Chitinase Transcripts Research Articles

Molecular characterization of chitinase from polyphagous pest Helicoverpa armigera

Chitinase from a polyphagous pest, Helicoverpa armigera, has been cloned and expressed. The Helicoverpa chitinase cDNA is 2870 bp in length and contains an open reading frame of 1767 bp. The cDNA encodes a polypeptide of 588 residues with a predicted molecular weight of 66 kDa and a p I of 5.99. The polypeptide has distinct catalytic and substrate binding domains at the N- and the C termini, respectively. The two domains are held together by a proline, threonine rich linker region. The catalytic and the substrate binding domains shared a high level of homology with other lepidopteran chitinases, but the proline and threonine rich region is longer in H. armigera chitinase than in other lepidopteran chitinases. The transcription of chitinase at different developmental stages and in different tissues was analysed by RT-PCR. Chitinase transcript was found in the integument, gut, and fat bodies but was absent in the haemocytes. The levels of chitinase mRNA were abundant at the moulting stages and a basal level of transcript was maintained throughout the development of the insect. Interestingly, Western blot analysis of total proteins from the integument and the gut showed the presence of chitinase in the moulting stages but was absent in the intermoult periods, suggesting post-transcriptional control. The chitinase cDNA was expressed in bacteria and in insect cells. The insect cell expressed chitinase was glycosylated and catalytically active against the simple and complex substrates. The chitinase gene spans about 6.8 kb of genomic DNA and is organized into 10 exons and 9 introns. The 6.8 kb genomic clone of chitinase revealed a high degree of conservation in the position and size of the exons with other lepidopteran insects.

Plant development affects the cold-induced expression of plant defence-related transcripts in winter wheat

In winter cereals, low temperature hardening, plant age and genotype are known to influence the expression of resistance to snow mould diseases. A study was undertaken to determine the effects of genotype, plant age and duration of cold hardening on the temporal expression of the PR-protein and other defence-related protein transcripts under controlled environment conditions, and in the field during autumn, winter, and early spring. The plant defence-related proteins studied were classified into two groups based on their patterns of expression. The first group consisted of γ-thionin, chitinase, lipid transfer protein (LTP) and phenylalanine ammonia lyase (PAL) whereby transcript levels increased to maximum levels during November–December, but decreased gradually throughout the winter and early spring. Higher transcript levels were observed in late seeded treatments compared with early seeded treatments. Under controlled conditions, transcript levels accumulated rapidly in response to hardening, reaching maximum levels following 1-week and generally maintained levels throughout the hardening treatments. The second group consisted of β-1,3-glucanase, peroxidase and PR-1a whose transcripts increased to maximum levels in the field during November–December, then fluctuated throughout the winter and early spring. Higher transcript levels were observed in early seeded treatments compared to later seeded treatments. Under controlled environment conditions, these transcripts gradually accumulated in response to hardening at 2°C and reached maximum levels after 6 weeks. PR-1a, PAL, and LTP transcript levels were generally higher in the snow mould resistant cultivars PI181268 and D+ throughout the winter and early spring compared to the cold-hardy, snow mould susceptible cultivar, Norstar. Conversely the chitinase and γ-thionin transcripts were expressed at higher levels in the susceptible cultivar Norstar. These results demonstrate that the temporal expression of cold-induced, plant defence-related transcripts in winter wheat is differentially regulated among genotypes and during different plant development stages, and are the first to implicate LTPs in the expression of genotypic-based snow mould resistance in wheat. Potential plant defence signalling pathways involved in snow mould resistance induced at low temperatures during natural acclimation of winter wheat are discussed.

Pathogen challenge, salicylic acid, and jasmonic acid regulate expression of chitinase gene homologs in pine.

To better understand the molecular regulation of defense responses in members of the genus Pinus, we tested the expression of various chitinase homologs in response to pathogen-associated signals. PSCHI4, a putative extracellular class II chitinase, was secreted into liquid medium by pine cells and was also secreted by transgenic tobacco cells that ectopically expressed pschi4. Extracellular proteins of pine were separated by isoelectric focusing; PSCHI4 was not associated with fractions containing detectable beta-N-acetylglucosaminidase or lysozyme activities. However, other fractions contained enzyme activities that increased markedly after elicitor treatment. The pschi4 transcript and protein accumulated in pine seedlings challenged with the necrotrophic pathogen Fusarium subglutinans f. sp. pini, with the protein reaching detectable levels in susceptible seedlings concomitant with the onset of visible disease symptoms. Additional chitinase transcripts, assigned to classes I and IV based on primary sequence analysis, were also induced by pathogen challenge. Jasmonic acid induced class I and class IV but not class II chitinase, whereas salicylic acid induced all three classes of chitinase. These results show that multiple chitinase homologs are induced after challenge by a necrotrophic pathogen and by potential signaling molecules identified in angiosperms. This suggests the potential importance of de novo pathogenesis-related (PR) gene expression in pathogen defense responses of pine trees.

Class I chitinases in cotton ( Gossypium hirsutum): characterization, expression and purification

Chitinases help defend plants from pathogens. We have previously reported the isolation of a cDNA clone and a genomic clone of Class I chitinase genes from cotton ( Gossypium hirsutum). Here we report the results of further investigations of Class I chitinase genes in cotton. These include the characterization of an additional cDNA clone, an estimate of the number of copies in the cotton genome, and experimental evidence for ethylene-induced expression. The Class I chitinase genes have very similar nucleotide sequences (over 98% identity). Based on Southern analysis, we estimate that there are approximately four copies of the cotton chitinase per haploid genome. Analysis of total RNA extracted from ethylene-treated plantlets showed that chitinase transcript levels increase after 12 h of treatment. A ∼31 kDa protein present in ethylene treated extracts has chitinolytic activity and cross-reacts with a rabbit antiserum raised against a cotton chitinase fusion protein. Using chitin affinity chromatography of ethylene treated extracts, we purified a 31.5 kDa protein band with chitinolytic activity. This protein band has been confirmed to be composed of Class I cotton chitinase by N-terminal peptide sequence analysis. The first 20 amino acids are identical to those predicted for the Class I cotton chitinases based on our cDNA and genomic nucleotide data. The purified Class I cotton chitinase preparation was analyzed by two dimensional electrophoresis. Three different isoelectric isomers were present in stained 2-D gels, and all three proteins cross-reacted with the cotton chitinase fusion protein antiserum. The predominant constituent of the affinity purified 31.5 kDa preparation has an isoelectric point of ∼7.0 and is glycosylated. There are additional isoelectric isomers with a p Is of 6.2 and 5.8 that are not glycosylated.

The requirements for Ca 2+, protein phosphorylation and concurrent protein synthesis for zeatin signaling of acidic chitinase transcript accumulation in Cucumis sativus L.

We have previously reported that both Ca2+ and staurosporine-sensitive protein kinase(s) are involved in the cytokinin zeatin induction of cucumber chitinase activity and its protein content (Barwe et al. 2001). To further characterize signal transduction events involved in this cytokinin induction of chitinase gene expression, Northern hybridizations of total RNAs prepared from excised, dark-grown cucumber cotyledons treated with cytokinins and/or various agonists and antagonists of signal transduction components, were carried out using a cucumber acidic chitinase (CACHT) cDNA probe (Metraux et al. 1989). CACHT mRNA increased by approximately 5- to 6-fold in response to exogenous zeatin (Z), zeatin riboside (ZR), and benzyladenine (BA) treatment, but failed to accumulate in response to kinetin (K). Among the cytokinins tested, Z was most effective. The Z-induced accumulation of CACHT mRNA was inhibited by a plasma membrane Ca2+ channel blocker verapamil. Treatment of cotyledons with exogenous CaCl2 and calcium ionophore A23187 in the presence and absence of cytokinin enhanced CACHT mRNA accumulation. These two observations suggest the participation of extracellular calcium in signaling Z-induction. Furthermore, the presence of staurosporine (an inhibitor of protein kinase) in Z treatment reduced CACHT mRNA, suggesting the involvement of phosphorylation of one or more cellular proteins. In addition, we provide evidence that the Z-induction of CACHT mRNA is blocked by protein synthesis inhibitor cycloheximide treatment. Taken together, these results suggest that Ca2+ influx from extracellular space, protein phosphorylation, and concurrent protein synthesis events participate in cytokinin signaling during Z-induced CACHT transcript accumulation.

Cbk1p, a protein similar to the human myotonic dystrophy kinase, is essential for normal morphogenesis in Saccharomyces cerevisiae.

We have studied the CBK1 gene of Saccharomyces cerevisiae, which encodes a conserved protein kinase similar to the human myotonic dystrophy kinase. We have shown that the subcellular localization of the protein, Cbk1p, varies in a cell cycle-dependent manner. Three phenotypes are associated with the inactivation of the CBK1 gene: large aggregates of cells, round rather than ellipsoidal cells and a change from a bipolar to a random budding pattern. Two-hybrid and extragenic suppressor studies have linked Cbk1p with the transcription factor Ace2p, which is responsible for the transcription of chitinase. Cbk1p is necessary for the activation of Ace2p and we have shown that the aggregation phenotype is due to a lack of chitinase expression. The random budding pattern and the round cell phenotype of the CBK1 deletion strain show that in addition to its role in regulating chitinase expression via Ace2p, Cbk1p is essential for a wild-type morphological development of the cell.

In situ localization of chitinase mRNA and protein in compatible and incompatible interactions of pepper stems with Phytophthora capsici

The induction of chitinase (CAChi2) mRNA started as early as 6h after inoculation and gradually increased in the incompatible interaction of pepper stems with Phytophthora capsici. In the compatible interaction, the induction of the chitinase transcripts was detected later than that in the incompatible interaction. The CAChi2 mRNA was usually localized in the vascular tissues and their expression was constricted in the phloem-related cells. These results showed that the spatial pattern of CAChi2 mRNA expression was similar in both compatible and incompatible interactions but the temporal patterns were different from each other. In addition, the early induction ofCAChi2 mRNA was quite distinct in the incompatible interaction. Immunogold labelling data showed specific labelling of chitinase on the cell wall of the oomycete in both compatible and incompatible interactions at 24h after inoculation. In particular, numerous gold particles were deposited on the cell wall of P. capsici with a predominant accumulation over areas showing signs of degradation in the incompatible interaction. Chitinase labelling was also detected in the intercellular space and the host cytoplasm. However, healthy pepper stem tissue was nearly free of labelling.

Expression of plant defence-related (PR-protein) transcripts during hardening and dehardening of winter wheat

The pattern of expression of the pathogenesis-related (PR) proteins PR-1, chitinase, β-glucanase, and peroxidase, and phenylalanine ammonia lyase (PAL) transcripts was studied in five winter wheat cultivars differing in resistance to snow mould, under controlled conditions following growth at 20°C, during hardening at 2°C and dehardening at 20°C. The expression of these transcripts was also studied in two cultivars of winter wheat under natural conditions during autumn, winter, and early spring of 1997–98 at a field site in Lethbridge. The relative abundance of transcripts was similar in both the field and controlled environment studies: chitinase was the most abundant transcript followed by PAL, β-1,3-glucanase, PR-1, and peroxidase. Under field conditions, all PR-protein transcripts exhibited the same basic pattern of expression during the autumn, winter and spring sampling dates; transcripts were expressed during the late autumn, reached high levels by mid-winter, then decreased before reaching maximum levels during the spring. Conversely, PAL expression was low or absent in autumn, reached the highest levels by mid-winter, and then gradually decreased during the spring. Under controlled environment conditions, transcripts encoding for PAL and the PR-proteins, except chitinase, were constitutively expressed to varying extents in the unhardened treatments. The general pattern of expression fell into two groups: (1) transcripts of chitinase, β-1,3-glucanase, and PAL, were weakly expressed in the unhardened treatments, strongly up-regulated following exposure to hardening conditions, and disappeared or remained stable following dehardening; and, (2) PR-1 and peroxidase transcripts were down-regulated upon initial exposure to hardening, increased slightly following prolonged exposure to hardening conditions, and remained stable during dehardening. Under controlled conditions, the pattern of expression of any of the PR-proteins did not appear to be associated with known genotypic levels of snow mould resistance among cultivars but, under field conditions, expression levels were generally higher for all PR-proteins in the snow mould resistant cultivar, Cl14106 than in the moderately susceptible cultivar, Norstar. These results demonstrate that different PR-protein and PAL transcripts in winter wheat are expressed in the field throughout the winter and are differentially induced in response to exposure of plants to low, hardening temperatures under both field and controlled environment conditions.

Regulation of the chitinase gene expression in suspension-cultured rice cells by N-acetylchitooligosaccharides: differences in the signal transduction pathways leading to the activation of elicitor-responsive genes.

Expression patterns of chitinase transcripts induced by N-acetylchitooligosaccharide elicitor were analyzed by northern blot hybridization in order to reveal a signal transduction pathway leading to the activation of class I chitinase genes (Cht-1 and Cht-3), which may play an important role in producing N-acetylchitooligosaccharide elicitor. The transcription level of both genes was enhanced in response to N-acetylchitooligosaccharides larger than pentaose at subnanomolar concentrations. These structure and dose dependencies were consistent not only with those for a 75 kDa high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane, but also with other series of cellular responses including phytoalexin production and the expression of elicitor-responsive genes (EL2, EL3). Therefore, the elicitor signal to evoke these cellular responses including the activation of the chitinase genes could be common and transmitted into cells through the 75 kDa protein. However, the signal transduction pathway for the activation of the chitinase gene appeared to diverge from those for the other elicitor-responsive genes shortly after the signal perception. It was shown that the induction of chitinase expression by N-acetylchitooligosaccharide would require protein phosphorylation, but not de novo protein synthesis. The oxidative burst was demonstrated not to be necessary for transcriptional induction of the all four elicitor-responsive genes (Cht, PAL, EL2, EL3) by N-acetylchitooligosaccharide.

Expression of beta-1,3-glucanase and chitinase in healthy, stem-rust-affected and elicitor-treated near-isogenic wheat lines showing Sr5-or Sr24-specified race-specific rust resistance.

Pathogenesis-related expression of the two antifungal hydrolases beta-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) was studied in wheat (Triticum aestivum L.) as part of the defence response to stem rust (Puccinia graminis f.sp. tritici, Pgt), mediated by the semi-dominantly acting resistance genes Sr5 and Sr24. Complete resistance (infection type 0), mediated by the Sr5 gene in cultivar Pre-Sr5, closely correlates with the hypersensitive response of penetrated cells at early stage of the interaction, when the first haustorium is formed. In contrast, cultivar Pre-Sr24 shows intermediate resistance (infection type 2-3) which is not directly linked to cell death. In both cases, the plant response included a rapid increase in beta-1,3-glucanase activity between 24 and 48 h after inoculation. One main extracellular 30-kDa isform of beta-1,3-glucanase was present in both lines, as shown by polyacrylamide-gel electrophoresis. Two additional minor isoforms (32 and 23 kDa) were detected only in Pre-Sr24, and only at later time points. Increased enzme activity and the appearance of new isoforms in the resistance lines was preceded by accumulation of mRNAs encoding beta-1,3-glucanase and chitinases. However, there were no changes in chitinase activity or isoforms. A high constitutive level of chitinase activity was observed in all wheat genotypes. Serological studies indicated the presence of a class II chitinase of 26 kDa. Accumulation of beta-1,3-glucanase and chitinase transcripts was detected before the pathogen penetrated the leaves through stomata and approximately 16 h before the typical hypersensitive response was observed, indicating that signal(s) for defense gene activation were recognised by the host plant long before a tight contact between the pathogen and a host cell is established. A glycoprotein (Pgt elicitor) derived from hyphal walls, strongly induced beta-1,3-glucanase. We discuss the possible role of the elicitor in the early signalling mediating Sr5- and Sr24-specified resistance in wheat.

Developmentally regulated silencing and reactivation of tobacco chitinase transgene expression

The tobacco class I chitinase gene CHN48 driven by CaMV 35S RNA expression signals introduced into Nicotiana sylvestris can inactivate its own expression as well as expression of homologous host genes at the mRNA level. Comparison of the silencing of chitinase and β‐1,3‐glucanase transgenes with the same expression signals and breeding experiments with transgenes inserted in different chromosomes showed that chitinase‐gene silencing depends on transcribed sequence hornology and trans‐gene dose. The CHN48 transgene silenced host class I but not class II chitinases suggesting that greater than about 60% identity of sequences encoding the catalytic domain is required for silencing. Although steady‐state mRNA contents were greatly reduced in silent plants, the levels of nascent chitinase transcripts detected in nuclear run‐on experiments were the same in nuclei from high‐expressing and silent plants. This strongly suggests that chitinase‐gene silencing is a post‐transcriptional phenomenon. Silencing and resetting to high‐level expression of chitinase genes is developmentally regulated. Silencing occurs stochastically in 25–100% of seedlings at the 6–10 leaf stage of development and is preceded by a marked, transient increase in transgene‐encoded chitinase. Resetting of silent genes to the high‐expressing state is a non‐stochastic event that occurs in developing seeds 8–11 days after pollination. Experiments with lateral buds and plants regenerated from cultured leaf disks show that once established, competence for silencing can persist in dormant, actively growing, and de novo established shoot meristems. Some plants show a variegated pattern of silencing. Analyses of these patterns indicate that chitinase‐gene silencing is not a clonal event. Thus, it is likely that communication between cells rather than the lineage of the cells is important in stabilizing the silent state. Possible mechanisms for developmentally regulated post‐transcriptional silencing are discussed.

Characterization of a wheat class Ib chitinase gene differentially induced in isogenic lines by infection with Puccinia graminis

Plant chitinases have been recognized to be involved in defense against pathogens. A class Ib genomic chitinase gene was isolated from a wheat ( Triticum aestivum L.) genomic library using a wheat chitinase sequence amplified from DNA by PCR. The nucleotide sequence analysis showed that the wheat chitinase gene ( Wchl) contains a 960-bp long coding region with no intron. The derived polypeptide includes a signal peptide, a cysteine-rich domain and a catalytic domain without a carboxy terminal extension. Primer extension experiments identified a single transcription start site 33 bp upstream of the translation initiation site. Southern blot analysis indicated that Wchl is one member of a small gene family in wheat. Northern blot hybridization and histological investigation of wheat leaves infected by Puccinia graminis f. sp. tritici revealed a close correlation between the accumulation of the chitinase transcripts and the inhibition of fungal growth when two isogenic wheat lines (Prelude Sr6 and Prelude sr6) differing at the Sr6 locus for race-specific incompatibility with the fungus were studied. The massive accumulation of the chitinase mRNA occurred only in the incompatible Sr6/P6 interaction, in parallel with a severe inhibition of mycelial growth in the leaves. In contrast, in the compatible sr6/P6 interaction the fungus grew rapidly throughout the leaf tissue and the chitinase transcripts were not detected. The chitinase mRNA was detectable in non-infected stems but not in non-infected leaves or roots.

Spatial and temporal accumulation of defense gene transcripts in bean (Phaseolus vulgaris) leaves in relation to bacteria-induced hypersensitive cell death.

Primary leaves of 7- to 9-day-old Red Mexican bean plants were inoculated with virulent or avirulent isolates of Pseudomonas syringae pv. phaseolicola, or saprophytic P. fluorescens either by vacuum infiltration of the whole leaf lamina, or by syringe-inoculation of selected leaf panels. In the incompatible combination, resistance was associated with a hypersensitive response (HR). Syringe-inoculated leaves were sampled in three zones: zone 1, the inoculated leaf area; zone 2, the surrounding 0.5-0.7 cm of leaf tissue; and zone 3, the remainder of the leaf. Northern blots of RNA from zones 1, 2, and 3 were probed with bean cDNAs for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chitinase (CHT), and lipoxygenase (LOX). Accumulation of PAL, CHS, and CHT transcripts was more rapid and generally of greater magnitude in the incompatible than in the compatible interaction and, in both cases, was observed essentially only in zone 1 tissues. Similarly, antibacterial phytoalexins were only detected in zone 1 from the incompatible interaction. Young primary leaves have a background level of LOX transcripts, which declines as leaves age. This decline was accelerated over the first 12 hr postinoculation (hpi) with avirulent bacteria, whereas a weak transient induction, peaking at 5-6 hpi, was observed in the compatible interaction. A subsequent, strong accumulation of LOX transcripts was seen in both the compatible and incompatible interactions outside the inoculation site starting about 14 hpi. LOX transcripts did not accumulate at the inoculation site itself in the incompatible interaction compared to a relatively strong induction in the compatible interaction. Interestingly, inoculation of leaves with cells of the saprophyte P. fluorescens also induced the accumulation of transcripts for CHS, CHT, and LOX, but generally to a lesser degree than in the incompatible interaction. No HR occurred and no macroscopic cell damage was apparent in leaves inoculated with P. fluorescens. However, at the microscopic level individual, trypan blue-stained, necrotic plant cells were visible. In spite of this and the accumulation of CHS transcripts, no phytoalexin accumulation was found up to 48 hr after inoculation. The spatial and temporal relationship of the hypersensitive reaction to defense gene transcript and phytoalexin accumulation is discussed.

Cloning and characterization of a pathogen-induced chitinase in Brassica napus.

A chitinase cDNA clone from rapeseed (Brassica napus L. ssp. oleifera) was isolated. The cDNA clone, ChB4, represents a previously purified and characterized basic chitinase isozyme. The longest open reading frame in ChB4 encodes a polypeptide of 268 amino acids. This polypeptide consists of a 24 amino acid N-terminal signal peptide, a cysteine-rich domain and a catalytic domain. The primary structure of the mature ChB4 shows a low degree of identity with class I and II chitinases, 43-48% and 35%, respectively. In contrast, ChB4 shows 62% identity to a basic sugar-beet chitinase and 63% identity to an acidic chitinase from bean. The expression of chitinase messenger RNA (mRNA) in response to infection with Phoma lingam (Tode ex. Fr.) Desm. was examined by northern hybridization and scintilation counting. A differential induction was seen between resistant and susceptible cultivars where 3-fold higher chitinase transcript levels were estimated one day after inoculation in resistant as compared to susceptible cultivar. This difference diminished eight days after inoculation. Southern hybridization analysis indicates that the chitinase is encoded by a small family of genes.

Parallel pathways of gene regulation: homologous regulators SWI5 and ACE2 differentially control transcription of HO and chitinase.

Two independent pathways of transcriptional regulation that show functional homology have been identified in yeast. It has been demonstrated previously that SWI5 encodes a zinc finger DNA-binding protein whose transcription and cellular localization both are cell cycle regulated. We show that ACE2, whose zinc finger region is nearly identical to that of SWI5, shows patterns of cell cycle-regulated transcription and nuclear localization similar to those seen previously for SWI5. Despite their similarities, SWI5 and ACE2 function in separate pathways of transcriptional regulation. SWI5 is a transcriptional activator of the HO endonuclease gene, whereas ACE2 is not. In contrast, ACE2 is a transcriptional activator of the CTS1 gene (which encodes chitinase), whereas SWI5 is not. An additional parallel between the SWI5/HO pathway and the ACE2/CTS1 pathway is that HO and CTS1 both are cell cycle regulated in the same way, and HO and CTS1 both require the SWI4 and SWI6 transcriptional activators. Overproduction of either SWI5 or ACE2 permits transcriptional activation of the target gene from the other pathway, suggesting that the DNA-binding proteins are capable of binding in vivo to promoters that they do not usually activate. Chimeric SWI5/ACE2 protein fusion experiments suggest that promoter specificity resides in a domain distinct from the zinc finger domain.

Isolation and characterization of a rice gene encoding a basic chitinase

Chitinase, which catalyzes the hydrolysis of the beta-1,4-N-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin, is involved in inducible defenses of plants. A basic chitinase genomic sequence was isolated from a rice (Oryza sativa L.) genomic library using a bean chitinase gene fragment as a probe. The complete nucleotide sequence of the rice chitinase RCH10 gene was determined, and shown to contain an open reading frame with no introns, encoding a polypeptide of 336 amino acids. This polypeptide consists of a 21 amino acid signal peptide, a hevein domain, and a chitinase catalytic domain. The RCH10 gene has 63% identity at the nucleotide level and 75% identity at the amino acid level with chitinase genes from dicotyledonous plants such as bean, potato, and tobacco. A gene fusion of trpE and the coding region of RCH10 expressed in Escherichia coli gave a product that reacted with antiserum to bean chitinase, confirming the identity of RCH10 as a rice chitinase gene. Primer extension analysis identified two transcription start sites 53 bp and 55 bp upstream from the translation initiation codon. The 5' flanking region contains TATA and CAAT boxes, and the 3' region contains an AATAA polyadenylation signal. Southern blot hybridization indicated that there is a family of chitinase genes in the rice genome. Northern blot analysis showed that the RCH10 chitinase gene is induced in suspension cultured cells by a fungal cell wall elicitor. Rice chitinase transcripts accumulate to a high level in roots, but only low levels are found in stem and leaf tissue.

CDNA cloning and characterization of a putative 1,3-?-D-glucanase transcript induced by fungal elicitor in bean cell suspension cultures

Synthetic oligonucleotides based on similarity between tobacco 1,3-beta-D-glucanase and barley 1,3-1,4-beta-D-glucanase were used to prime the synthesis and amplification of a 162 bp bean (Phaseolus vulgaris L.) beta-glucanase cDNA by the polymerase chain reaction (PCR). The PCR product was used to isolate a near full-length beta-glucanase cDNA corresponding to an approximately 1400 bp full-length transcript, from a library containing cDNA sequences complementary to mRNA from fungal elicitor-treated bean cells. At the amino acid level, the bean beta-glucanase cDNA was 59% similar to tobacco 1,3-beta-D-glucanase, 46% similar to barley 1,3-beta-D-glucanase and 46% similar to barley 1,3-1,4-beta-D-glucanase. At the nucleotide level, the similarities were 65, 50 and 53% respectively. The beta-glucanase appeared to be encoded by a single gene with similar genomic organization in bean cultivars Canadian Wonder, Imuna and Saxa. On the basis of predicted Mr, isoelectric point, sequence similarity, and comparisons of rate of transcript appearance with induced enzyme activity, it was concluded that the cDNA encodes the basic bean endo-1,3-beta-D-glucanase. Glucanase transcripts were induced, from very low basal levels, with similar kinetics to chitinase transcripts in elicitor-treated bean cell suspension cultures.

Chitinase cDNA Cloning and mRNA Induction by Fungal Elicitor, Wounding, and Infection

Chitinase, which catalyzes the hydrolysis of beta-1,4 N-acetylglucosamine linkages of the fungal cell wall polymer chitin, is a component of the inducible defenses of plants. We show that chitinase synthesis is stimulated in bean (Phaseolus vulgaris L.) cell suspension cultures treated with fungal cell wall elicitors and in hypocotyls in response to infection with the fungus Colletotrichum lindemuthianum. Chitinase cDNA clones were isolated by antibody screening of a lambdagt11 cDNA library containing sequences complementary to poly A(+) RNA from elicited cells. The identity of these clones was confirmed by nucleotide sequence analysis and comparison of the deduced amino acid sequence with that determined for the amino-terminal sequence of bean chitinase. Elicitor causes a very rapid activation of chitinase transcription with a 10-fold stimulation after 5 minutes and 30-fold increase within 20 minutes. This leads to a marked, transient accumulation of chitinase transcripts with maximum levels 2 hours after elicitor treatment, concomitant with the phase of rapid enzyme synthesis. Chitinase transcripts also markedly accumulate in wounded and infected hypocotyls. Chitinase cDNA sequences hybridize to several genomic fragments suggesting there are several chitinase genes in the bean genome.