Profile of the in vitro secretome of the barley net blotch fungus, Pyrenophora teres f. teres

Physiological and Molecular Plant Pathology 109 (2020) 101451

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Profile of the in vitro secretome of the barley net blotch fungus, Pyrenophora teres f. teres

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M. Jordi Muria-Gonzaleza,∗, Katherine G. Zulaka, Eef Allegaertb, Richard P. Olivera, Simon R. Ellwooda a b

Centre for Crop and Disease Management, Curtin University, Western Australia, Australia Biomedical Sciences Group, KU Leuven, Leuven, Belgium

A R T I C LE I N FO

A B S T R A C T

Keywords: Pyrenophora teres Hordeum vulgare Proteomics Effectors LC-MS

Plant pathogen interactions are governed by a molecular interchange in which effectors, secreted by the pathogen, are key players. Effectors are yet to be characterised in Pyrenophora teres f. teres, an important pathogen of barley. Proteins contributing to culture filtrate phytotoxicity were identified by chromatographic fractionation followed by mass spectrometry-based proteomics. Among the most prevalent proteins in the active fractions were glycosidases (20%), peptidases (8%), other hydrolases (8%) and a high proportion of proteins without functional annotations (30%). Furthermore, additional proteins were predicted to be similar to known chitin binding, ribonuclease-like and kinase effectors along with a hypothetical protein which was predicted to be an effector. We discuss the known properties of the groups of proteins identified in this study and their possible roles in the plant-pathogen interaction. The dataset of proteins presented is a valuable resource for discovering potential effector proteins in net blotch disease, and informs further genetic and in planta studies to dissect the molecular interactions of barley and P. teres f. teres.

1. Introduction Net blotch is a major disease of barley (Hordeum vulgare) worldwide that is caused by the fungal pathogen Pyrenophora teres. Two different forms are recognised, net type of net blotch (NTNB) and spot type of net blotch (STNB), produced by P. teres f. teres (PTT) and P. teres f. maculata (PTM), respectively [1]. PTT and PTM are considered as different forms of the same species but may be regarded as genetically distinct populations [2], although rare hybridisation events are known to occur [3]. Despite sharing a common niche and being closely related, resistance to PTT and PTM is governed by different host genes suggesting different pathogenicity factors influence the two diseases [4]. While genetic and genomic approaches have identified pathogenicity QTLs [5–7] and histological studies of the development of this fungi in planta describe the infection process [8], the underlying molecular interactions between PTT and barley are still largely obscure. Diverse mechanisms have evolved in pathogens allowing the subversion of plant defence including the secretion of proteins, small molecules and small RNAs [9]. In net blotch, early research discovered the phytotoxic compounds pyrenolines [10], pyrenolides [11] and three peptide alkaloids, aspergilomarasmine A (toxin C) and its derivatives,

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toxin A and toxin B [12]. The pyrenolines and pyrenolides are general toxins affecting different plants, but the peptide alkaloid toxins behave in a more host selective manner, causing chlorosis, rather than the classical net blotch necrosis, after infiltration of the pure compound. These effects correlated to the degree of susceptibility of different barley lines to the net blotches [13]. Later research on Australian isolates has suggested that proteinaceous toxins or effectors contribute to the establishment of net blotch of barley [14]. While effectors are commonly defined as small, cysteine-rich and secreted proteins for historical reasons, the term effector should not define a particular type of molecule but rather a particular function within a pathosystem. The definition we use here is: molecules secreted by a host-associated organism which exert an effect on the host response leading to the proliferation of the secreting organism or the restriction of its growth [15]. Examples of non-protein effectors include “small molecules” such as T-toxin, HC-toxin and victorin from Cochliobolus species [16], and small RNAs such as Bc-siR3.1, Bc-siR3.2, and Bc-siR5 from Botrytis cinerea [17]. Net blotch is a hemibiotroph exhibiting complex plant-pathogen interactions and does not conform to either the standard or inverse gene for gene hypotheses [4,18,19]. On the one hand, early research found

Corresponding author. E-mail address: [email protected] (M.J. Muria-Gonzalez).

https://doi.org/10.1016/j.pmpp.2019.101451 Received 26 July 2019; Received in revised form 27 September 2019; Accepted 8 November 2019 Available online 09 November 2019 0885-5765/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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PTT W1-1 mycelial homogenate. Flasks were placed in an orbital shaker incubator (Thermoline Scientific, Sydney, Australia) at 24 °C and 100 rpm in the dark for three days prior to static incubation in the dark at room temperature for one month. After static incubation, cultures were filtered through gauze, followed by two rounds of vacuum filtration through 4 layers of filter paper (Whatman™ 1, GE Healthcare, Little Chalfont, UK), then vacuum filtration through a 0.2 μm filter (Corning, New York, USA).

major dominant resistant genes in barley which is a signature of the classical gene-for-gene interaction [18,20], whilst on the other hand, there is also evidence of dominant susceptibility genes in barley as in the inverse gene for gene interaction observed in some necrotrophic infections [19,21]. Furthermore, the presence of multiple virulence QTLs per interaction and across pathotypes of the population adds complexity to the pathosystem (Anke Martin, USQ, pers. comm.). To investigate the molecular interchange between the fungus and the host, previous 2D-gel based proteomic studies correlated the secretion of several PTT and PTM proteins in vitro and symptom development in barley [22–25], identifying xylanases, glucanases, spherulins, cutinases, ceratoplatanins, and proteases as possibly playing a role in virulence. In this study, the protein fraction of the whole culture filtrates was also found to be responsible for necrotic symptoms of PTT isolate W1-1 when infiltrated into leaves. To our knowledge, there are only two previous proteomic studies on proteins secreted by PTT and both used gel-based separation of the culture filtrate [22,24]. The main objective of this study was identify the range of proteins present in phytotoxic fractions from the in vitro secretome of PTT. To select for proteins contributing activity and to reduce the complexity of the whole culture filtrate, anion exchange chromatography was performed, followed by size exclusion chromatography on pooled fractions. Liquid chromatography-tandem mass spectrometry (LC-MS2) was used as a more sensitive method to detect low abundance proteins as well as low molecular weight and hydrophobic proteins. Finally, proteins identified from peptide matches against the genome of PTT W1-1 were annotated by their putative functional gene ontologies and in silico probability of being effectors, and are discussed in comparison to similar proteins in the literature.

2.3. Separating small molecules from macromolecules and protease treatment

2. Materials and methods

To separate the proteins from the smaller metabolites, 20 ml samples from the PTT W1-1 whole culture filtrate of four flasks were independently filtered through Ultracel 3 KDa Amicon centrifugal filters (Merck Millipore, Tullagreen, Ireland) in a centrifuge (5810 R, Eppendorf AG, Hamburg, Germany) at 3200 RCF and at 4 °C for 60 min. The small molecule fraction contained the bulk of the culture media while the macromolecular fraction was reconstituted up to 20 ml in potassium phosphate buffer (0.1 M pH 5.8). The phytotoxicity of each fraction was evaluated by infiltration into barley cv. Baudin. Aliquots of 1 ml from each of the macromolecular fractions were transferred into 2 ml microcentrifuge tubes to digest the contained proteins. To each tube, 25 μl of a freshly prepared aqueous solution of 20 mg/ml Pronase protease (Calbiochem, San Diego, CA, USA) was added to the flask and incubated at 37 °C for 2 h in a Thermo Mixer C (Eppendorf AG, Hamburg, Germany). To inactivate the Pronase the temperature was raised and maintained at 80 °C for 20 min. The aliquots were centrifuged at 9000 rcf during 1 min in a Microfuge 20 centrifuge (Beckman Coulter, Krefeld, Germany) before infiltration into barley leaves.

2.1. Plant and fungal material

2.4. Chromatographic fractionation of culture filtrates

PTT W1-1 was used in this study since this Western Australian isolate has a high quality genome sequence which is publicly available [26]. W1-1 was maintained on V8-PDA solid media (150 ml/l V8 juice (Campbell's Soups Australia, Lemnos, VIC, Australia), 10 g/l Difco™ potato dextrose agar (BD, Sparks, MD, USA), 3 g/l CaCO3 and 10 g/l agar (Oxoid Ltd., Basingstoke, UK)) and in Fries 2 liquid media (5 g/l ammonium tartrate, 1 g/l ammonium nitrate, 0.5 g/l MgSO3·7H2O, 1.3 g/l KH2PO4, 2.6 g/l K2HPO4, 30 g/l sucrose (10 mM), 1 g/l yeast extract (BD, Sparks, MD, USA), 334 μg/l LiCl, 214 μg/l CuCl2·2H2O, 160 μg/l CoCl2·4H2O, 144 μg/l MnCl2·4H2O, 68 μg/l H2MoO4). Given that the objective of this study is to profile those proteins exerting a phytotoxic response in barley irrespective of their cultivar specificity, a susceptible net blotch cultivar (cv.), Baudin [27], was used in infiltration experiments with W1-1 proteins. The whole culture filtrate of W1-1 was also infiltrated into 17 other barley cultivars to observe cultivarspecific responses. The following cultivars were used: 2ND25389, CBSS95M00804T-4Y, Flagship, ND24260-1, ND24388-1, NRB06059-1, Maritime, Grout, Fairview, Beecher, Prior, Skiff, CI 20019 Russ, CIho 5791, CIho 9825, Mackay and Scope. In all cases, plants were sown in propagation trays with 5 cm2 cells (Garden City Plastics, Melbourne, Australia) containing vermiculite, fertilised with 1 teaspoon of Thrive all-purpose fertiliser (Yates, Auckland, New Zealand). Plants were maintained at room temperature, watered every three days (or before trays dried out) and grown under a 12 h light:12 h dark cycle for two weeks before infiltration.

Protein chromatography was performed in an AKTA Purifier UPC10 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) controlled with the Unicorn 5.31 workstation. For anionic exchange chromatography (AEC), five batches of culture filtrate were dialysed together for 8 h in 0.4 M Tris buffer pH 8.8 using a Snake Skin dialysis tubing with a 3.5 KDa cut-off (Thermo Scientific, Rockford, IL, USA). 170 ml of dialysed culture filtrate was chromatographically fractionated a 5 ml HiTrap QFF anion column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Separation was performed in a step gradient starting at 100% buffer A (40 mM Tris pH 8.8) followed by five increments of 20% buffer B (40 mM Tris pH 8.8 + 1 M NaCl) lasting five column volumes each until 100% solvent B was reached using a flow of 5 ml/min while collecting 5 ml fractions. A first fractionation was performed to evaluate the biological activity of each step; fractions per step were pooled together and concentrated using Ultracel 3 KDa Amicon centrifugal filters (Merck Millipore, Tullagreen, Ireland) centrifuged in a refrigerated centrifuge (5810 R, Eppendorf AG, Hamburg, Germany) at 3200 RCF and at 4 °C for 60 min. The phytotoxicity of each pooled fraction was evaluated by infiltration into barley leaves. A second separation was done using the same conditions as the previous with a new set of five culture filtrates, then the head and body of the active peak collected (fractions AEC 1–2). For the size exclusion chromatography (SEC), fractions AEC 1–2 were pooled together with a 3 KDa Amicon filter (4 °C, 3200 RCF, 60 min). To wash the proteins, samples were reconstituted in phosphate buffer pH 7 and centrifuged, and then reconstituted again in the same volume of buffer. To concentrate the samples a final centrifugation step was performed and the proteins reconstituted in 1 ml of 20 mM sodium phosphate buffer pH 7 + 0.15 M NaCl. The concentrated sample (5 mg) was injected into a HiPrep 16/60 Sephacryl S-200HR 120 ml column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) using a 1 ml loop and an isocratic flow rate of 0.5 ml/min using 20 mM phosphate buffer

2.2. In-vitro fungal culture PTT W1-1 mycelia was collected with a sterile scalpel from 5 day old V8-PDA cultures and homogenised in 600 μl of sterile water using a 1.5 ml microcentrifuge tube and a micro pestle. 250 ml Erlenmeyer flasks containing 70 ml of Fries 2 media were inoculated with 100 μl of 2

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Blast hits 5; word size 3; low complexity filter on; HPS length cutoff 90; HPS-hit coverage 60; filter by description, no) followed by a GO mapping (Goa version 2019.02), and GO annotation (annotation cutoff, 55; GO weight, 5; E-Value hit filter, 1 × 10−6; HPS-hit coverage cutoff, 0; hit filter, 500; ISS, ISO, ISA, ISM, IBA, IBD, IKR, RCA, and NAS, 0.8; IGC, IRD, and IEA, 0.7; TAS and IC, 0.9; ND, 0.5; NR, 0; experimental evidence codes: all 1). An EMBL-EBI InterPro analysis was also performed (parameters: Families, Domains, Sites and Repeats: all on, except PatternScan; Structural Domains: all on; other sequence features: MobiDBLite and Phobious on) and Interpro terms merged to the annotation.

pH 7 + 0.15 NaCl. 1 ml fractions were collected from SEC 35 to SEC 82 and grouped in sets of three yielding 16 pooled fractions which were concentrated using 3 KDa Amicon filters (60 min, 4 °C, 3200 RCF). These pooled fractions were then infiltrated into barley cv. Baudin leaves to evaluate its phytotoxicity and select the active ones for proteomic analysis. 2.5. Infiltration of barley leaves The second leaf of two week old barley seedlings was infiltrated with 50–100 μl of the culture filtrate or the chromatographic fractions, and sterile media or the correspondent phosphate buffer used as controls. The liquid to be infiltrated was loaded into a needleless 1 ml syringe and the tip placed on the adaxial side of the leaf; the liquid was infiltrated with a gentle continuous pressure to the plunger, opposed by a gentle pressure of a finger in the abaxial side of the leaf. Unless otherwise stated, all symptoms were recorded at 7 days post infiltration. For the analysis of the whole culture filtrate, and the small molecule and macromolecule fractions, four biological replicates were used to infiltrate a minimum of five seedlings each. When testing the chromatographic fractions, each was infiltrated into a minimum of five plants. For each experiment, controls were also infiltrated into a minimum of five plants. Whole culture filtrate, small molecule and protein fractions, anion exchange chromatography fractions, and size exclusion chromatography fractions were tested independently as separate experiments. Reproducible symptoms were observed following infiltration, and therefore visual symptoms were sufficient for recording or selecting active fractions for downstream analysis.

3. Results and discussion 3.1. W1-1 culture filtrate activity lies within the macromolecular fraction Culture filtrates from PTT and PTM are known to produce necrotic lesions and have been associated with either small molecules or secreted proteins [13,14]. To distinguish symptoms caused by metabolites from those caused by proteins, membrane filters were used to separate the small molecule fraction from the macromolecular fraction containing masses higher than 3 KDa. When the PTT W1-1 small molecule fraction was infiltrated into the second leaf of barley seedlings (cv. Baudin) no symptoms were observed; however, clear and defined necrotic lesions developed on leaves infiltrated with the macromolecular fraction (Fig. 1). Pronase treatment of this fraction abolished the observed activity (Fig. S1), indicating the involvement of proteins. These results are consistent with previous reports showing that the necrotising activity of culture filtrates from Australian Pyrenophora teres isolates is caused by proteins [14]. However, these observations alone are not enough to rule out the involvement of specialised metabolites (also known as secondary metabolites) in barley net blotch pathogenesis since in vitro gene expression may not mimic in vivo conditions inside the plant. In addition to the infiltration on the susceptible cv. Baudin, the effect of the whole culture filtrate of PTT W1-1 was also evaluated on 17 other barley varieties which showed a range of responses from mild chlorosis to strong necrosis (Fig. S2). This suggests that the active proteins secreted in vitro by W1-1 are cultivar-specific.

2.6. Proteomic analysis of the PTT W1-1 active fractions The active size exclusion chromatography fractions were sent to the Protein Discovery Centre, QIMR Berghofer (Brisbane, Queensland, Australia) for analysis. Protein concentration was determined using a 2D Quant assay (GE Healthcare Life Sciences, Piscataway, NJ, USA). Samples were reduced with tris (2-carboxyethyl) phosphine (TCEP), alkylated with iodoacetamide, then co-precipitated and digested with trypsin. Digested samples were then separated on a C18 column Ultra Performance Liquid Chromatography (UPLC) gradient run (nanoACQUITY, Waters Corporation, Milford, MA, USA) and analysed by high-resolution tandem mass spectrometry (LTQ Orbitrap Elite, Thermo Scientific, Bremen, Germany). Each sample was analysed by LC/MS2 using collision-induced dissociation (CID) with ion trap mass analyser and then by higher-energy collisional dissociation (HCD) with an Orbitrap. Data was analysed by searching a combination of the PTT genome (GCA_900232044.1) together with contaminant databases plus respective decoy databases (reversed databases) using the Mascot search engine v. 2.5 (variable modifications oxidation (M) and deamidation (N,Q); fixed modification, carbamidomethyl (C); precursor mass tolerance, 20 ppm; fragment mass tolerance, 0.8 Da (CID)/0.03 Da (HCD); enzyme used trypsin with 2 missed cleavages). Peptide and protein probabilities were assigned with Scaffold v. 4.4.1 using the built-in prophet-tools (http://peptideprophet.sourceforge.net/) from the Trans-Proteomic-Pipeline to accept peptide and protein identifications. Protein threshold parameters: 1% FDR; minimum number of peptides, 2; peptide probability 95%; charge state, +2/+3).

3.2. Multiple general elicitors are present in phytotoxic fractions In order to profile the PTT W1-1 culture filtrate components that may be responsible for necrotising activity, the secretome was enriched for active proteins by a bio-guided chromatographic fractionation. Two-

2.7. Gene ontology (GO) analysis and effector scoring Fasta files containing proteins identified from both the cell free culture filtrates and the active fractions were submitted to Blast2GO v 5.2.5 [28] to determine predicted functions based on the functional gene ontologies (GOs). EffectorP v2.0 [29] was used to evaluate the probability of each protein of being an effector. The data in Blast2Go was subject to an NCBI BLASTp search (parameters: Taxonomy Filter, fungi; E-value 1 × 10−15; number of

Fig. 1. Effect of small molecule (< 3 KDa) and macromolecular (> 3 KDa) fractions from the culture filtrate of P. teres f. teres W1-1 on barley leaves after infiltration. The second leaves of barley cv. Baudin were infiltrated with ~50 μl of each fraction; leaves were excised and the effects evaluated four days post infiltration. The black marks delimit the area of infiltration. Background was digitally darkened. 3

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observed, in a separate study xylanase activity was reported to cause cell death in maize and tobacco plants [34,35]. Furthermore, a study showed a differential expression in vitro of a PTT xylanase, PttXyn11A, between highly virulent and low virulence isolates [24]; however expression of PttXyn11A occurred at 168 h post inoculation and was not temporally correlated with in planta symptom development, which started at 64 h post inoculation. Hydrolytic enzymes in the fungal cultures may therefore play a role in the development of necrosis after leaf infiltration; but whether this is relevant for the onset of net blotch infection requires further investigation. Among the degrading enzymes that fungi secrete, peptidases are of particular interest in pathogenesis. Apart from the obvious direct role of processing substrates for gaining nutrients, there is extensive evidence that peptidases or peptidase-like proteins play important roles in the pathogenicity of diverse microbes [36]. Fungi possess a diverse array of peptidases depending on their lifestyle and the expression of some of these proteins has been found to correlate with the specific stages of the disease cycle in the wheat pathogen Zymoseptoria tritici [37]. The mechanisms of action of microbial proteases during the pathogenic interactions are diverse. In some cases the products of the proteins can be detected as PAMPs [38,39], but in others the proteases act as effectors triggering a hypersensitive response [40,41]. Peptidases can also interfere with the plant machinery suppressing the defence response [36]. Therefore, the peptidases detected in the active fractions may contribute to the observed necrotising activity but further characterisation of their significance is required. Among the top 20 most abundant proteins of each fraction, different proportions are present based on their functional gene ontology annotations (Table 1). Glycosidases are clearly over-represented in SEC 41–43 with 8 proteins (40%), while SEC 53–55 and SEC 62–64 have 3 (15%) and 4 (18%) respectively; peptidases were the second most abundant functional annotation in SEC 41–43 (15%) and SEC 53–55 (10%) but none were present in SEC 62–64. On the contrary, chitin binding proteins represent 10% of the 20 most abundant proteins in SEC 62–64 but none were found within the most abundant proteins of SEC 41–43 and SEC 53–55. Another difference among top 20 proteins between the active fractions is the proportion of proteins without functional annotation, 15% of SEC 41–43, 40% of SEC 53–55 and 45% of SEC 62–64. Interestingly, one of the top 20 most abundant proteins in fraction SEC 41–43, Ptt_W11.g0029 (homologous to NCBI PTT 0–1 EFQ88112.1 hypothetical protein PTT_16150), was reported previously to be expressed in vitro by a virulent isolate but not by an avirulent isolate, although in planta expression did not suggest involvement in disease onset [24].

stage fractionation of proteins was performed by anion exchange and size exclusion chromatography. Thirty 5 ml anionic exchange chromatography (AEC) fractions were obtained and pooled into five groups corresponding to each step of the anionic gradient. Pooled fractions were concentrated with 3 KDa Amicon filters and reconstituted in phosphate buffer before infiltration into barley leaves. Proteins from the first step of the gradient showed the most severe symptoms. A second fractionation of a new batch of whole culture filtrate was performed and fractions AEC 1 and 2, representing the bulk of the chromatographic peak of the first step, were selected for size exclusion chromatography (SEC), yielding 16 pooled sub-fractions which were desalted and infiltrated into barley cv. Baudin leaves. The activity was not concentrated in a small group of fractions but spread over several fractions, nonetheless three islands of stronger necrotising activity were observed on pooled sub-fractions SEC 41–42, SEC 53–55 and SEC 62–64 (Fig. S3). Proteomic analysis was performed on each of these groups of subfractions, identifying 182 unique proteins. One hundred and nineteen were present in SEC 41–44, 104 in SEC 53–55 and 75 in SEC 62–64 (Table S1). A GO search yielded no functional information for a third of the proteins identified (Table S1). From those with GO annotations (Fig. 2), the most abundant were glycosidases (27–33% of all proteins in each fraction) followed by peptidases (9–13%) and other hydrolases (9–11%). In SEC 41–43 and SEC 53–55, FAD binding domain oxidoreductases were also highly represented, at 10% and 12% respectively, but represented just 2% of the functionally annotated proteins in SEC 62–64. Three to five chitin binding proteins were found in each fraction, representing 4%, 7% and 8% in SEC 41–43, SEC 53–55 and SEC 62–64, respectively. Proteins binding to copper, manganese, proteins and carbohydrates were also identified. Other functions associated with some of the proteins in the fractions were transferases present at 5–8% of all proteins, and less abundant nucleases, lyases, kinases, oxygenases and ligases. Single proteins with unique functions were grouped into the ‘other function’ category. While fungi secrete a plethora of degrading enzymes for acquiring nutrients from complex substrates, many hydrolases, including glycosidases and proteases, and some transferases and lyases are involved in the degradation of the plant cell wall and play important roles in pathogenesis [30]. The plant cell wall is a physical barrier for invading microorganisms and the secretion of cell-wall degrading enzymes (CWDE) enables the pathogen to breach it; however, the products of the CWDE such as oligosaccharides, as well as the enzymes themselves, may interact with host receptors and elicit a defence reaction [31,32]. Aziz et al. [33] measured the response of grapevine to different oligosaccharides finding that after the application of these molecules, an oxidative burst was triggered and defence-related genes were activated, including plant chitinases. Although no hypersensitive response was

Fig. 2. Percentage of each functional gene ontology annotation in the necrosis-inducing size exclusion chromatography fractions from the culture filtrate of P. teres f. teres W1-1. The gene ontology search was performed with Blast2Go program [28]. The percentage (y-axis) is based on the number of proteins with a given annotation in each fraction, excluding proteins with no annotation.

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which is both responsible for necrosis on wheat and protecting the pathogen from plant chitinases due to its chitin binding domain [43]. The second highest score (86%) is a protein annotated as a ribonuclease, Ptt_W11.g07378, detected in SEC 53–55 and SEC 62–64. Ribonuclease-like effectors are common in powdery mildews and seem to interact non-catalytically with RNA because the active site amino acid residues are missing [44]. Two of the active amino acid residues of Ptt_W11.g07378 are mutated but experimental characterisation is needed to evaluate whether the enzyme remains catalytically functional and has a role in pathogenesis (Fig. 3). The mechanism of action of ribonuclease-like effectors is not known but in biotrophs they appear to be involved in a complex interaction resulting in the suppression of host cell death [45–47]. Although chitin binding and ribonuclease like effectors typically suppress the host cell death or the recognition of the pathogen, potentially playing a role on the early biotrophic stage of PTT, the possibility of these triggering a necrotising response such as snTox1 cannot be discarded. A third protein, Ptt_W11.g05879, annotated as a kinase and is present in all three analysed fractions, had a probability score of 82%. Kinases are key players in signal transduction and many have been identified as regulators of pathogenicity [48]. However, to our knowledge, a direct interaction between fungal kinases and plant components has not been reported. Furthermore, Ismail et al. [24] found this protein during a 2D-gel proteomic analysis comparing the in vitro secretomes of PTT virulent and avirulent isolates, but no correlation was observed between its in planta expression and the development of disease symptoms. The final protein with an effector probability score above 80% (81%), Ptt_W11.g06093, possessed no recognizable domains and was not functionally annotated. In addition, Ptt_W11.g04012, detected in all three samples, is a chitin binding protein with an effector probability score of 77%; however, instead of a LysM, it possesses a hevein or type 1 chitin binding domain. Interestingly, among the active proteins detected, Ptt_W11.g06727, a protein that is not obvious by its abundance or effector probability score (41%), has been recently confirmed by reverse genetic approaches as an effector of PTT, pttAvrHar, by Wyatt et al. [50]. The presence of this effector in the active fractions validates our approach; however, the low probability ranking by EffectorP shows the drawback of basing effector prediction approaches on sequence similarities, given

Table 1 Number (percentage) of proteins per functional annotation in the top 20 most abundant proteins found in the necrosis-inducing size exclusion chromatography fractions from the culture filtrate of P. teres f. teres W1-1. The gene ontology (GO) search was performed with Blast2Go program [28]. Functional GO terms

SEC 41-43

SEC 53-55

SEC 62-64

Glycosidases Peptidases Other hydrolases FAD-binding Chitin binding Other binding Transferases Lyases Nucleases Phosphatases Unspecified catalytic function No functional information

8 3 0 1 0 1 1 1 0 1 1 3

3 2 2 1 0 0 2 1 1 0 0 8

4 0 1 0 2 0 2 1 1 0 0 9

(40%) (15%) (0%) (5%) (0%) (5%) (5%) (5%) (0%) (5%) (5%) (15%)

(15%) (10%) (10%) (5%) (0%) (0%) (10%) (5%) (5%) (0%) (0%) (40%)

(18%) (0%) (5%) (0%) (10%) (0%) (10%) (5%) (5%) (0%) (0%) (45%)

3.3. Candidate effectors found within the active fractions Although several of the proteins discussed are known to be general elicitors, the fact that some of the barley varieties infiltrated with PTT W1-1 culture filtrate did not produce significant symptoms (Fig. S2), suggests that the contribution of non-host specific toxins in this experiment is not significant. While some protein classes may work as cultivar-specific toxins, such as some proteases [40,41], we also looked for predicted effectors among the active fractions. To complement the GO search annotations and to evaluate the probability of proteins being pathogenicity effectors, their sequences were submitted to EffectorP 2.0 [29]. Among the 182 unique proteins identified, 20 were flagged as possible effectors with probability scores above 55% (Table S1). Four proteins had a score higher than 80% and a protein with no functional annotation was present in all three samples and showed the highest score at 89%. This protein, Ptt_W11.g04687, possess a lysine motif (LysM) which is present in fungal effectors that bind chitin and are known to supress host recognition of the pathogen [42]. Interestingly, evidence has shown that effectors could have a dual action, for example SNTox1 from the related Parastagonospora nodorum,

Fig. 3. Protein alignment of ribonuclease-like candidate effector from P. teres f. teres W1-1, Ptt_W11.g07378, and ten curated fungal RNAses. Grey scale shading of amino acid residues corresponds to their degree of similarity, with the more similar the darker. Asterisks indicate amino acid residues important for ribonuclease activity. Analysis of the RNAse domain was performed with a NCBI conserved domain search and the Subfamily Protein Architecture Labeling Engine (SPARCLE) [49]. 5

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that relatively few effectors have been characterised to date and which may not be applicable to diverse species with different lifestyles such as PTT.

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4. Conclusions The analysis of the active fractions of the whole culture filtrate of PTT W1-1 revealed that a third of the proteins detected were unannotated, representing an interesting set for further functional characterisation to elucidate their possible role in pathogenesis. Furthermore, the annotation revealed a high percentage of glycosidases, peptidases, transferases and an array of enzymes with diverse activities. Among the fractions, ten diverse proteins showed high probability scores (> 75%) of being effectors including chitin binding, ribonuclease and kinase proteins along with an unannotated protein. In addition, while several proteins detected in this project have been highlighted as potential contributors to the symptoms observed after the infiltration of PTT W1-1 culture filtrate, further in planta studies are warranted to determine their possible roles in the development of net blotch disease. Starvation conditions in fungi, such as prolonged culturing time, have been reported to mimic in planta conditions [51] and there are several cases in which in vitro cultures have led to the discovery of effectors in other fungal species. However, attributing the observed bioactivity of PTT culture filtrates to cultivar-specific toxins (effectors) becomes difficult given the complexity of the secretome and the potential presence of defence response elicitors discussed above involved in processes such as hypersensitive response. It is clear, from this and other studies [21–23], that to understand the molecular interaction between PTT and its host from a proteomics perspective, without the confounding factors due to in vitro culturing, experiments to analyse the in planta secreted proteins are required. Nonetheless, the proteomic data set presented in this study provides a significant resource for investigating whether specific proteins are produced by all PTT isolates or are common in virulent pathotypes and present in virulence QTLs. Declaration of competing interest None of the authors has conflicts of interest to disclose. Acknowledgments This research was funded under Grains Research and Development Corporation (GRDC) grant CUR00023 P5. The study was supported by the Centre for Crop and Disease Management, a cooperative research vehicle of the GRDC and Curtin University. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.pmpp.2019.101451. References [1] V. Smedegård-Petersen, Pyrenophora teres F. Maculata F. Nov. And Pyrenophora teres F. Teres on Barley in Denmark, Yearbook of the Royal Veterinary and Agricultural University, Copenhagen), 1971, pp. 124–144. [2] S.R. Ellwood, R.A. Syme, C.S. Moffat, R.P. Oliver, Evolution of three Pyrenophora cereal pathogens: recent divergence, speciation and evolution of non-coding DNA, Fungal Genet. Biol. 49 (2012) 825–829. [3] B. Poudel, S.R. Ellwood, A.C. Testa, M. McLean, M.W. Sutherland, A. Martin, Rare Pyrenophora teres hybridization events revealed by development of sequence-specific PCR markers, Phytopathology 107 (2017) 878–884. [4] Z. Liu, S.R. Ellwood, R.P. Oliver, T.L. Friesen, Pyrenophora teres: profile of an increasingly damaging barley pathogen, Mol. Plant Pathol. 12 (2011) 1–19. [5] S.A. Carlsen, A. Neupane, N.A. Wyatt, J.K. Richards, J.D. Faris, S.S. Xu, R.S. Brueggeman, T.L. Friesen, Characterizing the Pyrenophora teres f. maculatabarley interaction using pathogen genetics, G3: Genes Genomes Genet. 7 (2017) 2615–2626.

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