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Purification and Characterization of AsES Protein

A SUBTILISIN SECRETED BY ACREMONIUM STRICTUM IS A NOVEL PLANT DEFENSE ELICITOR*
  • Nadia R. Chalfoun
    Footnotes
    Affiliations
    Instituto Superior de Investigaciones Biológicas, Consejo Nacional de Investigaciones Científicas y Técnicas, and Instituto de Química Biológica “Dr. Bernabé Bloj,” Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, T4000ILJ San Miguel de Tucumán, Argentina
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  • Carlos F. Grellet-Bournonville
    Footnotes
    Affiliations
    Instituto Superior de Investigaciones Biológicas, Consejo Nacional de Investigaciones Científicas y Técnicas, and Instituto de Química Biológica “Dr. Bernabé Bloj,” Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, T4000ILJ San Miguel de Tucumán, Argentina
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  • Martín G. Martínez-Zamora
    Footnotes
    Affiliations
    Instituto Superior de Investigaciones Biológicas, Consejo Nacional de Investigaciones Científicas y Técnicas, and Instituto de Química Biológica “Dr. Bernabé Bloj,” Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, T4000ILJ San Miguel de Tucumán, Argentina
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  • Araceli Díaz-Perales
    Footnotes
    Affiliations
    Unidad de Química y Bioquímica, Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28040 Madrid, Spain
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  • Atilio P. Castagnaro
    Footnotes
    Affiliations
    Sección Biotecnología, Estación Experimental Agroindustrial Obispo Colombres, T4101XAC Las Talitas, Tucumán, Argentina
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  • Juan C. Díaz-Ricci
    Correspondence
    To whom correspondence should be addressed. Tel./Fax: 54-381-4248921;
    Footnotes
    Affiliations
    Instituto Superior de Investigaciones Biológicas, Consejo Nacional de Investigaciones Científicas y Técnicas, and Instituto de Química Biológica “Dr. Bernabé Bloj,” Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, T4000ILJ San Miguel de Tucumán, Argentina
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  • Author Footnotes
    * This work was supported in part by Consejo de Investigaciones de la Universidad Nacional de Tucumán Grant 26/D423 and Agencia Nacional de Promoción Científica y Tecnológica Grant BID PICT-2008-2105.
    1 Members of Consejo Nacional de Investigaciones Científicas y Técnicas.
    2 Investigator of Universidad Politécnica de Madrid.
    3 Career Investigators of Consejo Nacional de Investigaciones Científicas y Técnicas.
Open AccessPublished:March 25, 2013DOI:https://doi.org/10.1074/jbc.M112.429423
      In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2̇̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

      Introduction

      Anthracnose caused by fungal species of the genus Colletotrichum represents one of the major fungal diseases in strawberry (Fragaria × ananassa Duch.) crops (
      • Smith B.J.
      • Black L.L.
      Morphological, cultural, and pathogenic variation among Colletotrichum species isolated from strawberry.
      ). Anthracnose can affect all plant tissues, e.g. fruits, flowers, leaves, runners, roots, and crowns, and the typical symptoms are described as irregular and black leaf spot, flower blight, and fruit and crown rot, bringing about serious losses in plant and fruit productions (
      • Freeman S.
      • Katan T.
      Identification of Colletotrichum species responsible for anthracnose and root necrosis of strawberry in Israel.
      ).
      We have previously reported a typical incompatible interaction between an avirulent isolate of Colletotrichum fragariae and strawberry plants of the cultivar (cv.) Pájaro (Fragaria × ananassa), and when strawberry plants were inoculated with this isolate prior to the challenge with a virulent strain (M11) of Colletotrichum acutatum, the former avirulent pathogen prevented the development of disease symptoms (
      • Salazar S.M.
      • Castagnaro A.P.
      • Arias M.E.
      • Chalfoun N.R.
      • Tonello U.
      • Díaz-Ricci J.C.
      Induction of a defense response in strawberry mediated by an avirulent strain of Colletotrichum.
      ). Further studies indicated that the anthracnose resistance was accompanied by the activation of plant defense mechanisms, including the production of reactive oxygen species (ROS)
      The abbreviations used are: ROS
      reactive oxygen species
      CF
      culture filtrate
      DAMP
      damage-associated molecular patterns
      dpi
      day post-inoculation
      dpt
      day post-treatment
      DSR
      disease severity rating
      hpt
      hour post-treatment
      IEF
      isoelectrofocusing
      IR
      induced resistance
      PAMP
      pathogen-associated molecular patterns
      pNA
      p-nitroanilide
      PR
      pathogenesis-related
      PS
      phenyl-Sepharose
      RACE
      rapid amplification of cDNA ends
      RH
      relative humidity
      Suc-AAPF-pNA
      succinyl-Ala-Ala-Pro-Phe-pNA
      NBT
      nitro blue tetrazolium
      RP
      reverse phase
      PAS
      periodic acid-Schiff.
      (i.e. H2O2 and O2̇̄), accumulation of salicylic acid, and transcriptional induction of pathogen-responsive genes encoding for pathogenesis-related (PR) proteins (
      • Grellet-Bournonville C.F.
      • Martinez-Zamora M.G.
      • Castagnaro A.P.
      • Díaz-Ricci J.C.
      Temporal accumulation of salicylic acid activates the defense response against Colletotrichum in strawberry.
      ,
      • Salazar S.M.
      • Grellet C.F.
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz-Ricci J.C.
      Avirulent strain of Colletotrichum induces a systemic resistance in strawberry.
      ). Recently, we have also shown that strawberry plants pretreated with culture filtrates derived from that avirulent isolate also acquired the resistance to the M11 isolate, confirming that this protection effect was due to a defense response induced by one or more proteinaceous elicitors contained in these extracts (
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz Ricci J.C.
      Induced resistance activated by a culture filtrate derived from an avirulent pathogen as a mechanism of biological control of anthracnose in strawberry.
      ).
      Plants have an innate immunity system to defend themselves against pathogens (
      • Jones J.D.
      • Dangl J.L.
      The plant immune system.
      ). The term elicitor is commonly applied to agents stimulating any type of defense response in intact plants or cultured plant cells, resulting in enhanced resistance to the invading pathogen (
      • Kamoun S.
      A catalogue of the effector secretome of plant pathogenic oomycetes.
      ). Elicitors of a diverse chemical nature have been characterized, including (poly)peptides, glycoproteins, lipids, glycolipids, and oligosaccharides, which can be derived from a variety of different phytopathogenic microbes (
      • Bonas U.
      • Lahaye T.
      Plant disease resistance triggered by pathogen-derived molecules: refined models of specific recognition.
      ,
      • Nürnberger T.
      • Brunner F.
      • Kemmerling B.
      • Piater L.
      Innate immunity in plants and animals: striking similarities and obvious differences.
      ) or host plants (
      • Mamaní A.
      • Filippone M.P.
      • Grellet C.
      • Welin B.
      • Castagnaro A.P.
      • Díaz Ricci J.C.
      Pathogen-induced accumulation of an ellagitannin elicits plant defense response.
      ).
      The activation of plant defense in incompatible plant-microbe interactions results from recognition by the plant of either cell surface components or molecules constitutively secreted by the pathogen or factors that are produced in the plant/pathogen interface upon contact with the host plant (
      • Nürnberger T.
      • Brunner F.
      • Kemmerling B.
      • Piater L.
      Innate immunity in plants and animals: striking similarities and obvious differences.
      ). With the primary immune system, plants can recognize pathogen-associated molecular patterns (PAMPs) of potential pathogens that activate a basal defense response (
      • Chisholm S.T.
      • Coaker G.
      • Day B.
      • Staskawicz B.J.
      Host-microbe interactions: shaping the evolution of the plant immune response.
      ). PAMPs, previously called “general elicitors,” are highly conserved molecular structures unique to microbes that play an essential role in the microbial lifestyle (
      • Boller T.
      • Felix G.
      A renaissance of elicitors: Perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors.
      ). Several PAMP-like proteins have been identified in plant pathogens, including flagellin and “harpins” from bacteria, xylanase from fungi, invertase from yeast, Pep13 and elicitins from oomycetes, and also NEP-like proteins that are widely distributed in diverse species (
      • Nürnberger T.
      • Brunner F.
      • Kemmerling B.
      • Piater L.
      Innate immunity in plants and animals: striking similarities and obvious differences.
      ). Furthermore, many phytopathogenic fungi secrete a mixture of hydrolytic enzymes (e.g. cellulases, xylanases, pectate lyases, proteases, polygalacturonases, and cutinases) to degrade and traverse the outer structural barriers of plant tissues. The products generated by the hydrolysis of plant components may function as “endogenous elicitors” and are called damage-associated molecular patterns (DAMPs) (
      • Boller T.
      • Felix G.
      A renaissance of elicitors: Perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors.
      ,
      • Lotze M.T.
      • Zeh H.J.
      • Rubartelli A.
      • Sparvero L.J.
      • Amoscato A.A.
      • Washburn N.R.
      • Devera M.E.
      • Liang X.
      • Tör M.
      • Billiar T.
      The grateful dead: damage-associated molecular pattern molecules and reduction/oxidation regulate immunity.
      ). The classic examples of DAMPs are plant cell wall fragments released by microbial xylanases, pectate lyases, and endopolygalacturonases and cutin monomers generated by cutinases (
      • Boller T.
      • Felix G.
      A renaissance of elicitors: Perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors.
      ).
      Filamentous microorganisms, such as fungi and oomycetes, secrete an arsenal of effector proteins that enable parasitic infection frequently by suppressing PAMP-triggered immune responses (
      • Kamoun S.
      A catalogue of the effector secretome of plant pathogenic oomycetes.
      ,
      • Chisholm S.T.
      • Coaker G.
      • Day B.
      • Staskawicz B.J.
      Host-microbe interactions: shaping the evolution of the plant immune response.
      ,
      • van't Slot K.A.
      • Knogge W.
      A dual role for microbial pathogen-derived effector proteins in plant disease and resistance.
      ,
      • de Wit P.J.
      • Mehrabi R.
      • van den Burg H.A.
      • Stergiopoulos I.
      Fungal effector proteins: past, present and future.
      ). However, these molecules can serve as elicitor signals for the plant to reinforce its defense when are specifically perceived by host cognate resistance proteins and are known as avirulence proteins or “specific elicitors” (
      • Kamoun S.
      A catalogue of the effector secretome of plant pathogenic oomycetes.
      ,
      • de Wit P.J.
      • Mehrabi R.
      • van den Burg H.A.
      • Stergiopoulos I.
      Fungal effector proteins: past, present and future.
      ,
      • Stergiopoulos I.
      • de Wit P.J.
      Fungal effector proteins.
      ). This dual activity of elicitor effectors has been broadly reported in plant-microbial pathosystems (
      • van't Slot K.A.
      • Knogge W.
      A dual role for microbial pathogen-derived effector proteins in plant disease and resistance.
      ). The role in virulence has been shown for a few fungal effectors, including Avr2 and Avr4 of leaf-mold fungus Cladosporium fulvum, which inhibit tomato cysteine proteases (Rcr3, Pip1, aleurain, and TDI65) and protect chitin in fungal cell walls against plant chitinases, respectively (
      • Stergiopoulos I.
      • de Wit P.J.
      Fungal effector proteins.
      ).
      Recent findings illustrate a diversity of effector structures and activities, as well as validate the view that effector genes are the target of the evolutionary forces that drive the antagonistic interplay between pathogen and host (
      • Kamoun S.
      Groovy times: filamentous pathogen effectors revealed.
      ). Biochemical, genetic, and bioinformatic strategies have been applied to the identification of elicitors from fungi. However, intrinsic function of most fungal elicitors remains elusive (
      • Stergiopoulos I.
      • de Wit P.J.
      Fungal effector proteins.
      ). Of the vast numbers of effector proteins that have been identified, only a few have been biochemically characterized (
      • Chisholm S.T.
      • Coaker G.
      • Day B.
      • Staskawicz B.J.
      Host-microbe interactions: shaping the evolution of the plant immune response.
      ). GP42 is an abundant 42-kDa cell wall glycoprotein isolated from Phytophthora sojae with Ca2+-dependent transglutaminase activity that functions as PAMP, which triggers transcriptional activation of defense genes, accumulation of phytoalexins in parsley, and cell death in potato (
      • Sacks W.
      • Nürnberger T.
      • Hahlbrock K.
      • Scheel D.
      Molecular characterization of nucleotide sequences encoding the extracellular glycoprotein elicitor from Phytophthora megasperma.
      ). Phytophthora elicitins encode structurally related small (<150 amino acids) extracellular proteins, which can induce a local necrosis called the hypersensitive response and a systemic acquired resistance in tobacco (
      • Kamoun S.
      A catalogue of the effector secretome of plant pathogenic oomycetes.
      ). It was demonstrated that class I elicitins (INF1) can bind sterols, such as ergosterol, and function as sterol-carrier proteins. In addition, phospholipase activity was assigned to elicitin-like proteins from Phytophthora capsici, suggesting a general lipid binding/processing role for the various members of the elicitin family (
      • Mikes V.
      • Milat M.L.
      • Ponchet M.
      • Panabières F.
      • Ricci P.
      • Blein J.P.
      Elicitins, proteinaceous elicitors of plant defense, are a new class of sterol carrier proteins.
      ). PcF is a secreted 52-amino acid peptide of Phytophthora cactorum that produces necrosis in tomato and strawberry. PcF was proposed to function as a toxin because it triggers responses that are similar to disease symptoms in host plants (
      • Orsomando G.
      • Lorenzi M.
      • Raffaelli N.
      • Dalla Rizza M.
      • Mezzetti B.
      • Ruggieri S.
      Phytotoxic protein PcF, purification, characterization, and cDNA sequencing of a novel hydroxyproline-containing factor secreted by the strawberry pathogen Phytophthora cactorum.
      ). Likewise, a dual function was found for CBEL (cellulose binding, elicitor, and lectin-like), a 34-kDa cell wall protein that was first isolated from Phytophthora parasitica var. nicotianae. It was reported that CBEL can elicit necrosis and defense gene expression in tobacco plants and, on the other side, is required for the attachment to cellulosic substrates such as plant surfaces (
      • Mateos F.V.
      • Rickauer M.
      • Esquerré-Tugayé M.T.
      Cloning and characterization of a cDNA encoding an elicitor of Phytophthora parasitica var. nicotianae that shows cellulose-binding and lectin-like activities.
      ). Therefore, deciphering the biochemical activity of elicitors to understand how pathogens successfully colonize and grow in their host plants has become a driving paradigm in the field of fungal pathology.
      Because a similar protection effect against C. acutatum (M11), as described previously by Chalfoun et al. (
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz Ricci J.C.
      Induced resistance activated by a culture filtrate derived from an avirulent pathogen as a mechanism of biological control of anthracnose in strawberry.
      ), was observed when strawberry plants were treated with culture filtrates of an avirulent isolate of the strawberry pathogen Acremonium strictum (SS71), we were interested to identify and biochemically characterize the defense elicitor secreted by the SS71 isolate that was also able to induce a systemic resistance against anthracnose in strawberry. The relationship between the intrinsic biochemical function of the elicitor and its ability to elicit a defense response is discussed.

      EXPERIMENTAL PROCEDURES

      Plant Material

      Strawberry plants (Fragaria × ananassa Duch.) of the cv. Pájaro used in experiments were kindly provided by the strawberry BGA (Strawberry Active Germplasm Bank at National University of Tucumán). Healthy plantlets were obtained from in vitro cultures in MS medium (Sigma), rooted in pots with sterilized substrate (humus and perlite, 2:1), and maintained at 28 °C, 70% relative humidity (RH), with a light cycle of 16 h (white fluorescent, 350 μmol photons m−2 s−1).
      Arabidopsis thaliana ecotype Col-0 seeds were obtained from the Arabidopsis Biological Resource Center (Columbus, OH) and germinated on MS medium plates supplemented with 1% sucrose and 0.8% agar. Seedlings at the two-leaf stage were then transplanted to soil and grown in a growth chamber (Sanyo) with 70% RH, under a regime of 16 h of light (white fluorescent, 200 μmol photons m−2 s−1) at 23 °C (
      • Penninckx I.A.
      • Thomma B.P.
      • Buchala A.
      • Métraux J.P.
      • Broekaert W.F.
      Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis.
      ). Plants of 4–5 weeks old were used for the experiments.

      Fungal Cultures

      Three local fungal isolates characterized in our laboratory were used in this paper as follows: M11 of C. acutatum (
      • Salazar S.M.
      • Castagnaro A.P.
      • Arias M.E.
      • Chalfoun N.R.
      • Tonello U.
      • Díaz-Ricci J.C.
      Induction of a defense response in strawberry mediated by an avirulent strain of Colletotrichum.
      ,
      • Grellet-Bournonville C.F.
      • Martinez-Zamora M.G.
      • Castagnaro A.P.
      • Díaz-Ricci J.C.
      Temporal accumulation of salicylic acid activates the defense response against Colletotrichum in strawberry.
      ,
      • Salazar S.M.
      • Grellet C.F.
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz-Ricci J.C.
      Avirulent strain of Colletotrichum induces a systemic resistance in strawberry.
      ,
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz Ricci J.C.
      Induced resistance activated by a culture filtrate derived from an avirulent pathogen as a mechanism of biological control of anthracnose in strawberry.
      ); B1 of Botrytis cinerea (
      • Salazar S.M.
      • Grellet C.F.
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz-Ricci J.C.
      Avirulent strain of Colletotrichum induces a systemic resistance in strawberry.
      ), and SS71 of A. strictum. M11 and B1 isolates are preserved in the culture collection available in the Instituto Superior de Investigaciones Biológicas (Consejo Nacional de Investigaciones Científicas y Técnicas-Universidad Nacional de Tucumán), and the SS71 isolate was deposited in the International Microorganism Bank DSMZ (Germany) under accession number DSM24396. Fungal isolates were propagated on potato dextrose agar as indicated previously (
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz Ricci J.C.
      Induced resistance activated by a culture filtrate derived from an avirulent pathogen as a mechanism of biological control of anthracnose in strawberry.
      ) and maintained on potato dextrose agar slants at 4 °C. To obtain elicitor, the SS71 isolate was cultivated in potato dextrose broth at 28 °C under continuous white fluorescent light (200 μmol photons m−2 s−1) without agitation for 21 days.

      Elicitor Protein Purification

      Elicitor protein was purified from 4 liters of 21-day-old SS71 A. strictum culture grown in potato dextrose broth. Mycelia and spores were removed from the exhausted medium by centrifugation at 10,000 × g for 30 min at 4 °C, followed by filtration through diatomaceous earth (Sigma) and membranes of 0.22 μm diameter (Millipore). This axenic supernatant is referred to as the nondiluted culture filtrate (CF 1×). The extract was concentrated 10-fold under vacuum and filtrated under gas nitrogen pressure through a molecular sieve (30-kDa cutoff, Amicon). The retentate over the membrane was recovered by washing the membrane surface with 20 mm Tris-HCl buffer, pH 7.5, and was subjected to two steps of chromatographic separation by anionic exchange and hydrophobic interaction.

      Q-Sepharose Fast Flow Chromatography

      The fraction adjusted to pH 7.5 was loaded onto a Q-Sepharose fast flow column (Q-FF 1 ml, Amersham Biosciences) previously equilibrated with 20 mm Tris-HCl, pH 7.5 (buffer A). After washing the column with buffer A, the elution was carried out with a discontinuous increasing gradient of NaCl with buffer A containing 1 m NaCl (buffer B). A 1 ml/min flow rate was maintained. The gradient conditions were as follows: 0% buffer B for 5 min; 0–24% B in 3 min; 24% B for 8 min, 24–38% B in 2 min, 38% B for 10 min, 38–100% B in 6 min, and then held at 100% B for 10 min. Eluted fractions were combined in four Q-FPLC pools, dialyzed (12-kDa cutoff, Sigma) against bidistilled water during 24 h at 4 °C, concentrated under vacuum (SpeedVac, Savant), and assayed on strawberry plants as described below. The Q-FPLC pool that showed eliciting activity was used to dissolve (NH4)2SO4 up to a final concentration of 1.5 m.

      Phenyl-Sepharose High Performance Chromatography

      This active Q-FPLC pool was applied to a FPLC phenyl-Sepharose high performance column (PS-HP 5 ml, GE Healthcare), previously equilibrated with 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, and 1.5 m (NH4)2SO4 (buffer A). The column was eluted at a flow rate of 1 ml/min with a discontinuous decreasing gradient of (NH4)2SO4 obtained with buffers A and B (50 mm Tris-HCl pH 7.5, 1 mm EDTA). The gradient conditions were as follows: 0% buffer B for 10 min; 0–22% B in 5 min; 22% B for 5 min; 22–30% B in 4 min; 30% B for 7 min; 30–40% B in 3 min; 40% B for 7 min; 40–50% B in 3 min; 50% B for 7 min; 50–70% B in 5 min; 70% B for 5 min; 70–80% B in 4 min; 80% B for 5 min; 80–90% B in 4 min; 90% B for 5 min; 90–100% B in 4 min and then held at 100% B for 10 min. Eluted fractions were combined in 10 PS-FPLC pools, desalted, concentrated as described above, and assayed on strawberry plants as described below.

      Bioassays for Eliciting Activity

      Elicitor activity of either crude fraction (CF) or purified fractions was routinely assayed on strawberry plants by analyzing the accumulation of ROS (i.e. H2O2 and O2̇̄) in foliar tissue and by evaluating the systemic resistance against the M11 isolate (see below).
      For biological experiments, protein concentration of CF and the pooled fractions from the purification steps were adjusted to 2.5 μg of protein ml−1 with sterile bidistilled water. In all cases, CF from M11, buffers, and bovine serum albumin (BSA; 10 μg ml−1) were used as negative controls, and the axenic CF from SS71 as positive control.

      Oxidative Burst

      Accumulation of hydrogen peroxide was detected by a peroxidase-dependent in situ histochemical staining procedure using 3,3′-diaminobenzidine according to Thordal-Christensen et al. (
      • Thordal-Christensen H.
      • Zhang Z.
      • Wei Y.
      • Collinge D.B.
      Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction.
      ) and superoxide ion using a superoxide-dependent reduction of nitro blue tetrazolium (NBT) according to Doke (
      • Doke N.
      Generation of superoxide anion by potato tuber protoplasts during the hypersensitive response to hyphal wall components of Phytophthora infestans and specific inhibition of the reaction by suppressors of hypersensitivity.
      ). Analyses were performed on plant leaves sprayed with each of the purified fractions (2.5 μg of protein ml−1) of CF SS71, 4 h after leaf aspersion, as described previously (
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz Ricci J.C.
      Induced resistance activated by a culture filtrate derived from an avirulent pathogen as a mechanism of biological control of anthracnose in strawberry.
      ).

      Induced Resistance (IR) against Anthracnose Disease

      To evaluate the resistance inducing activity of the different fractions, plants of the cv. Pájaro received a double treatment (
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz Ricci J.C.
      Induced resistance activated by a culture filtrate derived from an avirulent pathogen as a mechanism of biological control of anthracnose in strawberry.
      ). Briefly, only the youngest completely expanded leaf of each plant (isolated from the rest of the plant) was sprayed with each of the fractions purified from CF (or CFs from SS71 or M11 as controls), and then after 7 days, the whole plant was inoculated with a conidial suspension (1.5 × 106 conidia/ml) of the virulent isolate M11 of C. acutatum. Immediately after the second treatment, plants were placed in the infection chamber at 100% RH, 28 °C, in the dark for 48 h and then returned to the growth chamber at 70% RH, 28 °C, under white fluorescent light (350 μmol photons m−2 s−1) with a light cycle of 16 h per day, where they remained for 50 days.
      Susceptibility to anthracnose disease was measured by assessing Disease Severity Ratings (DSR), according to a scale described on petioles by Delp and Milholland (
      • Delp B.R.
      • Milholland R.D.
      Evaluating strawberry plants for resistance to Colletotrichum fragariae.
      ). The DSR was evaluated at 21 days post-inoculation (dpi). Experimental design and statistic analysis were achieved as described previously (
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz Ricci J.C.
      Induced resistance activated by a culture filtrate derived from an avirulent pathogen as a mechanism of biological control of anthracnose in strawberry.
      ). Experimental data were analyzed with the program Statistix (Analytical Software, 1996) for Windows. The LSD test was used to determine the arithmetic mean of the DSR values (significance level, 0.05) of each IR experiment, and the analysis of variance test was used to evaluate the data dispersion with respect to the mean values.

      Analysis of Proteins (SDS-PAGE and 2D-DE)

      In each step of purification, the total protein content was quantified according to Bradford (
      • Bradford M.
      A rapid and sensitive method for the determination of microgram quantities of protein utilizing the principle of protein-dye binding.
      ) using BSA as the standard. Protein profiles were routinely analyzed by SDS-PAGE according to Laemmli (
      • Laemmli U.K.
      Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
      ). Previously, protein samples were desalted through Sephadex G-25 superfine semi-dry matrix (Sigma). Aliquots containing 10 μg of desalted protein were heated to 100 °C for 5 min in a denaturating mixture (
      • Laemmli U.K.
      Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
      ) and loaded onto a 12% (w/v) polyacrylamide gel. Gels were run at 20 mA per gel constant current for 90 min.
      To study the purity of the sample, the biologically active fraction obtained by PS-FPLC was submitted to two-dimensional PAGE. Briefly, 30 μg of desalted protein were vacuum-dried and solubilized in 7 m urea, 2 m thiourea, 4% CHAPS, 30 mm DTT, and 0.5% biolytes (Bio-Rad). Insoluble material was removed by centrifugation (15 min at 15,000 × g), and the supernatant was applied onto IPG strips (7 cm, 3–10 linear pH gradient, Bio-Rad), which were rehydrated for 12 h at constant 50 V with 30 μg of protein in 125 μl. Isoelectrofocusing (IEF) was carried out in a PROTEAN IEF cell (Bio-Rad) at a constant current of 50 μA per strip until 10,000 V were reached. After IEF, the IPG strips were equilibrated according to Görg et al. (
      • Görg A.
      • Postel W.
      • Weser J.
      • Günther S.
      • Strahler J.R.
      • Hanash S.M.
      • Somerlot L.
      Elimination of point streaking on silver stained two-dimensional gels by addition of iodoacetamide to the equilibration buffer.
      ). Second dimension SDS-PAGE was performed on 12% polyacrylamide gels using Mini-PROTEAN III as indicated above. Gels were stained with the fluorescent dye ruthenium II tris-bathophenanthroline disulfonate (Sypro, Bio-Rad). The images were captured, digitized, and analyzed with PDQuestTM software (Bio-Rad) (
      • Jorge I.
      • Navarro R.M.
      • Lenz C.
      • Ariza D.
      • Porras C.
      • Jorrín J.
      The Holm Oak leaf proteome. Analytical and biological variability in the protein expression level assessed by 2-DE and protein identification by MS/MS de novo sequencing and sequence similarity searching.
      ). For each spot, the pI and the molecular mass were determined (
      • Jorge I.
      • Navarro R.M.
      • Lenz C.
      • Ariza D.
      • Porras C.
      • Jorrín J.
      The Holm Oak leaf proteome. Analytical and biological variability in the protein expression level assessed by 2-DE and protein identification by MS/MS de novo sequencing and sequence similarity searching.
      ). The two-dimensional gel analysis was repeated three times to avoid inconsistencies. For the detection of glycoside residues, 30 μg of desalted pure elicitor protein was separated by SDS-PAGE in 12% polyacrylamide gels and revealed by periodic acid-Schiff (PAS) staining according to Segrest and Jackson (
      • Segrest J.P.
      • Jackson R.L.
      Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulphate.
      ) with minor modifications. Albumin from chicken egg white (ovalbumin; Sigma) of 45 kDa was used as glycoprotein control. To detect the sensitivity of PAS staining method to glycoside residues, different amounts of ovalbumin (1, 5, and 10 μg) were loaded in the wells.

      Nano-LC and Ion Trap MS/MS Analysis of Tryptic Peptides

      Protein spots were excised from the gel and then digested with porcine trypsin (Promega) using in-gel digestion according to Schevchenko et al. (
      • Shevchenko A.
      • Wilm M.
      • Vorm O.
      • Mann M.
      Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.
      ) with minor variations. In parallel, 100 μg of pure elicitor protein (corresponding to desalted PS-FPLC pool) was proteolytically digested under highly denaturing conditions as described by Link et al. (
      • Link A.J.
      • Eng J.
      • Schieltz D.M.
      • Carmack E.
      • Mize G.J.
      • Morris D.R.
      • Garvik B.M.
      • Yates 3rd, J.R.
      Direct analysis of protein complexes using mass spectrometry.
      ). Both tryptic digests were vacuum-dried and used for MS analysis.
      MS analysis was performed by fingerprinting of the tryptic digests, using standard methods on a Biflex III spectrometer (Bruker-Franzen Analytik, Bremen, Germany) in a positive ion reflector mode. Selected tryptic peptides were on-line injected onto a C-18 reverse phase (RP) nano-column (Discovery® BIO Wide pore, Supelco) and analyzed in a continuous acetonitrile gradient consisting of 0–50% B in 45 min, 50–90% B in 1 min (A = bidistilled water; B = 95% acetonitrile, 0.5% acetic acid). A flow rate of ∼300 nl/min was used to elute peptides from the RP nano-column to a PicoTipTM emitter nano-spray needle (New Objective, Woburn, MA) for real time ionization and peptide fragmentation on an Esquire HCT IT (Bruker-Daltonics) mass spectrometer. A 3-Da window (precursor m/z ± 1.5), MS/MS fragmentation amplitude of 0.90 V, and a dynamic exclusion time of 0.30 min were used for peptide fragmentation. Nano-LC was automatically performed on an advanced nano-gradient generator (Ultimate nano-HPLC, LC Packings, Amsterdam, The Netherlands) coupled to an autosampler (Famos, LC Packings). The software Hystar 2.3 was used to control the whole analytical process. MS/MS spectra were batch-processed by using DataAnalysis 5.1 SR1 and BioTools 2.0 software packages and searched against the MSDB protein database using MASCOT software (Matrix Science) or sequenced de novo followed by MS BLAST alignment at EMBL with default settings.

      RP-HPLC and MALDI-MS Analysis of Intact Protein

      The absolute purity of the native elicitor protein obtained by PS-FPLC was verified using two different methods as follows: analytical RP-HPLC and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis. Briefly, 100 μg of lyophilized protein (corresponding to desalted PS-FPLC pool) was resuspended in bidistilled water and chromatographed on an HPLC μRPC C2/C18 column (GE Healthcare) using a ÄKTA purifier 10 system (GE Healthcare). The column was previously equilibrated in bidistilled water with 0.1% (v/v) trifluoroacetic acid and eluted with a linear gradient of acetonitrile with 0.1% (v/v) trifluoroacetic acid at 0.7 ml/min flow rate. Peaks were detected at λ = 285 nm. In parallel, 10 μg of lyophilized intact protein was resuspended in 50% acetonitrile with 0.1% (v/v) trifluoroacetic acid and analyzed on a Biflex III MALDI-TOF mass spectrometer with delayed extraction (Brucker-Franzen Analytik, Bremen, Germany) using standard methods. The instrument performed acquisition of a full-scan mass spectrum ranging from 500 to 100,000 Da.

      N-terminal Sequencing

      The biologically active purified protein was run on a 12% SDS-polyacrylamide gel and electroblotted onto a 0.1-μm pore size PVDF transfer membrane (Immobilon-PSQ, Millipore) at 4 °C applying constant voltage (at 45 V) for 12 h. Transfer buffer was 10 mm CAPS (3-(cyclohexylamino)propanesulfonic acid), pH 11.0, 10% methanol, and 5 mm DTT (
      • Matsudaira P.
      Sequence from picomole quantities of proteins electroblotted onto PVDF.
      ). Protein bands were visualized by staining with 0.1% Coomassie Brilliant Blue-R in 50% methanol, 1% acetic acid for 1 min. The membrane was unstained with 50% methanol. The band corresponding to the elicitor protein was excised and subjected to N-terminal amino acid sequencing by automated Edman degradation on an Applied Biosystems 477A gas-phase sequencer (Applied Biosystems, Foster City, CA).

      Fungal RNA Preparation

      Fresh SS71 A. strictum mycelium (1.2 g wet weight) was harvested by filtration from the liquid culture and pestle-homogenized in liquid nitrogen. Subsequently, total RNA was obtained and purified according to Iandolino et al. (
      • Iandolino A.B.
      • Goes da Silva F.
      • Lim H.
      • Choi H.
      • Williams L.E.
      • Cook D.R.
      High-quality RNA, cDNA, and derived EST libraries from grapevine (Vitis vinifera L.).
      ). RNA quantification was performed by spectrophotometry at 230, 260, and 280 nm. Reverse transcription reactions were carried out with 5 μg of DNase-treated total RNA, using SuperScript II RT (Invitrogen) following the recommendations of the manufacturer.

      Degenerate PCR

      The degenerate PCR primers used were designed from the less conserved regions of amino acid sequences de novo of tryptic peptides and N-terminal region of the elicitor protein, according to the following criteria: (a) frequency of codons usage in the 27 cDNA and 19 genes for Acremonium spp. reported so far, by using the program General Codon Usage Analysis, and (b) codons encoding identical amino acids to that sequenced in five homologous proteins of different fungal species identified by using BLASTP algorithm (
      • Altschul S.F.
      • Gish W.
      • Miller W.
      • Myers E.W.
      • Lipman D.J.
      Basic local alignment search tool.
      ). The combinations of degenerate oligonucleotide primers N1, N2, IF, IR, C1, and C2, used in three consecutive PCRs, are represented in Fig. 3. PCRs were carried out in 25-μl total volume containing 50 mm KCl, 20 mm Tris-HCl, pH 8.4, 1.5 mm MgCl2, 2 μm of each primer, 0.2 mm of each dNTP, and 0.75 units of TaqDNA polymerase (Invitrogen). 200 ng of cDNA were used as template for the initial PCR, and 1:125 dilution from the latter PCR product was used for the first nested PCR. The amplified band exhibiting the expected size was excised from the agarose gel, purified, and used as template for both second nested PCRs (Fig. 3). PCR amplifications were performed in a PTC-100 thermal cycler (MJ Research). Cycling conditions included an initial denaturizing step of 10 min at 94 °C, followed by 40 cycles of 45 s at 94 °C (melting), 30 s at 55 °C (annealing), 1.5 min at 72 °C (extension), and a final extension step of 10 min at 72 °C.
      Figure thumbnail gr3
      FIGURE 3Scheme of the isolation strategy used to obtain the elicitor-coding cDNA. Top, cDNA encoding for the elicitor protein, which has been obtained by overlapping the nucleotide fragments A–D, identified after nested PCR with degenerate primers and RACE experiments (see details under “Experimental Procedures”). A, 350-bp fragment isolated from expression library by nested PCR with the combinations N1/C2, N2/C1, and N2/IR of degenerate primers. B, 480-bp fragment isolated from expression library by nested PCR with the combinations N1/C2, N2/C1, and IF/C1 of degenerate primers. C, 5′-RACE fragment of 680 bp amplified with the S-5- and SN-5-specific nested primers. D, 3′-RACE fragment of 780 bp amplified with the S-3- and SN-3-specific nested primers. The oligonucleotide primers utilized were as follows: N1, 5′-GCNTAYACNACNCARGCNT-3′; N2, 5′-CAGGCBWSBGCBCCBTGG-3′; IF, 5′-ATYATYGCYGGYATYAACTAYG-3′; IR, 5′-CRTAGTTRATRCCRGCRATRAT-3′; C1, 5′-RATVACRCCVGTVGTVGCRAT-3′; C2, 5′-GTTVGGVGTRCCVWSVGGR-3′; S-5, 5′-GATGTTGTTGTCGATCAAGGACTTGG-3′; SN-5, 5′-TGCCTTGGTAGGAGACAAGCTGGAA-3′, S-3, 5′-AGCTTGTCTCCTACCAAGGCAGCAA-3′; SN-3, 5′-GGCCAAGTCCTTGATCGACAACAAC-3′. R-5 and RN-5 and R-3 and RN-3 primers were the nested anchor primer for ends 5′ or 3′, respectively, supplied by the 3′/5′ GeneRacer kit (Invitrogen).

      Cloning and Sequencing

      After electrophoretic separation of each nested PCR product, sharp bands of the expected size were excised from the gel and purified using the GFXTM PCR DNA and gel band purification kit (GE Healthcare). The purified cDNA obtained for each band was cloned using the plasmid pCRR4.1 of the TopoTA cloning kit (Invitrogen) following the manufacturer's recommendations and transformed into Escherichia coli DH5-α strain. Recombinant plasmids were extracted from 10 recombinant bacterial colonies using Wizard plus SV Minipreps DNA purification system (Promega) and digested with EcoRI to verify the presence of the expected insert. Sequences of PCR products were determined using an ABI PRISM TM 377 DNA sequencer (Applied Biosystems) (
      • Sanger F.
      • Nicklen S.
      • Coulson A.R.
      DNA sequencing with chain terminator inhibitors.
      ).

      RACE Experiments

      To isolate the full-length elicitor-coding cDNA, RNA ligase-mediated rapid amplification reactions of cDNA ends (RACE) were performed with the GeneRacer kit (Invitrogen) as recommended by the manufacturer, using total RNA purified from 150 mg of SS71 mycelium with the RNeasy minikit (Qiagen). cDNA synthesis was performed by SuperScript III RT (Invitrogen) from 4 μg of 5′-modified mRNA in the presence of GeneRacer oligo(dT) primer, for 3′-end extension.
      Based on nucleotide multiple sequence alignment with homologous proteins, less conserved regions were identified in the partial sequence of elicitor cDNA, and a new set of specific primers were designed following GeneRacer kit instructions. The primers S-5, SN-5, S-3, and SN-3 were then used for 5′ or 3′ cDNA ends cloning through nested PCRs as depicted in Fig. 3, by using High Fidelity Platinum TaqDNA polymerase (Invitrogen). The anchor primers, R-5, RN-5, R-3, and RN-3, were part of the kit (Fig. 3).
      For 5′-RACE, the single strand cDNA (1:5 dilution) was used as the template for PCR in the presence of the S-5 (0.2 μm) and R-5 (0.6 μm) primers. A nested PCR was carried out by diluting an aliquot of the previous PCR product (1:10 dilution) in a second PCR mixture containing the SN-5 and RN-5 nested primers to concentration 0.2 μm. Similarly, for 3′-RACE, the single strand cDNA was utilized as the template in the subsequent PCR and one nested PCR in the presence of the S-3 and R-3 primers and SN-3 and RN-3 nested primers, respectively (Fig. 3). First amplifications were performed using “touchdown” conditions (
      • Don R.H.
      • Cox P.T.
      • Wainwright B.J.
      • Baker K.
      • Mattick J.S.
      Touchdown PCR to circumvent spurious priming during gene amplification.
      ) as follows: 2 min at 94 °C; 5 cycles (30 s at 94 °C; 30 s at 72 °C; 4 min at 68 °C); 5 cycles (30 s at 94 °C; 30 s at 70 °C; 4 min at 68 °C); 25 cycles (30 s at 94 °C; 30 s at 66 °C; 4 min at 68 °C), and 10 min at 68 °C. Nested PCR conditions were as follows: 5 min at 95 °C; 30 cycles (30 s at 95 °C; 30 s at 69 °C; 2 min at 68 °C) and 10 min at 68 °C. The cDNA fragments of both 3′- and 5′-end extensions obtained from RACE experiments were purified from the agarose gel and ligated by a standard cloning procedure, as described above. After transformation of the E. coli DH5-α strain, 10 recombinant plasmid clones were purified and subjected to automated dideoxy chain termination sequencing as described above.

      Sequence Edition and Analysis

      Cloned sequences were trimmed of vector sequence contamination using VecScreen at NCBI (www.ncbi.nlm.nih.gov). Assemblage of cDNA sequences and translation to the predicted amino acid sequence were performed using the DNAMAN software (version 6.0). Identity of the fungal elicitor obtained was studied by comparisons of deduced amino acid and nucleotide sequences with sequences included in the GenBankTM NR database by using BLASTX and BLASTP algorithms (
      • Altschul S.F.
      • Gish W.
      • Miller W.
      • Myers E.W.
      • Lipman D.J.
      Basic local alignment search tool.
      ).
      Determinations of conserved structural motifs and domains in sequence were carried out with CDSearch (at NCBI) (
      • Marchler-Bauer A.
      • Bryant S.H.
      CD-Search: protein domain annotations on the fly.
      ), SMART 5 (
      • Letunic I.
      • Copley R.R.
      • Pils B.
      • Pinkert S.
      • Schultz J.
      • Bork P.
      SMART 5: domains in the context of genomes and networks.
      ), and Pfam (at Sanger Institute) (
      • Bateman A.
      • Coin L.
      • Durbin R.
      • Finn R.D.
      • Hollich V.
      • Griffiths-Jones S.
      • Khanna A.
      • Marshall M.
      • Moxon S.
      • Sonnhammer E.L.
      • Studholme D.J.
      • Yeats C.
      • Eddy S.R.
      The Pfam protein families database.
      ). Multiple sequence alignments were performed with Clustal X (
      • Thompson J.D.
      • Gibson T.J.
      • Plewniak F.
      • Jeanmougin F.
      • Higgins D.G.
      The CLUSTAL-X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.
      ) and edited with BOXSHADE. The SignalP server (
      • Emanuelsson O.
      • Brunak S.
      • von Heijne G.
      • Nielsen H.
      Locating proteins in the cell using TargetP, SignalP and related tools.
      ) was used to predict the presence and localization of signal peptide cleavage sites. Potential N- and O-glycosylation sites were identified by using NetNGlyc 1.0 and NetOGlyc 3.1, respectively. Molecular mass and pI were calculated by using PeptideMass program at the ExPASy server.

      Proteolytic Activity Determinations and Protease Inhibitor Assays

      Proteolytic activity was measured in fractions that exhibited eliciting activity obtained in each purification step using the chromogenic peptides N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA; Sigma) or Nα-benzoyl-dl-Arg-p-nitroanilide hydrochloride (Sigma), as specific substrates. Substrate hydrolysis was measured by adding 10 μl of each indicated substrate (5 mm) (final concentration of 0.1 mm) to an appropriate amount of protein sample dissolved in 20 mm Tris-HCl, pH 7.5, for a final volume of 500 μl. Reaction mixtures were incubated at 37 °C for 30 min, and the hydrolysis of the chromogenic substrates was monitored per min at 405 nm (
      • Moallaei H.
      • Zaini F.
      • Larcher G.
      • Beucher B.
      • Bouchara J.P.
      Partial purification and characterization of a 37-kDa extracellular proteinase from Trichophyton vanbreuseghemii.
      ). All activity assays were performed in triplicate. Subtilisin Carlsberg (subtilopeptidase A) of Bacillus licheniformis (Fluka) was used as reference of protease activity. The proteolytic activity of pure elicitor protein was calculated as the concentration of pNA liberated per min, where pNA concentration was determined spectrophotometrically to 405 nm using a molar ϵ405 nm of 9.62 mm−1 cm−1, at 37 °C and pH 7.5. Autoproteolysis rate of substrate Suc-AAPF-pNA was evaluated and subtracted to each measured value.
      For protease inhibitor studies, pure elicitor protein was preincubated with different protease inhibitors for 1 min at 37 °C, at the concentrations indicated for a final volume of 500 μl. Inhibition assays were performed against the substrate Suc-AAPF-pNA as described above.

      Protease Inhibitor Treatment of Elicitor Protein

      Pure elicitor protein (50 μg) resuspended in 1 ml of 20 mm Tris-HCl, pH 7.5, was treated with PMSF (to final concentration of 1 mm) at 37 °C for 30 min and then diluted 20-fold with the same buffer (until reaching a final concentration of 2.5 μg ml−1). PMSF-treated elicitor protein was used in IR experiments with strawberry plants as described above. PMSF adjusted to 0.05 mm with 20 mm Tris-HCl, pH 7.5, was used as negative control, and proteinase K from Tritirachium album (2.5 μg ml−1; Sigma) and subtilisin Carlsberg (2.5 μg ml−1) were used as control of protease activity.

      Expression Analysis of Defense-related Genes

      Total RNA from strawberry leaves was obtained, according to the protocol published by Iandolino et al. (
      • Iandolino A.B.
      • Goes da Silva F.
      • Lim H.
      • Choi H.
      • Williams L.E.
      • Cook D.R.
      High-quality RNA, cDNA, and derived EST libraries from grapevine (Vitis vinifera L.).
      ), with some modifications. One gram of fresh young leaves was sampled from elicitor-treated plants at different time points, from 0 to 72 h post-treatment (hpt). Amount and quality of obtained total RNA were monitored, and reverse transcription reactions were performed as described above. Semi-quantitative RT-PCR method was used to evaluate the expression of plant defense response-associated genes (
      • Grellet-Bournonville C.F.
      • Martinez-Zamora M.G.
      • Castagnaro A.P.
      • Díaz-Ricci J.C.
      Temporal accumulation of salicylic acid activates the defense response against Colletotrichum in strawberry.
      ). Expressions of FaPR1 (pathogenesis related-protein 1) and FaChi2-1 (class II chitinase) genes were studied using nucleotide sequences previously reported in GenBankTM (AB462752 and AF147091, respectively). The gene GAPDH1 (AF421492) was used as control (housekeeping) to adjust the amount of cDNA in each treatment and to attain the same exponential phase PCR signal strength. A volume of 25 μl of reaction mix containing Green GoTaq Reaction Buffer 1×, pH 8.5 (Promega), 1.5 units of GoTaq DNA polymerase (Promega), 1.5 mm MgCl2, 400 μm of each dNTPs 0.4 μm of each primer, was mixed with 5 μl of cDNA (properly diluted to adjust the amount according to housekeeping gene) in each PCR. Specific primers used were as follows: GAPDH1 (forward), 5′-CTACAGCAACACAGAAAACAG-3′, and GAPDH1 (reverse), 5′-AACTAAGTGCTAATCCAGCC-3′; FaPR1 (forward), 5′-TGCTAATTCACATTATGGCG-3′, and FaPR-1 (reverse), 5′-GTTAGAGTTGTAATTATAGTAGG-3′; and FaChi2-1 (forward), 5′-TCGTCACTTGCAACTCCTAA-3′, and FaChi2-1 (reverse), 5′-GGACTTCTGATTTTCACAGTCT-3′. The PCR program used was as follows: 7 min at 94 °C (initial step); cycles (see below) of 45 s at 94 °C, 1 min at different annealing temperature (see below), 1.5 min at 72 °C; and a final extension step of 10 min at 72 °C. The number of cycles used for each gene was adjusted to obtain the specific band amplified at the exponential phase of PCR. Annealing temperatures for GAPDH1, FaPR-1, and FaChi2-1 genes were 52, 57, and 55 °C, respectively. Amplified bands were visualized in ethidium bromide-stained (10 μg ml−1) agarose gel (2%) and photographed under UV light (340 nm) with a digital camera. Bands observed were digitalized and quantified using Total Lab Quant software (Nonlinear Dynamics Ltd., Newcastle, UK). Relative expression of studied genes was calculated as the change of the band intensity in treated leaves with respect to control leaves. To ensure the absence of genomic DNA in each cDNA sample, GAPDH1 primer sequences were designed to enclose an intronic region.

      IR Assay against Gray Mold Disease

      To evaluate the protection against B. cinerea, plants of the cv. Pájaro were sprayed with the elicitor protein purified from CF (or water as negative control), and then after 7 days were inoculated with the virulent isolate B1 of B. cinerea as described previously (
      • Salazar S.M.
      • Grellet C.F.
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz-Ricci J.C.
      Avirulent strain of Colletotrichum induces a systemic resistance in strawberry.
      ). Immediately after inoculation, plants were placed in the infection chamber at 100% RH, 20 °C under continuous white fluorescent light (50 μmol photons m−2 s−1) for 48 h, and then returned to the growth chamber under conditions indicated above, where they remained for 50 days. Susceptibility to gray mold disease was evaluated at 10 dpi by measuring necrotic lesion extension on leaves, according to a DSR scale described by Vellice et al. (
      • Vellicce G.R.
      • Díaz Ricci J.C.
      • Hernández L.
      • Castagnaro A.P.
      Enhanced resistance to Botrytis cinerea mediated by the transgenic expression of the chitinase gene ch5B in strawberry.
      ). Experimental design and statistical analysis were achieved as described above (
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz Ricci J.C.
      Induced resistance activated by a culture filtrate derived from an avirulent pathogen as a mechanism of biological control of anthracnose in strawberry.
      ).

      Tests of Eliciting Activity in Arabidopsis

      Defense responses were evaluated through histochemical staining techniques in leaves of A. thaliana plants sprayed with the pure elicitor protein (2.5 μg ml−1) or distilled water as control. Leaves were collected at different times post-treatment: from 0 to 12 hpt for burst oxidative and every 3 days up to 12 days post-treatment (dpt) for callose deposition. Evaluation was performed on 20 leaf tissues obtained from five plants that were equally treated.
      Hydrogen peroxide was detected using the 2′,7′-dichlorofluorescein diacetate (Invitrogen) probe according to Bozsó et al. (
      • Bozsó Z.
      • Ott P.G.
      • Szatmari A.
      • Czelleng A.
      • Varga G.
      • Besenyei E.
      • Sárdi É.
      • Bányai É.
      • Klement Z.
      Early detection of bacterium-induced basal resistance in tobacco leaves with diaminobenzidine and dichlorofluorescein diacetate.
      ) modified in our laboratory (
      • Grellet-Bournonville C.F.
      • Martinez-Zamora M.G.
      • Castagnaro A.P.
      • Díaz-Ricci J.C.
      Temporal accumulation of salicylic acid activates the defense response against Colletotrichum in strawberry.
      ), and superoxide anion by NBT staining (
      • Doke N.
      Generation of superoxide anion by potato tuber protoplasts during the hypersensitive response to hyphal wall components of Phytophthora infestans and specific inhibition of the reaction by suppressors of hypersensitivity.
      ), as described previously (
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz Ricci J.C.
      Induced resistance activated by a culture filtrate derived from an avirulent pathogen as a mechanism of biological control of anthracnose in strawberry.
      ). To detect callose deposition, leaves were treated and stained with 0.01% aniline blue (Sigma) according to Yun et al. (
      • Yun M.H.
      • Torres P.S.
      • El Oirdi M.
      • Rigano L.A.
      • Gonzalez-Lamothe R.
      • Marano M.R.
      • Castagnaro A.P.
      • Dankert M.A.
      • Bouarab K.
      • Vojnov A.A.
      Xanthan induces plant susceptibility by suppressing callose deposition.
      ). Samples were examined with an Olympus System Microscope model BX51 equipped with U-LH100HG reflected fluorescence system, setting blue excitation filter (U-MWB2). Images were captured by Olympus Video/Photo Adapter (Olympus, Hamburg, Germany), and callose depositions were quantified with Image Pro Plus software (Media Cybernetics).

      DISCUSSION

      In this paper, we report a novel elicitor protein named AsES that was obtained and purified from A. strictum cultures. It was characterized as a subtilisin-like serine protease that belongs to the proteinase K-like family (
      • Siezen R.J.
      • Leunissen J.A.
      Subtilases: the superfamily of subtilisin like serine proteases.
      ), and it shows extensive similarity to the widely known T. album proteinase K (
      • Gunkel F.A.
      • Gassen H.G.
      Proteinase K from Tritirachium album Limber. Characterization of the chromosomal gene and expression of the cDNA in Escherichia coli.
      ). AsES is synthesized as a 388-residue preprotein precursor, which undergoes proteolytic removal in two steps. A first event consists of the cleavage of the signal peptide that tags the protein to the extracellular medium, and the second event consists of the removal of the propeptide (inhibitor peptidase I9 domain). Subtilisin propeptides are known to function as intramolecular chaperones, assisting in the folding of the mature peptidase (
      • Takagi H.
      • Koga M.
      • Katsurada S.
      • Yabuta Y.
      • Shinde U.
      • Inouye M.
      • Nakamori S.
      Functional analysis of the propeptides of subtilisin E and aqualysin I as intramolecular chaperones.
      ), but they have also been shown to act as “temporary inhibitors” of the enzyme function because they are gradually removed by autoproteolytic cleavage activating the enzyme (
      • Fabre E.
      • Nicaud J.M.
      • Lopez M.C.
      • Gaillardin C.
      Role of the proregion in the production and secretion of the Yarrowia lipolytica alkaline extracellular protease.
      ). The mature and active protein therefore consists of a 283-amino acid polypeptide of 34 kDa comprising only the catalytic peptidase S8 domain, which in addition to the plant defense eliciting activity retains the protease activity. Because the purified AsES lacks glycoside residues, other post-translational modifications occurring in the protein may cause its aberrant mobility on SDS-PAGE and would explain the discrepancy between the experimental molecular mass and that predicted (28.187 kDa). A recombinant subtilisin from Pyrococcus furiosus has also been described to display aberrant mobility on SDS-gel due to high Glu/Asp content (
      • Ikram N.
      • Naz S.
      • Rajoka M.I.
      • Sadaf S.
      • Akhtar M.W.
      Enhanced production of subtilisin of Pyrococcus furiosus expressed in Escherichia coli using autoinducing medium.
      ).
      AsES has four Cys residues that probably form two disulfide bonds, which would contribute to the protein stability similarly to proteinase K (
      • Gunkel F.A.
      • Gassen H.G.
      Proteinase K from Tritirachium album Limber. Characterization of the chromosomal gene and expression of the cDNA in Escherichia coli.
      ). Although two putative Ca2+-binding sites have been detected in the mature elicitor protein, the lack of inhibition exerted by EDTA and the inhibitory effect exerted by 1,10-phenanthroline on its protease activity suggest that AsES may require the association with Fe2+.
      Additionally, among the proteins homologous to AsES include the nonpathogenic subtilisins secreted by endophytic and saprophytic fungi and the pathogenic subtilisins produced by fungal pathogens of plants, nematodes, and insects, whose amino acid sequence identity varies between 55–65% (data not shown). Particularly, subtilisin-like proteinases are considered to be important virulence factors in the infection process of entomopathogenic, nematophagous, and mycoparasitic fungi (
      • Wang B.
      • Liu X.
      • Wu W.
      • Liu X.
      • Li S.
      Purification, characterization, and gene cloning of an alkaline serine protease from a highly virulent strain of the nematode-endoparasitic fungus Hirsutella rhossiliensis.
      ,
      • Yang J.
      • Zhao X.
      • Liang L.
      • Xia Z.
      • Lei L.
      • Niu X.
      • Zou C.
      • Zhang K.Q.
      Overexpression of a cuticle-degrading protease Ver112 increases the nematicidal activity of Paecilomyces lilacinus.
      ), but little is known however about those produced by plant pathogenic fungi. Magnaporthe poae, a fungal pathogen of Kentucky bluegrass, expressed a subtilisin-like proteinase (Mp1) in infected roots, and its expression level correlated with the higher severity of disease symptoms (
      • Sreedhar L.
      • Kobayashi D.Y.
      • Bunting T.E.
      • Hillman B.I.
      • Belanger F.C.
      Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host.
      ). Also, Olivieri et al. (
      • Olivieri F.
      • Zanetti M.E.
      • Oliva C.R.
      • Covarrubias A.A.
      • Casalongué C.A.
      Characterization of an extracellular serine protease of Fusarium eumartii and its action on pathogenesis related proteins.
      ) reported an extracellular subtilisin-like serine protease produced by the phytopathogenic fungus Fusarium solani f. sp. eumartii that is able to degrade potato PR proteins as well as specific polypeptides of intercellular washing fluids and cell wall proteins from potato tubers.
      AsES also shows similarity (although at lower degree) with the other serine proteases identified in Acremonium spp. (
      • Liu C.
      • Matsushita Y.
      • Shimizu K.
      • Makimura K.
      • Hasumi K.
      Activation of prothrombin by two subtilisin-like serine proteases from Acremonium spp.
      ,
      • Isogai T.
      • Fukagawa M.
      • Kojo H.
      • Kohsaka M.
      • Aoki H.
      • Imanaka H.
      Cloning and nucleotide sequences of the complementary and genomic DNAs for the alkaline protease from Acremonium chrysogenum.
      ). A serine alkaline endoproteinase with a bound carbohydrate (At1) was purified from leaf sheath tissue of grass Poa species infected with the pathogen Acremonium typhinum, suggesting that its expression may be involved in the symbiotic interaction between the plant and the fungus (
      • Lindstrom J.T.
      • Belanger F.C.
      Purification and characterization of an endophytic fungal proteinase that is abundantly expressed in the infected host grass.
      ). Liu et al. (
      • Liu C.
      • Matsushita Y.
      • Shimizu K.
      • Makimura K.
      • Hasumi K.
      Activation of prothrombin by two subtilisin-like serine proteases from Acremonium spp.
      ) reported the only two subtilisin-like serine proteases from Acremonium spp. that were biochemically characterized, namely AS-E1 of 34.4 kDa and AS-E2 of 32 kDa, but only their N-terminal regions were sequenced. These authors have also reported that both enzymes were able to proteolytically activate prothrombin to meizothrombin(desF1)-like molecules and inhibit plasma clotting, possibly due to the extensive degradation of fibrinogen (
      • Liu C.
      • Matsushita Y.
      • Shimizu K.
      • Makimura K.
      • Hasumi K.
      Activation of prothrombin by two subtilisin-like serine proteases from Acremonium spp.
      ). An important conclusion we can arrive at is that although these proteases are homologous to AsES, none of them exhibits biological activity in plants or are involved in mechanisms of virulence or defense in other organisms.
      Because preliminary experiments showed that the active fraction was susceptible to heat and protease digestion (data not shown) suggesting that the elicitor molecule was a protein, two-dimensional gels yielded a single protein, and the MS/MS analysis did not display any strange peptide sequence or other spurious MS peak, we concluded that the AsES protein was pure. Sensitivity of elicitor activity to PMSF demonstrated that the AsES subtilisin is the one that exhibits such activity; however, the expression of AsES in a heterologous system would provide a direct link between the cloned cDNA and elicitor activity of its cDNA product.
      Because the first barrier that invading fungi have to overcome is the plant cell wall, fungal endohydrolytic enzymes have been suggested to act as elicitors of the PAMP-type (
      • Nürnberger T.
      • Brunner F.
      • Kemmerling B.
      • Piater L.
      Innate immunity in plants and animals: striking similarities and obvious differences.
      ). The latter could also occur with the AsES as it is able to induce a defense response in different plant species. Our results clearly show that AsES can trigger a strong defense reaction in strawberry, which is characterized primarily by a transient oxidative burst, then by a strong transcriptional induction of PR-1 (FaPR1) and class II chitinase (FaChi2-1), and finally manifested by an enhanced resistance against fungal pathogens of hemibiotrophic (i.e. C. acutatum) and necrotrophic (i.e. B. cinerea) lifestyle. The fact that the protection effect mediated by AsES was not restricted to pathogens of the genus Colletotrichum, let us further conclude that AsES is able to induce a broad-based resistance against pathogens (
      • Salazar S.M.
      • Grellet C.F.
      • Chalfoun N.R.
      • Castagnaro A.P.
      • Díaz-Ricci J.C.
      Avirulent strain of Colletotrichum induces a systemic resistance in strawberry.
      ).
      Synthesis of proteins directly associated with defense response in plants, as PR proteins, is essential to protect them against pathogen infection (
      • Nürnberger T.
      • Brunner F.
      • Kemmerling B.
      • Piater L.
      Innate immunity in plants and animals: striking similarities and obvious differences.
      ). Hence, the induction of PR genes such as PR-1, PR-2 (including β1,3-glucanases), PR-3 (including class II chitinases), among others, is clear evidence of defense mechanism activation (
      • van Loon L.C.
      • Rep M.
      • Pieterse C.M.
      Significance of inducible defense-related proteins in infected plants.
      ). Furthermore, it is well documented that the increase of the expression of PR-1 is strongly associated with systemic acquired resistance activation in pathogen-challenged or elicitor-treated plants, whereas induction of glucanase and chitinase genes is closely related to systemic defense activation against a broad pathogen spectrum (
      • van Loon L.C.
      • Rep M.
      • Pieterse C.M.
      Significance of inducible defense-related proteins in infected plants.
      ,
      • Pieterse C.M.
      • Leon-Reyes A.
      • Van der Ent S.
      • Van Wees S.C.
      Networking by small-molecule hormones in plant immunity.
      ). Particularly, the induction of many PR genes has been reported in strawberry plants challenged by pathogens (
      • Amil-Ruiz F.
      • Blanco-Portales R.
      • Muñoz-Blanco J.
      • Caballero J.L.
      The strawberry plant defense mechanism: a molecular review.
      ).
      Further studies revealed that AsES can also confer protection to different strawberry cultivars against different virulent isolates of C. acutatum (data not shown). We may also speculate that the production of ROS (i.e. H2O2 and O2̇̄) and the cell wall reinforcement due to callose deposition observed in Arabidopsis plants would also confer resistance to pathogens as suggested elsewhere (
      • Jones J.D.
      • Dangl J.L.
      The plant immune system.
      ,
      • Nürnberger T.
      • Brunner F.
      • Kemmerling B.
      • Piater L.
      Innate immunity in plants and animals: striking similarities and obvious differences.
      ,
      • Chisholm S.T.
      • Coaker G.
      • Day B.
      • Staskawicz B.J.
      Host-microbe interactions: shaping the evolution of the plant immune response.
      ).
      Our results suggested that the protease activity of AsES is essential for its elicitor function in strawberry plants. However, elicitor activity of another hydrolytic enzyme as the Trichoderma viride endoxylanase, a PAMP that elicits hypersensitive response, ethylene, and phytoalexin production in tobacco and tomato, was found to be independent of enzyme activity (
      • Sharon A.
      • Fuchs Y.
      • Anderson J.D.
      The elicitation of ethylene biosynthesis by a Trichoderma xylanase is not related to the cell wall degradation activity of the enzyme.
      ). Moreover, in few cases elicitor activity was found to be determined by small fragments of the intact elicitor molecule of PAMP, suggesting recognition of “epitope”-like structures by receptors at the plant cell surface (
      • de Wit P.J.
      Pathogen avirulence and plant resistance: a key role for recognition.
      ). Examples of the latter are an 8-amino acid glycopeptide fragment derived from yeast invertase (gp 8c) (
      • Basse C.W.
      • Fath A.
      • Boller T.
      High affinity binding of a glycopeptide elicitor to tomato cells and microsomal membranes and displacement by specific glycan suppressors.
      ) and an internal peptide of 13 amino acids (Pep-13) derived from 42-kDa cell wall glycoprotein of P. sojae that exhibits Ca2+-dependent transglutaminase activity (
      • Sacks W.
      • Nürnberger T.
      • Hahlbrock K.
      • Scheel D.
      Molecular characterization of nucleotide sequences encoding the extracellular glycoprotein elicitor from Phytophthora megasperma.
      ,
      • Brunner F.
      • Rosahl S.
      • Lee J.
      • Rudd J.J.
      • Geiler C.
      • Kauppinen S.
      • Rasmussen G.
      • Scheel D.
      • Nürnberger T.
      Pep-13, a plant defense inducing pathogen-associated pattern from Phytophthora transglutaminases.
      ).
      Defense inducing activity was detected in some proteases derived from both bacterial and fungal pathogens (
      • Chisholm S.T.
      • Coaker G.
      • Day B.
      • Staskawicz B.J.
      Host-microbe interactions: shaping the evolution of the plant immune response.
      ). In phytopathogenic bacteria of the genera Pseudomonas, Xanthomonas, Ralstonia, and Yersinia, four families of cysteine proteases (i.e. XopD, YopJ, YopT, and AvrRpt2), including many “effector” proteins, have been identified (
      • Mudgett M.B.
      New insights to the function of phytopathogenic bacterial type III effectors in plants.
      ). In fungi, however, a single group of proteases with defense eliciting activity was identified. Cultivar-specific Avr-Pita genes have been cloned and characterized from Magnaporthe oryzae (formerly known as Magnaporthe grisea) that is the causal agent of blast rice. Avr-Pita gene encodes a secreted 223-amino acid preprotein with homology to fungal zinc-dependent neutral metalloproteases that is further processed into an active 176-amino acid mature protein (
      • Valent B.
      • Chumley F.G.
      ). Point mutations in the putative protease catalytic residues of Avr-Pita176 resulted in gain of virulence on rice cultivars carrying the cognate resistance gene Pi-ta, although a direct biochemical evidence for protease activity of Avr-Pita is still missing (
      • de Wit P.J.
      Pathogen avirulence and plant resistance: a key role for recognition.
      ,
      • Orbach M.J.
      • Farrall L.
      • Sweigard J.A.
      • Chumley F.G.
      • Valent B.
      A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta.
      ). Furthermore, mutations in the putative catalytic residues of AvrBsT, a bacterial avirulence effector that displays structural similarity to the YopJ family of cysteine proteases, also abolished avirulence activity (
      • Orth K.
      • Xu Z.
      • Mudgett M.B.
      • Bao Z.Q.
      • Palmer L.E.
      • Bliska J.B.
      • Mangel W.F.
      • Staskawicz B.
      • Dixon J.E.
      Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease.
      ). We therefore hypothesized that AsES may function by releasing an active elicitor from a plant precursor molecule rather than being itself an elicitor. The latter has been described for the gene avrD from Pseudomonas syringae pv. tomato, whose product is responsible for the synthesis of syringolide elicitors (
      • Keen N.T.
      • Tamaki S.
      • Kobayashi D.
      • Gerhold D.
      • Stayton M.
      • Shen H.
      • Gold S.
      • Lorang J.
      • Thordal-Christensen H.
      • Dahlbeck D.
      • Staskawicz B.
      Bacteria expressing avirulence gene D produce a specific elicitor of the soybean hypersensitive reaction.
      ). Furthermore, some peptides that are considered elicitors, such as systemin, HypSys (hydroxyproline-containing glycopeptides), and RALF (rapid alkalinization inducing factor), would apparently come from protein precursors constitutively present in plant cell wall or cytoplasm and would be activated by microbial proteases or by intracellular proteases upon cell injury, acting as elicitors of the DAMP-type (
      • Yamaguchi Y.
      • Huffaker A.
      Endogenous peptide elicitors in higher plants.
      ).
      Because the elicitor activity was only found in AsES from SS71 A. strictum and not was found in a homologous subtilisin as proteinase K, we conclude that the proteolytic activity is necessary but not sufficient to induce defense, and we suggest that AsES might induce defense by means of proteolysis of one or multiple host proteins that are specific targets of this protease.
      Although the results presented in this paper confirm that the proteolytic activity exhibited by the AsES elicitor is required for the induction of the defense response in plants, further experiments using recombinant AsES protein, site-directed mutants, or synthetic (PAMP-like) peptides derived from AsES are necessary to elucidate its action mechanism and the resistance-causing signal perception process.
      In conclusion, we have purified and characterized AsES, a novel extracellular elicitor protein produced by the pathogenic fungi A. strictum, which exhibits defense inducing activity on its strawberry host Fragaria × ananassa and other nonhost species A. thaliana, and in vitro subtilisin-like proteolytic activity. AsES can be considered a new member of the fungal protein effectors. Despite the variety of examples showing that fungal proteases are involved in plant defenses, no subtilisin-like protease from a phytopathogen has been reported so far. This discovery is relevant in the plant-pathogen interaction knowledge and may contribute to envisioning possible strategies for controlling diseases in the field.

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