Fig 2: 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 test 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. H2O2 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.

Fig 3: 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 Sep6C144S‐GFP in appressorium. Bar, 5 µm. L) Percentage of septin‐ring formation in wild‐type (WT), ∆gsnor /SEP6, and ∆sep6/SEP6C144S 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 Sep6C144S‐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).

Fig 4: 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 wild‐type, 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 wild‐type (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 H2O2 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.