The multifunctional ascorbate peroxidase MoApx1 secreted by Magnaporthe oryzae mediates the suppression of rice immunity

Abstract

Fungi secrete effector proteins, including extracellular redox enzymes, to inhibit host immunity. Redox enzymes have been hypothesized to inhibit host reactive oxygen species (ROS); however, how they suppress host immunity remains unknown. We characterized an extracellular ascorbate peroxidase (MoApx1) that is secreted into rice chloroplasts by the rice blast fungus Magnaporthe oryzae. MoApx1 displays multifunctional capabilities that significantly contribute to fungal virulence. Firstly, MoApx1 neutralizes host-derived H2O2 within the chloroplast through its peroxidase activity, thereby inhibiting chloroplast ROS (cROS)-mediated defense responses. Secondly, MoApx1 targets the photosystem I subunit OsPsaD, disrupting photosynthetic electron transport to further suppress cROS production. Most importantly, MoApx1 has evolved a fungal-specific starch-binding domain that binds host starch, inhibiting its degradation and disrupting the energy supply required for host resistance. Our findings underscore the importance of a novel multifaceted strategy, potentially widely employed by other fungal pathogens, in suppressing host immunity during host-microbe interactions.

Figure 1.

MoApx1 is required for the virulence of the rice blast fungus. A) MoApx1 is required for the full virulence of M. oryzae. The virulence was tested using the conidial suspension spray assay. Diseased rice leaves were photographed after 7 d post-inoculation (dpi). Scale bar, 10 µm. B and C) The disease lesion numbers and the lesion sizes in A) were measured. The mean values of 3 determinations with Sds are shown. Significant differences were determined by Student's t-test and marked with asterisk (P < 0.01). D) MoApx1 is important for normal invasion growth. Invasive hyphae were examined at 24 and 48 hpi. The experiments were repeated independently with similar results at least 3 times. Scale bar, 10 µm. E) The ΔMoapx1 mutant cannot inhibit ROS bursts in rice cells. DAB (3,3′-diaminobenzidine) staining and inclusion observation on infected leaf sheaths at 24 and 48 hpi. The experiments were repeated independently with similar results at least 3 times. Scale bar, 10 µm. F) The ΔMoapx1 mutant infection induced the PR gene expression in rice. Examination of transcript levels of AOS2, CHT1, and PR1a genes in rice cultivar ‘CO39’ inoculated with the Guy11 and the ΔMoapx1 mutant at 36 h. The error bars represent Sd (n = 3). Significant differences were determined by 2-sided Duncan's new multiple-range tests, and marked with P values (P < 0.05).

Figure 2.

MoApx1 is a cytoplasmic effector secreted into the rice chloroplast. A) MoApx1 is a cytoplasmic effector secretory regulated by MoSwa2. The fungal transformants ΔMoapx1 and ΔMoswa2 expressing MoApx1:GFP at 30 hpi in the sheath cells of rice cultivar ‘CO39’ rice are shown as a projection of a confocal microscope. Arrows indicate the biotrophic interface complex. Scale bar, 10 µm. B) MoApx1 is secreted into extracellular space. The GFP-tagged MoApx1, MoAo1, and empty-GFP were expressed in the Guy11 strain, respectively. Total extracellular proteins were extracted from cultures respectively grown in MMN liquid culture for 4 d, and separated by SDS-PAGE followed by detection with the anti-GFP antibody. Coomassie brilliant blue (CBB) was used as the loading control. C) MoApx1 localizes in the chloroplast. GFP-tagged MoApx1 without signal peptide was expressed in the rice protoplast. The autofluorescence represents chloroplast. Scale bar, 5 µm. D) MoApx1 targets rice chloroplast. After the susceptible rice variety NPB inoculated with above strains for 48 h, the extracellular fluids, cytoplasmic, and chloroplast proteins were isolated. The proteins were extracted and separated by SDS-PAGE followed by detection with the anti-GFP antibody. Ponceau S (PS) was used as the loading control. E to G) MoApx1 can enter into rice chloroplast during infection stage. The wild-type strain Guy11 and Guy11/MoApx1-GFP transformed strain were inoculated to wild-type rice NPB or MoAPX1-OE rice leaves at 48 hpi, the immunogold labeling results showed that MoApx1-GFP gold particles were localized in rice chloroplasts. Scale bars, 1 μm.

Figure 3.

Interactions between MoApx1 and OsPsaD in vitro and in vivo. A) Yeast 2-hybrid assay between AD (pGADT7)-MoAxp1^ΔSP (without the signal peptide sequence) and BD (pGBKT7)-OsPsaD. Cells were plated on a SD-Leu-Trp medium and then transferred onto SD-Ade-Leu-Trp-His medium. B) Co-IP analysis of MoAPX1^ΔSP-GFP and OsPsaD-RFP in vivo. The MoAPX1-GFP or empty-GFP was co-expressed with OsPSAD-RFP genes in rice protoplast. The co-IP experiment was performed with the anti-GFP antibody, and the isolated protein was analyzed by immunoblot using an anti-RFP antibody to detect OsPsaD and an anti-GFP antibody to detect MoApx1. The experiments were repeated independently with similar results at least 3 times. C) Bimolecular Fluorescence Complementation (BiFC) assays in rice protoplast cells. Co-expression of MoApx1^ΔSP-nYFP and OsPsaD-cYFP showed that MoApx1 and OsPsaD co-localized in chloroplast. The relevant negative controls showed no fluorescence. Autofluorescence represents chloroplast. All the experiments were repeated independently with similar results at least 3 times. Scale bar, 5 µm.

Figure 4.

MoApx1 suppresses host immunity by competitively binding OsPsaD. A to C) A structural model for the MoApx1–OsPsaD interaction, predicted by ClusPro 2.0 server. The surface of the MoApx1–OsPsaD and the interaction amino residues were visualized by PyMOL. The yellow dotted line with the marked distance is hydrogen binding. L, loop; α, α-helix. D) α5 and L10 regions are required for the MoApx1–OsPsaD interaction. In vivo co-IP assay between OsPsaD with MoApx1 or interaction sites mutants. The MoApx1-GFP or mutants were co-expressed with OsPsaD-RFP genes in rice protoplast. The co-IP experiment was performed with the anti-GFP antibody, and the isolated protein was analyzed by immunoblot using an anti-RFP antibody to detect OsPsaD and an anti-GFP antibody to detect MoApx1. E) The α5 and L10 regions in MoApx1 are required for the full virulence of M. oryzae. The virulence was tested using the conidial suspension spray assay. Diseased rice leaves were photographed after 7 dpi. The disease lesion area was assessed using ImageJ software. The mean values of 3 determinations with Sds are shown. Significant differences were determined by 2-sided Duncan's new multiple-range tests, and marked with different letters (P < 0.01). F) The structural model of OsPsaC–OsPsaD. The OsPsaC–OsPsaD model generated using AlphaFold 3, closely resembles the structure found in red algae (PDB ID: 5ZGB). The cartoon structures were visualized by PyMOL. G) In vitro competitive binding assay between MoApx1 and OsPsaC with OsPsaD. The RFP-tagged OsPsaD and HA-tagged OsPsaC were expressed and purified from rice protoplasts. GST-MoApx1 was purified from E. coli BL21 (DE3) cell lysate. Equal amounts of OsPsaD-RFP and OsPsaC-HA were added to each reaction, along with a gradient dilution of GST-MoAxp1, and the mixtures were enriched using RFP beads. Eluted proteins were analyzed by immunoblot with anti-RFP, anti-HA, and anti-GST antibodies.

Figure 5.

Determination of resistance of OsPsaD knockout and overexpression transgenic lines. A) Infection phenotype of Ospsad-KO and OsPsaD-OE lines against M. oryzae. The leaves of 3-wk-old plants were infected with the wild-type Guy11 and the ΔMoapx1 strains using punch inoculation. Photos were taken at 7 dpi. The lesion length was measured by rule. The mean values of 3 measurements with Sds are shown. Significant differences were determined by 2-sided Duncan's new multiple-range tests, which were marked with different letters (P < 0.05). B) The OsPsaD overexpression lines accumulate a higher ion of superoxide anions when treated with an elicitor. The NPB, Ospsad-KO, and OsPsaD-OE rice plants treated with 0.1 μm flg22 were stained with NBT. The red autofluorescence represents chloroplast. Scale bar, 5 µm. C and D) The determination of O2.⁻. The content and production rate in rice plants when treated with or without flg22 were determined using the Superoxide Anion Content Assay Kit. The mean values of 3 measurements with Sds are shown. Significant differences were determined by 2-sided Duncan's new multiple-range tests and marked with different letters (P < 0.05). E) The determination of ETR. The ETR in NPB, Ospsad-KO, and OsPsaD-OE rice plants infected by Guy11 and ΔMoapx1 at 24 and 48 hpi was measured using the instrument DUAL-PAM-100. The mean values of 3 measurements with Sds are shown. Significant differences were determined by 2-sided Duncan's new multiple-range tests and marked with different letters (P < 0.05). F) The translation of OsPsaD was induced by the infection of M. oryzae. The protein level of OsPsaD in the different infectious stages was determined by immunoblotting using anti-PsaD polyclonal antibodies (AS09461, Agrisera). Protein loading is indicated with Ponceau staining.

Figure 6.

MoApx1 contains a C-terminal starch-binding domain and binds to starch. A) The structure model of MoApx1 was predicted by AlphaFold2, and aligned with the ascorbate peroxidase (PDB ID: 3RRW) from Arabidopsis thaliana and the starch-binding domain of glucoamylase from Rhizopus oryzae (PDB ID: 2DJM) by PyMOL. B) The MoApx1-His fusion proteins were examined for the ability to co-sediment with total starch. The empty-His proteins were used as control. The supernatant (S) and pellet (P) fractions were probed with an anti-His antibody. C and D) Biomembrane interferometry was employed to evaluate the binding capacity of MoApx1 with cyclodextrins (CDs). The sensorgrams display real-time binding responses, with different concentrations of α-CD and β-CD injected over immobilized MoApx1. The resulting curves indicate the binding kinetics, allowing for the calculation of association and dissociation rates. E and F) Microscale thermophoresis (MST) was employed to evaluate the binding capacity of MoApx1 with CDs. Ten-micrometer MoApx1-His or empty-His was labeled by RED-NHS. The raw data were integrated and fitted to a binding model using the MST analysis software. The recombinant proteins were contained in NT standard capillaries. The solid curve is the fit of the data points to the standard Kd-fit function. Kd, dissociation constant. Each binding assay was repeated 3 times independently (n = 3), and the bars represent Sd. G) Natural mutations in the SBD domain and the SBD deletion mutant showed significantly reduced α-CD binding abilities by MST assay. The mean values of 3 measurements with Sds are shown.

Figure 7.

MoApx1 inhibits starch-mediated resistance against M. oryzae. A and B) Iodine staining of the leaves of NPB and MoAPX1-OE rice plants inoculated with Guy11 strain at different infectious stages. ED represents the end of the day, EN represents the end of the night, and HB represents the complemented strain (ΔMoapx1/MoAPX1). C) The contents of total starch were determined in leaves of NPB and MoAPX1-OE rice plants inoculated with the Guy11 strain at different infectious stages. The mean values of 3 measurements with Sds are shown. Significant differences were determined by 2-sided Duncan's new multiple-range tests, which were marked with different letters (P < 0.05). D) The determination of glucose in rice leaves. The glucose content was measured in the leaves of NPB and MoAPX1-OE rice lines at 0, 24, 38, and 48 h following inoculation with the Guy11 strain, the ΔMoapx1 mutant, and the complemented strain. The mean values of 3 measurements with Sds are shown. Significant differences were determined by 2-sided Duncan's new multiple-range tests, which were marked with different letters (P < 0.05). E to G) The resistant phenotype of Oslesv-KO, OsLESV-OX, Osesv1-KO, and OsESV1-OX lines against M. oryzae the conidial suspension spray assay. Photos were taken at 7 dpi. The disease lesion area was assessed using ImageJ software, and the fungal growth was measured by quantifying M. oryzae genomic 28S rDNA relative to rice genomic Rubq1 DNA. The mean values of 3 determinations with Sds are shown. Significant differences were determined by 2-sided Duncan's new multiple-range tests, which were marked with different letters (P < 0.01).

Figure 8.

A proposed model of MoApx1 functioning to suppress host immunity. There are 3 roles of the ascorbate oxidase MoApx1 during the M. oryzae–rice interaction. (1) MoApx1 secreted by the rice blast fungus functions as an extracellular peroxidase to neutralize the host's H₂O₂ directly. (2) MoApx1 targets the PSI subunit OsPsaD, which is crucial for host immunity, thereby inhibiting cROS bursts. (3) MoApx1 features a starch-binding domain that effectively binds to the host's starch, potentially preventing its degradation and thereby disrupting the plant's energy supply and resistance to the disease.