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A Kazal-like Extracellular Serine Protease Inhibitor from Phytophthora infestans Targets the Tomato Pathogenesis-related Protease P69B

A Kazal-like Extracellular Serine Protease Inhibitor from Phytophthora infestans Targets the... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 25, Issue of June 18, pp. 26370 –26377, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Kazal-like Extracellular Serine Protease Inhibitor from Phytophthora infestans Targets the Tomato Pathogenesis-related Protease P69B* Received for publication, January 28, 2004, and in revised form, April 15, 2004 Published, JBC Papers in Press, April 19, 2004, DOI 10.1074/jbc.M400941200 Miaoying Tian, Edgar Huitema, Luis da Cunha, Trudy Torto-Alalibo, and Sophien Kamoun‡ From the Department of Plant Pathology, The Ohio State University, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691 host cells but later causes extensive necrosis of host tissue, a The oomycetes form one of several lineages within the eukaryotes that independently evolved a parasitic life- lifestyle that is known as hemibiotrophy. As with other biotro- style and consequently are thought to have developed phic plant pathogens, processes associated with P. infestans alternative mechanisms of pathogenicity. The oomycete pathogenesis are thought to include the suppression of host Phytophthora infestans causes late blight, a ravaging defense responses (3, 8, 9). In P. infestans, water-soluble glu- disease of potato and tomato. Little is known about pro- cans have been reported to suppress host defenses in a plant cesses associated with P. infestans pathogenesis, partic- cultivar-specific manner (10 –12). Nevertheless, the molecular ularly the suppression of host defense responses. We basis of suppression of host defenses by Phytophthora remains describe and functionally characterize an extracellular poorly understood (3). It is tempting to speculate that unique protease inhibitor, EPI1, from P. infestans. EPI1 con- classes of suppressor genes have been recruited to aid in infec- tains two domains with significant similarity to the tion and counteract host defenses during the evolution of Kazal family of serine protease inhibitors. Database pathogenesis in the oomycete lineage. searches suggested that Kazal-like proteins are mainly Parasitic eukaryotes often face inhospitable environments in restricted to animals and apicomplexan parasites but their hosts. For example, parasites that colonize or transit appear to be widespread and diverse in the oomycetes. through the mammalian digestive tract must adapt to the Recombinant EPI1 specifically inhibited subtilisin A diverse and abundant array of proteases secreted in the gastric among major serine proteases and inhibited and inter- juices (13–15). Some of these parasites secrete inhibitors that acted with the pathogenesis-related P69B subtilisin-like target host proteases and may aid in survival and colonization serine protease of tomato in intercellular fluids. The of the host. For instance, the apicomplexan obligate parasite epi1 and P69B genes were coordinately expressed and Toxoplasma gondii secretes TgPI-1 and TgPI-2, four-domain up-regulated during infection of tomato by P. infestans. serine protease inhibitors of the Kazal family (15–19), and the Inhibition of tomato proteases by EPI1 could form a novel type of defense-counterdefense mechanism be- intestinal hookworm Ancylostoma ceylanicum secretes an tween plants and microbial pathogens. In addition, this 8-kDa broad spectrum serine protease inhibitor of the Kunitz study points to a common virulence strategy between family (14). In plants, the apoplast (intercellular fluid) forms a the oomycete plant pathogen P. infestans and several protease-rich environment that is colonized by many patho- mammalian parasites, such as the apicomplexan Toxo- gens, including P. infestans and the fungus Cladosporium ful- plasma gondii. vum. In tomato, apoplastic proteases are integral components of the plant defense response. Serine proteases of the P69 subtilase family have long been tied to pathogen defense, and Parasitic and pathogenic lifestyles have evolved repeatedly two isoforms, P69B and P69C, are known as pathogenesis- in eukaryotes (1). Several parasitic eukaryotes represent deep related proteins (PR-7 class) (20 –22). More recently, an apo- phylogenetic lineages, suggesting that they feature unique mo- plastic papain-like cysteine protease, Rcr3, was shown to be lecular processes for infecting their hosts. One such lineage is required for specific resistance to C. fulvum (23). In addition, formed by the oomycetes, a group of fungus-like organisms that several C. fulvum extracellular proteins are processed or de- are distantly related to fungi but closely related to brown algae graded by host proteases in the apoplast, resulting in altered and diatoms in the Stramenopiles (1–3). One of the most noto- functionality (24, 25). rious and destructive oomycete is the Irish famine pathogen, Despite the importance of extracellular proteases in plant Phytophthora infestans. This species causes late blight, a re- defense, to date no protease inhibitor has been reported from emerging and ravaging disease of potato and tomato (4 –7). microbial plant pathogens. In this paper, we describe and func- During the early stages of infection, P. infestans requires living tionally characterize an extracellular protease inhibitor, EPI1, from P. infestans. EPI1 contains two domains with significant * This work was supported by the Ohio Agricultural Research and similarity to the Kazal family of serine protease inhibitors, Development Center Research Enhancement Grant Program and Syn- which also occurs in many animal species and in apicomplexan genta Biotechnology Inc. Salaries and research support were provided parasites. In vitro studies indicated that recombinant EPI1 by State and Federal Funds appropriated to the Ohio Agricultural (rEPI1) specifically inhibited subtilisin A among the major Research and Development Center, the Ohio State University. The costs of publication of this article were defrayed in part by the payment serine proteases. rEPI1 was further demonstrated to inhibit of page charges. This article must therefore be hereby marked “adver- and interact with tomato P69B subtilisin-like serine protease. tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: Dept. of Plant Pa- thology, The Ohio State University-OARDC, 1680 Madison Ave., The abbreviations used are: rEPI1, recombinant EPI1; BTH, benzo- Wooster, OH 44691. Tel.: 330-263-3847; Fax: 330-263-3841; E-mail: (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; EST, expressed [email protected]. sequence tag; RT, reverse transcriptase. 26370 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Protease Inhibitors from Phytophthora 26371 random primer labeling using gel-purified fragments digested or PCR- The epi1 and P69B genes were coordinately expressed and amplified from the corresponding cDNA clones (Ref. 41 and this study). up-regulated during infection of tomato by P. infestans. Overall Probe for tomato P69B was generated from a gel-purified RT-PCR these results suggest that inhibition of tomato proteases by fragment amplified from total RNA isolated from infected tomato tis- P. infestans EPI1 could form a novel type of defense-counter- TM sue. For RT-PCR, total RNA was treated with DNA-free (Ambion, defense mechanism between plants and microbial pathogens. Austin, TX) to remove contaminating DNA, and first-strand cDNAs TM In addition, this study points to a common virulence strategy were synthesized using the ThermoScript RT-PCR system from 5 g of total RNA following the instructions of the manufacturer (Invitro- between the oomycete plant pathogen P. infestans and mam- gen). PCR amplifications were carried out with 0.005% of the cDNA malian parasites, such as the apicomplexan T. gondii. product. The oligonucleotide primer pairs, P69A-RTF1 (5-TGGCAGG- TGGTGGAGTTCCGAGGG-3) and P69A-RTR1 (5-CATTGGATCAAC- MATERIALS AND METHODS AAAAGTGCAATTGG-3), P69B-RTF1 (5-CAGCACTCGGCCATGTA- Phytophthora Strains and Culture Conditions—P. infestans isolate GCCAATGTT-3) and P69B-RTR1 (5-CTAGGCAGACACAACTGCAA- 90128 (A2 mating type, race 1.3.4.7.8.9.10.11) was used throughout the TTGGACTTC-3), P69D-RTF1 (5-TGCGAAGTATAAGTCTTCTCAGA- study. P. infestans 90128 was routinely grown on rye agar medium GTTGC-3), and P69D-RTR1 (5-TCAGCAGACACTCTAACTGCAATT- supplemented with 2% sucrose (26). For RNA extraction, plugs of my- GGAC-3), were designed to be gene-specific based on the published P69 celium were transferred to modified Plich medium (27) and grown for gene sequences (21) and were used for the amplification of P69A, P69B, 2–3 weeks before harvesting. and P69D sequences, respectively. The oligonucleotides EPI1-F1 and Bacterial Strains and Plasmids—Escherichia coli XL1-Blue was EPI1-R1, previously used for cloning epi1 into pFLAG-ATS vector, were used in this study and was routinely grown at 37 °C in LB medium (28). used to detect epi1 transcripts by RT-PCR. Primer specificity was Plasmid pFLAG-EPI1 was constructed by cloning PCR-amplified DNA confirmed by sequencing the RT-PCR products. The expression of P69A, fragment corresponding to the mature sequence of EPI1 into the Hin- P69B, and P69D was controlled with primer pair EF1-F1 (5-GCT- dIII site of pFLAG-ATS (Sigma), a vector that allows secreted expres- GCTGTAACAAGGTTTGCTTTAATTCG-3) and EF1-R1 (5-CCAG- sion in E. coli. The oligonucleotides EPI1-F1 (5-GCGAAGCTTCAAA- CATCACACTGCACAGTTCACTTC-3), which are specific for the con- GCCCGCAAGTCATCAG-3) and EPI1-R1 (5-GCGAAGCTTATCCCT- stitutively expressed tomato elongation factor 1 gene (42). The CCTGCGGTGTC-3) were used to amplify the fragment. The intro- expression of epi1 was controlled with P. infestans elongation factor 2 duced HindIII restriction sites are underlined. The N-terminal se- gene using the primer pair described previously (43). quence of the processed FLAG-rEPI1 protein is DYKDDDDKVKLQS- SDS-PAGE and Western Blot Analyses—Proteins were subjected to PQVISPAP.... The FLAG epitope sequence is underlined, and the 10 –15% SDS-PAGE as previously described (28). Following electro- first 10 amino acids of mature EPI1 are shown in bold type. phoresis, the gels were stained with silver nitrate following the method Plant Growth, BTH Treatment, and Infection by P. infestans—To- of Merril et al. (44) or stained with Coomassie Brilliant Blue (28), or the mato (Lycopersicon esculentum) cultivar Ohio 7814 was used through- proteins were transferred to supported nitrocellulose membranes (Bio- out the study and grown in pots at 25 °C, 60% humidity, under 16 Rad) using a Mini Trans-Blot apparatus (Bio-Rad). Detection of anti- h-light/8 h-dark cycle. We used the salicylic acid analog BTH to mimic gen-antibody complexes was carried out with a Western blot alkaline pathogen infection. For BTH treatment, 10 ml of a 25 g/ml BTH phosphatase kit (Bio-Rad). Antisera to P69 subtilases were produced by solution was applied to 3-week-old tomato plants by soil drench. Plants immunizing rabbits with the keyhole limpet hemocyanin-conjugated treated with 10 ml of water were used as controls. Leaves from BTH- peptide, H2N-TTHTPSFLGLQQNC-amide. The sequence underlined is treated and control plants were detached for isolation of intercellular located at the N terminus of mature P69B and P69D proteins (21) and fluids 6 days after treatment. Time courses of P. infestans infection of was chosen for its highly antigenic characteristics and conservation tomato leaves were performed exactly as described earlier (29). 10-l among P69 proteins. Selection of peptides for highly antigenic charac- droplets containing 1,000 zoospores of P. infestans were used to inocu- teristics, peptide synthesis, and conjugation, as well as antisera pro- late the underside of detached tomato leaves. Leaf discs of similar sizes duction, was performed by Rockland Immunochemicals (Gilbertsville, were dissected from the inoculated regions while making sure that the PA). In Western blot analyses, the antisera to the P69 peptide reacted inoculation spot is in the center of sampled area. Leaf discs were frozen only with 70-kDa bands from tomato intercellular fluids. in liquid nitrogen and stored at 80 °C for later use in RNA extraction. Expression and Purification of rEPI1—Expression of rEPI from For isolation of intercellular fluids, tomato leaves were sprayed with pFLAG-EPI1 was conducted as described previously (45). Overnight zoospore suspensions at the concentration mentioned above (10 /ml), cultures of E. coli XL1-blue containing pFLAG-EPI1 were diluted (1: and the intact leaves were collected at different time points for imme- 100) in LB medium containing ampicillin (50 g/ml) and incubated at diate preparation of intercellular fluids. 37 °C. When the A of the cultures reached 0.6, isopropyl--D-thioga- Isolation of Intercellular Fluids—Intercellular fluids were prepared lactopyranoside was added to a final concentration of 0.4 mM. The using a 0.24 M sorbitol solution according to the method of de Wit and cultures were further incubated for 5– 6 h before processing. rEPI1 was Spikman (30). The intercellular fluids were filter-sterilized (0.22 M) recovered from the culture supernatant and was purified by immuno- and were used immediately or stored at 20 °C. affinity using gravity column packed with anti-FLAG M2 affinity gel Sequence Analyses—GC counting was performed as described else- (Sigma). The proteins were eluted with 0.1 M glycine, pH 3.5, and where (31). PexFinder and signal peptide predictions were performed as immediately equilibrated to neutral pH with 20 lof1 M Tris, pH 8.0, described by Torto et al. (32). Similarity searches were performed lo- for each 1-ml eluted fraction. The protein concentrations were deter- cally on an Intel Linux or a Mac OSX work station or through the mined using the Bio-Rad protein assay. To determine the purity of internet on the NCGR (www.ncgr.org) and Whitehead Institute web rEPI1, 0.5 g of the purified protein was run on a SDS-PAGE gel servers (www-genome.wi.mit.edu/resources.html). Search programs in- followed by staining with silver nitrate. cluded BLAST (33), and the similarity search programs implemented in Assays of Protease Inhibition—Inhibition assays of commercial ser- the BLOCKS (34), pfam (35), SMART (36), and InterPro (37) websites. ine proteases by rEPI1 were performed by the colorimetric Quanti- TM TM The examined sequence databases included GenBank nonredundant, Cleave Protease Assay Kit (Pierce). 20 pmol of rEPI1 was preincu- dBEST, and TraceDB (38), PGC (39), SPC, a proprietary database of bated with 20 pmol of trypsin (Pierce), chymotrypsin (Sigma), or Syngenta Inc. containing 75,000 ESTs from P. infestans (courtesy of subtilisin A (Carlsberg) (Sigma), in a volume of 50 l for 30 min at the Syngenta Phytophthora Consortium, Research Triangle Park, NC), 25 °C, followed by incubation with 100 l of succinylated casein (2 and the genome sequences of the fungal species Aspergillus nidulans, mg/ml) in 50 mM Tris buffer, pH 8, containing 20 mM CaCl at room Magnaporthe grisea, Neurospora crassa, and Fusarium graminearum temperature for 20 min. Protease activity was measured as absorbance available through the Whitehead Institute Fungal Genome Initiative at 405 nm using a HTS 7000 Bio Assay Reader (PerkinElmer Life Databases (www-genome.wi.mit.edu/resources.html). Multiple align- Sciences) 20 min after the addition of chromogenic reagent 2,4,6-trini- ments of the Kazal domains were conducted using the program trobenzene sulfonic acid, which reacts with the primary amine of di- CLUSTAL-X (40). The P. infestans and Phytophthora brassicae se- gested peptide and produces a color reaction that can be quantified by TM quences described in this paper were deposited in GenBank under absorbance reader. Detailed kinetic analysis of Subtilisin A inhibition accession numbers AY586273-AY586284 and AY589086-AY589087, re- by rEPI1 was performed as follows. 2 pmol of subtilisin A was preincu- spectively. Other sequences were obtained from the NCBI nr, dBEST, bated with increasing concentrations of rEPI1 in a volume of 50 l for or Trace Archive data bases (www.ncbi.nlm.nih.gov) (Table I). 15 min at 25 °C and was followed by the addition of 150 l assay buffer: RNA Isolation, Northern Blot, and RT-PCR Analyses—RNA isolation 50 mM Tris-Cl, pH 8.0, containing 2.5% Me SO, and 500 M subtilisin and Northern blot hybridizations were performed as described earlier chromogenic substrate Boc-Gly-Gly-Leu-pNA (Calbiochem, La Jolla, (32). Probes for epi1, actA, and tomato -tubulin were generated by CA). The experiments were performed three times and in triplicate each 26372 Protease Inhibitors from Phytophthora TABLE I Predicted Kazal-like proteins from the oomycete plant pathogens P. infestans, P. sojae, P. ramorum, P. brassicae, and P. halstedii TM GenBank Number of Signal Species Protein accession Expression stage Kazal-like P1 residue peptide number domains P. infestans EPI1 AY586273 Yes Infected tomato 2 Asp, Asp P. infestans EPI2 AY586274 Yes Mycelium, H O -treated 2 Asp, Asp 2 2 P. infestans EPI3 AY586275 Yes Genomic sequence 1 Glu P. infestans EPI4 AY586276 Yes Mycelium, nitrogen starvation 3 Thr, Asp, Asp P. infestans EPI5 AY586277 Yes Mating culture 1 Arg P. infestans EPI6 AY586278 NA Infected tomato 2 Asp, Asp P. infestans EPI7 AY586279 Yes Genomic sequence 1 Asp P. infestans EPI8 AY586280 Yes Genomic sequence 1 Asp P. infestans EPI9 AY586281 Yes Mycelium, non-sporulating growth 1 Arg P. infestans EPI10 AY586282 Yes Zoospores 3 Asp, Asp, Asp P. infestans EPI11 AY586283 Yes Mating culture 1 Asp P. infestans EPI12 AY586284 Yes Infected potato, germinating cysts 1 Ser P. infestans EPI13 317886987 Yes Genomic sequence 1 Glu P. infestans EPI14 317892389 Yes Genomic sequence 1 His P. sojae PsojEPI1 CF842223 Yes Infected soybean 4 Ala, Glu, Lys, Ala P. sojae PsojEPI2 AAO24652 Yes Mycelium 1 Glu P. sojae PsojEPI3 274204995 Yes Genomic sequence 3 Met, Asp, Glu P. sojae PsojEPI4 273523724 Yes Genomic sequence 3 Asp, Thr, Asp P. sojae PsojEPI5 273752552 Yes Genomic sequence 1 Arg P. sojae PsojEPI6 273759065 Yes Genomic sequence 1 Glu P. sojae PsojEPI7 324111439 Yes Genomic sequence 1 Asp P. sojae PsojEPI8 273566013 Yes Genomic sequence 1 Asp P. sojae PsojEPI9 274071280 Yes Genomic sequence 1 Arg P. sojae PsojEPI10 273704880 Yes Genomic sequence 1 Ala P. sojae PsojEPI11 324096913 Yes Genomic sequence 1 Asp P. sojae PsojEPI12 324106054 Yes Genomic sequence 1 Asp P. ramorum PramEPI1 303509335 Yes Genomic sequence 3 Asp, Met, Glu P. ramorum PramEPI4 324426165 Yes Genomic sequence 3 Asp, Thr, Asp P. ramorum PramEPI5 324427992 Yes Genomic sequence 1 Arg P. ramorum PramEPI9 303791515 Yes Genomic sequence 1 Arg P. ramorum PramEPI10 303447516 Yes Ggenomic sequence 3 Asp, Asp, Asp P. ramorum PramEPI11 303578321 Yes Ggenomic sequence 1 Asp P. brassicae PbraEPI1 AY589086 Yes Mycelium, nitrogen starvation 2 Asn, Met P. brassicae PbraEPI2 AY589087 NA Mycelium 1 His P. halstedii PhaEPI1 CB174657 Yes Infected sunflower 1 Arg NA, not available. Ti (Trace Identifier) number from NCBI Trace Archive (www.ncbi.nlm.nih.gov/Traces/trace.cgi). time. Initial reaction velocities were measured by monitoring the ab- cored from the gel, and protein digestion was carried out as previously sorbance change at 405 nm over reaction time using the HTS 7000 Bio described (47). The liquid chromatography-mass spectrometry system Assay Reader (PerkinElmer Life Sciences). K was determined fol- used is a Finnigan LCQ-Deca ion trap mass spectrometer system with i app lowing the method described by Morris et al. (15). The slope of the linear a Protana microelectrospray ion source interfaced to a self-packed 10 plot of [V /V ]  1 versus [I] was estimated as 1/ K . K was cm  75 m Phenomenex Jupiter C18 reversed-phase capillary chro- 0 i i app i app converted to K according to the formula K  K /(1  [S]/K ) (46). matography column. 2-l volumes of the peptide extract were injected, i i i app m Varying concentrations of substrate were incubated with 2 pmol of and the peptides were eluted from the column by an acetonitrile, 0.05 M subtilisin A in a total volume of 200 l under the conditions described acetic acid gradient at a flow rate of 0.2 l/min. The microelectrospray above, and the initial velocities were measured by monitoring the ion source was operated at 2.5 kV. The digest was analyzed using the absorbance at 405 nm. The K was determined graphically by double- data-dependent multitask capability of the instrument resulting in reciprocal Lineweaver-Burk plots of 1/[v] versus 1/[s]. 1000 collision-induced dissociation spectra of ions ranging in abun- Inhibition assays of plant proteases by rEPI1 were carried out with dance over several orders of magnitude. The data were analyzed by TM the QuantiCleave Protease Assay Kit (Pierce) and in-gel protease using all collision-induced dissociation spectra collected in the experi- assays using the Bio-Rad zymogram buffer system. For the first ment to search the NCBI nonredundant data base with the search method, 50 l of intercellular fluids were preincubated with or without program TurboSequest. All matching spectra were verified by manual 10 pmol of rEPI1 at 25 °C for 30 min, and the protease activities were interpretation. subsequently measured. For the in-gel protease assays, 10 pmol of rEPI1 were preincubated with 8 l of intercellular fluids for 30 min at RESULTS 25 °C and then mixed with zymogram sample buffer and loaded on a EPI1 Belongs to the Kazal Family of Protease Inhibitors—We 10% SDS-polyacrylamide gel without boiling or addition of reducing mined an EST data set generated from tomato leaves 3 days reagents. Following electrophoresis, the gel was incubated in 1 zymo- gram renaturation buffer for 30 min. Then the gel was incubated in 1 after infection with P. infestans using two methods: 1) GC zymogram development buffer for4hat37 °C before staining with 0.5% counting to distinguish between Phytophthora and tomato se- Coomassie Brilliant Blue. quences (31) and 2) PexFinder to identify cDNAs encoding Coimmunoprecipitation—Coimmunoprecipitation of rEPI1 and to- extracellular proteins (32). 488 of 2808 ESTs examined showed mato intercellular fluid proteins was performed using the FLAG-tagged a GC content higher than 53%. Of these 42 were predicted to protein immunoprecipitation kit (Sigma) following the manufacturer’s encode extracellular proteins using the criteria of Torto et al. instructions. 100 pmol of purified rEPI1 were preincubated with 200 l of tomato intercellular fluid for 30 min at 25 °C. 50 l of anti-FLAG M2 (32). These ESTs were then annotated by similarity and motif resin was added and incubated at 4 °C for 2 h with gentle shaking. The searches against public databases. One EST, PC064G6 (GC precipitated protein complexes were eluted in 60 l of FLAG peptide content, 57.4%), showed similarity to proteins of the Kazal solution (150 ng/l) and were analyzed by SDS-PAGE and Western blot serine protease inhibitor family. DNA sequencing of the full analyses. cDNA revealed an open reading frame of 450 bp corresponding Tandem Mass Spectrometric Sequencing—Tandem mass spectromet- to a predicted translated product of 149 amino acids (Fig. 1A). ric sequencing was performed at the proteomics facility of The Cleve- land Clinic Foundation (Cleveland, OH). The selected protein band was SignalP (48) analysis of the predicted protein identified a 16- Protease Inhibitors from Phytophthora 26373 genes were predicted to have signal peptides based on SignalP (48, 50). We used Clustal X (40) to generate a multiple alignment of representative oomycete Kazal domains with domains from signal crayfish PAPI-1 (the best hit in BLASTP searches TM against GenBank nonredundant database) and T. gondii TgPI1 (Fig. 1B). Amino acid residues defining the Kazal family signature, including the cysteine backbone, tyrosine, and as- paragine residues, were highly conserved. The oomycete do- main structure was usually C-X -C-X -C-X -Y-X -C-X -C- 3,4 7 6 3 6 X -C. The first EPI1 domain was atypical and lacked 9,12,13,14 3 6 Cys and Cys but retained the other four cysteines (Fig. 1A). The predicted active site P1, which is central to the specificity of Kazal inhibitors (51, 52), was variable with 10 different amino acids represented (Ala, Asp, Glu, His, Lys, Met, Asn, Arg, Ser, and Thr). Remarkably, half (28 of 56) the P1 residues, including those of EPI1, were aspartate (Asp), an uncommon P1 amino acid in other natural Kazal inhibitors. These results suggest that genes encoding proteins with Kazal domains are diverse and ubiquitous in plant pathogenic oomycetes. EPI1 Inhibits the Serine Protease Subtilisin A—To deter- mine whether EPI1 functions as a serine protease inhibitor as FIG.1. EPI1 belongs to the Kazal family of serine protease predicted by bioinformatic analyses, we expressed in E. coli and inhibitors. A, schematic representation of EPI1 structure. The signal peptide (SP) and two Kazal domains (EPI1a and EPI1b) are shown in affinity-purified rEPI1 as a fusion protein with the FLAG gray. The numbers indicate the positions of amino acid residues starting epitope tag at the N terminus. Silver staining of the purified from the N terminus. The cysteine residues corresponding to the two rEPI1 fraction after SDS-PAGE revealed a single band indicat- Kazal domains are indicated by C, and the disulfide linkages predicted ing high purity. Chymotrypsin, trypsin, and subtilisin A, rep- based on the structure of other Kazal domains are shown. The positions of the P1 aspartate residues are indicated by arrows. B, sequence resenting three major classes of serine proteases, were selected alignment of EPI domains with representative Kazal family inhibitor for inhibition assays with the purified rEPI1. Protease activity domains. Protein names correspond to protease inhibitors of the oomy- was measured with or without EPI1. In repeated assays, rEPI1 cetes P. infestans (EPI1a and EPI1b; this study), P. sojae (PsojEPI1a– d; TM was found to inhibit about 90% of the measured activity of this study), and P. halstedii (PhaEPI1; GenBank accession number CB174657), the crayfish Pacifastacus leniusculus (PAPI-1a– d; subtilisin A but did not cause apparent inhibition of the other CAA56043), as well as the apicomplexan T. gondii (TgPI-1a– d; two proteases (Fig. 2A). Time courses of chromogenic substrate AF121778). Amino acid residues that define the Kazal family protease hydrolysis by subtilisin A in the presence of increasing inhibitor domain are marked with asterisks. The predicted P1 residues amounts of rEPI1 were performed and indicated that rEPI1 are shown by the arrowhead. inhibition followed a typical dose-response pattern (Fig. 2B). The inhibitory constant (K ) for subtilisin A inhibition by rEPI1 amino acid signal peptide with a significant mean S value of was determined at 2.77  1.07 nM. These results suggest that 0.88 and hidden Markov model score of 0.97. Similarity epi1 encodes a functional protease inhibitor that specifically searches of the predicted protein against the nonredundant targets the subtilisin class of serine proteases. TM database of GenBank using the BLASTP program (33) re- EPI1 Inhibits BTH-induced Apoplastic Proteases from Toma- vealed significant matches to Kazal protease inhibitors with to—In tomato, some members of the subtilisin-like family P69, the best hit corresponding to the signal crayfish protease in- 10 namely P69B and P69C, are known to be induced by pathogens hibitor PAPI-1 (E value  10 ). Searches against the Inter- and stress treatments and are classified as PR proteins (PR-7 Pro database (37) revealed two domains similar to InterPro class) (20 –22). To test whether rEPI1 inhibits PR-like pro- IPR002350 for Kazal inhibitors (Fig. 1A). Based on these anal- teases in tomato, the salicylic acid analog BTH was applied to yses, we propose that the examined P. infestans cDNA is likely tomato plants to induce defense-related proteases. In-gel pro- to encode a two-headed Kazal serine protease inhibitor, and we tease assays of tomato leaf intercellular fluid from both H O- designated the cDNA epi1 (extracellular protease inhibitor 1). 2 treated and BTH-treated plants revealed that, as expected, Proteins with Kazal Domains Are Diverse and Ubiquitous in BTH induced the production of abundant extracellular pro- Oomycetes—We used EPI1 and other Kazal domain sequences teases in tomato that migrated as two separate but close bands to search for Kazal-like motifs in sequence databases from (Fig. 3A). Inhibition assays revealed that rEPI1 dramatically oomycetes and other microbial plant pathogens (see “Material inhibited these BTH-induced proteases as well as partially and Methods”). We failed to identify sequences similar to the inhibited a constitutive protease. The total endoprotease activ- Kazal domain in all examined fungal and bacterial databases, ity of tomato intercellular fluids was also measured in the except for a predicted protein from the ammonia-oxidizing TM absence or presence of rEPI1. Significant inhibition of endopro- bacterium Nitrosomonas europaea (GenBank accession tease activity was observed and corresponded to 28 and 27% of NP_841298) (49). On the other hand, we unraveled a total of 35 total activity in control and BTH-treated tomato, respectively different putative proteins with 56 predicted Kazal-like do- (Fig. 4). mains (range, 1– 4/protein) in five plant pathogenic oomycete EPI1 Interacts with Pathogenesis-related Subtilases of the species, P. infestans, Phytophthora sojae, Phytophthora ramo- Tomato P69 Subfamily—To identify the plant proteases tar- rum, P. brassicae, and the downy mildew Plasmopara halstedii (Table I). These oomycete Kazal motifs were identified in ESTs geted by rEPI1, coimmunoprecipitation was performed on to- mato intercellular fluid incubated with rEPI1 using FLAG from a variety of developmental stages, including host tissue infected with P. sojae and P. halstedii. We identified a putative antibody covalently linked agarose beads. In addition to rEPI1, two proteins were pulled down with the FLAG antibody only in full open reading frame sequence for 27 of the identified genes and the putative start codon for 33 of the genes. All of these 33 the presence of rEPI1 (Fig. 5A). These two proteins exhibited a 26374 Protease Inhibitors from Phytophthora FIG.4. rEPI1 inhibition of total protease activity from tomato intercellular fluids. Total protease activity of Intercellular fluids obtained from water-treated (BTH) and BTH-treated (BTH) tomato plants was measured in the absence (gray columns) or presence (black TM columns) of rEPI1 using the QuantiCleave protease assay kit as described under “Materials and Methods.” Activity is expressed as absorbance at 405 nm. The bars correspond to the means of three independent replications of one representative experiment of three performed. The error bars represent the standard errors calculated from the three replications. FIG.2. rEPI1 inhibits subtilisin A. A, protease activity of chymo- trypsin, subtilisin A, and trypsin in the absence (gray columns)or presence of rEPI1 (black columns). The activities were determined TM using the QuantiCleave protease assay kit as described under “Ma- terials and Methods.” Activity is expressed as a percentage of total protease activity in the absence of protease inhibitors. The bars corre- spond to the means of three independent replications of one represent- ative experiment of three performed. The error bars represent the standard errors calculated from the three replications. B, time course of substrate hydrolysis by subtilisin A in the presence of varying concen- trations of rEPI1. Protease activity was measured as absorbance at 405 FIG.5. Coimmunoprecipitation of rEPIs and P69 subtilases nm based on hydrolysis of a chromogenic substrate. The final concen- using FLAG antisera. A, eluates from coimmunoprecipitation of tration of subtilisin A is 10 nM. The concentrations of rEPI1 are indi- rEPI1 with proteins in tomato intercellular fluids were run on SDS- cated next to the curves. PAGE gel followed by staining with silver nitrate. The numbers on the left indicate the molecular masses of the marker proteins in kDa. rEPI1 indicates whether or not rEPI1 was added to the reaction mix. BTH indicates whether or not the intercellular fluids were obtained from plants treated with BTH. The lower molecular mass band corresponds to rEPI1, and the high molecular mass bands correspond to the rEPI1- interacting protein(s). B, the same eluate samples were run on SDS- PAGE gel followed by staining with Coomassie Brilliant Blue (Coom.)or immunoblotting with antisera raised against a peptide specific for the tomato P69 family (-P69), and FLAG (-FLAG), respectively. The top two panels (Coom. and -P69) correspond to the high molecular mass bands of A, whereas the bottom panel (-FLAG) corresponds to the low molecular mass band. similar molecular mass of 70 kDa (Fig. 5A) and were more FIG.3. rEPI1 inhibits BTH-induced tomato proteases. A, inter- cellular fluids obtained from water-treated () and BTH-treated () abundant in BTH-induced intercellular fluid (Fig. 5B). These tomato plants were run on SDS-PAGE gel followed by staining with results prompted us to test whether these proteins could be Coomassie Brilliant Blue (left panel) or were used in zymogen in-gel tomato P69 subtilisin-like proteases. Western blot analyses protease assays (right panel). The asterisks represent known pathogen- with antisera raised against a peptide specific to P69 subtilisin- esis-related proteins PR1, PR3, and PR2 (from bottom to top) and confirm the induction of defense responses by BTH. The arrows indicate like proteases strongly interacted with both bands, suggesting BTH-induced protease activities that migrated as two close bands. B, that rEPI1 interacts with P69 subtilases of tomato (Fig. 5B). To inhibition of tomato proteases by rEPI1. Intercellular fluids from BTH- confirm the results obtained with the Western blot and further treated tomato leaves were incubated in the absence ( rEPI1)or identify which P69 isoforms are the main targets of rEPI1, the presence of rEPI1 ( rEPI1) and then analyzed using zymogen in-gel protease assays. The arrows indicate the BTH-induced protease bands. two closely migrated protein bands (Fig. 5) were cored from the Protease Inhibitors from Phytophthora 26375 FIG.6. Tandem mass spectrometry identifies P69B as the main target of rEPI1. The amino acid sequence of P69B subtilisin-like protease precursor is shown with the signal peptide sequence in italics and the propeptide domain sequence in gray. The 21 peptides se- quenced by tandem mass spectrometry are shown in bold type. Each sequenced peptide ends with an Arg or a Lys residue, which is high- lighted in bold italics. Underlined sequences are specific to P69B among FIG.7. The epi1 and P69 genes are concurrently expressed the known P69 isoforms. during colonization of tomato by P. infestans. A, time course of expression of P. infestans epi1 and actA and tomato P69B and tubulin Coomassie Blue-stained SDS-PAGE gel as one sample and during colonization of tomato by P. infestans. Total RNA isolated from analyzed by tandem mass spectrometry. A total of 21 trypsin- infected leaves of tomato, 0, 1, 2, 3, or 4 days after inoculation, from digested peptides were sequenced and perfectly matched the noninfected leaves (To), and from P. infestans mycelium grown in syn- TM subtilisin-like protease P69B (GenBank accession number thetic medium (My) was hybridized with probes from the four genes. T07184 or CAA76725) (Fig. 6). Of these 21 peptides, 13 pep- The approximate sizes of the transcripts are 600 nucleotides for epi1, 1600 nucleotides for actA, and 2500 nucleotides for P69B and tubulin. tides were specific to P69B and did not match any of the other B, RT-PCR analysis of epi1, P69A, P69B, and P69D expression during five known P69 isoforms. At this stage it cannot be ruled out colonization by P. infestans. Total RNA from a time course similar to the that the two closely migrated protein bands contain other iso- one described in A was used in RT-PCR amplifications as described in forms in minor amounts, but the results from the tandem mass the text. Amplification of P. infestans elongation factor 2 (Pief2) and tomato elongation factor 1 (Toef1) were used as controls to determine spectrometry clearly showed that P69B is the main target of the relative expression of epi1 and P69 genes, respectively. C, Western rEPI1. blot analyses of tomato P69 subtilases during colonization by P. infes- The epi1 and P69B Gene Are Concurrently Expressed during tans. The time course is as described for A. Equal volumes of intercel- Infection of Tomato by P. infestans—Expression pattern of both lular fluids were obtained from infected tomato leaves, subjected to SDS-PAGE, and immunoblotted with P69 antisera (-P69). epi1 and P69 genes during infection of tomato by P. infestans was studied by Northern blot and RT-PCR analyses. The epi1 gene displayed the highest mRNA levels 3 days post-inocula- lence or avirulence molecules, known as effectors. In suscepti- tion and was moderately up-regulated (approximately 2 ble plants, biotrophic plant pathogens produce effectors that based on PhosphorImager quantification) compared with in promote infection by suppressing defense responses. Here, we vitro grown mycelium and relative to the constitutive actA gene describe EPI1, a two-domain extracellular protease inhibitor (Fig. 7A). Semi-quantitative RT-PCR analyses confirmed these from P. infestans that inhibits apoplastic subtilases of tomato, results (Fig. 7B). The expression of P69 protease genes was namely the PR proteins P69. Based on its biological activity induced after inoculation with P. infestans and attained the and expression pattern, EPI1 may function as a disease effector highest level 2 and 3 days after inoculation (Fig. 7A). Semi- molecule and may play an important role in P. infestans colo- quantitative RT-PCR amplifications using primers specific for nization of host apoplast. P69A, P69B, and P69D, indicated that the pathogenesis-re- Suppression of host defenses is thought to play a critical role lated P69B gene is the only gene that is up-regulated during in plant-microbe interactions, especially those involving biotro- interaction with P. infestans (Fig. 7B). We could not assess the phic pathogens that require live plant cells to establish a suc- expression of P69C, the other pathogenesis-related gene of the cessful infection (8, 53). Nonetheless, only a few pathogen mol- P69 family (21), because we repeatedly failed to amplify P69C ecules that suppress host defenses have been identified. from tomato cultivar Ohio 7814 based on published sequences. Examples include tomatinase, a saponin-detoxifying enzyme Increase in P69 protein during infection of tomato by P. infes- from the fungal pathogen Septoria lycopersici that was recently tans was also noted by Western blot analyses with P69 antisera shown to indirectly suppress host defense responses through of intercellular fluids obtained from a time course infection its degradation products (9). P. sojae secretes glucanase inhib- (Fig. 7C). Altogether, these results suggest that epi1 and P69 itor proteins that inhibit a soybean endo--1,3-glucanase and genes are concurrently expressed during infection and support are thought to function as counterdefensive molecules that the possibility of direct interaction between P. infestans EPI1 inhibit the degradation of -1,3/1,6-glucans in the pathogen cell and plant P69 proteases, particularly P69B, at the infection wall and/or the release of defense-eliciting oligosaccharides by interface. host endo--1,3 glucanases (54). P. infestans and other Phyto- DISCUSSION phthora species produce water-soluble glucans that suppress Plant pathogens manipulate biochemical and physiological induction of host defense responses (10 –12). Here, we describe processes in their host plants through a diverse array of viru- a novel class of pathogen suppressors of plant defense response, 26376 Protease Inhibitors from Phytophthora namely extracellular protease inhibitors that directly interact less are colonized by a variety of microbial pathogens. Appar- with and inhibit host proteases. This interaction could form ently, P. infestans and T. gondii, even though phylogenetically another type of defense-counterdefense mechanism between unrelated, have independently recruited secreted proteins of plants and microbial pathogens the Kazal family to inhibit host proteases and adapt to pro- TM tease-rich host environments. Interestingly, unlike the T. gon- We scanned GenBank and several other sequence data bases for the occurrence of Kazal-like domains. The examined dii inhibitors (15, 19), EPI1 does not inhibit trypsin and chy- mortrypsin, suggesting that coevolution between the inhibitors data sets included the full genome sequence of several plant and their target proteases may have shaped the inhibitor spec- pathogenic bacteria and fungi. A 235-amino acid protein from TM ificity. Future structural and functional characterization of the ammonia-oxidizing bacterium N. europaea (GenBank Kazal protease inhibitors from animal and plant pathogens will accession NP_841298) was the only bacterial or fungal protein shed some light on interesting questions on the evolution of with significant similarity to the Kazal motif. In sharp con- pathogenesis in eukaryotic microbes and the coevolution of trast, 56 Kazal-like motifs were detected in 35 predicted pro- pathogen effectors with host targets. teins of five plant pathogenic oomycete species, P. infestans, P. sojae, P. ramorum, P. brassicae, and P. halstedii (Table I). Acknowledgments—We are grateful to Caitlin Cardina, Shujing Interestingly, oomycete Kazal motif genes are often expressed Dong, Diane Kinney, and Kristin Wille for technical assistance; during host colonization. Five of the identified sequences were Margaret Redinbaugh and Saskia Hogenhout for valuable advice on the protein work; Tea Meulia and the staff of the Ohio Agricultural Re- from cDNAs obtained from infected plant tissue corresponding search and Development Center Molecular and Cellular Imaging Cen- to diverse oomycete pathosystems: P. infestans tomato/potato, ter for help with DNA sequencing; and Andrew Keightley and Mike P. sojae soybean, and P. halstedii sunflower. 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A Kazal-like Extracellular Serine Protease Inhibitor from Phytophthora infestans Targets the Tomato Pathogenesis-related Protease P69B

Tian, Miaoying; Huitema, Edgar; da Cunha, Luis; Torto-Alalibo, Trudy; Kamoun, Sophien
Journal of Biological Chemistry – Jun 1, 2004
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 25, Issue of June 18, pp. 26370 –26377, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Kazal-like Extracellular Serine Protease Inhibitor from Phytophthora infestans Targets the Tomato Pathogenesis-related Protease P69B* Received for publication, January 28, 2004, and in revised form, April 15, 2004 Published, JBC Papers in Press, April 19, 2004, DOI 10.1074/jbc.M400941200 Miaoying Tian, Edgar Huitema, Luis da Cunha, Trudy Torto-Alalibo, and Sophien Kamoun‡ From the Department of Plant Pathology, The Ohio State University, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691 host cells but later causes extensive necrosis of host tissue, a The oomycetes form one of several lineages within the eukaryotes that independently evolved a parasitic life- lifestyle that is known as hemibiotrophy. As with other biotro- style and consequently are thought to have developed phic plant pathogens, processes associated with P. infestans alternative mechanisms of pathogenicity. The oomycete pathogenesis are thought to include the suppression of host Phytophthora infestans causes late blight, a ravaging defense responses (3, 8, 9). In P. infestans, water-soluble glu- disease of potato and tomato. Little is known about pro- cans have been reported to suppress host defenses in a plant cesses associated with P. infestans pathogenesis, partic- cultivar-specific manner (10 –12). Nevertheless, the molecular ularly the suppression of host defense responses. We basis of suppression of host defenses by Phytophthora remains describe and functionally characterize an extracellular poorly understood (3). It is tempting to speculate that unique protease inhibitor, EPI1, from P. infestans. EPI1 con- classes of suppressor genes have been recruited to aid in infec- tains two domains with significant similarity to the tion and counteract host defenses during the evolution of Kazal family of serine protease inhibitors. Database pathogenesis in the oomycete lineage. searches suggested that Kazal-like proteins are mainly Parasitic eukaryotes often face inhospitable environments in restricted to animals and apicomplexan parasites but their hosts. For example, parasites that colonize or transit appear to be widespread and diverse in the oomycetes. through the mammalian digestive tract must adapt to the Recombinant EPI1 specifically inhibited subtilisin A diverse and abundant array of proteases secreted in the gastric among major serine proteases and inhibited and inter- juices (13–15). Some of these parasites secrete inhibitors that acted with the pathogenesis-related P69B subtilisin-like target host proteases and may aid in survival and colonization serine protease of tomato in intercellular fluids. The of the host. For instance, the apicomplexan obligate parasite epi1 and P69B genes were coordinately expressed and Toxoplasma gondii secretes TgPI-1 and TgPI-2, four-domain up-regulated during infection of tomato by P. infestans. serine protease inhibitors of the Kazal family (15–19), and the Inhibition of tomato proteases by EPI1 could form a novel type of defense-counterdefense mechanism be- intestinal hookworm Ancylostoma ceylanicum secretes an tween plants and microbial pathogens. In addition, this 8-kDa broad spectrum serine protease inhibitor of the Kunitz study points to a common virulence strategy between family (14). In plants, the apoplast (intercellular fluid) forms a the oomycete plant pathogen P. infestans and several protease-rich environment that is colonized by many patho- mammalian parasites, such as the apicomplexan Toxo- gens, including P. infestans and the fungus Cladosporium ful- plasma gondii. vum. In tomato, apoplastic proteases are integral components of the plant defense response. Serine proteases of the P69 subtilase family have long been tied to pathogen defense, and Parasitic and pathogenic lifestyles have evolved repeatedly two isoforms, P69B and P69C, are known as pathogenesis- in eukaryotes (1). Several parasitic eukaryotes represent deep related proteins (PR-7 class) (20 –22). More recently, an apo- phylogenetic lineages, suggesting that they feature unique mo- plastic papain-like cysteine protease, Rcr3, was shown to be lecular processes for infecting their hosts. One such lineage is required for specific resistance to C. fulvum (23). In addition, formed by the oomycetes, a group of fungus-like organisms that several C. fulvum extracellular proteins are processed or de- are distantly related to fungi but closely related to brown algae graded by host proteases in the apoplast, resulting in altered and diatoms in the Stramenopiles (1–3). One of the most noto- functionality (24, 25). rious and destructive oomycete is the Irish famine pathogen, Despite the importance of extracellular proteases in plant Phytophthora infestans. This species causes late blight, a re- defense, to date no protease inhibitor has been reported from emerging and ravaging disease of potato and tomato (4 –7). microbial plant pathogens. In this paper, we describe and func- During the early stages of infection, P. infestans requires living tionally characterize an extracellular protease inhibitor, EPI1, from P. infestans. EPI1 contains two domains with significant * This work was supported by the Ohio Agricultural Research and similarity to the Kazal family of serine protease inhibitors, Development Center Research Enhancement Grant Program and Syn- which also occurs in many animal species and in apicomplexan genta Biotechnology Inc. Salaries and research support were provided parasites. In vitro studies indicated that recombinant EPI1 by State and Federal Funds appropriated to the Ohio Agricultural (rEPI1) specifically inhibited subtilisin A among the major Research and Development Center, the Ohio State University. The costs of publication of this article were defrayed in part by the payment serine proteases. rEPI1 was further demonstrated to inhibit of page charges. This article must therefore be hereby marked “adver- and interact with tomato P69B subtilisin-like serine protease. tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: Dept. of Plant Pa- thology, The Ohio State University-OARDC, 1680 Madison Ave., The abbreviations used are: rEPI1, recombinant EPI1; BTH, benzo- Wooster, OH 44691. Tel.: 330-263-3847; Fax: 330-263-3841; E-mail: (1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; EST, expressed [email protected]. sequence tag; RT, reverse transcriptase. 26370 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Protease Inhibitors from Phytophthora 26371 random primer labeling using gel-purified fragments digested or PCR- The epi1 and P69B genes were coordinately expressed and amplified from the corresponding cDNA clones (Ref. 41 and this study). up-regulated during infection of tomato by P. infestans. Overall Probe for tomato P69B was generated from a gel-purified RT-PCR these results suggest that inhibition of tomato proteases by fragment amplified from total RNA isolated from infected tomato tis- P. infestans EPI1 could form a novel type of defense-counter- TM sue. For RT-PCR, total RNA was treated with DNA-free (Ambion, defense mechanism between plants and microbial pathogens. Austin, TX) to remove contaminating DNA, and first-strand cDNAs TM In addition, this study points to a common virulence strategy were synthesized using the ThermoScript RT-PCR system from 5 g of total RNA following the instructions of the manufacturer (Invitro- between the oomycete plant pathogen P. infestans and mam- gen). PCR amplifications were carried out with 0.005% of the cDNA malian parasites, such as the apicomplexan T. gondii. product. The oligonucleotide primer pairs, P69A-RTF1 (5-TGGCAGG- TGGTGGAGTTCCGAGGG-3) and P69A-RTR1 (5-CATTGGATCAAC- MATERIALS AND METHODS AAAAGTGCAATTGG-3), P69B-RTF1 (5-CAGCACTCGGCCATGTA- Phytophthora Strains and Culture Conditions—P. infestans isolate GCCAATGTT-3) and P69B-RTR1 (5-CTAGGCAGACACAACTGCAA- 90128 (A2 mating type, race 1.3.4.7.8.9.10.11) was used throughout the TTGGACTTC-3), P69D-RTF1 (5-TGCGAAGTATAAGTCTTCTCAGA- study. P. infestans 90128 was routinely grown on rye agar medium GTTGC-3), and P69D-RTR1 (5-TCAGCAGACACTCTAACTGCAATT- supplemented with 2% sucrose (26). For RNA extraction, plugs of my- GGAC-3), were designed to be gene-specific based on the published P69 celium were transferred to modified Plich medium (27) and grown for gene sequences (21) and were used for the amplification of P69A, P69B, 2–3 weeks before harvesting. and P69D sequences, respectively. The oligonucleotides EPI1-F1 and Bacterial Strains and Plasmids—Escherichia coli XL1-Blue was EPI1-R1, previously used for cloning epi1 into pFLAG-ATS vector, were used in this study and was routinely grown at 37 °C in LB medium (28). used to detect epi1 transcripts by RT-PCR. Primer specificity was Plasmid pFLAG-EPI1 was constructed by cloning PCR-amplified DNA confirmed by sequencing the RT-PCR products. The expression of P69A, fragment corresponding to the mature sequence of EPI1 into the Hin- P69B, and P69D was controlled with primer pair EF1-F1 (5-GCT- dIII site of pFLAG-ATS (Sigma), a vector that allows secreted expres- GCTGTAACAAGGTTTGCTTTAATTCG-3) and EF1-R1 (5-CCAG- sion in E. coli. The oligonucleotides EPI1-F1 (5-GCGAAGCTTCAAA- CATCACACTGCACAGTTCACTTC-3), which are specific for the con- GCCCGCAAGTCATCAG-3) and EPI1-R1 (5-GCGAAGCTTATCCCT- stitutively expressed tomato elongation factor 1 gene (42). The CCTGCGGTGTC-3) were used to amplify the fragment. The intro- expression of epi1 was controlled with P. infestans elongation factor 2 duced HindIII restriction sites are underlined. The N-terminal se- gene using the primer pair described previously (43). quence of the processed FLAG-rEPI1 protein is DYKDDDDKVKLQS- SDS-PAGE and Western Blot Analyses—Proteins were subjected to PQVISPAP.... The FLAG epitope sequence is underlined, and the 10 –15% SDS-PAGE as previously described (28). Following electro- first 10 amino acids of mature EPI1 are shown in bold type. phoresis, the gels were stained with silver nitrate following the method Plant Growth, BTH Treatment, and Infection by P. infestans—To- of Merril et al. (44) or stained with Coomassie Brilliant Blue (28), or the mato (Lycopersicon esculentum) cultivar Ohio 7814 was used through- proteins were transferred to supported nitrocellulose membranes (Bio- out the study and grown in pots at 25 °C, 60% humidity, under 16 Rad) using a Mini Trans-Blot apparatus (Bio-Rad). Detection of anti- h-light/8 h-dark cycle. We used the salicylic acid analog BTH to mimic gen-antibody complexes was carried out with a Western blot alkaline pathogen infection. For BTH treatment, 10 ml of a 25 g/ml BTH phosphatase kit (Bio-Rad). Antisera to P69 subtilases were produced by solution was applied to 3-week-old tomato plants by soil drench. Plants immunizing rabbits with the keyhole limpet hemocyanin-conjugated treated with 10 ml of water were used as controls. Leaves from BTH- peptide, H2N-TTHTPSFLGLQQNC-amide. The sequence underlined is treated and control plants were detached for isolation of intercellular located at the N terminus of mature P69B and P69D proteins (21) and fluids 6 days after treatment. Time courses of P. infestans infection of was chosen for its highly antigenic characteristics and conservation tomato leaves were performed exactly as described earlier (29). 10-l among P69 proteins. Selection of peptides for highly antigenic charac- droplets containing 1,000 zoospores of P. infestans were used to inocu- teristics, peptide synthesis, and conjugation, as well as antisera pro- late the underside of detached tomato leaves. Leaf discs of similar sizes duction, was performed by Rockland Immunochemicals (Gilbertsville, were dissected from the inoculated regions while making sure that the PA). In Western blot analyses, the antisera to the P69 peptide reacted inoculation spot is in the center of sampled area. Leaf discs were frozen only with 70-kDa bands from tomato intercellular fluids. in liquid nitrogen and stored at 80 °C for later use in RNA extraction. Expression and Purification of rEPI1—Expression of rEPI from For isolation of intercellular fluids, tomato leaves were sprayed with pFLAG-EPI1 was conducted as described previously (45). Overnight zoospore suspensions at the concentration mentioned above (10 /ml), cultures of E. coli XL1-blue containing pFLAG-EPI1 were diluted (1: and the intact leaves were collected at different time points for imme- 100) in LB medium containing ampicillin (50 g/ml) and incubated at diate preparation of intercellular fluids. 37 °C. When the A of the cultures reached 0.6, isopropyl--D-thioga- Isolation of Intercellular Fluids—Intercellular fluids were prepared lactopyranoside was added to a final concentration of 0.4 mM. The using a 0.24 M sorbitol solution according to the method of de Wit and cultures were further incubated for 5– 6 h before processing. rEPI1 was Spikman (30). The intercellular fluids were filter-sterilized (0.22 M) recovered from the culture supernatant and was purified by immuno- and were used immediately or stored at 20 °C. affinity using gravity column packed with anti-FLAG M2 affinity gel Sequence Analyses—GC counting was performed as described else- (Sigma). The proteins were eluted with 0.1 M glycine, pH 3.5, and where (31). PexFinder and signal peptide predictions were performed as immediately equilibrated to neutral pH with 20 lof1 M Tris, pH 8.0, described by Torto et al. (32). Similarity searches were performed lo- for each 1-ml eluted fraction. The protein concentrations were deter- cally on an Intel Linux or a Mac OSX work station or through the mined using the Bio-Rad protein assay. To determine the purity of internet on the NCGR (www.ncgr.org) and Whitehead Institute web rEPI1, 0.5 g of the purified protein was run on a SDS-PAGE gel servers (www-genome.wi.mit.edu/resources.html). Search programs in- followed by staining with silver nitrate. cluded BLAST (33), and the similarity search programs implemented in Assays of Protease Inhibition—Inhibition assays of commercial ser- the BLOCKS (34), pfam (35), SMART (36), and InterPro (37) websites. ine proteases by rEPI1 were performed by the colorimetric Quanti- TM TM The examined sequence databases included GenBank nonredundant, Cleave Protease Assay Kit (Pierce). 20 pmol of rEPI1 was preincu- dBEST, and TraceDB (38), PGC (39), SPC, a proprietary database of bated with 20 pmol of trypsin (Pierce), chymotrypsin (Sigma), or Syngenta Inc. containing 75,000 ESTs from P. infestans (courtesy of subtilisin A (Carlsberg) (Sigma), in a volume of 50 l for 30 min at the Syngenta Phytophthora Consortium, Research Triangle Park, NC), 25 °C, followed by incubation with 100 l of succinylated casein (2 and the genome sequences of the fungal species Aspergillus nidulans, mg/ml) in 50 mM Tris buffer, pH 8, containing 20 mM CaCl at room Magnaporthe grisea, Neurospora crassa, and Fusarium graminearum temperature for 20 min. Protease activity was measured as absorbance available through the Whitehead Institute Fungal Genome Initiative at 405 nm using a HTS 7000 Bio Assay Reader (PerkinElmer Life Databases (www-genome.wi.mit.edu/resources.html). Multiple align- Sciences) 20 min after the addition of chromogenic reagent 2,4,6-trini- ments of the Kazal domains were conducted using the program trobenzene sulfonic acid, which reacts with the primary amine of di- CLUSTAL-X (40). The P. infestans and Phytophthora brassicae se- gested peptide and produces a color reaction that can be quantified by TM quences described in this paper were deposited in GenBank under absorbance reader. Detailed kinetic analysis of Subtilisin A inhibition accession numbers AY586273-AY586284 and AY589086-AY589087, re- by rEPI1 was performed as follows. 2 pmol of subtilisin A was preincu- spectively. Other sequences were obtained from the NCBI nr, dBEST, bated with increasing concentrations of rEPI1 in a volume of 50 l for or Trace Archive data bases (www.ncbi.nlm.nih.gov) (Table I). 15 min at 25 °C and was followed by the addition of 150 l assay buffer: RNA Isolation, Northern Blot, and RT-PCR Analyses—RNA isolation 50 mM Tris-Cl, pH 8.0, containing 2.5% Me SO, and 500 M subtilisin and Northern blot hybridizations were performed as described earlier chromogenic substrate Boc-Gly-Gly-Leu-pNA (Calbiochem, La Jolla, (32). Probes for epi1, actA, and tomato -tubulin were generated by CA). The experiments were performed three times and in triplicate each 26372 Protease Inhibitors from Phytophthora TABLE I Predicted Kazal-like proteins from the oomycete plant pathogens P. infestans, P. sojae, P. ramorum, P. brassicae, and P. halstedii TM GenBank Number of Signal Species Protein accession Expression stage Kazal-like P1 residue peptide number domains P. infestans EPI1 AY586273 Yes Infected tomato 2 Asp, Asp P. infestans EPI2 AY586274 Yes Mycelium, H O -treated 2 Asp, Asp 2 2 P. infestans EPI3 AY586275 Yes Genomic sequence 1 Glu P. infestans EPI4 AY586276 Yes Mycelium, nitrogen starvation 3 Thr, Asp, Asp P. infestans EPI5 AY586277 Yes Mating culture 1 Arg P. infestans EPI6 AY586278 NA Infected tomato 2 Asp, Asp P. infestans EPI7 AY586279 Yes Genomic sequence 1 Asp P. infestans EPI8 AY586280 Yes Genomic sequence 1 Asp P. infestans EPI9 AY586281 Yes Mycelium, non-sporulating growth 1 Arg P. infestans EPI10 AY586282 Yes Zoospores 3 Asp, Asp, Asp P. infestans EPI11 AY586283 Yes Mating culture 1 Asp P. infestans EPI12 AY586284 Yes Infected potato, germinating cysts 1 Ser P. infestans EPI13 317886987 Yes Genomic sequence 1 Glu P. infestans EPI14 317892389 Yes Genomic sequence 1 His P. sojae PsojEPI1 CF842223 Yes Infected soybean 4 Ala, Glu, Lys, Ala P. sojae PsojEPI2 AAO24652 Yes Mycelium 1 Glu P. sojae PsojEPI3 274204995 Yes Genomic sequence 3 Met, Asp, Glu P. sojae PsojEPI4 273523724 Yes Genomic sequence 3 Asp, Thr, Asp P. sojae PsojEPI5 273752552 Yes Genomic sequence 1 Arg P. sojae PsojEPI6 273759065 Yes Genomic sequence 1 Glu P. sojae PsojEPI7 324111439 Yes Genomic sequence 1 Asp P. sojae PsojEPI8 273566013 Yes Genomic sequence 1 Asp P. sojae PsojEPI9 274071280 Yes Genomic sequence 1 Arg P. sojae PsojEPI10 273704880 Yes Genomic sequence 1 Ala P. sojae PsojEPI11 324096913 Yes Genomic sequence 1 Asp P. sojae PsojEPI12 324106054 Yes Genomic sequence 1 Asp P. ramorum PramEPI1 303509335 Yes Genomic sequence 3 Asp, Met, Glu P. ramorum PramEPI4 324426165 Yes Genomic sequence 3 Asp, Thr, Asp P. ramorum PramEPI5 324427992 Yes Genomic sequence 1 Arg P. ramorum PramEPI9 303791515 Yes Genomic sequence 1 Arg P. ramorum PramEPI10 303447516 Yes Ggenomic sequence 3 Asp, Asp, Asp P. ramorum PramEPI11 303578321 Yes Ggenomic sequence 1 Asp P. brassicae PbraEPI1 AY589086 Yes Mycelium, nitrogen starvation 2 Asn, Met P. brassicae PbraEPI2 AY589087 NA Mycelium 1 His P. halstedii PhaEPI1 CB174657 Yes Infected sunflower 1 Arg NA, not available. Ti (Trace Identifier) number from NCBI Trace Archive (www.ncbi.nlm.nih.gov/Traces/trace.cgi). time. Initial reaction velocities were measured by monitoring the ab- cored from the gel, and protein digestion was carried out as previously sorbance change at 405 nm over reaction time using the HTS 7000 Bio described (47). The liquid chromatography-mass spectrometry system Assay Reader (PerkinElmer Life Sciences). K was determined fol- used is a Finnigan LCQ-Deca ion trap mass spectrometer system with i app lowing the method described by Morris et al. (15). The slope of the linear a Protana microelectrospray ion source interfaced to a self-packed 10 plot of [V /V ]  1 versus [I] was estimated as 1/ K . K was cm  75 m Phenomenex Jupiter C18 reversed-phase capillary chro- 0 i i app i app converted to K according to the formula K  K /(1  [S]/K ) (46). matography column. 2-l volumes of the peptide extract were injected, i i i app m Varying concentrations of substrate were incubated with 2 pmol of and the peptides were eluted from the column by an acetonitrile, 0.05 M subtilisin A in a total volume of 200 l under the conditions described acetic acid gradient at a flow rate of 0.2 l/min. The microelectrospray above, and the initial velocities were measured by monitoring the ion source was operated at 2.5 kV. The digest was analyzed using the absorbance at 405 nm. The K was determined graphically by double- data-dependent multitask capability of the instrument resulting in reciprocal Lineweaver-Burk plots of 1/[v] versus 1/[s]. 1000 collision-induced dissociation spectra of ions ranging in abun- Inhibition assays of plant proteases by rEPI1 were carried out with dance over several orders of magnitude. The data were analyzed by TM the QuantiCleave Protease Assay Kit (Pierce) and in-gel protease using all collision-induced dissociation spectra collected in the experi- assays using the Bio-Rad zymogram buffer system. For the first ment to search the NCBI nonredundant data base with the search method, 50 l of intercellular fluids were preincubated with or without program TurboSequest. All matching spectra were verified by manual 10 pmol of rEPI1 at 25 °C for 30 min, and the protease activities were interpretation. subsequently measured. For the in-gel protease assays, 10 pmol of rEPI1 were preincubated with 8 l of intercellular fluids for 30 min at RESULTS 25 °C and then mixed with zymogram sample buffer and loaded on a EPI1 Belongs to the Kazal Family of Protease Inhibitors—We 10% SDS-polyacrylamide gel without boiling or addition of reducing mined an EST data set generated from tomato leaves 3 days reagents. Following electrophoresis, the gel was incubated in 1 zymo- gram renaturation buffer for 30 min. Then the gel was incubated in 1 after infection with P. infestans using two methods: 1) GC zymogram development buffer for4hat37 °C before staining with 0.5% counting to distinguish between Phytophthora and tomato se- Coomassie Brilliant Blue. quences (31) and 2) PexFinder to identify cDNAs encoding Coimmunoprecipitation—Coimmunoprecipitation of rEPI1 and to- extracellular proteins (32). 488 of 2808 ESTs examined showed mato intercellular fluid proteins was performed using the FLAG-tagged a GC content higher than 53%. Of these 42 were predicted to protein immunoprecipitation kit (Sigma) following the manufacturer’s encode extracellular proteins using the criteria of Torto et al. instructions. 100 pmol of purified rEPI1 were preincubated with 200 l of tomato intercellular fluid for 30 min at 25 °C. 50 l of anti-FLAG M2 (32). These ESTs were then annotated by similarity and motif resin was added and incubated at 4 °C for 2 h with gentle shaking. The searches against public databases. One EST, PC064G6 (GC precipitated protein complexes were eluted in 60 l of FLAG peptide content, 57.4%), showed similarity to proteins of the Kazal solution (150 ng/l) and were analyzed by SDS-PAGE and Western blot serine protease inhibitor family. DNA sequencing of the full analyses. cDNA revealed an open reading frame of 450 bp corresponding Tandem Mass Spectrometric Sequencing—Tandem mass spectromet- to a predicted translated product of 149 amino acids (Fig. 1A). ric sequencing was performed at the proteomics facility of The Cleve- land Clinic Foundation (Cleveland, OH). The selected protein band was SignalP (48) analysis of the predicted protein identified a 16- Protease Inhibitors from Phytophthora 26373 genes were predicted to have signal peptides based on SignalP (48, 50). We used Clustal X (40) to generate a multiple alignment of representative oomycete Kazal domains with domains from signal crayfish PAPI-1 (the best hit in BLASTP searches TM against GenBank nonredundant database) and T. gondii TgPI1 (Fig. 1B). Amino acid residues defining the Kazal family signature, including the cysteine backbone, tyrosine, and as- paragine residues, were highly conserved. The oomycete do- main structure was usually C-X -C-X -C-X -Y-X -C-X -C- 3,4 7 6 3 6 X -C. The first EPI1 domain was atypical and lacked 9,12,13,14 3 6 Cys and Cys but retained the other four cysteines (Fig. 1A). The predicted active site P1, which is central to the specificity of Kazal inhibitors (51, 52), was variable with 10 different amino acids represented (Ala, Asp, Glu, His, Lys, Met, Asn, Arg, Ser, and Thr). Remarkably, half (28 of 56) the P1 residues, including those of EPI1, were aspartate (Asp), an uncommon P1 amino acid in other natural Kazal inhibitors. These results suggest that genes encoding proteins with Kazal domains are diverse and ubiquitous in plant pathogenic oomycetes. EPI1 Inhibits the Serine Protease Subtilisin A—To deter- mine whether EPI1 functions as a serine protease inhibitor as FIG.1. EPI1 belongs to the Kazal family of serine protease predicted by bioinformatic analyses, we expressed in E. coli and inhibitors. A, schematic representation of EPI1 structure. The signal peptide (SP) and two Kazal domains (EPI1a and EPI1b) are shown in affinity-purified rEPI1 as a fusion protein with the FLAG gray. The numbers indicate the positions of amino acid residues starting epitope tag at the N terminus. Silver staining of the purified from the N terminus. The cysteine residues corresponding to the two rEPI1 fraction after SDS-PAGE revealed a single band indicat- Kazal domains are indicated by C, and the disulfide linkages predicted ing high purity. Chymotrypsin, trypsin, and subtilisin A, rep- based on the structure of other Kazal domains are shown. The positions of the P1 aspartate residues are indicated by arrows. B, sequence resenting three major classes of serine proteases, were selected alignment of EPI domains with representative Kazal family inhibitor for inhibition assays with the purified rEPI1. Protease activity domains. Protein names correspond to protease inhibitors of the oomy- was measured with or without EPI1. In repeated assays, rEPI1 cetes P. infestans (EPI1a and EPI1b; this study), P. sojae (PsojEPI1a– d; TM was found to inhibit about 90% of the measured activity of this study), and P. halstedii (PhaEPI1; GenBank accession number CB174657), the crayfish Pacifastacus leniusculus (PAPI-1a– d; subtilisin A but did not cause apparent inhibition of the other CAA56043), as well as the apicomplexan T. gondii (TgPI-1a– d; two proteases (Fig. 2A). Time courses of chromogenic substrate AF121778). Amino acid residues that define the Kazal family protease hydrolysis by subtilisin A in the presence of increasing inhibitor domain are marked with asterisks. The predicted P1 residues amounts of rEPI1 were performed and indicated that rEPI1 are shown by the arrowhead. inhibition followed a typical dose-response pattern (Fig. 2B). The inhibitory constant (K ) for subtilisin A inhibition by rEPI1 amino acid signal peptide with a significant mean S value of was determined at 2.77  1.07 nM. These results suggest that 0.88 and hidden Markov model score of 0.97. Similarity epi1 encodes a functional protease inhibitor that specifically searches of the predicted protein against the nonredundant targets the subtilisin class of serine proteases. TM database of GenBank using the BLASTP program (33) re- EPI1 Inhibits BTH-induced Apoplastic Proteases from Toma- vealed significant matches to Kazal protease inhibitors with to—In tomato, some members of the subtilisin-like family P69, the best hit corresponding to the signal crayfish protease in- 10 namely P69B and P69C, are known to be induced by pathogens hibitor PAPI-1 (E value  10 ). Searches against the Inter- and stress treatments and are classified as PR proteins (PR-7 Pro database (37) revealed two domains similar to InterPro class) (20 –22). To test whether rEPI1 inhibits PR-like pro- IPR002350 for Kazal inhibitors (Fig. 1A). Based on these anal- teases in tomato, the salicylic acid analog BTH was applied to yses, we propose that the examined P. infestans cDNA is likely tomato plants to induce defense-related proteases. In-gel pro- to encode a two-headed Kazal serine protease inhibitor, and we tease assays of tomato leaf intercellular fluid from both H O- designated the cDNA epi1 (extracellular protease inhibitor 1). 2 treated and BTH-treated plants revealed that, as expected, Proteins with Kazal Domains Are Diverse and Ubiquitous in BTH induced the production of abundant extracellular pro- Oomycetes—We used EPI1 and other Kazal domain sequences teases in tomato that migrated as two separate but close bands to search for Kazal-like motifs in sequence databases from (Fig. 3A). Inhibition assays revealed that rEPI1 dramatically oomycetes and other microbial plant pathogens (see “Material inhibited these BTH-induced proteases as well as partially and Methods”). We failed to identify sequences similar to the inhibited a constitutive protease. The total endoprotease activ- Kazal domain in all examined fungal and bacterial databases, ity of tomato intercellular fluids was also measured in the except for a predicted protein from the ammonia-oxidizing TM absence or presence of rEPI1. Significant inhibition of endopro- bacterium Nitrosomonas europaea (GenBank accession tease activity was observed and corresponded to 28 and 27% of NP_841298) (49). On the other hand, we unraveled a total of 35 total activity in control and BTH-treated tomato, respectively different putative proteins with 56 predicted Kazal-like do- (Fig. 4). mains (range, 1– 4/protein) in five plant pathogenic oomycete EPI1 Interacts with Pathogenesis-related Subtilases of the species, P. infestans, Phytophthora sojae, Phytophthora ramo- Tomato P69 Subfamily—To identify the plant proteases tar- rum, P. brassicae, and the downy mildew Plasmopara halstedii (Table I). These oomycete Kazal motifs were identified in ESTs geted by rEPI1, coimmunoprecipitation was performed on to- mato intercellular fluid incubated with rEPI1 using FLAG from a variety of developmental stages, including host tissue infected with P. sojae and P. halstedii. We identified a putative antibody covalently linked agarose beads. In addition to rEPI1, two proteins were pulled down with the FLAG antibody only in full open reading frame sequence for 27 of the identified genes and the putative start codon for 33 of the genes. All of these 33 the presence of rEPI1 (Fig. 5A). These two proteins exhibited a 26374 Protease Inhibitors from Phytophthora FIG.4. rEPI1 inhibition of total protease activity from tomato intercellular fluids. Total protease activity of Intercellular fluids obtained from water-treated (BTH) and BTH-treated (BTH) tomato plants was measured in the absence (gray columns) or presence (black TM columns) of rEPI1 using the QuantiCleave protease assay kit as described under “Materials and Methods.” Activity is expressed as absorbance at 405 nm. The bars correspond to the means of three independent replications of one representative experiment of three performed. The error bars represent the standard errors calculated from the three replications. FIG.2. rEPI1 inhibits subtilisin A. A, protease activity of chymo- trypsin, subtilisin A, and trypsin in the absence (gray columns)or presence of rEPI1 (black columns). The activities were determined TM using the QuantiCleave protease assay kit as described under “Ma- terials and Methods.” Activity is expressed as a percentage of total protease activity in the absence of protease inhibitors. The bars corre- spond to the means of three independent replications of one represent- ative experiment of three performed. The error bars represent the standard errors calculated from the three replications. B, time course of substrate hydrolysis by subtilisin A in the presence of varying concen- trations of rEPI1. Protease activity was measured as absorbance at 405 FIG.5. Coimmunoprecipitation of rEPIs and P69 subtilases nm based on hydrolysis of a chromogenic substrate. The final concen- using FLAG antisera. A, eluates from coimmunoprecipitation of tration of subtilisin A is 10 nM. The concentrations of rEPI1 are indi- rEPI1 with proteins in tomato intercellular fluids were run on SDS- cated next to the curves. PAGE gel followed by staining with silver nitrate. The numbers on the left indicate the molecular masses of the marker proteins in kDa. rEPI1 indicates whether or not rEPI1 was added to the reaction mix. BTH indicates whether or not the intercellular fluids were obtained from plants treated with BTH. The lower molecular mass band corresponds to rEPI1, and the high molecular mass bands correspond to the rEPI1- interacting protein(s). B, the same eluate samples were run on SDS- PAGE gel followed by staining with Coomassie Brilliant Blue (Coom.)or immunoblotting with antisera raised against a peptide specific for the tomato P69 family (-P69), and FLAG (-FLAG), respectively. The top two panels (Coom. and -P69) correspond to the high molecular mass bands of A, whereas the bottom panel (-FLAG) corresponds to the low molecular mass band. similar molecular mass of 70 kDa (Fig. 5A) and were more FIG.3. rEPI1 inhibits BTH-induced tomato proteases. A, inter- cellular fluids obtained from water-treated () and BTH-treated () abundant in BTH-induced intercellular fluid (Fig. 5B). These tomato plants were run on SDS-PAGE gel followed by staining with results prompted us to test whether these proteins could be Coomassie Brilliant Blue (left panel) or were used in zymogen in-gel tomato P69 subtilisin-like proteases. Western blot analyses protease assays (right panel). The asterisks represent known pathogen- with antisera raised against a peptide specific to P69 subtilisin- esis-related proteins PR1, PR3, and PR2 (from bottom to top) and confirm the induction of defense responses by BTH. The arrows indicate like proteases strongly interacted with both bands, suggesting BTH-induced protease activities that migrated as two close bands. B, that rEPI1 interacts with P69 subtilases of tomato (Fig. 5B). To inhibition of tomato proteases by rEPI1. Intercellular fluids from BTH- confirm the results obtained with the Western blot and further treated tomato leaves were incubated in the absence ( rEPI1)or identify which P69 isoforms are the main targets of rEPI1, the presence of rEPI1 ( rEPI1) and then analyzed using zymogen in-gel protease assays. The arrows indicate the BTH-induced protease bands. two closely migrated protein bands (Fig. 5) were cored from the Protease Inhibitors from Phytophthora 26375 FIG.6. Tandem mass spectrometry identifies P69B as the main target of rEPI1. The amino acid sequence of P69B subtilisin-like protease precursor is shown with the signal peptide sequence in italics and the propeptide domain sequence in gray. The 21 peptides se- quenced by tandem mass spectrometry are shown in bold type. Each sequenced peptide ends with an Arg or a Lys residue, which is high- lighted in bold italics. Underlined sequences are specific to P69B among FIG.7. The epi1 and P69 genes are concurrently expressed the known P69 isoforms. during colonization of tomato by P. infestans. A, time course of expression of P. infestans epi1 and actA and tomato P69B and tubulin Coomassie Blue-stained SDS-PAGE gel as one sample and during colonization of tomato by P. infestans. Total RNA isolated from analyzed by tandem mass spectrometry. A total of 21 trypsin- infected leaves of tomato, 0, 1, 2, 3, or 4 days after inoculation, from digested peptides were sequenced and perfectly matched the noninfected leaves (To), and from P. infestans mycelium grown in syn- TM subtilisin-like protease P69B (GenBank accession number thetic medium (My) was hybridized with probes from the four genes. T07184 or CAA76725) (Fig. 6). Of these 21 peptides, 13 pep- The approximate sizes of the transcripts are 600 nucleotides for epi1, 1600 nucleotides for actA, and 2500 nucleotides for P69B and tubulin. tides were specific to P69B and did not match any of the other B, RT-PCR analysis of epi1, P69A, P69B, and P69D expression during five known P69 isoforms. At this stage it cannot be ruled out colonization by P. infestans. Total RNA from a time course similar to the that the two closely migrated protein bands contain other iso- one described in A was used in RT-PCR amplifications as described in forms in minor amounts, but the results from the tandem mass the text. Amplification of P. infestans elongation factor 2 (Pief2) and tomato elongation factor 1 (Toef1) were used as controls to determine spectrometry clearly showed that P69B is the main target of the relative expression of epi1 and P69 genes, respectively. C, Western rEPI1. blot analyses of tomato P69 subtilases during colonization by P. infes- The epi1 and P69B Gene Are Concurrently Expressed during tans. The time course is as described for A. Equal volumes of intercel- Infection of Tomato by P. infestans—Expression pattern of both lular fluids were obtained from infected tomato leaves, subjected to SDS-PAGE, and immunoblotted with P69 antisera (-P69). epi1 and P69 genes during infection of tomato by P. infestans was studied by Northern blot and RT-PCR analyses. The epi1 gene displayed the highest mRNA levels 3 days post-inocula- lence or avirulence molecules, known as effectors. In suscepti- tion and was moderately up-regulated (approximately 2 ble plants, biotrophic plant pathogens produce effectors that based on PhosphorImager quantification) compared with in promote infection by suppressing defense responses. Here, we vitro grown mycelium and relative to the constitutive actA gene describe EPI1, a two-domain extracellular protease inhibitor (Fig. 7A). Semi-quantitative RT-PCR analyses confirmed these from P. infestans that inhibits apoplastic subtilases of tomato, results (Fig. 7B). The expression of P69 protease genes was namely the PR proteins P69. Based on its biological activity induced after inoculation with P. infestans and attained the and expression pattern, EPI1 may function as a disease effector highest level 2 and 3 days after inoculation (Fig. 7A). Semi- molecule and may play an important role in P. infestans colo- quantitative RT-PCR amplifications using primers specific for nization of host apoplast. P69A, P69B, and P69D, indicated that the pathogenesis-re- Suppression of host defenses is thought to play a critical role lated P69B gene is the only gene that is up-regulated during in plant-microbe interactions, especially those involving biotro- interaction with P. infestans (Fig. 7B). We could not assess the phic pathogens that require live plant cells to establish a suc- expression of P69C, the other pathogenesis-related gene of the cessful infection (8, 53). Nonetheless, only a few pathogen mol- P69 family (21), because we repeatedly failed to amplify P69C ecules that suppress host defenses have been identified. from tomato cultivar Ohio 7814 based on published sequences. Examples include tomatinase, a saponin-detoxifying enzyme Increase in P69 protein during infection of tomato by P. infes- from the fungal pathogen Septoria lycopersici that was recently tans was also noted by Western blot analyses with P69 antisera shown to indirectly suppress host defense responses through of intercellular fluids obtained from a time course infection its degradation products (9). P. sojae secretes glucanase inhib- (Fig. 7C). Altogether, these results suggest that epi1 and P69 itor proteins that inhibit a soybean endo--1,3-glucanase and genes are concurrently expressed during infection and support are thought to function as counterdefensive molecules that the possibility of direct interaction between P. infestans EPI1 inhibit the degradation of -1,3/1,6-glucans in the pathogen cell and plant P69 proteases, particularly P69B, at the infection wall and/or the release of defense-eliciting oligosaccharides by interface. host endo--1,3 glucanases (54). P. infestans and other Phyto- DISCUSSION phthora species produce water-soluble glucans that suppress Plant pathogens manipulate biochemical and physiological induction of host defense responses (10 –12). Here, we describe processes in their host plants through a diverse array of viru- a novel class of pathogen suppressors of plant defense response, 26376 Protease Inhibitors from Phytophthora namely extracellular protease inhibitors that directly interact less are colonized by a variety of microbial pathogens. Appar- with and inhibit host proteases. This interaction could form ently, P. infestans and T. gondii, even though phylogenetically another type of defense-counterdefense mechanism between unrelated, have independently recruited secreted proteins of plants and microbial pathogens the Kazal family to inhibit host proteases and adapt to pro- TM tease-rich host environments. Interestingly, unlike the T. gon- We scanned GenBank and several other sequence data bases for the occurrence of Kazal-like domains. The examined dii inhibitors (15, 19), EPI1 does not inhibit trypsin and chy- mortrypsin, suggesting that coevolution between the inhibitors data sets included the full genome sequence of several plant and their target proteases may have shaped the inhibitor spec- pathogenic bacteria and fungi. A 235-amino acid protein from TM ificity. Future structural and functional characterization of the ammonia-oxidizing bacterium N. europaea (GenBank Kazal protease inhibitors from animal and plant pathogens will accession NP_841298) was the only bacterial or fungal protein shed some light on interesting questions on the evolution of with significant similarity to the Kazal motif. In sharp con- pathogenesis in eukaryotic microbes and the coevolution of trast, 56 Kazal-like motifs were detected in 35 predicted pro- pathogen effectors with host targets. teins of five plant pathogenic oomycete species, P. infestans, P. sojae, P. ramorum, P. brassicae, and P. halstedii (Table I). Acknowledgments—We are grateful to Caitlin Cardina, Shujing Interestingly, oomycete Kazal motif genes are often expressed Dong, Diane Kinney, and Kristin Wille for technical assistance; during host colonization. Five of the identified sequences were Margaret Redinbaugh and Saskia Hogenhout for valuable advice on the protein work; Tea Meulia and the staff of the Ohio Agricultural Re- from cDNAs obtained from infected plant tissue corresponding search and Development Center Molecular and Cellular Imaging Cen- to diverse oomycete pathosystems: P. infestans tomato/potato, ter for help with DNA sequencing; and Andrew Keightley and Mike P. sojae soybean, and P. halstedii sunflower. 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