Defence-related gene activation during an incompatible interaction between Stagonospora (Septoria) nodorum and barley (Hordeum vulgare L.) coleoptile cells

Plant Molecular Biology 31:741-749 , 1996.

© 1996 KluwerAcademic Publishers. Printed in Belgium.

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Defence-re lated gene activation during an incompat ib le interact ion between Stagonospora (Septoria) nodorum and barley (Hordeum vulgare L.) coleopti le cells

Conrad Stevens',3, Elena Titarenko 1'4, John A. Hargreaves 2,* and Sarah J. Gurr I J Department of Plant Sciences, South Parks Road, University of Oxford, Oxford OX1 3RB, UK; 2 Cell Biology Department, IACR-Long Ashton Research Station, Universi~ of Bristol, Long Ashton, Bristol BS18 9 AF, UK (*author for correspondence); 3present address: Department of Biological Sciences, Wye College, University of London, W3'e, Ashford, Kent TN25 5AH, UK; 4present address: Centro Nacional De Biotechnologia, Conse]o Superior de Investigaciones Cientificas, Campus Universidad Autonoma, Cantoblanco, Madrid, Spain

Received 6 Februari 1996; accepted in revised form 17 april 1996

Key words: antifungal proteins, non-host resitance, pathogenesis-related protein, proteinase inhibitor, Septoria nodorum

Abstract

Two previously unidentified cDNA clones (bsil and bprl-1) were isolated by differential hybridization from a cDNA library of Stagonospora (Septoria) nodorum (Berk) Castellani & E.G. Germano (teleomorph Phaeosphaer- ia (Leptosphaeria) nodorum (E. Muller) Hedjaroude-challenged barley (Hordeum vulgare L.) coleoptiles, bsil encoded a cysteine-rich protein containing 89 amino acids (aa) with a relative molecular mass (Mr) of 9405. Protein sequence homologies showed that Bsil was very similar to an aluminium-induced protein from wheat and indicated that it was related to the Bowman-Birk-type proteinase inhibitors (BB-PIs). The predicted aa sequence of Bsil contained an N-terminal secretory signal sequence which implied that the protein was exported. The other clone, bprl-1, which was truncated at the 5 t end, encoded a type-1 pathogenesis-related (PR-1) protein. The complete sequence of bprl-1 was obtained after cloning a barley genomic DNA fragment and was shown to encode a basic protein containing 174 aa with a Mr of 18 859. The deduced aa sequence of bprl-1 contained both an N-terminal secretory signal sequence and a charged C-terminal extension. This latter sequence may represent a vacuolar targeting signal, bsil and bprl-I and four other defence-related genes (encoding 1,3-/3-glucanase, 3-hydroxy- 3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a homologue of a putative wheat peroxidase, and barley leaf-specific thionin), showed increased transcription levels in S. nodorum-challenged coleoptiles, although their pattern of accumulation varied after inoculation (a.i.). The potential role of these induced genes in defence against fungal attack is discussed.

Introduction

The inability of fungal pathogens to infect and col- onize non-host plants is associated with a variety of metabolic changes in challenged cells. Some of these changes, such as oxidative cross-linking of cell walt proteins [7], occur rapidly upon perception of the pathogen and do not appear to rely on induced gene

The nucleotide sequence data reported will appear in the EMBL Nucleotide Sequence Database under the accession numbers Z48728 (bprl- I) and Z48729 (bsil).

expression. Others, however, take longer to develop and are known to depend on transcriptional activation of specific sets of defence genes [10, 23]. In recent years, a number of genes have been found to be up- regulated in plant cells responding to fungal attack. Proteins encoded by these pathogen-induced genes include structural cell wall proteins [20], biosynthetic enzymes leading to the formation of antifungal sec- ondary metabolites [ 10] and pathogenesis-related (PR) proteins [24, 47]. This latter group represent a large and diverse collection of unrelated proteins which accumu-

742

late in plant cells in response to either stress or attack by a variety of microorganisms. Some PR proteins, such as 1,3-/%glucanases, chitinases and chitosanases, have hydrolic activities which may weaken the fungal cell wall and, thereby, lead to lysis of hyphae during penetration of the cell [ 14, 28]. Other PR proteins, including thionins [5, 6], PR-1 and type-5 PR proteins [29, 34, 51] and some proteinase inhibitors [26], have recog- nized antifungal properties and may, thus, contribute to the development of an inhibitory environment in the vicinity of penetrating hyphae.

Resistance to potential fungal pathogens, particularly in Graminaceous species [44], is often associated with structural modifications to the cell wall at the site of penetration. Polymeric material, including callose, lignin, polyphenolics and glycoproteins, is deposited on the inner surface of the cell wall beneath the penetration site and within the surrounding cell wall [22, 40]. In many cases, invading hyphae do not breach these modified cell walls and, thus, are prevented from gain- ing access to the plant cell. Understanding the molecular basis of the processes underlying this basic incompatible response will be an important step towards the development of transgenic crops able to resist infection by fungi. Previously, the interaction between a wheat- adapted biotype of S. nodorum and cereal coleoptile cells has been used to study cell wall modifications associated with defence against fungal attack [17, 18, 22]. Here we describe the isolation and identification of two previously unidentified barley genes and show that transcripts of these genes, along with those of four other defence-related genes, increased during the basic incompatible interaction between S. nodorum and barley coleoptile cells. A preliminary report of this work has recently appeared [49].

Materials and methods

Plant and fungal material

Barley (cv. Golden Promise) seeds were surface- sterilized (10% NaHC103 for 20 min), washed with sterile water and germinated on damp tissue paper laid on top of seed trays containing water-soaked vermi- culite. The trays were incubated at 25 °C in the dark for 5 days and then the etiolated seedlings exposed to light for 1 day prior to harvesting. The S. nodorum strain used in this work was isolated from infected wheat seed supplied by J.A. Hutcheon (IACR-Long Ashton Research Station, Bristol, UK). Cultures were

grown on CZ-V8 agar (200 ml Campbell's V8 juice, 33.4 g/1 Czapek-dox liquid medium (modified), 3 g/1 CaCO3 and 20 g/1 agar) under near UV light (Philips TL 8W/O8F8T5/BLB) at 22 °C for 6 days. Spores were suspended from the plates in sterile distilled water, adjusted to 106 spores/ml, then after adding 8.75% dimethyl sulfoxide snap-frozen in liquid nitrogen and stored at - 8 0 °C until required.

Inoculation technique

Sterile glass microscope slides were coated on one side with 2 ml molten minimal medium agar minus glucose [19]. When the agar had set, 200 #1 of spore sus- pension was spread over the surface of the agar. The slides were placed in a moist chamber and incubated overnight at 25 °C to allow the spores to germinate. Barley seedlings were excised at the base and then laid across a slide so that the coleoptiles were in contact with the germinating spores on the agar surface. A second slide was placed on top of the coleoptiles with the agar-coated surface facing downwards so that the coleoptiles were sandwiched between the agar sur- faces on which the spores had germinated. About 30 coleoptiles were infected per pair of glass slides. The emerging tips of the primary leaves were excised and the coleoptile-glass slide sandwiches placed in a moist chamber. Uninfected control coleoptiles were treated in a similar manner, except that they were placed between agar-coated slides which had not been seeded with S. nodorum spores. Sandwiched coleoptiles were incubated in the dark at 25 °C. Sites of attempted penetration and development of cell wall modifications were monitored by fluorescence microscopy as described by Hargreaves and Keon [17].

mRNA extraction and cDNA library construction

Coleoptile tissue was collected after removing the remnants of the primary leaf and total RNA isolated according to the method of Gurr and McPher- son [15]. RNA for construction of the cDNA library was extracted from S. nodorum-challenged coleoptiles between 16 and 24 h a.i. mRNA was purified from 500 #g total RNA by oligo(dT)-cellulose chromato- graphy as described by Sambrook et al. [42]. Pur- ified mRNA (2 #g) was used to synthesise cDNA using a cDNA Synthesis Kit (Pharmacia, Uppsala, Sweden) according to the manufacturers' instruc- tions. EcoRI/NotI adaptors were ligated to blunt-ended, double-stranded cDNA and the cDNA purified away

from excess adaptors by electrophoresis through a 2% low-melting temperature agarose gel (NuSieve, FMC, ME). DNA fragments (0.5-2.0 kb) were recovered from the agarose gel using a Qiagen DNA extraction kit. Purified cDNA (70 ng) was ligated to 1 #g EcoRI- predigested/phosphatased AZAPII arms (Stratagene, CA). The ligation reaction was incubated at 12 °C for 48 h and the ligation mixture packaged using a Gigapack II Gold packaging extract (Stratagene, CA). The resulting library consisted of 4.6 x 105 recombinant phage of which ca. 80% contained DNA inserts.

Plaque hybridization

Hybridization probes for differential screening employed 32p-labelled cDNA prepared from S. nodorum- challenged coleoptiles and from uninfected control tissue. Both sets of tissue were collected 16-24 h a.i. cDNA probes were prepared from 10 #g of total RNA essentially as described by Gurr and McPherson [15], with the exception that Moloney Murine Leuk- aemia Virus reverse transcriptase was used instead of Avian Myeloblastosis Virus reverse transcriptase. Duplicate plaque lifts were made using Hybond N nylon filters (Amersham, Bucks., UK). Hybridiza- tions were performed at 65 °C overnight in 250 mM (Na)zHPO4/NaHzPO4 buffer pH 7.0, 250 mM NaC1, 7% SDS, 1 mM EDTA and 10% PEG 6000. Fol- lowing hybridization, filters were washed at 65 °C sequentially with 3 / SSC, 0.1% SDS; 2x SSC, 0.1% SDS and 0.5 x SSC, 0.1% SDS. Selected clones were taken through two further rounds of differential hybridization, pBluescript phagemids were excised from )~ZAPII clones as described by the manufacturers.

Northern blot analysis

For northern blot analysis, total RNA was extracted from ca. 50 mg tissue and 10 #g purified RNA electro- phoresed through a 1.4% agarose gel containing 0.66M formaldehyde as described by Davis et al. [9]. Hybrid- izations were performed according to Gurr and McPh- erson [15]. Hybridization probes were either DNA fragments excised from plasmids containing the cloned gene (bsil, pET4; bprl-1, pET6; barley leaf-specific thionin, pHvDB4; wheat peroxidase (WIR3), pUW 13; 1,3-9 glucanase, p~13G), or in the case of HMG- CoA reductase, a PCR product amplified from barley genomic DNA using degenerate primers as described elsewhere [46]. The identity of the amplified DNA was confirmed by its size (ca. 450 bp) and by homo-

743

Figure I. Reaction sites on the surface of a S. nodorum-challenged barley coleoptile 48 h.a.i. Tissue was stained with toluidine blue O [17] (bar = 5 ~Lm).

logy of a partial DNA sequence to potato HMG-CoA reductase sequences. All other molecular biology techniques were as described by Sambrook et al. [421.

DNA sequence analysis

DNA sequencing was performed by the dideoxynucleotide- chain termination method [43] using a T7 Sequen- cing Kit (Pharmacia LKB, Upsalla, Sweden) and ct-[35S]-dATE All templates were alkali-denatured, double-stranded, plasmid DNA and both strands were sequenced by extension from either 17-met Mt3 forward and reverse primers or from 16-met specific primers. Translation of DNA sequences and other sequence analyses utilized the Staden programs (Staden DNA and Protein Analysis Sofware, Cambridge, UK). Searches of GenBank and EMBL DNA databases and of Swissprot, PIR, GenPept and Brookhaven Protein Data Bank were perlormed using either the BLAST [2] or the FASTA [37] programs. Sequences were aligned using the BESTFIT and PILEUP programs of the UWGCG package (Uni- versity of Wisconsin Genetics Computer Group soft- ware package, version 7.3).

744

18

38

58

78

~T~GC~C~TG~GGG~CCAAG~CGC~CGATTCTGATCCTCCAGGCCGTCCTGG 49 M K G T K L A A I L I L Q A V L V

T~T~GAGTCCT~CG~CGTG~CGCCGTCTATTTCCCCACGTGCTGCAAC~CTGCA 109 M G V L S A H V N A V Y F P T C C N N C R

GGTC~T~CC~GGTCGACGTCTGCGACGACGCCCATCCGCAGTGCCCCACCGGC~CT 169 S F S G V D V C D D A H P Q C P T G C S

CGGCGTGCCGCGTGGTGACCACG~CCCCeAGACGTTCCGCTGCGCCGACATGAAAGCCA 229 A C R V V T T N P Q T F R C A D M K A T

CCGTCGAC~CACCTGCGGCGGGCCATGC~G~GTACTGATCGCTCAGGCTCACATGAG 289 V D G T C G G P C K K Y *

ACTATTGTTCGCTTGGCGCCTAAAATAAAAGCTCGGATGAGATGACCGGACTCATCTTGC 349 CTATGCGTGTGTGTGTGGCC~CAAC~TATGTATACTGTTCCGTCGTGTCT~GTCACC 409 GTGGTTTCTTTCTCGAGAAATATCCCCCGTG~TATTGTGCGTGTCTACTC~T~CATG 469 ~TATTACTTGTATTTAT 487

Figure 2. Sequence of bsil. The deduced aa sequence is indicated below the respective codons. Numbers to the right indicate position relative to the An t of the initiation codon (ATG). Those to the left represent the aa position. The initiation codon and a potential polyadenylation signal which lies 45 nt downstream of the stop codon (TGA) are in bold. The putative N-terminal secretory signal sequence (italics) is thought to be cleaved after serine at position 22 (&).

Results

S. nodorum-challenged coleoptiles

The coleoptile 'sandwich' inoculation technique described by Hargreaves and Keon [17] was used so that sufficient quantities of tissue, enriched in cells responding in an incompatible manner to infection, could be collected for cDNA library construction, synthesis of cDNA probes and analysis of gene expression. This inoculation procedure resulted in a large propor- tion of the outer coleoptile epidermal cells being challenged by S. nodorum and many of the hyphae attempted more than one penetration (Fig. 1). Although syn- chronous infection could not be achieved, this method of inoculation resulted in many attempted penetrations that led to the formation of large numbers of papillae and associated modified cell walls.

Isolation of induced cDNA clones

A total of 40 000 recombinant plaques were screened by differential hybridization and six differentially expressed clones (ET1-ET6) were chosen for further examination. The cloned cDNAs were excised on pBluescript phagemids (pETI-pET6) and par- tially sequenced using M13 forward and reverse primers. Sequence analysis revealed that ET1, ET4 and ET5 shared identical stretches of nucleotide (nt) sequence and were, therefore, probably derived from the same gene. ET2 ~md ET3 both appeared to con- tain two tandem cDNA inserts (estimated to be 500 and 1500 bp, and 235 and 1560 bp long, respectively). The sequences of the two smaller fragments from both clones were found to be identical to the sequences of ET1, ET4, and ET5. Partial sequen-

cing of the larger cDNA fragment in pET2 revealed that it was identical to the 3' end of a barley gene encoding 1,3-1,4-/3-glucanase [25]. However, no sig- nificant homology was found to the partial sequence derived from the larger insert of pET3. Even though ET2 and ET3 were probably selected because of the presence of the DNA sequences that were similar to ET1, ET4 and ET5, mRNA of 1,3-1,4-/3-glucanase was found to be up-regulated, albeit to low levels, in S. nodorum-challenged coleoptiles [16]. pET4 was selected for detailed examination of the most abundant clone and the complete DNA insert was sequenced. The nt sequence and other relevant data are shown in Fig. 2. The cloned DNA in pET4 was 499 bp long and contained a complete open reading frame encoding a cysteine-rich protein 89 aa long with a Mr of 9405. The sequence started 11 bp upstream of the initiation codon (ATG) and continued 218 bp bey- ond the stop codon (TGA). A putative polyadenylation signal (AATAAA) was identified 46 bp downstream of the stop codon, however, polyadenylation was not observed. The sequence of this cDNA (bsil) exhibited 86% identity to wali5a, an aluminium-induced gene of unknown function from wheat roots [45]. This high level of homology extended into the 3' non-coding regions, indicating that the two genes were closely related. The deduced aa sequence of bsil was 87% identical and 91% similar to Wali5a (Fig. 3). The predicted aa sequence derived from both DNA sequences possessed a putative N-terminal signal sequence for translocation to the endoplasmic reticulum. Cleavage immediately after serine at position 22, as predicted by von Heijne [52], would yield a mature protein containing 67 aa with a Mr of 7153. Database searches also revealed that Bsil shared homology with BB-PIs [4]. The closest relative was a putative wound-induced

745

Bpil Wali5a WIPI

MKGTK.LAAILILQAVLVMGVLS .............. HVNAVYFPTCCNNC.RS L .............. DF K

SSPH VL C A FAALAKENAMVESKAIDI PGQL.K T NF

Bpi 1 WaliSa WIPI

* * * * * *

FSGVDVCDDAHPKCPQGCSACRV... VSTSP. EMWRCADMKSTVDGTCGGPCKKY Q T . . . T N .QTF A

LYT VKKD DPV KK v AVHA Y GNNKF T . . . FL M . . C

Figure 3. Alignment of the predicted aa sequence of Bsil with Wali5a, an unidentified aluminium-induced wheat protein [441, and WIPI, a presumed wound-induced Bowman-Birk proteinase inhibitor from maize [40]. Only the positions where the sequence of Bsil differ are listed for Wali5a and WIPh The putative active site is overlined and the positions of conserved cysteine residues are marked with an asterisk.

GGTGCATACATGAGTAGTACT~TACT~TT~TAGGAGT~TTGTTTCCACGCCTTAAA -469 CTTTGTCTA~TTAAAAATG~CGC~GT~GTGACAGATGGAGTAGTAGCGAGGA -409 CGC~GCATATTTTGT~CTTGCCCCTGCCGCCCAAGTGGATGTACGCTTTTC~CTTCG -349 CCCGTCC~TGAGGTTATGTTeTAG~ATA~A~GCAAACTC~CTGCTCAAAGT~ -289 TCGACC~TGTATATCGGGAAATCG~TGG~T~TCACTCGTTGTCAG~CTCAG~GT -229 ATTTATATGTACTCGAAATAAAGG~GCC~GCCA~TGT~CTAGC~TTCTTGATACA -169 TATGCATGCACTCGATTC~CCCCACGTCATCTTC~GCTAGCTTCCTTGGAAAATAAAG -109 ~CTATGGACTCCCTACAAAATTTTGC~CGGGCCCTTTCTATATGCT~TCCTATAAAT -49 ATCCATC~GTTC~GCACTCTTCATCGATCTGGAGCCACCACTAGT~T~CATCTTCC 12

M A S S ~GAGTAGTCTTGC~TGTTCGCACTAGCCATAGTCATGGCCGTGGTGGCCGGCGTCTCG 72

5 K S S L A M F A L A I V M A V V A G V S GCGCAG~CACCCCGCAGGACTTCGTC~CCTGCAC~CCGCGCCCGTGCTGTGGACGGC 132

2 5 A A Q N T P Q D F V N L H N R A R A V D G GTCGGCCCGGTGGCGTGGGAC~C~CGTGGCCAGGTTCGCACAG~CTACGCGGCGGAG 192

4 5 V G P V A W D N N V A R F A Q N Y A A E CGCGCCGGCGACTGTCGGCTGCAGCACTCCGGGGGGCCGTTCGGCGAG~CATCTTCTGG 252

6 5 R A G D C R L Q H S G G P F G E N I F W GGGTCCGGGCGGTCATGGACGGCCGCCGACGCCGTG~GCTGTGGGTGGACGAG~GCAG 312

8 5 G S G R S W T A A D A V K L W V D E K Q

~C~CCATCTTGACAGC~CACCTGC~CGCCGGC~GGTGTGCGGGCACTACACGCAG 372 1 0 5 N Y H L D S N T C N A G K V C G H Y T Q

GTG~GTGGCGC~GTCGATCCGCATCGCGTGCGCGCGGGTGGTCTGCGCTGGG~CCGG 432 1 2 5 V V W R K S I R I A C A R V V C A G N R

GGCGTCTTCATCACCTGC~CTACGATCCCCCGGGC~CTTC~CGGCGAGCGCCCGTTC 492 1 4 5 G V F I T C N Y D P P G N F N G E R P F

GCGTTCCTCACCCTTGACGCCG~GCC~GTAGTACTGTACGTGCGTGTG~CGTTGATC 552 1 6 5 A F L T L D A E A K *

ATATACATGCATACGTACGTGCGTGTGTTTATGTGTGTGCGGTTTG~TATTGATACGTC 612 TACAAACGTTACATACGTACGTA~AT~G~CA~CCCGCCTTCATGTATCGATGCGG 672 ATGTGTAGAGCGTGTAAATGT~TCCTATATATATGCGTCCATGCAAATGTCTTTCCTTT 732 TGT~T~TGTTTAAATCTTGGGCCTAGCCAAAT~T~CAT~T~TATTGGG~CCC 792 CTGGGAAAAAATGATACTTACGAAACGATACTTACACAAAACCAAATTTG~GTTGTGTT 852 C~TGAT~G~CCTGT~TTCTCCAAATAGGTTTATGCCATGCTTTATAAATAAAAC~ 912 CTTC 916

Figure 4. Sequence of bpr I - I. The deduced aa sequence is indicated below the respective codons. Numbers to the fight indicate position relative to the Ant of the initiation codon (ATG), those to the left represent the aa position. The initiation codon, potential CAAT and TATA boxes -91 and -54, respectively, relative to the A of the initiation codon and putative polyadenylation signals located 112, 240 and 380 nt downstream of the stop codon (TAG) are in bold. The N-terminal secretory signal sequence, which is thought to be cleaved after the alanine residue at position 25 (A), and the putative C-terminal vacuolar target sequence, are shown in italics. The position of the sequence derived from pET6 is underlined.

BB-PI, WIP1, from maize [41], which shared 41% identity and 49% similarity with Bs i l . The cysteine residues in the mature proteins were particularly well conserved (Fig. 3). BB-PIs are known to be inhibitors of serine proteinases and are found mainly in the seeds of legumes. BB-PIs have also been identified in seeds of non-legumes, including wheat [35] and barley [32]. However, Bs i l was not closely related to either of these cereal proteinase inhibitors.

The sequence of the other cDNA clone, ET6, was 661 bp long and appeared to lack part of the 5' end of the gene containing the initiation codon. A 2 kb

EcoRI genomic D N A fragment containing the com-

plete coding region was recovered f rom a barley AFIX

II barley genomic library (a gift of Dr A. Goldsbor-

ough) using ET6 as a hybridizat ion probe. This frag-

ment was subcloned into pBluescr ipt to create p G E T 6

and 1444 bp, including the regions flanking the coding

region, were sequenced. The nt sequence der ived f rom

pET6 and pGET6, and other relevant data are shown

in Fig. 4. The sequence started 528 bp upst ream of

the initiation codon (ATG) and finished 390 bp down-

stream f rom the stop codon (TAG). Potential C A A T

and TATA boxes were identified at posi t ions - 9 1 and

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Bpr-i MASsKSSLAMFALAIVMAwAGv.•S.AQNTPQDFvNLHNRARAVDGVGPvAWDNNvARFAQNYAAERAGDCRLQHSGGPFGENIFWGS.GRS HvPRIa ...METPKLAIL LA AAM. NL Q S YLSP A V A S STKLQAY S NQ I K Y A AD HvPRIb ...MQTPKLVIL LA SAAM. NL Q s Y SP A V A S STKLQA NQ IN K Y A AD prbl ...MQTPKLVIL LA SAAM. NL Q S Y SP A V A S STKLQA NQ IN K Y A AD PRbl-2 ...MQTPKLAIL LA AAM. NL Q s Y SP A S V A S STKLQA NQ IN K Y A AD PRbl-3 ...MQTPKLVIL LA SAAM. NL Q S Y SP A V A S STKLQA NQ IN K Y A AD

Bpr-i wTAADA•KLWvDEKQNYHLDSNTCNAGKvCGHYTQVvWRKSIRIGCARVvCAGNRGvFIT•NYDPPGNFNGERPFAFLTL DAEAK HvPRIa K KD DYG AG A TS NN G E A W QK Y HvPRIb K S NS S KD DYG A A TS NN E R IV QK Y prbl K S NS S KD DYG A A TS NN E R IV QK Y PRbl-2 K NS N KD NYG A A TS NN E R IV QK Y PRbl-3 K S NS S KD DYG A A TS NN E R IV QK Y

Figure 5. Comparison of the predicted aa sequence of Bprl-1 with other PR-l-type proteins derived from barley cDNA clones, HvPR-la and HvPR-Ib [8], prbl [30], PRbl-2 and PRbl-3 [31]. The sequences were aligned using the PILEUP program. The arrow marks the first aa of the mature protein predicted from the N-terminal sequence of maize ZMPR-1 [ 13], tobacco PR-la [36] and tomato P6/p 14 [27] PR-1 proteins. Only the positions where the sequence of Bprl-I differ are listed for the other PR-1 proteins.

-54 , respectively and three putative polyadenylation sites (AATAAA) were found 111, 239, and 378 bp downstream from the stop codon. The predicted protein encoded by this gene (bprl-1) was basic, with a deduced pI of 8.27, and a calculated Mr of 18 859. The aa sequence derived from bprl-1 was strikingly similar to 20 PR-1 proteins presently available on the EMBL/GenBank databases. Bprl-1, in keeping with other PR-1 proteins, contained an N-terminal secretory signal peptide that was predicted to be cleaved after alanine at position 25, indicating that this protein may, like Bsil, be secreted. However, Bprl-1 also possessed a 10 aa charged C-terminal extension, similar to that found in some other PR-1 proteins [12, 36, 38, 50], which may represent a cleavable vacuolar targeting signal sequence [33]. Cleavage of both the N-terminal and C-terminal regions would leave a mature peptide 139 aa long, with a calculated Mr of 15 403 and a predicted pI of 8.28. Five cDNA clones encoding PR-1 proteins have previously been isolated from barley leaves infected with the powdery mildew fungus, Erysiphe graminis f. sp. hordei [8, 30, 31]. However, all of these PR- 1 proteins differed significantly from Bprl-1, even though they exhibited greater than 90% identity to each other (Fig. 5). Two of these PR- 1 proteins were also basic [8], but neither contained an identifiable vacuolar targeting sequence, as found in Bprl- l . Thus, Bprl-1 would appear to conform with the tobacco model which suggests that basic PR proteins are transported to, and accumulate in, the vacuole [29]. These findings, therefore, place Bprl-1 in a sep- arate class of PR- 1 proteins to those already identified from barley.

Induced gene expression

Transcript levels of bsil and bprl-1, and four other known fungal-induced genes, were examined in S. nodorum-challenged coleoptiles. DNA probes used for this purpose were derived from clones ET4 and ET6 and from genes encoding a barley leaf-specific thionin [6], a wheat pathogen-induced peroxidase [39], 1,3-/3- glucanase [21] and HMG-CoA reductase, an enzyme known to be induced in potato by fungal elicitors [46]. Expression of these genes was examined in coleophile tissue collected 8, 16, 24, 32, and 48 h a.i. with S. nodorum and in uninoculated control coleoptiles collected after 16 h incubation between agar-coated glass slides (Fig. 6). In general, bsil, bprl-1, 1,3-/3- glucanase and HMG-CoA reductase showed similar patterns of induction. Transcripts of these genes were either present at low levels or absent in control uninfected coleoptiles, but progressively accumulated to high concentrations in S. nodorum-challenged coleoptiles during the first 16 h a.i. Levels of transcripts homo- logous to the wheat peroxidase gene also increased during this period but then declined with time a.i. In contrast, concentrations of barley leaf-specific thionin mRNA did not significantly increase until 24 h a.i., but then continued to increase during the duration of the experiment.

Discussion

Transcripts of a number of unrelated genes increased in barley coleoptile cells upon an incompatbile interaction with S. nodorum. Some of these genes were known to be pathogen-induced (leaf-specific thionin, peroxidase 1,3-fl-glucanase and HMG-CoA reductase) and

Figure 6. Transcription levels ofbsil, bprl- 1 and other defence genes encoding 1,3-~q-glucanase, HMG-CoA reductase, a homologue of a putative wheat peroxidase (WIR3) and leaf-specific thionin in uninfected control coleoptiles (C) and in coleoptiles collected 8. 12. 16, 24 and 48 h.a.i, with S. nodorum. Normalization of the amount of RNA loaded in each lane of the gels was based on the intensity of fluoroescence of ribosomal 28S and 18S RNA bands after staining with ethidium bromide

were thought to encode proteins that play either a direct or an indirect role in defence against fungal attack [3, 21, 46]. Two other genes (bsil and bprl-1), which had not previously been identified, were also found to be induced in S. nodorum-challenged coleoptile cells. bsil and bprl- 1 were shown by sequence homology to encode a protein related to cysteine-rich BB-PIs and a PR-1 protein, respectively. Taken together, these data indicate that a number of different cellular processes are activated during this particular incompatible interaction.

The multi-component nature of this response makes it difficult to assess the relative importance of each indi- vidual component contributing to the defence reaction. Indeed, the induction of a number of diverse cellular processes is perhaps surprising considering that S. nodorum hyphae rarely penetrate further than the outer layers of the cell wall [18, 22]. Signal molecules triggering this defence reaction may be elicitors eman-

747

ating from the penetrating hyphae or they may be endo- genous activators released from, or formed by, the plant as a consequence of pathogen activity [23]. In addition, it is likely that wound damage at the site of penetration triggers cellular repair messages that induce metabolic changes which are not specifically associated with fungal attack.

The finding that five out of six S. nodorum-induced clones recovered by differential screening contained fragments ofbsil indicated that transcripts of this gene were highly abundant in S. nodorum-challenged cells and implied that Bsil may play an important role in the incompatible response. However, bsil which appeared to be related to BB-PIs was highly homolog- ous to an aluminium-induced wheat gene, wali5a [45], and exhibited homology to a wound-induced protein, WIP1, from maize [11, 41]. This suggested that bsil may be activated by wound damage rather than by a pathogen-derived elicitor. Nevertheless, Bsil contained a putative N-terminal secretory signal sequence indicating that it may be externalized and could poten- tially accumulate within the cell wall. A number of plant proteinase inhibitors have been shown to possess antifungal activity [26] and this raises the pos- sibility that Bsil may function in preventing ingress of the pathogen by inhibiting growth of the penetrating hyphae within the cell wall. A similar role has been identified for barley leaf-specific thionin [3, 5], although transcripts of this gene were found to accumulate much later than those derived from bsil in this incompatible interaction. In this context, it is also worth noting that some proteinase inhibitors have been shown to enhance the antifungal activity of thionins [48].

The other S. nodorum-induced barley gene (bprl- 1) encoded a basic PR-I protein which differed significantly from other barley PR- 1 proteins previously identified from E. graminis-infected barley leaves [8, 30, 31]. Bprl-I contained a putative C-terminal aa extension indicating that, unlike Bsil and the other barley PR-I proteins, it may be targeted to the vacuole. If this were the case, then Bprl-I is unlikely to come into direct contact with invading hyphae of the pathogen until cell membranes lose their integrity, as occurs during a hypersensitive reaction. The role of Bprl-I in defence against infection by S. nodorum is, therefore, unclear. Although no enzymatic or biological function has been attributed to this group of PR-1 proteins, it has been shown recently that some basic PR-1 proteins possess antifungal activity [34]. Furthermore. transgenic tobacco plants overexpress-

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ing the acidic t obacco P R - l a p ro te in have e n h a n c e d

res i s tance to funga l infec t ion , i m p l y i n g tha t this type

o f p ro te in m a y also be inh ib i to ry [ 1 ]. However , in o rder

to impl i ca te e i the r B s i l or B p r 1-1 in the p l an t ' s a r sena l

aga ins t a t tack b y S. nodorum it wi l l be necessa ry to

show that these p ro te ins a c c u m u l a t e w h e r e and w h e n

funga l g rowth is res t r ic ted and to d e m o n s t r a t e tha t they

possess fung ic ida l activity.

Acknowledgements

We are ex t r eme ly gra te fu l to Dr R. D u d l e r for p U W 1 3

c o n t a i n i n g the W I R 3 gene , to Dr H. B o h l m a n n for

p H v D B 4 c o n t a i n i n g the leaf-speci f ic t h ion in gene and

to Dr A. G o l d s b o r o u g h for the 1 ,3- /3-glucanase gene

and for the bar ley g e n o m i c library. E.T. and C.S. were

f u n d e d t h r o u g h B B S R C L i n k resea rch gran t LR24 /567 .

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Documents

Defence-related gene activation during an incompatible interaction between Stagonospora (Septoria) nodorum and barley (Hordeum vulgare L.) coleoptile cells