M. oryzae TPC1 mutants show defects in development and pathogenicity

In order to identify novel infection-related genes we screened a total of 300 T-DNA transformants for their ability to infect rice roots using a M. oryzae insertional library[30]. The M1422 mutant developed very restricted disease lesions on roots and was selected for further characterization (Fig 1A). On leaves, M1422 produced only a small number of resistant-type lesions (Fig 1B and S1A Fig). Colonies of M1422 were also compact and reduced in size, when compared with the wild-type (Fig 1C).

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Fig 1. M1422 mutant shows severe defects in pathogenicity and vegetative growth together with increase tolerance to nitrogen depletion, calcofluor white (CFW) and hyperosmotic stress.

(A) M1422 shows reduced disease symptoms on rice roots. Photographs were taken 15 days after inoculation. Wild-type (WT) strain Guy11 presents maximum lesion severity (score 3) in 95% of the infected roots compared to the M1422-infected seedlings that show mild (score 1–2) or null (score 0) necrotic symptoms on roots. Lesions were scored on a scale 0–3, based on colour intensity and extension of the necrotic lesion[30]. (B) M1422 is strongly impaired in its ability to infect rice leaves. (C) M1422 displays impaired ability to grow on complete medium (CM) and minimal medium (MM) depleted of carbon (MM-C) and nitrogen (MM-N). Relative to strain growth on MM, WT is more affected than M1422 on MM-N. (D) Sustained growth ratio difference in M1422 and WT strains on CM with 10 μg ml^-1 Congo Red (CR) and 10 μg ml^-1 CFW. Relative to growth on CM, M1422 shows stronger growth defects on CR compared to WT. (E) Colony images of CM plates with increased concentrations of sodium chloride (NaCl) (left). Diameter of WT and M1422 colonies measured at 10 dpi reveals increased tolerance to NaCl (right graph). All error bars represent the standard deviation of at least three independent biological replicas. All colony images were captured at 10 days post inoculation (dpi).

https://doi.org/10.1371/journal.ppat.1006516.g001

The insertion site of the T-DNA within M1422 was located 0.9 kb downstream of the start codon of locus MGG_01285 in the M. oryzae genome (S1B Fig). This gene encodes a putative transcription factor that belongs to a Zn(II)₂Cys₆ binuclear cluster family. We named this gene Transcription factor for Polarity Control 1 (TPC1). The predicted coding region of TPC1 is ~2.6 kb long and encodes 839 amino acids (http://fungi.ensembl.org/index.html; MG8). The TPC1 predicted amino acid sequence includes a putative nuclear localisation signal (NLS) and one Zn(II)₂Cys₆ binuclear cluster DNA binding domain (S1B Fig).

A single T-DNA insertion in M1422 genome was detected by Southern blot hybridisation using the hygromycin phosphotransferase gene as a probe (S1C Fig). We also generated a second mutant in TPC1 by targeted gene replacement (S2A and S2B Fig). We complemented both M1422 and Δtpc1 with a C-terminal TPC1:GFP gene fusion under control of its native promoter. The complemented mutants recovered normal mycelial growth, colonial morphology and full virulence on rice (S1D, S1E and S2C Figs). We conclude that mutants M1422 and Δtpc1 are impaired in TPC1 function.

Two striking characteristics of M1422 and Δtpc1 were their impaired hyphal growth and colony morphology (Fig 1C, S1E and S2C Figs). Vegetative growth of Tpc1-lacking strains was severely compromised in both complete (CM) and minimal (MM) medium (p < 0.01), and showed compact colonies and non-invasive colony morphology (S1E, S1F and S2D Figs). In Neurospora crassa, a class of mutants with polarity defects also exhibited this type of colony morphology[31].

To analyse integrity of the cell wall, development of M1422 was evaluated in the presence of the anionic dyes, Congo Red (CR) and Calcofluor White (CFW), which interfere with fungal cell wall assembly by binding to β-1,4-glucan and chitin, respectively [32]. Additive growth defects were observed on M1422 development in the presence of CR but not in CFW (Fig 1D). In addition, mycelial growth was affected by NaCl-induced hyperosmotic stress (Fig 1E). High concentrations of NaCl (0.6M - 1.0M) changed the growth ratio in colonial size between wild-type and M1422, leading to an increase in the relative growth rate of M1422 compared to wild-type. Therefore, the lack of Tpc1 affected plant virulence, vegetative growth, colony morphology and hyperosmotic stress adaptation.

M. oryzae TPC1 is required for conidiogenesis and appressorium development

We observed that M1422 and Δtpc1 mutants sporulated poorly compared to wild-type (Fig 2A and S2E Fig). In addition, M1422 asexual spores showed defects in septation (numbers of cell per conidium) and conidial morphology (Fig 2B). Wild-type conidia were uniformly pyriform, three-celled spores. By contrast, in M1422, although the majority of conidia were three-celled (80%), a significant percentage of two-celled conidia (17%), single-celled (2%) and four-celled conidia (1%) were observed. Up to 26% of spores showed abnormal morphology in contrast to wild-type where less than 4% were misshapen (n> 300). We also found that appressorium development was affected in M1422 (Fig 2C). On hydrophobic coverslips, wild-type conidia germinated to form one germ tube that emerged from the apical cell and formed an appressorium within 4h-8h (Fig 2C and 2D). In M1422, 40% of conidia germinated from two cells. This percentage increased to 50%-60% with extended incubation time (4h-8h). Formation of two appressoria was rarely observed in wild-type conidia (Fig 2E). We conclude that M1422 is impaired in the normal spatial patterning of appressorium development.

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Fig 2. M. oryzae TPC1 is required for conidiogenesis and infection-related development.

(A) M1422 mycelia produces less conidia per cm² than mycelia of wild-type (WT) strain on CM (mean±SD, n>300, three independent experiments). (B) Defects in the number of cells per conidium and conidia morphology of M1422. The M1422 mutant produces 1-, 2-, 3- (normal and abnormal morphology) and 4-celled conidia (respectively 1c, 2c, 3c and 4c in the panel) compared to the 3-celled conidia uniformly produced by the WT. Scale bar = 20 μm. (C) M1422 conidia inoculated onto glass coverslips showed an increased frequency of conidia germination from two cells. Scale bar = 10μm. (D) Quantification of M1422 defects in germination rate and development of germ tubes per conidia on coverslips. Two germ tubes germinated frequently from M1422 conidia. (E) Quantification of M1422 defects in the formation of infection-related structures; germ tubes (gt) and appressoria (app). M1422 conidia form at least one appressorium during infection-related development on coverslips (mean from >300 conidia; three biological repeats).

https://doi.org/10.1371/journal.ppat.1006516.g002

Polarity is coupled to autophagy and glycogen/lipid degradation in M. oryzae

The impairment of appressorium-mediated plant infection by TPC1 mutants suggested that it might play a critical role in penetration peg development[33]. Appressorium function is known to depend on autophagic cell death of conidia, prior to appressorium maturation[4,34]. Therefore, we investigated whether infection-associated autophagy proceeded normally and if conidia underwent autophagic cell death. A GFP:MoATG8 construct was introduced into M1422 to determine the spatial and temporal dynamics of autophagy (Fig 3A). MoATG8 encodes an autophagic, ubiquitin-like protein involved in autophagosome function and has been shown to be a reliable marker for autophagy[4,34]. Compared to the wild-type Guy11 (33.5±4.4), GFP:MoATG8-labeled autophagosomes accumulated in M1422 conidia in significantly smaller numbers (21.6±5.5; p<0.01). In both strains, the number of conidial autophagosomes decreased during germination, appressorium maturation and at the onset of spore cell death and was significantly lower in M1422 conidia and germ tubes (Fig 3A). However, autophagosome numbers increased significantly during appressorium maturation (8h; 16.1±4.9) and dropped considerably after conidial death (24h; 5.0±1.8) in wild-type, whereas autophagosome number remained relatively constant in M1422 during appressorium maturation (8.4±4.1) and even after conidial cell death (7.5±3.3).

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Fig 3. Infection-associated autophagy and glycogen/lipid degradation are impaired in Tpc1-lacking strains.

(A) Impaired cellular localisation of autophagosomes in M1422 during infection-related development. Epifluorescence images of wild-type (WT) Guy11 and M1422 transformants expressing GFP:MoATG8. Scale bar = 10 μm. Bar charts show mean autophagosome numbers and trend lines present in conidium, germ tube, and appressorium at the time points indicated for each strain (mean ± SD; three biological repeats). Asterisks indicate significant differences of the medians with p values of <0.01 (*), <0.001(**) or <0.0001 (***) using the Mann-Whitney (Wilcoxon) W-test. (B) Glycogen metabolism is delayed in the M1422 mutant. Conidia from Guy11 and M1422 inoculated onto glass coverslips were exposed to potassium iodide (KI) solution at 0h, 2h, 4h, 6h, 8h and 24h after inoculation. The KI solution stains glycogen within the conidia, but not simple sugars such as glucose and fructose. Scale bar = 10 μm. Bar charts show the relative percentage of stained appressoria with KI solution for each strain at 24h after inoculation (three biological replicas). (C) Lipid droplet mobilization during appressorium maturation visualised using Nile red staining samples and confocal laser scanning microscopy. Fluorescence signals of all images were captured using the same parameters. Δtpc1structures showing consistently higher fluorescence signals than the WT are evident at 9h and 12h. (D) In cytorrhysis assays with glycerol, Δtpc1 appressoria collapse quicker than WT appressoria, suggesting that glycerol concentration inside Δtpc1 appressoria is lower (mean±SD; three biological repeats; n>500).

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Appressorium development is accompanied by rapid degradation of glycogen from conidia during germination and from appressoria during turgor generation[35,36]. We therefore determined glycogen levels during appressorium development using potassium iodide (KI). Comparative analysis of KI staining between wild-type Guy11 and M1422 showed differences during the onset and later stages of conidial cell death (8h and 24h; Fig 3B). In Guy11 glycogen depletion was observed (no staining) within both conidial cells and appressoria during development. In the M1422 mutant, conidial cells were also depleted of glycogen although the appressorium still contained high levels of glycogen (95%) during maturation. We also looked at lipid metabolism, which is an additional driver of turgor generation in M. oryzae. The triacylglycerol lipase degrades lipid bodies that move to the appressorium during development[24,37]. Accordingly, we followed lipid body distribution during appressorium maturation in Δtpc1 using Nile red (Fig 3C). We consistently visualised delayed degradation of lipid bodies in conidia and germ tubes in Δtpc1, which were evident at 9h and 12h after germination on coverslips. Using a cytorrhysis assay, in which hyperosmotic concentrations of a solute are applied to collapse appressoria, we estimated the internal solute concentration and turgor of appressoria of the two strains. We observed that Δtpc1 appressoria clearly collapsed at higher rates than the WT at 1M concentration of glycerol (Fig 3D). This suggests decreased in turgor within Δtpc1 appressoria, consistent with the observed delayed degradation of glycogen and lipid bodies. When considered together, these observations point that autophagy, and glycogen/lipid metabolisms are delayed during appressorium development in Tpc1-lacking strains.

Tpc1 is required for re-establishment of polarity during appressorium-mediated plant infection

Following maturation of the appressorium, a penetration peg emerges from the appressorial pore to penetrate the plant cuticle and successfully colonise the plant host. To assess whether repolarization was impaired in the M1422 mutant, a penetration assay was performed on onion epidermis and rice leaf sheath (Fig 4A). After 24h, 91% of wild-type conidia formed an appressorium effectively, penetrated and invaded onion epidermal cells. The majority of M1422 conidia (60%) germinated and produced an appressorium, but failed to penetrate and invade onion cells. Only 40% of M1422 appressoria formed a penetration peg, but were not able to invade the onion epidermal cells and spread away from the point of penetration. Similarly, on rice leaf sheath preparations 81% of wild-type conidia penetrated successfully compared to 21% of Δtpc1 mutant spores, which managed to develop a penetration peg but hardly ever spread to adjacent cells.

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Fig 4. M. oryzae Tpc1 is required for re-establishment of polarity in appressoria.

(A) Tpc1-lacking mutants are impaired in appressorium-mediated penetration. After 24 hpi wild-type (WT) appressoria formed on surface of the onion strip and leaf sheath have penetrated the underlying epidermal cell and formed invasive hyphae (IH). M1422 and Δtpc1 show a defective invasion of onion epidermis and leaf sheath, respectively. Scale bar = 10 μm. Bar charts show the relative percentage of appressorial-penetration of onion epidermis and leaf sheath for each strain at 24 hpi (three biological replicas). (B) Micrographs of F-actin ring organization visualized by expression of gelsolin:GFP and Sep:GFP in Guy11 and Δtpc1 strains. The Δtpc1 mutant produces aberrant septin and actin rings; mis-localization of Sep5:GFP is more severe. The linescan graphs show fluorescence in a transverse section of individual appressoria. White arrowheads point appressorial pores.

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To examine how formation of the germ tube and penetration peg was compromised, we investigated cellular organization of the F-actin cytoskeleton[38], using the actin-binding protein fimbrin tagged with GFP (S3 Fig). Once wild-type conidia attached to the surface, fimbrin:GFP spots were observed at the periphery of the germinating cells (0h). However, conidia harvested from M1422 instead localised F-actin randomly at the periphery of the three cells of conidia and not preferentially in the germinating cell (white arrowheads, S3 Fig). The most clear mis-localisation defects were observed in mutant appressoria. Fimbrin was localised in discrete puncta at the periphery of Guy11 appressoria, but in contrast was dispersed within appressoria of the mutant (6h). Furthermore, the F-actin network was more diffuse and several pores were observed in M1422 mature appressoria (white arrowheads, 24h). These results suggest that re-polarization of the appressorium is adversely affected in the M1422 mutant. To confirm this, we also tracked gelsolin:GFP and Sep5:GFP in Δtpc1 mutant. The use of gelsolin:GFP and Sep5:GFP to follow actin reorganization has helped to understand cytoskeleton dynamics during infection-related development[6]. The disorganisation of the appressorial cytoskeleton and actin ring was evident in Δtpc1. Sep5 was mis-localised in all mutant appressoria and only 26% of mutant appressoria formed an intact actin ring with a central pore (Fig 4B). Consequently, TPC1 is required for the correct penetration peg emergence in M. oryzae.

Tpc1 is a nuclear protein regulated by the Pmk1 signalling cascade

The Tpc1:GFP fusion protein co-localised with histone H1:RFP in nuclei of vegetative hyphae, attached conidia (30 min), and germinated conidia (Fig 5A). Moreover, whenever Tpc1:GFP was observed in nuclei, GFP fluorescence was never observed in the cytoplasm or other organelles within conidia. The results are consistent with TPC1 encoding a transcription factor that acts within the nucleus during the initial stages of spore germination and appressorium development, and correlate with the observed overexpression of TPC1 in these fungal structures [29].

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Fig 5. Tpc1 shows steady state nuclear localisation and is regulated by the Pmk1 signalling pathway.

(A) Nuclear localisation of Tpc1:GFP during infection-related development and vegetative hyphae by confocal microscopy. Conidia and mycelium from a WT strain expressing both histone H1:RFP and TPC1:GFP protein fusions. (B) Confocal images of conidia harvested from M. oryzae Δpmk1, Δatg1 and Δatg8 mutants expressing TPC1:GFP. Tpc1 is not visualized in the nuclei of Δpmk1 mutant. (C) Yeast two-hybrid experiment showing Tpc1 interacts with Mst12 but not with Pmk1. Yeast colonies co-transformed simultaneously with the pGAD-TPC1 (bait Tpc1), and pGBK-Pmk1 and pGBK-Mst12 (prey Pmk1 and Mst12) vectors grow in high stringency media (-His/-Ade/-Leu/-Trp/+X-α-Gal) at specified concentrations: 1 x 10⁶, 1 x 10⁵, 1 x 10⁴, 1 x 10³, 1 x 10² cells, each a 10μl droplet. The interaction of proteins expressed by prey and bait vectors generates a blue-coloured colony due to the activation of α-galactosidase expression in the presence of X-α-Gal. Positive control cells contain pGBKT7-53 and pGADT7-T plasmids co-transformed into Y2HGold.

https://doi.org/10.1371/journal.ppat.1006516.g005

To investigate whether TPC1 is associated with specific or multiple regulatory networks, TPC1:GFP localisation was observed in conidia of different mutant backgrounds (Fig 5B). In the Δpmk1 MAPK mutant[39], Tpc1:GFP was observed within the cytoplasm but not in nuclei. By contrast, strong GFP fluorescence was visualised in conidial nuclei of the autophagy-defective Δatg1 and Δatg8 mutants, compared to the fluorescence observed in M1422 complemented with TPC1:GFP or Guy11 expressing TPC1:GFP. These results suggest that Tpc1 activity is associated with the Pmk1 MAP kinase signalling pathway, which regulates appressorium formation[39], and the control of autophagy[34].

We further analysed the link with between Tpc1 and the Pmk1 pathway by looking at the ability of Tpc1 to interact with components of this pathway in a yeast two-hybrid system. Strikingly, we observed that Tpc1 interacted with Mst12, a transcription factor that functions downstream of Pmk1[40], although Tpc1 did not interact with Pmk1 itself (Fig 5C). The mis-localisation of Tpc1 in Δpmk1 and its interaction with Mst12 strongly support Tpc1 involvement in this pathogenicity-associated MAPK signalling cascade.

Tpc1-like transcriptional regulators are found exclusively in filamentous fungi

We investigated the phylogenetic relationship of Tpc1 to other putative Magnaporthe Zn(II)₂Cys₆ proteins and the closest orthologues of Tpc1 in other fungal species (S4 and S5 Figs). We observed that the six cysteine residues of the DNA-binding domain (DBD) in the Zn(II)₂Cys₆ proteins were ordered in a conserved pattern, CX₂CX₆CX_5-12CX₂CX_6-8C (S4A and S4B Fig). In M. oryzae, the Zn(II)₂Cys₆ binuclear cluster family is diverse and composed of 175 members (S2 Table). The closest orthologues of Tpc1 (MGG_01285) were identified using BLASTP and used to construct a phylogenetic tree (S4C and S5 Figs). Tpc1 clustered in a group with sequences from other Sordariomycetes, such as Fusarium graminearum, N. crassa, Chaetomium globosum and Podospora anserina. Based on this tree, Tpc1 is a single copy gene and has not been subject to paralogous duplications. Our phylogenetic analysis reflected the diversification of the Zn(II)₂Cys₆-containing proteins in the fungal kingdom. Interestingly, we did not find putative homologues of Tpc1 in S. cerevisiae or Schizosaccharomyces pombe using a BLASTP search.

In F. graminearum, it is remarkable that only 16% (46/296) of the mutants lacking Zn(II)₂Cys₆ transcription factors showed a phenotype, compared to the 42% (30/72) of N. crassa mutants or the 59% (61/104) of M. oryzae mutants[28,41,42]. Among the F. graminearum mutants with clear phenotypes is found the orthologue of M. oryzae TPC1 (FgTPC1 = FGSG_08769; GzZC108), which is required for plant infection, perithecia formation, synthesis of mycotoxins (ZEA, zearalenone; and DON, deoxynivalenol) and growth[41]. Similar to M. oryzae (Fig 1E and S2F Fig), the Δfgtpc1 mutant is more resistant than wild-type to hyperosmotic and oxidative stresses.

We further investigated functional conservation of Tpc1 in the saprotrophic filamentous fungus N. crassa, and characterized a N. crassa NcTPC1 deletion mutant (Δnctpc1; NCU05996), obtained from the Fungal Genetic Stock Centre[43]. The analysis of the alignment of M. oryzae and N. crassa Tpc1 proteins showed that they share 67% amino acid identity (S6A Fig). Strikingly, the Δnctpc1 mutant was severely reduced in vegetative growth compared to an isogenic wild-type strain (p <0.01) (S6B Fig), and its vegetative hyphae also formed compacted colonies. In addition, we observed that the Δnctpc1 mutant of N. crassa was less severely affected when exposed to increasing osmotic stress using NaCl, compared with the N. crassa wild-type strain (p <0.01) (S6C Fig). Similar tolerance effect was also found in F. graminearum Tpc1[41] and in M. oryzae Tpc1 (Fig 1E). Consequently, N. crassa Tpc1 also plays a significant role in growth and development of the fungus and its responses to abiotic stress.

Oxidation-reduction processes are significantly affected in the Δtpc1 mutant

Tpc1 contains a Zn(II)₂Cys₆ binuclear cluster DNA binding domain, which is found only in fungal proteins considered bonafide transcription regulators[13,14]. We carried out a comparative transcriptome analysis using the wild-type strain and the TPC1 deletion mutant to identify the biological processes and genes regulated by Tpc1. For this experiment, RNA was extracted from fungal material grown on cellophane on top of CM agar plates (S2D Fig). We considered it to be an optimal condition since fungal hypha is able to penetrate the cellophane, i.e. a change in polar growth occurs under these conditions, and allow us to obtain enough amount of RNA for subsequent microarray analysis. We identified 215 down-regulated genes and 185 genes to be up-regulated with at least a two-fold change in expression level in the Δtpc1 mutant (S3 Table). We classified all the genes that were up- and down-regulated into four functional groups according to potential roles in signalling (13 genes), cell wall biosynthesis or modulation of plant response (secreted proteins; 140 genes), metabolism (127 genes) and other functions (54 genes). Sixty-six genes encoded proteins that lacked any known domain. Remarkably, two gene ontology (GO) terms were found significantly enriched among these differentially expressed genes, the oxidation-reduction process (GO:0055114; 57 genes; p<0.001) and the oxidoreductase activity (GO:0016491; 58 genes; p<0.001).

Within the signalling functional group, two down-regulated genes encoded phosphatidyl ethanolamine-binding proteins (PEBP) that have been shown to regulate protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) pathways[44,45]. Amongst the up-regulated genes, eight of them encoded transcriptional regulators, which suggests a link between the gene networks controlled by these transcriptional regulators and Tpc1.

The largest group of mis-regulated genes comprised 140 genes coding for secreted or cell wall-related proteins. Within this group, more than half of the members (74 genes) had no matches in databases. However, twenty-four genes were potentially involved in cell wall remodeling, and encompassed different types of glycosyl hydrolases (GH10, GH18, GH32, GH43, GH61 and GH81), seventeen proteases and two secreted phospholipases A2. Three Mas3/Gas1 paralogues and several effector proteins such as a Bas2-like, Bas113 and avrPi54 were also found[46,47,48,49]. The up-down regulation of two CFEM G-protein coupled receptors[50], including PTH11[51], suggested an alteration in the ability of the Δtpc1 mutant to perceive external signals.

The second largest group of genes with altered expression levels encoded proteins related with primary and secondary metabolism (127 genes). We found a significant number of them participating in oxidation-reduction processes (46%) and transport (9%). Alteration in nitrogen and glycerol metabolism was evidenced by the expression changes of four NmrA-like regulatory proteins[52], enzymes involved in amino acid biosynthesis, a glycerol kinase and the glycerol dehydrogenase Gcy1, an enzyme also associated with redox regulation in yeast[53]. Down-regulation of an α-glucosidase supported the glycogen degradation delay of Tpc1-lacking strains.

The last functional group included 54 genes encoding proteins that carry a wide range of biochemical roles. The reduced expression of the autophagy gene ATG22 and the up-regulation of three small chaperones Hsp20-like suggested the unbalanced signals for survival and cell death existent in Δtpc1[54]. Microtubule-dependent vesicle trafficking and cell cycle were also affected in the mutant as inferred from the misregulation of two dynamins, one kinesin light chain, one Marvel protein, two cyclins and the Cdc26 subunit. Genes involved in silencing pathways, spliceosomal snRNP assembly, tRNA processing, RNA-mediated heterochromatin silencing and translational arrest were also misregulated in Δtpc1, highlighting alterations in other cellular processes that regulate gene expression.

Gene-deletion analysis of down-regulated transcripts in Δtpc1 identifies a major pathogenicity gene in M. oryzae

The majority of the Zn(II)₂Cys₆ binuclear cluster proteins are transcriptional activators and only few of them have been shown to act as repressors[14]. To identify novel pathogenicity genes we focused on genes that could play a role in TPC1-associated defects. Five out of the 133 down-regulated genes were selected for gene replacement (S3 Table; S7 Fig), including the conidiation-related gene CON6[55], a glycosyl transferase 18 gene (GH18) that undergoes a 50-fold increased expression in planta[48], and the two signaling-associated PEBP genes (S7 Fig). The PRO41/HAM-6 gene, which is required for hyphal fusion in Neurospora crassa and sexual development in Sordaria macrospora was also selected for the analysis[56,57]. We confirmed by RT-PCR that the five genes were down-regulated in the Δtpc1 mutant (S8A Fig).

Among the six deletion mutants generated, only Δpro41/Δham-6 displayed a severe pathogenicity-deficient phenotype (S7 Fig). Despite the links found between conidiogenesis and pathogenicity in M. oryzae[58,59,60], the Δcon6 mutant behaved like wild-type in planta. Similarly, Δgh18, Δpebp1, Δpebp2, and the double mutant Δpebp1Δpebp2 did not show any pathogenicity-associated defects possibly due to redundancy in related gene functions. Consequently, we selected Δpro41/Δham-6 mutants for further characterization.

M. oryzae NOXD/PRO41 is required for superoxide production, sexual development and plant penetration

The open reading frame of M. oryzae PRO41 was initially annotated in the EnsemblFungi database as HAM-6, a N. crassa gene required for cell fusion[61]. However, the orthologue of this protein was first characterized in S. macrospora and named Pro41[57,62]. Pro41 is a novel ER membrane protein required for fruiting body maturation in S. macrospora. Later, Pro41 was found to be the functional orthologue of the p22^phox subunit of the NADPH oxidase complex in both Podospora anserina and Botrytis cinerea[63,64]. Therefore, we renamed the Pro41/Ham-6 protein NoxD (S8B Fig).

We looked at the growth of the M. oryzae ΔnoxD mutant in different media and stress conditions (Fig 6A and S8C Fig). ΔnoxD grew slightly faster than the wild-type on CM and MM, under salt stress (0.2 M LiCl, 0.4 M NaCl) and in Congo Red (CR). However, we did not observe differences in growth under carbon starvation, calcofluor white (CFW) or basic conditions (pH 9.5). Increased resistance to CFW was previously observed for M. oryzae Δnox1 but not for Δnox2[65], suggesting NoxD and Nox2 fulfill similar roles during cell wall biogenesis. Growth of M. oryzae ΔnoxD and Δnox1Δnox2 mutants in 1mM methyl viologen, 1mM H₂O₂ and 5mM H₂O₂ was similar or improved when compared to wild-type (S8D Fig). Thus, the lack of NoxD did not affect fungal growth under oxidative stressors in contrast to the growth defects displayed by B. cinerea NADPH oxidase mutants[64].

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Fig 6. The M. oryzae NoxD protein is required for plant penetration and sexual reproduction.

(A) Eight-day old wild-type (WT) Guy11 and ΔnoxD strains grown on different media and stress conditions (additional information in S8C and S8D Fig). The ΔnoxD mutant showed accelerated growth rate compared to wild-type (WT) on CM. ΔnoxD growth is affected in MM-N media and shows increase resistance to salts (LiCl, ClNa) and CongoRed (CR). Colony edge is indicated with a black arrowhead. (B) Leaf and root infection assays showing ΔnoxD pathogenicity defects and functional complementation of Pr_RP27:NOXD:mCherry construct. (C) Penetration assays using leaf sheaths and onion epidermal cells. It is very unusual to observe ΔnoxD within the host cells. (D) Fertility assay in oatmeal agar plates pairing Guy11 and ΔnoxD with the tester strain of opposite mating type TH3. No perithecia were observed from the cross of ΔnoxD×TH3.

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The infection ability of ΔnoxD was severely affected on leaves and roots (Fig 6B and S7 Fig), in accordance with the strong penetration defects displayed by Δnox1 and Δnox2[65]. The penetration defect displayed by ΔnoxD was confirmed using rice leaf sheaths (81% in the wild-type versus 19% in the mutant) and onion epidermis penetration assays (74% in the wild-type versus 22% in the mutant; Fig 6C). Subsequently, we crossed the ΔnoxD mutant with the rice isolate TH3, a M. oryzae strain of opposite mating type (Fig 6D). The inability to produce perithecia indicated that NoxD is required for sexual reproduction in M. oryzae.

To define whether superoxide production was impaired in the ΔnoxD mutant, we used nitroblue tetrazolium (NBT), which forms a dark-blue water-insoluble formazan precipitate upon reduction by superoxide radicals[65,66]. In the ΔnoxD mutant, we observed an increase in superoxide production at hyphal tips and a significant reduction in appressoria based on mean pixel intensity measurements (p<0.01) (Fig 7A). This was previously described for Δnox1Δnox2 mutants[65], and supports the existence of alternative routes for cellular ROS generation in M. oryzae during hyphal development. Since Δtpc1 was affected in oxidation-reduction processes, we also included Δtpc1 in this analysis. Increased superoxide production was found in Δtpc1 hyphal tips but to a lesser extent than nox mutants, while in appressoria Δtpc1 showed the highest superoxide levels among the strains analyzed, indicating that the lack of Tpc1 affects superoxide production pathways in M. oryzae.

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Fig 7. M. oryzae NoxD is required for ROS production in appressoria, localizes in ER-derived vesicles and plasma membrane, and interacts with Nox1.

(A) Superoxide production is significantly affected in the mutants as shown by nitroblue tetrazolium (NBT) staining and quantification of pixel intensities in hyphal tips (mean±SD; n = 10). Both ΔnoxD and Δnox1Δnox2 produced less ROS in appressoria but higher amounts of ROS in hyphal tips compared to WT; Δtpc1 generated higher amounts of ROS in hyphal tips and appressoria. Addition of diphenylene iodonium (DPI) before NBT treatment abolishes the formation of dark precipitates associated with sites of superoxide generation. Values to calculate mean pixel intensity were for white 0 and black for 100. (B) A yeast two-hybrid screen reveals that NoxD interacts with Nox1. A yeast colony co-transformed simultaneous with the pGBK-NOX1 (bait Nox1) and pGAD-NOXD (prey NoxD) vectors grows in high stringency media (-His/-Ade/-Leu/-Trp/+X-α-Gal). The interaction of proteins expressed by prey and bait vectors generates a blue-coloured colony due to the activation of α-galactosidase expression in the presence of X-α-Gal. (C) Images of NOXD fusion constructs using GFP and mRFP under two different promoters, the NOXD promoter and the strong promoter of ribosomal protein Rp27, respectively. Both NoxD fusions localized in vesicles and plasma membrane. In conidia expressing NOXD:mRFP, fluorescence signal is observed in vesicles closely associated to ER membranes labelled with GFP containing the ER retention signal KDEL. After 8h, fluorescence signal is strongly detected at the plasma membrane of the appressoria. pEFGPKDEL encodes GFP with the ER retention signal KDEL. (D) Micrographs of F-actin ring organization visualized by expression of gelsolin:GFP and Sep:GFP in WT and ΔnoxD. This mutant produces aberrant septin and actin rings; mis-localization of Sep5:GFP is more severe. The linescan graphs show fluorescence in a transverse section of individual appressoria.

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A yeast two-hybrid assay was used to identify putative NoxD interactors. We found that M. oryzae NoxD interacts with the Nox1 NADPH oxidase subunit (Fig 7B) but not with Nox2 or NoxR, supporting previous work in B. cinerea and P. anserina[63,64].

NoxD is visualized in ER-associated vesicles and plasma membrane of appressoria and conidia

To localize NoxD we generated C-terminal mRFP (cherry variant) and GFP translational fusions under the control of strong or native promoters, respectively. Both constructs fully complemented ΔnoxD plant infection defects (Fig 6B), which indicated that the C-terminal tag does not affect NoxD function, although expression of NoxD:mRFP was clearly stronger. M. oryzae NoxD was mainly observed in subapical vesicles and the plasma membrane of appressoria and conidia (Fig 7C). Co-localization of NoxD:mRFP with GFP containing the ER retention signal KDEL showed that the vesicles are closely associated with the ER, overlapping with some of them (Fig 7, white arrowheads). The subapical vesicles observed near plasma membranes and septa in M. oryzae structures correlated with the localisation of NoxD in P. anserina [63]. In P.anserina, these vesicles co-localised with the GFP:Idi7 reporter protein, suggesting that they originate from the ER and travel towards the vacuolar system [63].

NoxD is required for septin ring assembly at the appressorial pore

The Nox2-NoxR complex is essential for septin-mediated cytoskeletal reorientation, whereas Nox1 is dispensable although may have important roles to play in maintenance and elongation of the penetration peg[6]. To test if NoxD was also involved in this process, we expressed the acting-binding protein gelsolin:GFP and Sep5:GFP in ΔnoxD. In the wild-type, both a septin and gelsolin ring was present at the appressorium pore[6] (Fig 7D). In the ΔnoxD mutant, however, Sep5:GFP formed a disorganized mass in the infection cell as previously reported for Δnox2 and ΔnoxR expressing Sep5:GFP[6]. Gelsolin:GFP rings in ΔnoxD also possessed distorted pores. Considering that gelsolin colocalizes with F-actin at the appressorial pore[6], the altered fluorescence pattern of gelsolin:GFP revealed that the toroidal F-actin ring was disorganized (Fig 7D). Previous reports showed that Sep5:GFP and gelsolin:GFP patterns in the Δnox1 mutant displayed normal conformation[6]. NoxD and Nox1 therefore appear to play alternative roles in cytoskeletal re-modeling in appressoria of M. oryzae.

NOXD expression is regulated by Tpc1 and the Pmk1 pathway

The down-regulation of NOXD in Δtpc1 suggested that this gene may be directly regulated by Tpc1. To investigate this idea, we carried out chromatin immunoprecipitation (ChIP) followed by qPCR (Fig 8A and 8B). We observed that the promoter region of NOXD comprising the NOXD1, NOXD2 and NOXD3 fragments immunoprecipitated with Tpc1:GFP, which indicated that NOXD expression is regulated in vivo by this transcription factor. In addition, we performed electrophoretic mobility shift assays (EMSA) with M. oryzae Tpc1 and Mst12 since both proteins can interact in yeast two-hybrid assays (Fig 5C). We found that Mst12 strongly recognised the probe 1 located between -1120 and -643 upstream of the start codon of the NOXD gene (Fig 8C). Mst12 also recognized probes 2 and 3, but less strongly. Mst12 bound to the probes produced multiple bands, possibly due to the presence of several protein molecules on the biotinylated DNA (Fig 8D). Intriguingly, Tpc1 itself was not capable of recognizing any of the three probes under the conditions tested (Fig 8C). However, the addition of Tpc1 to Mst12 increased its DNA-binding capacity (Fig 8E), which is consistent with both, the ability of these proteins to interact, and with Tpc1 as modulator of Mst12 DNA-binding affinity. Increasing amounts of Tpc1 did not alter significantly Mst12affinity. Importantly, the promoter regions tested using these in vitro DNA-binding assays correlated perfectly with the enriched fragments obtained in the ChIP analysis, which supports that Mst12 and Tpc1 are part of a complex that coordinately regulate NoxD expression. To further confirm these results, we checked NOXD expression levels in the Δmst12 mutant and corroborated that they were reduced (Fig 8F). We also observed that MST12 and TPC1 genes were overexpressed when the corresponding partner was not present in the fungal cell (Fig 8G). We conclude that Tpc1 regulates NoxD expression through its interaction with Mst12 and confirm the link between Tpc1 and the participation of the Pmk1 pathway in the regulation of NoxD expression.

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Fig 8. Tpc1 and Mst12 bind the promoter region of the NOXD gene.

(A) Genomic location of fragments used for chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA). Transcription start site (TSS) was identified by 5’-end RACE (rapid amplification of cDNA ends) followed by sequencing. The 3’UTR length was estimated using EST data. (B) ChIP analysis with anti-GFP antibody was used to identify Tpc1:GFP levels associated with NOXD promoter (means±SD, n = 4). DNA isolated from chromatin immunoprecipitated with anti-GFP antibody was subjected to qPCR. The DNA in ChIP fractions prepared without antibody was used as negative control. The ratio of DNA in ChIP fractions with respect to that in the untagged wild-type cell extracts was calculated for NOXD region and normalized with the values for the NOXD site. The normalized ratios were plotted in the lower panel. Probes indicated by asterisks are statistically significant according to the Wilcoxon-Mann-Whitney test and have a p value <0.05 (*) or <0.01 (**). (C) EMSA with Mst12 and Tpc1. Mst12 recognises strongly probe 1, and with less affinity probes 2 and 3. Tpc1 cannot directly bind to the NOXD promoter. The promoter region of a YdiU-containing protein was used as a negative control. (D) DNA-binding analysis of Mst12 to NOXD promoter. Dissociation constant (K_D) was estimated using bound probes against protein concentration and the fitting equation y = mx/(k+x). (E) DNA-binding experiments showing Tpc1 promoting Mst12 DNA-binding affinity to NoxD probe 1 using different amounts of Tpc1. BSA (bovine serum albumin) resuspended in Tpc1 buffer was used as a negative control; BSA does not stimulate Mst12 binding to the probe. (F) Analysis of NOXD transcript abundance in Δtpc1 and Δmst12 mutants by qPCR. (G) Transcript abundance of MST12 and TPC1 in Δtpc1 and Δmst12 backgrounds, respectively. cDNA concentration in the mutants was normalised using actin and wild-type levels of the genes analysed (referred as 1; mean±SD, n = 3).

https://doi.org/10.1371/journal.ppat.1006516.g008

Strains, media and plant infections

M. oryzae was routinely incubated in a controlled temperature room at 25°C with a 12h light/dark cycle. Media composition of complete medium (CM), minimal medium (MM), minimal medium without carbon (MM-C) or nitrogen (MM-N), and DNA extraction and hybridisation were all as previously described[76]. Growth tests were carried out with 7 mm plugs of mycelium from Guy11 and the M1422 mutant strains as initial inoculum. The wild-type Neurospora crassa strain and isogenic deletion mutant NCU05996 were obtained from the Fungal Genetics Stock Centre (FGSC, Kansas City, Missouri, USA). Vogel’s minimal medium was used for cultivation of N. crassa strains at 25°C with a 12h light/ dark cycle and for stock-keeping at 4°C (http://www.fgsc.net/Neurospora/NeurosporaProtocolGuide.htm). Growth tests were carried out on Vogel plates with 5 mm plugs of mycelium from N. crassa wild-type (wt) and NcTPC1 KO strains. Plates were incubated at 25°C for 2 days. M. oryzae leaf and root infection assays were carried out, as previously described [30,77].

Conidiation, onion/leaf sheath penetration assays, cytorrhysis assay and glycogen/Nile red staining

Conidia were harvested using 2 ml of sterile water per plate after fungal cultures were incubated at 25°C for a period of 10 days on CM. Calculations were then carried out to determine the number of conidia generated per cm² of mycelium using a Neubauer counting chamber. Values are the mean ± SD from >300 conidia of each strain, which were measured using the ImageJ software [78]. Photographs were taken using the Zeiss Axioskop 2 microscope camera using differential interference contrast (DIC) microscopy or epifluorescence. Conidia were stained with 5μl calcofluor white (CFW) solution (Fluka) and incubated at 25°C for 30 minutes. Cell number per conidium was determined by visualizing septa with CFW. Appressorium-mediated penetration of onion epidermal strips was assessed using a procedure based on Chida and Sisler[79]. A conidial suspension at a concentration of 1 x 10⁵ conidia mL^-1 was prepared and dropped onto the adaxial surface of epidermal layers taken from onion. The strips were incubated in a moist chamber at 25°C and penetration events scored 24h to 48h later by recording images with an Olympus IX81 inverted microscope system. Leaf sheath assays were carried out as previously described [10]. Glycogen staining solution contained 60 mg of KI and 10 mg of I₂ per milliliter of distilled water. Glycogen deposits are visible immediately. For cytorrhysis assays, 10⁵ spores were allowed to form appressoria for 18h on coverslips prior the addition of external glycerol (1M or 3M). After 10 minutes in glycerol ~500 appressoria were analyzed in each biological replica; experiment was carried out by triplicate. To visualize lipid droplets, conidia were allowed to germinate in water on coverslips. After 0h, 2h, 9h and 12h water was removed and conidia directly stained with Nile red (Nile Red Oxazone (9-diethylamino-5Hbenzo[alpha]phenoxazine-5-one; Sigma). Nile red was used to 2.5 mg/ml diluted in 50mM Tris/Maleate, pH 7.5 and polyvinylpyrrolidone (PVP) (2–3% w/v). Lipid droplets begin to fluoresce within seconds. Samples were visualized under a confocal laser scanning microscope using a 561 nm excitation wave length and a long pass emission filter (592–700 nm). All images were taken using the same parameters.

Generation of mutant strains by gene replacement

Gene deletion constructs were generated using multisite gateway technology (Invitrogen) as previously described[77,80]. TPC1, CON6, GH18, PEBP2 and NOXD coding sequences were replaced by the hygromycin resistance cassette and PEBP1 by the sulfonylurea resistance cassette in the gene replacement constructs. Primers for constructing entry plasmids are described in S4 Table. Fungal transformants generated by Agrobacterium-mediated transformation [81] were selected growing in DCM solid media supplied with 5-fluoro-2’-deoxyuridine (50μM) and 200μg/ml Hygromycin or 150μg/ml Chlorymuronethyl in the case of Δpebp1. DCM is 1.7 g yeast N-base without amino acids, 1.0 g NH₄NO₃, 2.0 g of L-asparagine and 10 g of D-glucose. Knockout strains were confirmed by PCR or Southern blotting using radioactive probes (³²P; primers listed in S4 Table). Sequence data and gene numbers used in this study were taken from EnsemblFungi (Magnaporthe oryzae MG8; http://fungi.ensembl.org/index.html).

Generation and cellular localisation of fluorescently tagged proteins

To determine the localisation of Tpc1, live-cell imaging was performed using a M. oryzae Guy11 strain containing two constructs, histone H1 tagged with red fluorescent protein (H1:RFP; tdTomato) to visualize nuclei [82], and TPC1:GFP. For the construction of a functional TPC1:GFP gene fusion, primers were designed in order to amplify the TPC1 (MGG_01285) promoter region and ORF from genomic DNA of M. oryzae Guy11 (S4 Table). The TPC1_GFP_F forward primer was designed approximately 1.3 kb upstream from the TPC1 start codon to include a substantial component of the promoter sequence. The TPC1_GFP_R reverse primer spanned the stop codon and contained a complementary region to the GFP sequence. GFP primers were designed to amplify the 1.4 kb sGFP:TrpC construct cloned in pGEMT. Both fragments were joined together by fusion nested PCR. The amplicons were cloned into pGEMT-easy digested with EcoRI. The 4.3 kb TPC1:GFP fragment was gel purified and cloned into pCB1532 that had previously been digested with EcoRI. The pCB1532 vector contains the 2.8 kb ILV1 gene, which encodes the acetolactate synthase-encoding allele bestowing resistance to sulfonylurea[83]. The resulting plasmid pCB1532-TPC1:GFP was used to transform protoplasts of M1422 mutant. For all rounds of PCR amplification, Phusion High-Fidelity DNA polymerase (Finnzymes, Thermo Fischer Scientific Inc.) was used, following the manufacturers’ guidelines for PCR conditions.

The GFP:MoATG8[34] and the FIM:GFP constructs were used to transform protoplasts of M1422 mutant. Protoplast generation and transformation were carried out as previously described[76]. The GFP:MoATG8 and the FIM:GFP protein fusion vectors were generated using the native M. oryzae MoATG8 gene (MGG_01062) and the native M. oryzae fimbrin-encoding gene (MGG_04478), respectively. Both fragments were cloned into pCB1532 vector that contains the 2.8 kb ILV1 gene, which encodes the acetolactate synthase allele conferring sulfonylurea resistance. Transformants showing identical growth and colony morphology to the background strain were selected for further examination using epifluorescence or confocal microscopy. At least three different transformants of each were independently analysed.

The TPC1:GFP gene fusion was cloned into pCB1532 vector (SUR^R) and used to transform protoplasts of Guy11 expressing Histone H1 fused with red fluorescent protein (H1:RFP)[33], and also introduced into isogenic Δpmk1, Δatg1 and Δatg8 mutants. Transformants were selected for further examination using confocal microscopy and verified as containing a single copy of the gene fusion construct by Southern blot hybridisation. At least three different transformants of each were used in all experiments.

RNA isolation and global gene expression profile using microarrays

Using a modified protocol of LiCl method[77], RNA was extracted from 8-day old fungal mycelia grown on cellophane placed on top of CM agar plates (S2E Fig). Two to three additional washes with phenol:chloroform were implemented to avoid RNA degradation from cellophane samples. RNA quality control was carried out with Agilent RNA 6000 Nano kit (ref. 5067–1504). Four biological replicates were independently hybridized for each transcriptomic comparison. Each of these replicates derived from three technical repetitions. Slides were Agilent Magnaporthe II Oligo Microarrays 4x44K (ref. 015060). Background correction and normalization of expression data were performed as previously described[77]. Hybridizations and statistical analysis were conducted by the Genomics Facility at the National Biotechnology Centre (Madrid, Spain). The GO term analysis was carried out with gProfiler[84]. Enriched motifs were not found when using the promoter regions of the 185 up-regulated genes. Microarray data are available in the ArrayExpress database (EMBL_EBI) under accession number E-MTAB-4127.

Yeast-two hybrid screen

In-Fusion Cloning based on in vitro homologous recombination was performed to generate vectors including NoxD and Tpc1 into the pGADT7 prey vector, and Nox1, Nox2 NoxR, Pmk1 and Mst12 into the pGBKT7 bait vector. Genes were amplified from M. oryzae cDNA derived from mycelia grown on liquid CM using primers with a 15bp overhang and restriction site complementary to the target vector (S4 Table). For NoxD, a 435bp fragment was amplified, for Nox1, a 1662bp fragment was amplified, for Nox2, a 1749bp fragment was amplified, and for NoxR, a 1578bp fragment was amplified. Respective fragments were cloned into pGBKT7 and pGADT7 plasmids linearized by digestion with EcoRI and SmaI. Yeast two-hybrid assays using pGADT7 or pGBKT7 (Clontech) based constructs were performed according to the manufacturer’s instructions (MATCHMAKER Gold Yeast Two-Hybrid System).

Imaging of fluorescent fusion proteins

For the construction of NoxD:GFP, primers were designed to amplify the ORF including 2kb upstream of the start codon, GFP and TrpC terminator with 15bp overhangs complementary to adjacent fragments (S4 Table). Fragments were ligated into pCB1532[83], which carries the sulphonyl urea resistance cassette and had been digested with BamHI and HindIII and this construct transformed into of the wild-type strain Guy11 using protoplasts[6]. The NoxD:mRFP construct was generated using multi-site gateway technology (Life Technologies) with the entry mCherry-withSTOP and destination SULPH-R3R4 vectors[77], and PCR fragments amplified from M. oryzae genomic DNA using Phusion DNA polymerase (NEB) and primers detailed in S4 Table. Appressorium development assays were performed on hydrophobic borosilicate glass coverslips (Fisher Scientific), as described previously[6]. For epifluorescence microscopy, conidia were incubated on coverslips and observed at each time point using an IX-81 inverted microscope (Olympus) and a UPlanSApo X100/1.40 oil objective. All microscopic images were analyzed using MetaMorph (Molecular Devices). Confocal imaging was performed with a Leica SP8 microscope.

qPCR and ROS detection

To confirm microarray results, the relative abundance of gene transcripts were analysed by qPCR (S4 Table). One μg of total RNA from 8-day old fungal mycelia grown on cellophane placed on CM agar was reverse transcribed using PrimeScript RT reagent Kit (Takara). The average threshold cycle (Ct) was normalized against actin transcript and relative quantification of gene expression was calculated by the 2^ΔΔCt method[85]. Primer efficiency was tested using dilutions of cDNA samples. qPCR reactions were carried out with 1 μl of reverse transcribed products and fast-start DNA master SYBR green I kit (Roche Diagnostics) in a final reaction of 20 μl using the following program: one cycle of 95°C for 4 min and 40 cycles of 94°C for 30 s and 60°C for 30 s. The Ct (threshold cycle) provided a measure for the starting copy numbers of the target genes. Three technical repetitions from three independent biological experiments were used for each gene. For ROS detection in M. oryzae fungal structures, NBT staining[65] and quantification method of pixel intensities in hyphal tips[86] were carried out as previously described.

Chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) analysis

Two strains, the Δtpc1 mutant expressing TPC1:GFP and M. oryzae wild-type Guy11 strain as negative control were used for this experiment. Mycelia were grown in liquid CM at 25°C for 48 h in a shaker (120 rpm), and collected using two layers of Miracloth. Harvested mycelia were washed extensively with sterile water. To crosslink DNA and proteins, one gram of each washed mycelium was treated with 1% formaldehyde in 20 mM HEPES pH 7.4 buffer for 20 min with continuous shaking at 100 rpm. Then, 0.125 M glycine was added and incubated at room temperature for an additional 10 min to stop crosslinking. Mycelia were harvested with Miracloth, rinsed with water removing excessive water by squeezing and immediately frozen in liquid nitrogen, grinded into a fine powder and stored at -80°C until used. ChIP was conducted according to published procedures with some modifications [87]. 600 mg of each mycelium powder was used for chromatin extraction and sonication. The powder was added into 10 ml of Extraction buffer 1 (0.4 M sucrose, 10 mM Tris-HCl pH 8, 10 mM MgCl₂, 5 mM β-mercaptoethanol/β-ME and Protease Inhibitors Complete-PIC/Roche) and mixed by vortexing. The solution was filtered through a double layer of Miracloth and centrifuged at 5000 g for 10 min at 4°C. The pellet was resuspended in 1 ml of Extraction buffer 2 (0.25 M sucrose, 10 mM Tris-HCl pH 8, 10 mM MgCl₂, 1% Triton X-100, 5 mM β-ME and PIC) and centrifuged at 5000 g for 10 min at 4°C. The pellet was resuspended in 300 μl of Extraction buffer 3 (1.7 M sucrose, 10 mM Tris-HCl pH 8, 0.15% Triton X-100, 2 mM MgCl₂, 5 mM β-ME and PIC) and, carefully layered on the top of additional 600 μl of extraction buffer 3. Then, samples were centrifuged at 16000 g for 60 min at 4°C. The chromatin pellet was resuspended in 300 μl of Nuclei Lysis Buffer (50 mM Tris-HCl ph 8, 10 mM EDTA, 1% SDS and PIC) and sonicated for 25 min at 4°C, operating a pattern of 30 sec ON and 30 sec OFF, at high power level in the Bioruptor Plus (Diagenode, Liege, Belgium) to obtain DNA fragments ranging from 500 to 1,000 bp. The chromatin solution was centrifuged at maximum speed for 5 min at 4°C to pellet cell debris. The supernatant was kept as chromatin solution and a small aliquot (10%) was stored as input DNA control. For each immunoprecipitation, 15 μl of Dynabeads Protein A magnetic beads (ref. 10001D, Life Technologies) was washed twice with 500 μl ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8, 167 mM NaCl and PIC). Then, anti-GFP antibody (ref. A6455, Life Technologies) was added and incubated with gentle rotation for 1h at 4°C in 50 μl ChIP dilution buffer. Prepared anti-GFP coated beads were washed twice with 500 μl ChIP dilution buffer and resuspended in 100 μl of ChIP dilution buffer. For each immunoprecipitation, the latter and 100 μl of chromatin solution were gathered together and diluted up to 1 ml of ChIP dilution buffer. All immunoprecipitations were incubated overnight at 4°C with gentle rotation, then washed with a serie of wash buffers (2 washes with Low Salt Wash Buffer: 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8; one wash with High Salt Wash Buffer: 500 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8; one wash with LiCl Wash Buffer: 0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8, 2 washes with TE Buffer: 10 mM Tris-HCl pH 8, 1 mM EDTA). Immunoprecipitated DNAs and Input DNA control were reverse-crosslinked at 95°C for 10 min with 200 μl of 10% chelex 100 resin to remove any trace of metals. DNA samples were treated with proteinase K that was inactivated afterwards. After centrifugation, supernatants of DNA samples were stored at -20°C until used. Immunoprecipitated chromatin was diluted 10 times for qPCR analysis (primers listed in S4 Table). This was performed using a Roche LightCycler 480 machine. qPCR reactions were carried out using either 2 μl of input DNA or 2 μl of immunoprecipitated chromatin in a final reaction of 12 μl with the following program: one cycle of 95°C for 5 min and 58 cycles of 94°C for 10 s, 60°C for 10 s and 72°C for 10 s. The Ct (threshold cycle) provided a measure for the starting copy numbers of DNA. Three technical repetitions from 4 independent biological experiments were used. Ct values were used to calculate ratios evaluating the fold difference between experimental samples (GFP-tagged or untagged wild-type strains) and normalized the input. We normalized with “Fold Enrichment Method” using the untagged strain. The Wilcoxon Mann Whitney test was applied to analyze the difference between two independent groups. Statgraphics software was used to make pairwise comparisons between GFP-tagged strain and untagged wild-type strain.

Protein purification and EMSA

M. oryzae MST12 and TPC1 cDNAs derived from mycelial RNA were cloned by PCR using a high fidelity Q5 DNA polymerase (NEB), primers (S4 Table) and the restriction enzymes BamHI-NotI and EcoRI- NotI for MST12 and TPC1 respectively, into a modified pET28 vector (5,667bp; Novagen). MST12- and TPC1-containing plasmids were transformed in E. coli Rosetta DE3 (Novagen) and colonies grown in LB medium containing chloramphenicol (34 μg/L) and kanamycin (50 μg/L) until reaching OD_600nm = 0.8. Protein expression was induced 4 hours at 28°C with 1 mM IPTG (Sigma-Aldrich). Centrifuged cell pellets (30 min at 7000g) were resuspended in lysis buffer (20 mM sodium phosphate pH 8, 300 mM NaCl and one tablet of PIC/50 ml, 1 mM PMSF and 50 μg/ml Dnase I), lysed by sonication and pelleted at 4°C and high speed (20 min at 20,000g). Recombinant proteins were purified from clear lysate by metal affinity chromatography (HisTrap HP 1 ml, #17-5247-01 GE Healthcare) in denaturing conditions using 6 M Urea and eluted with 250 mM imidazole containing buffer. Samples were desalted on PD10 column (#17085101 GE Healthcare) to remove urea and imidazole using buffer (20 mM sodium phosphate pH 8, 10% glycerol and PIC). Protein samples purity was evaluated by SDS-PAGE.

EMSA probes were generated as follows. Amplified by PCR fragments using primers listed in S4 Table were prepared using modified Biotin 3’end DNA labeling procedure (#89818 Thermo-Scientific). Briefly, each ~500pb purified PCR products was KpnI-digested, purified and labelled (5 pmol of each probe) with Biotin-11-UTP and Terminal Deoxinucleotidyl Transferase at 37°C for 1 hour. Biotinylated probes were purified by Chloroform:IAA (24:1) extraction and stored at -20°C until use. EMSA reactions (20 μl) contained 10 mM Tris HCl pH 7.5, 50 mM KCl, 16 mM DTT, 1 mM ZnCl₂, 1 mM MgCl₂, 1% Glycerol, 50 ng/μl Poly dI-dC (#20148E Thermo-Scientific), 10 μg BSA, Protease inhibitor complete (Roche), and 80 fmol of biotinylated probe. Before probe addition proteins (0–12 μM) were incubated in binding buffer for 10 min, then probe was added and incubated during 30 min at room temperature before loading. The EMSA gel (0.2% agarose, 5% polyacrylamide, 1% glycerol in TBE 0.5x) was run for 2h 100V in TBE 0.5x and then transferred to a Hybond-XL nylon membrane (#RPN203S GE Healthcare) at 400 mA for 1 hour. The membrane was UV crosslinked at 120mJ/cm². Detection was performed with stabilized streptavidin-horseradish peroxidase conjugate (#21134 Thermo-Scientific) and enhanced chemiluminescent substrates (#32106 Thermo-Scientific) following LightShift Chemiluminescent EMSA procedure (#20148 Thermo-Scientific).

Phylogenetic analysis of Tpc1

First, 141 M. oryzae protein sequences containing a fungal Zn(II)₂Cys₆ binuclear cluster domain (PF00172) were identified from the Magnaporthe sequence database at the Broad Institute (http://www.broadinstitute.org/annotation/fungi/magnaporthe) and the Fungal Transcription Factor Database (http://ftfd.snu.ac.kr/intro.php). HMMsearch from HMMER3[88] was used to screen the genome assembly of M. oryzae proteins with the fungal Zn₂Cys₆ profile hidden Markov model pHMM zn_clus_ls.hmm (PF00172.13) from Pfam database[89] (http://pfam.xfam.org/). Subsequently, gene numbers were updated using the MG8 genome version of EnsemblFungi database (http://fungi.ensembl.org/index.html). Out of these 141 sequences, only 113 had a full length zinc cluster domain, and extra six closest sequences were included to build S5 Fig. Additional Zn(II)₂Cys₆ proteins found in Lu et colleagues[28] were included in S2 Table. Basic Local Alignment Search Tool (BLAST) was used to find orthologous proteins of TPC1/MGG_01285 (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Protein sequences were pre-aligned using HMMalign and the pHMM zn_clus_ls.hmm (S4 Fig) from Pfam. The Zn(II)₂Cys₆ binuclear cluster domain region was extensively manually aligned in BioEdit (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html). Unambiguous aligned positions were used for the subsequent phylogenetic analyses. The maximum likelihood (ML) analyses were performed with the program PhyML version 3.0.1[90]. All trees were visualised using the program Figtree (http://tree.bio.ed.ac.uk/software/figtree/).

Accession numbers

M. oryzae sequence data from this article can be found in the GenBank/EMBL-EBI (EnsemblFungi) databases under the following accession numbers: TPC1 (MGG_01285), PMK1 (MGG_09565), MST12 (MGG_12958), ATG1 (MGG_06393), ATG8 (MGG_01062), CON6 (MGG_02246), GH18 MGG_04732, NOXD (MGG_09956), PEBP1 (MGG_06800), PEBP2 (MGG_14045), NOXR (MGG_05280), NOX1 (MGG_00750), NOX2 (MGG_06559), FIMBRIN (MGG_04478) GELSOLIN (MGG_10059), ACTIN (MGG_03982), YDIU (MGG_03159) and SEP5 (MGG_03087).