Phenazine biosynthesis protein MoPhzF regulates appressorium formation and host infection through canonical metabolic and noncanonical signaling function in Magnaporthe oryzae

Abstract

Microbe-produced secondary metabolite phenazine-1-carboxylic acid (PCA) facilitates pathogen virulence and defense mechanisms against competitors. Magnaporthe oryzae, a causal agent of the devastating rice blast disease, needs to compete with other phyllosphere microbes and overcome host immunity for successful colonization and infection. However, whether M. oryzae produces PCA or it has any other functions remains unknown. Here, we found that the MoPHZF gene encodes the phenazine biosynthesis protein MoPhzF, synthesizes PCA in M. oryzae, and regulates appressorium formation and host virulence. MoPhzF is likely acquired through an ancient horizontal gene transfer event and has a canonical function in PCA synthesis. In addition, we found that PCA has a role in suppressing the accumulation of host-derived reactive oxygen species (ROS) during infection. Further examination indicated that MoPhzF recruits both the endoplasmic reticulum membrane protein MoEmc2 and the regulator of G-protein signaling MoRgs1 to the plasma membrane (PM) for MoRgs1 phosphorylation, which is a critical regulatory mechanism in appressorium formation and pathogenicity. Collectively, our studies unveiled a canonical function of MoPhzF in PCA synthesis and a noncanonical signaling function in promoting appressorium formation and host infection.

Keywords: Magnaporthe oryzae; MoPhzF; MoRgs1 phosphorylation; appressorium formation; membrane recruitment; pathogenicity; phenazine biosynthesis; phenazine-1-carboxylic acid.

Fig. 1.. The MoPhzF domain structure and critical active site for its binding ability to trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA).

(a) A schematic representation of MoPhzF. PhzC-PhzF domain (blue rectangle) and the catalytic active site (Glu77, 77E, orange line) were predicted by UniProt (https://www.uniprot.org/uniprot/G4N988). aa, amino acid. (b) Structures of MoPhzF and MoPhzF^E77D predicted with AlphaFold2. (c) Docking analysis of MoPhzF and MoPhzF^E77D with DHHA. Binding affinity is obtained by AutoDock Vina. Glu77 and Asp77 are indicated by orange circles. DHHA is indicated by a blue circle.

Fig. 2.. MoPhzF is essential for phenazine-1-carboxylic acid (PCA) production.

(a) Mass spectrum of highly purified PCA in positive ion mode. The ion peaks pointed by the arrows indicate the presence of PCA with m/z 225.11 and the fragment m/z of 207.10. (b) The presence of a single peak pointed by the arrows at 2.27 min indicates that high purity PCA was used as the standard. (c) Liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis of phenazine-1-carboxylic acid in Guy11, ΔMophzf, ΔMophzf/MoPHZF^E77D, and ΔMophzf/MoPHZF. (d) A standard curve of PCA and the content of PCA in different strains. Data were represented by means ± standard deviations. Data sets marked with asterisks are significantly different from the Guy11 strain under the same conditions (Student’s t-test, ** p < 0.01). PCA, phenazine-1-carboxylic acid. All experiments were conducted with three biological repetitions and three replicates.

Fig. 3.. MoPhzF regulates appressorium formation and pathogenicity by mediating MoRgs1 protein phosphorylation.

(a) Yeast two-hybrid analysis of the interaction between MoPhzF and MoRgs1. MoPhzF and MoRgs1 cDNA was inserted into pGBKT7 and pGADT7, respectively. Yeast transformants co-expressing the bait and prey constructs were incubated on SD-Leu-Trp plates and replicated onto SD-Leu-Trp-His-Ade plates. Transformants co-expressing pGADT7-RECT and pGBKT7–53, and pGADT7-RECT and pGBKT7-Lam were used as the positive control and negative control, respectively. (b) Bimolecular fluorescence complementation assay showing the interaction between MoPhzF and MoRgs1. The transformant co-expressing the MoPhzF-YFP^N and MoRgs1-YFP^C was observed for fluorescence using confocal fluorescence microscopy (Zeiss LSM710, 63×oil) during the germ tube hooking stage (3 h). Strains co-expressing MoPhzF-YFP^N and empty YFP^C, MoRgs1-YFP^C and empty YFP^N, and empty YFP^C and empty YFP^N were used as negative controls. YFP, yellow fluorescent protein. Bar represents 10 μm. (c) Co-immunoprecipitation assay for the interaction between MoPhzF and MoRgs1. Proteins were extracted from transformants co-expressing MoPhzF-Flag and MoRgs1-GFP during the germ tube hooking stage (3 h) and incubated with anti-GFP agarose beads and eluted. Total and eluted proteins were analyzed by Western blotting, and the presence of MoRgs1 and MoPhzF were detected with anti-GFP and anti-Flag antibodies, respectively. GFP was used as the negative control. T: Total proteins. E: Eluted proteins. Coomassie brilliant blue (CBB) staining indicates loading controls. (d) In vivo phosphorylation analysis of MoRgs1 in Guy11 and the ΔMophzf mutant. MoRgs1-GFP proteins were extracted from transformants during the germ tube hooking stage (3 h) and treated with phosphatase or phosphatase inhibitors, and analyzed by Mn²⁺-Phos-tag SDS-PAGE and normal SDS-PAGE with the anti-GFP antibody, respectively. PI, Phosphatase inhibitor. Phosphorylated-MoRgs1-GFP, P-MoRgs1:GFP. (e) Appressorium formation assay. Conidia of Guy11, ΔMophzf, ΔMophzf/MoRGS1^5A, ΔMophzf/MoRGS1^5D, and ΔMophzf/MoPHZF were incubated on artificial inductive surfaces. Appressorium formation rates at different time points were calculated and analyzed. The percentage at a given time was recorded by observing at least 100 conidia for each strain, and calculated with three replicates. Data were represented by means ± standard deviations, and different letters indicate significant differences (Duncan’s new multiple range test, p < 0.05). (f) Quantification of intracellular cAMP levels. Intracellular cAMP from mycelia cultured in the liquid CM for 48 h were exacted, and levels were quantified by high-performance liquid chromatography (HPLC). Data were represented by means ± standard deviations, and different letters indicate significant differences (Duncan’s new multiple range test, p < 0.05). (g) Pathogenicity assay of Guy11, ΔMophzf, ΔMophzf/MoRGS1^5A, ΔMophzf/MoRGS1^5D, and ΔMophzf/MoPHZF. Rice (Oryza sativa cv. CO39) seedlings were sprayed with conidial suspensions (5×10⁴ spores/mL), and lesions were photographed at 7 days post-inoculation. (h) The disease lesion areas were assessed by Image J. Data were represented by means ± standard deviations, and different letters indicate significant differences (Duncan’s new multiple range test, p < 0.05). (i) Leaf sheaths of 3-week-old rice seedlings were inoculated with conidial suspensions (2×10⁵ spores/mL) of Guy11, ΔMophzf, ΔMophzf/MoRGS1^5A, ΔMophzf/MoRGS1^5D, and ΔMophzf/MoPHZF. Detailed observation and statistical analysis for invasive hyphae in rice sheath cells at 36 h post-inoculation. Appressorium penetration sites (n = 100) were observed, and invasive hyphae were rated from type 1 to 4 (type1, no penetration with only appressoria; type 2, only with a penetration peg or primary invasion hyphal; type 3, secondary invasive hypha extended but was limited in one plant cell; type 4, invasive hyphae extended to surrounding cells). Data were represented by means ± standard deviations. Bar represents 20 μm. All experiments were conducted with three biological repetitions.

Fig. 4.. MoPhzF is required for MoEmc2-mediated MoRgs1 membrane recruitment and phosphorylation.

(a) Yeast two-hybrid analysis of MoPhzF-MoEmc2 interaction. MoPhzF cDNA was inserted into pGBKT7, and MoEmc2 cDNA was inserted into pGADT7. Yeast transformants co-expressing the bait and prey constructs were incubated on SD-Leu-Trp plates and screened on SD-Leu-Trp-His-Ade plates. pGADT7-RECT and pGBKT7–53 were used as the positive control, and pGADT7-RECT and pGBKT7-Lam were used as the negative control. (b) Bimolecular fluorescence complementation assay for the interaction between MoPhzF and MoEmc2. The transformant co-expressing the MoPhzF-YFP^C and MoEmc2-YFP^N was observed with confocal fluorescence microscopy (Zeiss LSM710, 63×oil) during the germ tube hooking stage (3 h). Strains co-expressing MoPhzF-YFP^C and empty YFP^N, MoEmc2-YFP^N and empty YFP^C, and empty YFP^C and empty YFP^N were used as negative controls. YFP, yellow fluorescent protein. Bar represents 10 μm. (c) Co-immunoprecipitation assay for the interaction between MoPhzF and MoEmc2. Total proteins were extracted from transformants co-expressing MoPhzF-GFP and MoEmc2-RFP, incubated with anti-GFP agarose beads, and eluted. Total and eluted proteins were analyzed by Western blotting, and the presence of MoPhzF and MoEmc2 was detected by Western blotting with anti-GFP and anti-RFP antibodies, respectively. GFP was used as the negative control. T: Total proteins. E: Eluted proteins. Coomassie brilliant blue (CBB) staining indicates loading controls. (d) Co-immunoprecipitation assays for the interactions between MoRgs1 and MoEmc2 in Guy11 and ΔMophzf, respectively. Proteins were extracted and eluted as above. The presence of MoRgs1 and MoEmc2 was analyzed by Western blotting with the corresponding antibodies. T: Total proteins. E: Eluted proteins. Coomassie brilliant blue (CBB) staining indicates loading controls. (e,f) Subcellular localization of MoRgs1-GFP and MoEmc2-GFP was visualized in Guy11 and ΔMophzf during the germ tube hooking stage (3 h), respectively. White arrows indicated the regions where the fluorescence intensity was measured by line-scan analysis. The percentage of a pattern shown in the image was calculated by observation for 50 germinated conidia that were randomly chosen. PM, plasma membrane. Bar represents 10 μm. All experiments were conducted with three biological repetitions.

Fig. 5.. MoPhzF-mediated PCA production is required for the invasive hyphae growth and full virulence of M. oryzae.

(a) Two-week-old rice (Oryza sativa cv. CO39) seedlings were sprayed with conidial suspensions (5×10⁴ spores/mL) of Guy11, ΔMophzf, ΔMophzf/MoPHZF^E77D, and ΔMophzf/MoPHZF following 5 μg/L PCA treatment and photographed 7 days post-inoculation. DMSO treatment was used as a negative control. (b) Diseased leaf areas were assessed using ImageJ. Data were represented by means ± standard deviations, and columns marked with different letters indicate significant differences (Duncan’s new multiple range test, p < 0.05). (c) Rice leaf sheaths of 3-week-old rice seedlings were inoculated with conidial suspensions (2×10⁵ spores/ml) of Guy11, ΔMophzf, ΔMophzf/MoPHZF^E77D, and ΔMophzf/MoPHZF following 5 μg/L PCA treatment. Detailed observation and statistical analysis for infectious growth in rice sheath cells were made at 36 h post-inoculation. Appressorium penetration sites (n = 100) were observed, and invasive hyphae were rated from type 1 to 4 (type1, no penetration with only appressoria; type 2, only with a penetration peg or primary invasion hyphal; type 3, secondary invasive hypha extended but was limited in one plant cell; type 4, invasive hyphae extended to surrounding cells). Data were represented by means ± standard deviations. Bar represents 20 μm. PCA, phenazine-1-carboxylic acid. All experiments were conducted with three biological repetitions.

Fig. 6.. MoPhzF-mediated PCA production is involved in suppressing ROS accumulation in the host.

(a) DAB was used to stain leaf sheaths injected with conidia suspensions of Guy11, ΔMophzf, ΔMophzf/MoPHZF^E77D, and ΔMophzf/MoPHZF following 5 μg/L PCA treatment at 36 h post-inoculation. The DMSO solvent treatment was used as a control. Bar represents 20 μm. (b) Statistical analysis of infected rice cells stained by DAB. Error bars represent standard deviations, and different letters indicate significant differences (Duncan’s new multiple range test, p < 0.05). (c) CM-H₂DCFDA staining on infected leaf sheaths by Guy11, ΔMophzf, ΔMophzf/MoPHZF^E77D, and ΔMophzf/MoPHZF following 5 μg/L PCA treatment at 36 h post-inoculation. Percentages of leaf sheath cells stained by CM-H₂DCFDA were shown in the image. Data were represented by means ± standard deviations, and different letters indicate significant differences (Duncan’s new multiple range test, p < 0.05). Bar represents 20 μm. (d) The transcriptional level of OsRBOHD in the infected rice was assayed using RT-qPCR. RNA samples were collected from rice leaves sprayed with conidial suspensions (2×10⁵ spores/mL) of Guy11, ΔMophzf, ΔMophzf/MoPHZF^E77D, and ΔMophzf/MoPHZF following 5 μg/L PCA treatment at 48 h post-inoculation. RT-qPCR was used to evaluate gene expression, with OsACTIN as the internal reference gene. Data were represented by means ± standard deviations, and columns marked with different letters indicate significant differences (Duncan’s new multiple range test, p < 0.05). PCA, phenazine-1-carboxylic acid. All experiments were conducted with three biological repetitions.

Fig. 7.. A proposed model depicting an ancient event of PhzF acquisition and functions of MoPhzF in regulating the development, appressorium formation, and pathogenicity of Magnaporthe oryzae.

M. oryzae acquires the phenazine biosynthesis gene PHZF likely through an ancient horizontal gene transfer (HGT) event from bacteria, and MoPhzF plays dual roles in M. oryzae. M. oryzae-secreted phenazine-1-carboxylic acid (PCA) overcomes host immunity during infection by suppressing host ROS accumulation. At the same time, MoPhzF mediates MoRgs1 and MoEmc2 recruitment to the plasma membrane (PM), where MoRgs1 undergoes phosphorylation to regulate G-protein/cAMP signal transduction. GPCR: G-protein coupled receptor. Gα, Gβ, and Gγ: heterotrimeric G-protein α, β, and γ subunit, respectively.