MoHG1 Regulates Fungal Development and Virulence in Magnaporthe oryzae

Abstract

Magnaporthe oryzae causes rice blast disease, which threatens global rice production. The interaction between M. oryzae and rice is regarded as a classic model for studying the relationship between the pathogen and the host. In this study, we found a gene, MoHG1, regulating fungal development and virulence in M. oryzae. The ∆Mohg1 mutants showed more sensitivity to cell wall integrity stressors and their cell wall is more easily degraded by enzymes. Moreover, a decreased content of chitin but higher contents of arabinose, sorbitol, lactose, rhamnose, and xylitol were found in the ∆Mohg1 mutant. Combined with transcriptomic results, many genes in MAPK and sugar metabolism pathways are significantly regulated in the ∆Mohg1 mutant. A hexokinase gene, MGG_00623 was downregulated in ∆Mohg1, according to transcriptome results. We overexpressed MGG_00623 in a ∆Mohg1 mutant. The results showed that fungal growth and chitin contents in MGG_00623-overexpressing strains were restored significantly compared to the ∆Mohg1 mutant. Furthermore, MoHG1 could interact with MGG_00623 directly through the yeast two-hybrid and BiFC. Overall, these results suggest that MoHG1 coordinating with hexokinase regulates fungal development and virulence by affecting chitin contents and cell wall integrity in M. oryzae, which provides a reference for studying the functions of MoHG1-like genes.

Keywords: MoHG1; cell wall integrity; chitin; hexokinase; rice blast.

Figure 1

MoHG1 influences the fungal development. (A) The amino acid sequence alignment between MoHG1 and MGG_14388. (B) Phylogenetic analyses of MoHG1 homologous sequences in different host-infecting strains. (C) Colonic phenotypes of ∆Mohg1 at CM and MM plates. (D–H) The diameter of the colony, dry weight, spore production, spore germination, and appressorium formation. Each experiment was conducted with 3 biological repeats and statistically significant differences were calculated by Student’s t-test, ** p < 0.01. Error bars represent the means ± SD.

Figure 2

MoHG1 regulates fungal cell wall integrity in M. oryzae. (A,B) ∆Mohg1 mutants show more sensitivity to CR (600 μg/mL), SDS (100 μg/mL), and CFW (4 μg/mL). (C,D) ∆Mohg1 mutants show more sensitivity to sorbitol (1 mol/L), KCl (0.7 mol/L), and NaCl (0.7 mol/L). (E,F) The released protoplast in WT and ∆Mohg1 mutants. Each experiment was conducted with 3 biological repeats and statistically significant differences were calculated by Student’s t-test, * p < 0.05, ** p < 0.01. Error bars represent the means ± SD. (G) The fungal cell wall staining in ∆Mohg1 and WT.

Figure 3

MoHG1 plays an essential role in pathogenicity. (A,B) The pathogenicity of ∆Mohg1 mutants on rice. (C–G) The expressions of basal defense genes in rice inoculated by ∆Mohg1 and WT. (H) The fluorescent signal of MoHG1-GFP in M. oryzae during infection. Each experiment was conducted with 3 biological repeats and statistically significant differences were calculated by Student’s t-test, * p < 0.05, ** p < 0.01. Error bars represent the means ± SD.

Figure 4

Transcriptome analysis between WT and ∆Mohg1. (A) The volcano plot of DEGs in ∆Mohg1. GO enrichment and KEGG pathway analysis of downregulated (B) and upregulated (C) genes in ∆Mohg1. The DEGs involving glycerol synthesis (D), synthesis pentose (E), and MAPK pathway (F) in M. oryzae. The box background in green or red means the decrease or increase of gene expression according to the transcriptome, respectively.

Figure 5

The carbohydrate contents in ∆Mohg1. (A–H) The content of 8 carbohydrates. Each experiment was conducted with 3 biological repeats and statistically significant differences were calculated by Student’s t-test, * p < 0.05, ** p < 0.01. Error bars represent the means ± SD.

Figure 6

Overexpressing of MGG_00623 in the ∆Mohg1 mutant restored the fungal growth and chitin contents. (A) The relative expressions of MGG_00623 in ∆Mohg1. Each experiment was conducted with 3 biological repeats and statistically significant differences were calculated by Student’s t-test, ** p < 0.01. (B,C) Overexpression of MGG_00623 in ∆Mohg1 partially restored the fungal growth. Each experiment was conducted in 3 biological repeats. The different letters above each bar graph indicate significant differences (p < 0.05) calculated by ANOVA and Duncan’s test. Error bars represent the means ± SD. (D,E) Overexpression of MGG_00623 in ∆Mohg1 restored CWI staining and chitin contents. The scale bar represents 25 μm. The different letters above each bar graph indicate significant differences (p < 0.05) calculated by ANOVA and Duncan’s test. Error bars represent the means ± SD. (F) Yeast two-hybrid and (G) BiFC assays show MoHG1 interacts with MGG_00623. The scale bar represents 20 μm.