Methyl jasmonate induces expression of a novel Brassica juncea chitinase with two chitin-binding domains

We have cloned a 1.3 kb Brassica juncea cDNA encoding BjCHI1, a novel acidic chitinase with two chitin-binding domains that shows 62% identity to Nicotiana tabacum Chia1 chitinase. BjCHI1 is structurally unlike Chia1 that has one chitin-binding domain, but resembles Chia5 chitinase UDA1, the precursor of Urtica dioica agglutinin; however there is only 36.9% identity between them. We propose that BjCHI1 should be classified under a new class, Chia7. The spacer and the hinge region of BjCHI1 are proline-rich, like that of Beta vulgaris Ch1, a Chia6 chitinase with half a chitin-binding domain. Northern blot analysis showed that the 1.3 kb BjCHI1 mRNA is induced by wounding and methyl jasmonate (MeJA) treatment but is unaffected by ethylene, salicylic acid (SA) or abscisic acid (ABA). This is the first report on MeJA induction of chitinase gene expression and further suggests that wound-related JA-mediated signal transduction is independent of that involving SA. Western blot analysis using polyclonal antibodies against BjCHI1 showed a cross-reacting band with an apparent molecular mass of 37 kDa in wounded tissues of B. juncea, revealing that, unlike UDA1, BjCHI1 is not cleaved post-translationally at the hinge. Expression of recombinant BjCHI1 in Escherichia coli BL21(DE3) inhibited its growth while crude extracts from E. coli JM109 expressing recombinant BjCHI1 showed chitinase activity. Results from polymerase chain reaction (PCR) suggest that genes encoding chitinases with single or double chitin-binding domains exist in B. juncea.     

Functional analyses of the chitin-binding domains and the catalytic domain of <Emphasis Type="Italic">Brassica juncea</Emphasis> chitinase BjCHI1

We previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains. Synthesis of its mRNA is induced by wounding, methyl jasmonate treatment, Aspergillus niger infection and caterpillar Pieris rapae feeding, suggesting that the protein has a role in defense. In that it possesses two chitin-binding domains, BjCHI1 resembles the precursor of Urtica dioica agglutinin but unlike that protein, BjCHI1 retains its chitinase catalytic domain after post-translational processing. To explore the properties of multi-domain BjCHI1, we have expressed recombinant BjCHI1 and two derivatives, which lack one (BjCHI2) or both (BjCHI3) chitin-binding domains, as secreted proteins in Pichia pastoris. Recombinant BjCHI1 and BjCHI2, showed apparent molecular masses on SDS-PAGE larger than calculated, and could be deglycosylated using -mannosidase. Recombinant BjCHI3, without the proline/threonine-rich linker region containing predicted O-glycosylation sites, did not appear to be processed by -mannosidase. BjCHI1s ability to agglutinate rabbit erythrocytes is unique among known chitinases. Both chitin-binding domains are essential for agglutination; this property is absent in recombinant BjCHI2 and BjCHI3. To identify potential catalytic residues, we generated site-directed mutations in recombinant BjCHI3. Mutation E212A showed the largest effect, exhibiting 0 of wild-type specific activity. H211N and R361A resulted in considerable (>91) activity loss, implying these charged residues are also important in catalysis. E234A showed 36 retention of activity and substitution Y269D, 50. The least affected mutants were E349A and D360A, with 73 and 68 retention, respectively. Like Y269, E349 and D360 are possibly involved in substrate binding rather than catalysis.     

An agglutinating chitinase with two chitin-binding domains confers fungal protection in transgenic potato

Brassica juncea BjCHI1 is a unique chitinase with two chitin-binding domains. Here, we show that, unlike other chitinases, potato-expressed BjCHI1 shows hemagglutination ability. BjCHI1 expression in B. juncea seedlings is induced by Rhizoctonia solani infection, suggesting its protective role against this fungus. To verify this, transgenic potato (Solanum tuberosum L. cv. Desiree) plants expressing BjCHI1 generated by Agrobacterium-mediated transformation were challenged with R. solani. We also transformed potato with a cDNA encoding Hevea brasiliensis -1,3-glucanase, designated HbGLU, and a pBI121-derivative that contains cDNAs encoding both BjCHI1 and HbGLU. In vitro fungal bioassays using Trichoderma viride showed that extracts from transgenic potato lines co-expressing BjCHI1 and HbGLU inhibited fungal growth better than extracts from transgenic potato expressing either BjCHI1 or HbGLU, suggesting a synergistic effect. Consistently, in vivo fungal bioassays with soil-borne R. solani on young transgenic potato plants indicated that the co-expressing plants showed healthier root development than untransformed plants or those that expressed either BjCHI1 or HbGLU. Light microscopy and transmission electron microscopy revealed abundant intact R. solani hyphae and monilioid cells in untransformed roots and disintegrated fungus in the BjCHI1-expressing and the BjCHI1 and HbGLU co-expressing plants. Observations of collapsed epidermal cells in the co-expressing potato roots suggest that these proteins effectively degrade the fungal cell wall, producing elicitors that initiate other defense responses causing epidermal cell collapse that ultimately restricts further fungal penetration.     

Brassica juncea chitinase BjCHI1 inhibits growth of fungal phytopathogens and agglutinates Gram-negative bacteria

Brassica juncea BjCHI1 is a plant chitinase with two chitin-binding domains. Its expression, induced in response to wounding, methyl jasmonate treatment, Aspergillus niger infection, and caterpillar Pieris rapae feeding, suggests that it plays a role in defence. In this study, to investigate the potential of using BjCHI1 in agriculture, Pichia-expressed BjCHI1 and its deletion derivatives that lack one or both chitin-binding domains were tested against phytopathogenic fungi and bacteria. Transplastomic tobacco expressing BjCHI1 was also generated and its extracts assessed. In radial growth-inhibition assays, BjCHI1 and its derivative with one chitin-binding domain showed anti-fungal activities against phytopathogens, Colletotrichum truncatum, C. acutatum, Botrytis cinerea, and Ascochyta rabiei. BjCHI1 also inhibited spore germination of C. truncatum. Furthermore, BjCHI1, but not its derivatives lacking one or both domains, inhibited the growth of Gram-negative bacteria (Escherichia coli, Ralstonia solanacearum, Pseudomonas aeruginosa) more effectively than Gram-positive bacteria (Micrococcus luteus and Bacillus megaterium), indicating that the duplicated chitin-binding domain, uncommon in chitinases, is essential for bacterial agglutination. Galactose, glucose, and lactose relieved agglutination, suggesting that BjCHI1 interacts with the carbohydrate components of the Gram-negative bacterial cell wall. Retention of chitinase and bacterial agglutination activities in transplastomic tobacco extracts implicates that BjCHI1 is potentially useful against both fungal and bacterial phytopathogens in agriculture.     

A Brassica juncea chitinase with two-chitin binding domains show anti-microbial properties against phytopathogens and Gram-negative bacteria

In our recent paper in the Journal of Experimental Botany, we demonstrated that Brassica juncea BjCHI1 shows anti-fungal properties against phytopathogens, Colletotrichum truncatum, C. acutatum, Botrytis cinerea and Ascochyta rabiei. Furthermore, BjCHI1 which is an unusual plant chitinase with two (almost identical) chitin-binding domains, agglutinates Gram-negative bacteria, adversely affecting their growth. In contrast, BjCHI1 derivatives lacking one or both domains do not show agglutination activity, suggesting that both chitin-binding domains are essential for agglutination. Observations that agglutination could be relieved by addition of galactose, glucose or lactose, imply that BjCHI1 interacts with the carbohydrate components of the Gram-negative bacterial cell wall. We propose here, a model for BjCHI1-mediated agglutination between Gram-negative bacteria, through interaction of their adjacent cell walls mediated by the two chitin-binding domains of BjCHI1. BjCHI1 is a plant chitinase which has evolved towards acquiring an enhanced role in plant defense against fungi and Gram-negative bacteria. Hence, it is a promising candidate for applications against phytopathogens in plant genetic engineering via nuclear or plastid transformation.Key words: bacterial agglutination, chitin-binding domain, Indian mustard, lectin, phytopathogens, Pichia-expressed proteins, transplastomic tobacco     

Molecular cloning and characterization of the promoter for the multiple stress-inducible gene <Emphasis Type="Italic">BjCHI1</Emphasis> from <Emphasis Type="Italic">Brassica juncea</Emphasis>

We have previously isolated a Brassica juncea cDNA encoding a novel chitinase BjCHI1 with two chitin-binding domains (Zhao and Chye in Plant Mol Biol 40:1009–1018, 1999). The expression of BjCHI1 was highly inducible by methyl jasmonate (MeJA) treatment, wounding, caterpillar feeding, and pathogenic fungal infection. These observations suggest that the promoter of BjCHI1 gene might contain specific cis-acting elements for stress responses. Here, we report the cloning and characterization of the BjCHI1 promoter. A 1,098 bp BjCHI1 genomic DNA fragment upstream of the ATG start codon was isolated by PCR walking and various constructs were made by fusing the BjCHI1 promoter or its derivatives to β-glucuronidase reporter gene. The transgenic Arabidopsis plants showed that the BjCHI1 promoter responded to wounding and MeJA treatment, and to treatments with either NaCl or polyethyleneglycol (PEG 6000), indicating that the BjCHI1 promoter responses to both biotic and abiotic stresses. A transient gene expression system of Nicotiana benthamiana leaves was adopted for promoter deletion analysis, and the results showed that a 76 bp region from −695 to −620 in the BjCHI1 promoter was necessary for MeJA-responsive expression. Furthermore, removal of a conserved T/G-box (AACGTG) at −353 to −348 of the promoter greatly reduced the induction by MeJA. This is the first T/G-box element identified in a chitinase gene promoter. Gain-of-function analysis demonstrated that the cis-acting element present in the 76 bp region requires coupling with the T/G-box to confer full magnitude of BjCHI1 induction by MeJA.     

The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.     

兼具凝集素活性的芥菜几丁质酶BjCHI1

利用毕赤酵母表达系统表达芥菜几丁质酶基因BjCHI1及其两个衍生基因BjCHI2和BjCHI3,获得相应的蛋白质。经FPLC纯化后,测定了3种蛋白质的几丁质酶活性,发现它们均能降解CM-chitin-RBV和胶状几丁质。以CM-chitin-RBV为底物时的Km值分别为0.799mg/mL、0.544mg/mL和0.793mg/mL,差别甚微。而以胶状几丁质为底物时的Km值分别为0.281mg/mL、0.388mg/mL和1.643mg/mL,表现一定的差别,说明几丁质结合域影响了酶对不溶性底物的亲和力。3种蛋白中,只有BjCHI1在33μg/mL以上浓度具有凝集素活性,而BjCHI2和BjCHI3的浓度即使高达800μg/mL也无凝集素活性,表明2个几丁质结合域是BjCHI1具有凝集素活性的必需条件,这是植物中发现的第一个兼有几丁质酶和凝集素活性的蛋白质。      

The Saccharomyces cerevisiae chitinase,encoded by the CTS1-2 gene,confers antifungal activity against Botrytis cinerea to Transgenic tobacco

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene has recently been confirmed by in vitro tests to possess antifungal abilities. In this study, the CTS1-2 gene has been evaluated for its in planta antifungal activity by constitutive overexpression in tobacco plants to assess its potential to increase the plant's defence against fungal pathogens. Transgenic tobacco plants, generated by Agrobacterium-mediated transformation, showed stable integration and inheritance of the transgene. Northern blot analyses conducted on the transgenic tobacco plants confirmed transgene expression. Leaf extracts from the transgenic lines inhibited Botrytis cinerea spore germination and hyphal growth by up to 70% in a quantitative in vitro assay, leading to severe physical damage on the hyphae. Several of the F₁ progeny lines were challenged with the fungal pathogen, B. cinerea, in a detached leaf infection assay, showing a decrease in susceptibility ranging from 50 to 70%. The plant lines that showed increased disease tolerance were also shown to have higher chitinase activities.     

Properties of Manduca sexta chitinase and its C-terminal deletions

Manduca sexta (tobacco hornworm) chitinase is a molting enzyme that contains several domains including a catalytic domain, a serine/threonine-rich region, and a C-terminal cysteine-rich domain. Previously we showed that this chitinase acts as a biopesticide in transgenic plants where it disrupts gut physiology. To delineate the role of these domains further and to identify and characterize some of the multiple forms produced in molting fluid and in transgenic plants, three different forms with variable lengths of C-terminal deletions were generated. Appropriately truncated forms of the M. sexta chitinase cDNA were generated, introduced into a baculovirus vector, and expressed in insect cells. Two of the truncated chitinases (Chi 1-407 and Chi 1-477) were secreted into the medium, whereas the one with the longest deletion (Chi 1-376) was retained inside the insect cells. The two larger truncated chitinases and the full-length enzyme (Chi 1-535) were purified and their properties were compared. Differences in carbohydrate compositions, pH–activity profiles, and kinetic constants were observed among the different forms of chitinases. All three of these chitinases had some affinity for chitin, and they also exhibited differences in their ability to hydrolyze colloidal chitin. The results support the hypothesis that multiple forms of this enzyme occur in vivo due to proteolytic processing at the C-terminal end and differential glycosylation.     

Enhanced fungal resistance in Arabidopsis expressing wild rice PR-3 (OgChitIVa) encoding chitinase class IV

Oryza grandiglumis Chitinase IVa (OgChitIVa) cDNA encoding a class IV chitinase was cloned from wild rice (Oryza grandiglumis). OgChitIVa cDNA contains an open reading frame of 867 nucleotides encoding 288 amino acid residues with a predicted molecular weight of 30.4 kDa and isoelectric point of 8.48. Deduced amino acid sequences of OgChitIVa include the signal peptide and chitin-binding domain in the N-terminal domain and conserved catalytic domain. OgChitIVa showed significant similarity at the amino acid level with related monocotyledonous rice and maize chitinase, but low similarity with dicotyledoneous chitinase. Southern blot analysis showed that OgChitIVa genes are present as two copies in the wild rice genome. It was shown that RNA expression of OgChitIVa was induced by defense/stress signaling chemicals, such as jasmonic acid, salicylic acid, and ethephon or cantharidin and endothall or wounding, and yeast extract. It was demonstrated that overexpression of OgChitIVa in Arabidopsis resulted in mild resistance against the fungal pathogen, Botrytis cinerea, by lowering disease rate and necrosis size. RT-PCR analysis showed that PR-1 and PR-2 RNA expression was induced in the transgenic lines. Here, we suggest that a novel OgChitIVa gene may play a role in signal transduction process in defense response against B. cinerea in plants. J.-H. Pak and E.-S. Chung contributed equally to this work.     

Kinetics of prolyl hydroxylation,intracellular transport and C-terminal processing of the tobacco vacuolar chitinase

The dynamics of intracellular transport and processing of one of the vacuolar chitinases of tobacco (Nic-otiana tabacum L.), chitinase A (CHN A; EC 3.2.1.14), was investigated with pulse-chase experiments in conjunction with cell fractionation and immunoprecipitation. Mature CHN A is composed of two domains, the N-terminal cysteine-rich chitin-binding domain and the catalytic domain, linked by a short peptide spacer containing several hydroxyprolines. It is synthetized as a preproprotein with a signal peptide for cotranslational transport into the endoplasmic reticulum (ER) and a C-terminal, vacuolar targeting peptide (VTP) required for targeting to the vacuole, which is removed by proteolytic cleavage. We investigated transformed N. sylvestris plants constitutively expressing CHN A or a mutant CHN A lacking the chitin-binding domain and spacer (CS CHN A), as well as N. plumbaginifolia protoplasts transiently expressing the same constructs. Processing and transport in the two systems was very similar. A shift in the apparent molecular weight of chitinase, indicative of prolyl hydroxylation, was detectable only 30 min after appearance of newly synthesized prochitinase, indicating that it might occur in a post-ER compartment. In total, labelled chitinase was detected in the microsomal fraction for up to 90–120 min as a prochitinase, bearing the VTP. Later, it appeared only in the soluble fraction (comprising the vacuolar sap) as the mature CHN A without the VTP. In both systems, intracellular transport and processing of CS CHN A was faster than that of the wildtype form, indicating that correct folding of the cysteine-rich chitin-binding domain and/or prolyl hydroxylation of the spacer delays transport to the vacuole.Abbbreviations CBD chitin-binding domain - CHN A chitinase A - PBS phosphate-buffered saline - S proline-rich spacer - VTP vacuolar targeting peptide - CS deletion of CBD and S; - VTP deletion of VTP We thank M. Müller and T. Hohn, Friedrich Miescher-Institute, Basel, for the preparation of the protoplasts and F. Fischer, Friedrich Miescher-Institute, Basel, for the synthesis of the peptide. This work was supported by the Swiss National Science Foundation, Grants 31-26402.89 and 3100-037434.93.     

Transgenic Tobacco Plants Constitutively Overexpressing a Rice Thaumatin-like Protein (PR-5) Show Enhanced Resistance to Alternaria alternata

Overexpression of antifungal pathogenesis-related (PR) proteins in crop plants has the potential for enhancing resistance against fungal pathogens. Thaumatin-like proteins (TLPs) are one group (PR-5, permatins) of antifungal PR-proteins isolated from various plants. In the present study, a plasmid containing a cDNA of rice tlp (D34) under the control of the CaMV-35S promoter was introduced into tobacco plants through Agrobacterium-mediated transformation system. A considerable overproduction of TLP was observed in transformed tobacco plants by Western blot analysis. There was a large accumulation of tlp mRNA in transgenic plants as revealed by Northern blot analysis. Southern blot analysis of the DNA from transgenic tobacco plants confirmed the presence of the rice tlp gene in the genomic DNA of transgenic tobacco plants. Immunoblot analysis of intracellular and extracellular proteins of transgenic tobacco leaves using a Pinto bean TLP antibody demonstrated that the 23-kDa TLP was secreted into the extracellular matrix. T₂ progeny of regenerated plants transformed with TLP gene were tested for their disease reaction to Alternaria alternata, the brown spot pathogen. Transgenic tobacco plants expressing TLP at high levels showed enhanced tolerance to necrotization caused by the pathogen. This revised version was published online in July 2006 with corrections to the Cover Date.     

Properties of catalytic,linker and chitin-binding domains of insect chitinase

Manduca sexta (tobacco hornworm) chitinase is a glycoprotein that consists of an N-terminal catalytic domain, a Ser/Thr-rich linker region, and a C-terminal chitin-binding domain. To delineate the properties of these domains, we have generated truncated forms of chitinase, which were expressed in insect cells using baculovirus vectors. Three additional recombinant proteins composed of the catalytic domain fused with one or two insect or plant chitin-binding domains (CBDs) were also generated and characterized. The catalytic and chitin-binding activities are independent of each other because each activity is functional separately. When attached to the catalytic domain, the CBD enhanced activity toward the insoluble polymer but not the soluble chitin oligosaccharide primarily through an effect on the Km for the former substrate. The linker region, which connects the two domains, facilitates secretion from the cell and helps to stabilize the enzyme in the presence of gut proteolytic enzymes. The linker region is extensively modified by O-glycosylation and the catalytic domain is moderately N-glycosylated. Immunological studies indicated that the linker region, along with elements of the CBD, is a major immunogenic epitope. The results support the hypothesis that the domain structure of insect chitinase evolved for efficient degradation of the insoluble polysaccharide to soluble oligosaccharides during the molting process.     

Purification,cDNA cloning,and characterization of plant chitinase with a novel domain combination from lycophyte Selaginella doederleinii

Chitinase-A from a lycophyte Selaginella doederleinii (SdChiA), having molecular mass of 53 kDa, was purified to homogeneity by column chromatography. The cDNA encoding SdChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1477 nucleotides and its open reading frame encoded a polypeptide of 467 amino acid residues. The deduced amino acid sequence indicated that SdChiA consisted of two N-terminal chitin-binding domains and a C-terminal plant class V chitinase catalytic domain, belonging to the carbohydrate-binding module family 18 (CBM18) and glycoside hydrolase family 18 (GH18), respectively. SdChiA had chitin-binding ability. The time-dependent cleavage pattern of (GlcNAc)₄ by SdChiA showed that SdChiA specifically recognizes the β-anomer in the + 2 subsite of the substrate (GlcNAc)₄ and cleaves the glycoside bond at the center of the substrate. This is the first report of the occurrence of a family 18 chitinase containing CBM18 chitin-binding domains.

Abbreviations: AtChiC: Arabidopsis thaliana class V chitinase; CBB: Coomassie brilliant blue R250; CBM: carbohydrate binding module family; CrChi-A: Cycas revolute chitinase-A; EaChiA: Equisetum arvense chitinase-A; GH: glycoside hydrolase family, GlxChi-B: gazyumaru latex chitinase-B; GlcNAc: N-acetylglucosamine; HPLC: high performance liquid chromatography; LysM; lysin motif; MtNFH1: Medicago truncatula ecotypes R108-1 chitinase; NCBI: national center for biotechnology information; NF: nodulation factor; NtChiV: Nicotiana tabacum class V chitinase; PCR: polymerase chain reaction; PrChi-A: Pteris ryukyuensis chitinase-A; RACE: rapid amplification of cDNA ends; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SdChiA: Selaginella doederleinii chitinase-A.     

Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides: implications for the catalytic mechanism

This research describes four X-ray structures of Vibrio harveyi chitinase A and its catalytically inactive mutant (E315M) in the presence and absence of substrates. The overall structure of chitinase A is that of a typical family-18 glycosyl hydrolase comprising three distinct domains: (i) the amino-terminal chitin-binding domain; (ii) the main catalytic (α/β)₈ TIM-barrel domain; and (iii) the small (α + β) insertion domain. The catalytic cleft of chitinase A has a long, deep groove, which contains six chitooligosaccharide ring-binding subsites (−4)(−3)(−2)(−1)(+1)(+2). The binding cleft of the ligand-free E315M is partially blocked by the C-terminal (His)₆-tag. Structures of E315M-chitooligosaccharide complexes display a linear conformation of pentaNAG, but a bent conformation of hexaNAG. Analysis of the final 2F_o − F_c omit map of E315M-NAG6 reveals the existence of the linear conformation of the hexaNAG at a lower occupancy with respect to the bent conformation. These crystallographic data provide evidence that the interacting sugars undergo conformational changes prior to hydrolysis by the wild-type enzyme.     

A family 19 chitinase (Chit30) from Streptomyces olivaceoviridis ATCC 11238 expressed in transgenic pea affects the development of T. harzianum in vitro

Embryo axes excised from mature seeds of pea (Pisum sativum L.) cv. ‘Sponsor’ were used as explants for Agrobacterium-mediated transformation using pGreenII 0229 binary vectors. The vectors harbored a chimeric chitinase gene (chit30), driven by the constitutive 35S promoter or the elicitor inducible stilbene synthase (vst) promoter from grape (Vitis vinifera L.). The secretion signal of the bacterial chitinase gene from Streptomyces olivaceoviridis ATCC 11238 (DSM 41433) was replaced by the A. thaliana basic chitinase leader sequence. Functional properties of the recombinant gene were tested in tobacco as a model system before the long process of pea transformation was undertaken. Several transgenic pea clones were obtained and the transgenic nature confirmed by different molecular methods. The accumulation and activity of chitinase in stably transformed plants were examined by Western blot analysis and in-gel assays, which showed the presence of an additional 3 isoform bands. Using in vitro bioassays with Trichoderma harzanium as a model, we found an inhibition or delay of hyphal extension, which might indicate enhanced antifungal activity compared with non-transformed pea plants. Up to the 4th generation, the transgenic plants did not show any phenotypic alterations compared with non-transgenic control plants.