Accumulation of defence-related transcripts and cloning of a chitinase mRNA from pea leaves (Pisum sativum L.) inoculated with Ascochyta pisi Lib.

ELSEVIER SCIENTIFIC PUBLISHERS IRELAND Plant Science 92 (1993) 69-79 plan ience Accumulation of defence-related transcripts and cloning of a chitinase mRNA from pea leaves (Pisum sativum L.) inoculated with Ascochyta pisi Lib. Knud Vadt a, Eigil de Neergaard a, Kenneth Madriz-Orde~ana a, J0rn D. Mikkelsen b, David B. Collinge *a aSection for Plant Pathology, Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen Denmark hMaribo Seed Biotechnology, Danisco A/S, Langebrogade 1, DK-IO01 Copenhagen K, Denmark (Received 22 March 1993; revision received 29 April 1993; accepted 29 April 1993) Abstract The race specific resistance of pea to Ascochyta pisi Lib. was shown to be exhibited as a hypersensitive response associated with the production of polyphenolic substances in epidermal and mesophyll cells. The levels of transcripts representing a pathogenesis-related (PR) protein (chitinase) and an enzyme of phytoalexin biosynthesis (chalcone synthase) were shown to accumulate more rapidly during the hypersensitive response than during lesion development in the compatible interaction. A full-length (1143 bp) eDNA sequence of a pea chitinase (EC 3.2.1.14) (coding for an approx. 34 500 Da protein) was deduced by combining the overlapping sequences of three clones obtained following PCR amplification of eDNA prepared from mRNA isolated 24 h after inoculation of pea leaves with Ascochyta pisi. The combined sequences were identified as a class I chitinase corresponding to the basic A l-chitinase enzyme previous- ly isolated from pea leaves (Vad et al., Planta, 184 (1991) 24-29). Like class III and IV chitinases, the pea sequence differs from other class I chitinases in the absence of a hydrophobic C-terminal domain. Key words: Hydrolase (antifungal); Polymerase chain reaction; Pathogenesis-related protein; Extensin; Hypersensitive response; Chalcone synthase 1. Introduction Plants respond to pathogenic infection by the synchronised activation and expression of a num- * Corresponding author. iPresent address: Novo Nordisk A/S, Molecular Biology, Diabetes Research, Novo All~, DK-2880 Bagsvaerd, Copen- hagen, Denmark. bet of genes which are believed to have a role in defence [1,2]. Many of these genes encode enzymes involved in synthesis of products which retard or inhibit pathogenic invasion e.g. phytoalexins and lignin. Others encode proteins believed to act directly on the invading pathogen. These include certain pathogenesis-related (PR) proteins such as B-1,3-glucanase and chitinase which can cause 0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)0365 l-B 70 lysis of hyphal tips in vitro [see 3,4,5]. In particu- lar, endochitinases (EC 3.2.1.14) in plants have been subject to intensive study during recent years. The studies have been based on the, now largely supported, prediction that at least some chitinases possess antimicrobial activity in planta [4,6]. Others, such as hydroxyproline rich glycoproteins (HRGP) are believed to act by strengthening the cell wall [7,8]. The interaction between pea and the fungal pathogen Ascochyta pisi Lib. is characterized by physiological specificity where at least 6 races of the pathogen are recognized [9]. Previous studies have shown phytoalexin accumulation [10] and increased chitinase [11] activity in pea leaves inoculated with A. pisi. The accumulation of transcripts corresponding to the key enzymes of isoflavonoid phytoalexin biosynthesis, phenylala- nine ammonia-lyase and chalcone synthase, has also been demonstrated in pea following treatment with elicitors isolated from the fungus Mycos- phaerella pinodes [12]. We have previously purified three basic isoforms of chitinase and the partial N-terminal amino acid sequences were obtained [11]. A basic fraction A (comprising isoforms A1 and A2) accumulated after inoculation, whereas isoform B did not accumulate. We report cDNA cloning of chitinase A 1 from pea leaves and a com- parison of chitinase transcript accumulation in incompatible (hypersensitive) and compatible in- teractions, in relation to transcript levels of chalcone synthase, a key enzyme in the biosynthe- sis of isoflavonoid phytoalexins, and to a structur- al host cell wall hydroxyproline-rich glycoprotein (HRGP). 2. Material and methods 2.1. Plant and fungi Seeds of Pisum sativum L. cv. Birte and Ascochyta pisi Lib. isolates PFI (avirulent on cv. Birte) and PF5 (virulent on cv. Birte) were supplied by Pajb- jergfonden, Odder, Denmark. Growth of plants and fungi, and inoculation and harvest of the plants were performed as described previously [11], except for the use of increased light intensity in the growth chamber (approx. 100 t~E. s -~- m-Z), measured with a Quantum Sensor, (Li-Cor, Inc., Lincoln, Nebraska, USA). The Rap-2 resis- K. Vad et al./Plant Sci. 92 (1993) 69-79 tance gene [9] is present in the pea cultivar Birte. Pea plants possessing the Rap-2 gene express resis- tance towards all British pathotypes of A. pisi, but apparently not to the Danish A. pisi pathotype PF5, believed to represent race E (H. Jaiser, Uni- versity of Berlin and Else Bollerup, Pajb- jergfonden, Odder, Denmark, pers. commun.). 2.2 Cytohistological studies Seven days after inoculation, leaf pieces with lesions were cut, fixed in formalin-acetic acid- alcohol, dehydrated in ethanol and embedded in glycolmethylacrylate (Historesin) (Kulzer, Wehr- heim, Germany). Sections of 7 /zm were stained with Toluidine Blue O. 2.3. Extraction of plant RNA and preparation of cDNA Poly (A)+RNA was prepared from total RNA [13,14] isolated from pea leaves 24 h after inocula- tion with A. pisi, isolate PF 1, on oligo-dT-cellulose (GIBCO/BRL, Maryland, USA)according to [15] and cDNA synthesis utilized M-MLV reverse transcriptase for first-strand synthesis and RNaseH/polymerase I for second strand synthesis [16]. The cDNA was stored at -20°C for later PCR amplification. 2.4. PCR amplification of cDNA The PCR amplification was performed essen- tially according to [17]. Two units Taq DNA poly- merase (Promega, Madison, Wis., USA) were used in the amplification buffer which included 500 ~M dATP, dGTP, dTTP and dCTP; and 200 pmol of the oligo-dT primer (PI): 5'-AAGCTTGA- ATTC(T)20-3'. P1, which contains a HindIII and a EcoRI site at the 5'-end, was used as 3' primer. 300 pmol of a mixture of eight oligonucleotides, deduced from a region conserved in chitinase se- quences from bean [18], tobacco [19] and potato [20]; were used as 5' primer (P2): 5'-CCGCG GC(AGCT) CTC TGG TT(TC) TGG ATG AC- 3'. P2 includes a 5' SacII site. The PCR pro- gramme consisted of one cycle at 95°C for 5 min, 37°C for 2 min, 50°C for 1 min and 72°C for 10 min. Amplification proceeded with five cycles at 95°C for 1 min, 37°C for 2 min, and 72°C for 5 min, followed by 30 cycles at 95°C for 1 min, 42°C K. Vad et al./Plant Sci. 92 (1993) 69-79 71 for 2 min, and 72°C for 2 min, with a final 10 min elongation at 72°C. The PCR product was precipitated and sub- jected to electrophoresis through a 2% agarose gel, from which the amplified products were extracted and purified using the Geneclean kit (Bio 101 Inc., La Jolla, CA, USA) according to Wilson [21]. Half of the product was re-amplified using the same conditions as described above. After EcoRI/SaclI digestion, the products from the second amplifica- tion were ligated into the EcoRI-SaclI site of pBluescript SK (-) (Stratagene, La Jolla, CA, USA) and transformed into E. coli strain JM105. The clone was designated pPCH1. The antisense oligonucleotide 5'-AGAATTC ATG GCT TGA TGG CTT GTT TG-3' (P3), based on sequence data from pPCH1, was used for primer extension. A mixture of 64 different 17- mer-oligonucleotides: 5'-CCTCTA GA(AG) CA(AG) TG(TC) GG(AGTC) AA(TC) CA-3' (P4), which included a XbaI site at the 5'-end, was used in PCR amplification. P4 was deduced from the N-terminal amino acid sequences of basic chitinase isoenzymes of pea [11]. The amplified products were ligated into pBluescript SK (-) and transformed into E. coli strain JM105. This clone was designated pPCH2. The 5'-part of the cDNA including the chitinase signal peptide was cloned using rapid amplifica- tion of cDNA ends (RACE) according to Frohman et al. [22]. Primer extension was per- formed using the specific primer P3, followed by tailing of the cDNA with dATP and subsequent amplification of the cDNA utilizing PI, and a nested primer: 5'-GTT TCA TGA GAA GTT TG-3' (P5). The amplified products were ligated into pBluescript SK (-) and transformed into E. coli strain JM105; the longest clone obtained was designated pPCH3. The amplification conditions used for preparing the sequences pPCH2 and pPCH3 were as follows: one cycle at 95°C for 5 min, 40°C for 2 min, and 72°C for 20 min, follow- ed by 40 cycles at 95°C for l min, 52°C for 2 min, and 72°C for 3 min, with a final 10 min elongation at 72°C. 2.5. RNA and DNA blotting and hybridization All blotting and hybridization procedures were as described by Thordal-Christensen et al., [16]. Pea chalcone synthase cDNA, pCHS2 [23] was obtained from Dr. T.H.N. Ellis (John Innes Insti- tute, Norwich, UK), carrot extensin cDNA, pDCI 1 [24], was obtained from Dr. Joseph E. Varner (Department of Biology, Washington Uni- versity, St. Louis, USA). 2.6. DNA sequencing The dideoxynucleotide-chain termination reac- tions [25] were resolved on denaturing gradient gels [261 as described by Thordal-Christensen et al. [16]. Complete sequencing of both strands was performed, and all sequences were confirmed from at least two clones obtained from separate PCR reactions. The DNASIS and PROSIS software (Pharmacia LKB, Uppsala, Sweden) and Universi- ty of Wisconsin GCG package [27] were used for computer-assisted analysis of sequence data using the EMBL data base [281. 3. Results and discussion Cross sections of infected areas of pea leaves, stained with Toluidine Blue O, showed accumula- tion of polyphenolic substances at the edge of the lesions (Fig. 1). In the incompatible interaction, hyphae of the pathogen were observed in the col- lapsed epidermal cell layer, occasionally penetrating into the mesophyll, but not developing beyond the area of the restricted lesion (usually less than 1 mm in diameter). The mesophyll cells showed intense positive staining reaction for polyphenolics. Since this reaction to pathogen in- vasion is rapid and comprises cell death, it is inter- preted as a hypersensitive response (Fig. 2). In contrast, in the compatible interaction, the lesions develop to a maximum size of 5 mm in diameter with hyphae present in the disrupted leaf tissue, although polyphenolics seem to occur in the border zone proximal to the lesion (Fig. 1). In agreement with our observations, Brittain [29] showed that not only epidermal cells, but also me- sophyll cells are involved in resistance reactions of several pea lines towards A. pisi infection. Heath and Wood [10] found heavy deposition of poly- phenolic compounds at the edge of the infection areas and detected the important phenolic com- pound, the phytoalexin pisatin, in pea tissues 24 h after inoculation with A. pisi. The continued ac- 72 K. Vad et al./Plant Sci. 92 (1993) 69-79 la Fig. 1. (a) Lesion on a pea leaf caused by a compatible race of A. pisi (isolate PF5). Weak polyphenolic reaction (arrow). A transverse section stained with Toluidine Blue O. Bar = 100tzm. (b) detail of (a) showing accumulation of polyphenolic substances (green) at the edge of the lesion. Bar = 100#m. Fig. 2. Pea leaf inoculated with an incompatible race (isolate PF 1 ) of A. pisi. Hypersensitivity reacting epidermal cells, adjacent meso- phyll cells showing polyphenolics (stained green). Transverse section, stained with Toluidine Blue O. Bar = 200~m. K. Vad et al./Plant Sci. 92 (1993) 69-79 cumulation of pisatin was suggested as the main agent in inhibiting hyphal growth in the brown tis- sue surrounding the zone colonized by the pathogen. The association of pea resistance to A. pisi with the production of pisatin [10] and the induction of chitinase [11], prompted us to investigate whether the development of the hypersensitive response in plants exhibiting an incompatible interaction is as- sociated with a more rapid expression of specific defence related genes than in the compatible inter- action. As we had demonstrated that pea chitinase enzyme activity corresponding to isozymes A 1 and A2 accumulated more rapidly in resistant plants [11], we wished to examine the levels of chitinase 73 transcripts in plants inoculated with A. pisi. How- ever, none of the chitinase cDNA clones made available to us (from bean [30], sugar beet chitinase 1 [31] and tobacco [32] cross hybridized to the pea genome (data not shown)). Comparative analyses of basic chitinase se- quences from bean, tobacco and potato [18-20] revealed three highly conserved regions. A con- served motif: A I/L W F W M T (positions 237-243, Fig. 4) closest to the C-terminal was chosen for synthesis of oligonucleotides (P2). These were successfully used in PCR amplifica- tion, yielding approx, one-third of the whole transcript. Of fifteen clones obtained, three 408-bp clones from independent amplifications were se- 1 TTT TTA TCA CAA AAC CGA CAT ACT 1 76 CTA CCT TTG TTT CTTGGA TCT AAA 15 L P L F L G S K 151 TGC TGC AGC AAA T~ GGT TTT TGT 40 C C S K F G F C 226 TCT CCA ACA CCG ACA ATA CCA ACA 65 S P T P T I P T 301 GAC CAA ATG CTT AAA TAT AGA AAC 90 D Q M L K Y R N 376 GCT GCT AGA TCT TTC AAT GGA 115 A A R S F N G F (P5) 451 GCT CAA ACT TCT CAT GAA ACC ACA 140 A Q T S H E T T 526 GTG TCC GAA CAAAAC ACT CAG GAA 165 V S E Q N T Q E 601 GGA AGA GGA CCA Aq'9 CAA CTC ACT 190 G R G P I Q L T 676 AAC AAT CCG GAC TTA TTG TCC ACA 215 N N P D L L S T (P3) 751 GCA AAC AAG CCA TCA AGC CAT GAT 240 A N K P S S H D 826 GTC CCC GGA TAT GGT GTG ATC ACC 265 V P G Y G V I T 901 GAT GAT CGG G~'f GGA TTT TAC AAA 290 D D R V G F Y K 976 AAC CAA AGG TCA T~ GCT TAA GAG 315 N Q R S F A 1051 CAA TGA ATG ATC ~'FA CAT GTA AT~ (Pl) 1126 TTT AGT AAA AAA AAA AAA ACT TTC AAA ATG AAA AGA ACT CTA AAA GTA TCT TTC T'I'f ATA CTA TGT q'fA 75 M K R T L K V S F F I L C L 14 (P4) GCA GAG CAA TGC GGT AGC CAA GCT GGT GGA GCT GTA TGC CCA AAT GGG TTA 150 A E Q C G S Q A G G A V C P N G L 39 GGT AGC ACT GAT CCT TAC TGT GGA GAT GGT TGC CAA AGC CAA TGT AAA TCA 225 G S T D P Y C G D G C Q S Q C K S 64 CCT AGT ACT GGT GGT GGA GAT G~T GGA AGG CTF GTF CCT TCT TCT CTC TIT 300 P S T G G G D V G R L V P S S L F 89 GAT GGA CGG TGT GCT GGT CAT GGA TTT TAT ACC TAT GAT GCT TIT ATT GCC 375 D G R C A G H G F Y T Y D A F I A 114 GGT ACA ACT GGT GAT GAT AAC ACA AAG AAG AAG GAA C~'f GCT GCT TTC ~ 450 G T T G D D N T K K K E L A A F L 139 GGA GGG TGG CCA ACT GCA CCA GAC GGT CCA TAC GCG TGG GGA TAT TGC TI'F 525 G G W P T A P D G P Y A W G Y C F 164 GTC TAT TGT TCA CCT AAA GAT TGG CCA TGT GCT CCT GGT AAA AAA TAC TAT 600 V Y C S P K D W P C A P G K K Y Y 189 CAC AAC TAC AAT TAT GGT CTA GCG GGT CAA GCA A~'~ AAG GAA GAT TTA ATT 675 H N Y N Y G L A G Q A I K E D L I 214 (P2) AAT CCA ACT GTG TCA TTC AAG ACA GCC ATA TGG ~ TGG ATG ACC CCA CAG 750 N P T V S F K T A I W F W M T P Q 239 GTG A~I'F ACT GGA AGA TGG ACT CCA TCT GCT C-CT CAT AOC TCA GCA GGT CGG 825 V I T G R W T P S A A D S S A G R 264 AAC ATA ATC AAT GGC GGG ATT GAA TGC GGC CAC GGT CAG GAT AAT CGA GTC 900 N I I N G G I E C G H G Q D N R V 289 AGG TAT TGC CAA ATC TTC GGA GTG GAC CCT GGC GGT AAC ~ CAT TGC AAC 975 R Y C Q I F G V D P G G N L D C N 314 ** *** ** AAT TAC CCA TGT TAA TGC TAT CAA GTA TAA AGT ATA GAA ATA AAG GGA TCA 1050 320 * *** *** TC-C ATC ~ ATT GCT ATG TAA AAT AGC ~ CAT AAT GTT TCA ATA AAA CTG 1125 1143 Fig. 3. Nucleotide and deduced amino acid sequence of pea chitinase A 1. The sequence was obtained from mRNA isolated from pea leaves (cv. Birte) 24 h after inoculation with A. pisi. The five sequences used for primer annealing in PCR amplification and primer extension are indicated by underlining. Putative polyadenylation signals are indicated at positions 1037 and 1115. The sequence is available in the EMBL data bases under the accession number: X 63899 (cDNA) and H12697 (derived amino acid sequence). 74 quenced in their entirety and shown to be identical and designated pPCH1 (position 724 to 1143 in Fig. 3). These were identified as chitinase by com- parison to DNA and derived amino acid sequences from the EMBL data base. Interestingly, no other chitinase sequences were obtained although the K. Vad et al./Plant Sci. 92 (1993) 69-79 same conserved sequence motif was used recently for cloning type 4 chitinases in rape [33] and sugar beet [31] using primers identical at the 3 '-end, and we believe that, in view of its size and N-terminal sequence, chitinase B of pea [11] is likely to be a type IV chitinase. This may well reflect the fact Pea A1 ( Ib ) Tobacco ( Ia ) Arab idops is ( Ia ) Potato ( Ia ) Bean ( Ia ) Petun ia ( I I ) Tobacco ( I I ) Rape ( IV ) Sugar beet ( IV ) Pea B N- term. Pea A2 N- term. Pea A1 N- term. 1 - I0 1 i0 20 30 40 50 60 70 80 MKRTLKVS FF I LCLLPLP LG SKAEQCGSQAGGAVC PNGLCCS KFGFCG STDPYCGDG - CQ SQCKSS PTPT I PTPSTGGGD SLLLLSASA . . . . . . . . . . R .AS . . . . . . . . W. . N . ND. . . P .N . . . . . - . . . . .GG. . . P - . . . . MPPQKENHRTLNKMKTNLFLFL IFSLLLSLSSA . . . . R . . . . . L . . . . . . . . E. . W. . N . E. . . KQPG . . . . . TPGG . . - . . . . PGPT . . M RRHKEVNFVAYLLP SLLVLVSAALAQN . . . . G . . KA . AS . Q . . . . . . W. . N . ND. . . S . N . . . . . - - - . GGG - . G . GP - . . . MKKNRMMMMIWS~VVWMLLLVGGSYG . . . . R . . . . . L . . G . N . . . Q . . W . . . . TD. . . P. N . . . . - . . . . . . . GG. . PAPT . L MKFWG SVLAL S FVVFLFL TGTLA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . QN MEFSGS PMALFCCVFFLFLTG SLA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . QG MNQSTITQNMALTKLS . V . FLC . . . LYS . TVK . . NC. - - . APN . . . . Q . . Y . . . . . A . . . T . - . R . G PCR . . GG . PS P. - - . . . S MSSFGPIFA I LMALACMSSTLVVA . NC . - - .ASN . . . . R . . F . . . . . A . . . E . - . R E G P C R . . SS . . . . . . . . . . S . . . . R . . . . . T . . .N . . . . QY .Y . . . . . T . . . . . L . .G . . .XXX. . . . . . N . . . . X.P . . . Pea A1 Tobacco Arab idops is Potato Bean Petun ia ac id ic Tobacco ac id ic Rape Sugar beet Pea A1 Tobacco Arab idops is Potato Bean Petun ia ac id ic Tobacco ac id ic Rape Sugar beet pPCH3,~ 90 I00 i i0 120 130 140 150 160 170 vGRLvPSSLFDQMLKYRNDGRCAGHGFYTYDA~IAAARSFNGFG~GDDNTKKKELAAFLAQTSHETI~GWPTAPDGPYAWGY~FVSEQN L .S I IS . .M . . . . . . H . . .NA.Q.K . . .S .N . . .N . . . . . P . . . . S .~TAR.R . . . . . F . . . . . . . . . . . A . . . . . . . . . . . . WLR. .G LSGI IS . .Q . .D . . .H . . .AA . PAR . . . . . N . . .T . .K . .P . . . . . . TA .R . . .V . . . FG . . . . . . . . . . A . . . . . . . S . . . . . KQ. . . L .SA ISN.M . . . . . . H . .ENS.Q.KN. .S .N . . .N . . . . . P . . . . S . I .AR .R . . . . . F . . . . . . . . . . . AS . . . . . . . . . . . . LR .RG SA- . ISR .T . . . . . . H . . . . A . PAK . . . . . . . . . . . . KAYPS. .N . .TA .R .R . . . . . . G . . . . . . . . . . A . . . . . . . . . . . . . . R .R . VGSI .T .D . .DQ. . .N . . .A . .FAVR . . . . . . . . . . . N . .P . . . . . . . TAR . . . . . . . FG . . . . . . . . . TLG- . . . . . . G . . . . LR .G- IGS I .TND. .NE . . .N . . . . . . PA . . . . . . . . . . . . . N . .P . . . . . . . TARR . . . . . . FG . . . . . . . . . SLS . - -E .FTG . . . . . R - . - . .S I .TQAF.NGI INQ-AG.G. . .KN. . .R .S . .N . .NT . PN.A . . . . NSVTRREI .TMF.HFT . . .GH . . . . . . . . . . . . . F .Y IE . I . .SSL .TDAF.NRI INQ-ASAS. . .KR . . .RAA.LS .L .FYPQ. .S - .SS .WRREV. . .F .HVT. . .GH . . . . . . . . . . . . . F .Y IE . I - pPCHI pPCH~ 180 190 200 210 220 230 240 260 ~EVYCSP- - -K -DWPCApGKKYYGRGPIQLTHNYNYGLAGQAIKEDL INNPDLLSTNPTVSFKTAIWFWMTPQ-ANKPSSHDVITGRW SPGD. .T . - - -SGQ . . . . . . R . .F . . . . . . IS . . . . . . PC .R . .GV. .L . . . . VA .D.V I . . .S .L . . . . . . . -SP . . .C . . . . I . . . PAS- . .E . - - -SAT . . . . S . .R . . . . . . M . .SW . . . . . . C .R . .GV. .L . . . . VANDAVIA . .A . . . . . . . A . -PP . . .C .A . .A .Q . NPGD. .P . - - -SSQ . . . . . . R . .F . . . . . . IS . . . . . . PC .R . .GV. .L . . . . VA .D.V I . . . . . L . . . . . . . -SP . . .C . . . . I . . . -PST . . .A - - -TPQF . . . . . QQ . . . . . . . . ISW . . . . . QC.R . .GV. .L .K . .VA .DSVI . . .S .L . . . . . A . -SP . . . . . . . . . S . . . . . . . . . . . . . . . . . . NQM.NG . . . . . . . . . . GQS. .D . . .K . .EQ. .V . . . . VA .DA . . . . . . . . . . . . . . . -G . . . . C . . . . . . . . . . . . . . . . . . . . . . . . NDQSDR . . . . . . . . . . NQN. .EK . .N . .RQ. .V . . . . VA .DA. I . . . . . . . . . . . . . -D . . . . . . . . . I .S . GASRDYCDENNR-QY . . . . . . G .F . . . . . . . SW . . . . . AC . .SLNLN. .GQPE.V .S . . . . A .R .GL . . . . N . . . . . . . .VRP .LN- - - -AKSTYC- -QSSAAF. .N .S .Q . . . . . . L . ITW . . . . I P . .RS .GF .GLNAPETVAN.AVTA.R .AF . . . . N . . . . . . . NVHS. .VNG- Pea A1 Tobacco Arab idops is Potato Bean Petun ia ac id ic Tobacco ac id ic Rape Sugar beet 2 270 280 290 300 310 320 TPSAADSSAGHVPGYGVITNI INGGIECGHGQDNRVDDRVGFYKRYCQIFGVDPGGNLDCNNQRSFA Q. .S . .RA .N .L . .F . . . . . . . . . . L . . .R .T .S . .Q. . I . . .R . . .S .L . .S . .D . .G . . . . . G -NGLLVDTM Q. .D . .RA . . . L . . . . . . . . . . . . . L . . .R . . .G . .A . . I . . .Q . . .N . . . . N . . . . . Y . . . . . V -NGLLEAAI N . .S . .RA .N .L . .F . . . . . . . . . . L . . .R .T . . . . Q . . I . . .R . . .S .L . .T. .D. .V. . .W.G-NALLVDTL . . .S . .VA .R .L . . . . TV . . . . . . . L . . .R . . .S . .Q . . I . . F . . . . DLL . .GY .N . .YS .TP .G-NSLLLSDLVTSQ . . . . . . QT .N . . . . . . . . . . . . . . . . . . . K . .NN. .E . . I .Y .R .NVS. I~ .A . .D . .Y . . .N . . -EV . . . . . . Q . .N .A . . . . . . . . . . . . . . . . . V .PNAA.E . . I .Y .R . . .GMLN.A . .D . .Y . . .N . . -QG . . . . . . . . . . . . Q .F .AT IRA. . - .M . .NG.NSGA.NA. IRY .RD. .GQL . . . . . P . .S . - . . . . . . . . . . . . . . . . . . Q .F .AS IRA. . - . . . . NG.NSAA.TA.VGY.~. . .QL . .S . .N . .R . - . . . . . . Ident i ty 68% 66% 65% 63% 53% 51% 43% 43% Fig. 4. Comparison of amino acid sequences between the AI pea chitinase and basic and acidic chitinases. The sequences are derivcd from cDNA clones representing class l a chitinases from tobacco [191, potato [20] and bean [18], acidic class II chitinases from petunia [37] and tobacco [36], and class IV chitinases from Rape [33], sugar beet [31] as well as a genomic clone representing a class la chitinase from Arabidopsis [35]. The conserved motif from which the P2 primer was designed for use in PCR is underlined, and posi- tion of overlap between clone pPCHI and pPCH2 is indicated. Arrow 1 indicates the putative signal peptide cleavage site. Arrow 2 indicates the putative signal peptide for vacuolar targeting. Identities calculated as fraction of identical residues within the region defined by the open reading frame of the combined pea chitinase clones are listed at the C-terminus. K. Vad et al./Plant Sci. 92 (1993) 69-79 that the critical 3' nucleotide is not conserved in the type 4 sequence, although it is present in the type II forms (see Fig. 4). A putative /3-1,3- glucanase transcript from bean cell suspension cul- tures has been cloned recently using a similar strat- egy [34]. Having cloned the 3'-end of the pea chitinase cDNA transcript, the primer extension and RACE procedures were applied, yielding a full-length clone in two steps. Amplification of a 669-bp frag- ment of the chitinase was obtained using an an- tisense oligonucleotide for primer extension. The products from three independent amplifications prepared using cDNA obtained following primer extension were cloned, sequenced and designated pPCH2 (position 103-770 in Fig. 3). The clones were identified as representing the same chitinase as pPCH1 from the overlapping region and con- firmed by Southern blotting performed using both pPCH1 and pPCH2 as probes (Fig. 5). A 470-bp product including the sequence representing the chitinase signal peptide was obtained independent- 75 ly; twice following the RACE protocol. The prod- ucts were cloned, sequenced and designated pPCH3 (position 1-470 in Fig. 3). Combination of the three pea cDNA sequences pPCHI, pPCH2 and pPCH3 results in a 1143 bp chitinase cDNA exhibiting an open reading frame coding for a 34 500 Da polypeptide including an approx. 2500 Da signal peptide (Fig. 3). The de- rived amino acid sequence from the combined clones matched the N-terminal amino acid se- quence of the Al-isoenzyme previously isolated from pea leaves inoculated with A. pisi with the ex- ception of the residues at the fifth and twelfth posi- tions of A1, which were determined with low scores (1 on a scale from 0 to 5) [11; Welinder, pers commun.]. Comparison of the total amino acid composition of chitinase A1 with that deduced from the combined cDNA clones clearly confirm- ed that the sequences represented cDNA for the Al-isoenzyme (data not shown). The combined cDNA clones obtained from the present study revealed a high degree of amino acid sequence (A) Kb (B) 23.0 9.4 6.6 4.3 2.3 2.0 Fig. 5. Genomic Southern blot hybridization of pea cv. Birte. Total DNA (30/~g) was digested with specific restriction enzymes as indicated, electrophoresed through a 0.7% agarose gel, blotted onto nitrocellulose filters and probed with pPCH1 (A) and pPCH2 (B). Washes were carried out under moderate conditions, IxSET at 65°C. 76 K. Vad et al./Plant Sci. 92 (1993) 69-79 identity to the basic chitinase class I sequences from tobacco (68%) [19], Arabidopsis (66%) [35], potato (65%) [20] and bean (63%) [18], as well as to two acidic class II chitinases from tobacco (51%) [36], Petunia (53%) [37] and the basic class IV chitinase from rape (43%) [33] and sugar beet (41%) [311 (Fig. 4). At 15 amino acids residues, the proline-rich hinge region (positions 64-79) is relatively long [3]. A short C-terminal extension of 6-10 amino acids present in the published basic class Ia chitinases is absent in acidic extracellular class II isoforms of chitinase analyzed from tobac- co and Petunia [36,37], and in the sequence of the basic class Ib pea protein. It has been demonstrated that a C-terminal extension of several PR-proteins including a tobacco basic class I chitinase functions as a signal-sequence for vacu- olar targeting [38,39]. This implies that the basic pea A l-chitinase is secreted into the extracellular space of the leaves, which is not common for basic chitinases. This was also indicated by im- munoblots performed on extracellular fluid from inoculated pea leaves which revealed differential accumulation of a product exhibiting similar ap- parent molecular weight as the chitinase A1 [11] and the deduced molecular weight of the combined chitinase cDNA sequence (data not shown). Re- cent results obtained with basic class IV chitinases from sugar beet support our findings: basic chitinases lacking the C-terminal extension are deposited in the intercellular space [31]. The chitinase cDNA inserts from pPCH1 and pPCH2 were used to probe filters carrying five dif- ferent digests of genomic DNA from pea cv. Birte. The two cDNAs revealed identical hybridization patterns (Fig. 5) confirming that these represent HOURS AFTER INOCULAT ION 0 3 6 12 24 72 150 c c i s c i s c i s c i s c i s c i A B I I I I I I C I I I I I I Fig. 6. Northern blot hybridization of total RNA P. sativum cv. Birte leaves inoculated with A. pisi. (i, incompatible interaction with isolate PFI; s, susceptible interaction with isolate PF5) and mock inoculated (c) at the times indicated (hours). 10 #g of total RNA were loaded in each lane. Filters were probed with A. chitinase probe pPCH2, B. carrot extensin probe pDCI 1 and C. pea-chalcone synthase probe pCHS2. The final wash was performed with 0.1xSSC at 42°C. K. Vad et al./Plant Sci. 92 (1993) 69-79 77 the same transcript. The Southern blots also in- dicated that the A 1-chitinase is part of a small gene family of at least three members, although the other members do not appear to be closely related to A1, since considerably less signal corresponding to these other species was detected. RNA samples from pea leaves isolated at dif- ferent timepoints following inoculation with A. pisi were blotted and hybridized with the chitinase cDNA clone pPCH2 (Fig. 6a), carrot extensin clone (Fig. 6b) and the pea CHS clone (Fig. 6c). Similar results were obtained from a further two inoculation experiments. Transcripts representing an approx. 1200 bp chitinase and 2000 bp chalcone synthase mRNAs were detected at elevated levels from 3 h after inoculation of the pea leaves. A generally lower level of chitinase mRNA was detected in mock-inoculated control plants at early time points than in inoculated plants. The RNA blotting data indicates that chitinase transcripts are induced not only by the pathogen but also by general stressing of the plants (by manipulating them in plastic bags at high humidity - - Fig. 6a), and confirm the observed patterns of protein ac- cumulation [11]. Elevated levels of chitinase transcript were prominent in inoculation controls during the first 24 h after inoculation when humid- ity levels were highest, and were lower at 3 and 5 days after inoculation. That the levels detected reflect real differences in transcript levels and not uneven loading of the gel was confirmed by meth- ylene blue staining of the filter prior to hybridiza- tion. Parallel hybridizations with the pea CHS clone, pCHS2, showed that CHS is strongly in- duced in inoculated plants but revealed no expres- sion in control plants indicating no stress induction (Fig. 6c). Chitinase transcript levels are distinctly, although faintly, increased 3 h after in- oculation in the incompatible interaction, whereas elevated levels of mRNA relative to the mock in- oculated control for that time point were first revealed as late as 24 h after inoculation in the compatible interaction (Fig. 6a). Carrot extensin mRNA levels apparently reflected the pattern observed for chitinase, except that a lower signal was generally detected; this may be attributable to the fact that the probe utilized in this hybridiza- tion (Fig. 6b) was not homologous. Accumulation of CHS mRNA was also detected from 3 h after in- oculation, and, again, considerable higher levels of transcript were detected in the incompatible inter- action than in the compatible interaction from 3 h after inoculation and up to 12 h after inoculation. At this time point, the compatible interaction ex- hibited higher levels of accumulation. No signal was detected in control plants (Fig. 6c) implying that there is no major role for the corresponding enzyme in healthy pea leaves. Differential regula- tion between levels of CHS transcript accumula- tion in susceptible and resistant plants is conclusively revealed. Thus the blots revealed syn- chronised increases in mRNA level of three dif- ferent categories of pathogen - - induced genes - - the chitinase gene representing a PR protein, the gene encoding for an enzyme of the isoflavonoid phytoalexin biosynthesis CHS and a cell wall strengthening component-indicating that a general resistance response is triggered during the hyper- sensitive response against A. pisi. These results imply that the resistance mechanism in pea is based on the rapid concomitant induction of func- tionally independent proteins. The same responses are also induced during the compatible interac- tion, but at a much later stage. That the high hu- midity necessary for the establishment of infection with A. pisi also stresses the plants and results in the induction of chitinase and HRGP sequences, would seem to support the view that phytoalexins are more important in this interaction. 4. Acknowledgements We wish to thank the following: P. Stougaard, Danisco A/S, Copenhagen, DK, for preparing oligonucleotides P1 and P2; Dr. Noel Ellis, John Innes Institute, Norwich, UK, for the CHS clone; Dr. Christopher J. Lamb, The Salk Institute for Biological Studies, La Jolla, CA. USA, for the bean chitinase clones; Prof. John Boi and Dr. Harry Hoge, Leiden University, Leiden, The Netherlands, for the tobacco chitinase clones; Dr. Lars Berglund, University of ]k rhus, DK, for the sugar beet chitinase clone; Else Bollerup and Tove Kufa, Pajbjergfonden, Odder, DK, for providing the pea seed and A. pisi isolates used in this study. We are indebted to Ulla Jagd and Tina Haar for 78 K. 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