Identification of a New Pathogen-induced Member of the Subtilisin-like Processing Protease Family from Plants

Abstract

By using biochemical, immunological, and molecular strategies we have identified and cloned a cDNA encoding a protease from tomato (Lycopersicon esculentum) plants (P69B) that is part of a proteolytic system activated in the plant as a result of infection with citrus exocortis viroid. This new protease is closely related, in terms of amino acid sequence and structural organization, to the previously identified pathogenesis-related subtilisin-like protease (Tornero, P., Conejero, V., and Vera, P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6332–6337). The 745-residue amino acid sequence of P69B begins with a cleavable signal peptide, contains a prodomain and a 631-residue mature domain which is homologous to the catalytic modules of bacterial subtilisins and eukaryotic Kex2-like proteases. Within the catalytic domain, the essential Asp, His, and Ser residues that conform the catalytic triad of this family of proteases are conserved in P69B. Northern blot and reverse transcriptase-polymerase chain reaction analysis demonstrated widespread induced expression of the 2.5-kilobase hybridizing mRNA in plant tissues as a consequence of viroid infection. We propose that P69B is a member of a complex gene family of plant Kex2/subtilisin-like proteases presumably involved in a number of specific proteolytic events activated during pathogenesis in plants and that takes place in the extracellular matrix. By using biochemical, immunological, and molecular strategies we have identified and cloned a cDNA encoding a protease from tomato (Lycopersicon esculentum) plants (P69B) that is part of a proteolytic system activated in the plant as a result of infection with citrus exocortis viroid. This new protease is closely related, in terms of amino acid sequence and structural organization, to the previously identified pathogenesis-related subtilisin-like protease (Tornero, P., Conejero, V., and Vera, P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6332–6337). The 745-residue amino acid sequence of P69B begins with a cleavable signal peptide, contains a prodomain and a 631-residue mature domain which is homologous to the catalytic modules of bacterial subtilisins and eukaryotic Kex2-like proteases. Within the catalytic domain, the essential Asp, His, and Ser residues that conform the catalytic triad of this family of proteases are conserved in P69B. Northern blot and reverse transcriptase-polymerase chain reaction analysis demonstrated widespread induced expression of the 2.5-kilobase hybridizing mRNA in plant tissues as a consequence of viroid infection. We propose that P69B is a member of a complex gene family of plant Kex2/subtilisin-like proteases presumably involved in a number of specific proteolytic events activated during pathogenesis in plants and that takes place in the extracellular matrix. Infection of plants with pathogens results in the induction of numerous host-specific biochemical responses, some of which are critical for the ability of the plant to withstand diseases (1Dixon R.A. Lamb C.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1990; 41: 339-367Crossref Scopus (557) Google Scholar). Physiological and pathological studies have examined diseased plants in the hope of uncovering the cause(s) of the pathogen-induced distress, and there is a plethora of studies describing the dramatic effect of pathogen infection upon different aspects of plant metabolism and catabolic disturbances (2Fraser R.S.S. Biochemistry of Virus-infected Plants. John Wiley & Sons, Chichester, United Kingdom1989Google Scholar, 3Zaitlin M. Hull R. Annu. Rev. Plant Physiol. 1987; 38: 291-315Crossref Google Scholar, 4Goodman R.N Novacky A.J. The Hypersensitive Reaction in Plants to Pathogens. APS Press, St. Paul, MN1994Google Scholar). What is lacking, however, is a clear understanding of how different pathogens promote the often deleterious symptoms observed by switching on a common cascade of cellular events resulting in the disease syndrome and the accompanying resistant character to subsequent pathogenic attacks. Viroids are the smallest known plant infectious agents, made up of nude, circular, single-stranded RNA molecules of a few hundred nucleotides which do not code for any protein (5Diener T.O. The Viroids. Plenum, New York1987Google Scholar, 6Semancik J.S. Viroids and Viroid-like Pathogens. CRC Press, Boca Raton, FL1987Google Scholar), and thus they are an adequate model to analyze the physiological and molecular basis of plant responses to pathogen infection. This is more relevant if we consider that the viroid elicited responses resemble those resulting from infection by more complex type of pathogens or different kind of stresses (7Semancik J.S. Conejero V. Semancik J.S. Viroid and Viroid-like Pathogens. CRC Press, Boca Raton, FL1987: 72-120Google Scholar, 8Conejero V. Bellés J.M. Garcı́a-Breijo F. Garro R. Hernández-Yago J. Rodrigo I. Vera P. NATO ASI Ser. Ser. H Cell Biol. 1990; 14: 233-261Google Scholar). It has been shown previously (8Conejero V. Bellés J.M. Garcı́a-Breijo F. Garro R. Hernández-Yago J. Rodrigo I. Vera P. NATO ASI Ser. Ser. H Cell Biol. 1990; 14: 233-261Google Scholar) that plants infected with viroids produce de novo synthesis of a set of host-encoded proteins termed pathogenesis-related (PR) 1The abbreviations used arePRpathogenesis-related;RTreverse transcription;PCRpolymerase chain reaction;PAGEpolyacrylamide gel electrophoresis. proteins. The function of some PR proteins as hydrolytic enzymes (e.g. chitinases or β-1,3-glucanases) has been demonstrated in many plant species irrespective of the nature of the attacking pathogen, and they appear to play a role in the induced defense response of the plant to combat pathogens (9Bol J, F. Linthorst H.J.M. Cornelissen B.J.C. Annu. Rev. Phytopath. 1990; 28: 113-138Crossref Google Scholar, 10Bowles D.J. Annu. Rev. Biochem. 1990; 59: 873-907Crossref PubMed Scopus (866) Google Scholar). pathogenesis-related; reverse transcription; polymerase chain reaction; polyacrylamide gel electrophoresis. Protein degradation as well as protein processing and maturation are believed to be important events in the plant defense response (11Tornero P. Conejero V. Vera P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6332-6337Crossref PubMed Scopus (164) Google Scholar, 12Schaller A. Ryan C.A. Plant Mol. Biol. 1996; 31: 1073-1077Crossref PubMed Scopus (81) Google Scholar, 13Pautot V. Holzer F.M. Reisch B. Walling L.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9906-9910Crossref PubMed Scopus (118) Google Scholar). In this regard, one of the viroid-induced PR proteins has been identified as a protease that was termed PR-P69 (14Vera P. Conejero V. Plant Physiol. 1988; 87: 58-63Crossref PubMed Google Scholar, 15Vera P. Conejero V. Physiol. Mol. Plant Pathol. 1989; 34: 323-334Crossref Scopus (32) Google Scholar). Recent molecular cloning (11Tornero P. Conejero V. Vera P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6332-6337Crossref PubMed Scopus (164) Google Scholar) indicated that P69 is a protein structurally related to the yeast processing protease Kex2, the prototypic member of the eukaryotic subtilisin-like protease family. This new finding broadens our understanding of defense responses activated by microbes, and opens a new perspective to unravel how plants perceive pathogenic insults and activate signaling processes that results in a resistant character to subsequent pathogenic challenges. In this paper we have have explored the existence of additional proteolytic activities activated in tomato plants following viroid infection. We performed comparative chromatographic fractionation of cell homogenates and determined differences in proteolytic profiles between healthy and infected plants. Using specific antibodies raised against the previously identified P69 protease, we identified a distinct, but immunologically related P69-like proteinase, that accumulates also in infected tissues. These studies were followed by the cloning of an inducible gene encoding a new subtilisin-like proteinase member related to the previously identified P69 protease (11Tornero P. Conejero V. Vera P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6332-6337Crossref PubMed Scopus (164) Google Scholar). We also discuss possible functions for this inducible protease family during pathogenesis in plants. Conditions for growth of tomato plants (Lycopersicon esculentum), and method for inoculation with citrus exocortis viroid have been described (8Conejero V. Bellés J.M. Garcı́a-Breijo F. Garro R. Hernández-Yago J. Rodrigo I. Vera P. NATO ASI Ser. Ser. H Cell Biol. 1990; 14: 233-261Google Scholar). Tissues were harvested 4 weeks after inoculation and stored at −80 °C. Restriction enzymes and modification enzymes were obtained from Boehringer. DEAE Sepharose CL-6B, T7-sequencing, and Ready-to-go DNA labeling kits were obtained from Pharmacia. Radioactive compounds were from Amersham Corp. All other commonly available reagents were of analytical grade. All operations were carried out on ice or at 4 °C. Leaf tissue (10 g) showing symptoms of viroid infection was frozen in liquid nitrogen, thawed, and homogenized in a mortar and pestle with 30 ml of buffer A (50 mm Tris-HCl, pH 7.21, 2 mm dithiothreitol, 1 mmCaCl2, 1 mm MgCl2). The homogenate was filtered through cheesecloth and centrifuged at 15,000 ×g for 20 min. Supernatants were immediately used or stored at −70 °C. The supernatant was adjusted to 20% with and centrifuged at 15,000 for min. The supernatant was adjusted to with and centrifuged as described The resulting was in ml of buffer (50 mm Tris-HCl, pH and in in buffer A. The proteins were to a in buffer A. with the was with ml of a of in the were for proteolytic using as as described previously (14Vera P. Conejero V. Plant Physiol. 1988; 87: 58-63Crossref PubMed Google Scholar) and the protein by were carried out in polyacrylamide as described previously (14Vera P. Conejero V. Plant Physiol. 1988; 87: 58-63Crossref PubMed Google Scholar). were with used were and of proteins from was performed as described previously P. Conejero V. Physiol. Mol. Plant Pathol. 1989; 34: 323-334Crossref Scopus (32) Google Scholar). The were for as described previously using (11Tornero P. Conejero V. Vera P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6332-6337Crossref PubMed Scopus (164) Google Scholar) and the with and A cDNA was from mRNA from tomato P. P. Conejero V. Mol. 1993; Google Scholar). The was at as described by and Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) with a cDNA encoding the protein (11Tornero P. Conejero V. Vera P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6332-6337Crossref PubMed Scopus (164) Google Scholar), and were by P. P. Conejero V. Mol. 1993; Google Scholar). cDNA were from cloned and by using a polymerase DNA sequence analysis was performed on by the chain method M. Biochem. 1987; Scopus Google Scholar). of the cDNA cloned in were using an and were using and of the of J. P. 1987; Scopus Google Scholar). RNA was from different tomato plant tissues as described J. J. Biochem. 1987; PubMed Scopus Google Scholar). RNA gel blot of RNA were on and & of RNA was by of the gel to the the RNA were with a cDNA for the gene to DNA was from as described A.J. R.A. Plant The Scholar). DNA gel blot of DNA was with enzymes and in a gel and RNA and DNA gel were with the cDNA or with the of cDNA which was by using polymerase and of were at as described Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). cDNA of the and reverse were as described S. J.M. Plant 1996; Google Scholar). The (50 and were used to an of the in single-stranded cDNA from the different mRNA in a DNA was for 30 with of for 1 for 2 and for min. The DNA were in or with a cDNA The of to the closely related protease sequence was in that of DNA the cDNA as were carried out in of DNA the P69B to proteolytic activities induced in tomato plants upon infection with citrus exocortis tissue homogenates were by on 1 chromatographic profiles from tissue homogenates from healthy and citrus exocortis plants after with a of differences in proteolytic activities were observed between of was used as for the of proteolytic In profiles from plants 1 the of different of proteolytic were observed with the proteolytic from healthy plants 1 with the and at by of proteins in the differences in the protein In the of plants 1 the of a set of proteins to the previously identified inducible PR proteins P. Conejero V. Physiol. Mol. Plant Pathol. 1989; 34: 323-334Crossref Scopus (32) Google Scholar) was These proteins in the of proteolytic and the protein previously identified as a of proteolytic (11Tornero P. Conejero V. Vera P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6332-6337Crossref PubMed Scopus (164) Google Scholar, P. Conejero V. Plant Physiol. 1988; 87: 58-63Crossref PubMed Google P. Conejero V. Physiol. Mol. Plant Pathol. 1989; 34: 323-334Crossref Scopus (32) Google Scholar). of proteins in to from infected plants not differences with healthy in an additional protein which was not in the from healthy plants. The inducible nature and in molecular of the proteins in and to for between proteins in and and the proteins from healthy were to blot analysis using an This that the antibodies the protein in and from infected was observed in the from healthy plants. the chromatographic was on the of the the of the that the previously identified P69 proteinase in homogenates from plants (14Vera P. Conejero V. Plant Physiol. 1988; 87: 58-63Crossref PubMed Google Scholar) was in a of different with biochemical and but with different chromatographic of antibodies proteins in proteolytic and from infected plants or the from healthy plants. are shown on the on the of between we cloning of the new P69 member by with antibodies and also by at with the cDNA previously described (11Tornero P. Conejero V. Vera P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6332-6337Crossref PubMed Scopus (164) Google Scholar) as a in cDNA from of a cDNA from tomato plants P. P. Conejero V. Mol. 1993; Google Scholar) not any result of the cDNA with a cDNA a of cDNA Restriction and sequence analysis of cDNA indicated that of the previously identified P69 the cDNA a different of cDNA was and in The sequence and the amino acid sequence from the in are shown in The cDNA contains nucleotides the and RNA gel blot using the cDNA as a a of in that the cDNA is an The with an at that is by that the sequence conserved for in M. PubMed Scopus Google Scholar), and at the at of the The is by an the that the is that at The protein has a molecular of and a of amino which is that the proteins by antibodies This was by in of the cDNA in which the synthesis of an protein not the of the sequence not and of amino acid sequence of the indicated the existence of a This of a signal at the The as by 14: PubMed Scopus Google Scholar), of this signal after This is followed by a acid which is a of proteases of the family and for the proteolytic of the is an important in the of the protease from the A. J. Biol. PubMed Scopus Google Scholar). The amino acid of the mature protein is identified by with other plant subtilisin-like proteases (11Tornero P. Conejero V. Vera P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6332-6337Crossref PubMed Scopus (164) Google Scholar, J. Biol. PubMed Google Scholar, A. A. Plant PubMed Scopus Google Scholar), and is indicated in the mature contains amino with a molecular of The amino acid for this mature protein is with that determined previously for the P69 proteinase and this structural the observed of other using antibodies upon the of we to this identified P69 proteinase as the previously identified one be as The amino acid sequence of P69B with that of is in of the P69B with other subtilisin-like the sequence of the P69B is with sequence differences in the The and the amino the catalytic triad in the and the conserved are by in which are to those of P69B are indicated by were to of of P69B to the amino acid sequence the catalytic residues of Kex2, and The of the amino acid of the proteins is indicated by the to of to those of P69 are indicated by residues are indicated acid residues of protease are from the Within the mature P69B protein the amino acid residues and common to subtilisin-like were The the catalytic are to the catalytic triad essential for of the subtilisin-like to function as and with A and the protein has an that has been to be conserved in this and is important in the subtilisins PubMed Scopus Google Scholar, S. J. Biol. PubMed Google Scholar). the the amino of the there is an of a sequence amino between the and the to other subtilisin-like which are by This has also been observed in the other subtilisin-like identified from plants (11Tornero P. Conejero V. Vera P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6332-6337Crossref PubMed Scopus (164) Google Scholar, J. Biol. PubMed Google Scholar, A. A. Plant PubMed Scopus Google Scholar). The of a but at a of the enzymes from plants. The expression of P69B was determined in different tissues from healthy and plants by Northern blot The were at with the cDNA The results that the of P69B which has a of is in and in tissue from infected the mRNA from healthy plants a with the and after of the with the The that the observed in P69B gene expression in infected tissues be by with the mRNA for the previously identified gene was also an to that the expression of the P69B gene was induced in infected plants In single-stranded from mRNA of and from healthy or plants were by in a with to a and and specific for of P69B but not for the were used in to P69B gene an of the RNA from and tissues of tomato but not the RNA of healthy the The were also by blot analysis with a cDNA not a the P69B cDNA or cDNA were and the the P69B cDNA the This that the observed using and also that P69B is induced during The of in from healthy plants that the mRNA species observed in Northern from healthy plants expression of different subtilisin-like with other the existence of other in tomato more analysis was using DNA and cDNA DNA gel blot shown in was performed to the of of the A blot tomato DNA with enzymes was with the cDNA and demonstrated the of a number of and of different that P69 conform a family of This was also but at a a DNA was with a cDNA the the catalytic domain of the enzymes used in this the this result to that the P69 gene family is of at but closely related of which of and have been identified In this we structural and on a member of a family of plant proteases induced during the response of tomato plants to pathogen P69B a new plant subtilisin-like protease on amino acid sequence and structural PubMed Scopus Google Scholar, S. J. Biol. PubMed Google Scholar) which is related amino to the previously identified PR-P69 protease (11Tornero P. Conejero V. Vera P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6332-6337Crossref PubMed Scopus (164) Google Scholar) as The of P69B that the protein is as a of a acid signal peptide, a acid and a acid mature that is the of the that accumulates in Within the mature the amino acid and is the of P69B and are closely related to those of the catalytic of the subtilisin-like proteases. from analysis of the different with the conserved amino acid the catalytic we have P69B as a new member of the subtilisin-like family of In this of proteases is also to These are involved in the proteolytic processing of and other at of of amino acid residues PubMed Scopus Google Scholar). the of as the subtilisin-like protease for processing in the yeast A. J. Proc. Natl. Acad. Sci. U. S. A. 1989; PubMed Scopus Google Scholar), which in the processing of protein and protein were identified and and J. M. M. M. R. Proc. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar) for one of the that Kex2/subtilisin-like have also been and U. M. de J. Biol. Google Scholar, de J. Scopus Google Scholar, J.S. J. PubMed Google Scholar) with sequence to which sequence to Mol. 14: PubMed Scopus Google Scholar). In molecular of the gene from which was shown by analysis of to be essential for the of this has that also gene related to the Kex2/subtilisin-like protease M. PubMed Scopus Google Scholar). sequence analysis protein which have been and These proteins a common amino but at the and are from In was available to the existence of proteases A. Ryan C.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) a subtilisin-like protease was from J. Biol. PubMed Google Scholar). cDNA encoding subtilisin-like proteases have been identified in other plant species the previously identified from tomato plants (11Tornero P. Conejero V. Vera P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6332-6337Crossref PubMed Scopus (164) Google Scholar). a cDNA encoding a subtilisin-like protease was shown to be during of A. A. Plant PubMed Scopus Google Scholar). a gene encoding a closely related subtilisin-like protease has been identified and expression A. A. Plant PubMed Scopus Google Scholar). that the gene family has been conserved of plants. with the of expression observed in plants for the different of this family of the tomato plant are during analysis that many more are in the of tomato and that additional to be be at of an of the of and with the of related that through gene thus that subtilisin-like enzymes biochemical some of which are important during is an that a one to the of subtilisin-like proteases. In for subtilisin-like proteases have been identified PubMed Scopus Google Scholar, S. J. Biol. PubMed Google Scholar), but has been observed that the different are from and in many of proteases are to the This the of a the family in and how this be has been by the expression and of the which to be a function of the structural differences between family member J. PubMed Scopus Google Scholar). has been shown that in be by expression to tissues and also of the enzymes to specific R. J. A. M. Mol. PubMed Scopus Google Scholar), and is a for functions which to a that in plants for the specific of the different subtilisin-like or pathogenic or other the of this family of proteases plant subtilisin-like enzymes described have in common some that from the of other eukaryotic In as with the of plant the of a sequence amino between the conserved and the Ser residues of the catalytic to other subtilisin-like proteases. The of a conserved but important functions in the of this of subtilisin-like enzymes are as a which is believed to function as an the of the protease domain and from the is in the A. J. Biol. PubMed Scopus Google Scholar). in other eukaryotic the and the catalytic are by the to from the and have an additional conserved at the A. J. Biol. PubMed Scopus Google Scholar). In the of plant the at is the is This that the plant mature and also have with to the presumably as a to cellular through the In this regard, other eukaryotic function specific of the the plant enzymes function after to the of the This also for subtilisins from studies of the P69 protease P. Hernández-Yago J. Conejero V. Plant Physiol. 1989; PubMed Google Scholar) that the in the and with the biochemical that if not of the be in an the P. Hernández-Yago J. Conejero V. Plant Physiol. 1989; PubMed Google Scholar). the of of plant be the extracellular matrix. This more if we consider that the extracellular the cell has been to be part of the of the plant cell as by P. The of Plant Scholar). The that the P69 proteolytic system described is induced during pathogenesis in plants is also in with the of This with the extracellular of the mature new for the of possible of and a role of proteases to the of the plant cell with the extracellular is a of in that between extracellular proteases and important protein during processes related to signal as tissue or in disease Cell of Press, New Google Scholar, Cell Biol. PubMed Scopus Google Scholar). In of in specific proteases and of growth and other important molecules through the on specific In this we have identified a extracellular protein that is by the P69 proteolytic system in plants P. Conejero V. Vera P. Plant J. 1996; PubMed Scopus (81) Google Scholar). to the conserved family of proteins that molecular processes in the extracellular of eukaryotic to different signaling processes B. J. Biochem. Sci. PubMed Scopus Google Scholar). the physiological from the processing of this is that of the family are in proteolytic processes in the plant cell a Kex2-like protease has been to in the and processing of the signaling processes during response in plants A. Ryan C.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and thus of of plant subtilisin-like proteases in processing of important signaling proteolytic degradation of the extracellular by the of encoding proteolytic the subtilisin-like proteases that and be as a by which plant during different processes and in pathological and which a number of different cellular processes important for the of the we the that proteases also as the attacking pathogens or by proteins by the In encoding a or an protease are also activated and during the defense response of tomato plants A. Ryan C.A. Plant Mol. Biol. 1996; 31: 1073-1077Crossref PubMed Scopus (81) Google Scholar, 13Pautot V. Holzer F.M. Reisch B. Walling L.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9906-9910Crossref PubMed Scopus (118) Google Scholar). is that expression of encoding different protease, or with different common strategies by plants to against as a defense or through the modification of proteins that in the of defense the that a extracellular protease the of P69 proteases and related proteins I. Vera P. Conejero V. J. Biochem. 1989; PubMed Scopus Google Scholar, I. Vera P. Conejero V. Plant Physiol. PubMed Scopus Google Scholar), that a of between different as proteolytic enzymes or as for the of other in plants. This new of that be in the for a understanding of how is in plants. studies on the role of pathogen-induced extracellular proteases in as well as and cloning of additional family and of and are one of our for the