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Protease Activities Triggered by Ralstonia solanacearum Infection in Susceptible and Tolerant Tomato Lines

2018; Elsevier BV; Volume: 17; Issue: 6 Linguagem: Inglês

10.1074/mcp.ra117.000052

1535-9484

Marc Planas‐Marquès, Martí Bernardo-Faura, Judith K. Paulus, Farnusch Kaschani, Markus Kaiser, Marc Valls, Renier A. L. van der Hoorn, Núria S. Coll,

Legume Nitrogen Fixing Symbiosis

Activity-based protein profiling (ABPP) is a powerful proteomic technique to display protein activities in a proteome. It is based on the use of small molecular probes that react with the active site of proteins in an activity-dependent manner. We used ABPP to dissect the protein activity changes that occur in the intercellular spaces of tolerant (Hawaii 7996) and susceptible (Marmande) tomato plants in response to R. solanacearum, the causing agent of bacterial wilt, one of the most destructive bacterial diseases in plants. The intercellular space -or apoplast- is the first battlefield where the plant faces R. solanacearum. Here, we explore the possibility that the limited R. solanacearum colonization reported in the apoplast of tolerant tomato is partly determined by its active proteome. Our work reveals specific activation of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs) in the leaf apoplast of the tolerant tomato Hawaii 7996 on R. solanacearum infection. The P69 family members P69C and P69F, and an unannotated lipase (Solyc02g077110.2.1), were found to be post-translationally activated. In addition, protein network analysis showed that deeper changes in network topology take place in the susceptible tomato variety, suggesting that the tolerant cultivar might be more prepared to face R. solanacearum in its basal state. Altogether this work identifies significant changes in the activity of 4 PLCPs and 27 SHs in the tomato leaf apoplast in response to R. solanacearum, most of which are yet to be characterized. Our findings denote the importance of novel proteomic approaches such as ABPP to provide new insights on old and elusive questions regarding the molecular basis of resistance to R. solanacearum. Activity-based protein profiling (ABPP) is a powerful proteomic technique to display protein activities in a proteome. It is based on the use of small molecular probes that react with the active site of proteins in an activity-dependent manner. We used ABPP to dissect the protein activity changes that occur in the intercellular spaces of tolerant (Hawaii 7996) and susceptible (Marmande) tomato plants in response to R. solanacearum, the causing agent of bacterial wilt, one of the most destructive bacterial diseases in plants. The intercellular space -or apoplast- is the first battlefield where the plant faces R. solanacearum. Here, we explore the possibility that the limited R. solanacearum colonization reported in the apoplast of tolerant tomato is partly determined by its active proteome. Our work reveals specific activation of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs) in the leaf apoplast of the tolerant tomato Hawaii 7996 on R. solanacearum infection. The P69 family members P69C and P69F, and an unannotated lipase (Solyc02g077110.2.1), were found to be post-translationally activated. In addition, protein network analysis showed that deeper changes in network topology take place in the susceptible tomato variety, suggesting that the tolerant cultivar might be more prepared to face R. solanacearum in its basal state. Altogether this work identifies significant changes in the activity of 4 PLCPs and 27 SHs in the tomato leaf apoplast in response to R. solanacearum, most of which are yet to be characterized. Our findings denote the importance of novel proteomic approaches such as ABPP to provide new insights on old and elusive questions regarding the molecular basis of resistance to R. solanacearum. Bacterial wilt caused by the soil-borne pathogen Ralstonia solanacearum is one of the most destructive and economically damaging bacterial diseases, affecting over 200 plant species, including important crops such as tomato, potato and peanut (1.Mansfield J. Genin S. Magori S. Citovsky V. Sriariyanum M. Ronald P. Dow M. Verdier V. Beer S.V. Machado M.A. Toth I. Salmond G. Foster G.D. Top 10 plant pathogenic bacteria in molecular plant pathology.Mol. Plant Pathol. 2012; 13: 614-629Crossref PubMed Scopus (1149) Google Scholar, 2.Hayward A. Bacterial Wilt Caused By Pseudomonas solanacearum.J. Plant Pathol. 1991; 95: 237-245Google Scholar). Yield losses caused by R. solanacearum on tomato can reach up to 90% in some countries (3.Elphinstone J. Allen C. Prior P. Hayward A. Bacterial wilt disease and the Ralstonia solanacearum species complex. American Phytopathological Society, St. Paul2005: 9-28Google Scholar). Management of bacterial wilt remains difficult because of R. solanacearum aggressiveness, its broad geographical distribution and its long persistence in soil and water (1.Mansfield J. Genin S. Magori S. Citovsky V. Sriariyanum M. Ronald P. Dow M. Verdier V. Beer S.V. Machado M.A. Toth I. Salmond G. Foster G.D. Top 10 plant pathogenic bacteria in molecular plant pathology.Mol. Plant Pathol. 2012; 13: 614-629Crossref PubMed Scopus (1149) Google Scholar, 4.Genin S. Molecular traits controlling host range and adaptation to plants in Ralstonia solanacearum.New Phytol. 2010; 187: 920-928Crossref PubMed Scopus (182) Google Scholar). Historically, grafting approaches using tolerant cultivars as rootstocks have been the most effective method to control bacterial wilt (5.Grimault V. Prior P. Grafting Tomato Cultivars Resistant or Susceptible to Bacterial Wilt - Analysis of Resistance Mechanisms.J. Phytopathol. 1994; 141: 330-334Crossref Scopus (21) Google Scholar, 6.Peregrine W.T.H. bin Ahmad K. Grafting—A simple technique for overcoming bacterial wilt in tomato.Trop. Pest Manag. 1982; 28: 71-76Crossref Scopus (19) Google Scholar, 7.Rivard C.L. O'Connell S. Peet M.M. Louws F.J. Grafting Tomato to Manage Bacterial Wilt Caused by Ralstonia solanacearum in the Southeastern United States.Plant Dis. 2012; : 973-978Crossref PubMed Scopus (62) Google Scholar). In tomato, the Hawaii cultivar series-particularly Hawaii 7996- has been proven to be the most effective source of resistance against various R. solanacearum strains under different environmental conditions (8.Grimault V. Anais G. Prior P. Distribution of Pseudomonas solanacearum in the stem tissues of tomato plants with different levels of resistance to bacterial wilt.Plant Pathotogy. 1994; 43: 663-668Crossref Scopus (58) Google Scholar, 9.Wang J.F. Hanson P. Barnes J. Prior P. Allen C. Elphinstone J. Bacterial Wilt Disease: Molecular and Ecological Aspects. Berlin, Springer1998: 269-275Crossref Google Scholar, 10.Prior P. Bart S. Leclercq S. Darrasse a Anais G. Resistance to bacterial wilt in tomato as discerned by spread of Pseudomonas (Burholderia) solanacearum in the stem tissues.Plant Pathol. 1996; 45: 720-726Crossref Scopus (47) Google Scholar). In Arabidopsis thaliana, two major resistance genes, RRS1-R (Resistance to Ralstonia solanacearum 1) and RPS4 (Resistance to Pseudomonas syringae 4), were shown to provide resistance to R. solanacearum as a dual R-gene system, recognizing the type III effector PopP2 and triggering defense (11.Deslandes L. Olivier J. Peeters N. Feng D.X. Khounlotham M. Boucher C. Somssich I. Genin S. Marco Y. Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus.Proc. Natl. Acad. Sci. 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A plant immune receptor detects pathogen effectors that target WRKY transcription factors.Cell. 2015; 161: 1089-1100Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). However, no major R-genes have been identified in tomato (15.Kim B.S. French E. Caldwell D. Harrington E.J. Iyer-Pascuzzi A.S. Bacterial wilt disease: Host resistance and pathogen virulence mechanisms.Physiol. Mol. Plant Pathol. 2015; 95: 37-43Crossref Scopus (30) Google Scholar), where resistance has been reported to be mainly quantitative, involving two major quantitative trait loci (QTLs) 1The abbreviations used are: QTL, Quantitative Trait Locus; ABPP, Activity-based protein profiling; PLCP, Papain-like Cysteine Protease; PAE, Pectinacetylesterase; Pip1, Phytophthora inhibited protease 1; Rcr3, Required for C. fulvum resistance 3; SCP, Serine Carboxypeptidases; SH, Serine Hydrolase; SLP, Subtilisin-like Protease. 1The abbreviations used are: QTL, Quantitative Trait Locus; ABPP, Activity-based protein profiling; PLCP, Papain-like Cysteine Protease; PAE, Pectinacetylesterase; Pip1, Phytophthora inhibited protease 1; Rcr3, Required for C. fulvum resistance 3; SCP, Serine Carboxypeptidases; SH, Serine Hydrolase; SLP, Subtilisin-like Protease. (Bwr-12 and Bwr-6), and three minor loci (Bwr-3, Bwr-4 and 8 Bwr-8) (16.Carmeille A. Caranta C. Dintinger J. Prior P. Luisetti J. Besse P. Identification of QTLs for Ralstonia solanacearum race 3-phylotype II resistance in tomato.Theor. Appl. Genet. 2006; 113: 110-121Crossref PubMed Scopus (76) Google Scholar, 17.Mangin B. Thoquet P. Olivier J. Grimsley N.H. Temporal and multiple quantitative trait loci analyses of resistance to bacterial wilt in tomato permit the resolution of linked loci.Genetics. 1999; 151: 1165-1172Crossref PubMed Google Scholar, 18.Thoquet P. Olivier J. Sperisen C. Rogowsky P. Laterrot H. Grimsley N. Quantitative trait loci determining resistance to bacterial wilt in tomato cultivar Hawaii7996.Mol. Plant. Microbe. Interact. 1996; 9: 826-836Crossref Scopus (113) Google Scholar, 19.Thoquet P. Olivier J. Sperisen C. Rogowsky P. Prior P. Anais G. Mangin B. Bazin B. Nazer R. Grimsley N. Polygenic resistance of tomato plants to bacterial wilt in the French West Indies.Mol. Plant. Microbe. Interact. 1996; 9: 837-842Crossref Scopus (90) Google Scholar, 20.Wang J.F. Olivier J. Thoquet P. Mangin B. Sauviac L. Grimsley N.H. Resistance of tomato line Hawaii7996 to Ralstonia solanacearum Pss4 in Taiwan is controlled mainly by a major strain-specific locus.Mol. Plant. Microbe. Interact. 2000; 13: 6-13Crossref PubMed Scopus (107) Google Scholar, 21.Wang J.F. Ho F.I. Truong H.T.H. Huang S.M. Balatero C.H. Dittapongpitch V. Hidayati N. Identification of major QTLs associated with stable resistance of tomato cultivar "Hawaii 7996" to Ralstonia solanacearum.Euphytica. 2013; 190: 241-252Crossref Scopus (69) Google Scholar). These QTLs, defined in the cultivar Hawaii 7996, were found to be both strain- and environment-specific (16.Carmeille A. Caranta C. Dintinger J. Prior P. Luisetti J. Besse P. Identification of QTLs for Ralstonia solanacearum race 3-phylotype II resistance in tomato.Theor. Appl. Genet. 2006; 113: 110-121Crossref PubMed Scopus (76) Google Scholar, 21.Wang J.F. Ho F.I. Truong H.T.H. Huang S.M. Balatero C.H. Dittapongpitch V. Hidayati N. Identification of major QTLs associated with stable resistance of tomato cultivar "Hawaii 7996" to Ralstonia solanacearum.Euphytica. 2013; 190: 241-252Crossref Scopus (69) Google Scholar). R. solanacearum infects plants through wounds in the roots, at secondary root emerging sites and at root tips, and migrates intercellularly through the apoplast until it reaches the xylem vessels, where it multiplies and spreads systemically (4.Genin S. Molecular traits controlling host range and adaptation to plants in Ralstonia solanacearum.New Phytol. 2010; 187: 920-928Crossref PubMed Scopus (182) Google Scholar, 22.Vasse J. Frey P. Trigalet A. Microscopic studies of intercellular infection and protoxylem invasion of tomato roots by Pseudomonas solanacearum.Mol. Plant. Microbe. Interact. 1995; 8: 241-251Crossref Scopus (262) Google Scholar). More than two decades ago, Grimault & Prior (23.Grimault V. Prior P. Bacterial wilt resistance in tomato associated with tolerance of vascular tissues to Pseudomonas solanacearum.Plant Pathol. 1993; 42: 589-594Crossref Scopus (60) Google Scholar), and later on McGarvey & collaborators (24.McGarvey J.a. Denny T.P. Schell M.a Spatial-temporal and quantitative analysis of growth and EPS I production by Ralstonia solanacearum in resistant and susceptible tomato cultivars.Phytopathology. 1999; 89: 1233-1239Crossref PubMed Scopus (67) Google Scholar), reported limited bacterial growth in the root, collar and middle stem of tolerant tomato cultivars. Hawaii 7996 showed the least bacterial colonization, and immunostaining analysis demonstrated lower levels of bacteria in the root apoplast (24.McGarvey J.a. Denny T.P. Schell M.a Spatial-temporal and quantitative analysis of growth and EPS I production by Ralstonia solanacearum in resistant and susceptible tomato cultivars.Phytopathology. 1999; 89: 1233-1239Crossref PubMed Scopus (67) Google Scholar). Later studies pointed to the importance of physical barriers and intercellular spaces in tomato defense against R. solanacearum (25.Grimault V. Gélie B. Lemattre M. Prior P. Schmit J. Comparative histology of resistant and susceptible tomato cultivars infected by Pseudomonas solanacearum.Physiol. Mol. Plant Pathol. 1994; 44: 105-123Crossref Scopus (64) Google Scholar, 26.Nakaho K. Hibino H. Miyagawa H. Possible mechanisms limiting movement of Ralstonia solanacearum in resistant tomato tissues.J. Phytopathol. 2000; 148: 181-190Crossref Scopus (49) Google Scholar, 27.Nakaho K. Inoue H. Takayama T. Miyagawa H. Distribution and multiplication of Ralstonia solanacearum in tomato plants with resistance derived from different origins.J. Gen. Plant Pathol. 2004; 70: 115-119Crossref Scopus (55) Google Scholar) but a deep understanding of the molecular mechanisms involved in resistance is still lacking. The apoplast is thus the first battlefield where the plant has to face the pathogen before it reaches the xylem. In this narrow compartment both plants and pathogens secrete a diverse set of molecules that ultimately determine the outcome of the infection. Recent research provides evidence of plant apoplastic proteases playing an important role in immunity, with their activity often targeted by pathogen-derived effectors (28.Gupta R. Lee S.E. Agrawal G.K. Rakwal R. Park S. Wang Y. Kim S.T. Understanding the plant-pathogen interactions in the context of proteomics-generated apoplastic proteins inventory.Front. Plant Sci. 2015; 6: 352Crossref PubMed Scopus (65) Google Scholar, 29.Jashni M.K. Mehrabi R. Collemare J. Mesarich C.H. de Wit P.J. G.M. The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant-pathogen interactions.Front. Plant Sci. 2015; 6: 584Crossref PubMed Scopus (137) Google Scholar). In this study we explore the possibility that the limited R. solanacearum colonization of intercellular spaces depicted by tolerant tomato cultivars (24.McGarvey J.a. Denny T.P. Schell M.a Spatial-temporal and quantitative analysis of growth and EPS I production by Ralstonia solanacearum in resistant and susceptible tomato cultivars.Phytopathology. 1999; 89: 1233-1239Crossref PubMed Scopus (67) Google Scholar, 27.Nakaho K. Inoue H. Takayama T. Miyagawa H. Distribution and multiplication of Ralstonia solanacearum in tomato plants with resistance derived from different origins.J. Gen. Plant Pathol. 2004; 70: 115-119Crossref Scopus (55) Google Scholar) is partly determined by the molecular environment of their apoplast. We have dissected the dynamic changes in protein activities that take place in the apoplast of tolerant (Hawaii 7996) and susceptible (Marmande) tomato in response to R. solanacearum using activity-based protein profiling (ABPP). ABPP is a technique that identifies the active proteins in a proteome. It is based on the use of small labeled molecules that react with the active site of proteins in an activity-dependent manner (30.Cravatt B.F. Sorensen E.J. Chemical strategies for the global analysis of protein function.Curr. Opin. Chem. Biol. 2000; 4: 663-668Crossref PubMed Scopus (144) Google Scholar, 31.Verhelst S.H.L. Bogyo M. Dissecting protein function using chemical proteomic methods.QSAR Comb. Sci. 2005; 24: 261-269Crossref Scopus (20) Google Scholar). ABPP has made important contributions to the understanding of immune responses in plants, allowing the identification of differential activities at the plant-pathogen interface (32.Kołodziejek I. van der Hoorn R.A.L. Mining the active proteome in plant science and biotechnology.Curr. Opin. Biotechnol. 2010; 21: 225-233Crossref PubMed Scopus (31) Google Scholar). Changes in the activities of the papain-like cysteine protease (PLCPs) and the serine hydrolase (SH) protease families are of interest, because they have been reported in the apoplast of tomato and other plant-pathogen systems (reviewed by Kołodziejek & van der Hoorn) (32.Kołodziejek I. van der Hoorn R.A.L. Mining the active proteome in plant science and biotechnology.Curr. Opin. Biotechnol. 2010; 21: 225-233Crossref PubMed Scopus (31) Google Scholar). PLCPs are usually 23–30 kDa in size, and use a catalytic cysteine residue to cleave peptide bonds in protein substrates. They have been shown to be required for full plant resistance against various bacterial, fungal, and oomycete pathogens, inducing a broad spectrum of defense (33.Misas-Villamil J.C. van der Hoorn R.A.L. Doehlemann G. Papain-like cysteine proteases as hubs in plant immunity.New Phytol. 2016; 212: 902-907Crossref PubMed Scopus (84) Google Scholar). Some PLCPs are required for defense-related program cell death, like the protease cathepsin B from Nicotiana Benthamiana. Silencing of cathepsin B prevented cell death and compromised nonhost disease resistance caused by Erwinia amylovora and Pseudomonas syringae pv. tomato (34.Gilroy E.M. Hein I. Van Der Hoorn R. Boevink P.C. Venter E. McLellan H. Kaffarnik F. Hrubikova K. Shaw J. Holeva M. López E.C. Borras-Hidalgo O. Pritchard L. Loake G.J. Lacomme C. Birch P.R.J. Involvement of cathepsin B in the plant disease resistance hypersensitive response.Plant J. 2007; 52: 1-13Crossref PubMed Scopus (130) Google Scholar). PLCPs can also act as co-receptors in the recognition of pathogen effectors. This is the case of Rcr3 (Required for Cladosporium fulvum Resistance 3), which is required by the tomato receptor-like protein Cf-2 for the perception of the Cladosporium fulvum effector Avr2 (35.Krüger J. Thomas C.M. Golstein C. Dixon M.S. Smoker M. Tang S. Mulder L. Jones J.D.G. A Tomato Cysteine Protease Required for Cf-2-Dependent Disease Resistance and Suppression of Autonecrosis.Science. 2002; 296: 744-747Crossref PubMed Scopus (322) Google Scholar) or the allergen-like effector protein Gr-VAP1 from the potato cyst nematode Globodera rostochiensis (36.Lozano-Torres J.L. Wilbers R.H.P. Gawronski P. Boshoven J.C. Finkers-Tomczak A. Cordewener J.H.G. America A.H.P. Overmars H.A. Van 't Klooster J.W. Baranowski L. Sobczak M. Ilyas M. van der Hoorn R.A.L. Schots A. de Wit P.J. G.M. Bakker J. Goverse A. Smant G. Dual disease resistance mediated by the immune receptor Cf-2 in tomato requires a common virulence target of a fungus and a nematode.Proc. Natl. Acad. Sci. USA. 2012; 109: 10119-10124Crossref PubMed Scopus (179) Google Scholar), acting as a decoy and hence triggering cell death. SHs, on the other hand, comprise a large collection of enzymes from different structural classes that carry an activated serine residue in their catalytic site. SHs fulfill diverse biochemical roles and are involved in a wide range of physiological processes including plant immunity (37.Kaschani F. Gu C. Niessen S. Hoover H. Cravatt B.F. van der Hoorn R.aL. Diversity of Serine Hydrolase Activities of Unchallenged and Botrytis-infected Arabidopsis thaliana.Mol. Cell. Proteomics. 2009; 8: 1082-1093Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In addition, members of the PLCP and SH protease families have been shown to be up-regulated on pathogen infection or targeted by pathogen-derived inhibitors (29.Jashni M.K. Mehrabi R. Collemare J. Mesarich C.H. de Wit P.J. G.M. The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant-pathogen interactions.Front. Plant Sci. 2015; 6: 584Crossref PubMed Scopus (137) Google Scholar). The aim of this work was to identify the active apoplastic proteases involved in the R. solanacearum infection of tomato. We describe a variety-specific induction of PLCP and SH protein activities in response to R. solanacearum. Altogether this work denotes the importance of novel proteomic approaches -such as ABPP- to provide new insights on old questions regarding the molecular basis of resistance to bacterial wilt. All assays were performed using a Ralstonia solanacearum GMI1000 (Phylotype I, race 1 biovar 3) reporter strain carrying the luxCDABE operon under the constitutive promoter PpsbA integrated in its chromosome. R. solanacearum was routinely grown at 28 °C in rich B medium (10 g/l bactopeptone, 1 g/l yeast extract and 1 g/l casaminoacids) using gentamicin (10 μg/ml) for selection. Tomato (Solanum lycopersicum) cultivars used were the susceptible cv. Marmande and the tolerant cv. Hawaii 7996. All plants were grown underlong-day light conditions and a light intensity of 120–150 μmol·m-2·s-1, at 25–26 °C and 60% relative humidity. For bacterial inoculation in the apoplast, three- to four-week-old tomato plants were first acclimatized by transferring them to a chamber at 28 °C with constant light conditions (12 h light, 12 h darkness). Two days later, plants were vacuum-infiltrated submerging the whole aerial part either in distilled water (mock) or in a 105 CFU/ml (OD600 = 0.0001) suspension of the pathogen for ∼20 s. In both cases, the adjuvant Silwet l-77 was added (80 μl/l suspension) to facilitate infiltration. After inoculation, plants were kept in the same conditions. Disease symptoms were evaluated using a scale measuring the affected surface of leaflets. Four levels of necrosis were defined: no necrosis (0% of affected surface), mild (< 25%), moderate (25–75%), and severe (> 75%). Leaflets from the third, fourth and fifth leaves of 24 plants per variety were evaluated. Apoplastic fluid isolation was performed following the protocol from Rico and Preston (38.Rico A. Preston G.M. Pseudomonas syringae pv. tomato DC3000 uses constitutive and apoplast-induced nutrient assimilation pathways to catabolize nutrients that are abundant in the tomato apoplast.Mol. Plant. Microbe. Interact. 2008; 21: 269-282Crossref PubMed Scopus (175) Google Scholar) and our previous experience (39.Zuluaga A.P. Puigvert M. Valls M. Novel plant inputs influencing Ralstonia solanacearum during infection.Front. Microbiol. 2013; 4: 1-7Crossref PubMed Scopus (46) Google Scholar). Briefly, tomato leaves were cut and vacuum-infiltrated with ice-cold distilled water. Infiltrated leaves were then blotted on a paper towel, rolled, and introduced into 5 ml tips (three-to-four leaves per tip), which were placed in 50 ml conical tubes (Falcon) containing 1.5 ml collection tubes (Eppendorf). Apoplast extract was collected by spinning the tubes at 3000 rpm for 15 min at 4 °C. For protein characterization, the supernatant was collected in new tubes, passed through a 0.22 μm filter to get rid of any bacteria, and stored at −80 °C. Bacterial growth was measured by plating 10-fold dilutions of apoplastic fluid from infiltrated leaves on B medium plates, which were then incubated at 28 °C for 1–2 days. Colony Forming Units (CFUs) were counted and bacterial growth was calculated as CFUs/ml of collected apoplastic fluid. Equal volumes of apoplastic fluid were labeled using specific activity-based probes to detect papain-like cysteine protease (PLCP) and serine hydrolase (SH). All labeling reactions were performed at room temperature in dark conditions in a final volume of 50 μl. For fluorescent PLCP labeling, plant extracts were incubated for 4h with 2 μm of the MV201 probe (40.Richau K.H. Kaschani F. Verdoes M. Pansuriya T.C. Niessen S. Stuber K. Colby T. Overkleeft H.S. Bogyo M. Van der Hoorn R. a. L. Subclassification and Biochemical Analysis of Plant Papain-Like Cysteine Proteases Displays Subfamily-Specific Characteristics.Plant Physiol. 2012; 158: 1583-1599Crossref PubMed Scopus (125) Google Scholar) in 50 mm sodium acetate (NaAc) pH 5, and 1 mm dithiothreitol (DTT) (Sigma-Aldrich). Fluorescent SH labeling was performed incubating for 1h plant extracts with 2 μm of a fluorophosphonate (FP)-based probe (41.Liu Y. Patricelli M.P. Cravatt B.F. Activity-based protein profiling: The serine hydrolases. Proc. Natl. Acad. Sci.USA. 1999; 96: 14694-14699Crossref Scopus (838) Google Scholar) in 50 mm NaAc pH 5. Labeling was stopped by adding gel loading buffer to the samples and boiling at 95 °C for 5 min before electrophoresis. Inhibition assays were performed by pre-incubating apoplastic fluids with specific inhibitors for 30 min previous to enzyme labeling. PLCPs were inhibited by 100 μm E-64 protease inhibitor, whereas a 200 μm mixture composed of diisopropyl fluorophosphate (DFP) (Sigma-Aldrich), 3,4-dichloroisocoumarin (DCI) (Sigma-Aldrich), and phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich) was used to inhibit SHs. Protein samples were separated on 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Fluorescently labeled proteins were detected by fluorescence on the gel using a Typhoon 9400 scanner (Amershan Biosciences, United Kingdom) and fluorescence intensity was measured using the ImageQuant TL software (GE Healthcare Life Sciences, United Kingdom). Gels were then fixed by two 15 min incubation in a 50% methanol 7% acetic acid solution. After fixation, proteins were stained overnight with SYPRO® Ruby (Invitrogen, MA) following the manufacturer's instructions. Finally, gels were rinsed under agitation with washing solution (10% methanol, 7% acetic acid) for 30 min. Fluorescent-stained gels were scanned using the Fujifilm LAS4000 image system. During staining and the subsequent steps, the gel was protected from light. Large-scale labeling was performed using biotinylated versions of the PLCP and SH activity probes, namely DCG-04 (42.Greenbaum D. Medzihradszky K.F. Burlingame A. Bogyo M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools.Chem. Biol. 2000; 7: 569-581Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 43.van der Hoorn R. a L. Leeuwenburgh M.A. Bogyo M. Joosten M.H. A.J. Peck S.C. Activity Profiling of Papain-Like Cysteine Proteases in Plants.Plant Physiol. 2004; 135: 1170-1178Crossref PubMed Scopus (114) Google Scholar) and FP-biotin (41.Liu Y. Patricelli M.P. Cravatt B.F. Activity-based protein profiling: The serine hydrolases. Proc. Natl. Acad. Sci.USA. 1999; 96: 14694-14699Crossref Scopus (838) Google Scholar) (Santa Cruz Biotechnology, TX). Both probes were mixed together in a 4 ml labeling reaction mixture (50 mm NaAc pH 5, 5 mm DTT, 4 μm of each probe) and incubated for 4 h. Proteins were then precipitated using the methanol-chloroform method. Briefly, 4 volumes (v) of cold methanol, 1 v of chloroform, and 3 v of cold MilliQ water were added to each sample, vortexing between every addition step. Samples were centrifuged at 3000 × g for 45 min and the aqueous layer was removed. Then 4 v of methanol were added to each sample and centrifuged again at 3000 × g for 45 min. The liquid phase was discarded, and the pellet dried at room temperature. Then 2 ml of 1.2% SDS-PBS was added, and the pellet was dissolved completely by pipetting. Proteins were denatured by incubating the samples at 90 °C in a water bath for 5 min, and then 12 ml 1x PBS were added to lower the SDS concentration to less than 0.2%. Avidin beads (Sigma Aldrich) were incubated with each sample for 1 h under rotation and then collected by spinning down at 400 × g for 10 min. The supernatant was removed, and the beads washed three times with 1.2% SDS-PBS, then with 1%SDS-PBS, then with 1× PBS, and finally with MilliQ water. The beads and remaining water were then transferred into 1.5 ml low binding protein tubes and spun down 400 × g for 10 min to remove the remaining liquid. Finally, on-bead trypsin digestion was performed as described by Weerapana and collaborators (44.Weerapana E. Speers A.E. Cravatt B.F. Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)–a general method for mapping sites of probe modification in proteomes.Nat. Protoc. 2007; 2: 1414-1425Crossref PubMed Scopus (170) Google Scholar). Tryptic samples were stored at −20 °C. In-solution digestion of protein samples was performed following the protocol by Kessler Lab-Proteomics (http://www.tdi.ox.ac.uk/protocols-and-tools). Briefly, proteins were reduced with 5 mm DTT for 45 min, alkylated with 20 mm iodoacetamide for 45 min, and then precipitated via methanol-chloroform. The protein pellet was re-suspended in 6 m urea-Tris buffer, pH 7.8, and sonicated. Finally, urea concentration was brought to less than 1 m with MilliQ water and proteins were digested with trypsin (incubation O/N at 37 °C). Tryptic samples were stored at −20 °C. Before mass spectrometry analysis, all peptide samples were purified using SEP-PAK C18 columns (Waters, MA) previously equilibrated with a solution of 65% acetonitrile (ACN) and 0.1% formic acid (FA) in Milli-Q water. The peptide digest samples were then added into a 2% ACN and 0.