1. Microbiology and Infectious Disease
  2. Plant Biology
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A self-balancing circuit centered on MoOsm1 kinase governs adaptive responses to host-derived ROS in Magnaporthe oryzae

  1. Xinyu Liu
  2. Qikun Zhou
  3. Ziqian Guo
  4. Peng Liu
  5. Lingbo Shen
  6. Ning Chai
  7. Bin Qian
  8. Yongchao Cai
  9. Wenya Wang
  10. Ziyi Yin
  11. Haifeng Zhang
  12. Xiaobo Zheng
  13. Zhengguang Zhang  Is a corresponding author
  1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, China
  2. The Key Laboratory of Plant Immunity, Nanjing Agricultural University, China
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Cite this article as: eLife 2020;9:e61605 doi: 10.7554/eLife.61605

Abstract

The production of reactive oxygen species (ROS) is a ubiquitous defense response in plants. Adapted pathogens evolved mechanisms to counteract the deleterious effects of host-derived ROS and promote infection. How plant pathogens regulate this elaborate response against ROS burst remains unclear. Using the rice blast fungus Magnaporthe oryzae, we uncovered a self-balancing circuit controlling response to ROS in planta and virulence. During infection, ROS induces phosphorylation of the high osmolarity glycerol pathway kinase MoOsm1 and its nuclear translocation. There, MoOsm1 phosphorylates transcription factor MoAtf1 and dissociates MoAtf1-MoTup1 complex. This releases MoTup1-mediated transcriptional repression on oxidoreduction-pathway genes and activates the transcription of MoPtp1/2 protein phosphatases. In turn, MoPtp1/2 dephosphorylate MoOsm1, restoring the circuit to its initial state. Balanced interactions among proteins centered on MoOsm1 provide a means to counter host-derived ROS. Our findings thereby reveal new insights into how M. oryzae utilizes a phosphor-regulatory circuitry to face plant immunity during infection.

Introduction

During co-evolution with pathogens, plants have developed an innate immune system by sensing pathogen-associated molecular patterns (PAMPs), such as flagellin and chitin produced, respectively, by bacteria and fungi (Jones and Dangl, 2006; Liu et al., 2014). Upon recognition, pattern-recognition receptors (PRRs) transduce the signals to the downstream components that initiate the immune response (Boutrot and Zipfel, 2017; Zipfel, 2014), which was referred to as PAMP-triggered immunity (PTI) (Boutrot and Zipfel, 2017; Jones and Dangl, 2006). This PRR to PTI processes often involves components of MAP kinase signaling pathways, callose deposition, ROS burst, and pathogenesis related (PR) gene expression.

The NADPH oxidase-mediated production of ROS is one of the earliest PTI responses restricting pathogen invasion. In Arabidopsis thaliana, phosphorylated BIK1 activates the NADPH oxidase RBOHD through protein phosphorylation to trigger ROS burst. In rice (Oryza sativa), the LysM domain of the chitin-elicitor binding protein (CEBiP) binds to chitin and associates with the bifunctional plant receptor OsCERK1 that phosphorylates the downstream receptor-like cytoplasmic kinase 185 (OsRLCK185) for the activation of PTI by causing the ROS burst (Yamaguchi et al., 2013). In addition, Rac1, the Rac/ROP small G-protein, which interacts with a defense-related NADPH oxidase RbohB that response to chitin elicitor for ROS production (Nagano et al., 2016).

During host infection, pathogens secrete numerous antimicrobial proteins, including superoxide dismutase, catalases, and peroxidases, to evade host immunity (Kawasaki et al., 1997; Lanfranco et al., 2005; Molina and Kahmann, 2007). In the corn smut fungus Ustilago maydis, the Protein Essential During Penetration 1 (Pep1) protein directly interferes with ROS generation by inhibiting peroxidase activities (Hemetsberger et al., 2012). In the tomato leaf mold fungus Cladosporium fulvum, the LysM domain-containing Effector Extracellular Protein 6 (Ecp6) circumvents chitin-induced immunity by sequestering host chitin oligomers (de Jonge et al., 2010). Similarly, M. oryzae secreted LysM Protein 1 (MoSlp1) competes with OsCEBiP for chitin binding, thereby preventing the activation of rice PTI (Chen et al., 2014; Mentlak et al., 2012). Recently, the rice tetratricopeptide repeat protein OsTPR1 was shown to interact with M. oryzae chitinase MoChia1 in the apoplast. In addition, the competitive binding of OsTPR1 by MoChia1 allows the accumulation of free chitin to reestablish the host immune response (Yang et al., 2019).

M. oryzae causes rice blast and is also a hemibiotrophic fungus in need of host nutrients for propagation (Wilson and Talbot, 2009; Zhang et al., 2016). How M. oryzae response to host-derived signals to circumvent plant immunity during infection remain a much-debated question. In M. oryzae and other fungal pathogens, G-protein/cAMP signaling plays an important role in the perception of host surface cues (Choi and Dean, 1997; Liu et al., 2007). The non-canonical G-protein coupled receptor (GPCR) Pth11 that functions upstream of G-protein/cAMP signaling is also important for surface perception in M. oryzae (DeZwaan et al., 1999; Kou et al., 2017). A previous study identified that the sensor kinase protein MoSln1 functions to sense glycerol and facilitates host penetration of M. oryzae (Ryder et al., 2019). In spite of any ROS receptor remaining to be identified, M. oryzae is known to contain several conserved MAP kinase pathways, including MoMst11-MoMst7-MoPmk1, MoMck1-MoMkk1-MoMps1, and MoSsk2-MoPbs2-MoOsm1 in conferring signal transduction during infection (Yin et al., 2016; Zhang et al., 2016). Among them, the Hog1 homolog, MoOsm1, which mediated the osmoregulation pathway is essential for the response to hyperosmotic stress through transcription factor MoMsn2 (Dixon et al., 1999; Zhang et al., 2014). Additional studies also found that the osmoregulation pathway is important for the response to oxidative species and resistance to fungicides (Kim et al., 2009).

Previous studies demonstrated that the bZIP transcription factor MoAp1 is important in response to oxidative stress by activating a suite of antioxidant genes during ROS stress (Guo et al., 2011), and MoAtf1 is also important in response to hyperosmotic stress and ROS stress (Guo et al., 2010). The ΔMoatf1 mutant was hypersensitive to oxidative stress, exhibited the reduced expression of several extracellular peroxidase and laccase genes, and failed to suppress the accumulation of ROS around the infection sites (Guo et al., 2010). To understand how M. oryzae responds to the ROS-mediated stress and triggered the downstream signaling pathway for ROS tolerance, we sought upstream to identify kinase which regulates MoAtf1 in response to ROS stress. We found that host-derived ROS induces the MoOsm1-mediated MAPK pathway to activate MoAtf1 phosphorylation. In addition, we identified phosphorylated MoAtf1 initiates the transcription of MoPTP1/MoPTP2 under ROS stress which function on the dephosphorylation of MoOsm1. The process of MoOsm1/MoPtps-mediated phosphor-regulatory feedback loop function as a switch which not only enhanced virulence of M. oryzae under ROS stress but also control the virulence that keep the rice cells alive during hemibiotrophic growth.

Results

M. oryzae infection induces ROS accumulation in rice

During M. oryzae infection, the pathogen and rice interaction results in either disease or host immunity. In infection of rice cultivar LTH by M. oryzae wild-type strain Guy11, the sequences of various developmental stages are as follows: primary hyphae to appressorium differentiation (<20 hpi), the penetration of epidermis (20 hpi), formation of the bulbous infection hyphae (IH) inside the host cell (24 hpi), and spreading into the neighboring cells (36 hpi) for further infection (48–72 hpi). In a moderate resistance cultivar-strain interaction, such as between K23 and Guy11, few and restricted lesions were present (Liu et al., 2018; Yin et al., 2020). The hyphae grew poorly in the leaf-sheath cells (24 and 36 hpi) and were restricted to the primary infected cells at 48 hpi until eventual spreading into adjacent cells (60 and 72 hpi) (Figure 1A).

Time-course images of ROS accumulation during rice sheath infection by M. oryzae.

(A) The conidial suspension of Guy11 (1 × 105 spores/ml) was inoculated in the excised rice sheath of 4-weeks-old rice seedlings LTH and K23. The invasive hyphae growth was observed at 20, 24, 36, 48, 60, and 72 hpi. Black asterisks represent the bulbous infection hyphae (IH). (B) DAB staining shows ROS accumulation in rice LTH and K23 cells at various time points following infection. Black asterisks represent infected cells without ROS and white asterisks represent cells stained by DAB. (C) Infected cells stained by DAB. Over 50 infected rice cells were calculated with three replicates each time. ‘%” represents to the rate of infected cells which stained by DAB in all infected cells. Three independent biological experiments were performed and yielded similar results. Error bars represent standard deviation, and asterisks represent significant differences between the different strains (p<0.01).

We used DAB staining to estimate ROS accumulation in response to M. oryzae infection. Rice cultivar K23 infected with Guy11 yielded reddish-brown precipitates around the appressoria and infected hypha at 24, 36, 48, and 60 hpi. Over 40% of the infected cells were stained brown at 24 hpi and/or 36 hpi. When observation was made at 60 hpi, nearly 10% of infected cells were still filled with ROS. In contrast, the accumulation of ROS was barely detectable in the susceptible LTH cultivar infected by Guy11. The rate of cells stained with DAB was no more than 20% at 24, 36, and 48 hpi (Figure 1B and C). These results indicated that rice elaborates ROS as a barrier to infection as early as 24 hpi, and scavenging of host ROS may be necessary for further expansion of M. oryzae during infection.

MoOsm1 phosphorylation in response to oxidative stress

MoOsm1 is an essential component of the osmoregulation pathway, and a previous study indicated that the deletion of the MoOSM1 gene resulted in hypersensitivity to oxidative stress (Dixon et al., 1999). To understand this MoOsm1-mediated ROS response, we constructed the strain expressing MoOsm1-GFP, in which the expression of the C-terminal GFP fusion protein is under the control of the native MoOSM1 promoter, and we tested phosphorylation of MoOsm1 using rice seedlings of both compatible pair (Guy11 and LTH) and the relative resistance pair (K23 and LTH). Total proteins were extracted at 0, 8, 20, 24, 48, and 72 hpi and the proteins bound to the anti-GFP beads were eluted and analyzed by anti-p38 MAPK (Figureure 2A and 2B). The results showed that the phosphorylation of MoOsm1 reached high levels at 24 and 48 hpi before dropping at 72 hpi in the K23 and Guy11 pair (Figureure 2B), but not in the LTH and Guy11 pair (Figureure 2A). Given the time of 24 and 48 hpi correlates with ROS levels, we speculated that ROS burst might have a role in MoOsm1 phosphorylation. When K23 was treated with 0.5 μM diphenyleneiodonium (DPI) that inhibits the activity of plant NADPH oxidases and thereby ROS (Bolwell et al., 1998; Grant et al., 2000; Zhang et al., 2009), MoOsm1 phosphorylation was significantly reduced at 24 hpi and 36 hpi (Figureure 2C). OsRbohA, an important NADPH oxidase, is critical for the ROS generation in rice, and OsRbohA-overexpressing transgenic plants exhibited higher ROS production (Wang et al., 2016). To further understand the function of MoOsm1 in response to host-produced ROS, we detected the phosphorylation level of MoOsm1 on the OsRbohA-ox line during the infection. The results showed that the phosphorylation of MoOsm1 was induced in the OsRbohA-ox line compared with the NPB lines (Figure 2D). To confirm that ROS levels were correlated with MoOsm1 phosphorylation, an in vitro assay was carried out, showing a similar result. When Guy11 was treated with 5 mM H2O2 for 0, 10, 30, and 60 min, an enhanced phosphorylated MoOsm1 was observed at 10 min (Figure 2—figure supplement 1A).

Figure 2 with 1 supplement see all
MoOsm1 phosphorylation and nuclear translocation in response to ROS stress.

(A) Phosphorylation of MoOsm1 in Guy11 in infection of LTH. Total proteins were extracted from LTH leaves 0, 8, 20, 24, 48, and 72 hr. Eluted proteins bound to the anti-GFP beads were analyzed by the antiphospho-p38 antibody, with the p38 antibody used as a control. The extent of phosphorylation was estimated by calculating the amount of antiphospho-p38 compared to the p38 (the histogram underneath the blot). Error bars represent standard deviation. (B) Phosphorylation of MoOsm1 in Guy11 in infection of K23. MoOsm1 phosphorylation was induced at 24 and 48 hpi. Error bars represent standard deviation, and asterisks represent significant differences between the different strains. Values are the means of 3 replications, and error bars represent the SD (n = 3). The asterisks indicate a significant difference (Duncan's new multiple range test, p<0.01). (C) DPI treatment in rice decreases the phosphorylation levels of MoOsm1 during infection. Total proteins were extracted from K23 leaves 0, 24, 48, and 72 hr with (+) or without (−) DPI treatment. MoOsm1 was purified and analyzed by the antiphospho-p38 antibody, with the p38 antibody used as a control. (D) Overexpression of OsRbohA induced the phosphorylation of MoOsm1. Total proteins were extracted from OsRbohA-ox leaves at 0, 48 and 72 hpi. MoOsm1 was purified and analyzed by the antiphospho-p38 antibody. NPB was used as the CK. R-ox represents OsRbohA-ox lines. (E) Localization of MoOsm1 under oxidative stress. Fluorescence observation of conidia treated with H2O2 for 10, 30, and 60 min. MoOsm1-GFP and H1-RFP were observed by confocal fluorescence microscopy. Bars = 5 μm. (F) Fluorescence intensity of MoOsm1-GFP/H1-RFP was observed with 10 min H2O2 treatment and CK. The green line represents MoOsm1-GFP while the red line represents H1-RFP. Insets highlight areas analyzed by line-scan. The number represents the quantification of GFP and RFP signals by ImageJ. (G) An equal amount (8 × 106 spore/ml x 60 ml) of conidia (with 10 min H2O2 treatment or not) were divided into three parts for extraction of the total, nuclear, and cytoplasm proteins. Equal amounts of the total, nuclear and cytoplasm proteins were separated by SDS-PAGE, and MoOsm1 was detected by western blotting using the anti-GFP antibody. Bands of MoOsm1-GFP were detected at 65kD. The intensity of western blotting bands was quantified with the ODYSSEY infrared imaging system (application software Version 2.1). The intensity of MoOsm1 was compared between the conidia without treatment (-) and conidia with 10 min of H2O2 treatment (+) among total proteins, nuclear proteins, and cytoplasmic proteins. H1 (a nucleus marker) and actin (a cytoplasm marker) were detected by western blotting analysis. Bars denote standard errors from three independent experiments. Asterisks indicate significant differences (Duncan's new multiple range test p<0.01).

In addition, we observed the localization of MoOsm1 in response to oxidative stress. Under normal conditions, MoOsm1 was equally present in both the cytoplasm and the nucleus in conidium and mycelium. Following treatment with 5 mM H2O2 for 10 min, an enhanced nuclear localization pattern was observed in conidia (76.32 ± 17.83%) and hypha (67.48 ± 19.33) (Figure 2E and S1B). We also fused a nuclear export signal (NES) sequence to MoOsm1 and found that 5 mM H2O2 did not exhibit any significant effect to the location of MoOsm1NES-GFP (Figure S1C). When H2O2 treatment was extended to 30 min, the localization of MoOsm1 recovered to the default distribution pattern (Figure 2E, Figure 2—figure supplement 1B). When a red fluorescent protein (RFP) was fused to histone H1 marking the nucleus, an enrichment of MoOsm1 in the nucleus following H2O2 treatment for 10 min was clearly visualized (Figure 2F). We also performed western blotting analysis using extracted nuclear proteins and found MoOsm1-GFP was significantly enriched in the nucleus compared with wild type upon 10 min H2O2 treatment (Figure 2G). These results suggested that MoOsm1 responds to oxidative stress by accumulating in the nucleus.

MoOsm1 undergoes dimer to monomer transition under oxidative stress-induced phosphorylation

As H2O2 induces phosphorylation and nuclear localization of MoOsm1, we hypothesized that the phosphorylation is relevant to its localization. To test this, we first identified the oxidation stress-dependent phosphorylation site of MoOsm1. We purified the MoOsm1-GFP protein from the ΔMoosm1/MoOSM1-GFP strain that was treated with H2O2 for 10 min and found that threonine (T) 171 and tyrosine (Y) 173 were the corresponding phosphorylation sites (Figure 3—figure supplement 1A) through mass spectrometry analysis. We then generated a constitutively activated phosphomimetic mutation of MoOsm1Y173D and an inactivated mutation of MoOsm1Y173A as a validation step. MoOsm1-GFP, MoOsm1Y173D-GFP, and MoOsm1Y173A-GFP were expressed in the ΔMoosm1 mutant, and the localization was observed using a fluorescence microscope. The results showed that the phosphomimetic mutation of MoOsm1Y173D had more nuclear accumulation relative to MoOsm1 and MoOsm1Y173A (Figures 3A, B and C). These findings indicated that the phosphorylation of MoOsm1 tyrosine 173 is important for its nuclear accumulation.

Figure 3 with 1 supplement see all
Phosphorylation-mediated monomerization of MoOsm1 results in its nuclear accumulation.

(A) Localization of MoOsm1, MoOsm1Y173D, and MoOsm1Y173A in conidia. MoOsm1-GFP, MoOsm1Y173D-GFP, MoOsm1Y173A-GFP, and H1-RFP were observed by confocal fluorescence microscopy. Bars = 5 μm. (B) Nuclear/cytoplasmic proteins harvested from conidia of ∆Moosm1/MoOsm1-GFP, ∆Moosm1/MoOsm1Y173D-GFP, and ∆Moosm1/MoOsm1Y173A-GFP strains were separated by SDS-PAGE. The intensity of MoOsm1 was compared among total proteins, nuclear proteins, and cytoplasmic proteins. ‘Y’ represents MoOsm1, ‘D’ represents MoOsm1Y173D, and ‘A’ represents MoOsm1Y173A. Bars denote standard errors from three independent experiments. Asterisks indicate significant differences (Duncan's new multiple range test p<0.01). (C) Fluorescence intensity of MoOsm1-GFP/H1-RFP in ∆Moosm1/MoOsm1-GFP, ∆Moosm1/MoOsm1Y173A-GFP, and ∆Moosm1/MoOsm1Y173D-GFP strains. The number represents the quantification of GFP and RFP signals by ImageJ. (D) Co-IP assay. Western blot analysis of total proteins (T) extracted from various transformants, suspension proteins (S), and elution proteins (E) eluted from anti-GFP beads. MoOsm1-GFP, MoOsm1Y173D-GFP, MoOsm1Y173A-GFP, and MoOsm1-FLAG were detected with respective antibodies. (E) Immunoblot analysis of proteins extracted from MoOsm1-GFP/∆Moosm1, MoOsm1Y173A-GFP/∆Moosm1, and MoOsm1Y173D-GFP/∆Moosm1 strains. (F) Gel-filtration chromatography assay of MoOsm1 dimerization. MoOsm1 was separated by the AKTA protein purification system (GE healthcare). Four protein peaks (I, II, III, and IV) were detected by western blot analysis. The brown line represents the ion peak. (G) BiFC assays for MoOsm1-nYFP and cYFP-MoOsm1 interactions. Conidia were treated with H2O2. MoOsm1Y173A-nYFP and cYFP-MoOsm1Y173A were used as control. (H) In vivo phosphorylation analysis of MoOsm1. Nuclear and cytoplasmic MoOsm1 phosphorylation was analyzed by Mn2+-Phos-tag SDS-PAGE and normal SDS-PAGE, respectively. Total proteins treated with alkaline phosphatase (phosphatase) and phosphatase inhibitor (inhibitor) were used as control.

As a previous study suggested that the p38 MAPK kinase forms dimers with swapped activation segments (Rothweiler et al., 2011), suggesting that MoOsm1 may also undergo changes in dimerization. To test this via a co-immunoprecipitation (co-IP) approach, we co-introduced the MoOSM1-FLAG, MoOSM1-GFP, and the point-mutation constructs into the protoplasts of Guy11. Total proteins were extracted from the transformants, and MoOsm1 was detected using the anti-FLAG and anti-GFP antibodies. In proteins eluted from MoOsm1 and MoOsm1 Y173A anti-GFP beads, MoOsm1-FLAG was also detected. However, when co-introduced MoOSM1 Y173D-FLAG and MoOSM1Y173D-GFP, the interaction was not found (Figure 3D). In addition, the interaction between MoOsm1 and MoOsm1T171D and the localization of MoOsm1T171D was also detected (Figure 3—figure supplement 1B and C). Collectively, the results suggested that phosphorylation of tyrosine 173 but not threonine 171 inhibits interaction. Using the native-PAGE analysis, we found that MoOsm1 is present in the form of both monomers and dimers, while only monomers were detected in the MoOsm1Y173D strains (Figure 3E). We also expressed the His-MoOsm1 protein in vitro and purified it by AKTA pure (GE healthcare) with gel-filtration chromatography. We separated four putative peaks for further verification by western blot. The results showed that only peaks I and II were identified at 110 kd and 55 kd, suggesting that MoOsm1 form dimer (Figure 3F). The dimerization was further verified by the bimolecular fluorescence complementation (BiFC) assay. cYFP-MoOSM1 and MoOSM1-nYFP pair, cYFP-MoOSM1Y173A and MoOSM1Y173A -nYFP pair fusion constructs were co-introduced into Guy11 protoplasts and transformants obtained. The recombined YFP fluorescence signal was detected in the cytoplasm containing the corresponding protein pairs (Figure 3G). Moreover, upon H2O2 treatment for 10 and 15 min, the YFP signal of cYFP-MoOSM1 and MoOSM1-nYFP pair was reduced in the nucleus, in contrast to cYFP-MoOSM1 Y173A and MoOSM1Y173A-nYFP pair that showed the default localization pattern, suggesting that the monomeric form of MoOsm1 is involved in the nuclear localization under the oxidative stress (Figure 3G).

Phosphorylation of MoOsm1 was further evaluated using Phos-tag gel electrophoresis. Total extracts were treated with either phosphatase or phosphatase inhibitor (PI), and the mobility shift was examined by immunoblotting proteins with the anti-GFP antibody. The induced MoOsm1 mobility shift was found in the phosphatase treated wild-type cells, but not in the PI-treated cells. A similar band shift was observed in the extracts from the cytoplasm with the phosphatase treated strain. The decreased mobility of MoOsm1-GFP purified from the nucleus was exhibited compared to the phosphatase treated strain, indicating a higher level of MoOsm1 phosphorylation in the nucleus (Figure 3H). These results suggested that MoOsm1 could be phosphorylated and transferred into the nucleus.

MoOsm1 phosphorylates MoAtf1 in vivo and in vitro

The MAPK kinase signaling pathways regulate developmental processes by targeting various downstream transcription factors or target genes. Several putative transcription factors were proposed to function downstream of MoOsm1, including MoAtf1, MoAp1, and MoMsn2 (Li et al., 2012; Zhang et al., 2014). To understand the function of MoOsm1 phosphorylation and translocation, we first validated the interaction between MoOsm1 and MoAtf1 by co-IP. The MoAtf1-FLAG, MoOsm1-GFP, MoOsm1Y173D-GFP, MoOsm1Y173A-GFP, and MoOsm1NES-GFP fusion constructs were introduced into the protoplasts of Guy11, and proteins were extracted from the transformants. MoAtf1 and MoOsm1 were detected using the anti-FLAG and anti-GFP antibodies. In proteins eluted from anti-GFP beads, MoAtf1 was detected in the elution among MoOsm1, MoOsm1Y173D, and MoOsm1Y173A, but not MoOsm1NES (Figure 4A), indicating that MoOsm1 interacts with MoAtf1. The interaction was further confirmed by the BiFC assay. The recombined YFP fluorescence signal was detected among MoOsm1-MoAtf1, MoOsm1Y173D-MoAtf1, and MoOsm1Y173A-MoAtf1 in the nucleus in comparison with MoOsm1NES (Figure 4B), suggesting that nuclear localization is important for MoOsm1 and MoAtf1 interaction.

Figure 4 with 1 supplement see all
MoOsm1 phosphorylates MoAtf1 in the nucleus.

(A) Co-IP assay. Western blot analysis of total proteins (T) extracted from MoOsm1-GFP/MoAtf1-FLAG, MoOsm1Y173A-GFP/MoAtf1-FLAG, MoOsm1Y173D-GFP/MoAtf1-FLAG, MoOsm1NES-GFP/MoAtf1-FLAG strains, suspension proteins (S), and elution proteins (E) eluted from anti-GFP beads. The presence of MoAtf1 and MoOsm1 was detected with the anti-GFP and anti-FLAG antibodies, respectively. MoOsm1NES-GFP was used as a negative control. (B) BiFC assays for the interaction between MoOsm1 and MoAtf1. Strains expressed MoAtf1-nYFP and empty cYFP, cYFP-MoOsm1 and empty nYFP, MoAtf1-nYFP and MoOsm1NES-GFP were used as negative controls. Bars = 5 μm. (C) Phosphorylation of MoAtf1 by MoOsm1. MoAtf1-GFP proteins treated with alkaline phosphatase and phosphatase inhibitors (PIs) were separated by Mn2+-Phos-tag and normal SDS-PAGE, respectively, and detected by the GFP antibody. (D) The localization of MoAtf1 in ∆Moosm1 and ∆Moatf1 mutants. MoAtf1-GFP was introduced in the ∆Moosm1 and ∆Moatf1 mutants, and the localization was observed by confocal fluorescence microscopy. (E) A model of MoAtf1 and mutants’ constructs. White bars indicate the conserved bZIP domain and nuclear localization signal (NLS) domain. The black triangle indicates putative serine and threonine sites and the numbers indicate amino acid positions. (F) Identification of the phosphorylation sites of MoAtf1 by MoOsm1. In vivo phosphorylation analysis of MoAtf1 and the inactivation mutants (S to A and T to A). MoAtf1-GFP proteins treated with alkaline phosphatase (MoAtf1-), PIs (MoAtf1+), and the phosphorylation inactivation mutants treated with PIs (S29A+, S117A+, S124A+, S152A+, T161A+, S308A+, and S334A+) were separated by Mn2+-Phos-tag SDS-PAGE and normal SDS-PAGE, respectively. (G) Pathogenicity assay. Four milliliters of conidial suspension (5 × 104 spores/ml) of each strain were used for spraying on LTH and K23 and photographed 5 d after inoculation. (H) Diseased leaf area analysis. Data are presented as a bar chart showing the percentage of lesion area analyzed by Image J. Error bars represent SD and asterisks represent significant differences (‘**” represents p<0.01).

Given that the phosphorylated MoOsm1 is translocated into the nucleus under ROS stress and interacts with MoAtf1, MoOsm1 could phosphorylate MoAtf1. To test this hypothesis, we generated a MoATF1-GFP construct and introduced it into both Guy11 and the ΔMoosm1 mutant strain and then analyzed MoAtf1-GFP using Phos-tag gel electrophoresis. Total extracts were treated with either a phosphatase or a PI, and the mobility shift was examined by immunoblotting proteins with the anti-GFP antibody. The induced MoAtf1 mobility shift was observed in the phosphatase treated wild-type cells but not in the untreated or PI-treated cells. A similar band shift was not observed in the extracts from the untreated ΔMoosm1 mutant. The increased mobility of MoAtf1-GFP in the untreated ΔMoosm1 mutant compared to the untreated wild-type strain indicated a higher level of MoAtf1 phosphorylation in the wild-type strain (Figure 4C). These results suggested that MoOsm1 regulates MoAtf1 through protein phosphorylation.

When observing MoAtf1 localization, we found that its nuclear localization remains unchanged in the ΔMoosm1 mutant, indicating that MoOsm1-mediated MoAtf1 phosphorylation does not seem to have an effect on its localization (Figure 4D). To understand the underlying mechanism, we tested whether any serine/threonine located in the front of the nuclear localization signal was involved. We generated the point-mutation mutants (S29A, S117A, S124A, S152A, T161A, S308A, and S334A) corresponding to six S (Ser) and one T (Thr) residues in MoAtf1 (Figure 4E). In vivo phosphorylation analysis showed that the phosphorylation level of MoAtf1 was dampened in the S124A mutation strain but not in other mutants (Figure 4F). In addition, MoAtf1-GST, the constitutively unphosphorylated MoAtf1S124A-GST, and MoOsm1-His fusion proteins were also obtained and analyzed. The results showed a relatively low level of MoAtf1S124A phosphorylation (Figure 4—figure supplement 1). These results collectively suggested that MoOsm1 phosphorylates MoAtf1 on serine 124.

Since MoAtf1 contributes to the full virulence of M. oryzae, we questioned if this phosphorylation on serine 124 could also regulate the virulence of M. oryzae. We then introduced the constitutively expressed MoAtf1S124D and MoAtf1S124A into ΔMoatf1, respectively. Virulence testing showed no differences between wild type (Guy11) and MoAtf1S124D expression strains on LTH cultivar, while both ΔMoatf1 and ΔMoatf1/MoAtf1S124A mutants exhibited restricted lesions. On the K23 cultivar, however, the wild-type strain caused few typical lesions (gray spots with brown margins), in contrast to the MoAtf1S124D expression strains that caused even more lesions and also larger lesion areas (Figure 4G and H). This result demonstrated that phosphorylation of MoAtf1 on residue 124 is important for the virulence of M. oryzae on rice.

MoAtf1 phosphorylation disrupts its interaction with MoTup1 that affects the expression of oxidoreduction- pathway factors

During screening for MoAtf1-interacting proteins, we identified MoTup1, a previously characterized conserved transcription repressor (Figure 5—figure supplement 1Chen et al., 2015). We found that MoAtf1 interacts with MoTup1 in yeast two-hybrid (Y2H), pull down and co-IP assay (Figure 5—figure supplement 2A, B and C). We then constructed the MoAtf1S124D and MoAtf1S124A alleles to verify whether the phosphorylation of MoAtf1 abolishes its interaction with MoTup1. Indeed, compared with the MoAtf1 and MoAtf1S124A, MoAtf1S124D could not interact with MoTup1 (Figure 5—figure supplement 2A, B and C), indicating that the phosphorylation of MoAtf1 on S124 causes its disassociation from MoTup1.

We have also performed a genome-wide ChIP-Seq assay to evaluate whether MoAtf1 has a role in regulating any pathogenicity-related genes. We found that MoAtf1 binds to the upstream regions of 574 open reading frames (ORFs) (Supplementary file 1). Among them, 16 were involved in the oxidation-reduction process, including six containing the signal peptide (Figure 5). We then validated ChIP-Seq findings by electrophoretic mobility shift assays and qRT-PCR. The results showed that these genes were down-regulated in the ΔMoatf1 mutant (Figure 5 and Figure 5—figure supplement 3A) indicated that MoAtf1 positively regulates the oxidoreduction pathway.

Figure 5 with 3 supplements see all
MoAtf1 is one of the regulators of the oxidation regulatory pathway.

ChIP-Seq assays showed that MoAtf1 binds to the promoters of 16 oxidation regulation pathway genes. Output- and input-DNA was visualized in blue and pink. The red arrow indicates gene direction. The green bar represents the promoter region covered during ChIP-Sequencing. Blank in the black dashed line indicates genes encoding proteins containing signal peptides. The EMSA assay was performed to evaluate the relevant promoter of these genes binding with MoAtf1. The purified MoAtf1 was mixed with DNA, incubated for 20 min at 25°C in binding buffer, and separated by 1.5% agarose gel. The qRT-PCR assay showed the expression of these genes in Guy11 and ∆Moatf1/MoAtf1S124D. Three independent biological experiments were performed, with three replicates each time that yielded similar results. Error bars represent standard deviation, and asterisks represent significant differences between the different strains (p<0.01).

Since MoAtf1 interacts with MoTup1, it is hypothesized that disruption of the MoAtf1-MoTup1 complex would reverse the suppression of genes involved in the oxidoreduction pathway. The phosphorylation at S124 of MoAtf1 dissociates the interaction between MoAtf1 and MoTup1 (Figure 5—figure supplement 2). The results showed that 13 out of 16 genes were upregulated after 10 min H2O2 treatment in Guy11, and 12 of 16 genes were upregulated at 24 hpi during infection (Figure 5—figure supplement 3B and C). The further qRT-PCR assay showed that H2O2 responsive genes were also upregulated in the ∆Moatf1/MoAtf1S124D strain (Figure 5). Collectively, the results showed that host-derived ROS induces the phosphorylation of MoOsm1 (Figure 2), which in turn phosphorylates MoAtf1 and disengages the MoAtf1-MoTup1 complex leading to the transcriptional activation of MoAtf1-regulating genes.

MoPtp1/2 involved in the dephosphorylation of MoOsm1

As a hemibiotrophic fungus, M. oryzae needs to maintain host cells alive during the biotrophic stage. We found that the pathogen utilizes MoOsm1 phosphorylated MoAtf1 and, in turn, phosphorylated MoAtf1 dissociated with MoTup1 in responding to host immunity. We speculated that MoOsm1 regulation might also involve functions of protein phosphatases. Based on the finding that the budding yeast Saccharomyces cerevisiae protein phosphatases Ptps are involved in the dephosphorylation of Hog1, a homolog of MoOsm1 (Lee et al., 2014; Murakami et al., 2008), we characterized whether the Ptp homologs MoPtp1 and MoPtp2 have a role in dephosphorylating MoOsm1.

We first generated and verified the respective ΔMoptp1 and ΔMoptp2 mutant strains (Figure 6—figure supplement 1). We showed that the deletion of MoPTP1 and MoPTP2 exhibited no defects in the vegetative growth (Figure 6—figure supplement 2A and B). Further microscopic observations showed that conidiation was significantly reduced in the ΔMoptp2 mutants, but not in the ΔMoptp1 mutants (Figure 6—figure supplement 2C and D). To examine the role of MoPtp1/2 in virulence, we inoculated conidial suspensions of wild type, ∆Moptp1, ∆Moptp2, and the complemented strains on the susceptible rice cultivar CO-39. The ∆Moptp2 mutant caused small and restricted lesions, compared to the ∆Moptp1 mutant that was fully virulent 7 days post-inoculation (dpi) (Figure 6—figure supplement 3A and B). Given the possibility that MoPtp1 and MoPtp2 may have redundant functions, independent ∆Moptp1Moptp2 double mutants were also constructed, and the result indicated that the double mutant was mostly similar to ∆Moptp2 in phenotypes (Figure 6—figure supplement 3A and B).

We used Y2H and co-IP assay to examine the interaction between MoOsm1 and MoPtp1/2 (Figure 6—figure supplement 3C and D). To test whether MoPtp1/2 regulates the activity of MoOsm1 through protein dephosphorylation, Mn2+-Phos-tag SDS-PAGE was performed. We found that the band of MoOsm1-GFP in both ΔMoptp1 and ΔMoptp2 mutants migrate as slow as that of the phosphorylated MoOsm1-GFP protein treated with the PI. MoOsm1-GFP in the wild-type strain displayed a migration pattern similar to proteins treated with the phosphatase (Figure 6—figure supplement 3E). These findings suggested that both MoPtp1 and MoPtp2 could dephosphorylate MoOsm1.

We further purified the MoOsm1-GFP protein from the ΔMoptp2/MoOSM1-GFP strain and found that tyrosine 173 was the phosphorylated (Figure 6—figure supplement 4A). So, we tested the phenotype of ΔMoosm1/MoOsm1Y173Dand found that it had a similar defect as the ΔMoptp2 mutant (Figure 6—figure supplement 4B), indicating that the constitutive phosphorylation of MoOsm1 results in virulence defect of M. oryzae.

MoPtp2 plays a role in suppressing host ROS accumulation

To further understand the function of the ∆Moptp1/2 mutants, we examined the appressorium formation and virulence. Appressorium formation of the ΔMoptp1/2 mutants was normal (Figure 6—figure supplement 5). We then examined the penetration and invasive hyphal extension in rice sheath cells. After incubation with conidia suspension for 36 hr, more than 76% invasive hyphae of Guy11 were found to spread freely into adjacent cells, while the invasive ∆Moptp2 hyphae (52%) and ∆Moptp1Moptp2 mutant (57%) were restricted in primary infected cells (Figure 6A and B). The ΔMoptp1 mutant exhibited similar invasive hyphal extension as the wild type. All these results indicated that MoPtp2 plays an important role in infection and host colonization.

Figure 6 with 5 supplements see all
MoPtps are important in host-derived ROS scavenging by M. oryzae.

(A) and (B) Excised rice sheaths from 3-week-old rice seedlings were inoculated with conidial suspension (1 × 105 spores/ml). Infectious growth was observed at 24- and 36-hr post-inoculation (hpi). Appressorium penetration sites (n = 100) were observed and invasive hyphae (IH) were rated from type 1 to 4 (type1, no hyphal penetration with only appressoria formation; type2, IH with 1 or two short branches; type3, IH with at least three branches but the IH are short and extending within a plant cell; type 4, IH that has numerous branches and fully occupies the plant cell or even extended to an adjacent plant cell). The experiment was repeated three times. (C) DAB staining of the excised leaf sheath of infected rice 24 hpi. 50 infecting hyphae were counted per replicate and the experiment was repeated three times. (D) The excised sheath of rice was inoculated with conidial suspension after treated with or without 0.5 μM DPI dissolved in DMSO by three independent experiments. The rice sheath was also inoculated with the conidial suspension after treating with or without 0.2 U Aspergillus niger catalase (CAG, Sigma) dissolved in 10 mM (NH4)2SO4. Samples were harvested and observed 36 hr after inoculation. (E) and (F) Quantification of the infection progress with DPI and CAG treatment, over 50 infecting hyphae were counted per replicate, and the experiment was repeated three times.

As the deletion of MoPTP2 caused defects in virulence, we speculated the increased accumulation of ROS around the infection sites accounts for the restricted growth. Using DAB staining, we found that the primary rice cells with the infectious hyphae of the ∆Moptp2 mutant were stained intensely after incubation with conidia suspension for 36 hr. In contrast, cells with the Guy11 infectious hyphae were not stained by DAB (Figure 6C).

To further evaluate the hypothesis that the decreased rate of hyphal growth in infected cells and reduced virulence of the ∆Moptp2 mutant was due to a lack of ROS scavenger production by the host plant, an excised leaf-sheath assay was performed using DPI. After incubation at 28°C for 36 hr, cells were observed under a light microscope. Without DPI treatment, the infectious hyphae of ∆Moptp2 mutants showed restricted growth in the primary infected cells, while those of Guy11 spread into the adjacent cells. Upon treatment with 0.5 μM DPI, the Moptp2 infectious hyphae spread into neighboring cells. Similar results were observed by using the Aspergillus niger catalase (CAG, Sigma) (Tanabe et al., 2009) that hydrolyzes ROS (Figure 6D, E and F). This observation indicated that MoPtp2 has an effect on suppressing accumulation of host-derived ROS during infection.

MoPtp1/2 are essential in suppressing MoOsm1 phosphorylation and function

To study whether the ROS accumulation caused by the deletion of MoPTP1 and MoPTP2 was dependent on their phosphatase activity, we generated the phosphatase activity inactivation mutant of MoPtp1 and MoPtp2 (Figure 7—figure supplement 1). The pathogenicity assay demonstrated that the phosphatase activity dead mutants showed a similar phenotype to the ΔMoptp1 and ΔMoptp2 mutants (Figure 7A). We further identified the phosphorylation pattern of MoOsm1 in these mutations. The results showed that, in both ∆Moptp1/MoPTP1ptpc and ∆Moptp2/MoPTP2ptpc mutants, the band of MoOsm1-GFP migrated as the phosphorylated MoOsm1-GFP protein treated with PI and as in ∆Moptp1/2 mutants (Figure 7B), indicating that the phosphatase activity is critical for the dephosphorylation of MoOsm1. We also detected the phosphorylation levels of MoOsm1 using the antiphospho-p38 antibody and observed increased MoOsm1 phosphorylation in both the ΔMoptp1 and ΔMoptp2 mutants (Figure 7—figure supplement 2). In addition, the phosphorylation of MoOsm1 remained at high levels even after H2O2 treatment at 60 min in the ΔMoptp2 mutants, while the wild type dropped to a low level at 10 min (Figure 7—figure supplement 2). Then we observed the localization of MoOsm1 in the ΔMoptp1, ΔMoptp2 and also ∆Moptp1/MoPTP1ptpc and ∆Moptp2/MoPTP2ptpc mutants. MoOsm1 was present in both the cytosol and the nucleus evenly, similar to that of the wild type. When treated with H2O2, MoOsm1 showed an enhanced nuclear translocation pattern. At 30 min following H2O2 treatment, MoOsm1 showed a nucleus to cytoplasm shifting in the wild-type strain but not in the ΔMoptp2 and ∆Moptp2/MoPTP2ptpc mutants (Figure 7C). These results further supported that MoPtp1/2-mediated dephosphorylation of MoOsm1 controls its nuclear-cytoplasm translocation that is important for the oxidation stress response in M. oryzae. As MoPtp1/2 function on the dephosphorylation of MoOsm1, we observed the formation of homodimer in MoOsm1 in these mutants. We found that the dimer exists in either ΔMoptp1 or ΔMoptp2 mutants; however, in the ΔMoptp1ΔMoptp2 double mutant, we observed monomerized MoOsm1 only, which is similar to that of MoOsm1Y173D (Figure 7D).

Figure 7 with 2 supplements see all
MoPtps-mediated dephosphorylation of MoOsm1 leads to its nuclear exporting and monomerization.

(A) Pathogenicity assay. Conidial suspensions were sprayed onto two-week-old rice seedlings (CO-39). Diseased leaves were photographed after 7 days of inoculation. (B) Dephosphorylation of MoOsm1 dependent on the phosphatase activity of MoPtp1/2. MoOsm1-GFP proteins of various sources treated with alkaline phosphatase and phosphatase inhibitors were separated by Mn2+-Phos-tag SDS-PAGE. (C) Fluorescence observation of conidia untreated (top panels) and treated with 5 mM H2O2 for 10, 30, and 60 min (lower panels). Bar = 5 μm. (D) The total proteins of MoOsm1-GFP/∆Moosm1, MoOsm1Y173A-GFP/∆Moosm1, MoOsm1Y173D-GFP/∆Moosm1, ∆Moptp1,Moptp1/MoPTP1ptpc, ∆Moptp2, and ∆Moptp2/MoPTP2ptpc were subjected to Native-PAGE followed by immunoblotting analysis. (E) Western blot analysis of MoAtf1-MoTup1 interactions in ∆Moptp1/2 mutant strains. The presence of MoAtf1-GFP and MoTup1-FLAG was detected with the anti-GFP and anti-FLAG antibodies, respectively. T: total proteins; S: suspension proteins; and E: elution proteins.

Additionally, the interaction between MoAtf1 and MoTup1 is also dependent on the phosphorylation of MoOsm1. When detected using co-IP, we found that the MoAtf1-MoTup1 complex disassociates in the ΔMoptp2 and ∆Moptp2/MoPTP2ptpc mutants; however, MoAtf1 remains interacting with MoTup1 in the ∆Moptp1 mutant (Figure 7E). These results suggested that MoPtp1 and MoPtp2 are involved in suppressing the overactivation of MoOsm1 upon ROS stress and that MoPtp2 may play a major role in the dephosphorylation of MoOsm1 in M. oryzae.

Identification of MoPtp1 and MoPtp2 as MoAtf1 targets

To identify MoAtf1 binding cis-elements in target promoters, we analyzed the ChIP data using the multiple EM for motif elicitation (MEME) program for 9 bp cis-elements containing the TGAY(C/T)R(G/A)W(T/A) motif (Figure 8—figure supplement 1A). We found that this cis-element exists in the promoters of MoAtf1-binding oxidoreduction-pathway genes (Figure 8—figure supplement 1B). According to the ChIP data, MoAtf1 binds with the promoter of MoPTP2, but not MoPTP1 (Figure 8—figure supplement 1C). However, the same cis-element was found in the promoter of both MoPTP1 and MoPTP2 (Figure 8—figure supplement 1D). To verify the relationship between MoAtf1 and MoPtp1/2, we observed the expression of MoPTP1 and MoPTP2 at various developmental stages that showed the transcription levels of MoPTP1 and MoPTP2 were dramatically decreased in the ∆Moatf1 mutant (Figure 8A and B). DNA containing the promoter sequence of MoPTP1 and MoPTP2 was retarded by the addition of the purified MoAtf1 protein and this retardation increased drastically as the amount of MoAtf1 increased (Figure 8C and D). To further confirm this binding, we used unlabeled DNA to compete with the Alex660-labeled DNA for the purified MoAtf1 protein’s binding sites and found that the binding of labeled DNA with MoAtf1 was decreased significantly with the rise in unlabeled DNA (Figure 8E and F). Further, we generated the putative cis-elements deletion promoter of MoPTP1 and MoPTP2 to confirm the binding with MoAtf1. After separated by polypropylene gel, we found that the band of the digoxin (DIG)-labeling oligomer binding with MoPTP1 and MoPTP2 promoters migrated slower than that of free DNA, and the mobility decreased with increasing concentration of MoAtf1 (Figure 8G and H). When deleted the putative cis-elements from MoPTP1 and MoPTP2 promoters, the oligomer band migrated as fast as the free DNA (Figure 8G and H). These results indicated that MoAtf1 regulates the expression of MoPTP1 and MoPTP2 by directly targeting their promoters.

Figure 8 with 1 supplement see all
MoAtf1 binds to the promoter regions of MoPTP1 and MoPTP2.

(A) Expression analysis of MoPTP1 in the ∆Moatf1 mutant and wild-type strains. The expression of MoPTP1 was analyzed by qRT-PCR and normalized to that of actin gene (MGG_03982). MY represents mycelium and CO represents conidium. Error bars represent the standard deviations and asterisks represent significant differences (p<0.01). (B) Expression analysis of MoPTP2 in the ∆Moatf1 mutant and wild-type strains. (C) and (D) The full-length promoter sequence of MoPTP1 and MoPTP2 was incubated in the absence (leftmost lane) or presence (second to the fourth lane with increasing amounts of MoAtf1) of purified MoAtf1 and GST protein (rightmost lane). The proteinase K was added after the incubation of MoAtf1 with the DNA (fifth lane). DNA-protein complexes were separated by electrophoresis on a 1.2% agarose gel. (E) and (F) The Alex660-labeled full-length DNA of promoter was incubated in the absence (leftmost lane) or presence (second to the sixth lane) of the purified MoAtf1 and GST protein (rightmost lane). Unlabeled DNA was added as a binding competitor with increasing amounts in lanes from third to sixth. DNA-protein complexes were separated by electrophoresis on a 1.2% agarose gel. (G) The DIG-labeled promoter of MoPTP1 was incubated with an increasing amount of MoAtf1 prior to separation by PAGE. pro-PTP1: full-length promoter. pro-PTP1: the cis-element deletion promoter. The white arrow represents free DNA, the black arrow represents migrated DNA. (H) The DIG-labeled promoter of MoPTP2 was incubated with an increasing amount of MoAtf1. The white arrow represents free DNA, the black arrow represents migrated DNA.

Phosphorylation of MoAtf1 is important for transcription initiation under oxidation stress

Given ROS burst activates MoOsm1, we hypothesized that MoPtp1/2-mediated dephosphorylation was also a response to oxidation stress. We then next examined the expression of MoPTP1 and MoPTP2 genes and found that the expression was induced upon H2O2 exposure. As MoAtf1 binds to the promoters of MoPTP1 and MoPTP2, we further analyzed whether the phosphorylation of MoAtf1 affects this expression. Even if the transcription levels were low, the expression of MoPTP1 and MoPTP2 genes increased and peaked after being treated with H2O2 for 10 min in the ΔMoosm1/MoATF1S124D strain, similar to the wild-type strain (Figure 9A and B). In the ΔMoosm1 mutants, whose phosphorylation level of MoAtf1 was low, however, the expression levels of MoPTP1 and MoPTP2 genes remain unchanged (Figure 9A and B). In addition, we evaluated the protein levels of MoPtp1 and MoPtp2 under oxidation stress. After treated with H2O2 for 10 min, the amount of MoPtp1 and MoPtp2 showed no significant difference in the ΔMoosm1 mutant (Figure 9C and D), but the protein levels of MoPtp1 and MoPtp2 were induced significantly in the ΔMoosm1/MoATF1S124D strain under stress (Figure 9E and F). Taken together, these results indicated that the phosphorylation of MoAtf1-mediated by MoOsm1 is required for the expression of MoPTP1 and MoPTP2 in response to oxidation stress.

MoAtf1 phosphorylation controls the transcription of MoPTP1 and MoPTP2 in response to oxidative stress.

(A) and (B) MoPTP1 and MoPTP2 expression analysis in Guy11, ∆Moosm1 mutant, and ∆Moosm1/MoAtf1S124D strains treated with H2O2 for 15, 30, and 60 min. Three independent biological experiments were performed, with three replicates each time, and yielded similar results in each independent biological experiment. Dotted lines represent the expression of MoPTP1 and MoPTP2 in these strains under H2O2 stress. Error bars represent standard deviation. (C) Total proteins were extracted from ∆Moosm1 treated with H2O2 for 15, 30, and 60 min. MoPtp1/2 was detected by western blotting analysis using anti-ptp1/2 antibodies. An anti-Actin antibody was used as control. (D) Western blotting bands were quantified with an ODYSSEY infrared imaging system (application software Version 2.1). Bars denote standard errors from three independent experiments. Asterisks indicate significant differences (Duncan's new multiple range test p<0.01). (E) Total proteins were extracted from ∆Moosm1/MoAtf1S124D strain for MoPtp1/2 detection. (F) Western blotting bands of MoPtp1 in ∆Moosm1/MoAtf1S124D strain was quantified with an ODYSSEY infrared imaging system.

Discussion

Upon pathogen infection, plants rapidly activate PTI through ROS burst and callose deposition. Meanwhile, pathogens secrete numerous extracellular proteins into the plant to circumvent host immunity (Doehlemann and Hemetsberger, 2013). Previous studies have identified several effector proteins, including those involved in oxidation-related functions; however, how these factors initiate their function to counter host immunity remains not fully understood. In this study, we discovered that MoOsm1 phosphorylates MoAtf1 that in turn dissociates from MoTup1 to regulate oxidation stress response pathways. We further revealed that phosphorylated MoAtf1 promotes the expression of MoPtp1/2 that could dephosphorylate MoOsm1, completing a feedback loop that controls the virulence of M. oryzae. Our results demonstrated MoOsm1-MoPtps-mediated feedback loop represents a previously unknown mechanism that balances the response to ROS stress and the hemibiotrophic growth of M. oryzae.

For the pathogen, ROS seems like a double-edged sword. During appressorial penetration, M. oryzae accumulates high levels of endogenous ROS to strengthen the appressorium cell wall. (Egan et al., 2007). Here, ROS accumulation is regulated by two fungal NADPH oxidases, which themselves are important for appressorium-mediated cuticle penetration (Dagdas et al., 2012). However, ROS is also one of the earliest responses by plants to microbial colonization and function a potent defense mechanism that limits fungal biotrophic growth (Torres, 2010; Torres et al., 2006). Rboh, which encodes NADPH oxidase, plays a key role in generating ROS upon pathogen challenge (Wong et al., 2007). Among them, both OsrbohA and OsrbohB are important for the production of ROS when rice are subjected to stresses (Wong et al., 2007). In addition, during M.oryzae infection, OsRBOHA and OsRBOHB were significantly induced in WT plants at 24 hpi, but decreased afterward (Yang et al., 2017). As the oxidative burst reaction occurs rapidly, how can the pathogen quickly respond to this stress? In M. oryzae, the MoOsm1-mediated osmoregulation pathway is essential for responding to oxidative stress, in addition to the previously demonstrated hyperosmotic stress. The deletion of MoOSM1 was shown to result in high sensitivity to oxidative stress (Dixon et al., 1999). We here showed that MoOsm1 phosphorylation also increases during the early stage of infection. Given that ROS burst is one of the earliest PTI responses during infection, ROS burst may activate osmoregulation MAPK signaling through MoOsm1 phosphorylation. Intriguingly, oxidative stress was also found to result in MoOsm1 cytoplasm to nucleus translocation.

How MoOsm1 translocated into the nucleus in response to oxidative stress? Previous studies showed that the dephosphorylation of MoHat1 led to its interaction with MoSsb1, causing a nucleus to cytoplasm translocation (Yin et al., 2019). Studies also showed that MoAp1 accumulated in the nucleus under oxidative stress (Guo et al., 2011). In S. cerevisiae, Ap1 protein forms disulfide bonds that dampen the recognition of the nuclear export protein Crm1, leading to its accumulation in the nucleus (Kuge et al., 2001) which prompted us to suggest a relevance between MoAtf1 phosphorylation and its nuclear localization. Arabidopsis regulatory protein NPR1 is involved in salicylic acid (SA)-mediated defense response in which SA triggers cytoplasmic NPR1 oligomer release that is required for its nuclear import (Mou et al., 2003). The SNF1-related protein kinase SnRK2.8 phosphorylate NPR1 prior to its nuclear trans-localization (Lee et al., 2015). In addition, the nitric oxide-induced S-nitrosoglutathione also causes NPR1 nuclear translocation (Lindermayr et al., 2010). Given these findings, we hypothesized and demonstrated that (1) phosphorylation of MoOsm1 inhibits the recognition of the nuclear export proteins causing its retention in the nucleus; (2) MoOsm1 forms homodimers in the cytoplasm, but MoOsm1 oligomers are released and imported into the nucleus when MoOsm1 was phosphorylated. We also showed that MoOsm1 forms homodimers that disintegrate upon phosphorylation of Y173 (Figure 3D–F). Further combined with the results that protein dimerization occurs only in the cytoplasm (Figure 3G) and MoOsm1 phosphorylation on Y173 leads to monomerization and nuclear localization, we thus considered that phosphorylation happening prior to nuclear import instead of the misrecognition of phosphorylated MoOsm1 in the nucleus that caused the nuclear accumulation of MoOsm1. Collectively, we concluded that the monomerization of MoOsm1 is important for its nuclear accumulation and Y173 phosphorylation is important for monomerization. Given the evidence indicating that the nuclear localization of MoOsm1 was more stable in the ΔMoptps mutant than Guy11 under ROS stress, it is plausibly that MoPtps function in the recycling of MoOsm1 to the cytoplasm. MoOsm1 recycling may have two benefits: (1) shutdown of phosphorylated MoOsm1 mediates signaling pathways that switch off virulence attack. (2) recycled MoOsm1 in the cytoplasm might respond quickly to external stress as there is no need for protein re-synthesing.

MoOsm1 co-localizes with and activates the transcription factor MoAtf1 through protein phosphorylation in the nucleus. MoAtf1 is one of the bZIP transcription factors control gene expression during plant infection (Kim et al., 2009; Proft et al., 2001; Tang et al., 2015). Here, we found that MoAtf1 positively regulates genes involved in oxidation response pathways (Figure 5—figure supplement 3). During infection, the expression of the oxidation responsive genes was significantly upregulated (Figure 5). This led us to identify MoTup1 as one of the proteins interacting with MoAtf1. MoTup1 was recently characterized in M. oryzae, and its deletion caused decreased pathogenicity (Chen et al., 2015). This study, together with those of other model organisms (Chen et al., 2013; García-Sánchez et al., 2005; Malavé and Dent, 2006; Smith and Johnson, 2000), suggests that Tup1 proteins do not bind directly to DNA but rather are brought to the promoters via interactions with sequence-specific regulatory proteins. We speculate that MoAtf1 recruits MoTup1 to repress transcription. Once MoAtf1 phosphorylates and no longer binds with MoTup1, MoAtf1 up-regulates or initiates the transcription of target genes, including those involved in oxidation regulated pathways.

During the interaction between M. oryzae and the host, the host induces ROS burst during the biotrophic growth stage of the fungus. Under ROS stress, the pathogen activates MoOsm1-mediated pathways that in turn activates the transcription factor MoAtf1. The phosphorylation of MoAtf1 causes the disintegration of the MoAtf1-MoTup1 complex to induce the expression of oxidation regulation pathway genes to further enhance virulence of M. oryzae. Phosphorylated MoAtf1 also initiates the transcription of the MoPTP1/2 genes to further dephosphorylate MoOsm1. Once the ROS stress was circumvented, such as that at the necrotrophic growth stage, MoPtps- mediated dephosphorylation of MoOsm1 shut off phosphor-regulatory circuitry to control the virulence (Figure 10).

A proposed model depicting MoOsm1/MoAtf1/MoTup1/MoPtp1/2 mediated ROS signaling and responses to host immunity.

Rice generates immunity, including ROS burst during its interaction with M. oryzae. Once host perception, M. oryzae induces MoOsm1 phosphorylation that disintegrates MoOsm1 dimerization leading to enhanced nuclear translocation of MoOsm1. MoOsm1 phosphorylates MoAtf1 uncoupling MoAtf1-MoTup1 interaction that induces the expression of oxidation regulation pathway genes. At the same time, the phosphorylated MoAtf1 promotes the expression of two phosphatases, MoPTP1 and MoPTP2, that dephosphorylate MoOsm1 to suppress MoAtf1-MoTup1 dissociation. MoPtp1/2-mediated MoOsm1 dephosphorylation provides an act balancing infection and its hemibiotrophic growth in rice.

Materials and methods

Strains and culture conditions

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The M. oryzae Guy11 strain was used as a wild type (WT) in this study. All strains were cultured on complete medium (CM) for 3–15 days in the dark at 28°C (Liu et al., 2016; Talbot et al., 1993). Mycelia were harvested from the liquid CM media with or without additional treatment for DNA, RNA, and total protein extractions. For conidia production, strains were maintained on straw decoction and corn (SDC) (100 g of straw, 40 g of corn powder, 15 g of agarin 1 l of distilled water) agar media at 28°C for 7 days in the dark followed by 3 days of continuous illumination under fluorescent light (Qi et al., 2016).

Targeted gene deletion and transformation

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The MoPTPs gene deletion mutants were generated using the standard one-step gene replacement strategy. First, two fragments with 1.0 kb of sequences flanking the targeted gene were PCR amplified with primer pairs. The resulting PCR products were digested with restriction endonucleases and ligated with the hygromycin-resistance cassette (HPH) released from pCX62. Finally, the recombinant insert was sequenced. The 3.4 kb fragment, which includes the flanking sequences and the HPH cassette, was amplified and transformed into Guy11 protoplasts. Putative mutants were first screened by PCR and later confirmed by Southern blotting analysis (Figure 6—figure supplement 1). Fragments for mutant complementation were amplified by PCR and inserted into pYF11 or pHZ126 before being introduced into the mutant strains through PEG-mediated transformation.

Virulence assay

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Conidia harvested from 10-day-old SDC agar cultures were filtered through two layers of Miracloth and resuspended to a concentration of 5 × 104 spores/ml in a 0.2% gelatin solution. Two-week-old seedlings of rice (Oryza sativa cv. CO39 and K23) was used for pathogenicity assays. For spray inoculation, 5 ml of a conidial suspension of each treatment was sprayed onto rice with a sprayer. Quantification of lesion types (0, no lesion; 1, pinhead-sized brown specks; 2, 1.5 mm brown spots; 3, 2–3 mm gray spots with brown margins; 4, many elliptical gray spots longer than 3 mm; 5, coalesced lesions infecting 50% or more of the leaf area) were measured according to Wang et al., 2013 Conidial germination and appressorium formation were measured on a hydrophobic surface as previously described (Qi et al., 2012). Appressorium induction and formation rates were also obtained as described previously (Li et al., 2017a).

For infection, conidia were harvested from 10-day-old SDC agar cultures, filtered, and resuspended to a concentration of 5 × 104 spores/ml in a 0.2% (w/v) gelatin solution. For the leaf assay, leaves from two-week-old seedlings of rice (Oryza sativa cv. CO39 or K23) and 7-day-old seedlings of barley were used for spray inoculation. For rice leaves, 5 ml of a conidial suspension of each treatment was sprayed. Inoculated plants were kept in a growth chamber at 25°C with 90% humidity and in the dark for the first 24 hr, followed by a 12/12 hr light/dark cycle. Lesion formation was observed daily and recorded by photography 7 days after inoculation (Yin et al., 2020).

For DAB staining, the leaf sheaths were immersed in 1 mg/ml solution of DAB in a buffer (pH = 3.8) at the indicated time after inoculation with M. oryzae. Samples were incubated at room temperature for 8 hr in the dark. When the brown spots appeared clearly, samples were bleached ethanol: acetic acid (95:5) for 1 hr. Images were captured using a microscope (Zeiss, Axio Observer A1).

Quantitative RT-PCR analysis

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For qRT-PCR, total RNA was reverse transcribed into first-strand cDNA using the oligo (dT) primer and HiScript II Q select RT SuperMix for qPCR (Vazyme, Nanjing, R233-01). The qRT-PCR was run on the Applied Biosystems 7500 Real-Time PCR System with ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, Q311-02). Normalization and comparison of mean Ct values were performed as previously described (Yin et al., 2020).

Epifluorescence microscopy

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M. oryzae cells (conidia) expressing fluorescent protein-fused chimera were incubated under appropriate conditions. The constructs including MoOsm1-GFP, MoAtf1-GFP, and other phosphorylation mutations were transformed into ΔMoosm1 mutant, ΔMoatf1 mutant or the wild-type Guy11 strain. Epifluorescence microscopy was performed using a Zeiss LSM710 (63x oil) microscope. H1-RFP was introduced into the MoOsm1-GFP transformants to visualize the nucleus.

Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation assays

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cDNA was respectively amplified with Super Fidelity DNA Polymerase. Amplified products were cloned into pGBKT7 or pGADT7 vectors, respectively. After sequence verification, they were introduced into yeast AH109 strain. Transformants grown on synthetic medium lacking leucine and tryptophan (SD–Leu–Trp) were transferred to synthetic medium lacking leucine, tryptophan, and histidine (SD–Leu–Trp–His).

For the BiFC assay, the cYFP-MoOSM1 fusion construct was generated by cloning MoOSM1 into pHZ68. Similarly, MoOSM1-nYFP, MoOSM1Y173D-nYFP, MoOSM1Y173A-nYFP and MoATF1-nYFP fusion constructs were generated into pHZ65, respectively. Construct pairs were introduced into the protoplasts of Guy11, respectively. Transformants resistant to both hygromycin and zeocin were isolated and confirmed by PCR.

Co-immunoprecipitation (co-IP) assay

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To confirm the interactions among MoOsm1-MoOsm1, MoOsm1-MoAtf1, and MoAtf1-MoTup1 in vivo, MoOsm1-GFP, MoOsm1-FLAG, MoTup1-GFP, MoAtf1-GFP, and all of the mutation protein fusion constructs were prepared by the yeast homologous recombination transformation. Different pairs of specific constructs were co-transformed into the protoplasts of the WT strain. Total hyphae proteins were isolated from different positive transformants and incubated with anti GFP agarose (Chromo Tek, gta-20) at 4°C for 2 to 12 hr with gently shaking. Proteins bound to the beads were eluted after a series of washing steps by 1 × PBS. Elution buffer (200 mM glycine, pH 2.5) and neutralization buffer (1 M Tris, pH 10.4) were used for the elution process. Total, suspension, and eluted protein were analyzed by western blot using GFP (mouse, 1:5000; Abmart, 293967) or FLAG (mouse, 1:5000; Abmart, M20018) specific antibodies.

GST-pull down

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GST, GST-MoAtf1, GST-MoAtf1S124D, and His-MoTup1 were expressed in Escherichia coli BL21-CodonPlus (DE3) cells. Cells were lysed in lysis buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM PMSF [Beyotime Biotechnology, ST506-2]) with a sonicator (Branson). Samples were centrifuged (13,000 g, 10 min) and the supernatants were transferred to a new 1.5 ml tube and stored at −70°C. The GST, GST-MoAtf1, and GST-MoAtf1S124D supernatants were then mixed with 30 μl glutathione sepharose beads (GE Healthcare, 10265165) and incubated at 4°C for 2 hr. The recombinant GST, GST-MoAtf1 or GST-MoAtf1S124D-bound to glutathione sepharose beads were incubated with E. coli cell lysate containing His-MoTup1 at 4°C for another 4 hr. Finally, the beads were washed with buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM PMSF, 1% Triton X-100) five times and eluted from the beads. Eluted proteins were then analyzed by immunoblot (IB) with monoclonal anti-His and monoclonal anti-GST antibodies (Li et al., 2017a), respectively.

Protein extraction and western blot analysis

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Mycelia were ground into a fine powder in liquid nitrogen for total protein extraction and resuspended in 1 ml lysis buffer (10 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) with 2 mM PMSF and proteinase inhibitor cocktail. The lysates were placed on the ice for 30 min and shaken once every 10 min. Cell debris was removed by centrifugation at 13, 000 g for 10 min at 4°C. The lysates were collected as to total proteins (Liu et al., 2019). For GFP- tagged protein detection, samples were separated by 8% SDS-PAGE and followed by western blotting analysis. For detecting phosphorylated MoOsm1, a p38 MAP kinase orthologue, the anti-pp38 (CST:9215S) and anti-p38 (CST:9212S) antibodies were used. For detecting MoPtp1 and MoPtp2, the anti-ptp1B antibody (ab244207) was used. Blot signals were detected and analyzed using the ODYSSEY infrared imaging system (Version 2.1).

Phosphorylation analysis through Phos-tag gel electrophoresis

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The MoAtf1-GFP fusion construct was introduced into the wild type (Guy11) and ΔMoosm1, and MoOsm1-GFP was introduced into Guy11, ΔMoptp1, and ΔMoptp2 mutant strains, respectively. The total protein extracted from mycelium was resolved on 8% SDS-polyacrylamide gels prepared with 50 µM acrylamide-dependent Phos-tag ligand and 100 µM MnCl2 as described (Li et al., 2017a). Gel electrophoresis was performed with a constant voltage of 80 V for 3–6 hr. Before transferring, gels were equilibrated in transfer buffer with 5 mM EDTA for 20 min two times and followed by transfer buffer without EDTA for another 20 min. Protein transfer from the Mn2+-phos-tag acrylamide gel to the PVDF membrane was performed for ~36 hr at 80 V at 4°C, and then the membrane was analyzed by western blotting using the anti-GFP antibody (Li et al., 2017b).

In vitro phosphorylation analysis

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The GST-MoAtf1, GST-MoAtf1S124A, and His-MoOsm1 were expressed in E. coli BL21-CodonPlus (DE3) cells and purified as described in the GST-pull down assays. The rapid and cost-effective fluorescence detection in tube (FDIT) method was used to analyze protein phosphorylation in vitro (Jin and Gou, 2016). The Pro-Q Diamond Phosphorylation Gel Stain, known as a widely used phosphor-protein gel-staining fluorescence dye, was used in this assay. For protein kinase reaction, 2 μg GST-MoAtf1, GST-MoAtf1S124A was mixed with MoOsm1 in a kinase reaction buffer (100 mM PBS, pH 7.5, 10 mM MgCl2, 1 mM ascorbic acid), with the appearance of 50 μM ATP at room temperature (RT) for 60 min, 10 folds of cold acetone was added to stop the reaction. For protein in tube staining, casein (Sango Biotech, T510256) was homogenized and suspended in Mili-Q water at the concentration of 0.2 μg/μl. For staining duration time analysis, 10 μl of casein was mixed with 100 μl of Pro-Q Diamond (Thermo Fisher Scientific, P33301) and kept in the dark at RT for 1 hr. The protein was then precipitated with 10 volumes of cold acetone, kept in a −20°C freezer, and centrifuged at 13,200 g for 1 hr at 4°C. The supernatant was carefully drained out and discarded without touching the protein pellet. The pellet was rinsed with 0.5 ml of cold acetone and centrifuge to remove the supernatant twice. The pellet was air-dried and dissolved in 200 μl of Mili-Q water and moved to a black 96 well plate (Corning, 3925). Fluorescence signal at 590 nm (excited at 530 nm) was measured in a Cytation3 microplate reader (Biotek, Winooski, VT, USA) (Yin et al., 2019; Yin et al., 2020).

Chromatin immunoprecipitation (ChIP)-Seq analyses

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ChIP was performed according to the described protocol with additional modifications. Briefly, fresh mycelia were cross-linked with 1% formaldehyde for 15 min and then stopped with 125 mM glycine. The cultures were ground with liquid nitrogen and resuspended in the lysis buffer (250 mM, HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% Deoxy Cholate, 10 mM DTT) and protease inhibitor (Sangon Co., Shanghai, China, A100754). The DNA was sheared into ~500 bp fragments with 20 pulses of 10 s and 20 s of resting at 35% amplitude (Qsonica*sonicator, Q125, Branson, USA). After centrifugation, the supernatant was diluted with 10 × ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl, pH 8.0 and 167 mM NaCl). Immunoprecipitation was performed using the monoclonal anti-GFP ab290 (Abcam, Cambridge, UK; 1:500 dilution) antibody. Following low salt wash (150 mM NaCl, 20 mM Tris-HCl (PH 8.0), 0.2% SDS, 0.5% TritonX-100, 2 mM EDTA), high salt wash (500 mM NaCl, 20 mM Tris-HCl (PH 8.0), 0.2% SDS, 0.5% TritonX-100, 2 mM EDTA), and LiCl and TE wash, DNA was eluted with elution buffer (1% SDS, 0.1 M NaHCO3). The eluants were precipitated by ethanol after washing and digested with proteinase K, and sequenced on an Illumina HiSeq2500 (Genergy Bio, Shanghai, China). The accession number for RNA-seq data reported in this paper is GSE144389 in GEO datasets (http://www.ncbi.nlm.nih.gov/gds).

Mass spectrometric analysis

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Proteins were separated by 10% SDS-PAGE To identify phosphorylation sites. The gel bands corresponding to the targeted protein were excised, reduced with 10 mM of DTT and alkylated with 55 mM iodoacetamide. In-gel digestion (or elution digestion) was carried out with the trypsin (Promega, V5113), GLU-C (Wako, 050–05941), or chymotrypsin (Sigma-Aldrich, C6423) in 50 mM ammonium bicarbonate at 37°C overnight. The peptides were extracted using ultrasonic processing with 50% acetonitrile aqueous solution for 5 min and with 100% acetonitrile for 5 min. The extractions were reduced in the volume by centrifugation. A liquid chromatography-mass spectrometry (LC–MS) system consisting of a Dionex Ultimate 3000 nano-LC system (nano UHPLC, Sunnyvale, CA, USA), connected to a linear quadrupole ion trap Orbitrap (LTQ Orbitrap XL) mass spectrometer (ThermoElectron, Bremen, Germany) and equipped with a nanoelectrospray ion source, was used for our analysis. For LC separation, an Acclaim PepMap 100 column (C18.3 μm, 100 Å) (Dionex, Sunnyvale, CA, USA) capillary with a 15 cm bed length was used with a flow rate of 300 nL/min. Two solvents, A (0.1% formic acid) and B (aqueous 90% acetonitrile in 0.1% formic acid), were used to elute the peptides from the nanocolumn. The gradient ranged from 5% to 40% B in 80 min and from 40% to 95% B in 5 min, with a total run time of 120 min. The mass spectrometer was operated in the data-dependent mode to automatically switch between Orbitrap-MS and LTQ-MS/MS acquisition. Survey full-scan MS spectra (from m/z 350 to 1800) were acquired in the Orbitrap with a resolution r = 60,000 at m/z 400, allowing the sequential isolation of the top ten ions, depending on signal intensity. The linear ion trap fragmentation used collision-induced dissociation at a collision energy of 35 V. Protein identification and database construction were processed using Proteome Discoverer software (1.2 version, Thermo Fisher Scientific, Waltham, MA, USA) with the SEQUEST model.

Electrophoretic mobility shift (EMSA) assays

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The GST-MoAtf1 protein was expressed and purified from E. coli strain BL21 using the pGEX4T-2 construct. The DNA fragments from the promoter of MoPTP1/2 were end-labeled with Alex660 by PCR amplification using the 5’ Alex660-labeled primer. The purified protein was mixed with Alex660-labeled DNA, incubated for 20 min at 25°C in binding buffer, and separated by agarose gel electrophoresis. Gels were directly visualized using an LI-COR Odyssey scanner with excitation at 700 nm (Wang et al., 2017).

Statistical analyses

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Results were presented as the mean ± standard deviation (SD) of three biology repeats. The significant differences between samples were statistically evaluated by using SDs in SPSS 2.0. The significant differences between treatments with a single factor random grouping model were statistically determined by one-way analysis of variance (ANOVA) comparison and followed by the F-test if the ANOVA result is significant at p<0.01.

Data availability

This information can be found in the corresponding figure legends and in the materials and methods section. And the Geo number "GSE144389" for the ChIP assay is available already, we mentioned it in the materials and methods section. All data generated or analysed during this study are included in the manuscript and supporting files. And the information can be found in the corresponding figure legends and in the materials and methods section.

The following data sets were generated

References

    1. Malavé TM
    2. Dent SY
    (2006) Transcriptional repression by Tup1-Ssn6
    Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire 84:437–443.
    https://doi.org/10.1139/o06-073

Decision letter

  1. Sylvain Raffaele
    Reviewing Editor
  2. Detlef Weigel
    Senior Editor; Max Planck Institute for Developmental Biology, Germany

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The rapid production of reactive oxygen species (ROS) is a widespread defense response in plants. How adapted pathogens overcome this defense response is a fascinating question given that maintaining an appropriate redox balance is critical for pathogen survival throughout infection. Through an extensive set of molecular biology and genetics experiments on the rice blast fungus Magnaporthe oryzae, this study uncovers a complete regulatory loop balancing the fungal response to ROS. A cascade of reversible phosphorylation reactions and protein complexes remodeling enable a dynamic response to the redox environment, each step of which contributes to the successful colonization of rice plants by this pathogen.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Host-derived ROS activates MoOsm1/MoPtp-dependent phosphorylation of MoAtf1 to orchestra virulence in Magnaporthe oryzae" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

We agree that your study is ambitious, but feel that the model you propose is composed of several pieces of evidence that are often incomplete. In some cases, important controls are missing or assumptions are made without justification. While it is admirable to attempt to obtain such a complete view of a signaling node, we suggest you consider focusing on one aspect of your model at a time, in order to put together a more focused and complete study, and submit elsewhere. We are sorry we cannot be more positive at this time.

Reviewer #1:

This manuscript proposes a novel counter-immunity signaling pathway in Magnaporthe oryzae during plant infection. This could represent a major advance but substantial revision would be necessary for this to be so. Overall, I felt that evidence was largely correlative rather than causative, conclusions often drawn from what appear to be rather minor differences, and that due-diligence has not been performed for a lot of experiments. This article needs to be revised by an English copy-editor, as the vast number of grammatical errors makes the article difficult to follow. A lot of information is missing from experimental design as described in the text and in figure legends. P-values are mentioned in figure legends but the statistical test is not described.

The Introduction and Discussion are difficult to follow, as many concepts are inadequately described or synthesized.

Are ROS mutants (rbohd or similar) available in the rice cultivars that can be used to validate the conclusion that it is host-derived ROS being detected by the fungus?

Has it been demonstrated that anti-pp38 and anti-p38 are able to recognize MoOSM1? If so please cite. If not, please demonstrate.

Enhanced phosphorylation in Figure 2A is not clear. Perhaps the authors can provide quantification from multiple blots.

Quantification of nuclear:cytoplasmic OsmI1 in Figure 2C-E, and later in Figure 3 is not overly convincing to me. The biological relevance of this sub-cellular movement should be assessed using NES and/or NLS localization/mutant constructs.

Phospho-memetic D/E mutations are not guaranteed to actually mimic phosphorylation. Did the authors also test phospho-ablative A/F mutations? What data support Osm1-Y173D is phosphomimetic? Same is true for Atf1-S124D presented in Figure 5.

The Phostag gel in Figure 4C is not clear, especially when compared to 4F.

In Figure 4E the authors describe “phosphorylation sites” upstream of the NLS: are these just serines, or have they been shown to be phosphorylated? If so, please cite. If not, rephrase.

It is clear that S124 is important for phosphostatus, but there is no evidence that this is due to phosphorylation by Osm1.

IP controls are lacking, and protein expression should be confirmed for the Y2H assays shown in Figure 5. It is possible that the mutant variants are not expressed.

The authors describe “hemibiotrophic fungi” as having a “symbiotic” stage with their plant host. I do not think this is accurate.

The authors demonstrate that ptp1/2 mutants are similar to ptp2 mutants and make the conclusion that PTP1 and PTP2 have differential functions. Are they expressed similarly?

In Figure 7E the legend describes “in vivo” phosphorylation but this described as being performed on protein extracts in the text. Is this really “in vivo”?

What makes a protein/residue hyper-phosphorylated? How can one tell this based on mass spec data without controls? The authors should provide more details about how this MS was performed, and how confident they are in the scores. No Mascot/proteomic confidence scores are provided for these data.

The conclusion that PTP-mediated desphosphorylation of Osm1 leads to nuclear export is not supported by direct evidence and is therefore speculative.

Reviewer #2:

The manuscript by Liu et al. describes molecular mechanisms by which the fungal plant pathogen Magnaporthe oryzae copes with reactive oxygen species (ROS) produced during the host immune response. It provides a complete view of one particular mechanism involving phosphorylation/dephosphorylation reactions, protein complex assembly and dissociation, protein relocalization to the nucleus and transcriptional regulation. The manuscript is organized with a straightforward logic, but requires extensive language editing to improve clarity. This is a very ambitious manuscript, featuring a lot of diverse experiments, apparently carefully executed, involving many different molecular players. It therefore has great potential to constitute a significant step forward in our understanding of fungal virulence. However, the relatively complex model the authors attempt to build is composed of pieces of evidence that sometimes remain incomplete. Several steps of the reasoning would require more thorough testing for the model to be fully convincing. An effort is also needed to synthesize these findings and present the conceptual novelty arising from the work. My specific major concerns are as follows:

1) I can see that there is a significant progress being made in understanding a particular ROS-response signaling pathway, the amount of work and new insights appear substantial, but the authors fail to unify their findings and convey the new concepts arising from their experimental work. After reading carefully the manuscript several times, I can tell the authors found new mechanisms for Magnaporthe to sense and respond to host-derived ROS, but I miss a clear statement on what the major novelty/originality is. Can the authors summarize the process they discovered in 1 or 2 simple sentences rather than a list of molecular players? The objectives of the study stated by the authors are:

– "How does M. oryzae percept and then overcome ROS stress?" a mechanism is proposed to mitigate the antifungal effect of host-derived ROS is provided, but how ROS are perceived is not directly addressed.

– "how MoOsm1 is linked to MoAp1 or MoAtf1 in stress response is unclear" This is a rather field- or organism-centered question. Some efforts are needed to place the study into a broader perspective an qualify in simple terms the novel concepts and mechanisms discovered.

2) "YFP signal of MoOSM1-cYFP and MoOSM1-nYFP pair was reduced in the nucleus, suggesting that the monomeric form of MoOsm1 is involved in the nuclear localization under the oxidative stress" > Several explanations could fit with this observation, the conclusion is not fully supported. A version of osm1 that remain in the dimer form (maybe a phospho dead mutant of Y173?) should not accumulate in the nucleus.

"Collectively, the results suggested that Y173D but not T171D phosphomimic mutation is required for the interaction"> I would rather conclude that Y173D but not T171D prevents Osm1 oligomer formation

BiFC "the empty vectors used as negative controls" > Better controls would be: Phospho-dead mutants of Osm1 or the Y171D mutant + MoOSM1 wt = positive / MoOsm1Y173D + MoOsm1 wt = negative

Subcellular localisation of MoOsm1Y171D-GFP is not analyzed/commented

3) "MoAtf1 interacts with MoOsm1" and following section> This is lacking a few controls to be fully convincing: what happens if osm is trapped outside the nucleus (nuclear exclusion signal or Y173 phospho dead mutant?) – Since the interaction assay was performed in the absence of ROS stress, what would be its relevance in response to ROS? What would be the function of Osm1 dimer dissociation and relocalization to the nucleus? Is Osm1-Atf1 interaction modified upon ROS stress? Similarly, the interaction between Atf1 and Tup1 has not been tested in the context of response to ROS.

4) "disruption of MoAtf1-MoTup1 complex would reverse the suppression of genes involved in virulence" > as far as I understand, disruption of this complex is achieved through Atf1 phosphorylation (S124D phosphomimic mutant) or deletion of Atf1. However, δ MoATF1 is less virulent than wild type (Figure 4G/H) while there is no Atf1-Tup1 complex and therefore no repression of virulence genes in this strain – Similarly, Tup1 deletion reduced virulence (Chen et al., 2015) – How can we explain these discrepancies? In these experiment, what is the virulence phenotype of Atf1S124D mutants? The reverse controls with phospho-dead mutants of Atf1 would be useful to support the conclusions. In which compartment of the cell (nucleus or cytoplasm) are these interactions happening?

5) Whether the genes under transcriptional control of the Atf1-Tup1 complex actually play a role in virulence is not described. Do they directly contribute to host-derived ROS detoxification?

Reviewer #3:

The article "Host-derived ROS activates MoOsm1/MoPtp-dependent phosphorylation of MoAtf1 to orchestra virulence in Magnaporthe oryzae" by Liu et al. propose to demonstrate the role of the MAPK kinase complex MoOsm/MoPtp in the regulation of transcription factors MoAtf1 in the fungus M. oryzae.

Overall, authors have provided evidence to show the role of ROS and its effect on one of the MAPK module, but they do not provide evidence to justify the title.

1) Exogenous application of H2O2 is NOT the same as host derived ROS. Authors have not provided evidence to show that host derived ROS can drive the changes in the MAPK complexes in the fungus outlined in this manuscript. Therefore, Authors should use host mutants that cannot make ROS to justify their claim.

2) Authors are aware that fungi have an equivalent ROS producing machinery that can be inhibited by both DPI and catalase (these are not specific to plant NADPH oxidases as claimed by the authors.

3) Authors describe PTI in their Introduction. How and more importantly when is ROS produced during PTI in a suc (LTH) vs res (K23) interaction? They need to define PTI in their pathosystem.

4) Authors need to detail their experimental protocols more thoroughly and edit the figure and figure legends so that they match.

The reviewer questions many of the experimental procedures used:

1) While the Co-IPs are done in hyphae and other time they use protoplasts; many of the localization studies are done in conidia. The authors do not make any distinction between these two. Conidia and hyphae are two distinct developmental stages of fungus as demonstrated by the authors in Figure 10A and authors cannot use interchangeably these two development stages to support their hypothesis

2) With respect to using protoplasts: where the Co-IPs performed from plasmids expressed transiently? No experimental procedure is described with respect to preparation, transformation, and efficiency.

3) What development stage of hyphae was used to extract proteins to perform Co-IPs. How much protein was used in each of the IP experiments?

4) Authors do not provide evidence that validates any of the constructs used in this manuscripts.

5) Monomer/Dimer (Figure 3E). should document this by gel-filtration chromatography and the native gel does NOT adequately demonstrate this.

6) Phosphorylation of MoATF-1 (Figure 4C) – not very convincing (compare 4c to 4F);

Should use ΔMoosm in 4G.

7) EMSA experiments: it is not appropriate it to use a 1 Kb fragment to perform gel shift analysis. An oligomer with the binding site and a mutated binding site should be used.

8) The legend for Figure 9A indicates 100 infecting hyphae were counted. How many of those were used for DAB staining in 9C?

Figures with no explanation or details:

1) Figure 3F: the figure clearly show conidia, but the text refers to it as protoplasts.

2) Figure 1H is referred in text as bulbous structure at 24hrs; not identified in the figure or legend

3) Figure 4H; Figure 5B; middle lane in Figure 7D; what are type 1.…typ4 infection in Figure 9A and 9B?

The reviewer questions some of the statements made in this manuscript:

Results section:

1) MoOsm1 was phosphorylated in response to oxidative stress: "These results suggested that MoOsm1 responds to oxidative stress by inducing a high phosphorylated level and increased accumulation in the nucleus."

This statement should be supported by Western with p-p38 Abs in Figure 2E.

2) MoOsm1 is reduced from a dimer to a monomer under oxidative stress-triggered phosphorylation: "Collectively, the results suggested that Y173D but not T171D phosphomimic mutation is required for the interaction"

Figure 3D clearly shows that wt MoOsm and the mutant MoOsm Y173D do not interact

Appropriate statement would be: phosphorylation of tyrosine 173 inhibits interaction

Figure 3 is a total mess. Legends do not match the Figures and therefore any interpretation from his figure is open to question.

Example: Figure 3 A says: (A) Localization of MoOsm1 and MoOsm1Y173D. The localization of MoOsm1 and MoOsm1Y173D was observed in conidia of the ΔMoosm1 mutants. MoOsm1-GFP and H1-RFP were observed by confocal fluorescence microscopy. Bars = 5μm.

We do not see any localization of MoOsm1 in the nucleus!

In the same legend: A and B is followed by E…..

Then C is refered as a Co-IP assay which clearly it’s not.

3) MoOsm1-triggered MoAtf1 phosphorylation inactivates MoAtf1-MoTup1 Interaction: "We screened MoAtf1-interacting proteins and identified a conserved transcription repressor, MoTup1 (Figure 4—figure supplement 1)."

No explanation or experimental procedures are given with respect to the identification of the interacting proteins. How did they verify that these are genuine interacting proteins?

4) The interaction between MoAtf1 and MoTup1 controls the expression of virulence Factors: "Collectively, the results showed that host-derived ROS accumulation induces the phosphorylation of MoOsm1…."

There is no evidence to support this statement.

5) MoPtps contribute to MoOsm1 dephosphorylation:

"As a hemibiotrophic fungus, M. oryzae also maintains a symbiotic relationship with rice without directly killing it."

The fungus has biotrophic phase but no symbiotic relationship.

" As the pathogen utilizes MoOsm1 phosphorylation as a means to control the interaction of MoAtf1-MoTup1 complex in responding to host immunity"

No evidence for this statement.

"We generated respective ΔMoptp1 and ΔMoptp2 mutant strains"

"independent ΔMoptp1ΔMoptp2 double mutants were also constructed"

No evidence is provided for either construction or validation of these mutant strains.

"We further purified the MoOsm1-GFP protein from the ∆Moptp2/MoOSM1-GFP

strain and found that tyrosine 173 was the hyperphosphorylated (Figure 6—figure supplement 1A)."

You can have protein that is hyperphosphorylated, but you cannot have a residue that is hyperphosphorylated…. Constitutively active is more appropriate

6) Identification of MoPtp1 and MoPtp2 as the targets of MoAtf1

"These results indicated that MoAtf1 regulated the expression of MoPTP1 and MoPTP2 by targeting their promoters directly during the growth and development of M. oryzae."

No evidence is provided to justify this statement.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "Host-derived ROS activates a phosphor-regulatory feedback circuitry to govern virulence in Magnaporthe oryzae" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Detlef Weigel as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

Plant pathogens face the accumulation of reactive oxygen species (ROS) during the colonization of their host. This study investigates the molecular mechanisms by which the fungal pathogen Magnaporthe oryzae responds to ROS during the infection of rice plants. The authors identified a ROS-sensing loop involving protein-protein interactions, subcellular translocation and transcriptional regulation controlled by differential phosphorylation. This thorough investigation proposes a mechanism for restoring fungal cells into a sensitive state after signaling.

Essential revisions:

1) There is no direct evidence that ROS perceived by M. oryzae are of plant origin. The authors are asked to provide experimental support for this claim and discuss putative functions of this ROS-sensing mechanism other than counteracting PTI.

2) The manuscript remains difficult to read due to its length and style. Reviewer 3 has made suggestions to streamline the manuscript. The authors should carefully edit the style of their manuscript and make better use of supplementary materials to simplify the main text.

3) Experimental procedures are not always sufficiently explained, justified, and lacking some controls. Please carefully address the reviewer comments related to this concern appended at the bottom of this message.

Reviewer #1:

The manuscript by Liu et al. is a revised version of a study aiming at deciphering the molecular mechanisms by which the fungal plant pathogen Magnaporthe oryzae copes with reactive oxygen species produced during the host immune response. I found this new version much improved compared to the previous one. The objectives of the work are much clearer, and the additional experiments improved the quality of the Results section.

There is a number of aspects in which the manuscript should be improved further:

1) Although it improved since the previous version, I would advise the authors need to think of a better title. First, whether ROS are produced by the plant during the interaction cannot be unambiguously established due to some limitations in the experimental system. Therefore the "host-derived" nature of ROS should be downplayed and discussed rather than presented as an established fact. Second, the authors show that ROS dissociate Mosm1 dimers, but it not clear how this is achieved. The use of "activates" may not be the most appropriate in this respect. Third, I feel like "phospho-regulatory circuitry" does not convey fully the originality and breadth of the findings from this manuscript.

2) The Discussion section could be improved by A) commenting on the source of ROS in the interaction. Could they be produced by the fungus instead of the plant? Is there any correlation between the expression of ROS-producing enzymes from Rice and the staining observed in Figure 1? B) commenting on how the current work related to the lifestyle and disease progression (e.g. relationship with hemibiotrophic growth) what is the link with either biotrophic or necrotrophic growth? or rather with virulence in general? C) For discussion: What would be the fitness advantage in recycling Osm1 instead of degrading it when phosphorylated?

3) In spite of real progress, there is still a need to carefully check spelling and grammar throughout the manuscript. (For instance: "To examine MoPtp1/2 in virulence" missing "the role of"; "that of the phosphorylated the MoOsm1-GF" too many "the"; "ROS accumulation around caused by" remove "around"; "pathogenicity assay exhibited that the" > demonstrated that)

Reviewer #2:

The work under revision disentangle the signalling pathway that activates ROS tolerance in a fungal pathogen. This pathway is shown to be critical for virulence and modulate plant immunity during infection. This ambitious work pursues to identify and determine the function of various members of the signalling pathway and the role of each of them in the regulation of ROS tolerance. The work substantially contributes to better understanding how virulence is regulated in plant pathogens. However, I consider that the authors should address the following concerns:

The virulence phenotype of MoTup1 does not fit either with the model. If it is a negative regulator, I would have thought that the knockout would be more virulent. There should be a discussion about this.

Figure 3: I think that there are some problems with the controls: Actin is missing in mutant D in Figure 3B. And also RFP is not detected in mutant A in the nuclear fraction

Finally, Materials and methods section is not complete. For example, I could not find the description on how DAB experiments were performed.

Reviewer #3:

In this manuscript the authors characterized a well conserved signaling pathway in rice blast fungus to show that it responds to changes in ROS, which they try to link to ROS production during immunity. The paper has far too many figures for someone to finish reading it in one go. Also it is a bit difficult to read due to the structuring of sentences.

My main concerns are:

1) I don't think their findings really demonstrate this is a signaling cascade evolved to adapt to counteract the ROS produced during infection. I think the data simply shows Osm1 and its regulators respond to ROS changes in environment. To be able to say it is in response to host produced ROS, they need to have rice NADPH Ox mutants, where Osm1 gains full pathogenesis. I assume this is not possible.

2) Since they are talking about plant infection, the data obtained during appressorium development, which are done on cover slips may not be very relevant. Also, the DPI inhibitor treatments could be problematic. Because the fungus also has NADPH oxidases and they are critical for infection. DPI would probably inhibit fungal enzymes as well.

3) Figure 2: Hydrogen peroxide treatments, the ROS response that the authors see happens 24 hours after inoculation. At that point, the mature appressoria are isolated from the rest of the hyphae. They should repeat these ROS treatment experiments in the right time points to see the response that is relevant to their infection conditions.

4) Do we know if the defects in pathogenesis is due to simple growth defects of the mutants? They should present plate growth assays to show that mutants grow similar to the wild type and only have issues during infection.

5) The text needs to be significantly shortened and focused. There is too much information flowing around, which makes it impossible follow the story. Results section

6) Model: the color code is confusing. On the left, the green squares represent rice cells. On the right, they represent fungal cells.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "A host-derived ROS inducible phosphor-regulatory circuitry centering on MoOsm1 governs virulence of Magnaporthe oryzae" for further consideration by eLife. Your revised article has been evaluated by Detlef Weigel (Senior Editor) , a Reviewing Editor and two reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The additional experiments with OsRbohA lines strengthened the manuscript by providing good support for the described mechanism to function in planta. We believe however that the style of the manuscript still requires attention.

To convey the complex set of results as clearly as possible, we would like to suggest the following revisions to the title and Abstract:

Title: "A self-balancing signaling circuit centered on the Osm1 kinase governs adaptive responses to host-derived reactive oxygen species in Magnaporthe oryzae"

Abstract

"The production of reactive oxygen species (ROS) is a ubiquitous defense response in plants. Adapted pathogens evolved mechanisms to counteract the deleterious effects of host-derived ROS and promote infection. How plant pathogens regulate such an elaborate response against ROS burst remains not fully understood. Using the rice blast fungus Magnaporthe oryzae, we uncovered a self-balancing circuit controlling response to ROS in planta and virulence. During infection, ROS induces the phosphorylation the high osmolarity glycerol pathway kinase Osm1 and its translocation to the nucleus. There, Osm1 phosphorylates the transcription factor Atf1 and dissociates Atf1-Tup1 complex. This releases Tup1-mediated transcriptional repression on oxidoreduction-pathway genes and activates the transcription of the Ptp1/2 protein phosphatases. In turn, Ptp1/2 dephosphorylate Osm1, restoring the circuit to its initial state. Balanced interactions among Osm1, Atf1, Tup1, and Ptp1/2 proteins provide a means to counter the ROS burst plant immune response. Our findings thereby reveal new insights into how M. oryzae utilizes a phosphor-regulatory feedback mechanism to face plant immunity during infection."

Please verify that this provides an accurate account of your work, and please correct and amend it as needed.

https://doi.org/10.7554/eLife.61605.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

This manuscript proposes a novel counter-immunity signaling pathway in Magnaporthe oryzae during plant infection. This could represent a major advance but substantial revision would be necessary for this to be so. Overall, I felt that evidence was largely correlative rather than causative, conclusions often drawn from what appear to be rather minor differences, and that due-diligence has not been performed for a lot of experiments. This article needs to be revised by an English copy-editor, as the vast number of grammatical errors makes the article difficult to follow. A lot of information is missing from experimental design as described in the text and in figure legends. P-values are mentioned in figure legends but the statistical test is not described.

The Introduction and Discussion are difficult to follow, as many concepts are inadequately described or synthesized.

We thank the reviewer for the overall positive response to our studies and for the critical but constructive comments. We have addressed all of the concerns raised previously, including data organization and interpretation, experimental design description, and English writing.

Are ROS mutants (rbohd or similar) available in the rice cultivars that can be used to validate the conclusion that it is host-derived ROS being detected by the fungus?

Currently, no ROS defective mutant rice is available to our studies and the common practice in the field has been employing Diphenyleneiodonium (DPI) that inhibits the activity of plant NADPH oxidases, thereby suppressing ROS production (Bolwell et al., 1998; Grant et al., 2000; Zhang et al., 2009). We have routinely treated rice with 0.5 μM DPI to suppress ROS in our studies.

Has it been demonstrated that anti-pp38 and anti-p38 are able to recognize MoOSM1? If so please cite. If not, please demonstrate.

The p38 MAP kinase is the mammalian orthologue of the yeast Hog kinase and the anti-pp38 (CST:9215S) and anti-p38 (CST:9212S) were successfully used for the detection of yeast Hog1. Since MoOsm1 is a Hog1 homolog, we used both antibodies anti-pp38 and anti-p38 to successfully detect MoOsm1 and its phosphorylation. We have since added the description in Materials and methods.

Enhanced phosphorylation in Figure 2A is not clear. Perhaps the authors can provide quantification from multiple blots.

We have repeated the experiments for the quantification (Figure 2D).

Quantification of nuclear:cytoplasmic OsmI1 in Figure 2C-E, and later in Figure 3 is not overly convincing to me. The biological relevance of this sub-cellular movement should be assessed using NES and/or NLS localization/mutant constructs.

Thank you for the suggestion. We have since obtained the MoOsm1-NES strain (which MoOsm1 fused with NES) and provided the localization of both MoOsm1-GFP and MoOsm1-NES strains under H2O2 treatment in Figure 2—figure supplement 1B.

Phospho-memetic D/E mutations are not guaranteed to actually mimic phosphorylation. Did the authors also test phospho-ablative A/F mutations? What data support Osm1-Y173D is phosphomimetic? Same is true for Atf1-S124D presented in Figure 5.

Thank you for the helpful suggestion. In the revised version, MoOsm1 Y173A strain was obtained. And we evaluated the function of MoOsm1Y173A in the localization and dimer’s formation.

We showed that MoOsm1Y173D was accumulated in the nucleus when compared with MoOsm1 (Figure 3A). Phosphorylation of MoOsm1 was further evaluated using Phos-tag gel electrophoresis. MoOsm1 mobility shift was found in phosphatase treated wild-type cells but not in phosphatase inhibitor-treated cells. A similar band shift was observed in cytoplasmic extracts of the phosphatase treated strain. A decreased mobility shift was found in nuclear MoOsm1-GFP when compared to phosphatase treated strain (Figure 3H). Based on these results, we considered that MoOsm1 Y173D is phosphomimic that induces the nuclear localization of MoOsm1.

MoAtf1 regulates the transcription of putative target genes (Figure 6 and Supplementary file 1). We also showed that these genes were induced in MoAtf1S124D, suggesting that MoAtf1 S124D is phosphomimic.

The Phostag gel in Figure 4C is not clear, especially when compared to 4F.

We have since improved the presentation by repeating the experiments (Figure 4C).

In Figure 4E the authors describe “phosphorylation sites” upstream of the NLS: are these just serines, or have they been shown to be phosphorylated? If so, please cite. If not, rephrase.

These are serine and threonine sites. We have revised the statements (Figure 4E).

It is clear that S124 is important for phosphostatus, but there is no evidence that this is due to phosphorylation by Osm1.

We showed that S124 is the important phosphorylation site of MoAtf1 (Figure 4F). We then used in vitro phosphorylation analysis to confirm the relationship between MoOsm1 and MoAtf1S124 (Figure 4—figure supplement 1). The result validated that MoOsm1 phosphorylates MoAtf1. In addition, when S124 is mutated, phosphorylation of MoAtf1 was reduced significantly, suggesting that S124 is also important for MoOsm1 mediated phosphorylation of MoAtf1.

IP controls are lacking, and protein expression should be confirmed for the Y2H assays shown in Figure 5. It is possible that the mutant variants are not expressed.

IP controls including MoOsm1-NES were provided in the revised version (Figure 4A). For the Y2H assay, we performed PCR to verify the transformants growing on SD-Leu-Trp and SD-Leu-Trp-His-Ade plates. We also used co-IP, BiFC, and GST pull down assays to confirm the interactions.

The authors describe “hemibiotrophic fungi” as having a “symbiotic” stage with their plant host. I do not think this is accurate.

We have since revised the statement.

The authors demonstrate that ptp1/2 mutants are similar to ptp2 mutants and make the conclusion that PTP1 and PTP2 have differential functions. Are they expressed similarly?

The expression levels of MoPTP1 and MoPTP2 are shown in Figure 10A. We have improved the description by stating that “Given the possibility that MoPtp1 and MoPtp2 may have redundant functions, independent ∆Moptp1Moptp2 double mutants were constructed whose phenotypes are mostly similar to ∆Moptp2 (Figure 7A and 7B)”.

In Figure 7E the legend describes “in vivo” phosphorylation but this described as being performed on protein extracts in the text. Is this really “in vivo”?

All of the proteins were extracted from M. oryzae, with alkaline phosphatase treatment (in vitro) as control, so we deemed that in vivo is more appropriate for the phosphorylation assay.

What makes a protein/residue hyper-phosphorylated? How can one tell this based on mass spec data without controls? The authors should provide more details about how this MS was performed, and how confident they are in the scores. No Mascot/proteomic confidence scores are provided for these data.

We agree and have since revised the statements by providing additional MS information (Figure 5—figure supplement 1).

The conclusion that PTP-mediated desphosphorylation of Osm1 leads to nuclear export is not supported by direct evidence and is therefore speculative.

We have generated the phosphatase domain deletion mutants of MoPtp1 and MoPtp2 and showed that the phosphatase domains were important for MoPtp1 and

MoPtp2 mediated dephosphorylation and also the nuclear exporting of MoOsm1 (Figure 9).

Reviewer #2:

The manuscript by Liu et al. describes molecular mechanisms by which the fungal plant pathogen Magnaporthe oryzae copes with reactive oxygen species (ROS) produced during the host immune response. It provides a complete view of one particular mechanism involving phosphorylation/dephosphorylation reactions, protein complex assembly and dissociation, protein relocalization to the nucleus and transcriptional regulation. The manuscript is organized with a straightforward logic, but requires extensive language editing to improve clarity. This is a very ambitious manuscript, featuring a lot of diverse experiments, apparently carefully executed, involving many different molecular players. It therefore has great potential to constitute a significant step forward in our understanding of fungal virulence. However, the relatively complex model the authors attempt to build is composed of pieces of evidence that sometimes remain incomplete. Several steps of the reasoning would require more thorough testing for the model to be fully convincing. An effort is also needed to synthecize these findings and present the conceptual novelty arising from the work. My specific major concerns are as follows:

1) I can see that there is a significant progress being made in understanding a particular ROS-response signaling pathway, the amount of work and new insights appear substantial, but the authors fail to unify their findings and convey the new concepts arising from their experimental work. After reading carefully the manuscript several times, I can tell the authors found new mechanisms for Magnaporthe to sense and respond to host-derived ROS, but I miss a clear statement on what the major novelty/originality is. Can the authors summarize the process they discovered in 1 or 2 simple sentences rather than a list of molecular players? The objectives of the study stated by the authors are:

– "How does M. oryzae percept and then overcome ROS stress?" a mechanism is proposed to mitigate the antifungal effect of host-derived ROS is provided, but how ROS are perceived is not directly addressed.

– "how MoOsm1 is linked to MoAp1 or MoAtf1 in stress response is unclear" This is a rather field- or organism-centered question. Some efforts are needed to place the study into a broader perspective an qualify in simple terms the novel concepts and mechanisms discovered.

We thank the reviewer for the very positive comments regarding our study and for the invaluable comments/suggestions. We have since revised the manuscript based on the reviewers’ comments in various areas to improve the clarity of the manuscript.

2) "YFP signal of MoOSM1-cYFP and MoOSM1-nYFP pair was reduced in the nucleus, suggesting that the monomeric form of MoOsm1 is involved in the nuclear localization under the oxidative stress" > Several explanations could fit with this observation, the conclusion is not fully supported. A version of osm1 that remain in the dimer form (maybe a phospho dead mutant of Y173?) should not accumulate in the nucleus.

"Collectively, the results suggested that Y173D but not T171D phosphomimic mutation is required for the interaction"> I would rather conclude that Y173D but not T171D prevents Osm1 oligomer formation

BiFC "the empty vectors used as negative controls" > Better controls would be: Phospho-dead mutants of Osm1 or the Y171D mutant + MoOSM1 wt = positive / MoOsm1Y173D + MoOsm1 wt = negative

Subcellular localisation of MoOsm1Y171D-GFP is not analyzed/commented

We have since revised the statements to improve data interpretation. We have obtained the MoOsm1Y173A strain and showed that MoOsm1 Y173A remains in the dimer form and is located in the cytoplasm upon ROS stress (Figure 2A and 2E). We have also employed the recommended BiFC control to evaluate the formation of MoOsm1 dimer under stress (Figure 2G). The localization of MoOsm1Y173D was revealed in Figure 3A.

3) "MoAtf1 interacts with MoOsm1" and following section> This is lacking a few controls to be fully convincing: what happens if osm is trapped outside the nucleus (nuclear exclusion signal or Y173 phospho dead mutant?) – Since the interaction assay was performed in the absence of ROS stress, what would be its relevance in response to ROS? What would be the function of Osm1 dimer dissociation and relocalization to the nucleus? Is Osm1-Atf1 interaction modified upon ROS stress? Similarly, the interaction between Atf1 and Tup1 has not been tested in the context of response to ROS.

We thank the reviewer for the helpful suggestion. By generating a MoOsm1NES mutant strain, we were able to show that the nuclear localization of MoOsm1 is important for its interaction with MoAtf1 (Figure 4A and 4B). We also assessed the interactions under H2O2 treatment that are also present. We reasoned that ROS may not induce all MoOsm1 into phosphor-MoOsm1. Given that deletion of MoPTP1/2 induces MoOsm1 phosphorylation, similar to being exposed to ROS stress, the interactions between MoOsm1-MoOsm1 and MoAtf1-MoTup1 were detectable in the Moptp1/Moptp2 mutants. The results showed that the deletion of MoPTP2 dissociates MoAtf1 from MoTup1 and MoOsm1 dimer formation is blocked in the ΔMoptp1ΔMoptp2 double mutant (Figure 9D and 9E).

4) "disruption of MoAtf1-MoTup1 complex would reverse the suppression of genes involved in virulence" > as far as I understand, disruption of this complex is achieved through Atf1 phosphorylation (S124D phosphomimic mutant) or deletion of Atf1. However, δ MoATF1 is less virulent than wild type (Figure 4G/H) while there is no Atf1-Tup1 complex and therefore no repression of virulence genes in this strain – Similarly, Tup1 deletion reduced virulence (Chen et al., 2015) – How can we explain these discrepancies? In these experiment, what is the virulence phenotype of Atf1S124D mutants? The reverse controls with phospho-dead mutants of Atf1 would be useful to support the conclusions. In which compartment of the cell (nucleus or cytoplasm) are these interactions happening?

We appreciate the insightful comments. We have assessed pathogenicity of MoAtf1S124D and MoAtf1S124A (Figure 4G and 4H) that failed to show any infection differences between wild type (Guy11) and MoAtf1S124D strains on cultivar LTH. The ΔMoatf1 and ΔMoatf1/MoAtf1S124A mutants exhibited restricted lesions. On cultivar K23, however, wild-type caused few typical lesions (gray spots with brown margins), in contrast to MoAtf1S124D that caused more lesions and larger lesion sizes (Figure 4G and 4H).

Given the function of MoAtf1 and MoTup1 in transcriptional regulation, deletion of MoAtf1 or MoTup1 is expected to result in wide changes in transcription levels. The defects of ΔMoatf1 and ΔMotup1 in pathogenicity may be not only solely dependent on MoAtf1-mediated phosphorylation. The conclusion we draw is that MoAtf1 phosphorylation is important for virulence of M. oryzae.

5) Whether the genes under transcriptional control of the Atf1-Tup1 complex actually play a role in virulence is not described. Do they directly contribute to host-derived ROS detoxification?

Functions of the putative target genes were predicted and those participate in the oxidoreduction pathway were selected (Figure 6). In MoAtf1 ChIP data, we identified several genes orchestrating host-derived ROS immunity, including MoChia1(MGG_08054) that suppresses chitin-triggered ROS burst by binding chitin.

Reviewer #3:

The article "Host-derived ROS activates MoOsm1/MoPtp-dependent phosphorylation of MoAtf1 to orchestra virulence in Magnaporthe oryzae" by Liu et al. propose to demonstrate the role of the MAPK kinase complex MoOsm/MoPtp in the regulation of transcription factors MoAtf1 in the fungus M. oryzae.

Overall, authors have provided evidence to show the role of ROS and its effect on one of the MAPK module, but they do not provide evidence to justify the title.

1) Exogenous application of H2O2 is NOT the same as host derived ROS. Authors have not provided evidence to show that host derived ROS can drive the changes in the MAPK complexes in the fungus outlined in this manuscript. Therefore, Authors should use host mutants that cannot make ROS to justify their claim.

We thank the reviewer for the insightful comments. Due to limitations in obtaining ROS defective rice cultivars, the research field traditionally employs 0.5 μM diphenyleneiodonium (DPI) to inhibit the activity of plant NADPH oxidases and thereby suppress ROS (Bolwell et al., 1998; Grant et al., 2000; Zhang et al., 2009). We also used DPI to suppress ROS generation in several of our previous studies. Here by treating rice (cultivar K23) with DPI, we found that MoOsm1 phosphorylation was significantly reduced at 24 hpi and 36 hpi (Figure 2C and Results section).

2) Authors are aware that fungi have an equivalent ROS producing machinery that can be inhibited by both DPI and catalase (these are not specific to plant NADPH oxidases as claimed by the authors.

True; both DPI and catalase were applied to rice sheaths prior to being inoculated with M. oryzae conidia to minimize fungal exposure to DPI/catalase.

3) Authors describe PTI in their Introduction. How and more importantly when is ROS produced during PTI in a suc (LTH) vs res (K23) interaction? They need to define PTI in their pathosystem.

Previous studies have shown that a moderate resistant cultivar-strain interaction, such as that between K23 and Guy11, produces few and restricted lesions (Liu et al., 2018; Yin et al., 2019b). As NADPH oxidase–mediated ROS production is one of the earliest PTI responses to pathogens, DAB staining is often used to estimate ROS accumulation. Rice cultivar K23 infected with Guy11 yielded reddish-brown precipitates around appressoria and infected hypha at 24, 36, 48, and 60 hpi. Over 40% of infected cells were stained brown at 24 hpi and/or 36 hpi (Figure 1B and 1C).

4) Authors need to detail their experimental protocols more thoroughly and edit the figure and figure legends so that they match.

The reviewer questions many of the experimental procedures used:

1) While the Co-IPs are done in hyphae and other time they use protoplasts; many of the localization studies are done in conidia. The authors do not make any distinction between these two. Conidia and hyphae are two distinct developmental stages of fungus as demonstrated by the authors in Figure 10 A and authors cannot use interchangeably these two development stages to support their hypothesis

We agreed that conidia and hyphae are the two distinct developmental stages. We used conidia for localization studies because of easy visualization; however, protein extraction from conidia was technically challenging and we thereby used hyphae as the source of protein. Interaction verification by BiFC indicated consistency between conidia and hyphae.

2) With respect to using protoplasts: where the Co-IPs performed from plasmids expressed transiently? No experimental procedure is described with respect to preparation, transformation, and efficiency.

Protoplasts were used for transformation and positive transformants were verified prior to protein extraction. We have provided more details in the Materials and methods section.

3) What development stage of hyphae was used to extract proteins to perform Co-IPs. How much protein was used in each of the IP experiments?

48 hours in liquid CM; this is considered active growing stages for fungal hyphae.

4) Authors do not provide evidence that validates any of the constructs used in this manuscripts.

We have improved the description in the Materials and methods section.

5) Monomer/Dimer (Figure 3E). should document this by gel-filtration chromatography and the native gel does NOT adequately demonstrate this.

We have performed a gel-filtration chromatography assay and the result is shown in Figure 3F.

6) Phosphorylation of MoATF-1 (Figure 4C) – not very convincing (compare 4c to 4F);

Should use ΔMoosm in 4G.

We have since repeated the experiment and an improved Figure 4C is presented.

7) EMSA experiments: it is not appropriate it to use a 1 Kb fragment to perform gel shift analysis. An oligomer with the binding site and a mutated binding site should be used.

We have repeated the experiment with the proper marker, as suggested (Figure 10G and 10H).

8) The legend for Figure 9A indicates 100 infecting hyphae were counted. How many of those were used for DAB staining in 9C?

50 infecting hyphae were counted per replicate for the DAB assay. We have added this information to the legend.

Figures with no explanation or details:

1) Figure 3F: the figure clearly show conidia, but the text refers to it as protoplasts.

We used conidia for protein localization and since corrected the error we made previously.

2) Figure 1H is referred in text as bulbous structure at 24hrs; not identified in the figure or legend

We have revised the statements in the legend.

3) Figure 4H; Figure 5B; middle lane in Figure 7D; what are type 1.…typ4 infection in Figure 9A and 9B?

Figure 4H, Figure 5B, and Figure 7D have been since revised. We have also added more details in the legends.

The reviewer questions some of the statements made in this manuscript:

Results section:

1) MoOsm1 was phosphorylated in response to oxidative stress: "These results suggested that MoOsm1 responds to oxidative stress by inducing a high phosphorylated level and increased accumulation in the nucleus."

This statement should be supported by Western with p-p38 Abs in Figure 2E.

We appreciate the suggestion. The decreased mobility of MoOsm1-GFP purified from the nucleus was observed when compared to the phosphatase treated strain, indicating a higher level of MoOsm1 phosphorylation in the nucleus (Figure 3H). These results suggest that MoOsm1 could be phosphorylated in the nucleus.

2) MoOsm1 is reduced from a dimer to a monomer under oxidative stress-triggered phosphorylation: "Collectively, the results suggested that Y173D but not T171D phosphomimic mutation is required for the interaction"

We have revised the statements.

Figure 3D clearly shows that wt MoOsm and the mutant MoOsm Y173D do not interact

Appropriate statement would be: phosphorylation of tyrosine 173 inhibits interaction

We have revised the statements.

Figure 3 is a total mess. Legends do not match the Figures and therefore any interpretation from his figure is open to question.

Example: Figure 3 A says: (A) Localization of MoOsm1 and MoOsm1Y173D. The localization of MoOsm1 and MoOsm1Y173D was observed in conidia of the ΔMoosm1 mutants. MoOsm1-GFP and H1-RFP were observed by confocal fluorescence microscopy. Bars = 5μm.

We do not see any localization of MoOsm1 in the nucleus!

In the same legend: A and B is followed by E…..

Then C is refered as a Co-IP assay which clearly it’s not.

We have revised the statements. We have provided Western blot analysis and the fluorescence intensity assay to evaluate the localization of MoOsm1 under H2O2 treatment (Figure 3).

3) MoOsm1-triggered MoAtf1 phosphorylation inactivates MoAtf1-MoTup1

Interaction: "We screened MoAtf1-interacting proteins and identified a

conserved transcription repressor, MoTup1 (Figure 4—figure supplement 1)."

No explanation or experimental procedures are given with respect to the identification of the interacting proteins. How did they verify that these are genuine interacting proteins?

All of the proteins were identified by utilizing the affinity purification approach. Following affinity purification, proteins bound to anti-GFP beads were eluted and analyzed by mass spectrometry (MS). We have provided more information on MoAtf1-interaction proteins in Figure 5—figure supplement 1.

4) The interaction between MoAtf1 and MoTup1 controls the expression of virulence

Factors: "Collectively, the results showed that host-derived ROS accumulation

induces the phosphorylation of MoOsm1…."

There is no evidence to support this statement.

We agree and have assessed MoOsm1 phosphorylation during infection under DPI treatment. The results showed that, when K23 was treated with 0.5 μM DPI, MoOsm1 phosphorylation was significantly attenuated at 24 hpi and 36 hpi (Figure 2C and Results section).

5) MoPtps contribute to MoOsm1 dephosphorylation:

We have performed a series of experiments to support this conclusion. We generated phosphatase activity inactivation mutants of MoPtp1 and MoPtp2 (∆Moptp1/MoPTP1ptpc and ∆Moptp2/MoPTP2ptpc mutant) and evaluated their MoOsm1 phosphorylation pattern. The results showed that in both ∆Moptp1/MoPTP1ptpc and ∆Moptp2/MoPTP2ptpc mutants, the band of MoOsm1-GFP migrated as the phosphorylated MoOsm1-GFP protein treated with an inhibitor (also in ∆Moptp1/2 mutants (Figure 9B)), indicating that the phosphatase activity is critical for the dephosphorylation of MoOsm1. We also detected the phosphorylation levels of MoOsm1 using the antiphospho-p38 antibody, and observed an increased MoOsm1 phosphorylation in both ΔMoptp1 and ΔMoptp2 mutants (Figure 7—figure supplement 2). In addition, the phosphorylation of MoOsm1 remains at high levels even after H2O2 treatment at 60 minutes in the ΔMoptp2 mutants, while the wild type decreased to a low level at 10 minutes (Figure 7—figure supplement 2). We then observed the localization of MoOsm1 in the ΔMoptp1, ΔMoptp2,Moptp1/MoPTP1ptpc, and ∆Moptp2/MoPTP2ptpc mutants. MoOsm1 was present in both the cytosol and the nucleus evenly, similar to that in wild type. When treated with H2O2, MoOsm1 shows an enhanced nuclear translocation pattern. At 30 minutes following H2O2 treatment, MoOsm1 shows a nucleus to cytoplasm shifting in the wild type strain but not in the ΔMoptp2 and ∆Moptp2/MoPTP2ptpc mutants (Figure 9C). These results further supported that MoPtp1/2-mediated dephosphorylation of MoOsm1 controls its nuclear-cytoplasm translocation.

"As a hemibiotrophic fungus, M. oryzae also maintains a symbiotic relationship

with rice without directly killing it."

The fungus has biotrophic phase but no symbiotic relationship.

We have corrected the error.

"As the pathogen utilizes MoOsm1 phosphorylation as a means to control the interaction of MoAtf1-MoTup1 complex in responding to host immunity"

No evidence for this statement.

We have made the proper correction.

"We generated respective ΔMoptp1 and ΔMoptp2 mutant strains"

"independent ΔMoptp1ΔMoptp2 double mutants were also constructed"

No evidence is provided for either construction or validation of these mutant strains.

Southern blot analysis was performed for both ΔMoptp1 and ΔMoptp2 mutant strains (Figure 6—figure supplement 1).

"We further purified the MoOsm1-GFP protein from the ∆Moptp2/MoOSM1-GFP

strain and found that tyrosine 173 was the hyperphosphorylated (Figure 6—figure supplement 1A)."

You can have protein that is hyperphosphorylated, but you cannot have a residue that is hyperphosphorylated…. Constitutively active is more appropriate

We have made the proper correction.

6) Identification of MoPtp1 and MoPtp2 as the targets of MoAtf1

"These results indicated that MoAtf1 regulated the expression of MoPTP1 and MoPTP2 by targeting their promoters directly during the growth and development of M. oryzae."

No evidence is provided to justify this statement.

We have revised the statements to “These results indicated that MoAtf1 regulates the expression of MoPTP1 and MoPTP2 by directly targeting their promoters”.

[Editors’ note: what follows is the authors’ response to the second round of review.]

Essential revisions:

1) There is no direct evidence that ROS perceived by M. oryzae are of plant origin. The authors are asked to provide experimental support for this claim and discuss putative functions of this ROS-sensing mechanism other than counteracting PTI.

We appreciate the critical comments. Previous studies demonstrate that RbohA functions as the NADPH oxidase critical for ROS generation in rice, and OsRbohA-overexpressing transgenic plants exhibit higher ROS production (Wang et al., 2016). We have provided the evidence that MoOsm1 phosphorylation levels were induced in the OsRbohA mutant (Figure 2D). And we have also shown that the treatment of DPI, which inhibits ROS generation in rice, resulted in a significant decrease in MoOsm1 phosphorylation (Figure 2C). These results revealed that MoOsm1 indeed responds to ROS stress during infection by M. oryzae. We have improved relevant statements in the revised version.

2) The manuscript remains difficult to read due to its length and style. Reviewer 3 has made suggestions to streamline the manuscript. The authors should carefully edit the style of their manuscript and make better use of supplementary materials to simplify the main text.

We appreciate the helpful suggestion and have improved manuscript presentation through English language editing.

3) Experimental procedures are not always sufficiently explained, justified, and lacking some controls. Please carefully address the reviewer comments related to this concern appended at the bottom of this message.

We have improved the presentation by adding additional descriptions and literature citation.

Reviewer #1:

The manuscript by Liu et al. is a revised version of a study aiming at deciphering the molecular mechanisms by which the fungal plant pathogen Magnaporthe oryzae copes with reactive oxygen species produced during the host immune response. I found this new version much improved compared to the previous one. The objectives of the work are much clearer, and the additional experiments improved the quality of the Results section.

There is a number of aspects in which the manuscript should be improved further:

1) Although it improved since the previous version, I would advise the authors need to think of a better title. First, whether ROS are produced by the plant during the interaction cannot be unambiguously established due to some limitations in the experimental system. Therefore the "host-derived" nature of ROS should be downplayed and discussed rather than presented as an established fact. Second, the authors show that ROS dissociate Mosm1 dimers, but it not clear how this is achieved. The use of "activates" may not be the most appropriate in this respect. Third, I feel like "phospho-regulatory circuitry" does not convey fully the originality and breadth of the findings from this manuscript.

Thank you for the helpful suggestion. Following new evidence of OsRboh overexpression inducing ROS generation and MoOsm1 phosphorylation, we thought that a “inducible” may better depict MoOsm1-MoAtf1-MoPtps in the phosphor-regulatory process, and thereby propose “A host-derived ROS inducible phosphor-regulatory circuitry centering on MoOsm1 governs virulence of Magnaporthe oryzae” as an updated title.

2) The Discussion section could be improved by A) commenting on the source of ROS in the interaction. Could they be produced by the fungus instead of the plant? Is there any correlation between the expression of ROS-producing enzymes from Rice and the staining observed in Figure 1? B) commenting on how the current work related to the lifestyle and disease progression (e.g. relationship with hemibiotrophic growth) what is the link with either biotrophic or necrotrophic growth? or rather with virulence in general? C) For discussion: What would be the fitness advantage in recycling Osm1 instead of degrading it when phosphorylated?

We appreciate the insightful comments. In the revised Discussion section, we discussed the following aspects:

A) For the pathogen, ROS seems like a double-edged sword. During appressorial penetration, M. oryzae accumulates high levels of endogenous ROS to strengthen the appressorium cell wall. Here, ROS accumulation is regulated by two fungal NADPH oxidases, Nox1 and Nox2, which themselves are important for appressorium-mediated cuticle penetration. However, ROS is also one of the earliest responses by plants to microbial colonization. This oxidative burst function a potent defense mechanism that limits fungal biotrophic growth (Torres, 2010). Rboh, which encodes NADPH oxidase, is the key host protein that generates ROS upon the pathogen challenge (Wong et al., 2007). Both OsrbohA and OsrbohB are important for this process (Wong et al., 2007). We have updated the relevant description.

B) During the interaction between M. oryzae and the host, the host induces ROS burst during the biotrophic growth stage of the fungus. Once sensing this stress, the pathogen activates MoOsm1-mediated pathways that in turn activates the transcription factor MoAtf1. The phosphorylation of MoAtf1 causes the disintegration of the MoAtf1-MoTup1 complex to induce the expression of oxidation regulation pathway genes to further enhance virulence of M. oryzae. Phosphorylated MoAtf1 also initiates the transcription of the MoPTP1/2 genes to further dephosphorylate MoOsm1. Once the ROS stress was circumvented, such as that at the necrotrophic growth stage, MoPtps mediated dephosphorylation of MoOsm1 shut off phosphor-regulatory circuitry to control the virulence.

C) Given the evidence indicating that the nuclear localization of MoOsm1 was more stable in the ΔMoptps mutant than Guy11 under ROS stress, it is plausibly that MoPtps function in the recycling of MoOsm1 to the cytoplasm. MoOsm1 recycling may have two benefits: 1) shutdown of phosphorylated MoOsm1 mediates signaling pathways that switch off virulence attack. 2) recycled MoOsm1 in the cytoplasm might respond quickly to external stress as there is no need for protein resynthesing. We have updated the relevant statements.

3) In spite of real progress, there is still a need to carefully check spelling and grammar throughout the manuscript. (For instance: "To examine MoPtp1/2 in virulence" missing "the role of"; "that of the phosphorylated the MoOsm1-GF" too many "the"; "ROS accumulation around caused by" remove "around"; "pathogenicity assay exhibited that the" > demonstrated that)

We have done so in the revised manuscript.

Reviewer #2:

The work under revision disentangle the signalling pathway that activates ROS tolerance in a fungal pathogen. This pathway is shown to be critical for virulence and modulate plant immunity during infection. This ambitious work pursues to identify and determine the function of various members of the signalling pathway and the role of each of them in the regulation of ROS tolerance. The work substantially contributes to better understanding how virulence is regulated in plant pathogens. However, I consider that the authors should address the following concerns:

The virulence phenotype of MoTup1 does not fit either with the model. If it is a negative regulator, I would have thought that the knockout would be more virulent. There should be a discussion about this.

Studies revealed that Tup1 is a critical transcription repressor that does not bind directly to DNA but is brought to the promoters via interactions with sequence-specific regulatory proteins to regulate the expression of genes. Here, the disrupted interaction between MoAtf1 and MoTup1 induces the expression of various genes. However, MoTup1 may regulate various other transcription factors in addition to MoAtf1, so it is plausible that the deletion of MoTUP1 causes defect in virulence expression.

Figure 3: I think that there are some problems with the controls: Actin is missing in mutant D in Figure 3B. And also RFP is not detected in mutant A in the nuclear fraction

Finally, Materials and methods section is not complete. For example, I could not find the description on how DAB experiments were performed.

We have provided additional information in the Materials and methods, including the DAB assay.

Reviewer #3:

In this manuscript the authors characterized a well conserved signaling pathway in rice blast fungus to show that it responds to changes in ROS, which they try to link to ROS production during immunity. The paper has far too many figures for someone to finish reading it in one go. Also it is a bit difficult to read due to the structuring of sentences.

My main concerns are:

1) I don't think their findings really demonstrate this is a signaling cascade evolved to adapt to counteract the ROS produced during infection. I think the data simply shows Osm1 and its regulators respond to ROS changes in environment. To be able to say it is in response to host produced ROS, they need to have rice NADPH Ox mutants, where Osm1 gains full pathogenesis. I assume this is not possible.

Thank you for the helpful suggestion. OsRbohA is an important NADPH oxidase critical for ROS generation in rice. Here, Prof. Kunming Cheng kindly provided us with the OsRbohA over-expression strain that allowed us to detect MoOsm1 phosphorylation during the infection (Figure 2D and subsection “MoOsm1 phosphorylation in response to oxidative stress”).

2) Since they are talking about plant infection, the data obtained during appressorium development, which are done on cover slips may not be very relevant. Also, the DPI inhibitor treatments could be problematic. Because the fungus also has NADPH oxidases and they are critical for infection. DPI would probably inhibit fungal enzymes as well.

Thank you for the help comments. M. oryzae produces the appressorium during infection and the evidence we provided were performed on rice infected with M. oryzae for 8 h (Figure 2). To further confirm the result on appressorium development, we provided the results at 20 hpi post infection on the revised version. We used the DPI inhibitor to treat rice sheath before infection with M. oryzae, and we agree that the fungus also produces NADPH oxidases critical for infection too. However, we showed that the wild type infection hyphae could expend into adjacent rice cells when treated with 0.5 μm DPI, indicating that DPI causes no defects on the infection ability of M. oryzae.

3) Figure 2: Hydrogen peroxide treatments, the ROS response that the authors see happens 24 hours after inoculation. At that point, the mature appressoria are isolated from the rest of the hyphae. They should repeat these ROS treatment experiments in the right time points to see the response that is relevant to their infection conditions.

We agreed that hydrogen peroxide treatment was not similar to that of infection. At 24 hpi, M. oryzae penetrated the epidermis and infected the host cells (Figure 1). As it is difficult to treat with rice sheath cell with H2O2 in vitro, we have provided the evidence that the overexpression line of OsRboha shows significant induced MoOsm1 phosphorylation levels than that of WT, indicating that ROS treatment induces MoOsm1 phosphorylation. In addition, we tried to treat appressoria with 5mM H2O2, however, it destroys appressoria. We thereby provide the results at 20 hpi post infection to confirm the phosphorylation levels of MoOsm1 at the appressorial stages.

4) Do we know if the defects in pathogenesis is due to simple growth defects of the mutants? They should present plate growth assays to show that mutants grow similar to the wild type and only have issues during infection.

We have provided the evidence through revision (Figure 6—figure supplement 2).

5) The text needs to be significantly shortened and focused. There is too much information flowing around, which makes it impossible follow the story.

We appreciate the helpful suggestions and have updated relevant descriptions, including the title.

6) Model: the color code is confusing. On the left, the green squares represent rice cells. On the right, they represent fungal cells.

Revised.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

The additional experiments with OsRbohA lines strengthened the manuscript by providing good support for the described mechanism to function in planta. We believe however that the style of the manuscript still requires attention.

To convey the complex set of results as clearly as possible, we would like to suggest the following revisions to the title and Abstract:

Title: "A self-balancing signaling circuit centered on the Osm1 kinase governs adaptive responses to host-derived reactive oxygen species in Magnaporthe oryzae"

Abstract

"The production of reactive oxygen species (ROS) is a ubiquitous defense response in plants. Adapted pathogens evolved mechanisms to counteract the deleterious effects of host-derived ROS and promote infection. How plant pathogens regulate such an elaborate response against ROS burst remains not fully understood. Using the rice blast fungus Magnaporthe oryzae, we uncovered a self-balancing circuit controlling response to ROS in planta and virulence. During infection, ROS induces the phosphorylation the high osmolarity glycerol pathway kinase Osm1 and its translocation to the nucleus. There, Osm1 phosphorylates the transcription factor Atf1 and dissociates Atf1-Tup1 complex. This releases Tup1-mediated transcriptional repression on oxidoreduction pathway genes and activates the transcription of the Ptp1/2 protein phosphatases. In turn, Ptp1/2 dephosphorylate Osm1, restoring the circuit to its initial state. Balanced interactions among Osm1, Atf1, Tup1, and Ptp1/2 proteins provide a means to counter the ROS burst plant immune response. Our findings thereby reveal new insights into how M. oryzae utilizes a phosphor-regulatory feedback mechanism to face plant immunity during infection."

Please verify that this provides an accurate account of your work, and please correct and amend it as needed.

Thank you for the critical comment on the title and Abstract. We agreed that the new title and Abstract convey the results much more clearly. In view of the word limitation of eLife, we made some modification to the title into “A self-balancing circuit centered on MoOsm1 kinase governs adaptive responses to host-derived ROS in Magnaporthe oryzae”.

In the Abstract, the new Abstract provides the accurate account of our work. As we used the M. oryzae as a model organism, we thought MoOsm1(MoAtf1, MoTup1 and MoPtp) should instead of Osm1(Atf1, Tup1 and Ptp) in the Abstract. We have revised the text.

https://doi.org/10.7554/eLife.61605.sa2

Article and author information

Author details

  1. Xinyu Liu

    Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    Contribution
    Conceptualization, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing - original draft, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
  2. Qikun Zhou

    Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    Contribution
    Conceptualization, Data curation, Software, Formal analysis, Funding acquisition, Validation, Visualization, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
  3. Ziqian Guo

    Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    Contribution
    Data curation, Software, Formal analysis, Validation, Visualization, Methodology, Writing - original draft
    Competing interests
    No competing interests declared
  4. Peng Liu

    Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    Contribution
    Data curation, Software, Formal analysis, Validation, Visualization, Methodology
    Competing interests
    No competing interests declared
  5. Lingbo Shen

    Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    Contribution
    Data curation, Software, Formal analysis, Validation, Methodology
    Competing interests
    No competing interests declared
  6. Ning Chai

    Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    Contribution
    Data curation, Software, Formal analysis, Validation, Methodology
    Competing interests
    No competing interests declared
  7. Bin Qian

    Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    Contribution
    Data curation, Software, Validation, Investigation, Methodology
    Competing interests
    No competing interests declared
  8. Yongchao Cai

    Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    Contribution
    Software, Validation, Investigation, Methodology
    Competing interests
    No competing interests declared
  9. Wenya Wang

    Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    Contribution
    Software, Validation, Investigation, Methodology
    Competing interests
    No competing interests declared
  10. Ziyi Yin

    Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    Contribution
    Conceptualization, Resources, Software, Validation, Writing - review and editing
    Competing interests
    No competing interests declared
  11. Haifeng Zhang

    1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    2. The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, China
    Contribution
    Conceptualization, Resources, Formal analysis, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
  12. Xiaobo Zheng

    1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    2. The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, China
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
  13. Zhengguang Zhang

    1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
    2. The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, China
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Project administration, Writing - review and editing
    For correspondence
    zhgzhang@njau.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8253-4505

Funding

Natural Science Foundation of China (31972979)

  • Xinyu Liu

Mobility program of NSFC and DFG (31861133017)

  • Zhengguang Zhang

Natural Science Foundation of China (31671979)

  • Xiaobo Zheng

InnovationTeam Program for NSFC (2017)

  • Zhengguang Zhang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This research was supported by the program of Natural Science Foundation of China (Grant No: 31972979, LX), NSFC-DFG (Grant No: 31861133017, ZZ), Natural Science Foundation of China (Grant No: and 31671979, ZX), and Innovation Team Program for NSFC (2017). We are grateful for Prof. Kunming Chen (North West Agriculture and Forestry University) and Prof. Mo Wang (Fujian Agriculture and Forestry University), who kindly provided the transgenic rice materials, including the OsRhobA-ox line. We also acknowledge professor Ping Wang for his critical comments during the preparation of this report.

Senior Editor

  1. Detlef Weigel, Max Planck Institute for Developmental Biology, Germany

Reviewing Editor

  1. Sylvain Raffaele

Publication history

  1. Received: August 10, 2020
  2. Accepted: November 26, 2020
  3. Version of Record published: December 4, 2020 (version 1)

Copyright

© 2020, Liu et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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