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. 2016 Sep 20;18(8):1075–1088. doi: 10.1111/mpp.12461

The regulatory factor X protein MoRfx1 is required for development and pathogenicity in the rice blast fungus Magnaporthe oryzae

Dandan Sun 1,†, Huijuan Cao 1,2,†, Yongkai Shi 1,†, Pengyun Huang 1, Bo Dong 3, Xiaohong Liu 2, Fucheng Lin 2, Jianping Lu 1,✉
PMCID: PMC6638216  PMID: 27434465

Summary

Magnaporthe oryzae is a cereal pathogen causing 20%–30% rice yield losses. Regulatory factor X transcription factors are highly conserved proteins with diverse functions among organisms. Here, we show that MoRfx1 is required for cell division, development and pathogenicity in M. oryzae. Deletion of MoRFX1 resulted in reduced growth and conidiation, decreased appressorium turgor and impaired virulence. ΔMorfx1 displayed increased sensitivity to UV light, four DNA‐damaging agents and three cell wall‐perturbing compounds. However, ΔMorfx1 showed decreased sensitivity to bleomycin, a DNA/cell wall‐damaging agent, and increased chitin content of the cell wall in vegetative mycelium. In addition, cell division speed was reduced in ΔMorfx1, and ΔMorfx1 did not produce three‐celled conidia. RNA‐sequencing and quantitative polymerase chain reaction analyses suggested that MoRfx1 has bipartite functions in the control of the expression of genes required for cell division and chitin metabolism, not only as a transcriptional repressor, but also as a transcriptional activator. In particular, the expression of chitin deacetylase genes MoCDA2 and MoCDA1 was greatly down‐regulated in ΔMorfx1, and deletion of MoCDA2 and MoCDA1, similar to ΔMorfx1, increased resistance to bleomycin. Taken together, our results indicate that MoRFX1 regulates development and pathogenicity by modulating the expression of genes involved in cell division and cell wall integrity.

Keywords: cell division, conidiation, Magnaporthe oryzae, pathogenicity, regulatory factor X, rice blast, transcription factor

Introduction

Magnaporthe oryzae is a major cereal pathogen that causes rice blast disease. This disease frequently leads to 20%–30% yield losses and can even cause complete crop loss during regional epidemics (Dean et al., 2012; Ou, 1980). Conidiogenesis is an important developmental process during the initiation of host infection and the spread of blast disease. Magnaporthe oryzae initiates its infection by releasing a three‐celled conidium, which lands and adheres to the rice surface, and then germinates and forms an appressorium with high turgor pressure (Howard et al., 1991; Talbot, 2003; Tucker and Talbot, 2001). The appressorium subsequently penetrates into the cuticle through a penetration peg that quickly develops into invasive hyphae. Cell division and cell wall integrity affect conidiation, appressorial differentiation and the pathogenicity of M. oryzae (Dagdas et al., 2012; Jeon et al., 2008; Kong et al., 2012; Saunders et al., 2010).

Many studies have reported that transcription factors (TFs) play important roles in the development and pathogenicity of M. oryzae (Chung et al., 2013; Guo et al., 2010, 2011; Liu et al., 2010; Mehrabi et al., 2008; Nishimura et al., 2009; Qi et al., 2012; Zhou et al., 2011), and the functional characterization of TFs has been accelerated by systematic gene deletion analyses in fungi (Colot et al., 2006; Kim et al., 2009; Kong et al., 2014; Son et al., 2011; Tang et al., 2014). Recently, we attempted to delete TF genes systematically in M. oryzae, and reported the functions of 104 Zn2Cys6 and 47 Cys2‐His2 TF genes in development and pathogenicity (Cao et al., 2016; Lu et al., 2014). In this trial, we also identified a TF deletion mutant (05B3‐1), which was reduced in virulence on barley excised leaves (Fig. S1, see Supporting Information). In the 05B3‐1 mutant, MGG_04000.7 encoding a regulatory factor X (RFX) DNA‐binding domain protein, namely MoRFX1, was deleted. RFX family TFs, first detected in mammals, are highly conserved, site‐specific, DNA‐binding proteins with diverse functions (Reith et al., 1990, 1994). In yeast and other fungi, RFX proteins have been reported to be major transcriptional repressors of DNA damage‐regulated genes, and essential for normal cell morphology and nuclear division (Bugeja et al., 2010; Hao et al., 2009; Huang et al., 1998; Min et al., 2014; Woolstencroft et al., 2006; Wu and McLeod, 1995); however, the functions of RFX proteins as transcriptional activators has not received sufficient attention.

In this study, we characterized the functions of MoRFX1, and found that MoRfx1 regulates cell division, development, appressorial formation and pathogenicity by bipartite functions, not only as a transcriptional repressor, but also as a transcriptional activator, in affecting the transcription of genes involved in cell division and chitin metabolism in M. oryzae.

Results

MoRFX1 is expressed in the life cycle of M. oryzae

MoRFX1 encodes a nuclear protein, as suggested by the co‐localization of MoRfx1‐GFP (green fluorescent protein) fusion proteins with H2B‐mCherry at hyphal nuclei in M. oryzae (Fig. 1A). The transcript levels of MoRFX1 during development and infection processes were assayed using quantitative polymerase chain reaction (qPCR). The expression of MoRFX1 was detected in eight tested developmental stages, including mycelium, spores, germinating spores, appressorium and invasive hyphae (Fig. 1B). The strongest MoRFX1 expression was observed in vegetative mycelia suffering nutritional deficiency. This expression profile implies that MoRfx1 functions in all developmental and infection stages.

Figure 1.

Figure 1

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Localization and expression pattern of MoRfx1 in Magnaporthe oryzae. (A) Co‐localization of MoRfx1‐GFP and H2B‐mCherry fusion proteins in the hyphal cells of the wild‐type. Bar, 10 μm. (B) Expression of MoRFX1 in eight developmental stages of the wild‐type: VH, mycelia grown in liquid complete medium (CM); VH‐S, mycelia grown in liquid CM and then cultured in H2O for 4 h; VH‐D, mycelia grown on CM plates in the dark; VH‐L, mycelia grown on CM plates under continuous light; CO, conidia; AP‐4h, appressoria at 4 h post‐inoculation (hpi); AP‐18h, appressoria at 18 hpi; IH, invasive hyphae in barley at 2 days post‐inoculation (dpi).

MoRfx1 is involved in the development of M. oryzae

To investigate the function of MoRfx1 in fungal development, we examined the mycelial growth, conidiation, conidial germination and appressorium formation in ΔMorfx1 (Fig. S2, see Supporting Information). ΔMorfx1 grew slowly (78.8% of the wild‐type) and produced fewer conidia (13.5% of the wild‐type) on complete medium (CM) (Fig. 2A–C). The aerial mycelia of ΔMorfx1 were whiter in colour than those of the wild‐type (Fig. 2A). The conidial germination and formation of appressoria were also reduced and delayed in the mutant relative to the wild‐type (Fig. 2D,E). However, MoRFX1 was not required for sexual reproduction in M. oryzae (Fig. S3, see Supporting Information). The rescued strain Morfx1‐c (Fig. S2D) showed recovery of the defects of ΔMorfx1 in mycelial growth, conidial germination and appressorial formation, and almost complete restoration of the defect in aerial hyphal differentiation and conidiation (Fig. 2).

Figure 2.

Figure 2

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Phenotypic analyses of ΔMorfx1. (A) Colonies of Magnaporthe oryzae strains grown on complete medium (CM). (B) Diameter of colonies of M. oryzae strains grown on CM medium. (C) Conidiation of M. oryzae strains. ΔMorfx1, the MoRFX1‐rescued strain Morfx1‐c and the wild‐type were cultured at 25 °C for 8 days (A–C). (D) Conidial germination rate of M. oryzae strains. Conidial suspensions were dropped onto plastic coverslips and incubated at 28 °C for 4 h. (E) Appressorial formation rate of M. oryzae strains. Conidial suspensions were dropped onto plastic coverslips and incubated at 28 °C for 24 h. Error bars represent standard deviation. The same capital letters above the columns indicate non‐significant differences as estimated by Duncan's test (P < 0.05).

MoRfx1 is required for cell division during conidiogenesis and vegetative growth in M. oryzae

We carefully examined conidiophore development and conidial morphology in ΔMorfx1. ΔMorfx1 differentiated fewer conidiophores in the aerial mycelia and produced fewer conidia on a conidiophore than the wild‐type or the MoRFX1‐rescued strain (Fig. 3A). More interestingly, ΔMorfx1 produced one‐celled conidia (50.4% ± 2.2%) and two‐celled conidia (49.6% ± 2.2%), but did not produce normal three‐celled conidia (Fig. 3A–C). The conidia of ΔMorfx1 were shorter in length (17.0 ± 3.5 μm) than those of the wild‐type (20.4 ± 3.2 μm) and the MoRFX1‐rescued strain (20.5 ± 3.1 μm). ΔMorfx1 conidia were slightly wider (9.2 ± 1.5 μm) than those of the wild‐type (8.4 ± 1.0 μm) and the MoRFX1‐rescued strain (8.4 ± 0.8 μm) (Fig. 3D). These results suggest that MoRFX1 is involved in conidial differentiation in M. oryzae, and that the function of MoRFX1 is particularly critical for the second and third cell divisions of conidiogenesis.

Figure 3.

Figure 3

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Conidial development assays of the wild‐type, ΔMorfx1 and Morfx1‐c (MoRFX1‐rescued) strains. (A) Conidiophores of Magnaporthe oryzae strains. Bar, 50 μm. (B) The conidial shape of M. oryzae strains. The conidia cell wall was stained with calcofluor white (CFW). (C) Percentage of conidia with one cell, two cells and three cells in M. oryzae strains. ΔMorfx1 failed to produce normal, three‐celled pyriform conidia. More than 450 conidia were counted for each strain. (D) Size of conidia in M. oryzae strains. More than 400 conidia were measured for each strain. Error bars represent standard deviation. The same capital letters above the columns indicate non‐significant differences in conidial length or width as estimated by Duncan's test (P < 0.05).

The septa and nuclei of the conidia and hyphae of M. oryzae were observed under a microscope (Figs 4A and S4, see Supporting Information). The septa in conidia and hyphae appeared similar between ΔMorfx1 and the wild‐type (Figs 3B, 4A and S4). Nuclei in most conidia and hyphae of ΔMorfx1 also appeared similar to those of the wild‐type, in which every conidial and hyphal cell had one nucleus with a normal shape (Figs S4 and 4A). However, a few micronuclei were visible in some of the ΔMorfx1 hyphal cells; no such micronuclei were observed in wild‐type cells (Fig. 4A). When treated with 5 μg/mL bleomycin, the hyphal nucleus and septum formation were not affected in the wild‐type, whereas cell division in ΔMorfx1 was notably impaired: zero or several nuclei/micronuclei appeared in 56.5% ± 3.3% of hyphal cells (Fig. 4A).

Figure 4.

Figure 4

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Nucleus, septum formation and cell division in hyphae. (A) Nucleus and septum formation in hyphae in wild‐type and ΔMorfx1. Histone H2B was tagged with red fluorescent protein (mCherry) to visualize nuclei. The cell wall was strained with calcofluor white (CFW). DIC, differential interference contrast. Nuclei & septa, overlays of CFW staining (cell wall) and red fluorescent protein (nuclei) images. Wild‐type + BLM and ΔMorfx1 + BLM, hyphae grown with 5 μg/mL bleomycin. Bar, 10 μm. Arrowheads indicate micronuclei; arrows indicate septa; asterisk indicates a cell without a nucleus. Cell number (B) and length of hyphae (C) initially generated from conidia. Conidia were inoculated on glass slides in complete medium (CM) and cultured at 28 °C for 3, 5 and 7 h, and the hyphae of 80–120 conidia were measured. (D) Size of hyphal cells in the mycelium of Magnaporthe oryzae strains. More than 400 cells were measured for each strain. Error bars represent standard deviation. The same capital letters above the columns indicate non‐significant differences between strains as estimated by Duncan's test (P < 0.01).

The cell division speed of M. oryzae strains was evaluated by counting the cell number of hyphae initially formed by conidia in CM at 3, 5 and 7 h post‐inoculation (hpi). At 3 hpi, the germ tubes of ΔMorfx1 and the wild‐type produced one‐celled hyphae; at 5 hpi, the wild‐type generated two‐celled hyphae, whereas the hyphae of ΔMorfx1 still contained one cell; at 7 hpi, the hyphae of ΔMorfx1 contained a fewer number of cells than those of the wild‐type (Fig. 4B). The length of hyphae formed by conidia before 5 hpi in ΔMorfx1 was slightly, but significantly, shorter than those in the wild‐type (P < 0.05); however, at 7 hpi, there were no significant differences in hyphal length between the two strains (Fig. 4C). Therefore, the cell division speed is reduced and the hyphal cell length is increased in ΔMorfx1. We then measured the size of vegetative hyphal cells in ΔMorfx1 and the wild‐type grown in liquid CM (Fig. 4D). The hyphal cell length (52.8 ± 21.8 μm) and width (2.3 ± 0.4 μm) in ΔMorfx1 were significantly larger than those in the wild‐type (41.3 ± 12.8 μm in length and 2.0 ± 0.3 μm in width) (P < 0.01). The rescued strain Morfx1‐c (48.4 ± 16.0 μm in length and 2.2 ± 0.3 μm in width of vegetative hyphal cells) showed part, but significant, downsizing of the oversized hyphal cells in ΔMorfx1 (P < 0.01). Therefore, the defects in cell division of ΔMorfx1, but not in cell size, probably reduce mycelial colony growth.

MoRFX1 deletion strain is sensitive to DNA damage treatments

To assess the role of MoRfx1 in response to DNA damage in M. oryzae, we examined the germination rate of conidia exposed to UV light (254 nm, 180 J/m2) and five DNA‐damaging agents, including hydrogen peroxide, hydroxyurea, methyl methanesulfonate, cisplatin and bleomycin (Fig. 5). The inhibition ratio of the germination rate of ΔMorfx1 conidia under UV exposure (22.3% ± 6.9%) was significantly increased relative to that of the wild‐type (10.0% ± 4.6%) and the MoRFX1‐rescued strain (6.0% ± 2.2%) (P < 0.05), indicating that the deletion of MoRFX1 led to increased sensitivity to DNA damage caused by UV light (Fig. 5A). The inhibition ratio of the germination rate of ΔMorfx1 conidia was also increased when exposed to 2 mM hydrogen peroxide, 200 mM hydroxyurea, 0.02% (v/v) methyl methanesulfonate and 0.1 mM cisplatin, indicating that the deletion of MoRFX1 led to increased sensitivity to DNA damage caused by these DNA‐damaging agents (Fig. 5B). However, ΔMorfx1 displayed reduced sensitivity to bleomycin when compared with the wild‐type and rescued strain (Fig. 5B). At 20 μg/mL bleomycin, 28.8% ± 1.5% of ΔMorfx1 conidia germinated, compared with only 4.4% ± 1.9% of the wild‐type and 2.0% ± 0.4% of the rescued strain. At 10 μg/mL bleomycin, the conidial germination rates were 60.3% ± 3.1%, 45.5% ± 0.2% and 46.1% ± 7.7% for ΔMorfx1, the wild‐type and the rescued strain, respectively.

Figure 5.

Figure 5

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Responses of Magnaporthe oryzae strains to DNA‐damaging and cell wall‐interfering agents. (A) Germination rates of conidia spread on agar plates and exposed to UV light (254 nm, 180 J/m2). (B) Germination inhibition rates of conidia spread on agar plates containing various DNA‐damaging agents: 2 mM hydrogen peroxide (H2O2), 200 mM hydroxyurea (HU), 0.02% methyl methanesulfonate (MMS), 0.1 mM cisplatin (CDDP), 10 μg/mL bleomycin (BLM‐10) and 20 μg/mL bleomycin (BLM‐20). (C) Growth inhibition rates of the strains cultured on minimal medium (MM) plates containing 10 mM H2O2, 20 mM HU, 0.02% MMS or 30 μg/mL BLM. (D) Colony of ΔMorfx1 cultured on stress medium containing 10 mM H2O2. Bar, 5 mm. Control, negative control without exposure to UV light (A, D). (E) The chitin content was assayed in 1‐day‐old vegetative mycelia cultured in liquid complete medium (CM) and 4‐day‐old sporogenous mycelia cultured on CM plates of the wild‐type and ΔMorfx1. The chitin content is evaluated as micrograms of glucosamine hydrochloride per milligram dry weight of fungal biomass. (F) Germination inhibition rates of conidia spread on agar plates containing 200 μg/mL calcofluor white (CFW), 50 μg/mL Congo red (CR) or 50 μg/mL sodium dodecylsulfate (SDS). Germination of conidia was observed under a microscope at 18 h post‐inoculation (hpi) (A, B, F). (G) Growth inhibition rates of strains cultured on MM plates containing 200 μg/mL CFW, 100 μg/mL CR or 100 μg/mL SDS. The M. oryzae strains are the wild‐type, ΔMorfx1 and Morfx1‐c (MoRFX1‐rescued strain). Error bars represent standard deviation. The same capital letters above the columns indicate non‐significant differences between different strains estimated by Duncan's test (P < 0.05).

The effects on the growth of ΔMorfx1 of methyl methanesulfonate, hydroxyurea, bleomycin and hydrogen peroxide were also evaluated (Fig. 5C). The inhibition rates of growth of ΔMorfx1 were increased when exposed to methyl methanesulfonate and hydroxyurea, and reduced when exposed to bleomycin, suggesting that the deletion of MoRFX1 led to increased sensitivity to methyl methanesulfonate and hydroxyurea, but reduced sensitivity to bleomycin. These results were in agreement with those of the conidial germination assays. However, the inhibition rate of growth of ΔMorfx1 when exposed to hydrogen peroxide was less than that of the wild‐type, and this result seemed to be different from that in conidial germination assays. We then carefully observed the mycelial colony of ΔMorfx1 exposed to hydrogen peroxide, and found that the colony of ΔMorfx1 consisted of sparse hyphae adhered on the medium surface with differentiation of a few aerial hyphae, which was different from the dense aerial hyphae observed in the wild‐type colony (Fig. 5D). This difference in aerial hyphae between the wild‐type and ΔMorfx1 implied that the differentiation of aerial hyphae in ΔMorfx1 was sensitive to hydrogen peroxide. These observations showed that the deletion of MoRFX1 led to sensitivity to DNA‐damaging treatments by UV light, hydrogen peroxide, hydroxyurea, methyl methanesulfonate and cisplatin, but resistance to treatment with bleomycin.

MoRfx1 is involved in chitin metabolism and cell wall integrity of M. oryzae

To evaluate the effect on chitin metabolism of MoRfx1, we measured the chitin content of vegetative and sporogenous mycelia of ΔMorfx1 using a previously described method (Chen and Johnson, 1983). In comparison with the wild‐type, ΔMorfx1 showed increased chitin content in vegetative mycelia, but no obvious change in chitin content of sporogenous mycelia (Fig. 5E). We further assessed the role of MoRfx1 in cell wall integrity by examining the germination rate of conidia and the growth rate of mycelia of the wild‐type and ΔMorfx1 exposed to Congo red (CR), calcofluor white (CFW) and sodium dodecylsulfate (SDS), which interfere with the formation and stress response of the cell wall (de Groot et al., 2001; Ram and Klis, 2006). When exposed to 200 μg/mL CFW, 50 μg/mL CR or 50 μg/mL SDS, the conidial germination of ΔMorfx1 was inhibited more severely than that of the wild‐type and the rescued strain (Fig. 5F). The inhibition rate of mycelial growth of ΔMorfx1 caused by 200 μg/mL CFW or 100 μg/mL CR was significantly, but mildly, greater than that of the wild‐type and the rescued strain. However, the inhibition of the mycelial growth of ΔMorfx1 by 100 μg/mL SDS was mildly less than that of the wild‐type and the rescued strain (Fig. 5G).

MoRfx1 is required for the pathogenicity of M. oryzae

The virulence of M. oryzae strains was tested by spraying a conidial suspension (1 × 105 spores/mL) on rice seedlings and observing disease symptoms after 7 days. Plants inoculated with ΔMorfx1 showed mild leaf lesions sparsely distributed on leaves, whereas plants of the wild‐type and the MoRFX1‐rescued strain showed densely distributed merged leaf lesions (Fig. 6A). The disease lesion area in a 5‐cm section of leaf was also calculated for statistical analysis. The disease lesion areas in leaves infected by ΔMorfx1 (12.6% ± 2.2%) were significantly less than those infected by the wild‐type and the MoRFX1‐rescued strain (48.4% ± 9.6% and 38.7% ± 4.5%, respectively) (P < 0.05). Consequently, ΔMorfx1 was reduced in virulence on rice relative to the wild‐type and MoRFX1‐rescued strain.

Figure 6.

Figure 6

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Pathogenicity‐related assays of Magnaporthe oryzae strains. Magnaporthe oryzae strains are the wild‐type, ΔMorfx1 and Morfx1‐c (MoRFX1‐rescued strain). (A) Virulence assays on rice. Rice seedlings were sprayed with conidial suspensions (1 × 105 conidia/mL) and cultured for 7 days. (B) Penetration rate (%) of M. oryzae appressoria on barley leaves; 20 μL of conidial suspension (5 × 104 conidia/mL) was inoculated on barley leaf explants (intact or abraded) and incubated for 24 h at 25 °C. (C) The barley leaf explants were inoculated with 20 μL of conidial suspension (5 × 104 conidia/mL) and cultured for 4 days. (D) Appressorium turgor was evaluated by incipient cytorrhysis analysis. Appressoria were allowed to form on plastic coverslips for 48 h, and the ratio of collapsed appressoria exposed to a series of glycerol solutions of various strengths was calculated from the recorded data. Error bars represent standard deviation; the same capital letters above the columns indicate non‐significant differences estimated by Duncan's test between M. oryzae strains (P < 0.05) (B, D). (E) Invasive growth of M. oryzae. Barley leaf explants were inoculated with 20 μL of conidial suspension (5 × 104 conidia/mL) and cultured for 24 and 48 h. The arrow indicates invasive hyphae; the triangle indicates an appressorium. Bar, 20 μm.

To explore the possible causes of the reduced virulence of ΔMorfx1, we examined the penetration ratios of appressoria into the barley cuticle. As shown in Fig. 6B, the penetration rate of ΔMorfx1 was significantly lower than that of the wild‐type and the MoRFX1‐rescued strain when inoculated on intact barley leaves at 24 hpi. As appressoria of M. oryzae penetrate the plant cuticle via mechanical force driven by huge turgor pressure (Howard et al., 1991; deJong et al., 1997), we further observed the penetration ability of ΔMorfx1 on cuticle abraded with a fine grade of abrasive paper. We found that the penetration rate of ΔMorfx1 increased significantly when inoculated on abraded barley leaves (Fig. 6B). As a result, ΔMorfx1 caused more severe lesions on abraded leaves than on intact leaves at 96 hpi (Fig. 6C). We then evaluated the turgor pressure of appressoria by measuring the ratio of collapsed appressoria in a series of glycerol solutions. The ratio of collapsed appressoria in ΔMorfx1 was greater than that of the wild‐type and the MoRFX1‐rescued strain for 0.5, 1 and 2 M glycerol solution treatments (Fig. 6D). We also examined the invasive growth of ΔMorfx1 on barley leaves, and found that the mutant's invasive hyphae lengthened at a slightly slower rate, and branched less frequently, than did those of the wild‐type and the MoRFX1‐rescued strain at 24 and 48 hpi (Fig. 6E). These results suggest that the weakened penetration ability of appressoria of the mutant is primarily caused by reduced appressorial turgor pressure, and that this is at least partly responsible for the impaired virulence observed for ΔMorfx1.

MoRFX1‐dependent genes in mycelium

As MoRFX1 was expressed at the highest observed levels in the vegetative mycelial samples suffering nutritional deficiency (Fig. 1B), we analysed the transcriptomes of the vegetative mycelia of ΔMorfx1 and the wild‐type cultured in water for 4 h with RNA‐sequencing (RNA‐seq). Transcripts of 10 864 and 10 864 genes were detected in the mycelia of ΔMorfx1 and the wild‐type, respectively. There were 1735 differentially expressed genes (DEGs) [false discovery rate (FDR) < 0.05, log2(fold change) > 0.4] (13.5% of all annotated genes in M. oryzae) between the two strains (Table S1, see Supporting Information). Among 1735 DEGs, 867 genes were up‐regulated and 868 genes were down‐regulated in ΔMorfx1 (Table S1). Putative changes of metabolic pathways in ΔMorfx1 were evaluated by assaying the functions of the mutant's DEGs using gene ontology (GO) enrichment analysis (Alexa et al., 2006). The expression of genes in many metabolic pathways was altered in ΔMorfx1 (Table S2, see Supporting Information). Several enriched GO terms implied that MoRfx1 is required for translation (GO:0043039, GO:0006399 and GO:0006412), response to DNA damage (GO:0006281 and GO:0006302), nucleotide metabolism (GO:0009259 and GO:0009117) and microtubule motor activity (GO:0003777) in M. oryzae (Table S2).

To assess the results revealed by RNA‐seq, the expression levels of 32 selected DEGs (Table S3, see Supporting Information), which are involved in DNA repair, cell division, transcription and cell wall formation in ΔMorfx1, were evaluated by qPCR in vegetative mycelia cultured in water for 4 h, and the significant changes in the transcript levels of 23 genes confirmed by qPCR were coincident with those in RNA‐seq (Fig. 7). We also measured the expression of 32 DEGs (Table S3) in sporogenous mycelia of ΔMorfx1 (Fig. 7). Six of the nine tested genes involved in DNA repair (MoKU70, MoMCM3, MoMCM4, MoMCM5, MoRAD51 and MoXLF) were significantly up‐regulated at the mRNA level in the vegetative mycelia of ΔMorfx1, whereas no significant changes in the transcript levels of the nine tested DNA repair genes were detected in the sporogenous mycelia (Fig. 7A). Three kinetochore Ndc80 complex genes (MoSPC25, MoNDC80 and MoNUF2) and four mitosis‐involved kinesin genes (MoKIP3, MoKAR3, MoKIF18A and MoKIF20A) were highly and significantly up‐regulated in the vegetative mycelia of ΔMorfx1 (Fig. 7A); however, only MoNDC80, MoNUF2 and MoKIF20A were significantly up‐regulated (albeit slightly) in sporogenous mycelia (Fig. 7A). Another kinesin gene, MoKLC4, was significantly down‐regulated in both the vegetative and sporogenous mycelia of ΔMorfx1 (Fig. 7A).

Figure 7.

Figure 7

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Fold changes in mRNA abundance in vegetative mycelia suffering nutritional deficiency and sporogenous mycelia of ΔMorfx1. (A) Relative fold changes of 18 genes involved in DNA repair and cell division. The transcript levels of eight transcription factor genes. (B) Relative fold changes of 18 genes involved in transcription and cell wall integrity. The relative transcript levels of target genes were analysed by RNA‐sequencing [false discovery rate (FDR) < 0.05, log2(fold change) > 0.4] or quantitative polymerase chain reaction (qPCR). The endogenous housekeeping gene β‐TUBULIN was used as the reference gene. Data are expressed as arbitrary units, where the transcript level of the wild‐type was set to unity. VG, vegetative mycelia suffering nutritional deficiency; SG, sporogenous mycelia; error bars represent standard deviation; asterisks above the columns indicate significant differences between the wild‐type and ΔMorfx1 estimated by Duncan's test (P < 0.05).

Of the eight tested TF genes required for conidiophore and conidium differentiation, four genes (COS1, MoLDB1, CNF1 and ACR1) were significantly down‐regulated in the vegetative mycelia of ΔMorfx1, and five genes (COS1, MoHOX2, CON7, CNF1 and ACR1) were significantly down‐regulated in the sporogenous mycelia (Fig. 7B), when compared with the wild‐type. Of the six cell wall‐related genes, CHS2, PAX1 and MoRHO4 were up‐regulated in the vegetative mycelia or sporogenous mycelia of ΔMorfx1, whereas MoCDA1, MoCDA2 and MoERG6 were down‐regulated in the vegetative or sporogenous mycelia of ΔMorfx1 (Fig. 7B).

Interestingly, MoCDA1 (MGG_14966.7) and MoCDA2 (MGG_08774.7), two chitin deacetylase genes, were greatly decreased in vegetative mycelia suffering nutritional deficiency (118.6‐fold and 1187.7‐fold, respectively) and sporogenous mycelia (6.3‐fold and 816.9‐fold, respectively) in ΔMorfx1 relative to the wild‐type. MoCDA2 is a highly expressed gene in vegetative and sporogenous mycelia of the wild‐type (Fig. S5, see Supporting Information). To determine the possible relation in biological function between MoRFX1 and MoCDA1 or MoCDA2, we deleted MoCDA1 and MoCDA2 in M. oryzae (Fig. S6, see Supporting Information). The growth of ΔMocda1, ΔMocda2 and the double‐gene deletion strain ΔMocda1ΔMocda2 was comparable with that of the wild‐type (Fig. S7A, see Supporting Information). ΔMocda1, ΔMocda2 and ΔMocda1ΔMocda2 displayed similar virulence on rice and barley to the wild‐type (Fig. S7B,C). However, ΔMocda2 and ΔMocda1ΔMocda2 produced significantly fewer conidia than the wild‐type (Fig. 8A). In addition, ΔMocda1, ΔMocda2 and ΔMocda1ΔMocda2 showed increased resistance to bleomycin and sensitivity to CR (Fig. 8B), similar to ΔMorfx1.

Figure 8.

Figure 8

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Characteristics of MoCDA1 and MoCDA2 deletion mutants. (A) The conidiation of Magnaporthe oryzae strains. The strains were cultured on complete medium (CM) plates at 25 °C for 8 days. (B) Germination inhibition rates of conidia spread on 1.5% agar plates containing 200 μg/mL calcofluor white (CFW), 50 μg/mL Congo red (CR) or 10 μg/mL bleomycin (BLM). Conidia were cultured at 28 °C for 18 h. Error bars represent standard deviation. The same capital letters above the columns indicate non‐significant differences between different strains estimated by Duncan's test (P < 0.05).

Discussion

RFX family TFs are highly conserved proteins in fungi and animals. However, the functions of Rfx TFs in various species are diverse. In animals, RFX TFs mediate the regulation of ciliary genes (Ashique et al., 2009) and major histocompatibility complex (MHC) class II genes (Reith et al., 1990, 1994). Yeast RFX1 is required for cell morphogenesis (Woolstencroft et al., 2006; Wu and McLeod, 1995). In Fusarium graminearum, FgRFX1 is involved in fungal growth, conidiation and virulence by regulating genome integrity (Min et al., 2014). In this study, we found that MoRFX1 in M. oryzae is involved in growth, conidial differentiation, appressorium maturation and pathogenicity.

Magnaporthe oryzae infects rice and barley through an appressorium that penetrates the plant cuticle using huge turgor pressure in excess of 8.0 MPa (Howard et al., 1991; deJong et al., 1997; Tucker and Talbot, 2001). On wounded leaves, M. oryzae conidia also infect plants via appressoria, but with less turgor pressure than on intact leaves. In ΔMorfx1, the turgor pressure was significantly reduced in appressoria when compared with the wild‐type. The penetration rate of the mutant appressoria on intact plant cuticles was decreased, but this rate almost returned to wild‐type levels on wounded leaves. ΔMorfx1 caused more severe lesions on wounded barley leaves than on intact leaves. Therefore, MoRfx1 is involved in plant penetration and virulence in M. oryzae by regulating the huge turgor pressure in appressoria.

In ΔMorfx1, the second cell septum formation of three‐celled conidia was blocked, the cell division of hyphae was delayed and hyphal cells were enlarged in size, suggesting that MoRfx1 is involved in cell division in M. oryzae. Chitin [(1‐4)‐β‐linked N‐acetylglucosamine] is a major structural cell wall component in ascomycetes, and most chitin is fully acetylated and associated with (1‐3)‐β‐/(1‐6)‐β‐glucan in an alkali‐insoluble complex (glucosaminoglycan) (Wessels, 1994). The chitin content in vegetative hyphal cells was increased in ΔMorfx1; ΔMorfx1 was sensitive to two chitin‐binding anionic dyes, CFW and CR, which inhibited the assembly of enzymes that connect chitin to (1‐3)‐β‐glucan and (1‐6)‐β‐glucan (Ram and Klis, 2006). Chitin synthase genes (CHSs) are involved in cell wall integrity, conidiation and pathogenicity in M. oryzae (Kong et al., 2012; Odenbach et al., 2007). Δchs2 displayed defects in growth, conidiation and cell wall integrity in M. oryzae (Kong et al., 2012). Pax1 is involved in cell wall integrity in M. oryzae (Li et al., 2014), and Rho4 is involved in cell wall integrity and the regulation of septum degradation in yeast (Santos et al., 2003). CON7 is required for conidial morphology, cell wall and chitin content in germinated spores (Cao et al., 2016; Odenbach et al., 2007). ACR1, CNF1, COS1 and MoHOX2 are TF genes controlling conidial differentiation in M. oryzae (Lau and Hamer, 1998; Lu et al., 2014; Kim et al., 2009; Zhou et al., 2009). In ΔMorfx1, TF genes ACR1, CNF1, CON7, COS1 and MoHOX2 were significantly down‐regulated in sporogenous mycelia, whereas cell wall‐related genes CHS2, PAX1 and MoRHO4 were up‐regulated in sporogenous mycelia, suggesting that the defects in conidial differentiation in ΔMorfx1 are probably caused by the comprehensive effect of the alteration in the expression level of many conidiation‐related genes.

Chitin deacetylase genes MoCDA1 and MoCDA2 were greatly down‐regulated in ΔMorfx1. Deletion of MoCDA2, and double deletion of MoCDA1 and MoCDA2, led to reduced conidial production in M. oryzae. The increased resistance to bleomycin and sensitivity to CR shown by ΔMorfx1, and its downstream gene deletion mutants ΔMocda1, ΔMocda2 and ΔMocda1ΔMocda2, implied that MoRfx1 probably regulates cell wall structure by affecting the expression of MoCDA1 and MoCDA2. Interestingly, the FgRFX1 deletion mutant is sensitive to bleomycin in F. graminearum (Min et al., 2014). Bleomycin, a DNA‐damaging agent, plays a role not only through the induction of DNA strand breaks (Hecht, 2000), but also through the blockage of fungal septum formation and cytokinesis by destroying cell wall components via an oxidative mechanism (Beaudouin et al., 1993; Lim et al., 1995; Moore et al., 2003). In F. graminearum, bleomycin inhibits hyphal septum formation and produces micronuclei, and bleomycin‐treated hyphae show many micronuclei and are largely aseptate, as in ΔFgrfx1 (Min et al., 2014). However, in M. oryzae, bleomycin does not produce micronuclei and does not inhibit hyphal septum formation in the wild‐type, but interferes with nuclear division and cytoplasmic division in ΔMorfx1. These diverse responses to bleomycin are probably caused by the differences in cell wall components and Rfx1‐dependent genes between M. oryzae and F. graminearum.

Generally, Rfxs have been found to be major transcriptional repressors of DNA repair genes in fungi (Bugeja et al., 2010; Hao et al., 2009; Huang et al., 1998; Min et al., 2014; Woolstencroft et al., 2006; Wu and McLeod, 1995), such as 95% Rfx1‐responsive genes up‐regulated in the FgRFX1 deletion mutant of F. graminearum. In M. oryzae, MoRFX1 is also required for the fungus to endure DNA‐damaging treatments by UV light, hydrogen peroxide, hydroxyurea, methyl methanesulfonate and cisplatin. However, 50.0% of Rfx1‐responsive genes were up‐regulated and 50.0% were down‐regulated in the MoRFX1 deletion mutant, suggesting that MoRfx1 is not only a transcriptional repressor, but also a transcriptional activator, in M. oryzae. As an example, MoRfx1 is a transcriptional repressor of many DNA repair genes and cell division genes, such as MoNUF2 and MoKIF20A, and is also a transcriptional activator of many cell wall‐related genes and conidiation‐related TF genes, such as MoCDA1 and MoCDA2, in M. oryzae.

In summary, this study demonstrates that TF MoRfx1 is required for growth, conidial differentiation, plant penetration and pathogenicity in M. oryzae, and MoRfx1 regulates the cell division in hyphal growth and conidial differentiation, at least partly, by affecting the expression of genes involved in DNA repair, cell division and chitin metabolism. Abnormal cell division and cell wall structure cause multiple defects in development, virulence and DNA damage responses in M. oryzae.

Experimental Procedures

Strains and nucleic acid manipulation

Magnaporthe oryzae wild‐type strain 70‐15, its null mutants ΔMorfx1, ΔMocda1, ΔMocda2, ΔMocda1ΔMocda2, the complementation strain Morfx1‐c, the wild‐type expressing both MoRFX1‐GFP and H2B‐mCherry, and the wild‐type and ΔMorfx1 expressing H2B‐mCherry were used in this study.

The target genes were deleted in M. oryzae using a method described previously (Lu et al., 2014), employing pKO1B and the primer sets listed in Table S4 (see Supporting Information). The sulfonylurea resistance gene (SUR) (to delete MoRFX1 and MoCDA2) and hygromycin B phosphotransferase gene (HPH) (to delete MoCDA1) were used as selective marker genes. The deleted MoRFX1 gene in ΔMorfx1 was complemented by introducing a native copy of MoRFX1, which was cloned in pKO1B‐HPH from genomic DNA of the wild‐type using the 05B3ComF/05B3ComR primer pair (Table S4), into the mutant via Agrobacterium tumefaciens‐mediated transformation (ATMT).

Magnaporthe oryzae strains with nuclei marked by the H2B‐mCherry fusion protein were constructed as follows. The GFP fragment was amplified from pEGFP (Clontech, Mountain View, CA, USA) with the P8GFPf/P8GFPr primer pair (Table S4) and cloned into the SmaI/XbaI sites of pKD7‐RED (Li et al., 2012), which contains the DsRED2 gene and the neomycin phosphotransferase II gene (NEO), using an In‐Fusion® HD Cloning Kit (Clontech) to produce the pKD7‐GFP vector. Then, pKD9‐GFP was constructed using HPH to replace NEO at the XhoI/EcoRI sites of pKD7‐GFP. GFP in pKD9‐GFP was replaced by mCherry amplified from pmCherry (Clontech) using the p9mcherryF/p9mcherryR primer pair (Table S4) to produce pKD9‐mCherry. Finally, the H2B gene was cloned from wild‐type genomic DNA using the P9H2B‐F/P9H2B‐R primer pair (Table S4) and inserted into the SmaI site of pKD9‐mCherry. pKD9‐H2B‐mCherry was transformed into both the wild‐type and ΔMorfx1 via ATMT.

To localize MoRfx1 in cells, the coding sequence of MoRFX1 was cloned from the wild‐type genomic DNA and inserted into the SmaI site of pKD8‐GFP (Li et al., 2012), and then transformed into the wild‐type strain which expresses H2B‐mCherry.

Phenotypic analysis of M. oryzae strains

The developmental characteristics of M. oryzae strains were analysed according to previously described methods (Lu et al., 2014). Each experiment was repeated three times, with either three or five replicates. Five‐millimetre mycelial blocks were inoculated on CM plates, and cultured at 25 °C under continuous fluorescent light. At 8 days, the diameter of the colonies was measured for colony growth, and the conidia in each plate were collected and counted for conidiation. Conidiophore development was monitored as described previously (Lau and Hamer, 1998). For conidial germination and appressorium formation assays, 40 μL of spore suspension (1 × 105 conidia/mL) was inoculated on a plastic coverslip and incubated in a moist chamber at 28 °C in the dark. The conidial germination rate or appressorium formation rate of 200–300 conidia was evaluated at 4 or 24 hpi.

The mating experiments between the M. oryzae strains 70‐15 or ΔMorfx1 and the opposite mating type strain Guy11 were performed according to a previously described method (Lu et al., 2009). Mycelium blocks of 70‐15, ΔMorfx1 and Guy11 were placed on an OMA medium (30 g oat in 1 L H2O) plate at about 5 cm apart from one another, and incubated at 25 °C for about 1 week until their colonies merged, and then further incubated at 22 °C under continuous light for 4 weeks.

The turgor pressure of appressoria was evaluated by incipient cytorrhysis (cell collapse) with a previously described procedure (Lu et al., 2009). Appressoria formed on plastic coverslips at 48 hpi were exposed to a series of glycerol solutions (0.5, 1, 2, 3 and 4 M), and the number of collapsed appressoria was recorded after 10 min of incubation in glycerol solution (n = 3, 200–300 appressoria per experiment).

DNA damage responses during conidial germination under stress were assessed as follows. Conidia were spread on 1.5% agar plates and immediately exposed to UV light (254 nm, 180 J/m2), or conidia were spread on agar plates containing different DNA‐damaging agents or wall‐perturbing compounds, and incubated at 28 °C in the dark. The germination of conidia was examined with a microscope at 18 hpi (n = 3, 200–300 conidia per experiment). The conidial germination inhibition rate was calculated as follows: conidial germination inhibition rate = (percentage of germinated conidia on agar plates – percentage of germinated conidia on stress agar plates)/percentage of germinated conidia on agar plates × 100.

The effects on fungal growth by DNA‐damaging agents and wall‐perturbing compounds were also analysed on CM plates containing DNA‐damaging agents, by inoculation with 5‐mm mycelial blocks of M. oryzae strains, followed by incubation at 28 °C in the dark for 7 days (n = 7). The growth inhibition rate was calculated as follows: growth inhibition rate = (colony diameter on CM – colony diameter on stress medium)/colony diameter on CM × 100. Each experiment was repeated three times.

Pathogenicity assay of M. oryzae strains

The virulence of the M. oryzae strains was assayed following previously reported protocols (Lu et al., 2009). Four millilitres of conidial suspension (1 × 105 conidia/mL) containing 0.2% (w/v) gelatin were sprayed onto rice seedlings (Oryza sativa cv. CO39) on three or four leaves using an artist's airbrush. Inoculated seedlings were placed in a wet, dark box at 25 ºC for 2 days, and then grown in a wet box with a photoperiod of 12 h using fluorescent light for 5 days. For virulence assays with leaf explants of barley (Hordeum vulgare) or rice, 5‐mm mycelial blocks from the strains were inoculated on leaves and incubated in a humid box at 25 °C for 4 days. Plant penetration by the appressoria of M. oryzae strains was examined at 24 and 48 hpi on leaf explants of barley inoculated with a droplet of conidia (5 × 104 conidia/mL) (Lu et al., 2009). Infected leaves were treated with methanol to remove chlorophyll, fixed in alcoholic lactophenol (95% alcohol–lactophenol = 2 : 1) and observed using a microscope.

Chitin content analysis

The chitin content of vegetative and sporogenous mycelia was determined by measuring the amount of glucosamine released by acid hydrolysis of the fungal cell wall according to reported procedures (Chen and Johnson, 1983; Kong et al., 2012). Freshly harvested mycelia were ground in liquid nitrogen and suspended in 20 mL of distilled water. After centrifugation at 18 000 g for 10 min, the pellets were freeze‐dried overnight. For each 5 mg of dried pellet, 1 mL of 6 M HCl was added. After hydrolysis at 100 °C for 4 h, the hydrolysate was neutralized with 500 μL of 10 M NaOH and evaporated to dryness at 50 °C. The dry hydrolysates were then redissolved in 200 μL of distilled water saturated with chloroform, added to 250 μL of 4% acetylacetone in 1.25 M sodium carbonate and heated for 30 min at 100 °C. After the addition of 2 mL of ethanol and 0.25 mL of Ehrlich reagent (Chen and Johnson, 1983), the mixture was heated for 1 h at 60 °C and centrifuged at 18 000 g for 10 min. The supernatant was measured for absorbance at 530 nm with a spectrophotometer. The chitin content (milligrams of glucosamine hydrochloride per milligram of dry weight of fungal cell wall biomass) was calculated by comparing the absorbance of glucosamine hydrochloride with that of a standard curve established using known amounts of glucosamine hydrochloride (Sigma, St. Louis, MO, USA).

RNA‐seq and qPCR analysis

The RNA‐seq analysis of ΔMorfx1 was performed concurrently with that of the Zn2Cys6 TF gene deletion mutants Δgpf1 and Δcnf2, and the C2H2 TF gene deletion mutants Δvrf1 and Δvrf2, detailed in previous reports (Cao et al., 2016; Lu et al., 2014). The experiments were performed with biological triplicates. The M. oryzae strains were initially grown in liquid CM at 25 ºC with shaking at 180 rpm for 2 days, and then incubated in water for another 4 h. Total RNA was extracted with an RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA), and mRNA was isolated with AMPure XP beads (Beckman, Brea, CA, USA). RNA‐seq libraries were constructed with a NEBNext RNA sample preparation kit (NEB, Ipswich, MA, USA) in accordance with the standard low‐throughput protocol. Samples were sequenced on an Illumina Hiseq2500 instrument with the TruSeq PE Cluster Kit v3‐cBot‐HS (Illumina, San Diego, CA, USA) and TruSeq SBS Kit v3‐HS (Illumina). The clean reads were mapped to the M. oryzae genome database (MG8) (www.broadinstitute.org) with Tophat software (Trapnell et al., 2009). Significant differences in FPKM (fragments per kilobase of exon per million fragments mapped fragments) between ΔMorfx1 mycelia and wild‐type mycelia were analysed using the Cuffdiff component of the Cufflinks package (Trapnell et al., 2010). GO enrichment analyses for DEGs were performed using hypergeometric tests with topGO (topgo.bioinf.mpi‐inf.mpg.de), and P values were adjusted with Bonferroni correction for multiple testing (Alexa et al., 2006). We selected FDR < 0.05 as the criterion for ‘enrichment’ among the terms.

The results of RNA‐seq for the expression level of the selected genes were confirmed by qPCR in vegetative mycelia suffering nutritional deficiency and in sporogenous hyphae with five replicates. The samples of vegetative mycelia suffering nutritional deficiency were prepared as described above. The sporogenous hyphae were prepared as follows: M. oryzae strains were grown on CM for 12 days, conidia were collected, spread on cellophane paper over CM plates, and cultured at 25 °C under continuous fluorescent light for 4 days; finally, the total RNA of mycelia was extracted and evaluated using qPCR. The primers used in qPCR are listed in Table S4.

The RNA‐seq data were deposited at Gene Expression Omnibus (Accession number GSE65296) (www.ncbi.nlm.nih.gov/geo).

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's website:

Fig. S1 Virulence assays. Barley leaf explants were inoculated with mycelial blocks of the wild‐type and ΔMorfx1, and cultured for 4 days.

Click here for additional data file. (888.4KB, tif)

Fig. S2 Knockout and complementation of MoRFX1 in Magnaporthe oryzae. (A) MoRFX1 deletion event in ΔMorfx1 confirmed by negative double polymerase chain reaction (PCR) (top) and positive PCR (bottom). M, DNA marker; Wild‐type, M. oryzae wild‐type strain; a, β‐TUBULIN; b, MoRFX1; c, unique recombinational DNA fragment (a marker of gene replacement event). (B) Copies of sulfonylurea resistance gene (SUR) in the quantitative PCR (qPCR)‐identified mutants after comparison with the wild‐type strain. ‘Single’ represents the targeted gene deletion without ectopic insertion. ΔMorfx1 was identified as a null mutant based on the following criteria: the mutant grew on positive selection plates, but did not emit green fluorescent protein (GFP) fluorescence; MoRFX1 was not detected by negative double PCR, but a unique recombinant DNA fragment was detected by positive PCR; only a single copy of SUR appeared in the mutant's genome. (C) Knockout of MoRFX1 in ΔMorfx1 was reconfirmed by Southern blot. Genomic DNAs were digested with XhoI and separated on 0.8% agarose gels. The size of the band detected by the probe was changed from 4.4 kb in the wild‐type to 7.0 kb in ΔMorfx1, indicating that homologous recombination occurred at a single site. (D) Complementation of MoRFX1 in ΔMorfx1 confirmed by reverse transcription‐polymerase chain reaction (RT‐PCR). The mutant was complemented with a native copy of MoRFX1 from the wild‐type. Total RNA was extracted from mycelia grown on complete medium (CM). 35 RT‐PCR cycles were performed. Morfx1‐c, the complementation strain; d, MoRFX1; e, β‐TUBULIN.

Click here for additional data file. (1.6MB, tif)

Fig. S3 Mating experiments between ΔMorfx1 or strain 70‐15 with strain Guy11 on OMA medium (30 g oat in 1 L H2O) at 22 °C. Magnaporthe oryzae wild‐type strain 70‐15 (MAT1‐1) and ΔMorfx1 were crossed with the opposite mating type strain Guy11 (MAT1‐2) on OMA medium at 22 °C for 5 weeks. Numerous perithecia were observed at the junctions between mated individuals.

Click here for additional data file. (2MB, tif)

Fig. S4 Nucleus and septum formation in conidia. Histone H2B was tagged with red fluorescent protein (mCherry) to visualize nuclei. DIC, differential interference contrast; Bar, 10 μm.

Click here for additional data file. (600.4KB, tif)

Fig. S5 Relative mRNA abundance of MoCDA1 and MoCDA2 in the wild‐type strain 70‐15. The endogenous housekeeping gene β‐TUBULIN was used as the reference gene. Error bars represent standard deviation.

Click here for additional data file. (88.3KB, tif)

Fig. S6 Knockout of MoCDA1 and MoCDA2 in Magnaporthe oryzae. (A) Gene deletion events in two transformants of ΔMocda1 (strains C1‐1 and C1‐4), ΔMocda2 (strains C2‐4 and C2‐7) and ΔMocda1ΔMocda2 (strains C1/2‐1 and C1/2‐2, in which MoCDA1 was deleted in ΔMocda2) confirmed by negative double polymerase chain reaction (PCR) (top) and positive PCR (bottom). M, DNA marker; a, β‐TUBULIN; b, MoCDA1 or MoCDA2; c, unique recombinational DNA fragments (a marker of gene replacement event). (B) Copies of inserted resistant gene [sulfonylurea resistance gene (SUR) or hygromycin B phosphotransferase gene (HPH)] in the quantitative PCR‐identified mutants after comparison with the wild‐type strain. ‘Single’ represents targeted gene deletion without ectopic insertion.

Click here for additional data file. (1.2MB, tif)

Fig. S7 Growth and virulence of MoCDA1 and MoCDA2 deletion mutants. (A) Diameter of colonies of Magnaporthe oryzae strains grown on complete medium (CM). The strains were cultured at 25 °C for 8 days. Error bars represent standard deviation. The same capital letters above the columns indicate non‐significant differences between different strains estimated by Duncan's test (P < 0.05). Barley leaf explants (B) and rice leaf explants (C) were inoculated with 5‐mm mycelial blocks of the wild‐type, ΔMocda1, ΔMocda2 and ΔMocda1ΔMocda2, and cultured for 4 days.

Click here for additional data file. (4.2MB, tif)

Table S1 Differentially expressed genes in ΔMorfx1 relative to the wild‐type strain [false discovery rate (FDR) < 0.05].

Click here for additional data file. (141.3KB, xlsx)

Table S2 Gene ontology (GO) terms enriched in ΔMorfx1 [false discovery rate (FDR) < 0.05].

Click here for additional data file. (33KB, xlsx)

Table S3 Thirty‐two selected genes for quantitative polymerase chain reaction (qPCR) analysis in Magnaporthe oryzae samples of vegetative mycelia suffering nutritional deficiency and sporogenous mycelia.

Click here for additional data file. (14KB, xlsx)

Table S4 Primers used in this study.

Click here for additional data file. (14.8KB, xlsx)

Acknowledgements

We thank Hangzhou Guhe Information and Technology Co., Ltd. for help in sequencing and bioinformatics analysis, and Dr John Hugh Snyder for critical reading of the manuscript. This study was supported by the National Basic Research Program of China (Grant Nos: 2012CB114002 and 2014CB541702) and the National Natural Science Foundation of China (Grant No: 31371891). The authors have no conflicts of interest to declare.

References

  1. Alexa, A. , Rahnenfuhrer, J. and Lengauer, T. (2006) Improved scoring of functional groups from gene expression data by decorrelating GO graph structure. Bioinformatics, 22, 1600–1607. [DOI] [PubMed] [Google Scholar]
  2. Ashique, A.M. , Choe, Y. , Karlen, M. , May, S.R. , Phamluong, K. , Solloway, M.J. Ericson, J. and Peterson, A.S. (2009) The Rfx4 transcription factor modulates Shh signaling by regional control of ciliogenesis. Sci. Signal, 2, ra70. [DOI] [PubMed] [Google Scholar]
  3. Beaudouin, R. , Lim, S.T. , Steide, J.A. , Powell, M. , McKoy, J. , Pramanik, A.J. , Johnson, E. , Moore, C.W. and Lipke, P.N. (1993) Bleomycin affects cell wall anchorage of mannoproteins in Saccharomyces cerevisiae . Antimicrob. Agents Chemother. 37, 1264–1269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bugeja, H.E. , Hynes, M.J. and Andrianopoulos, A. (2010) The RFX protein RfxA is an essential regulator of growth and morphogenesis in Penicillium marneffei . Eukaryot. Cell, 9, 578–591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cao, H. , Huang, P. , Zhang, L. , Shi, Y. , Sun, D. , Yan, Y. , Liu, X. , Dong, B. , Chen, G. , Snyder, J.H. , Lin, F. and Lu, J. (2016) Characterization of 47 Cys2‐His2 zinc finger proteins required for the development and pathogenicity of the rice blast fungus Magnaporthe oryzae . New Phytol. 211, 1035–1051. [DOI] [PubMed] [Google Scholar]
  6. Chen, G.C. and Johnson, B.R. (1983) Improved colorimetric determination of cell‐wall chitin in wood decay fungi. Appl. Environ. Microbiol. 46, 13–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chung, H. , Choi, J. , Park, S.Y. , Jeon, J. and Lee, Y.H. (2013) Two conidiation‐related Zn(II)2Cys6 transcription factor genes in the rice blast fungus. Fungal Genet. Biol. 61, 133–141. [DOI] [PubMed] [Google Scholar]
  8. Colot, H.V. , Park, G. , Turner, G.E. , Ringelberg, C. , Crew, C.M. , Litvinkova, L. , Weiss, R.L. , Borkovich, K.A. and Dunlap, J.C. (2006) A high‐throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc. Natl. Acad. Sci. USA, 103, 10 352–10 357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dagdas, Y.F. , Yoshino, K. , Dagdas, G. , Ryder, L.S. , Bielska, E. , Steinberg, G. and Talbot, N.J. (2012) Septin‐mediated plant cell invasion by the rice blast fungus, Magnaporthe oryzae . Science, 336, 1590–1595. [DOI] [PubMed] [Google Scholar]
  10. Dean, R. , Van Kan, J.A. , Pretorius, Z.A. , Hammond‐Kosack, K.E. , Di Pietro, A. , Spanu, P.D. , Rudd, J.J. , Dickman, M. , Kahmann, R. , Ellis, J. and Foster, G.D. (2012) The top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 13, 414–430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. de Groot, P.W. , Ruiz, C. , Vazquez de Aldana, C.R. , Duenas, E. , Cid, V.J. , Del Rey, F. , Rodríquez‐Peña, J.M. , Pérez, P. , Andel, A. , Caubín, J. , Arroyo, J. , García, J.C. , Gil, C. , Molina, M. , García, L.J. , Nombela, C. and Klis, F.M. (2001) A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae . Comp. Funct. Genomics, 2, 124–142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guo, M. , Guo, W. , Chen, Y. , Dong, S. , Zhang, X. , Zhang, H. , Song, W. , Wang, W. , Wang, Q. , Lv, R. , Zhang, Z. , Wang, Y. and Zheng, X. (2010) The basic leucine zipper transcription factor Moatf1 mediates oxidative stress responses and is necessary for full virulence of the rice blast fungus Magnaporthe oryzae . Mol. Plant–Microbe Interact. 23, 1053–1068. [DOI] [PubMed] [Google Scholar]
  13. Guo, M. , Chen, Y. , Du, Y. , Dong, Y. , Guo, W. , Zhai, S. , Zhang, H. , Dong, S. , Zhang, Z. , Wang, Y. , Wang, P. and Zheng, X. (2011) The bZIP transcription factor MoAP1 mediates the oxidative stress response and is critical for pathogenicity of the rice blast fungus Magnaporthe oryzae . PLoS Pathog. 7, e1001302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hao, B. , Clancy, C.J. , Cheng, S. , Raman, S.B. , Iczkowski, K.A. and Nguyen, M.H. (2009) Candida albicans RFX2 encodes a DNA binding protein involved in DNA damage responses, morphogenesis, and virulence. Eukaryot. Cell, 8, 627–639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hecht, S.M. (2000) Bleomycin: new perspectives on the mechanism of action. J. Nat. Prod. 63, 158–168. [DOI] [PubMed] [Google Scholar]
  16. Howard, R.J. , Ferrari, M.A. , Roach, D.H. and Money, N.P. (1991) Penetration of hard substrates by a fungus employing enormous turgor pressures. Proc. Natl. Acad. Sci. USA, 88, 11 281–11 284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huang, M. , Zhou, Z. and Elledge, S.J. (1998) The DNA replication and damage checkpoint pathways induce transcription by inhibition of the Crt1 repressor. Cell, 94, 595–605. [DOI] [PubMed] [Google Scholar]
  18. Jeon, J. , Goh, J. , Yoo, S. , Chi, M.H. , Choi, J. , Rho, H.S. , Park, J. , Han, S.S. , Kim, B.R. , Park, S.Y. , Kim, S. and Lee, Y.H. (2008) A putative MAP kinase kinase kinase, MCK1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae . Mol. Plant–Microbe Interact. 21, 525–534. [DOI] [PubMed] [Google Scholar]
  19. deJong, J.C. , McCormack, B.J. , Smirnoff, N. and Talbot, N.J. (1997) Glycerol generates turgor in rice blast. Nature, 389, 244–245. [Google Scholar]
  20. Kim, S. , Park, S.Y. , Kim, K.S. , Rho, H.S. , Chi, M.H. , Choi, J. , Park, J. , Kong, S. , Park, J. , Goh, J. and Lee, Y.H. (2009) Homeobox transcription factors are required for conidiation and appressorium development in the rice blast fungus Magnaporthe oryzae . PLoS Genet. 5, e1000757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kong, L.A. , Yang, J. , Li, G.T. , Qi, L.L. , Zhang, Y.J. , Wang, C.F. , Zhao, W.S. , Xu, J.R. and Peng, Y.L. (2012) Different chitin synthase genes are required for various developmental and plant infection processes in the rice blast fungus Magnaporthe oryzae . PLoS Pathog. 8, e1002526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kong, S. , Park, S.Y. and Lee, Y.H. (2014) Systematic characterization of the bZIP transcription factor gene family in the rice blast fungus, Magnaporthe oryzae . Environ. Microbiol. 17, 1425–1443. [DOI] [PubMed] [Google Scholar]
  23. Lau, G.W. and Hamer, J.E. (1998) Acropetal: a genetic locus required for conidiophore architecture and pathogenicity in the rice blast fungus. Fungal Genet. Biol. 24, 228–239. [DOI] [PubMed] [Google Scholar]
  24. Li, H.J. , Lu, J.P. , Liu, X.H. , Zhang, L.L. and Lin, F.C. (2012) Vectors building and usage for gene knockout, protein expression and fluorescent fusion protein in the rice blast fungus. J. Agric. Biotechnol. 20, 94–104. [Google Scholar]
  25. Li, Y. , Yue, X. , Que, Y. , Yan, X. , Ma, Z. , Talbot, N.J. and Wang, Z. (2014) Characterisation of four LIM protein‐encoding genes involved in infection‐related development and pathogenicity by the rice blast fungus Magnaporthe oryzae . PLoS One, 9, e88246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lim, S.T. , Jue, C.K. , Moore, C.W. and Lipke, P.N. (1995) Oxidative cell wall damage mediated by bleomycin‐Fe(II) in Saccharomyces cerevisiae . J. Bacteriol. 177, 3534–3539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Liu, W. , Xie, S. , Zhao, X. , Chen, X. , Zheng, W. , Lu, G. , Xu, J.R. and Wang, Z. (2010) A homeobox gene is essential for conidiogenesis of the rice blast fungus Magnaporthe oryzae . Mol. Plant–Microbe Interact. 23, 366–375. [DOI] [PubMed] [Google Scholar]
  28. Lu, J. , Cao, H. , Zhang, L. , Huang, P. and Lin, F. (2014) Systematic analysis of Zn2Cys6 transcription factors required for development and pathogenicity by high‐throughput gene knockout in the rice blast fungus. PLoS Pathog. 10, e1004432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lu, J.P. , Liu, X.H. , Feng, X.X. , Min, H. and Lin, F.C. (2009) An autophagy gene, MgATG5, is required for cell differentiation and pathogenesis in Magnaporthe oryzae . Curr. Genet. 55, 461–473. [DOI] [PubMed] [Google Scholar]
  30. Mehrabi, R. , Ding, S. and Xu, J.R. (2008) MADS‐box transcription factor mig1 is required for infectious growth in Magnaporthe grisea . Eukaryot. Cell, 7, 791–799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Min, K. , Son, H. , Lim, J.Y. , Choi, G.J. , Kim, J.C. , Harris, S.D. and Lee, Y.W. (2014) Transcription factor RFX1 is crucial for maintenance of genome integrity in Fusarium graminearum . Eukaryot. Cell, 13, 427–436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Moore, C.W. , McKoy, J. , Del Valle, R. , Armstrong, D. , Bernard, E.M. , Katz, N. and Gordon, R.E. (2003) Fungal cell wall septation and cytokinesis are inhibited by bleomycins. Antimicrob. Agents Chemother. 47, 3281–3289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nishimura, M. , Fukada, J. , Moriwaki, A. , Fujikawa, T. , Ohashi, M. , Hibi, T. and Hayashi, N. (2009) Mstu1, an APSES transcription factor, is required for appressorium‐mediated infection in Magnaporthe grisea . Biosci. Biotechnol. Biochem. 73, 1779–1786. [DOI] [PubMed] [Google Scholar]
  34. Odenbach, D. , Breth, B. , Thines, E. , Weber, R.W. , Anke, H. and Foster, A.J. (2007) The transcription factor Con7p is a central regulator of infection‐related morphogenesis in the rice blast fungus Magnaporthe grisea . Mol. Microbiol. 64, 293–307. [DOI] [PubMed] [Google Scholar]
  35. Ou, S.H. (1980) Pathogen variability and host resistance in rice blast disease. Annu. Rev. Phytopathol. 18, 167–187. [Google Scholar]
  36. Qi, Z. , Wang, Q. , Dou, X. , Wang, W. , Zhao, Q. , Lv, R. , Zhang, H. , Zheng, X. , Wang, P. and Zhang, Z. (2012) MoSwi6, an APSES family transcription factor, interacts with MoMps1 and is required for hyphal and conidial morphogenesis, appressorial function and pathogenicity of Magnaporthe oryzae . Mol. Plant Pathol. 13, 677–689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ram, A.F.J. and Klis, F.M. (2006) Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red. Nat. Protoc. 1, 2253–2256. [DOI] [PubMed] [Google Scholar]
  38. Reith, W. , Herrero‐Sanchez, C. , Kobr, M. , Silacci, P. , Berte, C. , Barras, E. , Fey, S. and Mach, B. (1990) MHC class II regulatory factor RFX has a novel DNA‐binding domain and a functionally independent dimerization domain. Genes Dev. 4, 1528–1540. [DOI] [PubMed] [Google Scholar]
  39. Reith, W. , Ucla, C. , Barras, E. , Gaud, A. , Durand, B. , Herrero‐Sanchez, C. , Kobr, M. and Mach B. (1994) RFX1, a transactivator of hepatitis B virus enhancer I, belongs to a novel family of homodimeric and heterodimeric DNA‐binding proteins. Mol. Cell Biol. 14, 1230–1244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Santos, B. , Gutierrez, J. , Calonge, T.M. and Perez, P. (2003) Novel Rho GTPase involved in cytokinesis and cell wall integrity in the fission yeast Schizosaccharomyces pombe . Eukaryot. Cell, 2, 521–533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Saunders, D.G. , Dagdas, Y.F. and Talbot, N.J. (2010) Spatial uncoupling of mitosis and cytokinesis during appressorium‐mediated plant infection by the rice blast fungus Magnaporthe oryzae . Plant Cell, 22, 2417–2428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Son, H. , Seo, Y.S. , Min, K. , Park, A.R. , Lee, J. , Jin, J.M. , Lin, Y. , Cao, P. , Hong, S.Y. , Kim, E.K. , Lee, S.H. , Cho, A. , Lee, S. , Kim, M.G. , Kim, Y. , Kim, J.E. , Kim, J.C. , Choi, G.J. , Yun, S.H. , Lim, J.Y. , Kim, M. , Lee, Y.H. , Choi, Y.D. and Lee, Y.W. (2011) A phenome‐based functional analysis of transcription factors in the cereal head blight fungus, Fusarium graminearum . PLoS Pathog. 7, e1002310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Talbot, N.J. (2003) On the trail of a cereal killer: exploring the biology of Magnaporthe grisea . Annu. Rev. Microbiol. 57, 177–202. [DOI] [PubMed] [Google Scholar]
  44. Tang, W. , Ru, Y. , Hong, L. , Zhu, Q. , Zuo, R. , Guo, X. , Wang, J. , Zhang, H. , Zheng, X. , Wang, P. and Zhang, Z. (2014) System‐wide characterization of bZIP transcription factor proteins involved in infection‐related morphogenesis of Magnaporthe oryzae . Environ. Microbiol. 17, 1377–1396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Trapnell, C. , Pachter, L. and Salzberg, S.L. (2009) TopHat: discovering splice junctions with RNA‐Seq. Bioinformatics, 25, 1105–1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Trapnell, C. , Williams, B.A. , Pertea, G. , Mortazavi, A. , Kwan, G. , van Baren, M.J. , Salzberg, S.L. , Wold, B.J. and Pachter, L. (2010) Transcript assembly and quantification by RNA‐Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tucker, S.L. and Talbot, N.J. (2001) Surface attachment and pre‐penetration stage development by plant pathogenic fungi. Annu. Rev. Phytopathol. 39, 385–417. [DOI] [PubMed] [Google Scholar]
  48. Wessels, J.G.H. (1994) Developmental regulation of fungal cell‐wall formation. Annu. Rev. Phytopathol. 32, 413–437. [Google Scholar]
  49. Woolstencroft, R.N. , Beilharz, T.H. , Cook, M.A. , Preiss, T. , Durocher, D. and Tyers, M. (2006) Ccr4 contributes to tolerance of replication stress through control of CRT1 mRNA poly(a) tail length. J. Cell Sci. 119, 5178–5192. [DOI] [PubMed] [Google Scholar]
  50. Wu, S.Y. and McLeod, M. (1995) The sak1+ gene of Schizosaccharomyces pombe encodes an RFX family DNA‐binding protein that positively regulates cyclic AMP‐dependent protein kinase‐mediated exit from the mitotic cell cycle. Mol. Cell Biol. 15, 1479–1488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhou, X. , Liu, W. , Wang, C. , Xu, Q. , Wang, Y. , Ding, S. and Xu . J.R. (2011) A MADS‐box transcription factor MoMcm1 is required for male fertility, microconidium production and virulence in Magnaporthe oryzae . Mol. Microbiol. 80, 33–53. [DOI] [PubMed] [Google Scholar]
  52. Zhou, Z. , Li, G. , Lin, C. and He, C. (2009) Conidiophore stalk‐less1 encodes a putative zinc‐finger protein involved in the early stage of conidiation and mycelial infection in Magnaporthe oryzae . Mol. Plant–Microbe Interact. 22, 402–410. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

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Fig. S1 Virulence assays. Barley leaf explants were inoculated with mycelial blocks of the wild‐type and ΔMorfx1, and cultured for 4 days.

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Fig. S2 Knockout and complementation of MoRFX1 in Magnaporthe oryzae. (A) MoRFX1 deletion event in ΔMorfx1 confirmed by negative double polymerase chain reaction (PCR) (top) and positive PCR (bottom). M, DNA marker; Wild‐type, M. oryzae wild‐type strain; a, β‐TUBULIN; b, MoRFX1; c, unique recombinational DNA fragment (a marker of gene replacement event). (B) Copies of sulfonylurea resistance gene (SUR) in the quantitative PCR (qPCR)‐identified mutants after comparison with the wild‐type strain. ‘Single’ represents the targeted gene deletion without ectopic insertion. ΔMorfx1 was identified as a null mutant based on the following criteria: the mutant grew on positive selection plates, but did not emit green fluorescent protein (GFP) fluorescence; MoRFX1 was not detected by negative double PCR, but a unique recombinant DNA fragment was detected by positive PCR; only a single copy of SUR appeared in the mutant's genome. (C) Knockout of MoRFX1 in ΔMorfx1 was reconfirmed by Southern blot. Genomic DNAs were digested with XhoI and separated on 0.8% agarose gels. The size of the band detected by the probe was changed from 4.4 kb in the wild‐type to 7.0 kb in ΔMorfx1, indicating that homologous recombination occurred at a single site. (D) Complementation of MoRFX1 in ΔMorfx1 confirmed by reverse transcription‐polymerase chain reaction (RT‐PCR). The mutant was complemented with a native copy of MoRFX1 from the wild‐type. Total RNA was extracted from mycelia grown on complete medium (CM). 35 RT‐PCR cycles were performed. Morfx1‐c, the complementation strain; d, MoRFX1; e, β‐TUBULIN.

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Fig. S3 Mating experiments between ΔMorfx1 or strain 70‐15 with strain Guy11 on OMA medium (30 g oat in 1 L H2O) at 22 °C. Magnaporthe oryzae wild‐type strain 70‐15 (MAT1‐1) and ΔMorfx1 were crossed with the opposite mating type strain Guy11 (MAT1‐2) on OMA medium at 22 °C for 5 weeks. Numerous perithecia were observed at the junctions between mated individuals.

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Fig. S4 Nucleus and septum formation in conidia. Histone H2B was tagged with red fluorescent protein (mCherry) to visualize nuclei. DIC, differential interference contrast; Bar, 10 μm.

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Fig. S5 Relative mRNA abundance of MoCDA1 and MoCDA2 in the wild‐type strain 70‐15. The endogenous housekeeping gene β‐TUBULIN was used as the reference gene. Error bars represent standard deviation.

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Fig. S6 Knockout of MoCDA1 and MoCDA2 in Magnaporthe oryzae. (A) Gene deletion events in two transformants of ΔMocda1 (strains C1‐1 and C1‐4), ΔMocda2 (strains C2‐4 and C2‐7) and ΔMocda1ΔMocda2 (strains C1/2‐1 and C1/2‐2, in which MoCDA1 was deleted in ΔMocda2) confirmed by negative double polymerase chain reaction (PCR) (top) and positive PCR (bottom). M, DNA marker; a, β‐TUBULIN; b, MoCDA1 or MoCDA2; c, unique recombinational DNA fragments (a marker of gene replacement event). (B) Copies of inserted resistant gene [sulfonylurea resistance gene (SUR) or hygromycin B phosphotransferase gene (HPH)] in the quantitative PCR‐identified mutants after comparison with the wild‐type strain. ‘Single’ represents targeted gene deletion without ectopic insertion.

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Fig. S7 Growth and virulence of MoCDA1 and MoCDA2 deletion mutants. (A) Diameter of colonies of Magnaporthe oryzae strains grown on complete medium (CM). The strains were cultured at 25 °C for 8 days. Error bars represent standard deviation. The same capital letters above the columns indicate non‐significant differences between different strains estimated by Duncan's test (P < 0.05). Barley leaf explants (B) and rice leaf explants (C) were inoculated with 5‐mm mycelial blocks of the wild‐type, ΔMocda1, ΔMocda2 and ΔMocda1ΔMocda2, and cultured for 4 days.

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Table S1 Differentially expressed genes in ΔMorfx1 relative to the wild‐type strain [false discovery rate (FDR) < 0.05].

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Table S2 Gene ontology (GO) terms enriched in ΔMorfx1 [false discovery rate (FDR) < 0.05].

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Table S3 Thirty‐two selected genes for quantitative polymerase chain reaction (qPCR) analysis in Magnaporthe oryzae samples of vegetative mycelia suffering nutritional deficiency and sporogenous mycelia.

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Table S4 Primers used in this study.

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