Decapeptide Inducer Promotes the Conidiation of Phytopathogenic Magnaporthe oryzae via the Mps1 MAPK Signaling Pathway

Abstract

Magnaporthe oryzae (M. oryzae) is a phytopathogenic fungus that inflicts damage on vital crops, particularly rice. Its asexual reproduction leads to the generation of numerous conidia, which is a critical factor contributing to the prevalence of rice blast disease. However, the molecules regulating the asexual reproduction of M. oryzae are unknown. In our study, to identify the molecules capable of regulating the asexual reproduction of M. oryzae, compositions of the complete medium (CM) were screened. Results showed that acid-hydrolyzed casein (AHC) could remarkably promote conidial production. One M. oryzae conidiation inducer was isolated from AHC using high-performance liquid chromatography (HPLC) under the guidance of bioassay. Its structure was further elucidated as a decapeptide compound (pyroGlu-EQNQEQPIR) by LC-MS/MS, chemical synthesis, and conidium-inducing assays, named M. oryzae conidiation inducer decapeptide (MCIDP). MCIDP could significantly promote the conidiation of M. oryzae and two other filamentous ascomycetes (Botrytis cinerea and Fusarium graminearum). The Mps1 MAPK cascade signaling pathway is crucial for conidiation, and the effect of MCIDP on this pathway was investigated to elucidate the mechanism underlying conidiation enhancement. qRT-PCR analysis demonstrated that MCIDP could remarkably upregulate the gene expression within the Mps1 MAPK cascade signaling pathway, especially the WSC2, WSC3, PKC1, MKK1, MPS1, and MIG1. Furthermore, the ΔMowsc1, ΔMowsc2, ΔMowsc3, and ΔMomid2 mutant strains were constructed. Bioassay results showed that MCIDP failed to promote conidial formation and hyphal growth in these mutant strains. These findings indicate that MCIDP promotes conidiation of M. oryzae by modulating the Mps1 MAPK signaling pathway.

Keywords: MCIDP; Magnaporthe oryzae; Mps1 MAPK signaling pathway; conidiation.

Figure 1

The impact of AHC, yeast extract, glucose, peptone, TE, vitamins, and nitrate salts on the production of M. oryzae conidium. The effects of 30, 100, and 300 μg/mL of AHC (A), yeast extract (B), glucose (C) and peptone (D), 0.03, 0.1, and 0.3 μL 1000 × trace elements solution (E), and 1000 × vitamin solution (F), 0.15, 0.5 and 1.5 μL 200 × solution (G) on the conidia formation of the M. oryzae strain Guy11 were tested on the 24-well plates; the negative control is sterilized water. The experiment was repeated three times. The data were expressed as a mean ± SEM. *, **, and *** represent a significant difference as compared to the negative group at p < 0.05, 0.01, and 0.001, respectively.

Figure 2

Chemical and biological characteristics of MCIDP. (A) The amino sequence of MCIDP. (B–C) The effects of 30 ng, 300 ng, 3 μg, 30 μg, and 100 μg/mL of isolated MCIDP (B) or synthetic MCIDP (C) on the production of conidia by the M. oryzae strain Guy11 were tested; the negative control was sterilized water. The experiment was repeated three times. The data were expressed as a mean ± SEM. *** represents a significant difference as compared to the negative group at p < 0.001. (D) Micrographs of conidia induced by 30 μg/mL of MCIDP in M. oryzae; (up): fluorescence micrograph; (down): photomicrograph. The fluorescence image of conidia stained with calcofluor white (CFW) was acquired with an Olympus IX53 inverted fluorescence microscope. Conidia were obtained after the filtration of hyphae and agar. BF: bright field. Scale bar: 20 μm.

Figure 3

The effect of MCIDP on conidial formation of different fungi. Effects of 30 μg/mL MCIDP on the production of conidia in the M. oryzae (A), B. cinerea (B), F. graminearum (C), and P. capsici (D) strains were tested on agar plates; the negative control is sterilized water. M. oryzae was tested on CM plates. B. cinerea and P. capsici were tested on V8 juice agar plates, and F. graminearum was tested on PDA plates. The experiment was repeated three times. The data were expressed as a mean ± SEM. * and ** represent a significant difference as compared to the negative group at p < 0.05 and p < 0.01.

Figure 4

MCIDP elevated the expression of genes associated with the MAPK signaling pathway. (A–D) The expression of transmembrane protein genes WSCs and MID2 after MCIDP treatment. (E–H) The expression of downstream core genes after treatment with MCIDP. (I–K) The expression of the main transcription factor genes is regulated by the Mps1 MAPK cascade after treatment with MCIDP. The experiment was repeated three times. The data were expressed as a mean ± SEM. *, **, and *** represent a significant difference compared to the control group at p < 0.05, p < 0.01, and p < 0.001.

Figure 5

The conidiation and mycelial growth of M. oryzae wild-type strain Guy11, ΔMomid2, ΔMowsc1, ΔMowsc2, and ΔMowsc3 mutants. Colony morphology (A), the number of conidia analysis (B), and colony diameter analysis (C) of M. oryzae wild-type strain Guy11, ΔMomid2, ΔMowsc1, ΔMowsc2, and ΔMowsc3 mutants. Each plate was cultured at 25 °C for 10 days in the dark. The experiment was repeated three times. The data were expressed as a mean ± SEM. * and *** represent a significant difference compared to the control group at p < 0.05 and p < 0.001, respectively.

Figure 6

WSCs and MID2 contribute to the efficacy of MCIDP in enhancing conidiation and hyphal growth. Micrograph of the conidia (A), the number of conidia (B), the aerial hyphae morphology (C), the relative hyphal density (D) of wild-type strain Guy11, the ΔMowsc1, ΔMowsc2, ΔMowsc3, and ΔMomid2 mutant with or without 30 μg/mL MCIDP treatment on CM agar plates. Scale bar: 200 μm. Each group was cultured at 25 °C for 10 days in the dark. The experiment was repeated three times. The data were expressed as a mean ± SEM. *, **, and *** represent a significant difference compared to the control group in the wild-type strain Guy11 at p < 0.05, p < 0.01, and p < 0.001. “ns” represents no significant difference compared to the control group.

Figure 7

Proposed potential mechanism of action for MCIDP. MCIDP promotes conidial formation by increasing the gene expression in the Mps1 MAPK cascade signaling pathway.