De‐nitrosylation Coordinates Appressorium Function for Infection of the Rice Blast Fungus

Abstract As a signaling molecule, nitric oxide (NO) regulates the development and stress response in different organisms. The major biological activity of NO is protein S‐nitrosylation, whose function in fungi remains largely unclear. Here, it is found in the rice blast fungus Magnaporthe oryzae, de‐nitrosylation process is essential for functional appressorium formation during infection. Nitrosative stress caused by excessive accumulation of NO is harmful for fungal infection. While the S‐nitrosoglutathione reductase GSNOR‐mediated de‐nitrosylation removes excess NO toxicity during appressorium formation to promote infection. Through an indoTMT switch labeling proteomics technique, 741 S‐nitrosylation sites in 483 proteins are identified. Key appressorial proteins, such as Mgb1, MagB, Sps1, Cdc42, and septins, are activated by GSNOR through de‐nitrosylation. Removing S‐nitrosylation sites of above proteins is essential for proper protein structure and appressorial function. Therefore, GSNOR‐mediated de‐nitrosylation is an essential regulator for appressorium formation. It is also shown that breaking NO homeostasis by NO donors, NO scavengers, as well as chemical inhibitor of GSNOR, shall be effective methods for fungal disease control.


Introduction
As an intriguing signal molecule, NO controls significant biological processes, such as signal transduction, response to stress, and development. [1]It can cause nitrosylation, nitrosative stress, and apoptosis through its target proteins.The balance of NO content in organisms is essential for maintaining normal cell growth. [2]or example, NO acts as a key regulator of fungal morphology, pathogen, and host interaction in fungi. [3]However, the detailed DOI: 10.1002/advs.202403894 regulatory mechanism of NO during these processes remains largely unknown.
S-nitrosylation is a redox-based posttranslational modification (PTM), in which the covalent attachment of a nitric oxide (NO) moiety to a reactive cysteine thiol of a protein to form an S-nitrosothiol (SNO). [4]he degree of S-nitrosylation of proteins in cells is largely determined by the level of S-nitrosoglutathione (GSNO), which is the main bioactive NO donor in living organisms.GSNO acts as a stable and circulating NO reservoir, efficiently transmitting NO signals, and partially transferring it to cysteine thiol groups, leading to S-nitrosylation. [5]Emerging evidence indicates that S-nitrosylation produces significant conformational changes in proteins, therefore plays a crucial role in regulating protein activities such as stability, biochemical activity, subcellular localization, and protein-protein interactions. [6]hus, protein S-nitrosylation provides a basis for physiological regulation based on changes in the redox status of cells. [7]he imbalance of NO content can cause the change of Snitrosylation modification level of many proteins, which eventually lead to abnormal cell function.S-nitrosoglutathione reductase (GSNOR), also known as class III alcohol dehydrogenase (ADH5) or glutathione-dependent formaldehyde dehydrogenase (FALDH), is believed to regulate the availability of active NO in cells by converting GSNO, the reaction product of glutathione (GSH) and NO, to ammonia (NH3) and glutathione disulfide (GSSG).Thus, it can protect the body from the influence of Snitrosylation stress. [8]The direct effect of NO on cellular pathways and the important regulation of protein S-nitrosylation are closely related to GSNOR regulation, and an increasing number of studies have identified this enzyme as an important target for the treatment of human diseases. [9]n recent years, many proteins have been identified as targets of S-nitrosylation in various species. [10]Biological functions regulated by S-nitrosylation of some important plant proteins have also been reported, such as GAPDH, NPR1, transcription factor TGA1, and NADPH oxidase. [11]10a,12] A large number of S-nitrosylated modified proteins have been identified in plants using biotin switch assay, nano Figure 1.NO regulates the formation of functional appressorium.A) Appressorium formation rates of the wild-type (WT) strains after treatment with 5 mm SNP or cPTIO.The spore suspensions treated with 5 mm SNP or cPTIO were incubated on the hydrophobic surface, and the appressarium formation rate was measured at 24 h.Data presented are the mean ± standard errors from three biological replicates (n = 3), and asterisks represent significant differences (** P < 0.01).B) Observation of appressorium turgor pressure.Conidial suspension droplets of wild-type (WT) strains treated with SNP or cPTIO were placed on the hydrophobic surface of a coverslip and treated with different concentrations of polyethylene glycol 8000 (PEG8000) at 24 h post-inoculation (hpi).C) Statistics of lipid percentage in conidia or appressarium during lipid development in wild-type (WT) strains treated with SNP or cPTIO.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the WT are indicated by an asterisk (** P < 0.01).D) Statistics of lipid percentage in conidia or appressarium during glycogen development in wild-type (WT) strains treated with SNP or cPTIO.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the WT are indicated by an asterisk (** P < 0.01).E) Observation on the formation of appressorium septin ring after treatment with 5 mm SNP or cPTIO.Bar, 5 μm.F) Percentage of septin-ring formation in wild-type (WT) strains treated with 5 mm SNP or cPTIO.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the WT are indicated by an asterisk (*** P < 0.001).
10b,13] However, the function of S-nitrosylation and its regulatory mechanism in fungi has not been well revealed.
The filamentous fungus Magnaporthe oryzae is a destructive pathogen affecting the yield of cultivated rice worldwide. [14]M. oryzae can penetrate the host epidermis by forming a special infection structure that called appressorium.Formation of functional appressorium and expansion of infectious hyphae are the key steps for M. oryzae to invade the host.Zhang et al. previously reported that SFA1, a GSNO reductase, regulates NO stress and formaldehyde detoxification in M. oryzae. [15]However, the mechanism of how GSNOR removes excess NO toxicity in fungal cells remains unknown.In particular, the importance of Snitrosylation during infection process is unknown.
In the present study, we set out to uncovered the regulatory mechanism of S-nitrosylation during M. oryzae infection.We found that NO-triggered S-nitrosylation played a negative role in appressorium formation, while the GSNOR-mediated de-nitrosylation played a positive role in this stage.Then we established a large-scale nitrosoproteomic analysis to identify S-nitrosylated proteins in M. oryzae.Some appressoriumassociated key proteins were proved to be de-nitrosylated by GSNOR.Notably, functions of the four septins were blocked by S-nitrosylation, which was removed by GSNOR for septin ring formation, and required for function of the appressorium.In conclusion, our findings reveal a conserved mechanism of GSNORmediated de-nitrosylation in the formation of fungal infection structure, and disrupting the NO homeostasis may be a feasible strategy for controlling diverse diseases in plants.

Excess NO is Harmful for Functional Appressorium Formation of M. oryzae
To explore the possible role of NO in M. oryzae, we detected the effect of NO on development and infection process of the wild-type strain P131.Sodium nitroprusside (SNP) is an effective NO donor that releases NO into the cell. [16]We treated the conidia suspensions of M. oryzae on hydrophobic surface with 5 mM SNP, which resulted in a significant reduction of appressorium formation ratio (Figure 1A).SNP-treatment also led to form immature appressoria, demonstrated by reduced turgor formation (Figure 1B), lipid and glycogen utilization (Figure 1C,D; Figure S1A,B, Supporting Information), and abnormal septin ring formation (Figure 1E,F).

Identification of an S-nitrosoglutathione Reductase GSNOR in M. oryzae
In order to infect the host successfully, M. oryzae must developed NO detoxification mechanism for appressorium formation.An S-nitrosoglutathione (GSNO) reductase (GSNOR) converts GSNO to ammonia (NH3) and glutathione disulfide (GSSG) is commonly used for nitrosative stress detoxification in eukaryotes (Figure 2A).It has been revealed that the GSNOR encodes a S-nitrosoglutathione reductase involved in the regulation of S-nitrosylation in Arabidopsis and humans. [18]To investigate appressorial NO detoxification mechanism in M. oryzae, we identified the homologous protein of GSNOR in M. oryzae (MGG_06011), which contains an alcohol dehydrogenase domain (ADH) at the N terminus and another ADH domain with a zinc finger at the C terminus (Figure S2A, Supporting Information).GSNOR is highly conserved in various pathogenic fungi, as well as in bacteria Escherichia coli, Saccharomyces cerevisiae, A. thaliana, Oryza sativa, Drosophila melanogaster, mammals and humans (Figure S2B, Supporting Information).We successfully disrupted the M. oryzae GSNOR gene in the wild-type strain P131 through a split-PCR method (Figure S3A, Supporting Information).The transformants were screened by PCR-mediated method, and RT-PCR results showed that two mutants (KO1 and KO2) were lack of GSNOR (Figure S3B, Supporting Information).The complementary transformants were also successfully obtained by introducing GSNOR driven by its native promoter into the Δgsnor mutant.
We detected expression profile of GSNOR in different developmental stages and infection processes.GSNOR was stably expressed at all stages, with a significant increase in AP_12 h, and the highest expression at IH_24 h and IH_48 h (Figure 2B).These data suggest that GSNOR could play more important roles in appressorium and infectious hyphae of M. oryzae.GFP-GSNOR and the mitochondrial marker Mito-tracker partially colocalize in the mycelium and conidia (Figure S4, Supporting Information), indicating that GSNOR mainly localizes in the mitochondria and cytoplasm, consistent with its predicted function as a class III alcohol dehydrogenase. [19]

GSNOR is Required for Equilibrium of NO in M. oryzae
To explore the possible role of GSNOR in NO detoxification process, we observed NO level of the wild-type strain and Δgsnor mutant by DAF-FM (4-amino-5-methylamino-2,7-difluorofluorescein diacetate) staining assay at different developmental stages.3a,20] The results showed that, compared with that of the wild-type strain, NO levels of the Δgsnor mutant in all tested samples, including mycelium, conidium, appressorium, and invasive hypha, were significantly increased (Figure 2C,D).The NO content in the mycelium was also determined using the improved Griess method, and it was found that the NO content in Δgsnor mutant significantly increased (Figure 2E).This suggests the presence of NO at various developmental stages of M. oryzae, indicating the importance of this signaling molecule in the life cycle of M. oryzae.The NO level of the Δgsnor mutant was reduced by addition of 1 mm NO scavenger cPTIO (Figure 2F,G).To investigate whether GSNOR responds to NO level, we examined the expression level of GSNOR after SNP treatment.The qRT-PCR results indicate that NO can induce the expression of GSNOR in M. oryzae (Figure 2H).These results showed that deletion of GSNOR resulted in a significant increase of NO level in M. oryzae, suggesting that GSNOR is required for detoxification of intracellular NO to keep its balance.

NO and GSNOR Conversely Regulates S-nitrosylation
In cells, NO and its main bioactive donor S-nitrosoglutathione (GSNO) can transduce NO signals to a cysteine thiol through S-nitrosylation, [21] while GSNOR proteins are denitrosylases catalyzing de-nitrosylation process and converts GSNO into ammonia and oxidized glutathione (GSSG) (Figure 2a). [22]We hypothesized that the increase of NO level in M. oryzae should lead to a greater degree of S-nitrosylation of proteins.To test this hypothesis, the biotin-switch assay was used in this study to verify the difference in total S-nitrosylation levels.Total proteins of mycelia were extracted for the biotin-switch assay, the biotinylated proteins were then immunoblotted with an anti-biotin antibody to detect the S-nitrosoylation level.The result showed that total S-nitrosylation level in Δgsnor was much higher than that in WT (Figure 2I,J), suggesting that GSNOR was involved in denitrosylation of M. oryzae.
Since SNP and cPTIO can respectively increase and decrease the content of NO in the cells of M. oryzae, we suspect that SNP and cPTIO can also affect the S-nitrosoylation level of M. oryzae.We treated the hyphae of wild-type and Δgsnor with 1 mm SNP, 1 mm cPTIO, and 10 mm H 2 O 2 .The results showed that total S-nitrosylation levels of WT with 1 mm SNP were significantly increased, but evidently decreased in which treated with 1 mm cPTIO (Figure 2K,L).S-nitrosylation levels of Δgsnor treated with 1 mm SNP were also significantly increased, but evidently decreased in which treated with 1 mm cPTIO (Figure 2K,L).Therefore, we conclude that NO and GSNOR positively and negatively regulate S-nitrosylation modification, respectively.Samples of each strain were measured using the ImageJ software.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the WT are indicated by an asterisk (** P < 0.01).E) The concentration of NO in the mycelia of wild-type (WT) and Δgsnor was determined by the improved Griess method.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the WT are indicated by an asterisk (* P < 0.05).F) Observation of NO content in wild-type (WT) and Δgsnor during appressorium after SNP and cPTIO treatment.Bar,10 μm.G) The bar chart shows the fluorescence intensity of DA-FM DA staining at appressoria stage.Samples of each strain were measured using the ImageJ software.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the treatment are indicated by an asterisk (** P < 0.01).H) Expression of the GSNOR gene after SNP treatment in the wildtype, and the ACTIN gene was used as an internal control.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the SNP treatment are indicated by an asterisk (** P < 0.01).I) Total levels of S-nitrosylated proteins in wild-type (WT) and Δgsnor.Hypha at 7 days of age were used as samples for analysis.Asc, Sodium ascorbate; SNOs, S-nitrosylated proteins.J) Band quantification of the immunoblot showing a specific increase of S-nitrosylation for wild-type (WT) and Δgsnor proteins.K) Total levels of S-nitrosylated proteins in wildtype (WT) and Δgsnor after SNP treatment.Hypha at 7 days of age were used as samples for analysis.L) Total levels of S-nitrosylated proteins in wild-type (WT) and Δgsnor after cPTIO treatment.Hypha at 7 days of age were used as samples for analysis.M) Total levels of S-nitrosylated proteins in wild-type (WT) and Δgsnor after H 2 O 2 treatment.Hypha at 7 days of age were used as samples for analysis.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with no treatment are indicated by an asterisk (** P < 0.01).ns, not significant.

GSNOR-Mediated
www.advancedscience.commycelia of WT with 10 mm H 2 O 2 , and stained with DAF FM to detect the NO level.We found that treatment of H 2 O 2 caused an NO burst in wild-type cells (Figure S5A-C, Supporting Information), suggesting that H 2 O 2 stimulates NO production in M. oryzae.The degree of S-nitrosylation of WT was also slightly enhanced in H 2 O 2 condition (Figure 2M).
Since GSNOR-mediated S-nitrosylation is a redox modification, GSNOR may be involved in maintaining redox homeostasis of M. oryzae.Glutathione is either reduced glutathione (GSH) or oxidized glutathione disulfide (GSSG) forms.GSSG is reduced to GSH by glutathione reductase.Reduced glutathione plays an important role in maintaining the proper redox state of sulfhydryl groups in proteins and is a key antioxidant.Our data showed that the content of GSH in ∆gsnor mutant was significantly lower than that of the wild type, and the ratio of GSH/GSSG was also significantly decreased (Figure S5D,E, Supporting Information).Moreover, exogenous GSH did not impact the level or content of NO in M. oryzae (Figure S5F,G,H, Supporting Information), likely due to the fact that GSH is an inactive NO donor.These results indicate the importance of GSNOR-mediated NO balance in regulating the redox status of M. oryzae.

GSNOR is a Positive Regulator of Virulence
Subsequently, we detected the effect of GSNOR deletion on the virulence of M. oryzae.Compared with WT and the complementary strains, the virulence of ∆gsnor was significantly reduced on barley and rice (Figure 3A,B), and the lesions caused by ∆gsnor to rice wounds were significantly smaller (Figure 3C).In conclusion, these results demonstrate that GSNOR is important for the infection and full virulence of M. oryzae.
Since cPTIO treatment caused a decrease in the S-nitrosylation level of Δgsnor, we hypothesized that cPTIO might rescue the defects of Δgsnor.Inoculation of Δgsnor after cPTIO treatment showed that cPTIO could partially restore the infection ability of Δgsnor (Figure S6A,B,C, Supporting Information).At the same time, we observed that cPTIO reduced the NO level in the infectious hyphae of Δgsnor (Figure S6D,E, Supporting Information), and therefore, this may be caused by the removal of excess NO content in Δgsnor by cPTIO.These results suggest that cPTIO was able to partially restore the infection defect of Δgsnor.

GSNOR Plays an Important Role in Appressorium Maturation
Considering that deletion of GSNOR significantly resulted in accumulation of appressorial NO, we wonder if GSNOR is important for functional appressorium formation.There was no significant difference in the formation of appressorium between WT and ∆gsnor (Figure 3D), but appressorium maturation of the ∆gsnor mutants was significantly affected.The turgor pressure was tested by using PEG8000-treatment showed that the appressorium of ∆gsnor mutants was much easier to collapse than that of WT (Figure 3E,F), indicating that accumulation of turgor pressure in appressorium of the mutant was evidently reduced.
Glycogen and lipid stores in conidia are essential nutrients for the appressorium maturation of M. oryzae.Since ∆gsnor is defective in accumulation of turgor, we speculate that glycogen and lipid utilization may be blocked in ∆gsnor mutants.Therefore, I 2 /KI and Nile Red staining were used to observe the distribution of glycogen and lipid in cells at different time points.The results showed that after 18 hpi, the glycogen and lipid could be still detected in the ∆gsnor mutant but not in WT (Figure S7A-D, Supporting Information), indicating that blocking NO metabolism also affects glycogen and lipid metabolic processes.Taken together, GSNOR plays an important role in appressorium maturation of M. oryzae.

GSNOR-Mediated de-nitrosylation is Essential for NO Detoxification During the Development of Appressorial in M. oryzae
Since a suitable dose of NO is required during appressorial formation in M. oryzae, high doses of NO are detrimental to M. oryzae (Figure 1A-D).To better understand the function of GSNOR in NO detoxification in pathogen, we investigated the dynamics of NO during appressorial development in M. oryzae.We found that NO levels reached a maximum during appressorial germination in the wild-type and that NO was gradually and completely metabolised during subsequent development and maturation (Figure 3G-I).Notably, this dynamic is similar to the ROS metabolism process of appressorial in the wild type M. oryzae, suggesting that RNS metabolism is also present in fungal cells.Green fluorescence was more intense in the germ tubes and early appressorial formation in the ∆gsnor mutant than in the wildtype, while NO was still detectable in the ∆gsnor mutant at later stages of appressorial development (Figure 3-I).These results suggest that GSNOR is a key regulator of NO detoxification during appressorial development and pathogenesis of M. oryzae.

GSNOR is Required for Oxidative Stress Detoxification
We investigated whether GSNOR is required for response to various environmental stresses.The results showed that ∆gsnor was not sensitive to cell wall inhibitors of (0.1 mg ml −1 Calcofluor White [CFW], 0.2 mg ml −1 Congo Red [CR]), and osmotic stresses (0.5 m NaCl, 1.0 m Sorbitol).Notably, we found that the ∆gsnor mutant was more sensitive to oxidative stress (10 mm H 2 O 2 ) (Figure S8A,B, Supporting Information), with a concentration dependent (Figure S8C,D, Supporting Information).These results suggested that GSNOR is required for oxidative stress detoxification.

Identification of S-nitrosylated Proteins by indoTMT Switch Labeling
In order to reveal the regulatory mechanism of S-nitrosylation in pathogenesis through the target proteins, we performed a proteome analysis to identify S-nitrosylation modified proteins.We used an iodoTMT labeling method combining with tandem mass spectrometry to characterize the S-nitrosylated proteins in M. oryzae.A mixed samples containing mycelia, conidia, appressoria and invasive hyphae, as well as 1 mM SNP-treated mycelia was used for total protein extraction (Figure 4A).In The lesion number was examined at 5 days (dpi) after inoculation.B) Lesions formed by wild-type (WT) and Δgsnor on rice seedlings at 5 days (dpi) after inoculation.The lesion number was examined at 5 days (dpi) after inoculation.C) Lesions formed by wild-type (WT) and Δgsnor on wounded rice leaves.Hyphal agar plugs (5 mm diameter) were placed on rice leaves treated with wounds and incubated for 4 days (dpi).The lesion length was examined at 4 days (dpi) after inoculation.D) Statistical analysis of appressorium formation rate in 24 h period.Data presented are the mean ± standard errors from three biological replicates (n = 3).E) Cytorrhysis assay for appressorium turgor pressure.Data presented are the mean ± standard errors from three biological replicates (n = 3), and asterisks represent significant differences (** P < 0.01).F) Observation of appressorium turgor pressure.Conidial suspension droplets were placed on the hydrophobic surface of a coverslip and treated with different concentrations of polyethylene glycol 8000 (PEG8000) at 24 hpi.G) The levels of NO during conidial to appressorial formation were detected by DAF-FM DA.The conidia were stained after inoculation on hydrophobic cover slips for 2, 6, 10, 12, 16, and 24 h.H) The fluorescence intensity of NO levels at the indicated regions during conidial to appressorial formation in wild-type and Δgsnor strains was measured using DAF-FM DA staining at different time points.I) Statistics of the percentage of wild-type (WT) and Δgsnor strains stained by DAF-FM DA in conidia or appressorial during NO metabolism.Data in (A-C) are displayed as box and whisker plots with individual data points: center line, median; box limits, and asterisks represent significant differences (** P < 0.01, *** P < 0.001).

Functional Classification of the S-nitrosylated Proteins
Functional structural domain enrichment analysis showed that spermine/spermidine synthase, ribosomal proteins, nucleotide-binding domain, GTPase domain, proteasomal subunit domain, septin domain, pyrimidine binding domain, anticodon-binding of tRNA domain, and aminotransferase classv domain, etc. were enriched (Figure 4E).Interestingly, proteins containing spermine/spermidine synthase (Sps1) and septin domains (septin proteins) have been found to be important for appressorium function in M. oryzae. [23]ene ontology (GO) analysis revealed S-nitrosylated target proteins were involved in numerous biological processes, mainly involving the carboxylic acid biosynthetic and metabolic process, cellular amino acid metabolic process, nucleoside phosphate metabolic process, ribose phosphate biosynthetic and metabolic process, amide biosynthetic process, hexose biosynthetic and metabolic process, glucose metabolic process, peptide metabolic process, glyceraldehyde-3-phosphate metabolic process, etc.
(Figure 4F).For cellular components, the highest proportions of cytosol, cytosolic ribosome, proteasome core complex, and cell wall were found.For molecular functions, oxidoreductase activity, structural constituent of ribosome, transaminase activity, nuclear import signal receptor activity, and hydro-lyase activity were enriched (Figure 4F).
The KEGG enrichment analysis showed that some mitochondria-related pathways were enriched, including pentose phosphate pathway, citrate cycle, methane metabolism, amino acid biosynthesis and metabolism, pantothenate and CoA biosynthesis, glyoxylate and dicarboxylate metabolism, pyruvate metabolism, etc. (Figure 4G).This S-nitrosylation pattern suggested that S-nitrosylation plays an important role in cell biosynthesis and energy metabolism in M. oryzae.

Analysis of S-nitrosylation-Mediated Protein-Protein Interaction Network
Protein-protein interaction (PPI) network analysis showed that a large number of S-nitrosylation target proteins interacted and clustered into proteasome components, ribosome components, tRNA synthetases, key enzymes of the tricarboxylic acid cycle and lipid metabolism-related enzymes (Figure 4H), These data suggested that S-nitrosylated may play key roles in the proteasome system, ribosome system and mitochondrial system through simultaneously modifying key components.
To gain a deeper understanding of the potential mechanisms by which S-nitrosylation could impact the structural characteristics of its target proteins, proteins of Mgb1, MagB, Sps1, and Cdc42 were used for molecular dynamics simulations.S-nitrosylation of the cysteine (CYS) residues at the modification sites of target proteins resulted in S-nitroso-cysteine (SNC).In general, the Root Mean Square Deviation (RMSD) ≤0.3 nm during a 20 ns MD run indicates strong complex stability. [24]adius of gyration (Rg) value was used to describe the structural integrity and folding behavior of the proteins. [25]We found that the conformations of the S-nitrosylation target proteins have evidently changed.Cdc42 CYS8SNC exhibited a lower Rg value compared to Cdc42, indicating that the S-nitrosylated Cdc42 protein adopts a more compact structure, resulting in a reduced solvent-accessible surface area and an increase in the hydrophobic area (Figure 5C).The RMSD analysis of the target proteins during molecular dynamics simulation process showed that the simulation had basically reached equilibrium (Figure 5D).Similar result was also found in Mgb1.While, the S-nitrosylated MagB and Sps1 exhibited a higher Rg value compared to their non-nitrosylated forms, adopted much looser structures, resulting in an increased solvent-accessible surface area and an increase in the hydrophilic area.This alteration may affect the interactions between the target protein and substrate.

Functions of Septin Proteins are Negatively Regulated by S-nitrosylation
23c,26] Notably, three septin proteins have also been identified as Snitrosylated proteins (Sep4 at C39, Sep5 at C40, and Sep6 at C144) according to our S-nitrosoproteomic analysis (Figure 5A).We speculate that Sep3 may be also regulated by S-nitrosylation.Co-immunoprecipitation and yeast two hybrid experiments both showed that all the septins could interact with GSNOR (Figure 6A-D; Figure S9, Supporting Information) and that the interactions of GSNOR with septin proteins in vivo were enhanced by SNP treatment (Figure 6E,F).These results indicate that S-nitrosylation modifications modulate protein interactions in M. oryzae.The biotin-switch assay showed that all four septin proteins were S-nitrosylated and the degree of modification in ∆gsnor mutant were all significantly higher than that of the wildtype strain (Figure 6G-J), suggesting that GSNOR could induce de-nitrosylation of septin proteins.
In order to explore the possible biological significance of Snitrosylation in septin proteins, as an example, we analyzed the subcellular localization of Sep6 protein in the wild-type strain and ∆gsnor mutant.We observed that in the wild-type strain, the GFP signal accumulated in the center of appresorium as a ring, while in the ∆gsnor mutant, GFP-Sep6 was distributed throughout the appresorium mainly in the form of granules without forming a ring (Figure 6K,L).Overall, we confirmed that septin proteins is the target of S-nitrosylation, and that whose function can be regulated by S-nitrosylation through affecting their subcellular localization.

Structural Analysis of S-nitrosylation Sites in Septin Proteins by Molecular Dynamics Simulation
To gain a deeper understanding of the potential mechanisms by which S-nitrosylation could impact the structural characteristics of septins, we also conducted molecular dynamics simulations.We found that Sep5 CYS40SNC exhibited a higher Rg value compared to its non-nitrosylated forms, indicating that the Sep5 CYS40SNC adopts a much looser structure, resulting in a decreased in solvent-accessible surface area and an increase in the hydrophobic area (Figure 7A-C).Conversely, Sep3 CYS177SNC ,Sep4 CYS39SNC , and Sep6 CYS144SNC exhibited a lower Rg value compared to theirs non-nitrosylated forms, indicating these septins adopts a more compact structure.Resulting in a decreased solvent-accessible surface area and an increase in the hydrophobic area of Sep3 CYS177SNC , Sep4 CYS39SNC , and Sep6 CYS144SNC have an increased solvent-accessible surface area and an increase in the hydrophilic area.Taken together, S-nitrosylation changes the Rg values of target proteins and the accessible areas of the protein solvent, thus affecting the interaction between the target protein and the substrate.These changes could affect the accessible area of septins, thus affect the interaction between each other and consequently affect septin ring formation.

S-nitrosylation Site C144 of Sep6 Protein is Important for Virulence
S-nitrosoproteomic analysis and target validation confirmed that septin proteins are targets of S-nitrosylation, then we selected Sep6 protein for functional analysis of the S-nitrosylation sites.To validate the identified S-nitrosylation modification site of Sep6 (C144), we mutated this site by changing C to S and transformed the mutated construct into the ∆sep6 mutant.The biotin conversion assay showed reduced S-nitrosylation modification in the S-nitrosylation modification site mutant ∆sep6/SEP6 C144S compared to the ∆sep6/SEP6 strain (Figure 6M), suggesting that GSNOR may regulate de-nitrosylation of Sep6 through recognition of the C144 site.The C144 S-nitrosylation site of Sep6 protein plays an important role in the regulation of the growth In proteins eluted from anti-FLAG beads, GFP-Sep3, GFP-Sep4, GFP-Sep5, and GFP-Sep6 bands were also detected by anti-GFP antibody in the transformant expressing the FLAG construct.E,F) Co-immunoprecipitation analyses of GSNOR with Sep5 (E) and Sep6 (F).Proteins were extracted from the wild-type strains containing Sep5-GFP, Sep6-GFP, and GSNOR-3×FLAG constructs in liquid CM with or without SNP treatment, and were immunoprecipitated with anti-FLAG beads.Input protein levels were also determined using anti-FLAG antibody.The bands of GFP-Sep5 and GFP-Sep6 with or without SNP treatment were detected by anti-GFP antibody.G-J) Accumulation of S-nitrosylated Sep3-GFP (G), Sep4-GFP (H), Sep5-GFP (I), and Sep6-GFP (J) in WT and Δgsnor.The blots were detected by anti-GFP antibody.Input protein levels were also determined using anti-GFP antibody, respectively.Asc, sodium ascorbate; SNO, S-nitrosylated proteins.A quantitative analysis of the data is shown below the blot.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the WT are indicated by an asterisk (* P < 0.05; ** P < 0.01).K) Localization of Sep6-GFP and Sep6 C144S -GFP in appressorium.Bar, 5 μm.L) Percentage of septin-ring formation in wild-type (WT), ∆gsnor /SEP6, and ∆sep6/SEP6 C144S strains.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the WT are indicated by an asterisk (** P < 0.01).M) S-nitrosylation verification of Sep6-GFP and Sep6 C144S-GFP proteins in vivo.N) Diseased spots formed by different strains on barley leaves.Spore solution was sprayed on barley plants and cultured for 5 days to observe the disease spots.O) Lesions formed on scratched rice leaves by different strains.Hyphal agar plugs were placed onto rice leaves with treatment of wounds and incubated for 5 d.Data in (N-O) are displayed as box and whisker plots with individual data points: center line, median; box limits, and asterisks represent significant differences (** P < 0.01).
and conidia formation of M. oryzae (Figure S10A-C, Supporting Information).Moreover, C144 of Sep6 was also required for pathogenicity and disease spot expansion (Figure 6N,O).Further analysis showed that the infection process of the mutant strain ∆sep6/SEP6 C144S was slowed down (Figure S10D,E, Supporting Information).Importantly, C144S mutation also resulted in Sep6 distributing as some granules without forming a ring (Figure 6K,L).Taken together, these results showed that the S-nitrosylation site C144 is a key site for the biological functions of Sep6.

GSNOR Inhibitor N6022 Effectively Suppresses the Infection of M. oryzae
N6022 is a novel drug with strong inhibitory activity against GSNOR, which is important for maintaining NO homeostasis.To provide ideas for the development of fungal inhibitors and disease control strategies based on NO signaling pathway, we tested the effects of N6022 and SNP on the pathogenicity of M. oryzae.Spray experiments on barley and rice by adding 1 mm N6022 or SNP into conidial solution of the wild-type strain were performed.We found that the necrotic lesions on rice and barley leaves were significantly reduced when treated with 1 mm N6022 or 1 mm SNP (Figure 8A,B).Inoculation of N6022 or SNP into conidial solution of the wild-type strain onto the scratched rice leaves showed that N6022 or SNP-treatment both severely reduced the lesion expansion, which was consistent with those of spray experiments (Figure 8C).
As the septin proteins can be regulated by S-nitrosylation, we then investigated whether N6022 or SNP treatment could affect the subcellular localization of septins in appressorium.Our results showed that after treatment with different concentrations of N6022 and SNP, Sep6 failed to aggregate into a complete ring and both N6022 and SNP all reduced the frequency of septin ring formation of in appressorium by ≈40%−60% (Figure 8D,E).These results suggest that N6022 and SNP destabilized the formation of the septin ring.Overall, our findings suggest that N6022 and SNP disrupted the NO homeostasis in M. oryzae, thereby affecting appressorium-related functions and ultimately blocking infection.To assess the selectivity of the GSNOR inhibitor N6022, we explored its interactions with GSNOR from various species.Employing surface plasmon resonance (SPR), we directly analyzed the binding between N6022 and the GSNOR protein.The findings revealed that N6022 binds to M. oryzae with a binding affinity of 55.8 μm.This interaction is less potent than that observed with human GSNOR (as a positive control with an affinity of 1.5 μm) but significantly stronger than its interaction with rice GSNOR (with an affinity of 288 μm), as depicted in Figure 8F.

Discussion
Although the functions and regulatory mechanisms of Snitrosylation in other species have been widely revealed, they were largely unknown in fungi.In this study, we found that in M. oryzae, intracellular NO equilibrium was essential for development and infection processes, especially the functional appressorium formation (Figures 1 and 3).The S-nitrosoglutathione reductase GSNOR-mediated de-nitrosylation process played a key role in scavenging excessive intracellular NO accumulation or nitrosative stress.Based on iodoTMT switch labeling approach, we Figure 8. Effects of N6022 and SNP on infection structure and host pathogenicity.A) Lesions formed on barley leaves after treatment with 1 mm SNP or N6022.The lesions are observed and the number of lesions is counted after 5 days of inoculation.Asterisks indicate significant differences (*** P < 0.001).B) Lesions formed on rice leaves after treatment with 1 mm SNP or N6022.The lesions are observed and the number of lesions is counted after 5 days of inoculation.Asterisks indicate significant differences (*** P < 0.001).C) Virulence on wounded rice leaves.Rice leaves were gently scraped with a needle and inoculated with spore solution treated with 1 mm SNP or 1 mm N6022.The length of lesions was measured and recorded after 4 days of inoculation.Asterisks indicate significant differences (** P < 0.01,*** P < 0.001).D) The percentage of septin-ring formation was calculated for each treatment.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the WT are indicated by an asterisk (** P < 0.01, *** P < 0.001).E) Observation on the formation of appressorium septin ring after treatment with 0.5 and 2 mm SNP or N6022.Bar, 5 μm.F) SPR analysis of N6022 binding to GSNOR of M. oryzae and that of rice/human.G) A proposed model of de-nitrosylation mediated appressorium formation in M. oryzae.During infection of M. oryzae, the appressorium formation accompanied with accumulation of massive NO.H 2 O 2 also contributes to the accumulation of NO.NO and its bioactive donor S-nitrosoglutathione (GSNO) modify appressorium (AP) proteins through S-nitrosylation (-SNO), leading them to inactive proteins.While this process is reversed by the S-nitrosoglutathione reductase GSNOR-mediated de-nitrosylation process, which converts the modification site of -SNO into -SH form, and oxidized glutathione (GSSG), resulting an increased ratio of GSH/GSSG.The de-nitrosylated AP proteins are activated for full function, which facilitates appressorium-related cellular processes and appressorium maturation, leading to a successful infection.
were able to identify a large number of S-nitrosylated proteins in M. oryzae, some of which were reported key proteins for functional appressorium formation.We further found that GSNOR was required to activate these key appressorial proteins by removing S-nitrosylation (Figure 5).Notably, proper protein structure and septin ring formation of the septin proteins were required by removing S-nitrosylation (Figures 6 and 7).Our work provided the comprehensive understanding of S-nitrosylation regulatory mechanism in the plant pathogenic fungi.

GSNOR-Mediated de-nitrosylation is Essential for NO Homeostasis and Appressorium Formation
We revealed the biological significance of NO-mediated Snitrosylation, a new type of post-translational modification, in the pathogenic process of M. oryzae.8b,18a] We found that M. oryzae GSNOR also mediates intracellular NO level through regulating the de-nitrosylation process.GSNORmediated de-nitrosylation is important for asexual development, stress response, appressorium formation and invasive growth of M. oryzae.Currently, there are few reports on the roles of NO in plant pathogenic fungi, including in M. oryzae and C. minitans.For example, low dose of NO is found to be required in the germination and appressorium formation of M. oryzae, [27] and it is also required for asexual spore formation of C. minitans. [28]n this study, we found that high concentration of NO (addition of 5 mm SNP) is harmful for appressorium formation.Deletion of GSNOR led to a serious increase of NO level, which was also harmful for appressorium formation, but the NO scavenger cPTIO-treatment can reverse this phenomenon (Figure 1A-E).Consistent with this, the addition of exogenous SNP and cPTIO respectively increased and decreased the protein S-nitrosylation level in M. oryzae.Therefore, GSNOR is essential for keeping intracellular NO homeostasis for development and appressorium formation.Plants can employ nitrosative stress (NO burst) to suppress fungal infection. [29]Coordinately, fungi also developed NOdetoxification system to adapt to nitrosative stress, such as M. oryzae S-(hydroxymethyl)-glutathione dehydrogenase Sfa1 and flavohemoglobin MoFhb1 mediated NO detoxification [15,30] and F. graminearum transcription factor FgAreB. [3a] However, the synthetic pathway of NO in M. oryzae remains elusive, this notion awaits proof by more genetic studies.

Proteomics Analysis Identified Infection-Associated S-nitrosylation Proteins
The main physiological role of NO is the protein S-nitrosylation.However, less is known about the mechanism by which Snitrosylation regulates signaling in fungi.To date, more than thousands of S-nitrosylated proteins have been identified by proteomics methods in mammal and plants. [31]However, genomewide identification of S-nitrosylated proteins has not been performed in fungi.We used an iodoTMT switch labeling approach to investigate the role of S-nitrosylation modifications in M. oryzae.By using different samples from developmental stages and SNP treatment conditions, we were able to identify 483 Snitrosylated proteins with 741 S-nitrosylation sites (Figure 4A).Interestingly, a large number of previously reported infectionassociated proteins were identified as S-nitrosylated proteins, providing abundant information for future deciphering regulatory mechanism of S-nitrosylation.

GSNOR-Mediated de-nitrosylation Regulates Functional Appressorium Formation
A number of proteins crucial for appressorium formation were identified as S-nitrosylated proteins, including Pmk1-MAPK signaling pathway upstream proteins (Figure 5A).These appressorium-associated proteins suggested a crucial role of Snitrosylation during appressorium stage.However, we propose that the regulation of S-nitrosylation to these proteins may be negative.S-nitrosylated AP proteins are normally inactive, during appressorium formation, they are activated by GSNOR-mediated de-nitrosylation.Then activated AP proteins regulate glycogen and lipid metabolism, turgor accumulation, septin ring formation to form functional appressorium (Figure 8E).Consistent with this, the Δgsnor mutant showed defects of appressorium formation and maturation, as well as glycogen and lipid utilization, turgor accumulation, septin ring formation and appressorial adhesion.
We found that the S-nitrosylation of septin proteins is a novel regulatory mechanism for the formation of septin ring, which is essential for penetration during infection of M. oryzae. [26,32]Our study proved four septin proteins (Sep3, Sep4, Sep5, and Sep6) as S-nitrosylated proteins.GSNOR can interact with all of four septin proteins, and the septin ring formation was also affected in the ∆gsnor mutant, indicating that de-nitrosylation of septin proteins positively regulates their function.S-nitrosylation may block cysteine site and affecting formation of disulfide bonds, then affect intramolecular or intermolecular interactions intermolecular interactions of septin proteins, therefore affect septin ring construction.As a case study, we showed that the C144 Snitrosylation site of Sep6 protein was important for septin ring formation and infection of M. oryzae.PTM level regulation of septin proteins have been found to be important for assembly of septin ring in yeast and fungi. [33]33b] Here, we found a novel PTM, S-nitrosylation, also modified core septins, providing novel insight into the regulation of fungal septin ring assembly.

Disturbing of Appressorial NO Homeostasis Could be an Effective Disease Control Strategy
We propose that targeted breaking NO homeostasis by NO donors, NO scavengers, as well as chemical inhibitors of GSNOR, should be effective methods for fungal disease control.Studies have reported that NO was involved in pathogenicity in animal pathogens and controlled spore germination and infection structure development in plant pathogenic fungi. [34]Our study also showed that the NO homeostasis is also essential for development and infection of M. oryzae.Pathogenicity tests showed that both NO donor SNP and GSNOR inhibitor N6022 can reduce the virulence of M. oryzae, although this process does not exclude the contribution of N6022 to plant resistance.Our results suggest that disrupting the NO homeostasis in M. oryzae has a negative impact on the growth and development of the pathogen and the formation of infection structures (septin ring) (Figures 1  and 8G).Therefore, the use of NO donors or chemical inhibitors of GSNOR are effective in rice blast control.Considering that harmful of excessive NO to different fungal pathogens could be similar, and that both septins and GSNOR are conserved in fungi, this strategy may be a broad-spectrum strategy for controlling of fungal diseases.It is worth to mention that, the GSNOR inhibitor, such as N6022, is likely to be toxic to various organisms, including the plant host rice and barley.While on the other hand, the plant host could be more tolerant to N6022 than the fungus, when we treated rice or barley seedlings with 5 or 10 mm N6022 for one week, the growth of the plant was not affected (Figure S11, Supporting Information).Considering that 10 mm is much higher than that of 1 mm used for fungal control, it is possible for utilization.However, standard safety evaluation to human and environment should be tested in the future.It is also possible to develop more fungal-specific inhibitors for GSNOR, for example, based on the structural differences or by gene silencing strategy based on distinct RNA sequences in different organisms.

Experimental Section
Gene Knockout and Complementation: P131 was used as a wild-type strain of M. oryzae.The GSNOR gene was deleted using a split-PCR strategy. [35]Information of all strains and vectors was shown in Tables S3  and S4 (Supporting Information).To test the stress response of the mutants, different strains were inoculated on CM plate supplanted with 0.1 mg ml −1 Calcofluor White [CFW], 0.2 mg ml −1 Congo Red [CR], 0.5 m NaCl, 1.0 m Sorbitol or different concentrations of H 2 O 2 .The colony diameter was measured after 5-day post-inoculation (dpi).
Quantitative Real-Time PCR Analysis: To detect the expression of GSNOR gene in M. oryzae, total RNA was extracted from samples collected from different developmental stages (mycelium, conidium, appressorium at 3 and 12 h, and invasive hyphae at 18, 24, and 48 hpi).Then the cDNA template was prepared for qRT-PCR analysis using a SYBR Green PCR Master Mix Kit (Takara, Dalian, China) on an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA).
Staining Assays: The mycelia were stained with 10 μg ml −1 CFW (Sigma-Aldrich, St. Louis, MO, USA) for 5 min and examined under a fluorescence microscope (Ni90, Nikon) to observe the cell length.For glycogen and lipids staining, conidial suspensions were dropped onto hydrophobic coverslip and incubated for 0, 2, 4, 6, 8, 12, 18, and 24 hpi in a dark moist chamber at 28 °C, followed by staining with KI/I 2 solution (50 mg ml −1 KI, 5 mg ml −1 I 2 ) or Nile red solution (2.5 μg ml −1 Nile red, pH 7.5).For NO staining, samples from different developmental stages of M. oryzae were stained using 5 μm DAF-FM DA (Biyuntian, Beijing, China) dye and incubated at 37 °C for 20 min, followed by washing off the dye.Photographs were taken under a laser confocal microscope TCS SP8 (Leica Microsystems, Mannheim, Germany).For mitochondrial staining, spores and mycelial samples of GFP-GSNOR strain were stained using MitoTracker Red CMXRos (Coolaber, China) dye, incubated at 37 °C for 20 min, and then washed to remove the dye.Images were captured using a laser confocal microscope TCS SP8 (Leica Microsystems, Mannheim, Germany).
Yeast Two-Hybrid Experiment: The full-length cDNA of SEP3, SEP4, SEP5, and SEP6 were amplified and connected to the yeast expression vector pGBKT7.The full-length cDNA of GSNOR was amplified and connected with the yeast expression vector pGADT7.The AD and BD vectors were co-transformed into yeast strain AH109.The bacterial solution packets were inverted on SD/-Leu/-Trp and SD/-Leu/-Trp/-His (Clontech, San Francisco, CA, USA) dishes and cultured at 30 °C for 3 days to observe the growth of colonies.The grown single colonies were transferred to a new SD/-Leu/-Trp/-His medium, stained with 5 μl X--gal, and cultured at 30 °C for 2 days in dark to observe the results.The interaction between pGBKT7-53 and pGADT7-T were used as a positive control, and the interaction between pGBKT7-Lam and pGADT7-T were used as a negative control.
SNP, cPTIO, and N6022 Treatment: To observe the effects of SNP (Biyuntian, Beijing, China), cPTIO (Sigma-Aldrich, St. Louis, MO, USA), and N6022 (MedChemExpress, USA) treatments, spore suspension (2 × 10 5 spores ml −1 ) of different strains was added with each reagent, then dropped on the hydrophobic coverslip to calculate the appressorium formation rate at 24 hpi.To observe infectious hyphae, spore suspension (1 × 10 5 spores ml −1 ) containing each reagent was dropped on the barley epidermis, and the expansion was observed at 28 hpi.For rice inoculations, spore suspension (5 × 10 4 spores ml −1 in 0.025% Tween 20) containing each reagent was sprayed onto the leaves of one-month-old rice seedlings (Oryza sativa cultivar "LTH").The rice plants were then incubated at 28 °C for 5 days under full humidity to evaluate disease level.For scratch experiment, rice seedlings were punctured for inoculating spore suspension (1 × 10 5 spores ml −1 in 0.025% Tween 20) containing each reagent, and disease lesions were observed at 5 hpi at 28 °C in full humidity.
Assay for GSH, GSSG, and NO: According to the manufacturer's instructions, a GSH and GSSG assay kit from Solarbio, China, was used to assess the amount of GSH in the cortex.Thermo Scientific Multiskan FC, OD = 593 nm, colorimetric microplate reader was used to measure the content.Total Glutathione-GSSG×2 was used to calculate the GSH content of the test samples.GSH content was given as μg g −1 .The NO content was determined using a NO content assay kit (Abbkine Scientific, Wuhan) based on the improved Griess method principle.
Proteomics Analysis of S-nitrosylation Proteins: Protein labelling was performed according to the manufacturer's protocol for Iodo-TMT kit.Briefly, 1 mg of the protein extracts was blocked for 1 h at room temperature using a blocking reagent with 1% SDS and 50 mm iodoacetamide (IAA) and free thiols were blocked.The sample was precipitated with 3 volumes of cold acetone and washed three times.The S-nitrosothiols in the protein were reduced to free thiols by the addition of the reducing reagent sodium ascorbate (20 mm, 37 °C) and the samples were subsequently labeled with IodoTMT (Thermo Fisher Scientific, Rockford, USA) for 1 h at room temperature and then precipitated with acetone.The protein pellet was dissolved in wash buffer, all these steps were performed in the dark.Biotinylated proteins were digested with trypsin (Promega, Madison, WI, United States) overnight at 37 °C then mixed with 30 μL of streptavidin beads (Thermo Fisher Scientific, Rockford, USA) and incubated overnight at 4 °C.Wash the beads four times with a washing buffer (neutralization buffer plus 600 mm NaCl).S-nitrosylated proteins could be purified with streptavidin beads (anti-TMT antibody resin), which bind biotin with very high affinity and specificity.The proteins were then eluted with 20 mL of elution buffer and followed by mass spectrometry analysis using an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham MA, USA) connected to an Easy-nLCII (Thermo Fisher Scientific, Bremen, GA, USA).
Biotin Labeling of S-nitrosylated Proteins: S-nitrosylated proteins were analyzed using a kit (Cayman, USA) based on the biotin-switch assay as described previously. [36]Briefly, ≈100 to 200 uL of the protein extracts was blocked for 1 h at room temperature using a blocking reagent whose free thiols were blocked.The sample was precipitated with cold acetone and washed three times.The S-nitrosothiols in the protein were reduced to free thiols by the addition of the reducing reagent sodium ascorbate and the samples were subsequently labeled with maleimide-biotin for 1 h at room temperature and then precipitated with acetone.The protein pellet was dissolved in wash buffer, all these steps were performed under indirect light.The biotinylated proteins were then mixed with 30 μL of Streptavidin beads (ABclonal Technology) and incubated overnight at 4 °C.Wash the beads four times with a washing buffer.S-nitrosylated proteins could be

Figure 2 .
Figure 2. NO and GSNOR conversely regulates S-nitrosylation of M. oryzae.A) Cellular NO detoxification pathway.B) Expression of GSNOR gene in different developmental and infection stages of M.oryzae.HY: Mycelial hyphae; CO: Conidia; AP_3h: appressoria at 3 hpi (hours post inoculation); AP_12h: appressoria at 12 hpi; IH_18h: invasive hyphae at 18 hpi; IH_24h: invasive hyphae at 24 hpi; IH_48h: invasive hyphae at 48 hpi.Error bars represent standard errors.C) Observation of NO content in wild-type (WT) and Δgsnor at different developmental stages after DAF-FM DA staining.Bar,10 μm.D) The bar chart shows the fluorescence intensity of DA-FM DA staining at different developmental stages.Samples of each strain were measured using the ImageJ software.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the WT are indicated by an asterisk (** P < 0.01).E) The concentration of NO in the mycelia of wild-type (WT) and Δgsnor was determined by the improved Griess method.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the WT are indicated by an asterisk (* P < 0.05).F) Observation of NO content in wild-type (WT) and Δgsnor during appressorium after SNP and cPTIO treatment.Bar,10 μm.G) The bar chart shows the fluorescence intensity of DA-FM DA staining at appressoria stage.Samples of each strain were measured using the ImageJ software.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the treatment are indicated by an asterisk (** P < 0.01).H) Expression of the GSNOR gene after SNP treatment in the wildtype, and the ACTIN gene was used as an internal control.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the SNP treatment are indicated by an asterisk (** P < 0.01).I) Total levels of S-nitrosylated proteins in wild-type (WT) and Δgsnor.Hypha at 7 days of age were used as samples for analysis.Asc, Sodium ascorbate; SNOs, S-nitrosylated proteins.J) Band quantification of the immunoblot showing a specific increase of S-nitrosylation for wild-type (WT) and Δgsnor proteins.K) Total levels of S-nitrosylated proteins in wildtype (WT) and Δgsnor after SNP treatment.Hypha at 7 days of age were used as samples for analysis.L) Total levels of S-nitrosylated proteins in wild-type (WT) and Δgsnor after cPTIO treatment.Hypha at 7 days of age were used as samples for analysis.M) Total levels of S-nitrosylated proteins in wild-type (WT) and Δgsnor after H 2 O 2 treatment.Hypha at 7 days of age were used as samples for analysis.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with no treatment are indicated by an asterisk (** P < 0.01).ns, not significant.

Figure 3 .
Figure3.GSNOR is responsible for full virulence.A) Lesions formed by wild-type (WT) and Δgsnor on barley leaves at 5 days (dpi) after inoculation.The lesion number was examined at 5 days (dpi) after inoculation.B) Lesions formed by wild-type (WT) and Δgsnor on rice seedlings at 5 days (dpi) after inoculation.The lesion number was examined at 5 days (dpi) after inoculation.C) Lesions formed by wild-type (WT) and Δgsnor on wounded rice leaves.Hyphal agar plugs (5 mm diameter) were placed on rice leaves treated with wounds and incubated for 4 days (dpi).The lesion length was examined at 4 days (dpi) after inoculation.D) Statistical analysis of appressorium formation rate in 24 h period.Data presented are the mean ± standard errors from three biological replicates (n = 3).E) Cytorrhysis assay for appressorium turgor pressure.Data presented are the mean ± standard errors from three biological replicates (n = 3), and asterisks represent significant differences (** P < 0.01).F) Observation of appressorium turgor pressure.Conidial suspension droplets were placed on the hydrophobic surface of a coverslip and treated with different concentrations of polyethylene glycol 8000 (PEG8000) at 24 hpi.G) The levels of NO during conidial to appressorial formation were detected by DAF-FM DA.The conidia were stained after inoculation on hydrophobic cover slips for 2, 6, 10, 12, 16, and 24 h.H) The fluorescence intensity of NO levels at the indicated regions during conidial to appressorial formation in wild-type and Δgsnor strains was measured using DAF-FM DA staining at different time points.I) Statistics of the percentage of wild-type (WT) and Δgsnor strains stained by DAF-FM DA in conidia or appressorial during NO metabolism.Data in (A-C) are displayed as box and whisker plots with individual data points: center line, median; box limits, and asterisks represent significant differences (** P < 0.01, *** P < 0.001).

Figure 4 .
Figure 4. Identification of S-nitrosylated proteins by indoTMT switch labeling.A) Schematic illustration of S-nitrosoproteomic analysis procedure and its parameter statistics.For detailed notes on these proteins, see Table S1 (Supporting Information).B) Statistics of the number of individual protein modification sites discovered by S-nitrosoproteomic analysis.C) Presumptive consensus sequences of the S-nitrosylation motifs derived from the analysis of 741 S-nitrososylated protein sites.D) Localization and classification of S-nitrosylation target proteins.E) Enrichment of S-nitrosylation target protein domains.F) GO analysis of significantly altered S-nitrosylated proteins by cellular component, molecular function and biological process.G) Enrichment analysis of S-nitrosylated proteins KEGG signaling pathway.H) Protein-protein interaction network of the S-nitrosylated proteins.

Figure 5 .
Figure 5. S-nitrosylation regulates proteins related to appressorium function.A) S-nitrosylated proteins associated with the pathogenesis of rice blast fungus.B) Accumulation of S-nitrosylated MAGB-FLAG, MGB1-FLAG, SPS1-FLAG and CDC42-FLAG in wild-type (WT) and Δgsnor.The blots were detected by anti-FLAG antibody.Input protein levels were also determined using anti-FLAG antibody, respectively.Asc, sodium ascorbate; SNO, Snitrosylated proteins.A quantitative analysis of the data is shown at the right.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the WT are indicated by an asterisk (* P < 0.05; ** P < 0.01).C) Analysis of protein radius of gyration (Rg, left) and solvent-accessible surface area (right) of proteins during molecular dynamics simulations.D) Analysis of root mean square deviation (RMSD) of protein backbone during molecular dynamics simulations.E) Structural alignment of proteins with or without S-nitrosylation by dynamics simulations.

Figure 6 .
Figure6.S-nitrosylation regulates functions of septins in M. oryzae.A-D) Co-immunoprecipitation analyses between GSNOR and Sep3 (A), Sep4 (B), Sep5 (C), and Sep6 (D).In proteins eluted from anti-FLAG beads, GFP-Sep3, GFP-Sep4, GFP-Sep5, and GFP-Sep6 bands were also detected by anti-GFP antibody in the transformant expressing the FLAG construct.E,F) Co-immunoprecipitation analyses of GSNOR with Sep5 (E) and Sep6 (F).Proteins were extracted from the wild-type strains containing Sep5-GFP, Sep6-GFP, and GSNOR-3×FLAG constructs in liquid CM with or without SNP treatment, and were immunoprecipitated with anti-FLAG beads.Input protein levels were also determined using anti-FLAG antibody.The bands of GFP-Sep5 and GFP-Sep6 with or without SNP treatment were detected by anti-GFP antibody.G-J) Accumulation of S-nitrosylated Sep3-GFP (G), Sep4-GFP (H), Sep5-GFP (I), and Sep6-GFP (J) in WT and Δgsnor.The blots were detected by anti-GFP antibody.Input protein levels were also determined using anti-GFP antibody, respectively.Asc, sodium ascorbate; SNO, S-nitrosylated proteins.A quantitative analysis of the data is shown below the blot.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the WT are indicated by an asterisk (* P < 0.05; ** P < 0.01).K) Localization of Sep6-GFP and Sep6 C144S -GFP in appressorium.Bar, 5 μm.L) Percentage of septin-ring formation in wild-type (WT), ∆gsnor /SEP6, and ∆sep6/SEP6 C144S strains.Data presented are the mean ± standard errors from three biological replicates (n = 3), and significant differences compared with the WT are indicated by an asterisk (** P < 0.01).M) S-nitrosylation verification of Sep6-GFP and Sep6 C144S-GFP proteins in vivo.N) Diseased spots formed by different strains on barley leaves.Spore solution was sprayed on barley plants and cultured for 5 days to observe the disease spots.O) Lesions formed on scratched rice leaves by different strains.Hyphal agar plugs were placed onto rice leaves with treatment of wounds and incubated for 5 d.Data in (N-O) are displayed as box and whisker plots with individual data points: center line, median; box limits, and asterisks represent significant differences (** P < 0.01).

Figure 7 .
Figure 7. Structural modeling of septin proteins.A) Analysis of protein radius of gyration (Rg, left) and solvent-accessible surface area (right) of proteins during molecular dynamics simulations.B) Analysis of root mean square deviation (RMSD) of protein backbone during molecular dynamics simulations.C) Structural alignment of proteins with or without S-nitrosylation by dynamics simulations.

NO Balance is Required for Maintaining Redox Homeostasis in M. oryzae H
2 O 2 and NO interact with each other as endogenous signaling molecules in plants and animals.In order to elucidate the relationship between H 2 O 2 and NO in M. oryzae, we treated the