Melanin Promotes Spore Production in the Rice Blast Fungus Magnaporthe oryzae

Abstract

The rice blast pathogen, Magnaporthe oryzae, spreads through spores and invades rice through appressoria. Melanin is necessary for an appressorium to penetrate plant cells, but there are many unknown aspects of its role in fungal conidiation. In this study, we confirmed that melanin promotes spore production in M. oryzae, and that this effect is related to the background melanin content of wild-type strains. In the wild-type 70-15 strain with low melanin content of aerial hyphae, increased melanin synthesis promoted sporulation. In contrast, increased melanin synthesis in the wild-type Guy11 strain, which has higher melanin content, did not promote sporulation. The transcription factor Cnf1 (conidial production negative regulatory factor 1), which negatively regulates melanin synthesis, has opposite effects in conidiophore differentiation of Guy11 and 70-15. Deletion of CNF1 did not abolish the defects of Δcos1 and Δhox2 (where COS1/conidiophore stalk-less 1 or HOX2/homeodomain protein 2 was deleted) in conidiation, while increased the conidiation of Δgcc1 and Δgcf3 (where GCC1/growth, conidiation and cell wall regulatory factor 1, or GCF3/growth and conidiation regulatory factor 3 was deleted). Pig1 (pigment of Magnaporthe 1) regulates the melanin synthesis of hyphae but not of conidiophores, spores, or appressoria. Deletion of the same gene in different wild-type strains can lead to different phenotypes, partly because of differences in melanin content between fungal strains. Overall, this study reveals the functional diversity and complexity of melanin in different M. oryzae strains.

Keywords: Magnaporthe oryzae; conidia; melanin; rice blast; sporulation; transcription factor.

FIGURE 1

The different functions of Pig1 and Cnf1 on hyphal melanin synthesis in the rice blast fungus. (A) Colonial morphology and color. Bar = 5 mm. (B) Melanin contents in aerial mycelia of Guy11 and its mutants. (C) Melanin contents in aerial mycelia of 70-15 and its mutants. (D,E) Relative expression levels of four melanin synthesis genes (ALB1, HNR1, RSY1, and BUF1) in aerial mycelia of Guy11 (D) and 70-15 (E) with α-ACTIN and 40S as reference genes. (B–E) Error bars represent ± SD. Different lowercase letters represent significant differences between four strains as estimated by Tukey’s HSD test (p < 0.05).

FIGURE 2

The functions of Pig1 and Cnf1 on melanin synthesis in spores and conidia. (A) Images showing spore color. (B) Images of appressorium morphology. Bar = 10 μm. (C,D) Appressorium formation rates in Δpig1, Δcnf1Δpig1, and Δcnf1 derived from the wild-type strains Guy11 and 70-15. (C,D) Error bars represent ± SD. Different lowercase letters represent significant differences between four strains as estimated by Tukey’s HSD test (p < 0.05).

FIGURE 3

The different functions of Pig1 and Cnf1 on conidiation in M. oryzae strains Guy11 and 70-15. (A) Spore production of the wild-type Guy11 and its derived mutants (Δpig1, Δcnf1Δpig1, and Δcnf1). (B) Spore production of the wild-type 70-15 and its derived mutants (Δpig1, Δcnf1Δpig1, and Δcnf1). Error bars represent ± SD. Different lowercase letters represent significant differences between four strains as estimated by Tukey’s HSD test (p < 0.05). (C) Aerial hyphae and conidiophores formed by the wild-type Guy11 and 70-15, and their derived mutants (Δpig1, Δcnf1Δpig1, and Δcnf1). The aerial hyphae were dyed blue by lactophenol aniline blue. Red arrows indicate conidiophores, and black arrows indicate aerial hyphae. Bar = 50 μm. (D) Conidiophores and conidia of the wild-type Guy11 and 70-15, and their derived mutants (Δpig1, Δcnf1Δpig1, and Δcnf1). Red arrows indicate conidiophores, and white arrows indicate conidia. Bar = 20 μm.

FIGURE 4

Melanin promotes conidiation in M. oryzae. (A) Images of colonial morphology and color of the wild-type 70-15 and its derived mutants (Δcnf1, Δrsy1, Δcnf1Δrsy1, and Δcnf1-rsy1c, a complementation strain of RSY1 in Δcnf1Δrsy1). Bar = 5 mm. (B) Spore production in the wild-type 70-15 and its derived mutants. Error bars represent ± SD. Different lowercase letters represent significant differences between four strains as estimated by Tukey’s HSD test (p < 0.05).

FIGURE 5

Diverse roles of five transcription factors in regulating conidiation and melanin synthesis in M. oryzae. (A) Conidiation in the wild-type 70-15, and its derived mutants (Δcnf1, Δgcc1, Δcnf1Δgcc1, Δgcf3, Δcnf1Δgcf3, Δcos1, Δcnf1Δcos1, Δhox2, and Δcnf1Δhox2). Error bars represent ± SD. Different lowercase letters represent significant differences between strains as estimated by Tukey’s HSD test (p < 0.05). (B) Images of colonial morphology and color of the wild-type 70-15 and its derived mutants. Bar = 5 mm. (C) Spore morphology of the wild-type 70-15 and its derived mutants. Upper, percentage of spores with one cell, two cells, and three cells. More than 120 spores were counted for each strain. Error bars represent ± SD. Lower, spore morphology. Bar = 20 μm.

FIGURE 6

Analysis of phosphorylation level of Mps1 and Pmk1 in the wild-type 70-15 and its derived mutants (Δpig1, Δcnf1, and Δcnf1Δpig1) by western blot. Phosphorylated Pmk1 and Mps1 were detected using an anti-Phospho-p44/42 MAPK antibody. Total Pmk1 and Mps1 were detected using an anti-ERK1/ERK2 MAPK antibody. GAPDH was detected using an anti-GAPDH antibody. Numbers represent the protein contents of phospho-Pmk1 or phospho-Mps1 relative to total Pmk1 or Mps1.

FIGURE 7

Pmk1 is required for regulation of melanin synthesis and conidiation. (A) Images of colonial morphology and color of the wild-type 70-15 and its derived mutant Δpmk1_70–15. Bar = 5 mm. (B) Conidiation in the wild-type 70-15 and its derived mutant Δpmk1_70–15. (C) Relative expression levels of two genes involved in conidiation (COS1 and HOX2) and six melanin synthesis-related genes (CNF1, PIG1, HNR1, ALB1, RSY1, and BUF1) in aerial mycelia of Δpmk1_70–15. The data were shown after comparison with the wild-type 70–15. β-TUBULIN and H3 were used as reference genes. (B,C) Error bars represent ± SD. Significant differences between the wild-type 70-15 and Δpmk1_70–15 were estimated by Tukey’s HSD test (*p < 0.05, **p < 0.01, and ****p < 0.0001).

FIGURE 8

A schematic diagram of melanin and transcription factors regulating sporulation.