In the Ensembl Fungi release 52 (Dec 2021 ©EMBL-EBI) resource section for M. oryzae,¹ the amino acid sequences of both aspartate transaminase AAT1(YKL106W) and AAT2 (YLR027C) from the brewer’s yeast Saccharamycess cerevisiae were used to search the aspartate transaminases (ASTs) of M. oryzae. Three putative amino acid sequences that encode ASTs were found, termed as MoAST1 (MGG_06530), MoAST2 (MGG_04156), and MoAST3 (MGG_05067), respectively. The three obtained MoAST amino acids were used for a blastP search to identify other interested AST amino acid sequences in National Centre of Biotechnology Information.² The established phylogenetic tree showed two major clades among the selected proteins. All the fungal and yeast ASTs are in one clade, and AST1 proteins and AST2 proteins are in two neighbor-jointing subclades, respectively. MoAST3 together with the so-called CgAST3 is significantly far from AST1 and AST2 in genetic relationship (Figure 2A). Further analysis revealed that MoAST1, MoAST2, and MoAST3 shared a close ancestor with CgAST1, CgAST2, and CgAST3 of C. graminicola (Cg), respectively (Figure 2A). In addition, MoAST2 shares 78% identity and 88% similarity with CgAST2, and shares 71% identity and 82% similarity with FoAST2, and 52% identity and 66% similarity with ScAST2, suggesting a comparatively close genetic relationship with filamentous fungal phytopathogens.

To evaluate the biological activities of the three MoAST genes, we analyzed the abundance of MoAST1, MoAST2, and MoAST3 transcripts at different stages of M. oryzae development and pathogenesis. In comparison with that of the mycelia, MoAST1 expression was relatively higher at conidial, and appressorial stages but was downregulated and at low levels during in planta infection from 24 h post-inoculation (hpi) to 144 hpi (Figure 2B). MoAST3 expression remained almost unchanged at cultivate stage prior to infection except for a significant increase at conidial and appressorial stages but downregulated and was in extremely significant low levels at earlier infection stage (Figure 2D). Compared with MoAST1 and MoAST3, MoAST2 was drastically upregulated during appressorial formation and maturation and showed up to a three-fold increase as the appressoria matured (Figure 2C); from 24 to 72 hpi, MoAST2 was sharply downregulated to an almost undetected expression level; however, from 96 to 144 hpi, it returned to higher level in the appressoria (Figure 2C). Interestingly, C. graminicola AST2 possessed a similar expression profile with MoAST2 (Figure 2E). Therefore, MoAST2 together with the CgAST2 was reckoned to be a pathogenic related factor. Thereafter, we bioinformatically identified the MoAST2.

Based on the genomic DNA sequence, the MoAST2 gene is located on the Magnaporthe chromosome 6, with two introns and a length of 1374 bp open reading frame (ORF) encoding a protein of 457 amino acids. MoAST2 contains one predicted aminotransferase catalytic domain (EC Number: 2.6.1.1), together with which 13 of PLP binding sites and 2 of catalytic active sites were predicted (Figure 3A).

The tertiary (3D) structure of MoAST2 protein was predicted based on the web-based server I-TASSER.³ Among the studied ASTs, although there are variations in sequence identities, the overall domain folds of ASTs are highly similar. Like the SpAST protein structure (Jeong et al., 2019), the MoAST2 protein forms a very stable homodimer with an unusually extended interface (Figure 3B). The subunits of the homodimer are tightly combined in the exactly opposite direction through the salt bridges and hydrogen bonds present between side chains as well as between the main chain and the side chain, and many hydrophobic residues are aligned at the interface contributing hydrophobic interaction between two molecules of dimer. The MoAST2 homodimer contains four active sites in the vicinity of subunit interfaces (Figure 3B), which are associated with binding to the cofactor PLP and substrate. According to this structure with active and binding site residues, it appears that aspartate and α-ketoglutarate as substrates bound to the MoAST2 at the binding sites cause the catalytic amino acid transaminase reaction.

Aspartate transaminases (ASTs) reported were subcellularly localized in cytoplasm or mitochondria (Jaussi et al., 1982). When MoAST2 protein sequence was input in the Euk-mPLoc 2.0 web system⁴ performing the prediction of subcellular localization, MoAST2 was specifically localized in cytoplasm. Further confirmation was carried out through fluorescent microscopic observation of the DsRED or GFP tagged MoAST2 proteins.

We constructed a gene deletion vector for targeted gene replacement of the MoAST2 gene (Supplementary Figure 1A). Then, the deletion mutant strains (ΔMoast2) were created by replacing the MoAST2 ORF with the hygromycin phosphotransferase (HPH) gene. Based on the ΔMoast2 strain, we subsequently created the complementary stain with a MoAST2-GFP fusion gene driven by its native promoter (Supplementary Figure 1B). Fluorescent microscopic observation was carried out in the growing hyphae (6d). Green fluorescence signals of the MoAST2-GFP protein were detected with a rather strong green fluorescence signal in growing hyphae (Figure 4A). By comparison, in the wild-type or untransformed strains, the background green fluorescence was too weak to be detected (data not shown). In parallel, we generated the complementary stain of ΔMoast2 harboring DsRED-tagged MoAST2 in the ΔMoast2 background (ΔMoast2/DsRED-MoAST2) (Supplementary Figure 1C). The DsRED fluorescence in the hyphae of ΔMoast2/DsRED-MoAST2 indicated that the DsRED-tagged MoAST2 proteins were distributed in the cytoplasm (Figure 4B), confirming the cytosolic protein of MoAST2.

To investigate the contribution of MoAST2 gene to vegetative growth of M. oryzae, we inoculated the ΔMoast2, ΔMoast2/MoAST2, and the wild-type strains on complete medium (CM). After 7 days of cultivation, mycelial growth was analyzed according to the colony diameter among the three strains. As a result, the growth of ΔMoast2 strains was reduced to a certain extent in CM (Supplementary Figures 2A,B); but no colony morphology alterations were observed except that the ΔMoast2 strains appeared to be lower pigmentation on CM solid and liquid media than the wild-type and the complemented strains (Supplementary Figures 2C,D).

To analyze the role of the MoAST2 gene in sporulation in the rice blast fungus, the ΔMoast2, ΔMoast2/MoAST2, and wild-type strains were cultured on OMA plates for 10 days, and then conidiophore stalk and conidia were observed on microscope. The result indicated that the ΔMoast2 strains hardly produced conidia on OMA plates; however, ΔMoast2/MoAST2 produced more conidia like the wild type (Figures 4C,D). To harvest enough conidia for inoculation assays, we had to prepare for 20 times of wild type plates. Although ΔMoast2 strains were impaired in sporulation, their germination was normal. In terms of appressorium formation, ΔMoast2 was severely affected despite being induced by artificial hydrophobic film or by onion epidermis surface (Figure 4E). These data suggest that MoAST2 is involved in fungal growth and development prior to plant infection.

To understand the effects of the MoAST2 on infection, we inoculated intact rice leaves with spores of the wild type, ΔMoast2/MoAST2, and MoAST2. ΔMoast2 caused restricted lesions on the compatible cultivar at 144 hpi, although the wild type and ΔMoast2/MoAST2 have resulted in enlarged lesions (Figure 5A).

To observe the infection process during plant–pathogen interaction, we performed rice leaf sheath assays. At 24 hpi, the wild type and ΔMoast2/MoAST2 had invaded more than 70% of rice cells; similarly, nearly 70% of rice cells had been invaded (Figures 5B,C). By 48 h, the three strains, ΔMoast2,ΔMoast2/MoAST2, and wild type were all capable of elaborating invasive hyphae in the first infected cell. By 72 h, the majority of wild type and ΔMoast2/MoAST2 have spread to the adjacent cells around the infection sites, but the invasive hyphae of ΔMoast2 were still restricted to the primary infected rice cells (Figures 5B,C). These data indicated that the ΔMoast2 was unaffected during earlier biotrophic development but switched to be defective at the necrotrophic phase, which may be directly responsible for the reduced lesion size or limited necrotic zones. Therefore, MoAST2 as a player in host–pathogen interactions contributes to the development of rice blast disease.

Some pathogenic factors are responsible for regulating resistance to adverse environmental factors that fungal pathogens may encounter during their life cycle. Sensitivity to oxidative stress is associated with metabolic requirements such as purine biosynthesis and glutamine synthesis (Fernandez et al., 2013; Aron et al., 2021a). To investigate the contributions of the MoAST2 gene in oxidative stress tolerance, we performed the fungal hyphal growth under H₂O₂ stress on CM media supplemented with 2.5-mM and 5-mM concentrations of H₂O₂. As expected, the hyphal growth of ΔMoast2 was severely affected, but not as much in the wild-type and complementary strains (Figures 6A,B). The ΔMast2 strains were hypersensitive to 5 mM H₂O₂ concentrations (Figures 6A,B), implying the involvement of MoAST2 in the tolerance of oxidative stress in M. oryzae.

Also, amino acid metabolisms were reported to be relevant to cell wall integrity. In fact, cell wall defects had been proved to be associated with the MoGLN2 deletion in the rice blast fungus (Aron et al., 2021a). We analyzed the hyphal growth of ΔMoast2 on CM medium supplemented with cell wall stressors Congo Red (CR) and Sodium Dodecyl Sulfate (SDS). However, similar to the ΔMoast2/MoAST2 and wild-type strains, the ΔMoast2 strains were jaded to cell wall stressors (Figures 6C,D), revealing the complicated relationship between amino acid metabolism and biological function.

Generally, the double directions of the reversible reaction of glutamate/aspartate are all catalyzed by AST. To evaluate MoAST2 function involved in tolerance to nitrogen starvation, ΔMoast2, ΔMoast2/MoAST2, and the wild type were inoculated onto MM or MM supplemented with the indicated single amino acid as sole nitrogen source. The ΔMoast2 mutant strains, compared to the ΔMoast2/MoAST2 or wild-type strains, were almost unable to grow at Day 7 when applied with 10 mM aspartate or asparagine as the sole nitrogen source (Figure 7A), reflecting metabolism of aspartate or asparagine was blocked. However, when glutamate or glutamine was supplemented to MM, the defect in growth of ΔMoast2 was restored to the level of the wild-type or ΔMoast2/MoAST2 strains (Figure 7A). Meanwhile, we checked other members of aspartate family and glutamate family. As a result, the ΔMoast2 strains could grow in the supplemented MM with individual member of glutamate family, but not grow in aspartate family MM. According to the aspartate metabolic pathway, we proposed that the direction of the AST-catalyzed reaction is from aspartate to glutamine.

To address whether exogenous amino acids could restore the ΔMoast2 in H₂O₂ tolerance, we inoculated the three strains onto the CM, supplemented, respectively, with 5mM glutamate, glutamine, and proline under treatment of 5mM H₂O₂. The result indicated that exogenous amino acids of glutamate family could partially restore the growth of ΔMoast2 (Figure 7B). Accordingly, rice seedling disease assays were performed by spraying the ΔMoast2 spore suspension mixed with 5 mM glutamate. The result demonstrated that the replenishment of glutamate partially restored the ability to cause blast disease (Figure 7C).

The developmental process of C. graminicola at least involves the vegetative mycelium growth and the conidial and infection stages in one disease cycle. Compared to the mycelial stage and infection stage, CgAST2 exhibited much higher transcriptional activities in the conidial stages. In addition, the expression level of CgAST2 gene was up-regulated within 72 h after infection (Figure 2E). The similar expression patterns of CgAST2 and MoAST2 at different development stages implied their similar roles in development and pathogenesis.

Based on the significance of glutamate biosynthesis to virulence of M. oryzae, another hemibiotrophic fungal pathogen should be selected to check whether the AST2 gene is conserved in pathogenesis. We therefore analyzed the maize anthracnose pathogen C. graminicola and identified CgAST2. We focused on investigating the biological functions of CgAST2. The ΔCgast2 mutant strains were generated (Supplementary Figure 3A). The ΔCgast2 strains were unable to grow on MM supplemented with aspartate (Figure 8A). When the ΔCgast2 strains were cultured on MM supplemented with 10 mM glutamate, like the ΔMoast2, the growth defects of ΔCgast2 could be reversed (Figure 8A). The result indicated that CgAST2 is required for glutamate biosynthesis.

In order to further examine the role of CgAST2 in the infection process, we carried out microscopic observation after inoculating leaves. Like ΔMoast2, ΔCgast2, together with ΔCgast2/CgAST2 and the wild type M1.001, all could form in planta invasive hyphae in the first infected cell. However, the ΔCgast2 strains, different from the wild type, were limited in the primary infected maize cells (Figure 8B). Even at 144hpi, ΔCgast2 could not result in typical symptoms on maize leaves (Figure 8C), suggesting AST2 in pathogenesis is conserved.

The wild-type strain M. oryzae JL0910 was previously isolated and purified from the rice cultivar Jijing88, which is widely planted in Jilin Province, China. All strains, including the four strains generated in this study, were cultured on CM agar plates and stored on filter paper at −20°C.

The strains were cultured for 7 days on CM for assessment of their growth rates. Each test was repeated at least three times. Liquid CM was used to prepare the mycelia for DNA and RNA. The mycelia used for nucleic acid or protein extraction were prepared by growing the relevant strains in 100 ml liquid CM for 3 days at 25°C with gentle rocking at 150 rpm under bright light. For conidiation, the strains were inoculated on oatmeal-tomato agar medium (OTA) and incubated at 25°C for 7 days in the dark (Saint-Macary et al., 2015). After the aerial hyphae of the colonies had been washed away using sterilized distilled water, the strains were continually grown for 3 days under a fluorescent light.

The C. graminicola M1.001 strain used as wild type (WT) in this study was a gift from Professor H.B. Deising (Institute of Agricultural and Nutritional Sciences, Martin Luther University Halle-Wittenberg, Halle, Germany). The wild-type, mutant, and complementation strains were cultured at 25°C. PDA was used for the growth and conidia production of the strains. Phenotypic tests were performed on complete minimal media. All the fungal strains used were maintained on paper filters at −20°C. Agrobacterium tumefaciens AGL-1 and Escherichia coli strains DH5α were cultured in lysogeny broth media. The pXEH2.0 vector to construct a recombinant vector, which was transformed into A. tumefaciens (AGL-1) competent cells. The AGL-1 strain was transformed with wild-type M1.001 using an A. tumefaciens-mediated transformation (ATMT) protocol (Münch et al., 2011).

Total RNA or DNA was extracted using RNA or DNA extraction kits (Sangon, Shanghai, China). The rice or maize leaves inoculated with M. oryzae or C. graminicola were collected at 24, 48, 72, 96, 120, and 144 hpi as samples (100 g) for total RNA extraction. First-strand cDNA was synthesized from 2.0 μg of total RNA using Avian Myeloblastosis Virus reverse transcriptase (Promega, Madison, WI, United States). The cDNA samples were diluted 10-fold and used as templates for PCR.

Quantitative real-time RT-PCR was run on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) following the manufacturer’s instructions. Reactions were performed in a 20-μl volume system. Transcriptions of genes were analyzed and the ACTIN gene was used as an endogenous control. Fold changes were calculated as 2^–ΔΔCt to analyze the relative abundance of transcripts. Quantitative real-time RT-PCR was repeated in triplicate with three independent biological experiments, and the primer pairs used in this section are listed in Supplementary Table 1.

To generate the MoAST2 or CgAST2 deletion strain ΔMoast2 or ΔCgast2, the MoAST2 or CgAST2 gene was replaced by the hygromycin resistant cassette (HPH). To construct the replacement vector, the flanking sequences were amplified with their corresponding primer pairs (MoAST-LB F/R and MoAST-RB-F/R) and fused with the HPH cassette in a pXEH knockout vector. The construct was confirmed using RT-PCR with the upstream forward and downstream reverse primers MoAST-F/R and MoAST-QF/R, and the confirmed construct was introduced into wild-type protoplasts. The MoACTIN gene (MGG_03982.6) was amplified with the primers MGG-Actin-Q-F/R to serve as an endogenous reference. The complementation fragments, which contain the entire MoAST2 genes were amplified using PCR and inserted into the pKD7-RED (G418-resistance) vector or pCAMBIA1303 vector to complement ΔMoast2 (Supplementary Figure 1).

To construct the DsRED-tagged MoAST2 vector, MoAST2 gene, a DNA fragment containing the full-length MoAST2 open reading frame sequence was amplified with primers MoAST-PKD7-F/R. pKD7-RED contains the DsRED gene as a subcellular localization tag and the G418 resistance gene as a selection marker. To construct the RFP-tagged MoAST2 vector, MoAST2 gene, a DNA fragment containing the full-length MoAST2 open reading frame sequence was amplified with primers MoAST1303-F/R. pCAMBIA1303-GFP contains the Green fluorescent protein tag genes as a subcellular localization tag and the G418 resistance gene as a selection marker (Supplementary Figure 1).

The same construction strategy was used to generate CgAST2 deletion strains (ΔCgast2). The primer pairs CgAST-LB-F/R and CgAST-RB-F/R were used to amplify the both flanking sequences, and the right DNA fragments were, respectively, fused with the HPH cassette in a pXEH knockout vector, obtained the deletion vector. For construction of the complementary vector of the ΔCgast2 strains, the pKD7-RED (G418-resistance) vector was used (Supplementary Figure 3). The corresponding PCR amplification primer pairs are CgAST-PKD7-F and CgAST-PKD7-R. All the primer sequences are listed in Supplementary Table 1.

After 10 days of cultivation on OTA, conidia were collected with 5 ml of distilled water, filtered through three layers of lens paper, and counted with a hemacytometer under a microscope. Conidial germination and appressorium formation were measured on a hydrophobic surface (plastic cover slips or Gel-bond films). Conidia suspensions of 30 μl (1 × 10⁵ spores/ml) were dropped onto a hydrophobic surface and were placed in a moistened box at 25°C. Appressorium formation rate was then calculated under the microscope at 12 hpi. More than 100 appressoria were counted for each strain and the experiment was repeated three times. Photographs were taken at 24 hpi.

In order to observe the subcellular localization of the MoAST protein, MoAST2-DsRED or MoAST2-GFP was cultured for 7–14 days in the dark in a Potato Dextrose Agar (PDA) plate into which coverslips were obliquely inserted. The samples were observed using fluorescence microscopy until the mycelium extended to the coverslips.

To detect the effect of MoAST2 under exogenous H₂O₂, the wild-type, mutant, and complementation strains were continuously cultured on CM plates with concentrations of 0, 2.5, and 5.0 mM H₂O₂ in the dark for 7 days at 25°C, and the fungal colonies were observed and measured. Five-millimeter mycelial plugs were inoculated on CM plates with Congo red (CR) of 100 or 200 μg/mL, and SDS of 0.005% or 0.01% (w/v), respectively, and cultured in the dark at 25°C for 7 days to test the integrity of the cell wall. The growth of the colonies was observed, and the diameters were measured. These experiments were performed in triplicate and repeated three times for each strain.

To determine the effect of selected amino acid auxotrophy on mycelium growth and pathogenicity, media and conidial suspension liquid were supplemented with 10 or 5 mM individual amino acid, at 28°C. Experiments were performed in triplicate.

The rice cultivar Jijing88 was used for infection assays in identifying pathogenesis of M. oryzae. For the inoculation of the intact rice leaves, a conidial suspension (1 × 10⁵ conidia/ml) was sprayed onto the leaves using an air sprayer. The inoculated plants were placed in a high humidity chamber at 25°C for 24 h in the dark then transferred to a growth chamber with a 16-h light/8-h dark photoperiod. For microscopic observation of cuticle penetration and infectious hyphae growth, leaf sheaths and inoculation were prepared and inoculated with 100 ml of conidial suspension (1 × 10⁵ conidia/mL) on the inner leaf sheath cuticle cells. After 24, 48, and 72 h and incubation under humid conditions at room temperature, the leaf sheaths were observed under a microscope.

The corn cultivar used in the pathogenicity experiment was “Xianyu 335,” which is a variety that is planted widely in north China. The leaf spot inoculation test utilized 10 μl 5 × 10⁵ conidia/mL (2% gelatin, w/v) droplets from the wild-type, mutant, and complementation strains that were placed separately on the surfaces of third corn leaves. After inoculation, the leaves were placed in an artificial climate box with a relative humidity of 90% at 25°C and incubated in the dark for the first 24 h and then cultivated continuously for 6 days in the dark followed by light for 12 h each before examination. The leaves after spot inoculation were sampled at 24, 48, and 72 h, respectively, to microscopically observe the infection process. The samples were then sliced and observed with an optical microscope for pathogenicity.

All experiments were repeated at least three times. The mean ± standard deviation of the colony diameter, germination rate, and relative expression were determined using the Prism version 7.00 software (GraphPad, San Diego, CA, United States). The data were analyzed using InStat3 (GraphPad). The threshold for statistical significance was p < 0.05.