Evidence for a third structural class of β-1,3-glucanase in tobacco

Plant Molecular Biology 15: 797-808, 1990. © 1990 Kluwer Academic Publishers. Printed in Belgium. 797

Evidence for a third structural class of p-l,3-glucanase in tobacco

George Payne, Eric Ward, Thomas Gaffney, Patricia Ahl Goy 1, Mary Moyer, Andrew Harper, Frederick Meins, Jr. 2 and John Ryals* CIBA-GEIGY Agricultural Biotechnology Research Unit, P.O. Box 12257, Research Triangle Park, NC, 27709, USA; ICIBA-GEIGY Limited, Agricultural Division, CH-4002 Basel, Switzerland; 2Friedrich Miescher-Institut, P.O. Box 2543, CH-4002 Basel, Switzerland (* author for correspondence)

Received 5 July 1990; accepted in revised form 23 August 1990

Key words: glucanase, cDNA cloning, Nicotiana tabacum, pathogenesis-related proteins, plant defense genes

Abstract

Glucan endo-l,3-fl-glucosidases (fl-l,3-glucanases) have been implicated in several developmental processes and they may also play a direct role in the plant's defense against fungal pathogens. In an effort to characterize the glucanase gene family, complementary DNA clones encoding an acidic form of fl-l,3-glucanase have been isolated from tobacco. The cDNA was expressed in E. coli and shown to encode a fl-1,3-glucanase activity. The protein sequence encoded by the cDNA was found to match the partial protein sequence of PR-35, a previously characterized fl-1,3-glucanase [29]. The protein encoded by the cDNA was purified from the extracellular fluid of TMV-infected tobacco leaves and found by immunological methods to correspond to glucanase PR-Q' [ 10]. From a detailed analysis of the cDNA it is clear that this glucanase represents a third structural class of enzyme which differs substantially from both the basic, vacuolar glucanase and the acidic, extracellular forms (PR-2, PR-N and PR-O). It has previously been demonstrated that the basic form of fl-1,3-glucanase is synthesized as a pre-pro-enzyme and upon maturation the 21 amino acid signal peptide and a 22 amino acid carboxy-terminal peptide are removed. This processing event has been proposed to be involved with the vacuolar localization of the enzyme. By comparing the deduced protein structure of PR-Q' to that of the basic form it is evident that this extracellular enzyme is missing the carboxy-terminal 22 amino acids. The role of a conserved phenylalanine-glycine dipeptide in the processing of glucanases and other pathogenesis-related proteins from tobacco is discussed.

Introduction

The physiological functions of glucan endo- 1,3-fl-glucosidases (fl- 1,3-glucanase; EC 3.2.1.39) in seed plants are not well understood. Presuma- bly, these enzymes are involved in the breakdown and turnover of fl-l,3-glucans which are found

in several plant structures including the wall of pollen tubes, endosperm cell walls, the end walls of sieve elements, and as a deposit in response to a number of mechanical or physiological stresses [9]. This involvement implicates the enzymes in developmental processes such as pollen tube growth, coleoptile development, regulation of

The nucleotide sequence data reported in this revision will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X54456.

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transport through vascular tissue, and cell-wall biosynthesis. Of particular interest is the observa- tion that glucanase and chitinase are coordinately induced in response to pathogen infection [31 ]. A direct role for glucanase in defense against pathogens has been proposed because many fungal pathogens contain/3-1,3-glucan in their cell-wall [32] and combinations of/%l,3-glucanase and chitinase have pronounced fungicidal activity in vitro [17]. It has also been suggested that glucanase might act indirectly by degrading polysaccharides to oligosaccharides which act to elicit the plant's defense system [3].

Two distinct classes of/~-l,3-glucanase have been identified in tobacco [29]. The best characterized are the basic isoforms, which are induced by either pathogen infection or ethylene treatment [ 19, 20, 31 ], exhibit developmental regulation [8], and appear to be localized primarily in the central vacuole of the cell [18, 29]. These enzymes are encoded by a small gene family and cDNA clones encoding three isoforms have been isolated [26]. The second class of//-1,3-glucanase, which in- cludes the pathogenesis-related proteins PR-2, PR-N, PR-O and PR-35 [12, 29, 30], are also induced by pathogens and appear to be localized predominantly in the extracellular compartment. These proteins have a low isoelectric point and are often referred to as acidic/%l,3-glucanases. The genes encoding this class of enzymes have not been described and detailed structural com- parisons and studies of gene regulation have not been possible.

In this report, we describe the isolation and characterization of cDNA clones encoding an acidic/~-l,3-glucanase from tobacco. This extracellular glucanase, designated PR-Q', corresponds to the previously described protein, PR-35 [29], and is induced by tobacco mosaic virus (TMV) infection. A detailed sequence comparison of PR-Q' , the vacuolar isoforms of [3-1,3-glucanase [26] and the the partial protein sequence of PR-36 and PR-37, two extracellular isoforms of /%l,3-glucanase [29] suggests that the glucanase family is composed of at least three structurally distinct classes.

Comparison of the carboxy terminal end of the

basic and acidic forms ofglucanase, chitinase and PR-1 cDNAs and proteins suggests a possible mechanism for processing the C-terminus of these proteins involving recognition and cleavage at a conserved phenylalanine-glycine dipeptide. This C-terminal processing ofglucanase has been suggested to be involved with subcellular localization [29]. We suggest that this type of mechanism may not be restricted to the glucanases, but may also apply to the other PR-proteins in tobacco.

Materials and methods

Plant material and virus infection

Plants of Nicotiana tabacum cv. Xanthi.nc were grown in a greenhouse and infected with the com- mon strain (U1) of tobacco mosaic virus as described previously [22].

Isolation of cDNA clones encoding the acidic glucanase

A cDNA library was prepared from RNA ex- tracted from tobacco leaves 5 days after infection with TMV. The library was screened by the differential hybridization technique essentially as described [22] using as a probe a tobacco basic /~-l,3-glucanase cDNA (pGL43 [26]). The cDNA inserts were subcloned into the Bluescript plasmid (Stratagene) and their sequence determined by dideoxy sequencing of double-stranded templates [ 11].

RNA and DNA blotting experiments

Total RNA was prepared from TMV-infected leaves [ 16] and northern blot analysis was performed as described [2]. Total DNA was ex- tracted as described [2] and Southern blots [24] were carried out using as a probe the acidic /%l,3-glucanase eDNA insert in the plasmid pBSGL6e. The hybridization and wash conditions for both Southern and Northern analysis were as described for genomic sequencing [5].

Protein purification

PR-proteins were partially purified as described previously [22]. Fraction 6 from DEAE-Sephacel chromatography contained a mixture of PR- gmajor, PR-Rminor, PR-P, PR-Q, PR-O and several minor components. This fraction was further purified by HPLC using a reverse-phase phenyl column. PR-Q' eluted from this column at 55 minutes and represents a minor protein in the fraction.

Peptides were generated by LysC digestion and purified by reverse-phase HPLC. Automated Edman degradations were performed using the Applied Biosystems 470A gas phase sequencer. PTH amino acids were identified using an Applied Biosystems 120A PTH analyzer. Details of the protein purification and protein sequencing are available upon request.

Subcloning of the glucanase cDNA into an E. coil expression vector

The glucanase cDNA was subcloned into pCIB3300, a derivative of pKK233-2 [1] in a two-step procedure. First, the 5' half of the cDNA was subcloned from pBSGL6e as a S m a I - H i n d l I I fragment in frame with the initiating ATG and then the 3' end of the cDNA was subcloned as Hind III fragment. To subclone the 5' half of the cDNA, pCIB3300 was digested with Nco I and the resulting 5' ends were filled in using the Klenow fragment of DNA polymerase I. Following heat inactivation of the polymerase, the vector was digestedwithHind III and purified. The Sma I-Hind III fragment from pBSGL6e was ligated into this vector and one plasmid in which the glucanase cDNA was subcloned in-frame with the initiating ATG was selected as pCIB3301, pCIB3301 was digested with Hind III and the 550 bp Hind III fragment containing the 3' end of the glucanase cDNA from pBSGL6e was ligated into this site. Two plasmids were selected, pCIB3302 and pCIB3303, which contain the Hind III fragment in either the sense or anti-sense orientation, respectively. For fl-1,3-

799

glucanase assays, these plasmids were trans- formed into the E. coli strain JM109 [33].

[3-1,3-glucanase enzyme assays

Overnight bacterial cultures were subcultured into 300ml of Luria broth (supplemented with 25 #g/ml ampicillin for plasmid-containing strains) and incubated at 37 °C. 1.2ml of 100 mM IPTG was added when the cultures reached a density approaching OD6o o = 1.0. Incubation was continued for eight hours. Cells were pelleted by centrifugation for 5 min at 10000 x g, washed once in one-fifth volume of 2 0 m M phosphate buffer (10mM Na2HPO4; 10 mM KH2PO4) , and resuspended in 5 ml of the 20 mM phosphate buffer. Bacterial extracts were prepared by sonication of the cell suspensions for 75 seconds with the microtip of a Branson sonifier and centrifugation of the sonicates for 15 min at 10 000 x g. The bacterial extracts were incubated at room temperature for 24 hours to inactivate any native E. coli enzymes which might act upon pro- ducts generated by a [3-1,3-glucanase. Extracts were assayed for [3-1,3-glucanase activity essentially by the procedure of Kombrink et al. [ 15]. Enzyme assay reaction mixtures contained, in 1.0 ml total volume, 100#1 bacterial extracts, 2.0 mg of the [3-1,3-glucan laminarin (Sigma), and 100 mM potassium acetate, pH 5.3. At timed intervals, 200 #1 of each reaction mixture was added to 3.0ml of a 0.5~o solution of the colorimetric reagent p-hydroxybenzoic acid hy- drazide (Sigma) and heated in boiling water for 6 minutes. After cooling on ice, the absorbance of each sample was read at 410 nm and compared with the absorbance of a control sample lacking enzyme.

Results

Isolation of a cDNA encoding an acidic protein with homology to [3-1,3-glucanase

We have shown previously that cDNA clones encoding acidic forms of chitinase could be iso-

800

lated using a differential hybridization strategy in which plaques were isolated based on their ability to bind a probe derived from the gene for the basic chitinase under low-, but not high-stringency hybridization conditions [22]. This strategy was used to isolate an acidic form of/3-1,3-glucanase from tobacco. A cDNA library was prepared using RNA isolated from TMV-infected leaves five days after infection. The library was screened at 50 °C in 6x SSC with the basic /~-l,3- glucanase cDNA, pGL43 [26]. Seven plaques were purified and four of these would not bind the probe at 65 °C, 0.2 M sodium chloride. These plaques were further purified, the cDNA inserts subcloned into Bluescript and partial DNA sequences determined. When the sequences were compared to the sequence of the basic/%l,3-glucanase cDNA by dot-matrix analysis, two of the clones, pBSGL5g and pBSGL6e, were found to

I I pBSGL5

pBSQL6 I I

m ~ . ~ pBSGLSb-12

100bp I

Fig. la.

be significantly homologous. The inserts from these clones were completely sequenced and found to be independent clones from the same mRNA. However, by dot-matrix analysis comparison with the basic fl-l,3-glucanase cDNA they appeared truncated at the 5' end. The library was rescreened under high stringency using the cDNA insert from pBSGL6e as a probe and six more clones were isolated. One of these, pBSGL5b-12, contained a cDNA extended by 90 bases on the 5' end (Fig. la). The sequence com- piled from pBSGL5b-12, pBSGL6e and pBSGL5g is shown in Fig. lb with the deduced amino acid sequence shown below the DNA sequence. The cDNA encodes a protein with 53 ~ identity to the basic, vacuolar form of/~-1,3- glucanase. Translation may initiate either at the ATG at position 25 or more 5' on the mRNA at an ATG that is not contained on the cDNA clone.

Fig. 1. DNA sequence and complexity of the acidic glucanase from tobacco. A. The cDNA clones pBSGL5g, pBSGL6e and pBSGL5b-12 are diagrammed with the 3' untranslated sequence ( ), the coding sequence (I-l) and the signal peptide (m) depicted. B (next page). The DNA sequence of the acidic glucanase cDNA is shown with the protein translation below. The arrow indicates the predicted start of the mature protein. The dotted lines indicate amino acid sequence of peptides derived from the PR-35 protein [29] and the solid line indicates the amino acid sequence of peptides determined in this study. C. Genomic blot of tobacco DNA using the cDNA from pBSGL6e as a

probe. Fig. lc.

801

10 30 50 70 90

CT~-A~TT~-1~-~-rA~cTTACAAATGGCACATTTAATTGTCACA~TGC~Ftt.T~CTTAGTGTACTTA~~C~T~A~~

Q F L F S L Q M A H L I V T L L L L S V L T L A T L D F T G A Q A

110 130 150 170 190

AGGAGTTTGTTA TTACCATCTC CAGCAGATGTTGTGTCG CTATGCAACCGAAACAACATTCGTAGGATGAGAATATATGAT

G V C Y G R Q G N G L P S P A D V V S L C N R N N I R R M R I ¥ D

210 230 250 270 290

C~CAGCCAACT~CCAACATTOAGCTCATGCTAGGTGTCCCGAATCCGGAC~~AGCCAAGCCA

P D Q P T L E A L R G S N I E L M L G V P N P D L E N V A A S Q A N

310 330 350 370 390 ,

A~TACTTGGG~AAAAcAATG~AGGAA~A~T~TC-AAGTrCAGGTATAT~TTGGAAATGAAGTT~TcC~TGAA~

A D T W V Q N N V R N Y G N V K F R Y I A V G N E V S P L N E N S

410 430 450 470 490

TAAGTATGTACCTGTCCTTCTCAACG~cATGCGAAACATTcAAACTGCCATATC1T~TGCTy3GTCTTGGAAACCAGAT~AAAGTCTCCA~A~

K Y V P V L L N A M R N I Q T A I S G A G L G N Q I K V S T A I E

5 2 . 9 510 530 550 570 590

ACTGGACTTACTAcAGACACTTCTC~T~CAT~AAATGGGAGATT~AAA~TGATGTT~GAC-AGTTTATAGAGC~AT~T~ C ~ ~ T C

T (3 L T T D T S P P S N G R F K D D V R Q F I E P I I N F L V T N R

4 1 . 8 A 6 9 . 5 610 630 650 670 690

GC'GCCC~.~x-x L ~ ~ C ~;~-~-L'ATCCTTAL.-x-*-L~AATAGC.AAA~TATTAAGCTTGAGTATGC.A~.~-~-~-~-rAC.AT C CTCTGAAGTTG~T

A P L L V N L ¥ P ¥ F A I A N N A D I K L E Y A L F T S S E V V V

710 730 750 770 790

A A A T G A T ACC'GAAA¢~.-~-~-~-~-~ATC~CATC'TTAGATGCCACATACTC'GGCC CTTGAAAAGGCTAGTGGCTC'GT~-~-L-~~ N D N G R G Y R N L F D A I L D A T Y S A L E K A S G S S L E I V

8 2 . 3 810 830 850 870 890

G T A T ~ ~ C T T .CAATTAACATCC.ATTGAC.AATGC~CTTATAACAACAACTTGATTAGTCACGTGAAGGGAG

V S E S G W P S A G A G Q L T S I D N A R T Y N N N L I S H V K G G

4 1 . 8 b 910 930 950 970 990

GGAGTCCCAAAAGC~CTTCCGGTCCAATAGAGACCTACG L • • i CGCTCTG~TGAAGATCAGAA~FaACCC'TGAAATTGAGAAGCk ~ ~ ~ rGGACTATT

S P K R P S G P I E T Y V F A L F D E D Q K D P E I E K H F G L F

65.7 1010 1030 1050 1070 1090

TTCAGcAAACATGC-AA~AAAGTA~CAGAT~Au~L~L~.AA~TAG~AA~CATTAATAGGAAT~Lu~r~C~rr~TATGAAGAGAA S A N M Q P K Y Q I S F N *

4 9 . 2 1110 1130 1150 1170 1190

AGTAGTCCATTGGC-ACTATGTACTGAAACTATATATCATGCTC-ATAAAGAAAGCAGTTATTACAATAATGAAACA~A~ AGCCATCAA

Fig. lb.

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However, based on translation initiation at position 25, the encoded protein has a predicted molecular weight of 37 862 Da. Comparison with other PR-proteins from tobacco and with the basic fl-l,3-glucanase suggests that there is a signal peptide of at least 24 amino acids which would be cleaved between the alanine and glutamine residues leaving an acidic protein with a calculated pI of 5.1 and a molecular mass of 34488 Da.

To determine the complexity of this gene family, the pBSGL6e cDNA was used as a probe in blot analysis of tobacco genomic DNA (Fig. 1C). There are no restriction sites for Bam HI, Sty I or BgllI within the cDNA sequence while Hind III cleaves roughly in the middle of the cDNA. The data are consistent with the interpretation that this acidic fl-l,3-glucanase gene family contains a relatively small number of members comprised of possibly two strongly hybridizing members and two weakly hybridizing members.

Expression of glucanase activity in E. coli

To determine whether the cDNA encoded a protein with fl-1,3-glucanase activity, the cDNA was expressed in E. coli and bacterial extracts were assayed for enzyme activity. The cDNA encoded in pBSGL6e is truncated at the 5' end but still encodes a protein two amino acid residues longer than the predicted size of the mature protein. This cDNA was subcloned into the E. coli expression vector, pKK233-2 [1] in such a way that one plasmid, pCIB3302 (Fig. 2A), contained the cDNA in the sense orientation and a second plasmid, pCIB3303, contained the first half of the cDNA in the sense orientation and the second half of the cDNA in the anti-sense orientation. The results offl-1,3-glucanase assays on bacterial extracts are shown in Fig. 2B. Extracts derived from bacteria containing the pCIB3302 plasmid had at least 10-fold higher enzyme activity than extracts from bacteria containing either the pCIB3303 plasmid or no plasmid.

i~Nc*I~ F--e,tl---iFEcoRI~ # . . . • •

M G L Q E F P G A O A /

~ I I I ~ , ~ / Hind NI E c o R y ~ ~ I ~

/ ~ / EH~n°dR Illr

30C

20(

10(3

I I I I '~"'0 40 60 80 I00 120

>- I -

I -

,< Z

.._1 (.9 i

i

TIME

Fig. 2. Expression of glucanase activity in E. coil A (top). The structure of the fl-l,3-glucanase expression plasmid, pCIB3302, is shown. The arrow, Ptrc, represents the position and direction of transcription from the trp/lac promoter [ 1]. rrnBT~T2 represents the position of ribosomal RNA termi- nation sites. The coding cDNA sequence is represented by the striped box. The fusion of the acidic glucanase cDNA and the trc promoter is shown above the plasmid. The coding sequence for the glucanase begins at the arrow. The mature protein is represented by italics. The Shine-Dalgarno sequence is boxed in. The recombinant protein has an extension of nine amino acids on the amino terminus, relative to the mature protein. B (bottom). fl-1,3-glucanase activity of bacterial extracts from strains containing the empty vector (o symbols), the pCIB3302 plasmid (A symbols) and the pCIB3303 plasmid ( I symbols) after induction by IPTG. fl-1,3-glucanase activity is expressed as nanomoles of glucose equivalents released from laminarin per milligram of protein

in the bacterial extracts.

Identification of the acidic glucanase proteOl

It has been demonstrated that the pathogenesis- related proteins PR-2, PR-N and PR-O have r-1,3-glucanase activity [ 12 ]. To find out whether pBSGL5b-12 encoded PR-2, PR-N or PR-O, several acidic glucanases were purified from TMV-infected tobacco leaves and partial amino acid sequences were determined. Neither PR-2, PR-N, or PR-O was found to correspond to the protein encoded by the isolated clone (data not shown); however, a minor protein fraction obtained in the purification did correspond to the protein encoded by pBSGL5b-12. Figure 3 shows the purification of several PR-proteins using reverse-phase HPLC on a phenyl column. The amino acid sequences of peptides derived from the protein eluting at 55 minutes (PR-Q') were determined and found to correspond to the protein sequence in Fig. lb. These peptides are underlined on the cDNA in Fig. 1 by a solid line. When the purified protein was electrophoresed on native polyacrylamide gels using partially purified

324

240

o (~0 156

a~

c~5

I~-Q I c

' ' g o ' 3 o ' ' 4 o ' ' ' '

TIME

Fig. 3. Purification and peptide sequence of the acidic glucanase protein. The DEAE-Sephacel Fraction 6 containing a mixture of PR-proteins was separated by HPLC using a reverse-phase phenyl column. Class 3 PR-proteins (PR-Q and PR-P) co-eluted at 44 minutes. Class 4 PR-proteins eluted at about 35 minutes. Class 5 PR-proteins (PR-Rmajo r and PR-Rminor) eluted at 39 and 41 minutes respectively. PR-O eluted at 51 minutes and PR-Q' eluted at 54 minutes.

The remaining minor peaks have not been identified.

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PR-proteins as standards the protein usually migrated between PR-P and PR-Q (data not shown), but there was some variability due to aggregation of the purified protein. However, it was clear that this protein was not PR-2, PR-N or PR-O, but instead represented a minor com- ponent in the PR-protein preparation. In immuno- blotting experiments with purified glucanase PR-O' and PR-Q' as standards, and using either antisera specific for PR-2, PR-N, PR-O and PR-Q' or antisera specific for PR-O', it was apparent that this protein corresponds to PR-Q' ([ 10, 12], Dr. Michel Legrand, personal commu- nication).

Expression of PR-Q' RNA in TMV-infected leaves

It has been shown previously that the PR-proteins accumulate in TMV-infected tissue [30, 4] and that this accumulation is coordinately regulated at the level of mRNA for PR-la, PR-lb, PR-lc, PR-P, PR-Q, PR-Rmajo r and PR-Rmino r [22]. Figure 4 shows the time course of PR-Q' mRNA accumulation in TMV-infected leaves as determined by northern blot analysis of total RNA. In order to differentiate the PR-Q' mRNA from related mRNA encoding other forms of glucanase, cDNA clones encoding PR-Q', PR-2 (E. Ward et al., submitted) or the basic glucanase (pGL43 [26]) were subcloned into Bluescript plasmids such that RNA could be synthesized in vitro.

Fig. 4. Time course of TMV induction of PR-Q' mRNA. Total RNA was isolated from TMV-infected tobacco leaves and PR-Q'-specific mRNA identified by probing northern blots with the pB SGL6e cDNA. The numbers represent days post inoculation with TMV. The right lane contains RNA

from buffer-treated control plants.

804

t 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 3 5 0 0 4 0 0 0

/ /

/

> i

/

O 0 0

500

I~ ~\\\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \"~ BASIC GLUCANASE

Fig. 5. Dot-matrix comparison of the PR-Q' e D N A to the sequence of the tobacco basic genomic clone (GL-A). The structures of the gene and e D N A are represented by a checkered bar for the signal peptide, a hatched bar for the coding sequence, an open

bar for the intron in the basic glucanase gene, and a stippled bar for 3'-untranslated sequence.

RNA was prepared using these plasmids and an aliquot of each included in RNA blots as controls to verify that the hybridization and wash conditions described were specific for PR-Q' tran- scripts (see Materials and methods). PR-Q' mRNA begins to accumulate one day after infection and accumulates to high levels 3 to 5 days after TMV infection. This accumulation is quali- tatively similar to the other PR-proteins in which the RNA begins to accumulate from very low levels within 3 days. However, expression of the PR-Q' RNA decreases earlier than for the other

PR-proteins. This result is also in qualitative agreement with protein data which shows unde- tectable levels of protein in healthy leaf tissue and accumulation in TMV-infected tissue (data not shown).

Homology between fl-l,3-glucanases in tobacco

The structure of the PR-Q' eDNA was compared with that of GL-A, a genomic clone of one of the basic isoforms offl-1,3-glucanase (Sperisen et al.,

1 50 I00

pR-01 QFLFSLQM kHLIVTLLLL SVLTLATLDF TGAQAGVCYG RQGNGLPSPA DWSLCNRNN IRRMRIYDPD QPTLEALRGS NIELMLGVPN PDLENVAASQ F~7 IANNLPSDQ DVINLY IYYPE TNVF Pr~ HAI~NLPSDQ DVINLY IYNPD TNVFNALRGS NIEIILDVPL DQLSL TDPS ~a$i¢ MSTSHKHNTP QMAAITLLGL LLVASSIDIA GAQSIGVCYG MLGNNLPNHW EVIQLYKSRN IGRLRLYDPN HGALQALKGS NIEVMLGLPN SDVKHIASGM

150 200

PR-O I ANADTWVQNN VRNYG NVKF RYIAVGNEVS PLNENSKYVP VLLNAMP.NIQ TAISGAGLGN QIKVSTAIET GLTTDTSPPS NGRFKDDVHQ FIEPIINFLV Pr~7 ANGWVQDN IINGFP YIAVGNEVS P NEI Pr~ R ANGWVQDN IIN FPDVK pAMQNVY NALAAAGLQD QIK B&$i¢ EHARWWVQKN VKDFWPDVKI KYIAVGNEIS PVTGTSYLTS FLTPAMVNIY KAIGEAGLGN NIKVSTSVDM TLIGNSYPPS QGSFRNDARW FVDPIVGFLR

250 300

~R-O I TNRAPLLV2qL ypYFAIANN ADIKLEYALF TSSEV~DN GRGYRNLFDA ILDATYSALE KASGSSLEIV VSESGWPSAG AGQLTSIDNA RTYNNNLISH Pr37 AGGPNVEII VSESGWPSEG NSAAT NLIDH Pr36 AGGQNVEII VSESGWPSEG NSAAT Basic DTKAPLLVNI YPYFSYSGNP GQISLPYSLF TAPNVVVQDG SRQYP~NLFDA MLDSVYAALE RSGGASVGIV VSESGWPSAG A~GATY DNA ATYLRNLIQH

350 373

PR-Q' VKGGSPKKPS GPIETYVFAL FDEDQKDPEI EKHFGLFSA/q MQPKYQISFN * Pr37 vK LFNM FDENQK HFGLFSPD QR YQLNFN Pr36 AM FDEDQKDPEI EK YQLNFN Basic AKEGSPRKP GPIETYIFAM FDENNKNPEL EKHFGLFSPN KQPKYNINFG VSGGVWDSSV ETNATASLVS EM*

Fig. 6. Alignment ofglucanase protein sequences. The top line indicates the deduced amino acid sequence for the PR-Q' protein. The peptide sequence for the PR-36 and PR-37 proteins [29] is shown below the PR-Q' sequence aligned for maximum homology. The bottom line of protein data is the deduced protein sequence from the tobacco basic protein encoded by the GL-A

gene.

submitted) by dot-matrix analysis (Fig. 5). This comparison demonstrates that the homology between the two proteins begins at the coding sequence of the mature proteins and continues with discrete segments of highly conserved sequence near the carboxy end of the molecules.

Van den Bulcke et al. [29] have reported the isolation and partial characterization of three acidic glucanases, PR-35, PR-36 and PR-37, from the intercellular fluid of P. syringae infected and salicylic acid treated tobacco leaves. A comparison of the amino acid sequence of peptides obtained from these proteins and the deduced amino acid sequences of PR-Q' and the basic /3-1,3-glucanase is shown in Fig. 6. The com-

805

parison indicates that the PR-Q' cDNA encodes a protein that corresponds to PR-35. Based on the percent of positions in the aligned sequences with identical amino acids, PR-36 and PR-37 are highly related proteins which share approximately 91 ~o identity, but exhibit only approximately 55 ~o identity with the basic t-1,3-glucanase. In further studies it has been demonstrated that PR-36 corresponds to the PR-2 protein and PR-37 corresponds to PR-O (E. Ward et aL, submitted). PR- Q' is structurally distinct from both the basic fl-l,3-glucanase (55~o identity) and the PR- 36/PR-37 group ( 5 4 - 5 9 ~ identity). Therefore, there are at least three structural classes of/3-1,3- glucanase in tobacco.

GL-A GL-B C-Terminal

PR-Q' C-Terminal

GLUCANASE

F SPNKQPKYNLNFGVSGGVWD S SVETNATASL I SEM* F SPNKQPKYNINFGVSGGVWDS SVETNATASLVSEM* FSPNKQPKYNiNF_GG

FSANMQPKYQISFN* YQISFN

Class 1 PR-P PR-Q

CHITINASE

PGDNLDCGNQRSFGNGLLVDTM* PGENLDCYNQRNFAQG* PGDNLDCYNQRNFGQG*

Basic PR-IA PR-IB PR-IC

PR-IA C-Term. PR-IB C-Term.

PR-I

YDPPGNFIGQRPFGDLEEQPFDSKLELPTDV* YDPPGNYRGESPY* YDPPGNVIGQSPY* YDPPGNVIGKSPY*

PPGNYRGESPY PPGNVIGQSPY

Fig. 7. Comparison of the carboxy terminus of acidic and basic glucanases, chitinases and PR-1. For the glucanases, the deduced protein encoded in two genomic clones is designated GL-A and GL-B. The C terminal sequence for GL-A and GL-B is derived from amino acid sequence analysis of purified protein [26]. The amino acid sequence for PR-Q' is deduced from the cDNA sequence from this study and the carboxy-terminal sequence is from the PR-35 protein [29]. For the chitinases, the deduced protein sequence for the tobacco class I cDNA was derived from Shinshi e t al. [25] and the sequence for PR-P and PR-Q is from Payne e t al. [22]. For PR-1 the deduced protein sequence for the basic clone is from Payne e t aL [21], the deduced protein sequence for PR-la, PR-lb and PR-lc were from our unpublished results and Pfitzner e t aL [23]. The C-terminal peptide data

was taken from Payne e t al. [21].

806

Carboxy-terminal differences

Comparison of the deduced amino acid sequences of PR-Q' and the basic form of ~-l,3-glucanase showed that the basic form was extended at the carboxy terminus by 22 residues (Fig. 7). Shinshi etal. [26] have shown that this extension is removed during the maturation of the enzyme. In contrast, PR-Q' does not undergo carboxy- terminal processing. The carboxy terminal end of the protein deduced from the cDNA and by sequencing the protein [29] are identical. It is striking that processing of the basic form occurs immediately after the amino acid pair phenylalanine-glycine (FG) and at the position corre- sponding to the end of the PR-Q' coding sequence. A similar alignment could be made for both chitinase and PR-1, in which the basic forms extend beyond the acidic forms by 6 and 18 amino acids, respectively. Furthermore, the three basic proteins had an FG pair at similar positions relative to the end of the acidic forms.

Discussion

Prior to this study, the primary sequence of a basic/~-l,3-glucanase had been established from cDNA clones and partial amino acid sequencing of the mature enzyme [26]. We have isolated an extracellular, acidic form of /%l,3-glucanase, PR-Q' , and complete cDNA clones encoding this protein. Comparison of sequences deduced from these cDNA clones with partial protein sequence indicates that there are at least three structural classes of ]%l,3-glucanase in tobacco. The first class consists of at least three basic proteins that are predominantly, if not exclusively, localized in the vacuole [ 13, 29]. The second class consists of extracellular, acidic proteins which appear to include the pathogenesis-related proteins PR-2 (PR-36), PR-N and PR-O (PR-37). The present study demonstrates that there is a third structural class of/%l,3-glucanase represented by PR-Q' (PR-35), which is also an extracellular, acidic protein. Based on the limited sequence available, it appears that the three classes are related by 50 to

60~o identity at the amino acid level. It is of particular interest that chitinases, which are induced under the same conditions as /~-l,3-glucanases, also fall into three distinct structural classes: a class of basic proteins localized in the vacuole and two classes of acidic proteins secreted into the extracellular space [3, 22, 27].

The existence of three classes of ~-l,3-glucanases with similar activities and discrete segments of structural identity complicates studies of function and regulation. For clarity, we propose, by analogy to the chitinases [27], the nomencla- ture: class I for vacuolar, basic glucanases, class II for extracellular, acidic/~-l,3-glucanases closely related to PR-2, PR-N and PR-O and class III for extracellular, acidic forms closely related to PR-Q' (PR-35).

The biological significance of different classes of/%l,3-glucanases is not clear./~-l,3-glucanase has been implicated in the induced response of plants to fungal infection [3, 4]. Mauch and Staehelin [18] have proposed that extracellular fi-l,3-glucanases have a defense function early in the infection process. They suggest that in combi- nation with the extracellular classes of chitinases the glucanases would have a direct fungicidal action on hyphae invading the extracellular spaces. These /~-l,3-glucanases could also act indirectly by releasing fungal elicitors which activate the chemical defenses of the host [3]. A l% 1,3-glucanase purified from soybean cotyledons has been shown to release elicitors from the cell walls of Phytophthora megasperma f.sp. glycinea which elicit synthesis of the phytoallexin glyceollin [ 14]. It is of particular interest that PR-Q' is 63 ~o identical at the amino acid level to the soybean elicitor-releasing enzyme [28] whereas class I and class II enzymes of tobacco are 55~o and 51 ~o identical, respectively.

It has been proposed that the vacuolar, class I enzymes function late in the infection process when cell breakage releases cellular contents into the extracellular compartment [18]. The class I enzymes might also act in a similar fashion as part of the constitutive defense system of the plant. These enzymes accumulate to high concentra- tions in the outermost, epidermal cells of lower

leaves even in uninfected plants [8, 13]. There- fore, wounding, which provides an addit ional entry route for fungi, would also release the enzymes into the wound region where they could act on the fungus.

The default pa thway for proteins in the endo- m e m b r a n e sys tem of plant cells appears to be secretion [6] and there is evidence that positive informat ion conta ined in the polypept ide is required to target the protein to the vacuole [7]. The striking correlat ion of the carboxy- terminal extension of the basic fo rm of/~-1,3-glucanase and vacuolar localization has led Van den Bulcke et al. [29] to suggest that these sequences may contain signals for vacuolar targeting. The vacuolar, class I /%l,3-glucanases of tobacco are synthesized as p roenzymes with an N-glyco- sylated carboxy- terminal extension which is lost during process ing [26]. The amino acids at the processing site, phenylalanine-glycine, are at the posi t ion where the amino acid sequence of the mature class I I I g lucanase ends (Fig. 7). This pair o f amino acids is also conserved at comparab le posi t ions in chit inase and PR-1 suggesting that these proteins, like class I f l- l ,3-glucanase, undergo process ing with the loss of a carboxy- terminal extension and that this dipeptide may be involved. The relat ionship between this form of process ing and targeting of proteins induced in response to pa thogens is an interesting area for further study.

Acknowledgements

We would like to thank Dr. Michel Legrand for his help in determining the identity of the acidic g lucanase as P R - Q ' . We would also like to thank J a m e s Ligon, T o m Luntz, Mike Koziel, and Mary-Del l Chilton for their careful review and commen t s on the manuscr ipt .

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Evidence for a third structural class of β-1,3-glucanase in tobacco

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