The M. oryzae strain P131 was used as a wild type (Chen et al., 2014). All of the wild-type strain and transformants used in this study were grown on Oatmeal Tomato Agar (OTA) plates at 28°C. To extract genomic DNA, RNA, protein, and isolate protoplasts, mycelia were incubated in liquid CM cultures (180 rpm) at 28°C for 36 h. Colony growth and conidiation were performed as described previously (Chen et al., 2014). Conidia harvested from 7-day-old OTA cultures were used for testing virulence and observing infection process.

To test stress sensitivities, colony diameter of different strains were measured at 5 days post inoculation (dpi) on CM plates added with 0.2 mg ml^-1 Congo Red (CR), 0.1 mg/ml Calcofluor White (CFW), 0.005% Sodium dodecyl sulfate (SDS), 0.5 M NaCl, 10 mM H₂O₂, or buffered at pH 8.0 with phosphate buffer. The diameters of the colonies were recorded and used for calculating the growth reduction rates (Chen et al., 2014).

To test carbon sources utilization, colony diameter of different strains were measured at 5 dpi on MM agar plates amended with 1% glucose, 5 mM sodium acetate, ethanol, or glycerol as sole carbon source. The diameters of the colonies were recorded for the calculation of the growth reduction rates (Kong et al., 2012).

To generate gene’s replacement construct, 1.5-kb upstream and downstream of the gene’s flanking sequences were amplified from the genomic DNA of the wild-type strain. Both flanking sequences of MoUBP14 were cloned into pKNH as the deletion vector, and then transformed into protoplasts of the wild type (Chen et al., 2014). For complementation, MoUBP14 gene containing 1.5 kb promoter region and 0.5 kb terminator region was amplified and cloned into pKN (Chen et al., 2014). The resulting constructs were transformed into the ΔMoubp14 mutant. CM plates supplemented with 250 μg ml^-1 hygromycin B (Roche, United States) was used to select deletion transformants, or supplemented with 400 μg ml^-1 neomycin (Amresco, United States) to select complementation transformants. The deletion transformants were verified by PCR and confirmed by Southern blot.

The eGFP:MoUBP14 fusion vector was generated by an overlap method. A PCR product including 1.5 kb of the promoter region, eGFP gene without termination codon, and ORF region of the MoUBP14 gene was sequentially ligated by an overlap-PCR method. Then this product was cloned into pKN. The resulting construct pKNG-UBP14 was transformed into the ΔMoubp14 mutant. Subsequent complementary strains were used to observe GFP fluorescence at different developmental stages under a confocal microscope Leica TCS SP8 (Leica Microsystems, Germany).

To test virulence of different fungal strains, 1-week-old barley leaves (Hordeum vulgare cv. E9) and 1-month-old rice seedlings (Oryza sativa cv. LTH) were sprayed with conidia suspensions (5 × 10⁴ conidia ml^-1) in 0.025% Tween 20. The inoculated plants were incubated at 28°C with full humidity, and the disease lesions were observed at 5 dpi. To observe the infection process, conidia suspension (1 × 10⁵ conidia ml^-1) was inoculated onto the lower barley epidermis, and then incubated in a dark chamber with full humidity at 28°C. Infection process was observed at different times after inoculation under a microscope (Nikon Ni90, Japan).

To detect gene expression levels at different developmental stages, samples were harvested to extract total RNA using TRIzol (Invitrogen, United States). Total RNAs were subsequently used to prepare the cDNA templates. Samples of mycelia were harvested from cultures incubated in liquid CM for 48 h. Samples of the germ tubes and appressoria were harvested form hydrophobic plastic surface at 3 and 12 hpi (2 × 10⁵ conidia ml^-1). Samples of infection hyphae were harvested by tearing down the lower barley epidermis at 18, 24, and 42 h after inoculation with conidia suspension (2 × 10⁵ conidia ml^-1). The qRT-PCR was performed on the ABI 7500 real-time PCR system (Applied Biosystems, United States) by using SYBR Green PCR Master Mix (Takara, Dalian, China) as manufacture’s instruction.

Conidium germination and appressorium formation were observed on a hydrophobic coverslip. Drops of conidial suspension (1 × 10⁵ conidia ml^-1) were inoculated onto a coverslip and incubated in a moistened chamber at 28°C. Germ tubes and appressoria formation ratios were calculated by a microscope with at least 100 conidia per replicate, and three replicates were used for per experiment.

Appressorium turgor was determined by cytorrhysis assay. Drops of conidial suspension (1 × 10⁵ conidia ml^-1) were inoculated onto a coverslip and incubated in a moistened chamber at 28°C for 24 h. Then the cover slides were immerse in different concentrations of PEG8000 (25, 30, 35, and 40%, wt/vol) for 15 min, and then washed by distilled water. Appressoria collapse ratios were subsequently calculated under a microscope with at least 100 conidia per replicate, and three replicates were used for per experiment.

For glycogen and lipid droplets staining, conidia suspension (1 × 10⁵ conidia ml^-1) were inoculated at different times on hydrophobic coverslip, and were then applied to staining solution (60 ml mL^-1 KI and 10 mg mL^-1 I₂) (Thines et al., 2000), or Nile Red solution (Sigma-Aldrich, United States) (50 mM Tris/maleate buffer, pH 7.5, with 20 mg mL^-1 polyvinylpyrrolidone and 2.5 μg ml^-1 Nile Red) (Greenspan et al., 1985) for 10 min, respectively. The stained germinating conidia and appressoria were observed and photographed under an epifluorescence microscope (Nikon Ni90, Japan).

For CFW staining, mycelia were harvested from CM medium and stained with 10 μg ml^-1 CFW (Sigma-Aldrich, United States) for 10 min in the dark, then rinsed twice with PBS buffer and observed under the microscope (Nikon Ni90, Japan). For conidial cell staining, the conidia were harvested from strains incubated for 5 days on OTA plates.

For DAB staining assay, epidermis of barley leaves infected by strains at 36 hpi were stained with 1 mg/ml DAB (Sigma-Aldrich, United States) solution (pH 3.8) for 8 h, and de-stained with an ethanol/acetic acid solution (ethanol/acetic acid; 94:4) for 1 h, then observed with an epifluorescence microscope (Nikon Ni90, Japan).

Conidia and appressoria formed at 12 hpi on barley leaves were processed for transmission electron microscopy (TEM), with processing and imaging of the TEM samples performed as described previously (Soundararajan et al., 2004).

To purify MoUbp14-interacting proteins, a strain expressing eGFP-MoUbp14 under the constitutive promoter RP27 was obtained. Mycelia of this strain was harvested from liquid CM incubated for 48 h and ground into powder for extracting total proteins in lysis buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100] containing 10 μg/ml each of leupeptin and pepstatin A, and 1 mM PMSF. Resulting mixture was centrifuged at 4°C for 30 min at 15,000 rpm. The supernatant was incubated with anti-GFP affinity resins (Sigma-Aldrich, United States) and washed by several times. Then the proteins were eluted with 0.1% Rapigest as described (Zhou et al., 2012). The elution proteins were subjected to trypsin digestion and liquid chromatography tandem mass spectrometry (LC-MS/MS) as described (Zhou et al., 2012). The resulting MS/MS data were used to search against the M. oryzae protein database at NCBI. Two biological replicates were used for this assay.

The 3 × Flag fused MoFBP1 or MoPCK1 construct was constructed. Both constructs were transformed into the wild type or ΔMoubp14 mutant, respectively. Subsequent transformants were performed with Western blot analysis with an anti-Flag primary antibody (1:5000, Sigma, United States) and an anti-rabbit horseradish peroxidase secondary antibody (1:10000, Sigma, United States). Results were visualized with the ECL detection system (Amersham Biosciences, United Kingdom). To test catabolite degradation of MoFbp1 and MoPck1, the wild type and the ΔMoubp14 mutant strains expressing Fbp1:3 × Flag or Pck1: 3 × Flag were incubated firstly in CM medium for 42 h, and then transferred the mycelia into the liquid medium with NaAc as the sole carbon source for 12 h, and then added glucose into the medium for incubating for 2 h. Total ubiquitination levels of the wild type, the ΔMoubp14 mutant and the complementation strain cUBP14 were detected by an anti-ubiquitin antibody (Abcam, United Kingdom).

For all of the biological analyses, means and SE were calculated from three independent replicates. Significant differences are derived from t-test with P < 0.01 or P < 0.05.

The MoUbp14 (MGG_08270) was identified via a search of the M. oryzae genome database (Ensembl Fungi¹) by using the S. cerevisiae Ubp14 protein as a query. The MoUbp14 is predicted to encode a protein of 787 amino acids with a putative RING-type zinc finger domain at its N-terminus and a ubiquitin specific protease (USP) domain at its C-terminus (Supplementary Figure S1A). Phylogenetic tree analysis by using MEGA version 5.10 demonstrated the Ubp14 protein is well conserved among eukaryotes. Among the analyzed organisms, Neurospora crassa (XP_960037.1) and Fusarium graminearum (XP_011318590.1) Ubp14 are the closest match to MoUbp14 (Supplementary Figure S1B). Multiple sequence alignment was also performed to evaluate the conservation of the Ubp14 protein among eukaryotes. The MoUbp14 protein shares a 72% positive amino acid identity with that of N. crassa, 71% with F. graminearum, 36% with A. thaliana, 34% with Oryzae sativa, 34% with S. cerevisiae, and 37% with Homo sapiens (Supplementary Figure S2) at the protein level with more than 95% query coverage.

To evaluate potential roles of MoUBP14 in M. oryzae, the transcription profile was examined by quantitative real-time PCR (qRT-PCR). MoUBP14 was highly expressed during mycelial growth, appressorium formation and the late invasive growth at 48 h after inoculation (hpi), while was severely decreased during conidial germination and the early invasive growth at 18 and 24 hpi (Supplementary Figure S3). This result suggested that the expression of MoUBP14 gene is fine tuned for development and infection processes.

To determine the roles of MoUBP14, a gene replacement construct was obtained to perform a homologous recombination strategy-mediated disruption (Supplementary Figure S4A). This construct was then transformed into the wild-type strain P131. Two independent Δubp14 null mutants with similar phenotypes were obtained and confirmed by Southern blot analysis (Supplementary Figure S4B). One of the null mutants, ubp14KO, was randomly selected for further analysis. To confirm the phenotypic defects of the mutants were resulted from the disruption of MoUBP14, complementation transformants were also generated by transforming the native promoter drived MoUBP14 into the Δubp14 null mutant. All of the complementation strains was recovered in all the phenotypic defects, and one of which, termed cUBP14, was randomly selected for further study.

To investigate whether the function of MoUBP14 is associated with vegetative growth in M. oryzae, we observed colony morphology and size on oatmeal agar plate (OTA). The growth diameter of ΔMoubp14 was significantly reduced compared with that of the wild type, and the colony was evidently whiter than the wild type (Figures 1A,B). When the ΔMoubp14 mycelia was stained with Calcofluor White (CFW), we found the average lengths of apical hyphal cells were significantly reduced, and the location of septa were abnormal, compared with the wild-type (Figure 1C). These results indicated that MoUBP14 is required for fungal vegetative growth and mycelial pigmentation.

As conidia is very important for spread of the rice blast fungus, we next assessed the role of MoUBP14 in conidiation. Conidiation measurements showed that the ΔMoubp14 mutant produced only around 1.5% conidia of that produced by the wild type and the complementation strain (Figure 2A). Microscopic observation found that, compared with conidiophores with dense conidia in the wild-type and complementation strain, the ΔMoubp14 mutant formed sparse conidia on the conidiophores after 10 days of continuous exposure to light (Figure 2B). Moreover, we found that conidia of the ΔMoubp14 mutant were abmormal in conidial cell number. For the wild-type strain, more than 80% of the conidia were three-celled. While for the ΔMoubp14 mutant, only 53.5% of the conidia were three-celled and 47.5% of the conidia were two-celled (with one septum) or one-celled (without septum). These defects were recovered in the complementation strain cUBP14 (Figures 2C,D). Further TEM observation demonstrated that a large number of contents, which were supposed to be glycogen, lipid body, etc., can be observed in conidia of the wild type, but only a few contents were found in most conidia of the ΔMoubp14 mutant (Figure 2E). These results indicated that deletion of MoUBP14 affects conidiation, conidiophore formation, conidia morphology and conidial deposit formation.

To investigate whether the deletion of MoUBP14 could affect pathogenicity, susceptible seedlings (O. sativa cv. LTH) at fifth leaf stage were inoculated by spraying conidia suspension. Compared with the wild type and complementation strains which caused numerous typical necrotic lesions, the ΔMoubp14 mutant was completely non-pathogenic, (Figure 3A). A similar result can be also observed on the barley leaves infected by the same strains mentioned above (Figure 3B).

When inoculating the mycelial agar plugs onto the wounded rice leaves, the wild type and complementation strains can spread well and cause disease symptoms, but the ΔMoubp14 mutant spread much slower (Figure 3C). This result indicated that deletion of MoUBP14 affects host colonization. We also compared major infection steps of the ΔMoubp14 mutant with those of the wild-type strain by observing conidia adhesion, conidia germination, appressoria formation, and host penetration and invasive growth on barley leaves as shown in below.

To determine conidia germination and appressorium formation ability of the ΔMoubp4 mutant, we observed the germination process. Conidia harvested from 10-day-old OMA culture plates were inoculated on hydrophobic coverslips. Nearly 85% conidia of ΔMoubp14 can form appressoria at 24 hpi, close to that of the wild type and cUBP14. However, the germination and appressorium formation processes of the mutant were evidently delayed (Figures 4A,B). At 4 hpi, compared with the wild type, most of the ΔMoubp14 conidia formed long germ tubes, only 21% of ΔMoubp14 conidia formed appressoria, and most of which were immature. At 12 hpi, only 65% of ΔMoubp14 conidia formed appressoria, compared with 91% of the wild type (Figure 4B). In order to determine whether the ΔMoubp14 mutant formed dysfunctional appressorium, we carried out the cytorrhysis assay. When treated with different concentrations of PEG8000, appressoria formed by the ΔMoubp14 mutant were collapsed more easily than those of the wild type and the complementation strain (Figure 4C), suggesting turgor pressure in the mutant was significantly reduced. TEM observation also showed that the content of the ΔMoubp14 mutant was severely reduced, and the melanin layer was light-colored than the wild type, suggesting it formed a looser melanin layer (Figure 4D). Taken together, these results demonstrated that disruption of MoUBP14 affects conidium germination and appressorium maturation during infection of M. oryzae.

To further reveal why deletion of the MoUBP14 resulted in loss of pathogenicity, we observed infection process in barley epidermal cells. We found that appressoria formed by the ΔMoubp14 mutant were severely blocked in penetration and invasive growth in host cell (Figures 3D,E). At 24 hpi, more than 90% of the wild-type appressoria penetrated into the plant cells and 64% of them developed branched infection hyphae (IH). In contrast, seldom ΔMoubp14 appressoria formed primary IH. At 30 hpi, 82% of the wild-type IH formed branched IH, whereas it was only 7% in the ΔMoubp14 mutant (Figures 3D,E). The wild type and cUBP14 strains can well penetrate and colonize in the invaded host cells (Figures 3D,E). These results indicate that deletion of MoUBP14 significantly affects appressorium-mediated penetration and invasive growth in M. oryzae.

Next, we evaluated the effect of MoUBP14 disruption on stress tolerance in M. oryzae. The results revealed that the ΔMoubp14 mutant was more sensitive to a series of stresses, including the cell wall perturbing reagents [0.1 mg/ml Calcofluor White (CFW), 0.2 mg/ml Congo Red (CR) and 0.005% Sodium dodecyl sulfate (SDS)], the osmotic stress (0.5 M NaCl and 1 M Sorbitol), alkaline pH (pH 8.0), oxidative stress (10 mM H₂O₂), and high temperature (32°C) (Figures 5A–C). Notably, the mutant exhibited severe sensitivity to high temperature (32°C), but at the same time, the wild type and complementation strain were slightly affected. These data indicated that MoUBP14 is required for response to kinds of stresses.

We also tested whether the disruption of MoUBP14 can affect carbon source utilization. The wild-type, ΔMoubp14 mutant and complementation strains were cultured on MM plates supplemented with each of glucose, NaAc, ethanol and glycerol as the sole carbon source. After 5 days culture at 28°C, colony diameter of the ΔMoubp14 mutant was significantly reduced when grown on all of these conditions compared with itself growth on the CM plates. The mutant exhibited reduction of 39, 59, 56 and 48% in glucose, NaAc, ethanol, and glycerol conditions, respectively. In contrast, the reduction rates of the wild-type and the complementation strain were only around 6% in these carbon sources compared with themselves in the CM condition (Figures 6A,B). These demonstrated that the carbon sources utilization ability of the ΔMoubp14 mutant was significantly reduced, suggested that MoUBP14 is also relevant to carbon source utilization.

Since the conidial storage provides nutrient utilization during the appressorium-mediated infection, we next detected the cellular distribution of glycogen and lipid. I₂/KI solution was used to stain the glycogen. In wild type, the glycogen was abundant in conidia, along with the appressorium formation, the glycogen was translocated from conidia to nascent appressoria, and rapidly reduced in conidia and appressoria after 8 h. Then the glycogen was nearly disappeared in the mature appressoria after 12 h. In the ΔMoubp14 mutant, glycogen was disappeared slowly in the conidia and appressoria, and noticable amount of glycogen can be still detected at 18 h, indicating the glycogen was not well utilized in the appressoria (Figure 7A). We also detected the lipid droplets utilization during appressorium formation by using Nile Red staining. In wild type, lipid droplets were also abundant in conidia, and rapidly transported to the incipient appressorium for degradation, and nearly diappeared at 12 h. In the ΔMoubp14 mutant, degradation of the lipid droplets was slower than that of the wild type, and a large amount of lipid droplets can be still detected at 24 h in the conidia and germ tubes (Figure 7B). These results indicated deletion of MoUBP14 affects glycogen and lipid metabolism, which is important for conidia storage utilization and functional appressorium formation in M. oryzae.

For the ΔMoubp14 mutant was blocked in host cell growth, we explored whether the ΔMoubp14 mutant resulted in host ROS accumulation. We detected ROS accumulation in the barley epidermis cells by staining with 3,3′-diaminobenzidine (DAB) at 30 hpi. Most of the barley epidermis cells infected by the ΔMoubp14 mutant were intensely stained by DAB, which were detected with abundant reddish brown precipitates around the appressoria in the infected cells, while the host cells infected by the wild-type strain were not well stained (Supplementary Figure S5), indicating host ROS was accumulated in ΔMoubp14 infected cells.

In order to investigate localization of MoUbp14, the eGFP:MoUBP14 fusion construct was obtained and transformed into the ΔMoubp14 mutant, and the subsequent transformants were recovered in the development and pathogenicity. One of these transformants, termed Ubp14-GFP, was used for observing the subcellular localization. Conidial suspension of Ubp14-GFP was dropped on the hydrophobic cover glass and barley epidermis for fluorescence microscopic observation. GFP signals were highly expressed and detected in cytoplasm at different development stages (Supplementary Figure S6), suggesting MoUBP14 is required in all development and infection processes of M. oryzae.

In S. cerevisiae, Ubp14 protein functions as a deubiquitinating enzyme involved in removing ubiquitin from protein substrates (D’Andrea and Pellman, 1998). Thus, we compared the total ubiquitination levels of the wild type, the ΔMoubp14 mutant and the complementation strain cUBP14. Total proteins were extracted from mycelia incubated in liquid CM medium. Western blot analysis was performed and ubiquitination levels were detected with an anti-ubiquitin antibody. Compared with that of the wild type and the complementation strain, the total ubiquitination level of the ΔMoubp14 mutant was evidently increased. Notably, the monoubiquitin and polyubiquitin bands of Ub₁, Ub₂, Ub₃, and Ub₄ were massively accumulated in the ΔMoubp14 mutant (Figure 8A), indicating that loss of MoUBP14 blocked general protein degradation and chain turnover, which is consistent with previous observation in S. cerevisiae (Eisele et al., 2006).

We also analyzed proteins that were pulled down by Ubp14-GFP in the affinity purification analysis for identifying potential Ubp14 substrates. After filtering the background interacting proteins using the wild-type strain expressing GFP protein, a total of 31 proteins linked to Ubp14:GFP were identified in two experimental replicates (Supplementary Table S1). Interestingly, some proteins involved in carbohydrate metabolism, stress response, and two E3 ligases ptr1 and hulA, were identified as the Ubp14-interacting proteins (Supplementary Table S1), which is consistent with the roles of Ubp14 in development, nutrient utilization, stress response, and pathogenesis in M. oryzae.

Among the MoUbp14 interacting proteins, MoFbp1 and MoPck1 are two key rate-limiting enzymes in gluconeogenesis. To validate whether MoUbp14 is required for degradation of MoFbp1 and MoPck1, the constructs expressing the Fbp1:Flag and Pck1:Flag fusion proteins were introduced into the wild-type strain and the ΔMoubp14 mutant, respectively. Total proteins from the subsequent strains were purified and immuno-blotted with anti-Flag antibody. As expected, beside Fbp1:Flag protein itself, larger bands can be detected in proteins extracted from both of the wild type and the ΔMoubp14 mutant expressing Fbp1:Flag, however, more protein bands with massive products can be detected in the ΔMoubp14 mutant (Figure 8B). Similar phenomenon can be also found for the MoPck1 protein (Figure 8B). These results suggested that in the ΔMoubp14 mutant, ubiquitination levels of MoFbp1 and MoPck1 could be elevated.

In S. cerevisiae, when glucose is added into cells grown for around 10 h on potassium acetate as the non-fermentable carbon source, Fbp1 suffers rapid catabolite degradation by the ubiquitin proteasome pathway (UPS) (Hung et al., 2004). In order to clarify whether MoFbp1 and MoPck1 are degraded by the UPS pathway, we incubated the wild type and the ΔMoubp14 mutant expressing Fbp1:Flag or Pck1:Flag firstly in CM medium for 42 h, and then transferred the mycelia into the liquid medium with NaAc as the sole carbon source for 12 h, and subsequently add glucose into the medium for incubating for 2 h. Total proteins from the samples were extracted and immuno-blotted by anti-Flag antibody. For both of the wild type expressing Fbp1:Flag and Pck1:Flag, when adding glucose for 2 h, protein amounts of the target proteins were significantly reduced, but some larger bands were detected or increased in amounts, compared with that of 0 h. In contrast, for the ΔMoubp14 mutant expressing Fbp1:Flag or Pck1:Flag, protein amounts of the target proteins were significantly increased, as well as many larger bands (Figure 8C). This suggested that in the ΔMoubp14 mutant, both of the MoFbp1 and MoPck1 can’t suffer rapid catabolite degradation, perhaps by the result of failing in ubiquitin recycle. These results also suggested when the glucose was supplied to M. oryzae grown in low glucose media for a short period of time (∼12 h), both of the MoFbp1 and MoPck1 are degraded. This process could require MoUbp14.