Ethylene-regulated gene expression: Molecular cloning of the ... - PNAS

Proc. Natl. Acad. Sci. USA Vol. 83, pp. 6820-6824, September 1986 Botany

Ethylene-regulated gene expression: Molecular cloning of the genes encoding an endochitinase from Phaseolus vulgaris (phytohormone/cDNA clones)

KAREN E. BROGLIE*, JOHN J. GAYNORt, AND RICHARD M. BROGLIE* Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399

Communicated by Charles J. Arntzen, May 27, 1986

they subsequently purified to homogeneity. The purified Mr -30,000 protein was found to exhibit both chitinase and lysozyme activities. As a prelude to investigating the molecular mechanisms responsible for regulating chitinase gene expression, we have constructed a cDNA library complementary to mRNA isolated from ethylene-treated bean seedlings.. Here, we report the identification and complete nucleotide sequence of a full-length chitinase cDNA clone. This information has allowed us to deduce the pritnary structure of the chitinase polypeptide and its amino-terminal signal peptide. Using this clone as a hybridization probe, we present evidence that ethylene treatment results in an increase in steady-state chitinase mRNA levels, indicating that ethylene control of chitinase gene expression occurs at the level of gene transcription.

A full-length copy of bean leaf chitinase ABSTRACT mRNA has been cloned. The 1146-base-pair insert of pCH18 encodes the 27-residue amino-terminal signal peptide of the precursor and 301 residues of the mature protein. Utilizing pCH18 as a hybridization probe, we have shown that the increase in translatable chitinase mRNA seen upon ethylene treatment of bean seedlings is due to a 75- to 100-fold increase in steady-state mRNA levels. Southern blot analysis of bean genomic DNA revealed that chitinase is encoded by a small, multigene family consisting of approximately four members. From our nucleotide sequence analysis of five additional chitinase cDNA clones, it appears that at least two of these genes are expressed. Three of the bean chitinase genes have been isolated from a Sau3A genomic library and partially characterized.

The development of disease resistance in higher plants is manifested by the accumulation of a number of host-synthesized polypeptides that are produced in response to pathogen attack. Among these are the following: (i) enzymes involved in the synthesis of phytoalexins (secondary metabolites that are toxic to bacteria and fungi) (1), (ii) enzymes leading to the formation of physical barriers to fungal invasion through modifications of the plant cell wall (2), (iii) inhibitors of serine endoproteases (2, 3), and (iv) lytic enzymes (e.g., chitinase and ,-1,3-glucanase) that are capable of degrading fungal cell walls (4, 5). While inhibitor studies indicate that host RNA and protein synthesis are required for the induction of these proteins (6), there is a paucity of information concerning the regulation and expression of the genes involved. The activities of several of these enzymes (2, 4, 7) can be increased by exposure to exogenous ethylene. Increased synthesis of this phytohormone has been associated with various kinds of plant stresses including mechanical wounding (8) and infection by bacteria and fungi (9, 10). These observations have led to the suggestion that ethylene may mediate the host response to pathogen attack (2, 4, 5, 11). Because of its potential role in plant defense, we have undertaken an investigation of ethylene regulation of gene expression in higher plants. As a model system for these studies, we have selected the enzyme chitinase (EC 3.2.1.14). Chitinase is a basic protein that catalyzes the hydrolysis of the P-1,4 linkages of N-acetyl-D-glucosamine polymers of chitin, a major component of fungal cell walls. Since higher plants do not contain a substrate for this enzyme, it has been proposed that chitinase functions as a defense against chitincontaining pathogens (4, 5). Chitinase activity has been detected in both woody and herbaceous plants including a number of important crop species (4). Boller et al. (4) have shown that the major (95%) ethylene-induced chitinolytic activity in bean leaves is attributable to an endochitinase that

MATERIALS AND METHODS Reagents and Enzymes. L-(2-Aminoethoxyvinyl)glycine was a generous gift from Hoffmann-La Roche Co. Deoxyand dideoxyribonucleotides were purchased from P-L Biochemicals. Reverse transcriptase from avian myeloblastosis virus was obtained from Life Sciences (St. Petersburg, FL). M13 pentadecamer sequencing primer was purchased from New England Biolabs. DNase I was from Cooper Biochemicals (Malvern, PA). Restriction endonucleases and DNA modifying enzymes were obtained from Bethesda Research Laboratories, New England Biolabs, or Boehringer Mannheim. Plant Growth Conditions. Seeds of Phaseolus vulgaris L. cv. Saxa were purchased from Samen Mauser, Zurich, Switzerland. Plants were grown in controlled environmental chambers with a day/night photoperiod of 16 (22°C):8 (18°C) and used 7-10 days after imbibition of dry seeds. Ethylene treatment was achieved by spraying with a solution of ethephon (1 mg/ml) (2-chlorethylphosphonic acid; Sigma) and enclosing the plants in plastic bags. Similar results have been obtained by exposing plants to 10 ppm gaseous ethylene (unpublished results). Preparation of Chitinase Antibodies, Chitinase was purified from ethylene-treated leaves by affinity chromatography (14) and applied to preparative NaDodSO4 slab gels. The 30-kDa band was excised, and the protein was eluted by electrodialysis. Antibodies to purified chitinase were raised in rabbits (12). Protein Analysis. Soluble protein was extracted (12) and analyzed by two-dimensional polyacrylamide gel electrophoresis using nonequilibrium pH gradient electrophoresis (13)in Abbreviations: kb, kilobase(s); bp, base pair(s). *Present address: The E. I. du Pont de Nemours & Co., Inc., Central Research and Development Department, Experimental Station, Wilmington, DE 19898. tPresent address: Department of Botany, Rutgers University, Newark, NJ 07102.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 83 (1986)

the first dimension and 10% NaDodSO4/polyacrylamide in the second dimension (12). Immunoblots were by the method of Blake et al. (14) at an antibody concentration of 85 ,pg/ml. RNA Isolation. Poly(A)+ RNA was isolated as described (15) and translated using a wheat germ cell-free system. In vitro synthesized polypeptides were analyzed by polyacrylamide gel electrophoresis and fluorography (16). Immunoprecipitation of translation products was performed with anti-chitinase IgG. Construction and Screening of a Bean cDNA Library. Double-stranded cDNA was synthesized from poly(A)+ RNA (17) and cloned into the EcoRI site of Xgtll (18). The recombinant DNA molecules were packaged in vitro and used to infect Escherichia coli RY1088. cDNA clones corresponding to ethylene-induced mRNAs were identified using a combination of differential plaque hybridization (19) and hybridization to size-fractionated mRNA (20). Chitinase cDNA clones were identified by hybridization-selection and translation (21). DNA Sequence Analysis. Chitinase cDNA inserts were excised by EcoRI digestion and inserted into pEMBL8' (22). Two different approaches were employed to determine the nucleotide sequence of bean chitinase cDNA. Progressive deletions were constructed as described by Barnes et al. (23) and sequenced using the dideoxy method (24). For chitinase cDNA clones (pCH6, pCH18, pCH20, and pCH28) restriction enzyme fragments, generated by Taq I and HindII digestions, were inserted into the M13 vectors mplO and mpll and sequenced by the dideoxy method. In all cases, the sequence of complementary strands was determined. Amino Acid Sequence Analysis. A partial amino-terminal amino acid sequence was determined by subjecting 3 nmol of gel-purified chitinase to automated Edman degradation in a Beckman Model 890B Sequence Analyzer using the Dilute Quadrol program. The phenylthiohydantoin derivatives from each cycle were identified by HPLC (Hewlett-Packard Model 1084A). Isolation of Chitinase Genomic Clones. Total DNA was isolated from etiolated leaves (25) and purified by two cycles of CsCl/ethidium bromide density gradient centrifugation. Purified DNA was partially digested with Sau3A and fragments 10-20 kilobases (kb) long were cloned in XEMBL4 (26). The bean genomic library (5 x 105 recombinant phage) was screened for chitinase genomic sequences using nicktranslated (26) cDNA clone pCH18. RNA and DNA Filter Hybridizations. RNA was denatured with glyoxal, separated by electrophoresis on a 1% agarose H

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gel, and blotted onto nitrocellulose filters (27). DNA samples were digested with the appropriate restriction enzyme, fractionated by agarose gel electrophoresis, and transferred to nitrocellulose filters (28). Filters were hybridized with nicktranslated probes and washed as described (25).

RESULTS Chitinase activity in bean seedlings is increased 30-fold following exposure to exogenous ethylene (4, 5). When soluble leaf proteins are analyzed by two-dimensional polyacrylamide gel electrophoresis, approximately 20-25 additional polypeptides are found to accumulate to stainable levels with ethephon treatment. By immunological methods, at least two of these have been identified as isoelectric forms of chitinase (Fig. iB). In vitro translation of mRNA extracted from treated and untreated plants reveals a translation product, precipitated by anti-chitinase IgG and observed only after ethephon treatment (Fig. 2, lane 4). No chitinase polypeptide can be detected in the translation products from control mRNA (Fig. 2, lane 2). Thus, the elevation of chitinase activity, observed in earlier studies, is reflected at the mRNA level, where an increased amount of translatable chitinase mRNA directs the synthesis and accumulation of the polypeptide following ethylene treatment. To examine more closely the ethylene effect on chitinase gene expression, we constructed a cDNA library using poly(A)+ RNA from ethephon-treated leaves as template. cDNA clones corresponding to mRNAs expressed after ethephon treatment were selected by a combination of differential plaque hybridization and hybridization to an enriched mRNA fraction (19). Using these procedures, two cDNA clones (pCH4 and pCH6) were isolated that, upon hybrid-selection, yielded a 35-kDa translation product that was precipitated by chitinase antiserum (Fig. 2). The 850base-pair (bp) EcoRI insert of one of these recombinants, pCH6, was nick-translated and used as a probe to rescreen the cDNA library. This second screen produced four additional clones harboring chitinase cDNA inserts (pCH18, pCH20, pCH22, and pCH28). The six recombinant clones were found to contain chitinase cDNA inserts ranging in size from 750 to 1200 bp in length. The nucleotide sequence of a full-length cDNA clone (pCH18) shows that the 1146-bp insert contains a short, A+T-rich segment (33 bases) of the 5'-untranslated region and a 984-nucleotide open reading frame that terminates 115 nucleotides from the poly(A) tail (Fig. 3). The TGA termi* OH-

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FIG. 1. Two-dimensional profiles of total soluble leaf protein extracted from control (A) and ethephon-treated (B) bean seedlings. The positions of molecular size standards in kDa are indicated in the vertical axis at the left. The two major isoelectric forms of chitinase (indicated by the arrows in B) were identified by immunoblot analysis as described (14) using an IgG concentration of 85 /.g/ml.

polypeptide (pCh) and identification of cDNA clone pCH6.

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contains an open reading frame of 935 nucleotides and possesses 115 nucleotides of 3'-untranslated sequence. Deduced Amino Acid Sequence of Chitinase. The 984-bp open reading frame of pCH18 encodes a protein of Mr 35,400, a size consistent with that observed upon NaDodSO4/polyacrylamide gel electrophoresis of the in vitro translation product (Fig. 2). This value is larger than that determined for the isolated protein (4) and suggests that chitinase is initially synthesized as a larger precursor. A partial amino acid sequence of 30 residues has been obtained from the amino terminus of bean chitinase (30). By comparing the deduced amino acid sequence of pCH18 with data obtained from the purified protein, we have localized the amino terminus of mature chitinase to the 28th residue of the precursor polypeptide. pCH18, therefore, encodes the entire 301-amino acid residues of the mature protein and a 27residue amino-terminal sequence (Fig. 3). This leader sequence displays the primary structural properties characteristic of other eukaryotic signal sequences (31, 32). Although the biosynthetic pathway of chitinase has yet to be elucidated, the presence of a signal sequence suggests its synthesis on membrane-bound ribosomes. Similar results have been obtained for two protease inhibitors that accumulate in vacuoles of tomato leaves upon wounding (3). The deduced amino acid sequence of pCH18 was found to differ from pCH4 by six nucleotide changes in the open reading frame. At residue 141 an alanine is exchanged for threonine while at residues 61 and 293 there are conservative replacements of methionine with valine and of leucine with phenylalanine, respectively. The changes at residues 33 and 34 involve substitution of a neutral amino acid with a basic and an acidic residue, respectively. Despite the amino acid differences, the net charge of the two polypeptides is not altered. The remaining nucleotide changes in the open reading frame reside in the third base of the codon and are silent.

FIG. 2. Immunoprecipitation of chitinase precursor

kDa

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Proc. Natl. Acad. Sci. USA 83 (1986)

Botany: Broglie et A

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Lanes 1 and 3, translation profile of poly(A)+ RNA isolated from control and ethephontreated plants, respectively. immunoselectLanes 2 and 4,products. Lane ed translation of mRNA translation 5, profile hybrid-selected by 10 gg of pCH6 bound to nitrocellulose filters (21). Lane 6, immunoprecipitation of hybrid-selected translation products. The

positions of molecular size

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standards are indicated.

nation codon, at position 1018 is repeated again 54 residues downstream. Three hexanucleotide consensus sequences (29) for polyadenylylation (AAUAAA) are present in the 3'-untranslated region. Two of these sequences at positions 1038 and 1042 overlap, while the third is found at nucleotide 1113. Both of these potential poly(A) addition sites are used in bean chitinase mRNA. Five of the six analyzed clones (pCH4, pCH6, pCH18, pCH20, and pCH28) employ the most downstream site, while the remaining clone (pCH22) uses the more upstream site. DNA sequence analysis of the chitinase cDNA clones indicates that they are derived from two different chitinase mRNAs. Five clones (pCH4, pCH6, pCH20, pCH22, and pCH28) have identical DNA sequences and differ from the remaining clone, pCH18, by 16 nucleotides. Of these differences, 12 occur within the open reading frame. While pCH18 is a full-length clone, the largest insert representing the first mRNA species is 1060 bp long (pCH4). This cDNA insert CACCTrATCATTTAGAGGAAAAGAGAGAGAGAA

-10 -20 -27 met lys lys asn arg met met met met ile trp ser val gly val val trp mt lu leu leu ATG AAG AAG MAT AGG ATG AT ATG ATG ATA TOG AGC GTA GGA GTG GTG TGG ATG CTO TIM TTG

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val gly gly mer tyr glyVglu gin cys gly arg gin ala gly gly ala leu cys pro gly gly asn cys cys ser gin phe gly trp cys GTT GGA GGA AGC TAC GGA GAG CAG TGT GGA AGG CAA GCA GGA GGT GCA CTC TGC CCA GGG GGC AAC TGT TGC AGC CAM TTC GGG TOG TGC

50 40 30 gly ser thr thr asp tyr cys gly pro gly cym gin ser gin cys gly gly pro ser pro ala pro thr asp leu ser ala leu iie mer GGC TCC ACC ACC GAC TAC TGC GGC W GGT TGC CAG AGC CAG TGC GGG GGA CCG TCT CCT GCT CCT ACT GAT CTC AGC GCC CTC ATA TCC

80 70 60 phe asp gln met leu lys his arg asn asp gly ala cys pro ala lys gly phe tyr thr tyr asp ala ph. ile ala ala ACC TAC TAC GAT GCC TTC ATC GCC GCC CCA GCC GGC TTC AAC GCC GAC GGA TGC AM AGG TCC ACC TTC GAC CMG ATG CTC AAM CAT CGC arg ser thr

110 100 90 ala lys ala tyr pro mer phe gly asn thr gly asp thr ala thr arg lys arg glu ile ala ala phe leu gly gln thr ser his glu GCC AAG GCT TAC CCC AGC TTC GGA AAC ACC GGA GAC ACG GCC ACT CGC AAG AGG GAG ATT GCG GCC TTC TTG GGG CAM ACG TCT CAC GAA 140 130 120 thr thr gly gly trp ala thr ala pro asp gly pro tyr ala trp gly tyr cys phe val arg glu arg asn pro ser thr tyr cys ser TOO GCC ACT GCG CCC GAC GOA CCA TAC GCA TGG GGA TAC TGC TTC GTG AGO GAG CGG MC CCC AGI ACG TAC TGC TCC

ACA ACC GGG GGA

170 160 150 ala thr pro gin ph. pro cys ala pro gly gin gin tyr tyr gly arg gly pro ile gin He ser trp asn tyr asn tyr gly gin cys GCC ACT CCC CAG TTC CCC TGC CCC CCT 0G0 CAG CAG TAC TAC GGC AGG GOT CCC ATC CAG ATA TCC TGG MC TAC MC TAI GOT CMG TGC 200 190 180 gly arg ala ile gly val asp leu leu asn lys pro asp leu val ala thr asp ser val ile ser phe lys ser ala leu trp ph. trp TCC CTC TOG TTC TGO TCT GTC ATC TCC TTC AAG CCC MA GTC ACT GAC CCT CAT CTA MC CCC CTC OTT GAC TTt GGA AGM GCC ATT GGG 220

210

met thr ala gln ser pro lym pro ser ser his asp val ile thr ser arg trp thr pro ATS ACC CCA CAG TCC CCC AAG CCT TCC TCC CAC GAC GTC ATC ACC TCT CGA TGG ACC CCC

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230 ser ser ala asp val ala ala arg arg leu TCC TCT GCC GAC GTC 0CC GCC CGC CGO CTT

260 250 240 pro gly tyr gly thr val thr amn ile il- asn gly gly leu glu cys gly arg gly gin asp ser arg val gln asp *rg il gly ph. CCC GCC TAC GGC ACT MTG ACG MC ATC ATC MC GGA GGC CTG GAG TGC GGG CCA GGA CAG GAC AGC AGG GTT CAP GAC CCC ATC GGA TIC 280

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lys arg tyr cys asp l-u leu gly val gly tyr gly asn asn leu asp cys tyr ser gin thr pro phe gly asn ser leu leu leu TTC AM AGA TAC TGT OAT CTC CTT GGA GTC GGT TAT GGC MC AAC CTT GAC TGC TAC TCT CAG ACT CCA TTT GGA AAT TCA CTC TTAA CTC

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smr asp lou val thr mer gin OP TCT GMC CTT GT ACC TCT CMG TGA CACTG TCATCCCATCAGMTAAATAAACTCATMAGTCTOTOTrCACTITCGATCACAACTTTCTATA.CTTTCCcTCA A A .A A TCAATMAATCACTACTCTATMAAAAAAAAAAMA ccAc

FIG. 3. Nucleotide sequence of cDNA clone pCH18. The deduced amino acid residues are shown above the nucleotide triplets. Nucleotides in pCH18 that differ from the other five clones analyzed are indicated by (A) below the nucleotide; those changes that affect the amino acid codon are discussed in the text. Putative polyadenylylation signals are underlined. The alternative polyadenylylation site used by pCH22 is indicated by the arrow A.

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