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In Planta Proteomics and Proteogenomics of the Biotrophic Barley Fungal Pathogen Blumeria graminis f. sp. hordei>

2009; Elsevier BV; Volume: 8; Issue: 10 Linguagem: Inglês

10.1074/mcp.m900188-mcp200

1535-9484

Laurence V. Bindschedler, Timothy A. Burgis, Davinia J. Mills, Jenny T. C. Ho, Rainer Cramer, Pietro D. Spanu,

Plant Pathogens and Fungal Diseases

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. Fungi are the most prevalent cause of disease in plants, and failure to control them results in widespread damage and severe harvest loss. In particular, the powdery mildew Blumeria graminis attacks the cereals wheat and barley, which are two of the main food crops in Western agriculture. The nature of cereal powdery mildew infection and its detrimental effect on crop health require a continual effort aimed in fighting the disease through the use of fungicides and the breeding of novel resistant varieties. Both these approaches are currently effective albeit with very significant economic and ecological impact. However, the gradual withdrawal of some fungicides driven by concerns for their environmental impact, the constant threat of the emergence of fungicide resistance, and new virulence alleles that overcome breed resistance require new tools for disease control. This will entail the development of new classes of fungicides, introgression of novel disease resistance genes, and possibly the engineering of a specific biological control through enhancement of disease tolerance and resistance. Understanding the biology and virulence of plant pathogenic fungi is essential to meet this challenge (1Marris E. Dodds P. Glover J. Hibberd J. Zhang J.H. Sayre R. Agronomy: five crop researchers who could change the world.Nature. 2008; 456: 563-568Crossref PubMed Scopus (13) Google Scholar). Powdery mildews are easily recognizable as white pustules on leaves and stems and are some of the most conspicuous plant diseases affecting a wide range of hosts (2Glawe D.A. The powdery mildews: a review of the world's most familiar (yet poorly known) plant pathogens.Annu. Rev. Phytopathol. 2008; 46: 27-51Crossref PubMed Scopus (273) Google Scholar). Powdery mildews are obligate biotrophs, i.e. they have an absolute requirement of a host to grow and complete their life cycle. Blumeria shows a very high degree of host specificity; for example B. graminis f. sp. hordei grows exclusively on barley. In addition to being an economically important pathogen, B. graminis f. sp. hordei is also the best studied powdery mildew and a model for research into these interactions. Most of the life cycle of Blumeria is vegetative and is spent as a rapid succession of asexual cycles in which the airborne conidia germinate on the surfaces of leaves and stems to produce first a primary germ tube and then a secondary germ tube that develops a swollen adhesion structure called an appressorium. Within 8–12 h a thin peg emerges from the underside of the appressorium, breaches the plant cell wall, and penetrates the cell. Inside the plant cell a highly branched feeding structure, the haustorium, develops surrounded by an intact host membrane (3Both M. Spanu P. Blumeria graminis f. sp. hordei, an obligate pathogen of barley.in: Talbot N. Plant Pathogen Interactions. Blackwell Publishing, Oxford2004: 202-218Google Scholar). The haustorium has long been known to actively take up nutrients (4Bushnell W.R. Physiology of fungal haustoria.Annu. Rev. Phytopathol. 1972; 10: 151-176Crossref Google Scholar), but like other obligate pathogens, it is now also believed to control host perception and defense (5Catanzariti A.M. Dodds P.N. Ellis J.G. Avirulence proteins from haustoria-forming pathogens.FEMS Microbiol. Lett. 2007; 269: 181-188Crossref PubMed Scopus (87) Google Scholar), enabling the invading pathogen to survive, avoid, and suppress rejection responses. How this is achieved is still unknown, but current thinking postulates that this control is mediated by protein "effectors" secreted by the pathogen into the host cells (6Hogenhout S.A. Van der Hoorn R.A. Terauchi R. Kamoun S. Emerging concepts in effector biology of plant-associated organisms.Mol. Plant Microbe Interact. 2009; 22: 115-122Crossref PubMed Scopus (493) Google Scholar). To understand the biology and virulence of plant pathogenic fungi, a systematic analysis of the genomes and transcriptomes of plant pathogenic fungi is a top priority, and in recent years it has been actively pursued by a number of institutions and research consortia worldwide (Broad Institute Fungal Genome Initiative and the United States Department of Energy Joint Genome Institute). We are coordinating the sequencing of the barley powdery mildew fungus B. graminis f. sp. hordei genome (Blumeria Genome Sequencing Project) and have extensively researched the transcriptome dynamics of Blumeria during in vivo pathogenic development (7Both M. Eckert S.E. Csukai M. Müller E. Dimopoulos G. Spanu P.D. Transcript profiles of Blumeria graminis development during infection reveal a cluster of genes that are potential virulence determinants.Mol. Plant Microbe. Interact. 2005; 18: 125-133Crossref PubMed Scopus (63) Google Scholar, 8Both M. Csukai M. Stumpf M.P. Spanu P.D. Gene expression profiles of Blumeria graminis indicate dynamic changes to primary metabolism during development of an obligate biotrophic pathogen.Plant Cell. 2005; 17: 2107-2122Crossref PubMed Scopus (111) Google Scholar). A comparable effort to analyze the proteome in plant pathogenic fungi is missing despite the fact that it is essential for our understanding of complex biological systems at the post-transcriptional level because transcript levels do not always reflect protein levels and protein activities. This is further complicated by evidence of RNAs acting directly or indirectly with mRNA molecules to influence the expression and, indirectly, the activities of proteins (9Selbach M. Schwanhäusser B. Thierfelder N. Fang Z. Khanin R. Rajewsky N. Widespread changes in protein synthesis induced by microRNAs.Nature. 2008; 455: 58-63Crossref PubMed Scopus (2766) Google Scholar). It is also generally accepted that the biological activity of proteins also depends on post-translational modifications and protein-protein interactions. Furthermore a proteogenomics approach can contribute to validating and discovering new ORFs on annotated genomes. In fact, even in well studied and annotated genomes such as the Arabidopsis genome, it has been suggested that around 13% of the Arabidopsis proteome is incomplete because of missing or incorrect gene models or ORFs (10Castellana N.E. Payne S.H. Shen Z. Stanke M. Bafna V. Briggs S.P. Discovery and revision of Arabidopsis genes by proteogenomics. Proc. Natl. Acad. Sci.U.S.A. 2008; 105: 21034-21038Crossref Scopus (230) Google Scholar, 11Ansong C. Purvine S.O. Adkins J.N. Lipton M.S. Smith R.D. Proteogenomics: needs and roles to be filled by proteomics in genome annotation.Brief. Funct. Genomic. Proteomic. 2008; 7: 50-62Crossref PubMed Scopus (122) Google Scholar). It is therefore imperative to add a proteomic dimension in an integrated analysis to study gene function, protein expression, and localization. So far no large scale comparative study of proteomes of phytopathogenic fungi during in planta colonization has been undertaken to unravel key players of the infection, initiation, and establishment of a susceptible interaction with the host, in particular during a biotrophic interaction. There are published reports that analyze the leaf proteome of plants infected by pathogenic fungi, but few of these describe fungal proteins within total extracts from infected leaves, and only a very limited number of fungal proteins have been identified in these tissues (12Rampitsch C. Bykova N.V. McCallum B. Beimcik E. Ens W. Analysis of the wheat and Puccinia triticina (leaf rust) proteomes during a susceptible host-pathogen interaction.Proteomics. 2006; 6: 1897-1907Crossref PubMed Scopus (87) Google Scholar, 13Zhou W. Eudes F. Laroche A. Identification of differentially regulated proteins in response to a compatible interaction between the pathogen Fusarium graminearum and its host, Triticum aestivum.Proteomics. 2006; 6: 4599-4609Crossref PubMed Scopus (120) Google Scholar). In one instance intercellular washing fluids were extracted to detect secreted fungal proteins in the apoplast (14Paper J.M. Scott-Craig J.S. Adhikari N.D. Cuomo C.A. Walton J.D. Comparative proteomics of extracellular proteins in vitro and in planta from the pathogenic fungus Fusarium graminearum.Proteomics. 2007; 7: 3171-3183Crossref PubMed Scopus (178) Google Scholar). In the particular case of B. graminis f. sp. hordei, two recent studies have identified a few hundred proteins from conidia (15Noir S. Colby T. Harzen A. Schmidt J. Panstruga R. A proteomic analysis of powdery mildew (Blumeria graminis f.sp. hordei) conidiospores.Mol. Plant Pathol. 2009; 10: 223-236Crossref PubMed Scopus (42) Google Scholar) and isolated feeding structures (haustoria) (16Godfrey D. Zhang Z. Saalbach G. Thordal-Christensen H. A proteomics study of barley powdery mildew haustoria.Proteomics. 2009; 9: 3222-3232Crossref PubMed Scopus (48) Google Scholar) of B. graminis. Here we describe the analysis of three different in planta expressed proteomes of Blumeria. We compared proteins extracted from conidia, sporulating epiphytic hyphae growing on the host, and intracellular haustoria embedded in the host epidermis. Because of the impossibility of obtaining growth of obligate biotrophs in vitro, the analysis of the proteomes of biologically significant life stages must be carried out in the plant host. On the one hand, this analysis is technically challenging as the fungal proteins are embedded in and diluted by the plant proteome; on the other hand, the proteins will be identified in vivo, i.e. under conditions that are biologically relevant to the interaction. The availability of the recently sequenced B. graminis f. sp. hordei genome now allows the undertaking of a large scale shotgun proteomics approach as the peptides detected by mass spectrometry can directly be matched to the genomic sequence contigs thus avoiding cumbersome and difficult de novo peptide sequencing. At the same time the experimental proof of proteins expressed by Blumeria represents an invaluable proteogenomic resource for the creation and validation of ab initio gene-finding software for the functional annotation of the genome itself. Barley plants (Hordeum vulgaris cultivar Golden Promise) were grown in soil as described previously (7Both M. Eckert S.E. Csukai M. Müller E. Dimopoulos G. Spanu P.D. Transcript profiles of Blumeria graminis development during infection reveal a cluster of genes that are potential virulence determinants.Mol. Plant Microbe. Interact. 2005; 18: 125-133Crossref PubMed Scopus (63) Google Scholar). B. graminis f. sp. hordei strain DH14 was maintained as described previously (7Both M. Eckert S.E. Csukai M. Müller E. Dimopoulos G. Spanu P.D. Transcript profiles of Blumeria graminis development during infection reveal a cluster of genes that are potential virulence determinants.Mol. Plant Microbe. Interact. 2005; 18: 125-133Crossref PubMed Scopus (63) Google Scholar). Protein extraction in denaturing conditions was performed in a denaturing buffer consisting of 7 m urea, 2 m thiourea, 2% CHAPS, and 20 mm DTT. Conidia were recovered from infected plants 7 days postinoculation (dpi), 1The abbreviations used are:dpidays postinoculationCMchloroform-methanolCONconidiaEHepidermis and haustoriaFDRfalse discovery rateHYhyphaenLCnano-LCnESInano-ESITATCA-acetoneTLtotal leafmgfMascot generic formatLTQlinear trap quadrupoleESTexpressed sequence tagsNCBInrNational Center for Biotechnology Information non-redundantHCThigh capacity trapGOGene OntologyPANTHERProtein Analysis through Evolutionary RelationshipsRubiscoribulose-bisphosphate carboxylase/oxygenaseQDEquelling-deficient. transferred to a 1.5 ml-polypropylene microcentrifuge tube, and stored at −80 °C until protein extraction. Denaturing extraction buffer (350 µl) was added to 35 mg of conidia. The suspension was transferred with a pipette as small droplets into a small mortar containing liquid N2 and a small amount of quartz sand. After grinding, the powder was transferred to a microcentrifuge tube, left at room temperature to melt, and then incubated for 5 min with regular mixing. Insoluble particulates were removed by two successive 10-min centrifugations at 17,860 × g at room temperature. days postinoculation chloroform-methanol conidia epidermis and haustoria false discovery rate hyphae nano-LC nano-ESI TCA-acetone total leaf Mascot generic format linear trap quadrupole expressed sequence tags National Center for Biotechnology Information non-redundant high capacity trap Gene Ontology Protein Analysis through Evolutionary Relationships ribulose-bisphosphate carboxylase/oxygenase quelling-deficient. Sporulating hyphae were sampled from ∼200 infected primary leaves 5 dpi as described elsewhere (7Both M. Eckert S.E. Csukai M. Müller E. Dimopoulos G. Spanu P.D. Transcript profiles of Blumeria graminis development during infection reveal a cluster of genes that are potential virulence determinants.Mol. Plant Microbe. Interact. 2005; 18: 125-133Crossref PubMed Scopus (63) Google Scholar) and stored at −80 °C. Sporulating hyphae, embedded in the cellulose acetate peels, were transferred to a mortar and pestle and ground with a little sand in liquid N2. The powder was transferred to a centrifuge tube, and 3 ml of denaturing extraction buffer was added. The tissue was left to thaw at room temperature with occasional vortexing. Particulates were removed by two successive 10-min centrifugations at 17,860 × g at 4 °C. After removing hyphae from primary infected barley leaves (7–10 dpi) as described above, the abaxial epidermis from these primary leaves was stripped using watchmaker forceps. This tissue was defined as the epidermis-haustorial tissue. Epidermal strips were resuspended in 5–10 volumes of precooled denaturing buffer and homogenized in a glass homogenizer on ice. After transfer of the slurry to microcentrifuge tubes, sand was added, and the epidermis was further homogenized with a hand-driven Potter homogenizer. Insoluble particulates were removed as described for the conidia extracts. Sporulating hyphae (biological replicates HY1–3) and epidermis-haustoria (biological replicates EH1–4) protein extracts were further cleaned up and concentrated by either TCA-acetone (TA) precipitation (samples HY1, HY2, and EH1) or chloroform-methanol (CM) precipitation (samples HY3, EH2, and EH3) or both (sample EH4). For TA precipitation, 19 volumes of cold TA solution (10% (w/v) TCA in cold acetone and 0.07% β-mercaptoethanol) was added to 1 volume of protein extract in a microcentrifuge tube. After 30-min incubation at −20 °C, a protein pellet was recovered following 20-min centrifugation at 17,860 × g at 4 °C. To remove the excess salts the protein pellet was washed three times with cold acetone containing 0.07% β-mercaptoethanol and recovered by 10-min centrifugation at 17,860 × g at 4 °C. For CM precipitation, 400 µl of methanol was added to 100 µl of protein extract and vortexed. Then 100 µl of chloroform was added and vortexed. Finally 300 µl of double distilled H2O was added, and the mixture was vortexed. The two solvent phases were separated by 2-min centrifugation at 17,860 × g. The upper phase was removed, and proteins in the interphase were precipitated by addition of 400 µl of methanol and 5-min centrifugation at 17,860 × g. The pellet was further washed with cold acetone containing 0.07% β-mercaptoethanol, and the pellet was recovered by 3-min centrifugation at 17,860 × g. All liquid was removed, and the pellets were allowed to dry on the bench for 2 min, avoiding complete dryness. Pellets were resuspended in denaturing buffer. Total leaf (TL) tissue from infected plants (7–10 dpi) was also harvested, flash frozen in liquid N2, and stored at −80 °C until protein extraction. Leaves were ground with some sand in liquid N2 using a mortar and pestle. Soluble proteins were extracted in 3 volumes of ice-cold 50 mm Tris, pH 7.6, 0.33 m sucrose, 1 mm DTT, 1 mm MgCl2, 1% (w/v) poly(vinylpolypyrrolidone), and 0.4% (v/v) protease inhibitor mixture VI (Calbiochem/Merck). Samples were thawed, mixed, and kept on ice for 5 min. Particulates were removed by two successive 10-min centrifugations at 3,300 × g and 17,860 × g at 4 °C. Proteins were selectively precipitated by the addition of ammonium sulfate (520 mg/ml of protein solution). After 20-min incubation on ice, a protein pellet was recovered by 20-min centrifugation at 15,000 × g. Samples were desalted by buffer replacement with 1× PBS buffer (150 mm NaCl, 10 mm NaH2PO4, pH 7.4) in a VivaspinTM 20-ml ultrafiltration device and centrifugation at 3,300 × g (Vivascience, Sartorius, Epsom, UK) with a molecular mass cutoff of 3 kDa. Protein concentrations were estimated with the Bradford method (Bio-Rad) using BSA as protein standard. For each proteome analyzed, up to 50 µg of protein was separated on a 1-mm-thick SDS-polyacrylamide minigel (Mini-Protean III, Bio-Rad; conidia ("CON"), 30 µg; HY1, 45 µg; EH1, 50 µg). Alternatively bigger gels with a thickness of 1.5 mm were used for scale-up (Protean II, Bio-Rad), and 70–150-µg amounts of protein were loaded (HY2, 70 µg; HY3, 90 µg; EH3, 150 µg; EH4, 75 µg). All samples were separated on 12% acrylamide gels except for HY2 and HY3, which were separated on a 15% acrylamide gel. Gels were stained with colloidal Coomassie (17Candiano G. Bruschi M. Musante L. Santucci L. Ghiggeri G.M. Carnemolla B. Orecchia P. Zardi L. Righetti P.G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.Electrophoresis. 2004; 25: 1327-1333Crossref PubMed Scopus (1580) Google Scholar). In-gel tryptic digestion was performed as described previously (18Bindschedler L.V. Palmblad M. Cramer R. Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study.Phytochemistry. 2008; 69: 1962-1972Crossref PubMed Scopus (84) Google Scholar). Tryptic digests were then reconstituted in 12–20 µl of 0.2% TFA depending on gel band stain intensity. Four microliters of sample was loaded on a 10-mm trap column packed with 3.5-µm C18 particles (LC Packings/Dionex, Amsterdam, The Netherlands), washed with 0.2% TFA for 5 min at a flow rate of 30 µl/min, and eluted in 0.2% formic acid for 155 min in a 2–27% ACN gradient and for another 30 min in a steeper 27–50% ACN gradient at a flow rate of 250 nl/min on a 15-cm × 75-µm PepMap C18 reverse phase analytical column (3.5-µm particles; LC Packings/Dionex) using an UltimateTM nano-LC (nLC) system (LC Packings/Dionex). The LC system was coupled to an nESI-MS/MS three-dimensional ion trap mass spectrometer (HCT Esquire, Bruker Daltonics, Bremen, Germany) using a 10-cm-long stainless steel emitter (Proxeon, Odense, Denmark) in the nESI source. The LC system and the ion trap were controlled through HyStar (3.1 build 52.2) and Esquire Control (5.3. build 11) modules in the Compass software suite (Bruker Daltonics, Coventry, UK). Mass spectra were acquired from m/z 300 to 2,000 using parameters optimized at m/z 850 with the trap ion charge control set at 150,000 and a maximum acquisition time of 200 ms averaging three scans per spectrum. The three most abundant 2+ or 3+ ions were selected for MS/MS with a signal threshold of 5,000, the isolation window had a width of m/z 4, and the fragmentation amplitude was 1 V. The selected precursor ions were actively excluded for 45 s after two selections. Samples CON, HY1, HY2, EH1, and EH2 were analyzed under these conditions. Raw LC-MS/MS data were batch-processed in DataAnalysis 3.3.147 (Bruker Daltonics). For each LC run corresponding to one gel band tryptic digest up to 6,000 2+ and 3+ compounds (retention time restriction of 5–210 min) with a signal-to-noise ratio above 5 were extracted and exported as Mascot generic format (mgf) files. The resuspended digests of the samples HY3, EH3, and EH4 were analyzed by LC-MS/MS using a quaternary LC pump coupled to a hybrid linear ion trap-orbitrap (LTQ-Orbitrap, Thermo Fisher Scientific Bremen, Germany). Depending on the intensity of the Coomassie-stained band, 2–10 µl of sample solution was loaded on a peptide Captrap (Michrom Bioresources, Auburn, CA) with an Accela LC pump (Thermo Fisher Scientific) for 5 min at 10 µl/min using water containing 0.1% formic acid. Peptide separation was performed using a pulled tip column (15 cm × 100-µm inner diameter) containing C18 Reprosil 5-µm particles (Nikkyo Technos, Tokyo, Japan). The flow was passively split from 300 µl/min to ∼250 nl/min before the analytical column. Gradient elution was performed from 0 to 50% ACN containing 0.1% formic acid over 100 min. The eluent was directly sprayed into an LTQ-Orbitrap XL mass spectrometer, and a data-dependent top 5 method was used for data acquisition. For each cycle, one full MS scan in the orbitrap at 60,000 resolution and an automatic gain control target of 100,000 was followed by five MS/MS acquisitions in the LTQ at an automatic gain control target of 5,000 on the five most intense ions. Selected ions were excluded from further selection for 60 s. Singly charged or unassigned ions were also rejected. Maximum ion accumulation times were 500 ms for full MS scans and MS/MS scans. For ion trap MS/MS the normalized collision energy was set to 30, activation Q was set to 0.25, and activation time was set to 30 ms. Raw data generated with the LTQ-Orbitrap XL were converted to mgf files using ProteomeDiscoverer 1.0 software (Thermo Fisher Scientific) excluding singly charged ions and non-deconvoluted MS peaks or peaks with a signal-to-noise ratio below 3. The first line "monoisotopic mass" of the mgf file was removed using Microsoft Wordpad to achieve compatibility for batch processing with Mascot Integra 1.4 (Matrix Science, London, UK). For protein identification, the integrated analytical work flow software package Mascot Integra 1.4 (Matrix Science) was used for batch searches with Mascot 2.2.2 on an in-house server and the batch processor Mascot Daemon 2.2.2 (Matrix Science). Data were searched against the Blumeria genome ("Genoscope" draft assembly) and EST databases (Blumeria Genome Sequencing Project). The genomic Blumeria contigs longer than 50,000 bp were fractionated to comply with the Mascot software. The Blumeria DNA database used for the protein identification search consisted of numbered subcontigs rather than annotated and defined ORFs; therefore the percentage of sequence coverage, Mr, and pI of contigs were ignored. Searches were reiterated on a curated database from which repeats were excluded. The EST Blumeria database contains a collection of 17,869 ESTs from different studies. Some barley sequences were filtered out manually from the Mascot data obtained when searching with this database. Infected TL extracts and epidermal strips (epidermis and haustoria ("EH")) were also searched against the UniProtKB/Swiss-Prot rice database downloaded from the European Molecular Biology Laboratory-European Bioinformatics Institute Web site (version 23214, Oryza sativa Nipponbare, July 22, 2008) and the NCBInr database restricting the taxonomy to higher plants. The updated versions of the publically available databases (NCBInr) were downloaded onto the in-house Mascot server on a weekly basis. The rice genomic database was fused to the Blumeria genomic database for the estimation of the false discovery rate (FDR) of identified proteins in protein samples of infected EH tissues. The rice pseudomolecules database version 5.0 was downloaded from the Michigan State University Rice Genome Annotation Project Database and Resource. Initially each *.mgf file was searched individually with Mascot. In a second identical search, each mgf file generated from protein extracts from the same tissue (CON, hyphae ("HY"), or EH) and analyzed on the same MS/MS instrument (HCT or LTQ-Orbitrap) were merged on the Integra server to perform the Mascot search resulting in a single Mascot result file. The Mascot search parameters for the HCT Esquire data were the following: 1.2-Da error tolerance in MS mode and 0.4-Da error tolerance in MS/MS mode, allowing up to two tryptic missed cleavages and considering 2+ and 3+ ions. Cysteine carbamidomethylation was set as a fixed modification; methionine and proline oxidation (hydroxyproline) were included as variable modifications. Mascot search parameters for the orbitrap data were identical to the parameters described for the HCT except that 10-ppm error in MS mode and 0.8-Da error in MS/MS mode were tolerated. Every peptide identified with a Mascot score above the confidence limit (p < 0.05) was extracted from independent comma-separated value-formatted Mascot result files and aligned on the Blumeria genomic sequence. Genes and their proteolytic peptides were defined through individual exons of an ORF. Protein hits were validated if they had at least two different unique peptides with a score greater than the identity score (p < 0.05). In the case of the haustorial proteome, proteins identified with a single significant peptide (singletons) were also accepted if the same protein was identified in another tissue through multiple peptide identifications and the MS/MS data for the common peptide were found to be similar by manual inspection. These MS/MS spectra were extracted and visualized in the Mascot Applet of the Integra software (Matrix Science). To be accepted, the identification required that most of the intensive peaks from the b- and y-ion series were present in both MS/MS spectra with a similar intensity pattern. To annotate a group of peptides within a putative ORF within a contig, a homology search against the UniProt database using BlastP (19Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. Basic local alignment search tool.J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69652) Google Scholar) was used to find homologous proteins against the concatenated amino acid sequences of the identified peptides of that group, keeping their relative positions. The BlastP search parameters were: -F, F (turn off low complexity filtering); -W, 2 (use a smaller word size); -M, PAM30 (scoring matrix recommended for short queries). The DNA sequence around a group of peptides identified for the haustorial proteins was downloaded from the genome assembly (Genoscope version) as a FASTA format file to predict ORFs. Putative ORFs were determined with the FGENESH prediction program using either the Leptosphaeria maculans or the Sclerotinia sclerotiorum ORF prediction models (SoftBerry, Inc.). Putative classical secretion proteins (with signal peptide) and non-classical secretion proteins were predicted with the SignalP (Hidden Marker Model minimum probability of 0.9) and the SecretomeP (Neural Network-Score cutoff, 0.5) algorithms, respectively. The homology BlastP search was then reiterated with the predicted ORFs using the default settings and the NCBInr or Swiss-Prot databases. Using the GO terms associated with the UniProt entries homologous to the identified proteins, we applied the "GO to PANTHER mappings" to map the protein functions to the PANTHER ontology. This provided a one-to-one mapping of GO terms to PANTHER terms and indicated wh