Chitinase and β-1,3-glucanase activities in chickpea (Cicer arietinum). Induction of different isoenzymes in response to wounding and ethephon

Chitinase and β-1,3-glucanase activities were assayed in roots, stems and leaves of 12-day-old chickpea ( Cicer arietinum L.) plants. While glucanase activity was higher in roots than in the aerial parts of the plant, leaves had higher Chitinase activity. Both glucanase and chitinase activities were induced in roots and stems in response to wounding (excision into 1-cm pieces), with activity increasing 6 h after treatment, reaching a maximum between 24 and 48 h, and thereafter remaining nearly constant up to 72 h. Ethephon treatment also induced β-1,3-glucanase and chitinase activities in stems but not in roots. Both enzymes occurred in root and stem tissues as a complex mixture of isoenzymes. At least four different peaks with glucanase and chitinase activities could be resolved by gel filtration chromatography on Sephacryl S-200 and chromatofocusing on PBE 94 (pH 4–7). Following ammonium sulfate precipitation and ion exchange on CM- and DEAE-Trisacryl, three β-1,3-glucanase and chitinase fractions, referred to as basic, neutral and acidic, were separated on the basis of their chromatographic behaviour. Most of the total protein (75%) of stem extracts was found in the acidic fraction, whereas the major glucanase (53%) and chitinase (62%) activities were in the basic and neutral fractions, respectively. While wounding resulted in an increase in the neutral glucanase and chitinase activities, the activities of the acidic fractions were promoted by ethephon.     

Characterization, induction by wounding and salicylic acid, and activity against Botrytis cinerea of chitinases and β‐1,3‐glucanases of ripening grape berries

In order to better understand the defense strategy of grape berries ( Vitis vinifera L. cv. Pinot noir) as they mature, the activities of the defense‐related proteins, chitinase (CHV, EC 3.2.1.14) and β‐1,3‐glucanase (laminarinase, EC 3.2.1.39) were first estimated in berries at different maturation stages. Chitinase levels rose proportionally to the berry reducing sugar content, an indicator of the berry ripening degree, up to values 10 times higher than the ones seen in resting grapevine leaves. This rise in activity was due to the accumulation of two isoforms, CHV 5 and CHV 11. One more chitinase isoform, CHV 12, appeared in senescent berries. Conversely, no glucanase activity could be detected in berries at any maturation stage. Accumulation of chitinases and (β‐1,3‐glucanases could be stimulated by wounding the berry peduncle. Adding salicylic acid to the wounded berries only potentiated the wounding effect on the berry chitinase activity. The most active chitinase isoform, CHV 5, was purified to homogeneity. It represented about 40% of the total extractable protein content of a ripe berry. Its molecular mass was estimated to be 31 kDa. The peptide sequencing of four of its tryptic fragments revealed strong homologies to several class IV chitinases. Finally, it was shown to inhibit the germination of conidia of Botrytis cinerea by 50% at a concentration of 7.5 µg ml⁻¹.     

Effects of fungal infection and wounding on the expression of chitinases and β-1,3 glucanases in near-isogenic lines of barley

Chitinases (EC 3.2.1.14) and β -1.3 glucanases (EC 3.2.1.39) have been known to play a vital role in the defense of plants against fungal pathogens. The pattern of induction of these two enzymes subsequent to infection by powdery mildew was studied in 10 pairs of near-isogenic lines of barley ( Hordeum vulgare L.) which possess powdery mildew resistance genes. These isogenic lines have been grotiped according to their reaction to the fungus. The induction patterns varied between the resistant and the susceptible cultivars within each group and between different groups. More tsozymcs were induced in susceptible varieties of highly resistant groups and the overall levels and the number of isozymes of chitinases and β -1.3 glucanases were lower in groups with low resistance. The effect of powdery mildew infection and mechanical wounding on the cellular localization of chitinases and β -1.3 glucanases in barley leaves has also been studied. The 31 kDa leaf chitinase, L-CH2, and trace amounts of a 25 kDa chitinase. L-CH3. were present in healthy leaves. Wounding increased the levels of L-CH3 within I ft h. Powdery mildew infection increased the levels of L-CH3 both in intercellular fluid and in intracellular extract of leaves. A /3-I.3 glucanase. GH, also increased after infection and wounding. In infected barley leaves, GL-1 was present both in intercellular space and intracellular extract. It is concluded that powdery mildew resistance genes exhibit qualitative and quantitative differences in the expression of chitinases and β -1.3 glucanases. Further, chitinases and β -1.3 glucanases appear to be a response to active infection rather than the factors responsible for disease resistance.     

Hyphal in vitro growth of the arbuscular mycorrhizal fungus Glomus mosseae is affected by chitinase but not by β-1,3-glucanase

Purified basic chitinase or #-1,3-glucanase or a combination of the two enzymes were applied to hyphae of the arbuscular mycorrhizal fungus Glomus mosseae grown in vitro. Chitinase applied to the hyphal tip produced an inhibition of hyphal extension, lysis of the apex and alterations of the growth pattern of the fungus. No effect was observed, however, when chitinase was applied to subapical parts of the hyphae or when glucanase was applied to any part of the hyphae. Application of a combination of the two enzymes to the hyphal tip produced an effect similar to that of chitinase alone.     

Fungus fruit body lytic enzyme from a myxomycete,Badhamia utricularis

Chitinase,β-1,3-glucanase, cellulase, xylanase and protease activity were detected in a crude enzyme preparation obtained from a slime mold (Badhamia utricularis) which was grown on autoclaved mycelia ofPholiota nameko in a petri dish. The optimal pH of the enzyme preparation for lytic activity against fruit bodies ofLentinus edodes was 4.0, and those ofβ-1,3-glucanase and cellulase were the same. On the other hand, chitinase and protease showed optimal activity at pH 5.0 and 8.0, respectively. The lytic activity was stable below 40°C but completely inactivated at 70°C, and was most stable at pH 5.0. The studies of the optimal pH, thermal stability, and pH stability, and isoelectric focusing analysis of the enzyme preparation suggest that chitinase,β-1,3-glucanase and cellulase activities may be responsible for lysis of fruit bodies of some mushrooms. The crude enzyme preparation from the slime mold lysed fruit bodies of several mushrooms more efficiently than did commercial lytic enzymes preparations (Driselase and Usukizyme).     

Variability in antifungal proteins in the grains of maize, sorghum and wheat

Crude protein extracts were made from kernels of 12 cultivars each of maize, sorghum and wheat. These preparations were fractionated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and subjected to Western blot analyses. Bands corresponding to chitinases and β-glucanases were identified immunologically (Western blots) and on activity gels. Ribosome Inactivating Protein(s) (RIP) and permatins were identified immunologically. In maize, two chitinase bands (25–29 kDa) were seen in all cultivars, and a third band of about 23 kDa was detected in 7 of the 12 cultivars. Two or three β-glucanase bands of sizes between 24 and 36 kDa (probably a mixture of 1,3–β- and 1,3–1,4-β-glucanases) were detected in blots of SDS gels, and one band was detected in activity gels (1,3-β-glucanase). In sorghum, one chitnase band of approximately 29 kDa, and two or three additional bands ranging in size from 21–24 kDa were observed. Only one β-glucanase band was identified, with an estimated molecular weight of 30 kDa. All bands that appeared on Western blots of SDS gels corresponded to bands detected on activity gels. In wheat, one chitmase band of around 20 kDa, one β-glucanase band of about 30 kDa and one RIP band of about 30 kDa were identified. Permatins (molecular weight about 22 kDa) were identified in maize, sorghum and wheat, with the different cultivars having varying amounts of permatins.     

Inhibition of fungal growth by plant chitinases andβ-1,3-glucanases

Summary Plant chitinases and -1,3-glucanases have been demonstrated to inhibit fungal growth in model experiments, both on agar plates or in liquid media. Here,Trichoderma longibrachiatum was taken as a model to study the morphological changes caused by chitinase and glucanase treatments, using cytochemical techniques in combination with fluorescence and electron microscopy. Chitinase, alone or in the presence of glucanase, arrested growth of the hypha: it affected the extreme tip of the fungus producing a thinning of the wall, a balloon-like swelling and a rupture of the plasma membrane. Chitin and glucans were present in the wall, as shown by lectinand enzyme-binding experiments, but they had a different susceptibility to chitinase and -1,3-glucanase. Chitin was present at the apex and in the inner parts of the lateral walls; it was more susceptible to chitinase at the tip than in the subapical part. Glucans mostly occurred on the outer layer where they were degraded by glucanase. The latter did not affect the inner hyphal skeleton. It is suggested that the growth inhibition ofTrichoderma by hydrolytic enzymes is the consequence of a thinning of the cell wall in the hyphal apex, leading to an imbalance of turgor pressure and wall tension which causes the tip to swell and to burst.Abbreviations WGA-FITC wheat germ agglutinin labelled with fluorescein isothiocyanate - ConA-FITC concanavalin A labelled with fluorescein isothiocyanate - PEG polyethylene glycol - SEM scanning electron microscopy - TEM transmission electron microscopy     

A chitinase indispensable for formation of protoplast of Schizophyllum commune in basidiomycete-lytic enzyme preparation produced by Bacillus circulans KA-304

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia. alpha-1,3-Glucanase, which was isolated from an ammonium sulfate fraction of 0-30% saturation of KA-prep, gave the protoplast-forming activity to an ammonium sulfate fraction of 30-50% saturation of KA-prep, which contained chitinase(s) and beta-glucanase(s) but was inactive in the protoplast formation. Chitinase(s) and beta-glucanase(s) in the ammonium sulfate fraction of 30-50% saturation were separated by DEAE-cellulofine A-500 column chromatography, and the protoplast-forming activity appeared when the chitinase preparation was mixed with the alpha-1,3-glucanase. The beta-glucanase preparation was not effective for the protoplast formation whereas its addition enhanced the protoplast-forming activity of the mixture of alpha-1,3-glucanase and the chitinase preparation. The chitinase preparation contained two chitinases (chitinase I and II). Chitinase I showed the protoplast-forming activity with alpha-1,3-glucanase, but chitinase II did not. Chitinase I, a monomeric protein with a molecular weight of 41,000, was active toward colloidal chitin and ethylene glycol chitin. Chitinase I produced predominantly N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose from colloidal chitin, and the enzyme was inactive to p-NP-beta-D-N-acetylglucosaminide, suggesting that it was an endo-type enzyme. The N-terminal amino acid sequence of chitinase I (A L A T P T L N V S A S S G M) had no sequential identity to those of known chitinases.     

Antifungal Hydrolases in Pea Tissue : I. Purification and Characterization of Two Chitinases and Two beta-1,3-Glucanases Differentially Regulated during Development and in Response to Fungal Infection

Chitinase and β-1,-3-glucanase activities increased coordinately in pea (Pisum sativum L. cv “Dot”) pods during development and maturation and when immature pea pods were inoculated with compatible or incompatible strains of Fusarium solani or wounded or treated with chitosan or ethylene. Up to five major soluble, basic proteins accumulated in stressed immature pods and in maturing untreated pods. After separation of these proteins by chromatofocusing, an enzymic function could be assigned to four of them: two were chitinases and two were β-1,3-glucanases. The different molecular forms of chitinase and β-1,3-glucanase were differentially regulated. Chitinase Ch1 (mol wt 33,100) and β-1,3-glucanase G2 (mol wt 34,300) were strongly induced in immature tissue in response to the various stresses, while chitinase Ch2 (mol wt 36,200) and β-1,3-glucanase G1 (mol wt 33,500) accumulated during the course of maturation. With a simple, three-step procedure, both chitinases and both β-1,3-glucanases were purified to homogeneity from the same extract. The two chitinases were endochitinases. They differed in their pH optimum, in specific activity, in the pattern of products formed from [³H]chitin, as well as in their relative lysozyme activity. Similarly, the two β-1,3-glucanases were endoglucanases that showed differences in their pH optimum, specific activity, and pattern of products released from laminarin.     

Purification,characterization and differential hormonal regulation of a β-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) cell-suspension cultures were used to isolate one -1,3-glucanase (EC 3.2.1.29) and two chitinases (EC 3.2.1.14). The -1,3-glucanase (M_r = 36 kDa) and one of the chitinases (M_r = 32 kDa) belong to class I hydrolases with basic isoelectric points (10.5 and 8.5, respectively) and were located intracellularly. The basic chitinase (BC) was also found in the culture medium. The second chitinase (M_r = 28 kDa), with an acidic isoelectric point of 5.7, showed homology to N-terminal sequences of class III chitinases and represented the main protein accumulating in the culture medium. Polyclonal antibodies raised against the basic -1,3-glucanase (BG) and the acidic chitinase (AC) were shown to be monospecific. The anti-AC antiserum failed to recognize the BC on immune blots, confirming the structural diversity between class I and class III chitinases. Neither chitinase exhibitied lysozyme activity. All hydrolases were endo in action on appropriate substrates. The BC inhibited the hyphal growth of several test fungi, whereas the AC failed to show any inhibitory activity. Expression of BG activity appeared to be regulated by auxin in the cell culture and in the intact plant. In contrast, the expression of neither chitinase was apparently influenced by auxin, indicating a differential hormonal regulation of -1,3-glucanase and chitinase activities in chickpea. After elicitation of cell cultures or infection of chickpea plants with Ascochyta rabiei, both system were found to have hydrolase patterns which were qualitatively and quantitatively comparable. Finally, resitant (ILC 3279) and susceptible (ILC 1929) cultivars of chickpea showed no appreciable differences with regard to the time and amount of hydrolase accumulation after inoculation with spores of A. rabiei.Abbreviations AC acidic chitinase - BC basic chitinase - BG = basic -1,3-glucanase - CM-Chitin-RBV carboxymethylated-chitin-remazol brilliant violet - 2,4-D 2,4-dichlorophenoxyacetic acid - ILC international legume chickpea - M_r relative molecular mass - pI isoelectric point - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis We thank the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie for financial support and ICARDA, Aleppo, Syria, for the provision of seed material. We also thank Dr. B. Fritig (Institut de Biologie Moléculaire des Plantes, CNRS, Straßbourg, France) and Dr. F. Meins, Jr. (Friedrich-Miescher-Institut, Basel, Switzerland) for their kind gifts of antibodies.     

Purification and characterization of a β-1,3-glucanase from the novel mycoparasite <Emphasis Type="Italic">Periconia byssoides</Emphasis>

An extracellular β-1,3-glucanase with antifungal properties was secreted by the novel mycoparasite, Periconia byssoides. The glucanase has a molecular mass of 35 kDa estimated by SDS-PAGE. Its optimum activity was at pH 6.0 and 50°C (over 2 h). The purified β-1,3-glucanase was capable of degrading cell walls, and inhibiting mycelia growth and spore germination of plant pathogenic fungi including Fulvia fulva, Fusarium sp. and Rhizoctonia solani. The N-terminal amino acid residues of the purified β-1,3-glucanase are LKNGGPSFGA, which do not have any homology with previously described glucanases, suggesting it may be a novel member of the fungal β-1,3-glucanases. Chao Lin and Jinkui Yang contributed equally to this work.     

Pathogenesis-related proteins protect extrafloral nectar from microbial infestation

Plants in more than 300 genera produce extrafloral nectar (EFN) to attract carnivores as a means of indirect defence against herbivores. As EFN is secreted at nectaries that are not physically protected from the environment, and contains carbohydrates and amino acids, EFN must be protected from infestation by micro-organisms. We investigated the proteins and anti-microbial activity in the EFN of two Central American Acacia myrmecophytes ( A. cornigera and A. hindsii ) and two related non-myrmecophytes ( A. farnesiana and Prosopis juliflora ). Acacia myrmecophytes secrete EFN constitutively at high rates to nourish the ants inhabiting these plants as symbiotic mutualists, while non-myrmecophytes secrete EFN only in response to herbivore damage to attract non-symbiotic ants. Thus, the quality and anti-microbial protection of the EFN secreted by these two types of plants were likely to differ. Indeed, myrmecophyte EFN contained significantly more proteins than the EFN of non-myrmecophytes, and was protected effectively from microbial infestation. We found activity for three classes of pathogenesis-related (PR) enzymes: chitinase, β-1,3-glucanase and peroxidase. Chitinases and β-1,3-glucanases were significantly more active in myrmecophyte EFN, and chitinase at the concentrations found in myrmecophyte EFN significantly inhibited yeast growth. Of the 52 proteins found in A. cornigera EFN, 28 were annotated using nanoLC-MS/MS data, indicating that chitinases and glucanases contribute more than 50% of the total protein content in the EFN of this myrmecophyte. Our study demonstrates that PR enzymes play an important role in protecting EFN from microbial infestation.     

Only Specific Tobacco (Nicotiana tabacum) Chitinases and [beta]-1,3-Glucanases Exhibit Antifungal Activity

Different isoforms of chitinases and [beta]-1,3-glucanases of tobacco (Nicotiana tabacum cv Samsun NN) were tested for their antifungal activities. The class I, vacuolar chitinase and [beta]-1,3-glucanase isoforms were the most active against Fusarium solani germlings, resulting in lysis of the hyphal tips and in growth inhibition. In additon, we observed that the class I chitinase and [beta]-1,3-glucanase acted synergistically. The class II isoforms of the two hydrolases exhibited no antifungal activity. However, the class II chitinases showed limited growth inhibitory activity in combination with higher amounts of class I [beta]-1,3-glucanase. The class II [beta]-1,3-glucanases showed no inhibitory activity in any combination. In transgenic tobacco plants producing modified forms of either a class I chitinase or a class I [beta]-1,3-glucanase, or both, these proteins were targeted extracellularly. Both modified proteins lack their C-terminal propeptide, which functions as a vacuolar targeting signal. Extracellular targeting had no effect on the specific activities of the chitinase and [beta]-1,3-glucanase enzymes. Furthermore, the extracellular washing fluid (EF) from leaves of transgenic plants expressing either of the secreted class I enzymes exhibited antifungal activity on F. solani germlings in vitro comparable to that of the purified vacuolar class I proteins. Mixing EF fractions from these plants revealed synergism in inhibitory activity against F. solani; the mixed fractions exhibited inhibitory activity similar to that of EF from plants expressing both secreted enzymes.     

Aphid infestation induces PR-proteins differently in barley susceptible or resistant to the birdcherry-oat aphid (Rhopalosiphum padi)

The effect of infestation by the birdcherry-oat aphid ( Rhopalosiphum padi L.), on induction of PR-proteins was investigated in barley ( Hordeum vulgare L.), using barley lines susceptible or resistant to R. padi. The PR-proteins PR-1a (unknown function), PR-5a (acidic thaumatin) and peroxidase (EC 1.11.1.7) were not affected, whereas one chitinase (EC 3.2.1.14) and 4 β -1,3-glucanases (EC 3.2.1.39) were induced by the aphid treatment. In the resistant breeding line CI 16145, but not in the susceptible cultivar Golf, accumulation of one basic chitinase and two acidic β -1,3-glucanases increased with time from 2 until 11 days after infestation, as determined by western blots, with antibodies raised against purified chitinase (PR-3a) and β -1,3-glucanase (PR-2a) from barley. By isoelectric focusing, two additional basic β -1,3-glucanases were detected, which increased after infestation in both the resistant and the susceptible barley. The basic chitinase was only detected at days 7 and 11 in the susceptible cultivar, but already at day 2 in the resistant line. The induction was localized to the infested leaf. The PR-proteins PR-3a and PR-2a were also induced by the fungal pathogen ( Blumeria [syn. Erysiphe ] graminis f. sp. hordei ), methyl salicylate and, to a lower extent, by wounding with tweezers and methyl jasmonate (MeJA). Needle wounding performed to mimic aphid stylet penetration did not induce chitinase or β -1,3-glucanase. It is concluded that the fungal pathogen and the aphid infestation induce both similar and different responses, and that the aphid induction is not due to wounding only. The different responses in resistant and susceptible lines indicate that the induced enzymes may play a role in the resistance against aphid infestation.     

Primary structure and expression of mRNAs encoding basic chitinase and 1,3-β-glucanase in potato

Infection of potato leaves (Solanum tuberosum L. cv. Datura) by the late blight fungus Phytophthora infestans, or treatment with fungal elicitor leads to a strong increase in chitinase and 1,3--glucanase activities. Both enzymes have been implicated in the plant's defence against potential pathogens. In an effort to characterize the corresponding genes, we isolated complementary DNAs encoding the basic forms (class I) of both chitinase and 1,3--glucanase, which are the most abundant isoforms in infected leaves. Sequence analysis revealed that at least four genes each are expressed in elicitor-treated leaves. The structural features of the potato chitinases include a hydrophobic signal peptide at the N-terminus, a hevein domain which is characteristic of class I chitinases, a proline- and glycine-rich linker region which varies among all potato chitinases, a catalytic domain, and a C-terminal extension. The potato 1,3--glucanases also contain a N-terminal hydrophobic signal peptide and a C-terminal extension, the latter comprising a potential glycosylation site. RNA blot hybridization experiments showed that basic chitinase and 1,3--glucanase are strongly and coordinately induced in leaves in response to infection, elicitor treatment, ethylene treatment, or wounding. In addition to their activation by stress, both types of genes are regulated by endogenous factors in a developmental and organ-specific manner. Appreciable amounts of chitinase and 1,3--glucanase mRNAs were found in old leaves, stems, and roots, as well as in sepals of healthy, untreated plants, whereas tubers, root tips, and all other flower organs (petals, stamen, carpels) contained very low levels of both mRNAs. In young leaves and stems, chitinase and 1,3--glucanase were differentially expressed. While chitinase mRNA was abundant in these parts of the plant, 1,3--glucanase mRNA was absent. DNA blot analysis indicated that in potato, chitinase and 1,3--glucanase are encoded by gene families of considerable complexity.     

Antifungal Hydrolases in Pea Tissue : II. Inhibition of Fungal Growth by Combinations of Chitinase and beta-1,3-Glucanase

Chitinase and β-1,3-glucanase purified from pea pods acted synergistically in the degradation of fungal cell walls. The antifungal potential of the two enzymes was studied directly by adding protein preparations to paper discs placed on agar plates containing germinated fungal spores. Protein extracts from pea pods infected with Fusarium solani f.sp. phaseoli, which contained high activities of chitinase and β-1,3-glucanase, inhibited growth of 15 out of 18 fungi tested. Protein extracts from uninfected pea pods, which contained low activities of chitinase and β-1,3-glucanase, did not inhibit fungal growth. Purified chitinase and β-1,3-glucanase, tested individually, did not inhibit growth of most of the test fungi. Only Trichoderma viride was inhibited by chitinase alone, and only Fusarium solani f.sp. pisi was inhibited by β-1,3-glucanase alone. However, combinations of purified chitinase and β-1,3-glucanase inhibited all fungi tested as effectively as crude protein extracts containing the same enzyme activities. The pea pathogen, Fusarium solani f.sp. pisi, and the nonpathogen of peas, Fusarium solani f.sp. phaseoli, were similarly strongly inhibited by chitinase and β-1,3-glucanase, indicating that the differential pathogenicity of the two fungi is not due to differential sensitivity to the pea enzymes. Inhibition of fungal growth was caused by the lysis of the hyphal tips.     

Differential Induction and Accumulation of β-1,3-Glucanase and Chitinase Isoforms in the Intercellular Space and Leaf Tissues of Pepper by Xanthomonas campestris pv. vesicatoria Infection

The virulent strain Ds 1 of Xanthomonas campestris pv. vesicatoria multiplied in pepper (cv. Hanbyul) leaves better than did the avirulent strain 81–23, which formed localized necrosis at the onset of pathogenesis. Infection of pepper leaves by X. campestris pv. vesicatoria induced the synthesis and accumulation of β-1,3-glucanase and chitinase in the intercellular space and leaf tissue of pepper plants. In the uninoculated controls, the two hydrolases remained at a very low level. High levels of the two enzymes were found in an incompatible interaction of pepper leaves with X. campestris pv. vesicatoria . In particular, chitinase activity in the intercellular washing fluids (IWF) was higher in the incompatible than in the compatible interactions. The direct detection of acidic β-1,3-glucanases on 10% native PAGE gels revealed only two isoform bands (Ga 1 and Ga 2). Isoelectric focusing identified two acidic β-1,3-glucanase isoforms with pl 5.0 and 5.2, and four basic isoforms with pl 7.1, 7.4, 7.9, and 8.8 in the IWF and extracts of infected leaf tissues. Some of the isoforms disappeared during pathogenesis and the others appeared during symptom expression. The acidic chitinase isoforms (Ca 1, Ca 2, and Ca 3) were located primarily in the intercellular spaces. Synthesis of high levels of the acidic isoform Ca 3 in infected pepper leaves was seen. Several basis chitinase isoforms accumulated only in diseased leaf tissue, and especially more in the incompatible than the compatible interaction. By using isoelectric focusing, the three acidic and seven basic chitinase isoforms in the IWF and leaf extracts were detected on chitin overlay gels.     

Stability of transgene integration and expression in subsequent generations of doubled haploid oilseed rape transformed with chitinase and β-1,3-glucanase genes in a double-gene construct

A double-gene construct with one chitinase and one β-1,3-glucanase gene from barley, both driven by enhanced 35S promoters, was transformed into oilseed rape. From six primary transformants expressing both transgenes 10 doubled haploid lines were produced and studied for five generations. The number of inserted copies for both the genes was determined by Southern blotting and real-time PCR with full agreement between the two methods. When copy numbers were analysed in different generations, discrepancies were found, indicating that at least part of the inserted sequences were lost in one of the alleles of some doubled haploids. Chitinase and β-1,3-glucanase expression was analysed by Western blotting in all five doubled haploid generations. Despite that both the genes were present on the same T-DNA and directed by the same promoter their expression pattern between generations was different. The β-1,3-glucanase was expressed at high and stable levels in all generations, while the chitinase displayed lower expression that varied between generations. The transgenic plants did not show any major impact on fungal resistance when assayed in greenhouse, although purified β-1,3-glucanase and chitinase caused retardment of fungal growth in vitro.     

Local and Systemic Induction of β-1,3-Glucanase and Chitinase in Coffee Leaves Protected Against Hemileia vastatrix by Bacillus thuringiensis

β-1,3-glucanase and chitinase activities were induced locally and systemically 4–25 and 11–25 days, respectively, after spraying the surface of the third pair of coffee leaves from the apex of 8-month-old plants with a 50 mg/ml aqueous suspension of Bacillus thuringiensis in a commercial formulation (Thuricide HP-Sandoz). The treatment also induced local and systemic resistance against Hemileia vastatrix after the application of the inducer. Within 14–18 days of application of the Thuricide inducer, the β-1,3-glucanase activity in the locally and systemically-protected unchallenged leaves reached maximum levels of 226% and 279% higher levels respectively, than in control plants. The chitinase activity reached maximum levels of 224% and 181% respectively, within 18–21 days after treatment with the inducer. Two β-1,3-glucanase bands were detected by native PAGE electrophoresis in extracts from locally-and systemicallyprotected unchallenged coffee leaves.