Transcriptome and Quasi-Targeted Metabolome Analyze Overexpression of 4-Hydroxyphenylpyruvate Dioxygenase Alleviates Fungal Toxicity of 9-Phenanthrol in Magnaporthe oryzae

Abstract

Magnaporthe oryzae, the causal agent of rice blast disease, produces devastating damage to global rice production. It is urgent to explore novel strategies to overcome the losses caused by this disease. 9-phenanthrol is often used as a transient receptor potential melastatin 4 (TRPM4) channel inhibitor for animals, but we found its fungal toxicity to M. oryzae. Thus, we explored the antimicrobial mechanism through transcriptome and metabolome analyses. Moreover, we found that overexpression of a gene encoding 4-hydroxyphenylpyruvate dioxygenase involved in the tyrosine degradative pathway enhanced the tolerance of 9-phenanthrol in M. oryzae. Thus, our results highlight the potential fungal toxicity mechanism of 9-phenanthrol at metabolic and transcriptomic levels and identify a gene involving 9-phenanthrol alleviation. Importantly, our results demonstrate the novel mechanism of 9-phenanthrol on fungal toxicity that will provide new insights of 9-phenanthrol for application on other organisms.

Keywords: 4-hydroxyphenylpyruvate dioxygenase; 9-phenanthrol; Magnaporthe oryzae; overexpression; tyrosine degradative pathway.

Figure 1

The inhibitory effects of 9-phenanthrol on blast growth and pathogenicity. (A) The effect of 9-phenanthrol on mycelial growth (A,B) and appressorial formation (C). Application of 9-phenanthrol decreases the pathogenicity and biomass of M. oryzae (D,E). The morphological changes of hypha with 9-phenanthrol treatment (F). Values are presented as the mean of results of triplicate experiments ± SD. Bars with different capital letters indicate significant differences between treatments at a p-value < 0.01 using a Duncan statistics method.

Figure 2

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of differently expressed genes (DEGs) in M. oryzae treated with 9-phenanthrol. The GO enrichment of upregulation (A) and downregulation (B) of DEGs. The KEGG analysis of upregulation (C) and downregulation (D) of DEGs.

Figure 3

Metabolome analysis reveals the differently changed compounds with 9-phenanthrol in M. oryzae. (A) The volcano diagram of identified metabolites under 9-phenanthrol treatment. The class (B) and KEGG pathways (C) of significantly different metabolites of M. oryzae under 9-phenanthrol treatment.

Figure 4

Overexpression of 4-hydroxyphenylpyruvate dioxygenase (HPPD) enhances the tolerance of 9-phenanthrol in M. oryzae. (A) The colony appearance of HPPD overexpression strains and WT under 9-phenanthrol treatment. (B) The inhibitory rates of hyphal growth of M. oryzae under 9-phenanthrol treatment. Values are presented as the mean of results of triplicate experiments ± SD. The significant differences between treatments were calculated by Student t-test, ** p < 0.01.

Figure 5

The inhibitory effects of 9-phenanthrol on different plants and fungi. Polygenetic analyses of HDDP in fungi, bacteria, and plants (A). Inhibitory rates of 9-phenanthrol on plant germination (B) and other fungal pathogens (C,D). Values are presented as the mean of results of triplicate experiments ± SD. Bars with different letters indicate significant differences between treatments at a p-value < 0.01 using a Duncan statistics method.

Figure 6

HPPD interacted with gene network and metabolites on the tyrosine degradative pathway. Red nodes represent upregulated genes, and green nodes represent downregulated genes. The size of the node means the absolute value of gene expression. The blue arrows indicate the gene encoding enzyme involving catalyzed reaction among tyrosine degradative pathway. The histograms beside compounds mean the relative contents from metabolome data.