevels of ER stress.\" Additionally, to improve image quality, we have repeated the\nlocalization experiments for both proteins, reacquired the images, and replaced the\ncorresponding panels in the figure (Supplementary Figure S4 in the tracked version).\n6.The quality of some micrograph images presented is not suitable for publication.\nDIC images are essential for assessing hyphal health and the context of fluorescent\nsignal. And some ambiguous arrows and line charts hinder data interpretation.\nColocalization can be quantified via Pearson’s correlation coefficient or Manders’\noverlap coefficient, and provided methodological details (background correction,\nthresholding).\nResponse: Thank you for your valuable suggestions. We have carefully re-examined\nthe quality of all micrographs and replaced or adjusted those with suboptimal clarity.\nAdditionally, we now provide corresponding DIC (differential interference contrast)\nimages for all fluorescence micrographs, with some included in the supplementary\nmaterials due to space constraints in the main figures (Supplementary Figure S27A-\nS27H). Furthermore, we have optimized the presentation of arrows and line graphs in\nthe figures and incorporated additional statistical analyses where appropriate.\n7.The statistical analysis for Figure 2 C, D should be carefully reviewed to ensure\naccuracy and reliability.\n8.The nomenclature of the MoTcb1 and its mutant in Figure 4 A-D is inappropriate.\n9.Latin scientific names should be italicized. (line 128-129 in supp.)\nResponse: Thank you for raising these concerns. The specific figures involved have\nbeen amended to correct the improper annotations, the modes of representation, and\nthe use of italics for certain Latin names.\nPoint-by-point response to reviewers' comments\n1. Subcellular colocalization of MoPh1 and MoIre1 presented in Figure 6H. Please\nprovide additional evidence for colocalization. We request additional images to show\nreproducibility and higher resolution. Please include quantitative measures of\ncolocalization.\nResponse: Thank you for your valuable suggestions. Following your advice, we re-\nexamined the co-localization of MoIre1 and MoPh1 under ER stress and captured\nhigher-resolution images. The results confirmed a high degree of co-localization\nunder ER stress conditions induced by both DTT and TM, which was supported by\nboth quantitative analysis of co-localization points and fluorescence intensity\nmeasurements. We have updated Figure 6H with higher-quality images and further\nincluded statistical analysis of co-localization percentages as well as fluorescence\nintensity curve analysis.\n2. Apparent discrepancy between Figure 6H and Figure 3A. The localization of\nMoPh1 after DTT/TM treatment is shown in both figures. The localization of Ph1 at\nthe surface in control conditions followed by internalization after treatment is clear in\nfigure 3A but not Figure 6H. After TM treatment in particular the expression of Ph1\nappears restricted to a very small area which is not the case in Figure 3A. Please\nconsider repeating this experiment and providing additional, higher quality images.\nResponse: Thank you for pointing out this issue. We have repeated the experiment\ncorresponding to Figure 6H and have provided clearer images to ensure the\nconsistency and reliability of the results."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have carefully addressed all previous comments, and the revisions have substantially improved the clarity and quality of the work. All concerns have been adequately resolved, and I find the current version suitable for publication.", "reviewer_3": "The authors answered all my quesions and I am happy to suggest to accept the manuscript be published in NC."}, "author_response": ""}]

Decision letter