Balancing of the mitotic exit network and cell wall integrity signaling governs the development and pathogenicity in Magnaporthe oryzae

Abstract

The fungal cell wall plays an essential role in maintaining cell morphology, transmitting external signals, controlling cell growth, and even virulence. Relaxation and irreversible stretching of the cell wall are the prerequisites of cell division and development, but they also inevitably cause cell wall stress. Both Mitotic Exit Network (MEN) and Cell Wall Integrity (CWI) are signaling pathways that govern cell division and cell stress response, respectively, how these pathways cross talk to govern and coordinate cellular growth, development, and pathogenicity remains not fully understood. We have identified MoSep1, MoDbf2, and MoMob1 as the conserved components of MEN from the rice blast fungus Magnaporthe oryzae. We have found that blocking cell division results in abnormal CWI signaling. In addition, we discovered that MoSep1 targets MoMkk1, a conserved key MAP kinase of the CWI pathway, through protein phosphorylation that promotes CWI signaling. Moreover, we provided evidence demonstrating that MoSep1-dependent MoMkk1 phosphorylation is essential for balancing cell division with CWI that maintains the dynamic stability required for virulence of the blast fungus.

Fig 1. The balance of mitosis and cell wall integrity (CWI) is vital for vegetative and infectious growth of M. oryzae.

(A) Liquid cultured hyphae (LH) and infectious hyphae (IH) of the Guy11 transformant expressing H1-RFP were examined by epifluorescence microscopy. Bar, 10 μm. (B) Statistical analysis of the number of nuclei per 100 μm of hyphae of (A). One hundred hyphae were counted for each type and the experiment was repeated three times. Error bars represent the standard deviations. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (C) Phosphorylation analysis of MoMkk1 in LH and IH. MoMkk1-GFP proteins were extracted in the presence of PMSF (PF), phosphatase (PE), as well as phosphatase inhibitors (PI), respectively, and then detected by the anti-GFP antibody. MoMkk1 phosphorylation was estimated by calculating the amount of phosphorylated-MoMkk1 (P-MoMkk1) compared to the total amount of MoMkk1 (the numbers underneath the blot). (D) Pathogenicity test in rice. The spore suspension of Guy11 was divided into two parts, one was treated with Nocodazole, and another without. Two-week-old rice seedlings were inoculated with these conidial suspensions treated with or without Nocodazole, and photographed at 7 days post-inoculation (dpi). (E) Diseased leaf area analysis of (D). Data were presented as a bar chart showing the percentage of lesion areas. Error bars represented the standard deviations from three independent experiments. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (F) Wild-type hyphae treated with or without Nocodazole were stained with CFW and observed by epifluorescence microscopy. Bar, 10 μm. (G) Phosphorylation analysis of MoMkk1 in Guy11 treated with or without Nocodazole (the steps are similar to C). (H) Hyphae of Guy11 and the ΔMomck1, ΔMomkk1, ΔMomps1 mutants expressing the H1-RFP construct were stained with CFW and examined by epifluorescence microscopy. Bar, 10 μm.

Fig 2. The Mitosis Exit Network (MEN) kinase MoSep1 interacts with MoMkk1.

(A) Schematic representation of MoSep1 and its two segments MoSep1^STK and MoSep1^BACK. S_TKc domain was predicted using the SMART program (http//smart.embl-heidelberg.de/). aa, amino acid. (B) Yeast two-hybrid analysis of the interaction between MoMkk1 and MoSep1. MoMkk1 was inserted into vector pGADT7, two segments of MoSep1 (MoSep1^STK and MoSep1^BACK), and MoSep1 full-length were inserted into pGBKT7. AD-MoMkk1 and BD-MoSep1, AD-MoMkk1 and BD-MoSep1^STK, AD-MoMkk1, and BD-MoSep1^BACK were co-introduced into yeast AH109 strain, respectively, and then incubated on SD-Leu-Trp (as control) and SD-Leu-Trp-His-Ade (for selection) for 5 days. (C) In vitro pull-down assay of GST-MoMkk1 and His-MoSep1, His-MoSep1^STK, His- MoSep1^BACK. Recombinant GST-MoMkk1 bound to glutathione Sepharose beads was incubated with the bacterial cell lysate containing His-MoSep1, His-MoSep1^STK, His-MoSep1^BACK, respectively. Eluted protein was analyzed by immunoblot (IB) with anti-His and anti-GST antibodies. GST, glutathione transferase. His, histidine. (D) Co-IP assay for the interaction between MoMkk1 and MoSep1. Plasmids of MoMkk1-RFP and MoSep1^STK-GFP, MoMkk1-RFP, and MoSep1^BACK-GFP were co-expressed in wild-type Guy11, respectively, and proteins were detected using anti-RFP and anti-GFP antibodies. Lysed hyphal proteins were allowed to bind to RFP beads at 4°C for 4 h and then analyzed by IB with appropriate antibodies.

Fig 3. MEN components MoSep1, MoDbf2, and MoMob1 are involved in vegetative growth and conidiation of M. oryzae.

(A) The ΔMosep1, ΔModbf2, and ΔMomob1 mutants displayed significantly reduced mycelial growth on complete media (CM), minimal media (MM), oatmeal media (OM), and straw decoction and corn media (SDC) after incubation at 28°C for 7 days in darkness. (B) Statistical analysis of colony diameters from Guy11, ΔMosep1, ΔModbf2, and ΔMomob1 mutants, and their corresponding complemented strains on different media. Error bars represent standard deviations from three independent experiments. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (C) The first line shows aerial hyphae (AH) growth was reduced in the ΔMosep1, ΔModbf2, and ΔMomob1 mutants after incubation for 10 days. The second line shows conidia formation under a light microscope 24 h at room temperature after induction of conidiation under coverslips. Bar, 50 μm. The bottom line shows conidia of Guy11, the ΔMosep1, ΔModbf2, and ΔMomob1 mutants examined by differential interference contrast (DIC) and epifluorescence microscopy, respectively. NA, not available. Bar, 10 μm.

Fig 4. MoSep1, MoDbf2, and MoMob1 contribute to virulence.

(A) Pathogenicity assay in rice. Two-week-old rice seedlings were inoculated conidial suspensions of Guy11, the ΔMosep1, ΔModbf2, ΔMomob1 mutants, and their corresponding complemented strains and photographed at 5 days post-inoculation (dpi). (B) Quantification of lesion types (per 1.5 cm²) on susceptible rice spayed with conidia Guy11, the ΔMosep1, ΔModbf2, ΔMomob1 mutants, and their corresponding complemented strains. Lesions were quantified by a ‘lesion-type’ scoring assay which divided the lesions into 1–5 types according to their severity (type 1, pinhead-sized dark brown specks without visible centers; type 2, small brown lesions that are approximately 1 mm in diameter; type 3, 2–3 mm gray spots with brown margins; type 4, elliptical gray spots longer than approximately 3–4 mm; type 5, large eyespot lesions that coalesced infecting 50% or more of the leaf area). Error bars represent the standard deviations from three independent experiments. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (C) Pathogenicity was tested by rice injection assay with the conidia concentration of 5 × 10⁴ spores/ml and photographed at 7 dpi. The arrow points to the injection site. (D) Diseased leaf area analysis of (C). The data were presented as a bar chart showing the percentage of lesion areas. Error bars represent the standard deviations from three independent experiments. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (E, F) Statistical analysis of the infectious hyphal type (type 1, no penetration; type 2, with penetration peg; type 3, with a single invasive hypha; type 4, with extensive hyphal growth) on rice leaf sheaths. Rice leaf sheaths were inoculated with conidial suspensions and examined at 36 h post-inoculation (hpi). One hundred infectious hyphae were counted for each strain and the experiment was repeated three times. Error bars represent the standard deviations.

Fig 5. MoSep1, MoDbf2, and MoMob1 are involved in the Mitosis Exit Network (MEN).

(A) Hyphae of Guy11 and the ΔMosep1, ΔModbf2, ΔMomob1 mutants expressing the H1-RFP construct were stained with CFW and examined by epifluorescence microscopy. Bar, 10 μm. (B) Infectious hyphae of strains mentioned in (A) examined by epifluorescence microscopy. Bar, 10 μm. (C) Statistical analysis of the number of septa per 100 μm of hyphae in (A). One hundred hyphae were counted for each strain and the experiment was repeated three times. Error bars represent the standard deviations. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (D) Statistical analysis of the number of nuclei per cell of hyphae in (A). One hundred hyphae were counted for each strain and the experiment was repeated three times. Error bars represent the standard deviations. Asterisks denote statistical significance according to a Student’s test (p<0.01). (E) Statistical analysis of the number of nuclei per 100 μm of hyphae in (A). One hundred hyphae were counted for each strain and the experiment was repeated three times. Error bars represent the standard deviations. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (F) Statistical analysis of the number of nuclei per 100 μm of infectious hyphae of (B). One hundred infectious hyphae were counted for each strain and the experiment was repeated three times. Error bars represent the standard deviations. Asterisks indicate statistical significance according to a Student’s test (p<0.01).

Fig 6. Disruption of MEN results in defects in CWI signaling.

(A) Light microscopic examination of protoplast release of Guy11, ΔMosep1, ΔModbf2, and ΔMomob1 mutants after treatment with cell wall-degrading enzymes for 45 min at 30°C. Bar, 50 μm. (B) Statistical analysis of protoplast release of Guy11, and the ΔMosep1, ΔModbf2, ΔMomob1 mutants after treatment with cell wall-degrading enzymes for 30 min, 60 min, and 90 min at 30°C. Error bars represent the standard deviations from three independent experiments. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (C) The hyphal diameter of Guy11, ΔMosep1, ΔModbf2, and ΔMomob1 mutants examined 7 days after incubation on CM agar plates with different cell wall-perturbing agents including 400 μg/ml for CFW, and 400 μg/ml for Congo red. The experiments were repeated three times. Error bars represent the standard deviations from three independent experiments. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (D) Observation of chitin in the cell wall. Hyphae of Guy11, ΔMosep1, ΔModbf2, and ΔMomob1 mutants were stained with CFW for 5 min in darkness. Bar, 10 μm. CFW is widely used to stain cell wall chitin in fungi. (E) Linescan graph analysis of chitin stained with CFW of (D).

Fig 7. MoSep1 phosphorylates MoMkk1.

(A) Yeast two-hybrid analysis between CWI components (MoMCK1, MoMKK1, and MoMPS1) with MEN components (MoSEP1, MoDBF2, and MoMOB1). pGADT7 and pGBKT7 fused with specific genes were co-introduced into yeast AH109 strain, and transformants were plated on SD-Leu-Trp as control and on SD-Leu-Trp-His-Ade for selection. (B) In vivo phosphorylation analysis of MoMkk1 in Guy11 and the ΔMosep1 mutant. MoMkk1-GFP proteins treated with phosphatase inhibitors (PI), phosphatase (PE), and detected by the anti-GFP antibody. MoMkk1 phosphorylation was estimated by calculating the amount of phosphorylated-MoMkk1 (P-MoMkk1) compared to total MoMkk1 (the numbers underneath the blot). (C) In vitro phosphorylation analysis by the fluorescence detection in tube (FDIT) method. Purified proteins of GST-MoMkk1, His-MoSep1 were used for protein kinase reaction in the presence of 50 μm ATP in a kinase reaction buffer and then dyed with Pro-Q Diamond Phosphorylation Gel Stain. Fluorescence signal at 590 nm (excited at 530 nm) was measured in a Cytation3 microplate reader (Biotek, Winooski, VT, USA). Error bars represent the standard deviations from three independent experiments. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (D) MoMkk1 phosphorylation sites in the Guy11 compared with the ΔMosep1 mutant expressing MoMKK1 was identified by LC-MS/MS analysis.

Fig 8. MoSep1-dependent MoMkk1 phosphorylation is vital for vegetative growth, conidiation, and pathogenicity in M. oryzae.

(A) Growth of ΔMosep1/MoSEP1, ΔMosep1, ΔMosep1/MoMKK1^6D (ΔMosep1/MoMKK1^{S19D, T24D, S125D, S136D, T139D, T207D}), ΔMosep1/MoMKK1^S19D, ΔMosep1/MoMKK1^T24D, ΔMosep1/MoMKK1^S125D, ΔMosep1/MoMKK1^S136D, ΔMosep1/MoMKK1^T139D, ΔMosep1/MoMKK1^T207D mutant strains on CM medium. (B) Statistical analysis of conidia production on SDC medium cultured at 28°C for 7 days in the dark followed by 3 days of continuous illumination under fluorescent light. Error bars represent the standard deviations from three independent experiments. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (C) Pathogenicity analysis using rice spraying assays and photographed at 7 dpi. (D) Diseased leaf area analysis of (C). Data were presented as a bar chart showing the percentage of lesion areas. Error bars represented the standard deviations from three independent experiments. Asterisks denote statistical significance according to a Student’s test (p<0.01). (E) Statistical analysis of the infectious hyphal type (type 1, no penetration; type 2, with penetration peg; type 3, with a single invasive hypha; type 4, with extensive hyphal growth) on rice leaf sheaths. Rice leaf sheaths were inoculated with conidial suspensions and examined at 36 h post-inoculation (hpi). One hundred infectious hyphae were counted for each strain and the experiment was repeated three times. Error bars represented the standard deviations.

Fig 9. Disruption of MoSep1-dependent MoMkk1 phosphorylation leads to hyphal autolysis, reduced conidiation, and attenuated virulence.

(A) Autolysis observation of ΔMomkk1/MoMKK1, ΔMomkk1, ΔMomkk1/MoMKK1^6A (ΔMomkk1/MoMKK1^{S19A, T24A, S125A, S136A, T139A, T207A}), ΔMomkk1/MoMKK1^S19A, ΔMomkk1/MoMKK1^T24A, ΔMomkk1/MoMKK1^S125A, ΔMomkk1/MoMKK1^S136A, ΔMomkk1/MoMKK1^T139A, ΔMomkk1/MoMKK1^T207A mutant strains on CM medium. Hyphae autolysis is a process of self-digestion of hyphal cultures. The part of the aerial hyphae with autolysis appeared watery collapse. (B) Statistical analysis of conidia production on SDC medium cultured at 28°C for 7 days in the dark followed by 3 days of continuous illumination under fluorescent light. Error bars represent the standard deviations from three independent experiments. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (C) Pathogenicity test on rice leaves. Wounded rice leaves were incubated with different strains. Diseased leaves were photographed 4 days after inoculation. (D) Diseased leaf area analysis of (C). Data were presented as a bar chart showing the percentage of lesion areas. Error bars represent the standard deviations from three independent experiments. Asterisks indicate statistical significance according to a Student’s test (p<0.01).

Fig 10. MoSep1-dependent MoMkk1 phosphorylation is vital for MEN in M. oryzae.

(A) Hyphae of transformants of Guy11, the ΔMosep1, ΔMosep1/MoMKK1^6D, ΔMomkk1, ΔMomkk1/MoMKK1^6A mutants expressing the H1-RFP construct were stained with CFW and examined by epifluorescence microscopy. Bar, 10 μm. (B) Statistical analysis of the number of nuclei per cell of hyphae in (A). One hundred hyphae were counted for each strain and the experiment was repeated three times. Error bars represent the standard deviations. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (C) Statistical analysis of the number of septa per 100 μm of hyphae in (A). One hundred hyphae were counted for each strain and the experiment was repeated three times. Error bars represent the standard deviations. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (D) Conidia of transformants of Guy11, the ΔMosep1, ΔMosep1/MoMKK1^6D mutants expressing the H1-RFP construct were stained with CFW and examined by epifluorescence microscopy. The values mean proportion of corresponding type conidia in the total for each strain. Bar, 10 μm. (E) Infectious hyphae of Guy11, the ΔMosep1, ΔMosep1/MoMKK1^6D mutants expressing the H1-RFP construct examined by epifluorescence microscopy. Bar, 10 μm. (F) Statistical analysis of the number of nuclei per conidium in (D). One hundred conidia were counted for each strain and the experiment was repeated three times. Error bars represent the standard deviations. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (G) Statistical analysis of the number of septum per conidium in (D). One hundred conidia were counted for each strain and the experiment was repeated three times. Error bars represent the standard deviations. Asterisks indicate statistical significance according to a Student’s test (p<0.01). (H) Statistical analysis of the number of nuclei per 100μm infectious hypha in (E). One hundred infectious hyphae were counted for each strain and the experiment was repeated three times. Error bars represented the standard deviations. Asterisks indicate statistical significance (p<0.01).

Fig 11. M. oryzae ensures cell wall integrity and normal progress of cell division through MoSep1-mediated MoMkk1 phosphorylation.

Germination of conidia and expansion of infectious hyphae are accompanied by cell division and the cell wall synthesis. Normally, the MEN pathway cross talks with the CWI pathway through MoSep1 specific phosphorylation of MoMkk1, completing cell division and concurrently ensuring cell wall integrity during infection. However, the crosstalk between cell division and CWI cannot respond when the interaction of MoSep1 and MoMkk1 is blocked. Septa could not be normally formed, multiple nuclei per cell, and disordered distribution of cell wall chitin led to disrupted cell division and impaired CWI. The abnormality results in loss or attenuation of pathogenicity.