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

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.


Introduction
Rice (Oryza sativa) is an important crop widely grown in the world. However, rice production is seriously endangered by a variety of pathogens throughout the growing season, which threatens food security [1]. Rice blast disease, caused by ascomycetes fungus Magnaporthe oryzae, is a major constraint to rice production. M. oryzae has been listed as the top plant pathogen, and the wheat infecting pathotype causes the yield losses of global wheat production [2,3]. Therefore, developing new strategies for blast disease management is necessary.
9-phenanthrol, also named 9-hydroxyphenanthrene, phenanthrene-9-ol, or 9-phenanthrenol (C 14 H 10 O), is an aromatic compound from phenanthrene. 9-phenanthrol is a widely used TRPM4 (transient receptor potential melastatin 4) channel inhibitor for animals [4]. TRPM4 channel is a calcium-activated, phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 )-modulated, non-selective cation channel that belongs to the family of melastatin-related transient receptor potential (TRPM) channels. TRPM4 is involved in important physiological processes such as Ca 2+ -dependent immune response and human heart conduction dysfunction [5]. 9-phenanthrol is the degradation intermediate of phenanthrene, suggesting its risk of environmental toxicity. Previous results indicated that 9-phenanthrol has high sorption and heterogenous properties with lower risks for 2 of 12 9-phenanthrol to phenanthrene [6]. However, the antifungal activity of this compound was hardly reported. A paper reported that 9-phenanthrol could change the colony color of Stemphylium sarcinaeforme and inhibit the spores of Monilinia fructicola in the 1950s [7]. There were no other reports of 9-phenanthrol involving antifungal mechanisms, implying there are many novel characters about 9-phenanthrol waiting to be revealed.
In this paper, we tested the fungal toxicity of 9-phenanthrol on M. oryzae and rice blast disease management through transcriptome and metabolome and found that a gene encoding 4-hydroxyphenylpyruvate dioxygenase could play an important role in the tolerance of 9-phenanthrol in M. oryzae. Our results provide the antifungal mechanism of 9-phenanthrol, which could be considered a novel fungicide for disease management.

Results
2.1. 9-Phenanthrol Displayed Antifungal Activity against M. oryzae 9-phenanthrol was reported to have toxicity to the spores of M. fructicola; thus, we tested its antifungal activity against M. oryzae. The results showed that 9-phenanthrol application could significantly inhibit the mycelial growth ( Figure 1A,B). The formation of appressorium is a key step in deciding the infection of M. oryzae. We found that 9-phenanthrol disrupted the appressorium formation at 10 µg/mL ( Figure 1C). Moreover, the spore suspensions mixed with 9-phenanthrol were used to inoculate rice seedlings, and the results showed that the number of lesions was much less than those without 9-phenanthrol treatment ( Figure 1D). We calculated the fungal biomass of rice seedlings with different treatments, and similar results showed that the fungal biomass was significantly decreased treating with 10 µg/mL and 30 µg/mL 9-phenanthrol ( Figure 1E). We observed the increased branches and septum of hypha and abnormal mycelium after 9-phenanthrol treatment ( Figure 1F). Our results clearly suggested that 9-phenanthrol inhibits the fungal development and infection in M. oryzae. mune response and human heart conduction dysfunction [5]. 9-phenanthrol is the degradation intermediate of phenanthrene, suggesting its risk of environmental toxicity. Previous results indicated that 9-phenanthrol has high sorption and heterogenous properties with lower risks for 9-phenanthrol to phenanthrene [6]. However, the antifungal activity of this compound was hardly reported. A paper reported that 9-phenanthrol could change the colony color of Stemphylium sarcinaeforme and inhibit the spores of Monilinia fructicola in the 1950s [7]. There were no other reports of 9-phenanthrol involving antifungal mechanisms, implying there are many novel characters about 9-phenanthrol waiting to be revealed.
In this paper, we tested the fungal toxicity of 9-phenanthrol on M. oryzae and rice blast disease management through transcriptome and metabolome and found that a gene encoding 4-hydroxyphenylpyruvate dioxygenase could play an important role in the tolerance of 9-phenanthrol in M. oryzae. Our results provide the antifungal mechanism of 9-phenanthrol, which could be considered a novel fungicide for disease management.

9-Phenanthrol Displayed Antifungal Activity against M. oryzae
9-phenanthrol was reported to have toxicity to the spores of M. fructicola; thus, we tested its antifungal activity against M. oryzae. The results showed that 9-phenanthrol application could significantly inhibit the mycelial growth ( Figure 1A,B). The formation of appressorium is a key step in deciding the infection of M. oryzae. We found that 9-phenanthrol disrupted the appressorium formation at 10 μg/mL ( Figure 1C). Moreover, the spore suspensions mixed with 9-phenanthrol were used to inoculate rice seedlings, and the results showed that the number of lesions was much less than those without 9-phenanthrol treatment ( Figure 1D). We calculated the fungal biomass of rice seedlings with different treatments, and similar results showed that the fungal biomass was significantly decreased treating with 10 μg/mL and 30 μg/mL 9-phenanthrol ( Figure 1E). We observed the increased branches and septum of hypha and abnormal mycelium after 9-phenanthrol treatment ( Figure 1F). Our results clearly suggested that 9-phenanthrol inhibits the fungal development and infection in M. oryzae.

Transcriptome Analysis Reveals Gene Ontology Categories and KEGG Enrichment Analysis of DEGs
In order to reveal the inhibitory mechanism of 9-phenanthrol on M. oryzae. We conducted a high-throughput RNA sequencing to obtain transcripts from the M. oryzae Guy11 mycelia treated with 9-phenanthrol. Compared with control, a total of 6310 differentially expressed genes (DEGs) and 3102 upregulated and 3208 downregulated genes were identified in M. oryzae under 9-phenanthrol treatment (Table S1). To comprehend the function of DEGs, we performed a Gene Ontology (GO) enrichment analysis of the DEGs (Figure 2A,B, Tables S2 and S3). The upregulated genes involving biological processes were mainly associated with translation, ER (endoplasmic reticulum) to Golgi vesicle-mediated transport, aerobic respiration, and protein import into the mitochondrial inner membrane. In contrast, genes related to mycelium development, translation, ribosome biogenesis, and rRNA processing were downregulated. For cellular components, the upregulated DEGs were mainly related to the mitochondrion, endoplasmic reticulum, and Golgi apparatus, and downregulated DEGs were mainly enriched in the cytosol, nucleus, and cytoplasm. As for molecular functions, the upregulated DEGs were mainly related to integral components of membrane, metal ion binding, and RNA polymerase II transcription regulatory region sequence-specific DNA binding, while structural components of ribosomes and RNA binding were downregulated.
According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the upregulated DEGs were highly associated with pathways including endocytosis, protein processing in endoplasmic reticulum, oxidative phosphorylation, and ubiquitinmediated proteolysis ( Figure 2C, Table S4). The downregulated DEGs were involved in several pathways, including metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of amino acids, ribosome, carbon metabolism, RNA transport, and purine metabolism ( Figure 2D, Table S5). However, only endocytosis pathway was significantly enriched analyzed by upregulated genes, there were 10 pathways significantly enriched by downregulated genes and the biosynthesis of amino acids pathway was most significant. There are 116 genes on biosynthesis of amino acids pathway in M. oryzae, 95 genes were downregulated with 9-phenanthrol treatment, suggesting 9-phenanthrol disrupted the expressions of genes belonging to biosynthesis of amino acids pathway.

Quasi-Targeted Metabolomic Analyses Underlying the Compound Changes in M. oryzae with 9-Phenanthrol
We found that metabolic pathway-related genes were significantly changed with 9-phenanthrol treatment through transcriptome analysis. Thus, we used the quasi-targeted metabolome to analyze the variations of compounds in M. oryzae with 9-phenanthrol. There were 902 metabolites obtained, and 379 compounds were significantly down-or upregulated ( Figure 3A, Table S7). There were 39 classes identified from these significantly different metabolites and most of which were amino acids, organic acids, nucleotides, carbohydrates, and fatty acyls related compounds ( Figure 3B). KEGG analysis revealed that the top 10 enriched pathways were metabolic pathways, biosynthesis of secondary metabolites, purine metabolism, ABC transporters, pyrimidine metabolism, arginine and proline metabolism, amino sugar and nucleotide sugar metabolism, galactose metabolism, biosynthesis of alkaloids derived from ornithine, lysine and nicotinic acid, and aminoacyl-tRNA biosynthesis ( Figure 3C). Moreover, metabolic pathways, biosynthesis of secondary metabolites, purine metabolism, and pyrimidine metabolism were both enriched in the KEGG terms from transcriptome and metabolome results. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the upregulated DEGs were highly associated with pathways including endocytosis, protein processing in endoplasmic reticulum, oxidative phosphorylation, and ubiquitin-mediated proteolysis ( Figure 2C, Table S4). The downregulated DEGs were involved in several pathways, including metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of amino acids, ribosome, carbon metabolism, RNA transport, and purine metabolism ( Figure 2D, Table S5). However, only endocytosis pathway was sig-

9-Phenanthrol Inhibits Germination of Rice and Growth of Other Fungi
Due to the wide distribution of HPPD in diverse organisms, we aligned the amino sequences of HPPD in bacteria, plants, and fungi. To our surprise, the species with the most sequence identity of HPPD is Agrobacterium tumefaciens compared with that in M. oryzae. The polygenetic results showed that most HPPD sequences in fungi were clustered together, and HPPDs in plants belonged to another clade. For bacteria, the HPPDs in Dickeya chrysanthemi and Bacillus subtilis were clustered, but HPPDs in A. tumefaciens and Escherichia coli were spread into a fungal clade ( Figure 5A). We tested the inhibitory effects of 9-phenanthrol on plants and other fungi, and the results showed that 9-phenanthrol at 10 µg/mL could inhibit the radicle growth of rice and cabbage but had no effect on maize and cabbage ( Figure 5B). Moreover, 9-phenanthrol also inhibits the growth of other phytopathogens ( Figure 5C,D). While there were no significant inhibitory effects of 9-phenanthrol on E. coli and Pseudomonas aeruginosa, the D. chrysanthemi causing soft rot disease was inhibited at much higher concentrations ( Figure S1). Rhizoctonia solani and P. aeruginosa do not contain putative HPPDs through alignment, other 9-phenanthrol targets and detoxication are existed in R. solani and P. aeruginosa, respectively.

9-Phenanthrol Inhibits Germination of Rice and Growth of Other Fungi
Due to the wide distribution of HPPD in diverse organisms, we aligned the amino sequences of HPPD in bacteria, plants, and fungi. To our surprise, the species with the most sequence identity of HPPD is Agrobacterium tumefaciens compared with that in M. oryzae. The polygenetic results showed that most HPPD sequences in fungi were clustered together, and HPPDs in plants belonged to another clade. For bacteria, the HPPDs in Dickeya chrysanthemi and Bacillus subtilis were clustered, but HPPDs in A. tumefaciens and Escherichia coli were spread into a fungal clade ( Figure 5A). We tested the inhibitory effects of 9-phenanthrol on plants and other fungi, and the results showed that 9-phenanthrol at 10 μg/mL could inhibit the radicle growth of rice and cabbage but had no effect on maize and cabbage ( Figure 5B). Moreover, 9-phenanthrol also inhibits the growth of other phytopathogens ( Figure 5C,D). While there were no significant inhibitory effects of 9-phenanthrol on E. coli and Pseudomonas aeruginosa, the D. chrysanthemi causing soft rot disease was inhibited at much higher concentrations ( Figure S1). Rhizoctonia solani and P. aeruginosa do not contain putative HPPDs through alignment, other 9-phenanthrol targets and detoxication are existed in R. solani and P. aeruginosa, respectively.

Gene Co-Expression Network Analysis
To further analyze the expression pattern of DEGs, we constructed interacted network of DEGs (|foldchange| ≥ 1.5) with 9-phenanthrol treatment using String ( Figure S2, Table S9). There are 283 interacted genes, and the most significant interacted genes were RNA and amino acid-associated genes. KEGG analyses of interacted genes were meta-

Gene Co-Expression Network Analysis
To further analyze the expression pattern of DEGs, we constructed interacted network of DEGs (|foldchange| ≥ 1.5) with 9-phenanthrol treatment using String ( Figure S2, Table S9). There are 283 interacted genes, and the most significant interacted genes were RNA and amino acid-associated genes. KEGG analyses of interacted genes were metabolic pathways, biosynthesis of secondary metabolites, tyrosine metabolism, and biosynthesis of antibiotics that were similar to the KEGG results of downregulated DEGs. Moreover, the top 30 interacted genes were all downregulated, indicating the suppressed genes with 9-phenanthrol treatment develop a strong network to disrupt fungal growth. There were nine genes that interacted with HPPD ( Figure 6). The expressions of genes encoding sterol 24-C-methyltransferase (MGG_10860), salicylate hydroxylase (MGG_08293) and monothiol glutaredoxin-5 (MGG_01067) were upregulated, succinyl-CoA:3-ketoacid-coenzyme A transferase subunit A (MGG_11480), amino transferase (MGG_09919), fumarylacetoacetase (MGG_00317), nitrilase 2 (MGG_03280), homogentisate 1,2-dioxygenase (MGG_00431) and another salicylate hydroxylase (MGG_03764) were suppressed. Interestingly, HPPD, amino transferase, fumarylacetoacetase, and homogentisate 1,2-dioxygenase are the key catalyzed enzymes for the tyrosine degradation pathway [9]. We also searched the metabolic products in the metabolome results, and L-tyrosine, 4-hydroxyphenylpyruvate, fumaric acid, and acetoacetate were found. The relative content of 4-hydroxyphenylpyruvate was lower in M. oryzae with 9-phenanthrol compared with control. Thus, our results showed that 9-phenanthrol could disrupt the expressions of genes involving the tyrosine degradation pathway in M. oryzae.

Discussion
Rice crop is a staple food around the world. In order to meet its ever-increasing demand, high quantities of chemical pesticides and fertilizers are used. In this paper, we found a novel compound named 9-phenanthrol inhibiting fungal growth and pathogenicity and revealed the fungal toxicity mechanism through transcriptome and metabolome approaches.

Discussion
Rice crop is a staple food around the world. In order to meet its ever-increasing demand, high quantities of chemical pesticides and fertilizers are used. In this paper, we found a novel compound named 9-phenanthrol inhibiting fungal growth and pathogenicity and revealed the fungal toxicity mechanism through transcriptome and metabolome approaches.
3.1. 9-Phenanthrol Inhibits Fungal Growth through Transcriptomic Reprogramming 9-phenanthrol is widely used as a specific inhibitor of TRPM4, a Ca 2+ -activated nonselective cation channel, which is associated with cardiac electrical activity, exerts antiarrhythmic effects, and pharmacological effects [5]. As for antifungal activities, there was only one paper that suggested that 9-phenanthrol could change the colony color of S. sarcinaeforme and inhibit the spores of M. fructicola [7]. We found that 9-phenanthrol had the inhibitory ability to M. oryzae, and the inhibitory concentration was equivalent to most antibiotic compounds published [10]. Moreover, we aligned the amino acid sequence of TRPM4 in M. oryzae, and no significant similarity was found. Thus, deepening the inhibitory mechanism of 9-phenanthrol against M. oryzae is important for transcriptomic analyses (Figure 2A-D).
Endocytosis pathway-related genes were significantly upregulated and enriched according to KEGG analysis, which was involved in the transportation of secretory materials into the cell. SNAREs proteins are composed of major components mediating vesicle fusion, intracellular transport, and plasma membrane fusion involving endocytosis, development, and pathogenesis of M. oryzae [11,12]. Antifungal hexapeptide PAF26 shows fungicidal activity against Neurospora crassa through the endocytosis pathway, deletion mutants of the endocytic proteins RVS-161, RVS-167 and RAB-5 reduced the rates of PAF26 internalization and fungicidal activity [13]. These results indicate that the entrance of 9-phenanthrol into the cell might regulate the endocytosis pathway.

9-Phenanthrol Inhibits Fungal Growth through Disruption of Tyrosine Degradation Pathway
Transcriptome and metabolome analyses showed that the expression of HPPD and the content of 4-hydroxyphenylpyruvate were decreased ( Figure 6). Overexpressions of HPPD enhance the tolerance of blast fungus against 9-phenanthrol ( Figure 4). HPPD catalyzes the conversion of 4-hydroxyphenylpyruvic acid (HPPA) into homogentisic acid (HGA) in the tyrosine degradation pathway suggesting the relationship between 9-phenanthrol and HPPD. Many commercial herbicides targeting HPPD disrupt carotenoid biosynthesis and cause photosynthetic chlorophyll damage in weeds [20]. However, HPPD is widely conserved in plants, animals, and microbes. In Aspergillus nidulans, 4-HPPD deletion mutant could not grow in the presence of phenylalanine and accumulated increased concentrations of tyrosine and 4-hydroxyphenylpyruvic acid [21]. Moreover, we found a higher sequence similarity of HPPD among fungi, which was consistent with higher inhabitation to other fungi ( Figure 5). However, the HPPD in E. coli shows higher sequence similarity. There is no effect of 9-phenanthrol on the growth of E. coli and other bacteria ( Figure S4), implying there are extra detoxification mechanisms in procaryotic organisms.
Due to the role of HPPD on homogentisic acid production, HPPD is essential for melanin synthesis [22,23]. Melanin participates in maintaining turgor pressure and facilitates blast fungal infection. Interestingly, we found that three melanin synthesis-related genes, BUF1, ALB1, and RSY1 [8], were upregulated in M. oryzae with 9-phenanthrol treatment indicating higher expressions of these pigment genes might compensate for the disruption of HPPD by 9-phenanthrol (Table S6). However, there was no evidence that HPPD could directly catalyze 9-phenanthrol or form a complex for detoxification in M. oryzae; thus, the relationship between HPPD and 9-phenanthrol needs further research.
Among nine interacted genes, amino transferase, fumarylacetoacetase, and homogentisate 1,2-dioxygenase, together with HPPD, regulate the degradative pathway of tyrosine and were downregulated under 9-phenanthrol treatment ( Figure 6). Tyrosine is catalyzed by amino transferase to form 4-hydroxyphenylpyruvate, which can be converted into homogentisate by HPPD. Homogentisate 1,2-dioxygenase catalyzes homogentisate into maleylacetoacetate, and then maleylacetoacetate is converted to fumarylacetoacetate by maleylacetoacetase. Finally, fumarylacetoacetate is cleaved by fumarylacetoacetase to acetoacetate and fumarate [9]. Only maleylacetoacetase encoding gene was not found in our transcriptomic results. The other genes belonging to the tyrosine-degrative pathway were suppressed by 9-phenanthrol treatment. For the other interacted genes, monothiol glutaredoxin-5 is associated with iron homeostasis and iron-sulfur protein maturation [24]. Sterol 24-C-methyltransferase is essential for ergosterol biosynthesis and homeostasis in Cryptococcus neoformans [25,26]. Thus, upregulation of these two genes might be an emergency response to induce iron and ergosterol metabolism to cope with 9-phenanthrol stress.

Conclusions
In this paper, we illuminate the antifungal action mode of 9-phenanthrol on M. oryzae by disrupting the expression of HPPD and the content of its metabolite for amino acid dysfunction identified by transcriptome, metabolome, and gene overexpression. Our results provide a novel insight into the toxicity of 9-phenanthrol on M. oryzae, which could be referenced by other organisms.

Growth Condition
The rice blast strains stored at −80 • C are activated on the potato sugar agar media at 28 • C in the dark. The solid and liquid complete media supplemented with different compounds were used for the assessment of colony diameter and mycelial dry weight.
For the herbicide activity experiments, the plant seeds were sterilized with 70% (v/v) ethanol and washed 3 times with sterile water. The seeds were soaked into the sterile water adding 1 µg/mL, 5 µg/mL, 10 µg/mL, and 50 µg/mL of 9-phenanthrol. A total of 4 days after treatment, the lengths of radicle were measured.

RNA Sequencing
The sample preparation for RNA sequencing is referenced by the previous paper [27,28]. The mycelia of Guy11 were cultivated into liquid CM for 2 days and transferred into fresh liquid CM, adding 10 µg/mL 9-phenanthrol for 24 h for RNA extraction. Total RNA was extracted from the tissue using TRIzol ® Reagent, and genomic DNA was removed using DNase I (Takara, Tokyo, Japan). The RNA samples were sent to Shanghai Majorbio Technology Co., Ltd. for RNA sequencing based on Illumina HiSeq 6000 platform. The raw data have been deposited on NCBI (Project ID: PRJNA797246).

Quasi-Targeted Metabolomic Analyses
Quasi-targeted metabolomics analysis was performed by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). The sample preparation is consistent with RNA sequencing sample preparation. The harvest mycelium was individually grounded with liquid nitrogen and resuspended with prechilled 80% methanol. The samples were subsequently transferred to a fresh Eppendorf tube and then were centrifuged at 15,000× g, 4 • C for 20 min. Finally, the supernatant was injected into the LC-MS/MS system analysis.
LC-MS/MS analyses were performed using an ExionLC™ AD system (SCIEX) coupled with a QTRAP ® 6500+ mass spectrometer (SCIEX) in Novogene Co., Ltd. (Beijing, China). The positive transformants were selected and confirmed by PCR and Sanger sequencing. The detection of the experimental samples using MRM (multiple reaction monitoring) was based on a novogene in-house database. The Q3 was used for metabolite quantification. The Q1, Q3, RT (retention time), DP (declustering potential), and CE (collision energy) were used for the metabolite identification. The metabolites with variable importance in the projection, VIP ≥ 1 and |Log2FC| ≥ 1, were considered to be differential metabolites.

Overexpression Strain Construction
The vector pDL2 with the strong constitutive RP27 promoter was used for overexpressing vector construction. The full length of the candidate gene coding region was amplified from Guy11 genomic DNA and cloned into pDL2 to generate the recombinant vectors using the yeast gap repair approach (Table S9). The correct recombination vector was confirmed by Sanger sequencing and then transformed into M. oryzae YN2046 protoplast with PEG-mediated transformation.

Polygenetic Tree Construction
For alignment of HPPD in different organisms, we downloaded the amino acid sequences from NCBI through BLASTP programs, and the polygenetic tree was constructed by MEGA 6.0 using a maximum likelihood method.