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.

1. Introduction

Magnaporthe oryzae causes rice blast disease, which threatens global rice production. The pathogen can infect various grass hosts besides rice [1,2]. Genomic analyses revealed that there might be 10 distinct M. oryzae lineages and isolates from each lineage only infect a single host [3]. Recently, the emergence of M. oryzae Triticum (MoT) has caused serious losses in wheat production. The newest finding underlined that MoT was generated from the cross progeny of Eleusine- and Urochloa-infecting isolates and underwent a series of matings with a small number of individuals from three additional host-specialized populations [4]. However, phylogenetic analyses indicated a distant relationship between rice- and wheat-infecting populations. Our previous results also found a stronger selective sweep in the rice-infecting population than in the wheat-infecting population [5]. Thus, explorations of host selection mechanisms by M. oryzae favor decreasing the pathogen risk.

The fungal cell wall is composed of two layers. The inner layer has a cross-linked chitin–glucan matrix, and the outer layer possesses mannosylated proteins [6]. Chitin is a core component of the fungal cell wall, and UDP-N-acetylglucosamine is the substrate for chitin synthesis. Chitin is also a key elicitor to trigger plant immune response. Chitin modification in pathogens and recognition by the host are determinants of disease occurrence [7,8]. There are two effectors, MoChia1 and Slp1, which influence chitin modification and infection in M. oryzae [9,10]. Moreover, melanin is also found in the cell wall of numerous fungal pathogens, which mainly reinforces cell structure and maintains the pressure from appressorium [11,12]. So far, diverse chitin and melanin synthesis enzymes have been identified, most of which are essential for fungal development and infectious processes [7].

Carbon metabolism is essential for fungal cell wall development. Monosaccharides with glucosyl residues mainly take up 75% of monosaccharides of cell walls. Other monosaccharides are mannose (14%), N-acetylglucosamine (7%), galactose (2%), and traces of arabinose and xylose in M. oryzae [13]. The carbon catabolite repression (CCR) system is widely conserved in filamentous fungi and favors fungi to preferentially utilize a favorable carbon source [14]. In M. oryzae, Tps1 is responsible for glucose-6-phosphate sensing and triggers CCR via the inactivation of Nmr1-3 [15,16]. The CCR transcription factor MoCreA has been identified, which regulates the utilization of different carbon sources and gene expressions [17].

Many proteins on fungal cell walls preferentially perceive various cues from a host and regulate fungal infection. G-protein-coupled receptors Pth11 and WISH, hydrophobin proteins Mpg1 and Mhp1, transmembrane mucin Msb2, and membrane sensor MoSho1 were identified in M. oryzae [18,19,20,21]. These sensor proteins can transduce the signals for appressorium development in the of M. oryzae. Fungal cell wall integrity (CWI) proteins are essential for cell viability, morphogenesis, and pathogenesis. In M. oryzae, there are three mitogen-activated protein (MAP) cascade kinases responding to signal transduction, including MoMck1 (MAPKKK), MoMkk1 (MAPKK), and MoMps1 (MAPK) [22]. Diverse signal pathways identified have crosstalk with CWI, suggesting the central role of CWI in fungal growth and pathogenicity [23,24,25,26].

We previously found a gene encoding a hypothetical protein with a signal peptide and transmembrane domain by comparative genomics analysis, which might be involved in fungal virulence, but the functions of this gene were unknown. In this study, the gene-deletion mutants displayed compromised hypha growth, and the gene was named MoHG1 (hypha growth gene in M. oryzae). MoHG1 positively regulates fungal development and infection by maintaining CWI. Moreover, the decreased content of chitin was 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 yeast two-hybrid and BiFC. These results provide valuable references for the genes with similar structures in M. oryzae.

2. Materials and Methods

2.1. Phylogenetic Analyses

The homolog sequences of MoHG1 were downloaded from different rice- or non-rice-infecting isolates in the NCBI database. FastTree software (Version 2.1.10) was used for phylogenetic analysis. The phylogenetic tree plot was generated using ChiPlot (https://www.chiplot.online, accessed on 16 August 2024).

2.2. Culture Conditions

Wild-type M. oryzae YN125 and mutant strains conserved at Yunnan Agricultural University were cultured on a potato sucrose agar (PSA) medium for growth. Complete medium (CM) and minimum medium (MM) were used for growth assays with different stressor treatments. Oatmeal medium (OM) was used for sporulation. For quantitative analyses of spore production, germ tube, and appressorium formations, the conidia were harvested from OM and then filtered through two layers of Miracloth (Calbiochem, San Diego, CA, USA). The spore suspensions were resuspended twice in sterile water. Spore production in a given colony was counted using a hemocytometer. The spore suspension was dropped on a microscope cover glass (Fisher Scientific, 12545100, Waltham, MA, USA), and the rates of germ tube and appressorium formations were recorded using a microscope. The experiments were performed in three biological repeats.

2.3. Infection Assays

The susceptible rice variety Lijiangxintuanheigu (LTH) was used for fungal inoculation. A spore suspension (1 × 10⁵ spores/mL) was foliar-sprayed on 21-day-old rice seedlings. The inoculated seedlings were incubated in the dark for 24 h at 28 °C and transferred to the greenhouse for 6 days. At 7 days post-inoculation, the disease index was assessed. Fifteen seedlings were used for inoculation of each isolate, and there were three independent inoculation experiments. A disease score of 0–5 was assigned according to the lesion type. The disease index was calculated using the following formula.

$$\mathrm{D}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}={\displaystyle \frac{\sum (\mathrm{d}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{e}\times \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{d}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{s})}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{v}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{s}\times 5}}\times 100$$

2.4. Protoplast Release Assay

The fungi were grown in liquid yeast extraction and glucose media (YEG) for 2 days and then harvested. The dried mycelia were transferred into a lysis enzyme solution for 1 h. The released protoplast solution was dropped on a hemocytometer and the number of protoplasts was counted using a microscope [23,24,25,26].

2.5. Vector Construction and Transformation

The vector pCX62 was used for MoHG1 gene deletion, and the 1000–2000 bp sequences upstream and downstream of the MoHG1-coding sequences were inserted into two flanking regions of the maker gene (hygromycin phosphotransferase, HPT), respectively. The constructed deletion vector was transformed into protoplasts of YN125 using the PEG–CaCl₂-mediated method. The positive deleted transformants were confirmed using PCR with specific primers (Table S1). The native promoter and coding sequences of MoHG1 were inserted into a pYF11 vector carrying a green fluorescent gene and transformed into YN125 for MoHG1-GFP observation. For MGG_00623-overexpressing strain construction, coding sequences of MGG_00623 were inserted into pYF11 with the continuous expressing promoter RP27 and transformed into a ∆Mohg1 mutant. The overexpression of MGG_00623 was confirmed via qRT-PCR.

2.6. Carbohydrate Content Assay

Fungal strains were grown in liquid YEG for 2 days. The mycelia of WT and ∆Mohg1 were dried using vacuum freezing and then ground into powder, respectively. 30 mg of the powder from each sample was diluted into a 500 μL solution containing methanol/isopropanol/water (3:3:2 v/v/v), vortexed for 3 min, and subjected to ultrasound for 30 min. The extraction was centrifuged at 4 °C and 14,000 r/min for 3 min. 20 μL suspensions were transferred to a new tube and added to 20 μL ribitol (100 μg/mL) as the internal standard. For the derivatization treatment, the mixture was dried using nitrogen gas and added to a 100 μL solution of methoxyamine hydrochloride in pyridine (15 mg/mL) for incubation at 37 °C for 2 h. Then, 100 μL of BSTFA was added to the mixture and maintained at 37 °C for 30 min.

An Agilent 7890 (Santa Clara, CA, USA) gas chromatograph coupled with a 5975C mass spectrometer with a DB-5MS column (30 m length × 0.25 mm i.d. × 0.25 μm film thickness, J&W Scientific, Folsom, CA, USA) was used for sugar detection. Helium was used as the carrier gas at a flow rate of 1 mL/min. Injections were performed in the spitless mode, and the injected volume was 2 μL. The oven temperature was maintained at 70 °C for 1 min, raised to 112 °C at 30 °C/min and maintained for 3 min, raised to 175 °C at 15 °C/min and maintained for 1 min, raised to 190 °C at 3 °C/min and maintained for 2 min, then raised to 240 °C at 35 °C/min, and finally raised to 280 °C at 10 °C/min and maintained at this temperature for 2.5 min. All samples were analyzed in the selective ion-monitoring mode. The ion source and transfer line temperatures were 230 °C and 240 °C, respectively.

The standard curve for each sugar was generated using the different concentrations of standard sugar solutions and corresponding mass spectrometer peak data. The sugar content was calculated using the following equation: content (mg/g) = c × V1 × V2 ÷ V3 ÷ m ÷ 1000000 (c is the concentration generated by the standard curve according to the peak area of each sample; V1 is the constant volume; V2 is the extraction volume; V3 is the collected suspension volume; m is the sample weight).

2.7. RNA Isolation and Quantitative Real-Time (qRT)-PCR

The RNA isolations from fungi and rice leaves were performed using the UNlQ-10 Column TRIzol Total RNA Isolation Kit (Sangon Biotech, B511321, Shanghai, China). PrimeScript IV 1st strand cDNA Synthesis Mix (Takara, 6215A, Shiga, Japan) was used for cDNA synthesis. The primers used for qRT-PCR are listed in Table S1. The M. oryzae actin gene MGG_03982 and the rice actin gene were used as the endogenous controls for normalization, respectively. The relative expressions of target genes were calculated using the 2^−∆∆Ct method.

2.8. RNA Sequencing

The RNA sequencing methods for WT and ∆Mohg1 isolates were performed based on the previous protocols [27]. Fungal strains were grown in liquid YEG for 2 days and then the mycelia were collected for RNA isolation. Sequencing libraries were constructed using the Illumina TruseqTM RNA sample prep kit (San Diego, CA, USA) and sequenced on the Illumina Novaseq 6000 platform (San Diego, CA, USA). The raw data were uploaded to NCBI SRA under the BioProject number PRJNA1076595. Clean data were assembled using Cufflinks (Version 2.2.1) based on the reads mapped to the M. oryzae reference genome (https://www.ncbi.nlm.nih.gov/assembly/GCF_000002495.2, accessed on 16 August 2024), while unmapped reads were annotated with sequence alignment. The read count of each gene was generated by RSEM software (Version 1.3.3), DESeq2 (Version 1.10.1) was used to identify differentially expressed genes (DEGs), and the DEGs were filtered by |log₂FC| ≥ 1, with a Padjust value of <0.05. The GO and KEGG analyses were based on DAVID (https://david.ncifcrf.gov/home.jsp, accessed on 16 August 2024).

2.9. Cell Wall Staining and Chitin Content Assays

Calcofluor white (CFW) was used for fungal cell wall staining according to the methodology in a previous paper with a slight modification [23,24,25,26]. WT and ∆Mohg1 were grown in liquid YEG for 4 days and washed using ddH₂O. The dried mycelia were soaked in 10 μg/mL CFW solution for 5 min and rinsed with ddH₂O to wash off the dye. The cell wall staining was observed using a fluorescence microscope.

The chitin content assays for WT and ∆Mohg1 were conducted according to a previously published method [28]. WT and ∆Mohg1 were grown in liquid YEG for 4 days and washed using ddH₂O. The chitin content was determined by measuring the amount of glucosamine released after the hydrolysis of the cell walls.

2.10. Yeast Two-Hybrid and BiFC Assays

For yeast two-hybrid, the CDS fragment of MoHG1 was inserted into prey plasmid pPR3-N, while the CDS fragment of the MGG_00623 was cloned into bait plasmid pBT3-STE. The constructed vectors were co-transformed into competent cells of yeast NMY51 and incubated on a synthetic medium lacking leucine and tryptophan (SD–Leu–Trp) then were further transferred to a synthetic medium lacking leucine, tryptophan, adenine, and histidine (SD–Leu–Trp–Ade–His). For BiFC observation, the CDS regions of MGG_00623 and MoHG1 were cloned into pSm35s-nYFP and pSm35s-cYFP vectors, respectively. The pSm35s::nYFP::MGG_00623 and pSm35s::MoHG1::cYFP were co-inoculated into tobacco leaves through agrobacterium-mediated transformation and incubated for 2 days. The YFP was observed under a laser laser-scanning confocal microscope.

3. Results

3.1. MoHG1 Influence the Fungal Development

MoHG1 encodes 201 amino acids with a 24-amino acid signal peptide and possesses 3 transmembrane domains. There is one nonsynonymous substitution at the 106th amino acid locus between MoHG1 and MGG_14388 in the 70–15 reference strain genome (Figure 1A). We aligned the sequences of MoHG1 in the genomes of rice and non-rice lineage isolates and found that the sequences in rice lineage were generally clustered together while the sequences in non-rice lineages belonged to another subgroup (Figure 1B).

Two ∆Mohg1 mutants were screened using PCR (Figure S1), and the hyphal growth of both ∆Mohg1 mutants was compromised when they were grown on the CM and MM plates compared with the wild type. Moreover, spore production, germination, and appressorium formation were also decreased in the ∆Mohg1 mutants (Figure 1C–H). These results indicate that MoHG1 affects fungal development.

3.2. MoHG1 Regulates Fungal Cell Wall Integrity in M. oryzae

Congo red (CR), sodium dodecyl sulfate (SDS), and calcofluor white (CFW) are often used to assess fungal cell wall integrity. Mohg1 mutants were more sensitive to CR, SDS, and CFW than WT (Figure 2A,B). Similarly, osmotic stressors such as sorbitol, KCl, and NaCl show stronger inhibition in ∆Mohg1 mutants (Figure 2C,D). Moreover, fungal mycelia were treated with cell wall-degrading enzymes. The results showed more protoplasts were released by ∆Mohg1 mutants than WT at 1 h post-treatment (Figure 2E,F). The fungal cell wall of each strain was stained by CFW, and there was a stronger fluorescence signal observed in WT than in the ∆Mohg1 (Figure 2G). These results indicate that fungal cell wall integrity was disrupted in ∆Mohg1 mutants, and MoHG1 plays an important role in maintaining CWI.

3.3. MoHG1 Plays an Essential Role in Pathogenicity

To investigate the role of MoHG1 in pathogenesis, spore suspensions of WT and ∆Mohg1 mutants were sprayed onto rice seedlings. At day 7 post-inoculation, typically infectious lesions were formed by WT, while a smaller number of lesions were caused by the ∆Mohg1 mutants (Figure 3A). Pathogenicity assay showed that the disease index of WT was much higher than that of the mutants (Figure 3B). Moreover, some basal defense genes such as OsMPK5, OsMPK6, OsMPK12, OsPAD4, and OsEDS1 were upregulated in the rice inoculated with ∆Mohg1-31 (Figure 3C–G). The fragments containing MoHG1-GFP with its native promoter were transferred into an mCherry fluorescent protein tagged strain YN125. The fluorescent signal of MoHG1-GFP was observed in spore and infectious mycelia during infection (Figure 3H). These results suggest that the expressions of MoHG1 can be induced and affect fungal pathogenicity during infection.

3.4. Sugar and MAPK Pathways Are Regulated in ∆Mohg1 by Transcriptome Analysis

Transcriptomic analysis was performed to explore the expression patterns characterized by MoHG1. There were 2394 upregulated genes and 1581 downregulated genes in the ∆Mohg1 mutant compared with WT (Figure 4A). Based on the GO and KEGG results, the downregulated genes were mainly enriched in RNA transcription, an integral component of the membrane, nucleolus, MAPK signaling pathway, and cell cycle (Figure 4B). Meanwhile, the upregulated genes were related to translation, methylation, the carbohydrate metabolic process, ribosome, pentose, and glucuronate interconversion-related biological processes in the ∆Mohg1 mutant (Figure 4C). Based on the pentose and glucuronate interconversion pathway, there were many upregulated genes involved in the glycerol and xylitol metabolism (Figure 4D,E). Moreover, there were many downregulated genes in the MAPK pathway in the ∆Mohg1 mutant (Figure 4F). Thus, transcriptome analysis indicated that MoHG1 could regulate sugar and MAPK pathways to affect fungal growth.

3.5. MoHG1 Influences the Contents of Some Carbohydrates

According to the KEGG analyses, we found carbohydrate metabolism-related genes were upregulated in the ∆Mohg1 mutant. Thus, the contents of carbohydrates, including arabinose, fructose, sorbitol, glucose, inositol, lactose, rhamnose, and xylitol were measured. The results showed that the contents of arabinose, sorbitol, lactose, rhamnose, and xylitol were significantly higher in the ∆Mohg1 mutant than in WT (Figure 5A–H). The results show that MoHG1 influences the contents of some carbohydrates to regulate fungal growth in M. oryzae.

3.6. Overexpressing of MGG_00623 in ∆Mohg1 Mutant Restored the Fungal Growth and Chitin Contents

Transcriptomic analysis indicated that MoHG1 could affect cell wall integrity and carbohydrate metabolism. Coincidently, the contents of some carbohydrates and chitin were also disrupted in ∆Mohg1 compared with WT (Figure 2G and Figure 5). For the downregulated genes in ∆Mohg1, MGG_00623 encodes a hexokinase, which may be associated with carbohydrates and chitin metabolism. Thus, MGG_00623 was overexpressed in ∆Mohg1 mutants and two overexpression strains were obtained (Figure 6A). We found that the colony diameters of overexpressing strains were greater than ∆Mohg1 (Figure 6B,C). Moreover, the CFW staining and chitin contents were restored significantly in overexpressing strains, suggesting the overexpressing of MGG_00623 could promote the CWI (Figure 6D,E). We also found that MoHG1 could interact with MGG_00623 directly through yeast two-hybrid and BiFC (Figure 6F,G). Thus, our results showed that MoHG1 could interact with MGG_00623 and the overexpressing of MGG_00623 in the ∆Mohg1 mutant could restore the fungal growth and chitin contents.

4. Discussion

4.1. MoHG1 Is Required for Development and Pathogenicity of M. oryzae

In this study, we have identified a gene, MoHG1, involved in fungal development and pathogenicity in M. oryzae. MoHG1 in the M. oryzae Oryza pathotype is more conserved than in other pathotypes, indicating that this gene might be associated with the differentiation of the Oryza pathotype in M. oryzae populations (Figure 1B). MoHG1 possesses typical signal peptide and transmembrane domains, but the protein could not be secreted into the extracellular space and cytoplasm in rice under microscope observation (Figure 1A and Figure 3H). Moreover, previous secretome results did not identify this protein during the infection of M. oryzae [29]. A similar structural protein, PRO41, harboring a signal sequence and three transmembrane domains, was identified in Sordaria macrospora, which is located in the endoplasmic reticulum (ER) membrane and is essential for sexual development [30]. These results suggested that MoHG1 affected pathogenicity by regulating fungal development, not by being secreted into rice cells to disrupt host defense.

∆Mohg1 mutants display compromised fungal development and pathogenicity compared with the wild type (Figure 1C–H and Figure 3A). These results suggest that MoHG1 plays an important role in fungal development and infection. Based on transcriptome analysis (Table S2), many transcription factor (TF) genes were downregulated in the ∆Mohg1 mutant. Con7 regulates appressorium formation [31]. Homeobox TF family genes, such as MoHOX2 and MoHOX4, are essential for conidiogenesis [32]. Com1, Moatf1, and MoMcm1 are required for fungal development and virulence [33,34,35]. Thus, MoHG1 might affect the expression of some TF genes to regulate downstream genes to influence development and pathogenicity in M. oryzae.

Many innate immune-related genes were upregulated in rice inoculated with the ∆Mohg1 mutant (Figure 3C). The MAPK cascade genes OsMPK6 and OsMPK12 (OsBWMK1) were upregulated, which were associated with the WRKY45- and OsWRKY33-mediated SA signaling pathways, respectively [36]. Furthermore, OsPAD4 and OsEDS1 involved in the SA pathway were also induced by the ∆Mohg1 mutant. These results suggested that the activation of an SA pathway in rice enhances the resistance with ∆Mohg1. Effectors secreted by pathogens that interfere with the SA pathway have been identified. In M. oryzae, two nuclear effector gene deletion mutants, ∆Mohtr1 and ∆Mohtr2, also induce expressions of SA-related genes when they were inoculated [37]. Interestingly, many genes encoding cell death-inducing proteins (CDIPs) [38,39] were upregulated in the ∆Mohg1 mutant (Table S2) and cell death is regarded as an immunity response to pathogens. However, whether a ∆Mohg1 mutant could secret more cell death-inducing proteins into rice cells and trigger SA-mediated immunity needs further research.

4.2. MoHG1 Regulates Signal Sensors and Cell Wall Integrity in M. oryzae

In M. oryzae, signal transduction from the cell surface into the cell through a series of phosphorylation cascades, such as the cyclic AMP-dependent protein kinase A (cAMP/PKA) signaling pathway [40], the Pmk1 MAPK pathway [41,42], and the CWI pathway, have been well identified [22,43]. The crosstalk among these pathways can coordinate them with each other to regulate fungal behavior. According to the KEGG results, many downregulated genes were enriched in the MAPK signaling pathway (Figure 4B). We found that the expressions of four sensor genes, MoSho1, MoOpy2, Sln1, and PTH11 were downregulated in the ∆Mohg1 mutant (Table S2). MoSho1 functions by recognizing rice leaf waxes for Pmk1 activation and appressorium formation [21]. MoOpy2 participates in the Osm1 MAPK pathway and the Mps1 MAPK pathway to regulate fungal development, pathogenicity, and autophagy [44]. Sln1 is responsible for sensing the turgor threshold to affect penetration into rice leaves [45]. PTH11 as a G-protein-coupled receptor influences the MAPK Pmk1 and cAMP/PKA pathways to regulate appressorium [18]. In summary, MoHG1 influences the expressions of sensor genes to regulate signal transduction.

The ∆Mohg1 mutants showed more sensitivity to CWI stressors and more easily released protoplasts under lysis enzyme treatment (Figure 2). The CWI pathway is essential to pathogen development and infection. The core components of the CWI pathway have been well identified, but MoMck1, MoMkk1, and MoMps1 were not significantly regulated in the ∆Mohg1 mutant. However, MoSLN1, MoGln2, and Gel5 were reported to participate in CWI [13,46,47] and downregulated in the ∆Mohg1 mutant. Thus, MoHG1 may affect the expressions of CWI-related genes to maintain fungal cell wall integrity.

4.3. MoHG1 Interacts with a Hexokinase to Mediate Carbohydrate and Chitin Metabolism

Pentose and glucuronate interconversion pathway and glycolysis/gluconeogenesis pathway-related genes were significantly enriched in the ∆Mohg1 mutant (Figure 4C), and the contents of arabinose, sorbitol, lactose, rhamnose, and xylitol were also higher in ∆Mohg1 (Figure 5A,C,F–H). Interestingly, we also found many genes involving lactose, arabitol, and xylitol metabolism upregulated in the ∆Mohg1 mutant (Figure 4C–E). The glycolysis/gluconeogenesis pathway is responsible for the breakdown of glucose into pyruvate, which can be further converted into lactate by L-lactate dehydrogenase [48]. The ∆Mohg1 mutant was cultured in a nutrition-rich medium, which could provide sufficient glucose for growth, and thus the content of glucose in the ∆Mohg1 mutant is similar to that in WT. Arabinose and xylitol are the metabolites of the pentose catabolic pathway [49]. The expressions of the D-arabinitol dehydrogenase and D-xylulose reductase A were upregulated in the ∆Mohg1 mutant, and were consistent with higher contents of arabinose and xylitol in ∆Mohg1. These results suggest that MoHG1 negatively regulates some aspects of carbohydrate metabolism. However, the relationship between higher contents and growth defect regulated by MoHG1 in M. oryzae needs further research.

Fungal chitin is required for growth and virulence in M. oryzae. We found that the cell wall integrity was disrupted, and the content of chitin was also decreased in ∆Mohg1. There are seven chitin synthase (CHS) genes identified in M. oryzae [50]. Based on transcriptome analysis, CHS1 and CHS3 were downregulated, while CHS2 and CHS7 were upregulated in ∆Mohg1, which could affect chitin content (Table S2). Based on transcriptome results, many genes were downregulated that were involved in carbohydrate and chitin metabolism. These results might indicate that MoHG1 could be an upstream component to regulate fungal carbohydrate and chitin metabolism. For the downregulated genes, MGG_00623 encodes a hexokinase-1, which is regarded as the first enzyme to catalyze the conversion of glucose into glucose-6-phosphate in the glycolysis pathway, while glucose-6-phosphate can be converted into fructose-6-phosphate as the primary material for chitin synthesis in the hexosamine pathway. Many studies have shown that hexokinase-mediated glucose utilization can positively affect chitin synthesis in insects [51,52,53]. Our results indicate that MGG_00623 could play a more important role in MoHG1 mediating fungal growth and virulence via the sugar and chitin metabolism pathway. Moreover, MGG_00623 could interact with MoHG1 directly, which might remodel the MGG_00623 structure and enhance the hexokinase activity to increase the chitin production in M. oryzae. However, our results demonstrate that MoHG1 affects fungal growth and virulence through regulating CWI and chitin content. The molecular mechanisms of interactions among MoHG1, CWI, and chitin synthesis-related proteins still require further research.