Strains, growth conditions, and oligonucleotides

All mutants described in this study are derived from the Dutch field isolate Zymoseptoria tritici IPO323, used as a wildtype strain (CBS Fungal Collection, Utrecht, NL). IPO323 and the mutant strains were routinely cultivated at 18°C and 120 rpm in liquid medium YEG (Glucose 10 g/l, yeast extract 10 g/l, pH 6.5) or were grown at 18°C on YEG-agar medium (2%). As basis for phenotypical screening and stress based growth assays N-deprivation, MM, YEG and PDA agar media were used. For the stress assays, H₂O₂, SDS, Congo red, the phenylpyrrole fungicide fludioxonil, NaCl, and sorbitol were added to YEG medium at the concentrations indicated in the figure legends. The assays were carried out in triplicate and were repeated three times. Nitrogen starvation medium (N-deprivation) (pH 6.5), which represented the switch-inducing medium, contained 10 g/l glucose, 0.25 ml/l of a 0.01% biotin solution, 50 ml/l of a salt solution without nitrate salts (10.4 g/l KCl, 30.4 g/l KH₂PO₄, 10.4 g/l MgSO₄·7H₂O), 1 ml/l of a trace element solution (22 g/l ZnSO₄·7H₂O, 11 g/l H₃BO₃, 5 g/l MnCl₂·4H₂O, 5 g/l FeSO₄·7H₂O, 1.7 g/l CoCl₂·6H₂O, 1.6 g/l CuSO₄·5H₂O, 1.5 g/l Na₂MoO₄·2H₂O, 50 g/l Na₄·EDTA) and 1 ml/l of a 1% thiamine dichloride solution. Minimal medium (MM) was prepared based on nitrogen starvation medium and additionally contained a nitrogen source in the form of nitrate salt solution (10.4 g/l KCl, 30.4 g/l KH₂PO₄, 10.4 g/l MgSO₄·7H₂O and 120 g/l NaNO₃). Potato dextrose agar (PDA) was obtained from Carl Roth (Karlsruhe, Germany).

All oligonucleotides used in this study are listed in Supplementary materials (Tables A-E in S1 File) and were obtained from Eurofins-MWG-Operon (Ebersberg, Germany). All chemicals used were obtained from Sigma-Aldrich (Munich, Germany) unless otherwise stated.

Identification of dimorphic switch deficient transformants

The transformants generated and wildtype reference strain IPO323 were spotted (1.5 μl; 1×10⁸ spores/ml) onto N-deprivation solid medium and water agar. Colonies were analysed stereomicroscopically using binocular Zeiss Stemi 2000-C (Carl Zeiss Microscopy GmbH) after 7, 14 and 21 days of incubation at 18°C to monitor the dynamics of dimorphic switch of strains generated. Morphology scoring was based on macroscopic colony appearance at 14 days, with consideration of appearance at 21 days in questionable cases. The mutants were considered as dimorphic switch deficient when their growth and switching inability was comparable with that of ΔZthog1 mutant strain as reference after 7 days post inoculation of N-deprivation and water agar medium.

Nucleic acid manipulations

Unless otherwise specified, all DNA manipulations and molecular cloning procedures were carried out by standard protocols [27]. All restriction endonucleases and T4-DNA ligase were purchased from NEB. PCR amplification of fragments for cloning purposes was performed using Phusion High Fidelity Polymerase (New England Biolabs (NEB), Herts, UK). Diagnostic colony PCR was performed using Takara Sapphire Premix and all other PCR amplifications including those for genome walking were conducted using the Taq-Polymerase according to the manufacturer recommendations. Recombinant vectors were sequenced using the Sanger sequencing service at Eurofins Genomics (Ebersberg, Germany).

Plasmids were extracted from E. coli using the QIAprep spin mini-prep kit (Qiagen, Manchester, UK). Isolation of the genomic DNA from Z. tritici was routinely performed using Qiagen DNeasy Kit (Qiagen Sciences, Valencia, CA).

Transformations using ATMT for the generation of random / targeted /complementation mutants

Transformation of conidia and selection of hygromycin-resistant transformants of Z. tritici were performed using Agrobacterium tumefaciens-mediated transformation (ATMT) [28,29]. A. tumefaciens strain AGL1, containing the constructed gene inactivation vectors based on pCAMB0380 (CAMBIA, Canberra, Australia), was cocultivated with conidia of Z. tritici for 48 hours at 28°C on AIM medium. Prior to this cocultivation the Z. tritici culture was grown for 3 days in YEG at 18°C and finally centrifuged for 10 min at 4000 rpm in order to harvest conidia and finally resuspend them to final concentration of 5×10⁷/ml. For random mutagenesis we used pCAMB-HPT(SalI), a pCAMB0380-based vector, containing the hygromycin resistance gene HPT from pCB1003 [30] cloned as SalI fragment into the SalI site of the vector. The bacterial hygromycin B phosphotransferase (HPT) gene is expressed under the control of the strong constitutive Aspergillus nidulans trpC promoter, forming the HPT-cassette as selectable marker conferring the hygromycin resistance. For complementation of the mutant strains the vector pCAMB-BAR was used for ATMT. This vector is based on the plasmid pCAMB0380 and harbors a bialaphos resistance cassette cut with restriction enzyme SalI from the plasmid pCB1635 and ligated into the plasmid pCAMB0380 restricted with SalI. The detailed transformation procedure was previously described [31]. Selection of hygromycin resistant transformants containing the gene deletion constructs was performed using 200 mg/ml Hygromycin gold (Invivogen) on V8 agar, while selection of BASTA-resistant transformants produced within complementation of null mutants of Z. tritici was performed with 200 mg/l BASTA (glufosinate ammonium) on MM. Selection in both cases included 200 mg/l streptomycin and 350 mg/l cefotaxime.

Pathogenicity assays

For pathogenicity tests, conidia were harvested from 4-day-old YEG submerged cultures and resuspended at 1×10⁸/ml conidia/ml in 0.2% gelatine. Ten-day old seedlings of the susceptible wheat line cv. Riband were used for plant infection assays by spray-inoculation. Inoculation, incubation and examination of intact plants were carried out as previously described [31]. The plants were examined 21 days post inoculation.

Spore germination test

For the spore germination assay, conidia of Z. tritici strains were inoculated onto microscopic slides covered with a thin layer of water agar. For each strain 100 μl of spore suspension at final concentration of 10⁶/ml were spotted. The incubation was performed at 18°C in a moisture chamber with nearly full-saturated humidity for 72 h in the dark. The spores were directly microscopically analysed and the ratio of germination was calculated as the mean percentage of conidia germinated using three biological replicates for each strain.

Spore lysis test

To investigate cell wall defects, mutant strains of Z. tritici and the wildtype reference IPO323 were compared under conditions which lead to lysis of the cell wall. The strains selected for examination were grown for 3 days at 18°C and 120 rpm in liquid medium YEG. The conidial cells were pelleted by centrifugation at 4,000 rpm and washed three times in an isotonic buffered solution (0.15 M Na₂HPO₄, 0.08 M citric acid, 1.4 M KCl; pH 5.8). Finally the spores were resuspended in the isotonic buffer containing 30 mg of lysing enzymes from Trichoderma harzianum (Sigma) by adjusting 10⁷ spores/ml for each strain investigated, followed by incubation for 3 h at 30°C with a slight stirring. The lysing rate was determined by enumerating the remaining intact yeast-like conidia (or filaments in case of myco#5 mutant strain) using cell counting chamber (Neubauer improved, Carl Roth GmbH & Co.).

Analysis and recovery of HPT integration sites within the random-mutant genome

The DNA from the site of the T-DNA insertion was recovered using a “step-down” PCR-based approach [26] by two nested rounds of amplification using the “gene-specific” primers gspBa, gspB, gspAa, gspA and “adapter-specific” primers PP1 and PP2 (Table B in S1 File). The amplicons obtained were finally cloned into pGEMTeasy or pJET vectors (Promega) and sequenced using universal primers (M13–20, T7 and M13 rev -49).

Generation of a transformant library by ATMT and analysis of the mode of T-DNA integration into the genome of Zymoseptoria tritici

Using an insertional random mutagenesis as a forward genetics approach, we generated a collection of 10,000 transformants of the wheat pathogen Z. tritici. The mutagenesis program was conducted by Agrobacterium tumefaciens-mediated transformation (ATMT), representing a stable and reliable transformation method, which was previously established and approved in different reverse genetics studies with Z. tritici [31]. pCAMB-HPT(SalI) was used as a transformation vector (Fig 1C), containing hygromycin phosphotransferase gene (HPT) as a resistance marker derived from Escherichia coli under control of upstream trpC promoter from Aspergillus nidulans and downstream NOS terminator from Agrobacterium tumefaciens [31]. The suitability of random mutagenesis was proved by randomly selecting of 100 transformants and performing a Southern Blot analysis to verify the genomic integration of T-DNA and to assess the copy number of T-DNA. The isolated genomic DNA of the transformants which had been proved to be resistant to hygromycin B at 200 μg/ml, was fragmented with HindIII and probed with DIG-labelled HPT-fragment-probe (Fig 1A). This analysis revealed no bias towards multiple integrations of the T-DNA and confirmed a likely “randomized” distribution of integration sites, especially in view of the potential occurrence of so-called “hot spots” (which have been reported in the case of some fungi and plants). Overall, a nearly 72% frequency of single locus T—DNA integration had been observed with the site of insertion appearing to vary among transformants (Fig 1B and 1D).

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Fig 1. Analysis of Z. tritici random mutants generated by Agrobacterium tumefaciens-mediated transformation (ATMT).

(A) Map of T-DNA construct used for genomic integration, containing HPT cassette (promoter+ORF+NOS terminator) flanked by left- and right-borders. HindIII restriction enzyme was used for the restriction of genomic DNA derived from the random mutants prior to the following Southern Blot analysis. The red bar indicates the location of probe used for hybridization. (B) Southern Blot analysis of 40 representative random transformants of Z. tritici. Genomic DNA of respective strains was restricted with HindIII. Black asterisks represent most likely a single T-DNA integration event; two or more hybridization signals indicate that most likely two or multiple T-DNA integrations in the recipient genome at different sites might have occurred. M: DNA Molecular Weight Marker VII, DIG-labeled (Roche Applied Biosciences). (C) Map of pCAMB-HPT (SalI)-vector used for ATMT. (D) Pie chart showing the relative percentage of occurred T-DNA integration events in the generated random mutants (the estimated percentage refers to 100 randomly selected transformants).

https://doi.org/10.1371/journal.pone.0183065.g001

Screening and analysis of dimorphic switch deficient mutants

By cultivating the generated strains on nitrogen starvation medium, which represented the “dimorphic switch”-inducing medium by mimicking the natural condition encountered on the plant leaf, all the transformants were screened for reduced or complete inability to perform the yeast-to-hyphal transition. In a second round of screening the preselected transformants were assayed on water agar medium, also representing a strong switch-inducing condition in order to confirm the observed dimorphic switch deficiency of the generated transformants.

Thus, the outcome of the random mutagenesis approach resulted in 11 insertional mutants, exhibiting “dimorphic switch” deficiency, hence providing a recovery frequency of 0.11% when extrapolating from the total number of transformants screened. All of them remained mitotically stable, maintaining the hygromycin B resistance. Five successive passages of these transformants on YEG medium without hygromycin did not result in the loss of integrated T-DNA containing the HPT cassette. Thus, after five subcultures the transformants grew on V8 containing 200 μg/ml hygromycin B, suggesting that the HPT gene was stably integrated. The integration/insertion of HPT cassette was additionally confirmed in all mutants by colony-PCR (data not shown). Ten of the recovered mutants were growing predominantly yeast-like under “dimorphic switch”-inducing conditions (Fig 2).

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Fig 2. Analysis of Zymoseptoria tritici random mutant strains.

The figure shows generated mutant strains (1.5 μl; 1×10⁸ spores/ml) grown on water agar medium and nitrogen starvation medium (N-deprivation) after 21 days of incubation at 18°C. N-deprivation medium was used as screening medium. Water agar (H₂O), which provides no external nutrition source was used for second round of screening. The wildtype strain IPO323 grows filamentously and forms mycelium on N-deprivation medium and water agar medium. The strains myco#1, myco#3, myco#20, myco#26, myco#56, myco#76, myco#77, myco#100 and myco#5552 show an extreme reduction in mycelium formation on both screening media. The myco#4 mutant grows poorly on H₂O medium showing a drastically reduced mycelium formation, however being able to switch to filamentous growth on N-deprivation medium. In contrast, myco#5 displays a hyperfilamentation phenotype under tested cultivation conditions, exhibiting a severely increased filamentous growth. Scale bar corresponds to 1 cm.

https://doi.org/10.1371/journal.pone.0183065.g002

Within the screening pipeline the dimorphic switch deficiency was defined as a qualitative measure rather than quantitative one, since across the experimental replicates for each of the mutant strains with reduced dimorphic transition ability were considerable fluctuations. Thus, none of the mutants (even the reference strain ΔZthog1) completely failed to generate hyphae on inducing medium, indicating that dimorphic switch requires a complex and coordinated conditional expression of multiple genes or even gene networks. Although all of the mutants were drastically reduced in their ability of mycelium formation, some hyphal growth occurred occasionally, even in the ΔZthog1 used as reference strain. Thus, strains bearing a ΔZthog1-like growth behaviour under switch-inducing condition after 7 days of incubation were considered as dimorphic switch deficient. Hence, nine random mutants, which were designated myco#1, myco#3, myco#20, myco#26, myco#56, myco#76, myco#77, myco#100, myco#5552, exhibited a yeast-like propagation under switch-inducing condition by incubating them on N-deprivation medium or water agar at 18°C (Fig 2). One mutant, designated myco#5, appeared to be locked in one growth stage producing a high amount of mycelium even on complete medium. Interestingly, myco#3 appeared to produce slightly more mycelium after 14 days of incubation on water agar compared to other strains with a yeast-like growth behavior. In case of myco#4, one striking feature was apparent: within the first 7 days of cultivation on N-deprivation medium, the mutant strain grew exclusively yeast-like (data not shown). However, when following the prolonged incubation period of 21 days, the morphological conversion starting at 14 days of incubation from yeast-like towards filamentous growth was evident, indicating a delayed mode of transition compared to that of wildtype strain IPO323. Interestingly, when myco#4 was grown on water agar, no visible formation of hyphae could be observed, even when the cultivation time was extended to 21 days. Furthermore, the growth was very sparse, resulting in a significantly reduced colony density compared to wildtype (Fig 2). For the random mutant myco#5, a predominantly filamentous growth was detected, regardless of the cultivation condition. Thus, incubating the mutant strain on the YEG standard medium at 18°C, which normally supports the yeast-like propagation of the wildtype strain, revealed in contrast to the wildtype a filamentous growth accompanied with the formation of aerial mycelium. Interestingly, a very similar growth behavior was noticed in YEG liquid medium, resulting in the enhanced mycelium formation, which was not observed in the wildtype strain IPO323 and other strains (data not shown).

Moreover, the results of the phenotypic screen with the selected mutant strains also agreed with pathogenicity assays on whole plants (susceptible cultivar Riband) as well as detached leaf assay. Generally, defects in dimorphic transition affected pathogenicity and resulted in very few necrotic flecks on the host plants. On the contrary typical disease symptoms (leaf necrosis and pycnidia formation) were observed on plants inoculated with conidial suspensions of the wildtype strain IPO323 (data not shown). A very intriguing phenotype was detected in the case of myco#5. Although this mutant predominantly grows filamentously, no typical symptoms of successful colonization of the plant developed.

Molecular identification of T-DNA tagged gene loci

Genome walking analysis was carried out with all dimorphic switch deficient mutants in order to isolate DNA sequences flanking the T-DNA integration sites. Prior to genome walking analysis, the selected mutants were subjected to Southern Blot analysis to determine the T-DNA insertion copy number in their genomes. The isolated gDNA of each mutant strain obtained was restricted with endonucleases HindIII, PspOMI and NotI (were all predicted to cut frequently in the genome sequence) and probed with PCR-amplified HPT cassette fragment. Hence, in case of myco#1, myco#3, myco#4, myco#5, myco#26, myco#56, myco#76, myco#100 and myco#5552 single hybridization signals were detected, indicating only single genomic sites of T-DNA insertion. For the mutant strains myco#20 and myco#77 several hybridization signals were observed, suggesting multiple genomic T-DNA insertion sites (data not shown).

The T-DNA integration sites were recovered by “step-down”-PCR amplification of genomic DNA fragments acquired by endonuclease restrictions from two enzyme sets, followed by an adaptor ligation using a modification of the method previously described [26]. By interrogating the genome sequence of Z. tritici provided by JGI (Zymoseptoria Sequencing Project, Broad Institute of Harvard and MIT, http://www.broadinstitute.org/), these T-DNA flanking sequences allowed the localization of the T-DNA integration sites and identification of putative gene loci affected by these integration events (Fig 3).

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Fig 3. Results of genome walking analysis of recovered T-DNA disrupted gene loci.

Predicted T-DNA insertion sites for each random mutant within putative genomic loci containing annotated genes are shown. DNA sequences obtained from genome walking PCR are illustrated as red lines aligned to a corresponding genomic locus; arrow heads determine the sequencing direction. Restriction enzymes used to obtain each of the DNA fragments prior to sequencing are indicated. Genes are named in concordance with JGI annotation by their Protein ID. The asterisks indicate the start position of the region matching the partial sequence of HPT cassette, thus pointing to the site of T-DNA integration for each mutant strain-specific locus. In case when the portion of HPT cassette was not identified, the putative genomic position of T-DNA inserted was estimated by fragment length obtained from restriction analysis and Southern Blot results. Predicted sites of T-DNA insertions are marked by vertical arrowheads. The “house-intern” RNA-Seq data of IPO323 were used to verify the current JGI models by mapping the aligned reads to the IPO323 genome sequence. The incorrectly annotated gene models obtained from JGI, as well as spliced junctions and contiguous sequences according to RNA-Seq data are indicated.

https://doi.org/10.1371/journal.pone.0183065.g003

Overall, among the T-DNA insertions sites detected, two (in case of myco#3 and myco#56) were located inside predicted open reading frames (ORFs) of annotated genes, while the remaining nine genes (myco#1, myco#4, myco#5, myco#20, myco#26, myco#76, myco#77, myco#100 and myco#5552) were positioned in the intergenic regions, meaning upstream or downstream of predicted ORFs. In addition, we used the “house-intern” (unpublished) RNA-Seq data derived from IPO323 cultures grown for 7 days under dimorphic switch inducing condition (N-deprivation medium), as well as publically available RNA-Seq data (obtained from Sequence Read Archive (SRA) at NCBI), to evaluate the existing gene models obtained from JGI. For most of the genes the predicted models were found to be incomplete (Fig 3). In case of MYCO5-locus, one gene model appears to be entirely missing, however there was a clear evidence of transcription based on RNA-Seq read alignments. For the remaining gene loci obtained, the boundaries of the JGI gene models were found to exclude the full extent of the transcribed regions. Hence, for e.g. MYCO76 and MYCO100 the entire exons of the corresponding genes are missing (Fig 3). The JGI database IDs for the predicted genes derived from genome walking analysis as well as further information concerning homology analysis and genomic coordinates of T-DNA insertions sites are shown in Table 1. The following section describes the random mutants for which T-DNA insertion sites could be identified:

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Table 1. Summary of Z. tritici genes identified from T-DNA flanking sequences.

https://doi.org/10.1371/journal.pone.0183065.t001

Verification of random mutants by targeted gene inactivation and initial phenotypic characterization

Thus, to deliver a proof of concept and to validate the genome walking results, we selected two genes for inactivation, designated MYCO1 and MYCO4, the orthologs of which were previously characterized in other fungal organisms.

Hence, a detailed sequence analysis of the amplified flanks in case of mutant myco#1 revealed that the T-DNA integration occurred in the 5’-UTR region (at 327 bp according to JGI gene model) or in the predicted exon region (when considering the RNA-Seq data) of the annotated gene with the Protein ID 70181 according to JGI Institute database or MYCGR3_70181 according to Ensemble (Fig 4). The precise location of T-DNA insertion was determined on chromosome 3 at genomic position 2.499.750 (Table 1, Fig 3). BLASTp analysis of the deduced protein sequence indicated that this gene encodes a homolog of Ssk1p, a known regulator within the HOG1-pathway in fungi [35–37], based on similarity to the Magnaporthe oryzae homologous protein with significant E-value of 1.24e-153 and 99% identity. Myco1p is a predicted 589 amino acid protein harboring a Response_Reg domain (Pfam ID: PF00072) located between amino acids 329–406. Based on these finding we designated the gene ZtSSK1.

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Fig 4. Structural analysis of target genes.

The figure illustrates the structure of the predicted target genes ZtSSK1 (A) and MYCO4 (B) affected by random mutagenesis. T-DNA location sites (HPT-integration) assigned in the corresponding gene loci are indicated. The numbers over the bars indicate the length of the annotated genes according to JGI gene models. The highlighted bars represent the functional domains of each gene, determined by Pfam database interrogation using InterProScan and mapped to the corresponding gene model. Introns are indicated as black lines joining gene exons, the latter represented as gray bars. Dash lines depict flanking regions of the genes. Orange triangles mark identified position of T-DNA insertion for each target gene. UTR: untranslated region.

https://doi.org/10.1371/journal.pone.0183065.g004

For the myco#4 mutant, the genomic T-DNA insertion location was identified on chromosome 8 at genomic position 84.634 (Figs 3 and 4), about 50 bp upstream of a predicted gene (JGI Protein ID: 87000; Ensembl: MYCGR3_87000) with high similarity to the bifunctional Saccharomyces cerevisiae ADE5,7 (46% amino acid identity) required for the second and fifth step of de novo purine nucleotide biosynthesis (Table 1). When considering the information obtained from RNA-Seq data, the T-DNA insertion is suggested to interrupt the hypothetical 5’-UTR region of the gene. This gene was previously functionally characterized in the yeast S. cerevisiae and C. albicans encoding a bifunctional enzyme possessing aminoimidazole ribotide synthase and glycinamide ribotide synthase activities [38,39]. The similarity is largely confined to the region where the predicted functional domains are located. These domains include PRibGlycinamide_synth_N (PF02844), PRibGlycinamid_synth_ATP-grasp (PF01071) and PRibGlycinamide_synth_C-dom (PF02843). However, using alignment analysis, we noticed an absence of functional domains AIRS (PF00586) and AIRS_C (PF02769) typically present in other fungi including the yeast homolog, indicating that the Z. tritici counterpart is monofunctional (Fig B in S1 File). In the pre-screening the random mutant lacking the functional gene exhibited a poor vegetative colony growth on poor-nutrient media like N-deprivation and water agar, providing a nutrient-less medium. When grown on YEG standard complex medium a very similar phenotype to wildtype was observed. Also, no defects in spore morphology were visible when comparing to the wildtype strain IPO323. However when grown on MM or N-deprivation medium the colonies of myco#4 mutant exhibited a sparse growth compared to wildtype, prohibiting a considerable biomass increase, thus indicated auxotrophic properties of the mutant.

The two genes selected were successfully inactivated through replacement of their ORF-portion of with HPT cassette conferring hygromycin resistance (Figs C and D in S1 File). The physiological properties of generated targeted mutants (in the following indicated as ΔZtssk1 (= Δmyco1) and Δmyco4) were confirmed exhibited the same phenotype as the corresponding random transformants. Genetic complementation of the targeted inactivation mutants with the corresponding native gene loci also demonstrated the responsibility of the respective genes for the observed phenotype, as far as the wildtype phenotype could be recovered. Therefore, the mutation resulting in the observed phenotypes corresponded to the loci for which the sequence was obtained as the T-DNA flank. As expected, on the switch-inducing medium (N-deprivation medium), which was used for the screening of switch-deficient transformants, ΔZtssk1 and Δmyco4 showed an impaired ability for filamentous growth. This observation was also supported by results obtained from germination assays (Fig A(B) in S1 File). In the case of Δmyco4 the yeast-to-hyphal transition was observed, but it appeared in a delayed manner after two weeks of incubation under defined conditions in comparison to the wildtype-strain where transition was visible after one week of incubation. Furthermore growth of Δmyco4 under nutrient deprivation provided by N-deprivation and H₂O agar media was found to be reduced and resulted in a poor colony development associated with a reduced cell density. However incubation of the mutant on YEG restored these growth defects and a very similar phenotype to that of wildtype was apparent. On the water agar both mutants were found to grow yeast-like, even if the incubation period was prolonged up to 21 days.

Dimorphic switch as determinant factor for full pathogenicity

In order to verify whether dimorphic switch deficiency impacts the pathogenicity of the mutants, we inoculated the susceptible wheat cultivar Riband with the targeted mutant strains and IPO323 along with ΔZthog1 as the reference strain. Expectedly, we didn’t observe any symptoms of typical STB disease on plants inoculated with ΔZtssk1 and Δmyco4. Both developed only small chlorotic lesions, which were also partly noticed for mock control, indicating the senescence and aging of the host plants (Fig 5). In constrast, the wildtype developed at first chlorotic and later necrotic lesions in course of disease progression and formed pycnidia after 21 days post inoculation. Similar results were obtained for complemented strains ΔZtssk1/ZtSSK1 and Δmyco4/MYCO4 generated by reintegration of intact gene copy for which the targeted mutants were disrupted. For both complemented strains the wildtype-like virulence was recovered and after latent phase of infection they finalized their infection cycle by formation of pycnidia bearing asexual spores (Fig 5).

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Fig 5. Plant infection assay with selected mutant strains.

Effects of the gene inactivation on disease development in the susceptible wheat cv. Riband after 21 days after inoculation (21 dpi) are shown. WT IPO323 and ΔZthog1 are regarded as references and mock as negative control, meaning plant leaves were sprayed with water only. The incubation of infected plants was performed with the following parameters: temperature: 22°C, humidity: 80%, Lightcycle: 16 h / 8 h. Both ΔZtssk1 and Δmyco4 along with ΔZthog1 are drastically reduced in virulence. Meanwhile, typical symptoms of successful infection were observed for WT IPO323, forming mature pycnidia 21 dpi. Full virulence to the loss-of-function mutants was completely restored by retransformation of the strains with respective functional full-length gene copies.

https://doi.org/10.1371/journal.pone.0183065.g005

ΔZtssk1 is sensitive to osmotic and oxidative stress

To assess the implication of ZtSsk1p in canonical osmoregulation consistent with its role as a HOG-pathway constituent, the ΔZtssk1 mutants as well as wildtype and Δmyco4 as controls were incubated on PDA and YEG media supplemented with NaCl and sorbitol at different concentrations. Both, ΔZtssk1 and ΔZthog1 were drastically reduced in growth under all osmotic stress conditions tested compared to control strains, however less pronounced when incubating upon sorbitol stress (Fig 6A). As expected, the growth of ΔZthog1 was impaired compared to wildtype after 7-day incubation on YEG or PDA supplemented with 1 M sorbitol/NaCl, resulting in reduced conidial density. A very similar phenotype to that of ΔZthog1 was observed in the case of ΔZtssk1, which was accompanied by a drastic growth reduction on the agar media mentioned above. Moreover, both were found to lack the ability to yeast-to-hyphal transition when incubating on PDA amended with osmotic stressors. This was in contrast to wildtype, Δmyco4 and complementation strains exhibiting normal growth and dimorphic switch ability (Fig 6A).

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Fig 6.

(A) Influence of inactivation of target genes on growth under osmotic stress. Spores of the corresponding mutant strains shown were spotted on the indicated media in serial dilutions (1.5 μl; 10⁸ spores/ml). The plates were incubated for 7 days at 18°C. The phenotypes observed were verified by three independent experiments. (B) Examination of susceptibility of generated mutant strains to oxidative stress. Colony morphology of Zymoseptoria tritici IPO323 and generated mutants under oxidative stress growth conditions. The wildtype (WT) strain and indicated mutant strains of YEG cultures were spotted as dilution series (1.5 μl; 5×10⁷ spores/ml) and incubated on YEG plates containing various concentrations of H₂O₂. Plates were imaged after 7 days of incubation at 18°C. Strains lacking ZtSSK1 or ZtHOG1 are more susceptible to oxidative stress, compared to the rest.

https://doi.org/10.1371/journal.pone.0183065.g006

Furthermore, we tested the sensitivity to oxidative stress consistent with evidenced implication of HOG-pathway in oxidative stress response provided by previous studies with other fungal organisms. Hence, incubating both ΔZthog1 and ΔZtssk1 on YEG supplemented with hydrogen peroxide even at low concentration (4 mM) resulted in the anticipated sensitivity consonant with results previously described [21]. Both exhibited a considerably reduced growth in contrast to Δmyco4 and wildtype strain IPO323 as a control, displaying only a slightly reduced growth (Fig 6B).

Disruption of ZtSSK1 results in fungicide resistance

To investigate the role of the response regulator ZtSsk1p in fungicide sensitivity, in vitro growth assays were performed. The outcome of the test revealed that ZtSSK1 disrupted mutants along with ΔZthog1 strains maintained an increased resistance to phenylpyrrole fungicide fludioxonil, both exhibiting similar phenotypes (Fig 7). This observation coincides with previous reports by Mehrabi et al., showing resistance of ZtHOG1 deficient mutant strains against phenylpyrrole fungicides, fludioxonil and fenpiclonil, as well as dicarboximide fungicide iprodione [21]. Contrarily, incubation of IPO323 reference strain and complementation strain ΔZtssk1/ZtSSK1 on PDA supplemented with 30 μg/ml fludioxonil resulted in a strongly pronounced sensitivity against fludioxonil. This observation also coincides with previously reported results with Neurospora crassa, Colletotrichum lagenarium and Alternaria alternata, indicating that ZtSsk1p acts upstream of ZtHog1p and both contribute to fungicide sensitivity [40–42].

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Fig 7. In vitro fludioxonil fungicide sensitivity assay.

Prior to photographs the strains were incubated on PDA amended with fludioxonil (30 μg/ml, dissolved in EtOH) for 7 days at 18°C. Four times serial dilutions of the YEG-grown culture (1×10⁸ spores/ml) were dropped on the plate. The ΔZtssk1, ΔZthog1 as well as random myco#1 mutants display resistance to the phenylpyrrole fungicide fludioxonil. Meanwhile, the wildtype strain IPO323 and the ectopic transformant exhibit abolished growth upon fludioxonil treatment.

https://doi.org/10.1371/journal.pone.0183065.g007

ZtSSK1 and ZtHOG1 are required for proper morphology and cell wall biogenesis

Furthermore, ΔZtssk1 mutants were found to be affected in their morphology by forming partly ovoid/swollen conidia (Fig A(A) in S1 File). Conidia prepared from ΔZtssk1 mutants germinated at rates and magnitudes much slower than those of wildtype when assayed on microscopic slides covered with water agar. More than 80% of wildtype conidia germinated while less than 12% of conidia prepared from ΔZtssk1 mutants were able to form germ tubes (Fig A(B) in S1 File). Moreover, the length of germ tubes produced from mutant conidia was significantly shorter than those of wildtype. Conidia collected from wildtype were found to predominantly form multiple germ tubes. In contrast, the majority of ΔZtssk1 mutant conidia produced single germ tubes, indicating that mutation of ZtSSK1 affected the pattern of conidial germination (data not shown).

Furthermore, to investigate whether the mutants exhibit cell wall defects, the wildtype and ΔZtssk1 were plated on media containing cell wall / membrane perturbing agents Congo red and sodium dodecyl sulfate (SDS). Congo red interferes with cell wall by binding β (1–3) glucan, whereas SDS is known to disrupt the plasma membrane. Compared to the wildtype, the ΔZtssk1 strains showed increased sensitivity to SDS, indicating that ZtSSK1 is involved in plasma membrane biogenesis, whereas the sensitivity to Congo red was unaffected (Fig 8A). Moreover, compared to wildtype the strains ΔZtssk1 and ΔZthog1 were found to be frequently lysed when treated with CWDEs (Fig 8B). Neither ΔZtssk1 nor ΔZthog1 treated with CWDEs was able to generate viable protoplasts, forming numerous broken cell fragments (data not shown) indicative of cell autolysis.

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Fig 8. Involvement of the target genes ZtSSK1 and MYCO4 in maintaining the cell wall integrity.

(A) Sensitivity of mutant strains to cell wall perturbing agents. The reference and mutant strains were grown on YEG medium, supplemented with 0.01% SDS or 2 mg/ml Congo Red (CR), for 7 days at 18°C. Four times serial dilutions of the grown culture (1×10⁸ spores/ml) were dropped on the plate. (B) Lysis test to examine protoplast release of the wildtype IPO323 and generated mutant strains. Percentage of degraded conidia due to protoplast production of corresponding strains is shown. The mutant strains ΔZtssk1 and ΔZthog1 display an enhanced sensitivity towards treatment with cell wall degrading enzymes from Trichoderma harzianum compared to wildtype. Data presented are the mean (including standard deviations) of results from the 3 independent experiments.

https://doi.org/10.1371/journal.pone.0183065.g008

Mutant lacking MYCO4 gene exhibits adenine auxotrophy

Proceeding from the previous BLASTp analysis with the MYCO4 gene revealed its involvement in de novo purine biosynthesis, insofar as significant functional orthologs previously characterized in other fungi were identified. Consistent with the putative role for MYCO4 gene in the de novo purine biosynthesis, the incubation of MYCO4 defective mutant on N-deprivation medium without adenine resulted in a drastically impaired growth (Fig 9A). As expected, Δmyco4 also failed to grow on N-deprivation solid medium supplemented with 1 mM glutamine (Fig 9A), as well as glycine and aspartate (data not shown), which are normally required as precursors for de novo synthesis of purine nucleotides. Supplementation of N-deprivation medium with both, either adenine or hypoxanthine at 1 mM, restored the wildtype-like phenotype of the mutant completely, suggesting that the purine salvage pathway is operational in the mutant strain. The growth was also restored and was comparable to that of the wildtype strain when the native copy of the MYCO4 gene was reintroduced in the loss-of-function mutant, indicating that the growth defect of Δmyco4 was due to the lack of functional MYCO4 allele. Interestingly, using lower concentrations of adenine added to N-deprivation medium the growth defect of Δmyco4 mutant could be rescued and the colony development was similar to that observed for the wildtype strain IPO323. Furthermore, there was no difference in growth when Δmyco4 was incubated on N-starvation medium supplemented with higher concentration of adenine or hypoxanthine, suggesting a sufficient uptake of external purines from the medium. The same phenotype was observed by cultivating the Δmyco4 mutant strain on N-starvation medium supplemented with reduced amount of hypoxanthine (Fig 9A). Furthermore, as previously mentioned, the yeast-to-hyphal transition of Δmyco4 occurred after a lag period of approximately 14 days on N-deprivation medium and was completely abolished on water agar. With addition of external adenine, even at low concentration, the wildtype-like “dimorphic switch” ability of the mutant strain was restored (Fig 9B). Consequently, these observations indicate that MYCO4 is involved in de novo purine biosynthesis in Z. tritici, since the loss-of-function allele causes auxotrophic requirement for adenine.

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Fig 9. Growth of the Δmyco4 mutant under purine auxotrophic conditions.

(A) Inactivation of MYCO4 gene results in the auxotrophic, purine requiring mutant. MYCO4 deficient mutant is unable to grow on media, lacking the external source of adenine or hypoxanthine. Complementation of Δmyco4 mutant with the full-length copy of MYCO4 gene including its native promoter and terminator region or incubation of Δmyco4 in the presence of 0.01/0.1 mM adenine or 0.1 mg/ml hypoxanthine restored the wildtype growth. The examination of colony growth and morphology was performed after 7 days incubation on respective media at 18°C. The isolated spores were spotted as dilution series (1.5 μl; 10⁸ spores/ml). (B) Yeast-to-hyphal transition restored for Δmyco4 when incubating on water agar (WA) supplemented with 0.01 mM or 1 mM adenine for 7 days at 18°C.

https://doi.org/10.1371/journal.pone.0183065.g009

ZtSSK1 is involved in oxidative and osmotic stress responses and responsible for fludioxonile sensitivity

Thus, targeted inactivation of ZtSSK1 in Z. tritici resulted in a ROS-sensitive phenotype, similar to that observed in other fungal organisms previously reported [60,61,44]. Apart from ROS resistance, an important role for a ZtSSK1 response regulator in cellular tolerance to osmotic stress also was demonstrated in the present study. Thus, Z. tritici mutants impaired for ZtSSK1 display increased hypersensitivity to NaCl and sorbitol, a phenotype highly resembling the mutants lacking ZtHOG1 [21]. The involvement of ZtSSK1 in osmotic stress resistance is supported further by the fact that the ΔZtssk1 mutants produced very few or no protoplasts after incubating with cell wall-degrading enzymes. This stays in agreement with results previously reported by Mehrabi et al., demonstrating that ZtHOG1 deficient mutant strains of Z. tritici also failed to generate viable protoplasts. Generally, the finding that ΔZtssk1 mutants are impaired in their ability to cope with environmental stress is consistent with the prediction that ZtSSK1 is involved in stress response and regulation. In addition we could also confirm the previously reported requirement of HOG-pathway for fungal development and in particular for cell wall / plasma membrane biogenesis. We stated an enhanced lysis rate of conidia collected from ΔZthog1 and ΔZtssk1 mutant strains when treated with CWDE. This was also supported by in vitro growth test on SDS medium, showing elevated sensitivity of the mutant strains against this membrane perturbing agent compared to wildtype. Moreover, as previously shown for ΔZthog1, the inactivation of ZtSSK1 also resulted in resistance to fludioxonil fungicide, indicating the interaction among the signaling components in terms of fungicide sensitivity. These results also stay in concordance with those previously obtained for Alternaria alternata, showing that AaSSK1 is involved in ROS resistance, osmotic resistance, fungicide sensitivity and fungal virulence [44]. As expected, ZtSSK1 was also found to play an important role in the establishment of plant colonization because gene inactivation mutants were reduced in their ability to induce necrotic lesions on susceptible wheat cultivar Riband. The explanation for this observation is due to the impaired ability of ΔZtssk1 mutants to undergo dimorphic transition leading to decreased colonization of host tissue. In addition, the elevated sensitivity to ROS observed may be causative for this phenotype.

Inactivation of MYCO4 results in purine auxotrophy

An interesting phenotype was observed in case of the myco#4 random mutant. The gene in which the corresponding mutant was disrupted was found to encode a bifunctional phosphoribosylglycinamide synthetase (glycinamide ribotide synthase/aminoimidazole ribotide synthase; indicated as GART*), which is known for the second and fifth catalyzing step of de novo purine biosynthesis in Schizosaccharomyces pombe [62]. De novo purine biosynthesis has been extensively studied in different organisms, including fungi, bacteria, plants and animals, particularly in much detail in Escherichia coli and baker’s yeast S. cerevisiae and has been reported to be highly conserved in evolution, even between prokaryotes and eukaryotes [63–65].

Since our preliminary BLAST analysis indicated a high degree of conservation of purine biosynthetic enzymes in Z. tritici with those of distantly related Schizosaccharomyces pombe as well as closely related filamentous fungi such as Neurospora crassa, Magnaporthe oryzae, Aspergillus nidulans and Fusarium graminearum, it is highly conceivable that a similar pathway is present in Z. tritici.

Interestingly, the structural analysis of the deduced protein sequences revealed a diverged structure (and function) of the corresponding protein orthologs. The evidence for this is provided by comparison of deduced Myco4p with bifunctional homologs of other fungi including S. cerevisiae and the trifunctional mammalian homolog. Hence, InterProScan analysis revealed for Z. tritici and closely related fungi from Mycosphaerellaceae family only GARS domains responsible for conversion of 5-phospho-β-D-ribosylamine (PRA) to 5-phosphoribosylglycinamide (GAR), in contrast to other fungal representatives harbouring an additional AIRS domain (Fig B in S1 File). Meanwhile, the Homo sapiens homolog combines the functions of Ade5,7p, catalyzing the second and fifth steps of the de novo purine biosynthesis in analogy to fungal representatives, but owns additional domain responsible for the conversion of 5-phosphoribosylglycinamide (GAR) to 5-phosphoribosyl-N-formylglycinamide (FGAR). This finding is intriguing since the monofunctional members are only common for bacteria and plants. Hence, the three steps mentioned are catalyzed by three enzymes in bacteria and plants, but two in fungi (Fig 10). The reason for the predominant monofunctional variant of the GART* protein in Zymoseptoria clade is currently unknown and the biological role for this phenomenon remains to be elucidated. In light of the assumed structural difference, but at the same time retained functional conservation of the enzyme, the question remains, however, whether it is possible to identify inhibitors that are specific to the fungal GART*s.

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Fig 10. Schematic representation of the de novo purine biosynthesis in the yeast Saccharomyces cerevisiae.

Abbreviations: PRPP: 5-phospho-ribosyl-1α-pyrophosphate, PRA: 5-phospho-β-D-ribosylamine, GAR: 5-phosphoribosylglycinamide, FGAR: 5'-phosphoribosyl-N-formyl glycinamide, FGAM: 5'-phosphoribosyl-N-formylglycinamidine, AIR: 5'-phosphoribosyl-5-aminoimidazole, CAIR: 5'-phosphoribosyl-5-aminoimidazole-4-carboxylate, SAICAR: 5'-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole, AICAR: 5'-phosphoribosyl-4-carboxamide-5-aminoimidazole, FAICAR: 5'-phosphoribosyl-4-carboxamide-5-formamidoimidazole, IMP: inosine monophosphate, AMP: adenosine monophosphate, GMP: guanosine monophosphate. The S. cerevisiae gene ADE5,7 (depicted in red) is homologous to Z. tritici MYCO4. In contrast to bifunctional yeast counterpart, the Z. tritici has only GARS activity (monofunctional). The animal counterpart is predicted to encode a bifunctional enzyme complex, exhibiting GARS, AIRS and FGAR activities. The trifunctional human GART* is highlighted by blue box.

https://doi.org/10.1371/journal.pone.0183065.g010

However, this structural/functional difference observed may result in differentially coordinated regulation of the three enzymatic activities (assigned to three different enzymes in Z. tritici) with global impact on metabolic processes. The remaining two genes encoding AIRS (Protein ID: 74864) and GART (Protein ID: 73624) were found in Z. tritici genome.

The striking difference in conformational structure suggested between mammalian and fungal orthologs implies that Myco4p may be regarded as a potential therapeutic target for the development of novel antifungal drugs. In fact, because the human GART requires 10-formyl-tetrahydrofolate (THF) as a cofactor, the corresponding part of the trifunctional enzyme has been addressed as an anticancer target for THF analogues by inhibiting its activity [66]. This difference emphasizes the phylogenetic divergence of the fungal GART*s and their human counterparts. As expected, the targeted inactivation of the gene in Z. tritici resulted in auxotrophic phenotype of the generated mutant strains. Without supplemented adenine or hypoxanthine none of the targeted mutant strains were able to grow on N-deprivation basal medium, lacking the external source of purines. However when providing external purines, even at low concentration the wildtype growth could be restored, indicating that the salvage purine pathway was operational in the mutants. Collectively, our observations lead to the conclusion that the impaired purine (adenine) biosynthesis in MYCO4 deficient mutant strain affects cellular functions associated with growth and development, and appears to be causative for abolished pathogenicity. Previously, the role of de novo purine biosynthesis has also been addressed in dimorphic systemic fungi like C. albicans and C. neoformans, showing that disruption of genes involved in this pathway results in attenuated or complete loss of virulence in mammalian models and general growth defects [67,68]. Consequently, this implies that Myco4p represents a virulence factor necessary for disease establishment. Questionable is, however, the ability of mutant strain to use the exogenous purine nucleotides in planta, since our results indicate that growth of the mutant strain is restored by supplementing the synthetic media with exogenous adenine/hypoxanthine sources even at lower concentrations compared to those that are present in the plant apoplast. Therefore, further investigations should be carried out in order to analyze the ability/inability for the exogenous purine uptake in planta.

Additionally, the attributed traits concerning the auxotrophy of the mutant strain open up a further possibility which could be advantageous for the utilization within biotechnological and molecular biological applications. Complementation of the MYCO4-disruption strain with the intact copies of MYCO4 suggests that there could be a potential use of this gene as an auxotrophic selectable marker for genetic transformation in Z. tritici, as was previously shown for Giberella zeae (Fusarium graminearum) [69]. In general, genes responsible for adenine or arginine auxotrophic phenotype in other fungi have been broadly used as selection markers for transformation systems in Pichia pastoris, Aspergillus oryzae and Saccharomyces cerevisiae [70,71]. The usage of MYCO4 in a similar way would undoubtedly extend the arsenal of selection markers used today for transformation and functional genomics of Z. tritici.