Wound-inducible Expression Research Articles

Identification and validation of the wound and insect bite early inducible promoter from Arabidopsis thaliana.

The online version contains supplementary material available at 10.1007/s13205-022-03143-0.

A strong early acting wound-inducible promoter, RbPCD1pro, activates cryIAc expression within minutes of wounding to impart efficient protection against insects.

SummaryThe expression of insecticidal proteins under constitutive promoters in transgenic plants is fraught with problems like developmental abnormalities, yield drag, expression in unwanted tissues, and seasonal changes in expression. RbPCD1pro, a rapid, early acting wound‐inducible promoter from rose that is activated within 5 min of wounding, was isolated and characterized. Wounding increased transcript levels up to 150 and 500 folds within 5 and 20 min coupled with high translation as seen by histochemical GUS enzyme activity within 5–20 min. RbPCD1pro was activated by both sucking and chewing insects and showed wound‐inducible expression in various aerial tissues of plants representing commercially important dicot and monocot families. The promoter showed no expression in any vegetative tissue except upon wounding. Functionality of RbPCD1pro was tested by its ability to drive expression of the insecticidal protein gene cryIAc in transgenic Arabidopsis and tomato. Strong wound‐inducible CryIAc expression was observed in both plants that increased 100–350 fold (Arabidopsis) and 280–600 fold (tomato) over the unwounded background within 5 min and over 1000–1600 fold within 20 min. The unwounded background level was just 3–6% of the CaMV35S promoter while wound‐induced expression was 5–27 folds higher than the best CaMV35S line in just 5 min and 80‐fold higher in 20 min. Transgenic plants showed strong resistance even to larger fourth instar larvae of H. armigera and no abnormalities in development and general plant growth. This is one of the earliest acting promoters with wide biotechnological application across monocot and dicot plants.

Development of insect-resistant cotton lines with targeted expression of insecticidal gene

In order to address biosafety concerns regarding the constitutive expression of foreign genes in crops, we applied a strategy aimed at confining foreign gene expression in insect wounding sites of cotton. For this purpose, a plant expression construct was designed by cloning the AoPR1 promoter (pathogenesis-related protein gene isolated from Asparagus officinalis) upstream from the insecticidal gene cry1Ac. The Turkish cotton cultivar cv. STN-468 was transformed using the Agrobacterium tumefaciens strain LBA4404 containing the recombinant binary vector pRD400 harboring cry1Ac under a wound-inducible promoter. The neomycin phosphotransferase (nptII) gene was used as a selectable marker at a concentration of 100 mg/L. The primary transformants were analyzed for T-DNA integration and expression using standard molecular approaches. The efficacy of insecticidal gene control of the AoPR1 promoter was investigated using leaf bioassays with 2nd instar larvae of Helicoverpa armigera and Spodoptera littoralis. Positive primary transformants from T0 progeny were further raised under greenhouse conditions to obtain progeny (T1). The introduced gene was properly inherited and expressed in T1 progeny. The mechanical wounding of plants resulted in increased cry1Ac protein levels during 0-48 h of the wounding period. The transgenic lines exhibited appreciable levels of resistance against targeted insect pests in the leaf bioassays. The use of a wound-inducible promoter to drive insecticidal gene expression is a valuable insect resistant management strategy as gene expression will remain limited to the insect biting sites of plant and crop, food and environmental concerns can be minimized.

Wound-inducible expression of the OsDof1 gene promoter in a Ds insertion mutant and transgenic plants

The DNA binding with one finger (Dof) proteins are plant-specific transcription factors that contain a particular class of zinc-finger DNA-binding domain. Thirty-different Dof genes are predicted to exist in the rice genome based on in silico analysis. A member of the Dof transcription factor family, Dof1 of rice (Oryza sativa), was identified from a dissociation (Ds) insertion mutant population. The Ds-tagged rice mutant line osdof1-Ds was identified from a population of rice tagged with promoterless β-glucuronidase (GUS) in T-DNA, and showed wound-responsive GUS expression. Genomic Southern analysis revealed that this mutant line contained a single copy of the Ds gene trap. Four wound-response-like cis-acting elements (PI-II, EIRE, W box, and G box-like elements) were found in the OsDof1 promoter region. To analyze the cis-acting elements, we constructed fusion genes between the OsDof1 promoter and the GUS reporter gene and transformed Arabidopsis and rice plants with these constructs. GUS gene expression was induced in leaf, seed, culm, and flower tissues of the osdof1-Ds mutant after wounding. Furthermore, histochemical analysis of the OsDof1 promoter::GUS fusion transgenic plants showed an early response to wounding. In addition, OsDof1 mRNA was induced by pathogen treatment. These results suggest that OsDof1 is a transcriptional activator that regulates gene expression in response to wounding.

Upstream sequence of fatty acyl-CoA reductase (FAR6) of Arabidopsis thaliana drives wound-inducible and stem-specific expression

An Arabidopsis mutant line T90, exhibiting a stem-specific and wound-responsive GUS expression was identified from a population of Arabidopsis thaliana tagged with a promoterless β-glucuronidase (GUS) in the T-DNA. Sequence flanking the insertion from the right border was amplified by TAIL PCR and cloned. The insertion was located in the third chromosome, 57 bp upstream of the ATG start codon in 5' untranslated region (UTR) of the fatty acyl-CoA reductase 6 (FAR6) gene. RT-PCR analysis of the FAR6 gene revealed that the gene is expressed predominantly in stem tissue. Semi-quantitative RT-PCR showed that the expression is also induced by wounding in the epidermal layer of mature stem internodes. The transcription initiation site (TSS) was identified by 5' RACE PCR. Different 5' deletion fragments of the promoter sequences were developed and linked to the GUS reporter gene as transcriptional fusions and the expression patterns of GUS were histochemically analyzed in transgenic Arabidopsis plants. Sequences from -510 bp upstream to the transcriptional start site were sufficient to exhibit wound-inducible GUS expression in the stems. The addition of further upstream sequences (-510 to -958, -1,400 or -1,456) enhanced and extended the wound-inducible GUS expression throughout the mature stem.

The Nightshade Proteinase Inhibitor IIb Gene is Constitutively Expressed in Glandular Trichomes

The best known property of plant proteinase inhibitor II (PIN2) genes is their wound-inducible expression in leaves and constitutive expression in flowers. Here we show by promoter analysis in transgenic plants and in situ reverse transcription-PCR (RT-PCR) analysis that SaPIN2b, a member of the PIN2 gene family of nightshade (Solanum americanum), is also constitutively expressed in glandular trichomes. SaPIN2b promoter and its deletions were cloned and fused upstream of beta-glucuronidase (GUS) to transform the nightshade and tobacco (Nicotiana tabacum) plants. Histochemical staining assays indicated that SaPIN2b:GUS was expressed constitutively in glandular trichomes, predominantly in the gland cells, of both transgenic nightshade and tobacco plants. Constitutive expression of SaPIN2b in glandular trichomes was further confirmed by liquid phase in situ RT-PCR analysis of nightshade leaves. Deletion analysis from the 5' end of the SaPIN2b promoter revealed that separate regulatory elements control SaPIN2b expression in gland cells and stalk cells of glandular trichomes. Fluorometric GUS assays showed that SaPIN2b:GUS expression was significantly increased in transgenic plant leaves after mechanical wounding or methyl jasmonate treatment. The SaPIN2b promoter sequence contains six MYB-binding motifs and an L1 box that are involved in trichome differentiation and development. Overexpression of SaPIN2b in tobacco resulted in a significant increase in glandular trichome density and promotion of trichome branching. These results suggest that, as well as being an induced defensive protein of the well-known PIN2 family, SaPIN2b could also play roles in trichome-based defense by functioning as a constitutive component of trichome chemical defense and/or by regulating the development of glandular trichomes.

Comprehensive Analysis of Wound-inducible Genes from the Nicotiana glutinosa Leaves Using a Full-length cDNA Microarray

Wound-inducible gene expression in the the Nicotiana glutinosa leaves was studied by using a microarray with 9600 full-length CDNAS. As a result, 86 genes were identified as wound-inducible genes in the N. glutinosa leaves, including those encoding defense related proteins, such as heat shock proteins, glutathione S-transferase, ascorbate peroxidase and nonspecffic lipid-transfer proteins. Among 86 genes, 15 genes including 1 1 hypothetical protein genes and I unknown protein genes encode unknown functional proteins. Although the translational products of these genes have not been characterized, they are potential candidates for defense-related proteins toward wounding. The cluster analysis classified the genes into 6 groups on the basis of their expression patterns. It is likely that genes clustered in the same groups may be co-regulated by common transcriptional factors and also translational products belonging to the same clusters may share common functions in defense response to wounding.

상처에 의해서 유도되는 벼 calmodulin promoter의 transgenic 담배에서조직 특이적 발현

To study calmodulin (CaM) gene expression and its regulation, rice CaM promoter (RCaM-2) was isolated and fused to (GUS), reporter gene. X-Glue staining patterns revealed that GUS localization is high in meristemic tissues such as the stem apex, stolen tip, and vascular regions. GUS staining in the transverse sections of stem and petiole was restricted to the inside of the vascular system, and cortex and epidermis located outside of the vascular system usually did not show GUS staining even a plant that expressed strong activity. GUS activity was found to be tissue specific expressed and exhibited a dramatic transient increase in response to wounding. These results suggest that the 5`-flanking region of RCaM gene regulates wound-inducible expression.

Expression of the maize proteinase inhibitor (mpi) gene in rice plants enhances resistance against the striped stem borer (Chilo suppressalis): effects on larval growth and insect gut proteinases

The maize proteinase inhibitor (mpi) gene was introduced into two elite japonica rice varieties. Both constitutive expression of the mpi gene driven by the maize ubiquitin 1 promoter and wound-inducible expression of the mpi gene driven by its own promoter resulted in the accumulation of MPI protein in the transgenic plants. No effect on plant phenotype was observed in mpi-expressing lines. The stability of transgene expression through successive generations of mpi rice lines (up to the T(4) generation) and the production of functional MPI protein were confirmed. Expression of the mpi gene in rice enhanced resistance to the striped stem borer (Chilo suppressalis), one of the most important pests of rice. In addition, transgenic mpi plants were evaluated in terms of their effects on the growth of C. suppressalis larvae and the insect digestive proteolytic system. An important dose-dependent reduction of larval weight of C. suppressalis larvae fed on mpi rice, compared with larvae fed on untransformed rice plants, was observed. Analysis of the digestive proteolytic activity from the gut of C. suppressalis demonstrated that larvae adapted to mpi transgene expression by increasing the complement of digestive proteolytic activity: the serine and cysteine endoproteinases as well as the exopeptidases leucine aminopeptidase and carboxypeptidases A and B. However, the induction of such proteolytic activity did not prevent the deleterious effects of MPI on larval growth. The introduction of the mpi gene into rice plants can thus be considered as a promising strategy to protect rice plants against striped stem borer.

Systemic defense signaling in tomato

The wound-inducible expression of proteinase inhibitors (PIs) genes in tomato provides a powerful model system to elucidate the signal transduction pathway of systemic defense response. An increasing body of evidence indicates that systemin and jasmonic acid (JA)_work in the same signaling pathway to activate the expression of PIs and other defense-related genes. However, little is known about how systemin and JA interact to regulate cell to cell communication over long distances. Genetic analysis of the systemin/JA signaling pathway in tomato plants provides a unique opportunity to dissect the mechanism by which peptide and oxylipin signals interact to coordinate systemic expression of defense-related genes. Previously, it has been proposed that systemin is the long-distance mobile signal for systemic expression of defense related genes. However, recent genetic approach provided new evidence that jasmonic acid, rather than systemin, functions as the systemic wound signal, and that the peptide systemin works to regulate the biosynthesis of JA.

Wound-inducible Expression of the ipt Gene Stimulates Enhanced Lateral Shoot Development in Tobacco

The bacterial isopentenyl transferase (ipt) gene involved in cytokinin biosynthesis was fused with a promoter from the serine proteinase inhibiter (win3.12) gene of Populus x generosa and introduced into Nicotiana tabacum (cv. Havana). Transformants were confirmed by PCR reaction and Southern blot analysis, and then analyzed for phenotypic characteristics. RT-PCR analysis detected transcripts of the ipt gene following the wounding of win3.12:ipt transgenic plants. In win3.12: ipt transgenic plants, lateral shoot number and the diameter of lateral branches that developed following apical shoot removal increased relative to wild-type plants. However, the increase in cytokinin production following wounding appeared to be short lived. The potential utility of this construct in enhancing lateral branching in ornamental crops will be discussed.

Sugar acts as a regulatory signal on the wound-inducible expression of SbHRGP3::GUS in transgenic plants.

SbHRGP3 encodes an HRGP whose expression is correlated with the cessation of root elongation in soybean. The wound-inducible expression of SbHRGP3 interestingly requires sucrose although wounding alone induces the expression of many HRGP genes. To examine whether sugar serves as a specific signal on the wound-inducible expression or whether sugar is required to provide ATP, we examined SbHRGP3:: GUS expression in transgenic tobacco plants. Various oligosaccharides including non-metabolizable sugar induced SbHRGP3:: GUS expression in transgenic plants. The inhibitors of photosynthesis and of cellular respiration did not affect the wound-inducible expression of SbHRGP3:: GUS. However, the induction was significantly affected by PCMBS, an inhibitor of active apoplastic phloem loading of sucrose, suggesting that SbHRGP3:: GUS expression in phloem tissues requires translocated sucrose. We therefore propose that sugar acts as a specific regulatory signal on the wound-inducible expression of SbHRGP3, rather than acting as a simple provider of ATP.

Exciting prospects for plants with greater disease resistance

The spread of plant pathogens and insect pests is increasing worldwide. Several strategies have been used to increase crop resistance toward these pathogens, including expression of gene products that are toxic to pathogens or enhance the plants own natural resistance mechanisms. Such introduced genes are commonly placed under the control of promoters that lead to the production of these substances in all tissues of the plant. However, this can have detrimental effects on plant growth and crop yield. To reduce unwanted expression in uninfected tissues and to increase the chance of success of increasing disease resistance, we constructed synthetic promoters containing defined cis-acting elements, and showed that seven different elements direct high-level local expression around infection sites (Fig. 1Fig. 1) [1xSynthetic plant promoters containing defined regulatory elements provide novel insights into pathogen- and wound-induced signalling. Rushton, P.J. et al. Plant Cell. 2002; 14: 749–762Crossref | PubMed | Scopus (232)See all References[1]. The elements can be divided into three groups; W boxes, GCC-like boxes and Box D. Some also direct wound-inducible expression (Fig. 2Fig. 2).Fig. 1Transgenic Arabidopsis plants containing the synthetic promoter 4×W1::GUS two days after infection with Peronospora parasitica. Blue spots show the activity of the synthetic promoter at attempted infection sites. Scale bar = 2mm. Reproduced, with permission, from Ref. [1xSynthetic plant promoters containing defined regulatory elements provide novel insights into pathogen- and wound-induced signalling. Rushton, P.J. et al. Plant Cell. 2002; 14: 749–762Crossref | PubMed | Scopus (232)See all References[1].View Large Image | Download PowerPoint SlideFig. 2A transgenic Arabidopsis leaf containing 4×S::GUS that has been cut. Wound-induced expression is seen in a ‘wave’ around the cut sites. Scale bar = 5mm.View Large Image | Download PowerPoint SlideAn important discovery was that certain elements differ in their responses to pathogens. Some respond to all tested pathogens whereas others directed expression with only some of them. Crucially, the synthetic promoters are also active during compatible interactions. It is under such circumstances that drastic crop yield losses occur and in these situations biotechnology might be of great benefit. Currently, we are using these promoters to produce crop plants with increased disease resistance. We also showed that the synthetic promoters are not only active in the plant from which they originated but also in other species. This suggests that the synthetic promoters would be of general use and shows that signalling in plant defence is largely conserved across species boundaries.Our work also provides new insights into signalling during the plant defence response. It shows that the responses to wounding and pathogens can operate through the same DNA element and that similar elements, differing in only one base pair, can have drastically different expression patterns.

Copper amine oxidase expression in defense responses to wounding and Ascochyta rabiei invasion.

Wounding chickpea (Cicer arietinum) internodes or cotyledons resulted in an increase in the steady-state level of copper amine oxidase (CuAO) expression both locally and systemically. Dissection of the molecular mechanisms controlling CuAO expression indicated that jasmonic acid worked as a potent inducer of the basal and wound-inducible CuAO expression, whereas salicylic acid and abscisic acid caused a strong reduction of the wound-induced CuAO expression, without having any effect on the basal levels. Epicotyl treatment with the CuAO mechanism-based inhibitor 2-bromoethylamine decreased hydrogen peroxide (H(2)O(2)) levels in all the internodes, as evidenced in vivo by 3,3'-diaminobenzidine oxidation. Moreover, inhibitor pretreatment of wounded epicotyls resulted in a lower accumulation of H(2)O(2) both at the wound site and in distal organs. In vivo CuAO inhibition by 2-bromoethylamine after inoculation of resistant chickpea cv Sultano with Ascochyta rabiei resulted in the development of extended necrotic lesions, with extensive cell damage occurring in sclerenchyma and cortical parenchyma tissues. These results, besides stressing the fine-tuning by key signaling molecules in wound-induced CuAO regulation, demonstrate that local and systemic CuAO induction is essential for H(2)O(2) production in response to wounding and indicate the relevance of these enzymes in protection against pathogens.

Hydrogen Peroxide Acts as a Second Messenger for the Induction of Defense Genes in Tomato Plants in Response to Wounding, Systemin, and Methyl Jasmonate

The systemic accumulation of both hydrogen peroxide (H(2)O(2)) and proteinase inhibitor proteins in tomato leaves in response to wounding was inhibited by the NADPH oxidase inhibitors diphenylene iodonium (DPI), imidazole, and pyridine. The expression of several defense genes in response to wounding, systemin, oligosaccharides, and methyl jasmonate also was inhibited by DPI. These genes, including those of four proteinase inhibitors and polyphenol oxidase, are expressed within 4 to 12 hr after wounding. However, DPI did not inhibit the wound-inducible expression of genes encoding prosystemin, lipoxygenase, and allene oxide synthase, which are associated with the octadecanoid signaling pathway and are expressed 0.5 to 2 hr after wounding. Accordingly, treatment of plants with the H(2)O(2)-generating enzyme glucose oxidase plus glucose resulted in the induction of only the later-expressed defensive genes and not the early-expressed signaling-related genes. H(2)O(2) was cytochemically detected in the cell walls of vascular parenchyma cells and spongy mesophyll cells within 4 hr after wounding of wild-type tomato leaves, but not earlier. The cumulative results suggest that active oxygen species are generated near cell walls of vascular bundle cells by oligogalacturonide fragments produced by wound-inducible polygalacturonase and that the resulting H(2)O(2) acts as a second messenger for the activation of defense genes in mesophyll cells. These data provide a rationale for the sequential, coordinated, and functional roles of systemin, jasmonic acid, oligogalacturonides, and H(2)O(2) signals for systemic signaling in tomato plants in response to wounding.

The −689/+197 region of the maize protease inhibitor gene directs high level, wound-inducible expression of the cry1B gene which protects transgenic rice plants from stemborer attack

To investigate the activity of the regulatory region of the maize (Zea mays L.) proteinase inhibitor (mpi) gene, we transferred into rice (Oryza sativa L.) plants the −689/+197 (C1) fragment of the mpi genomic clone fused to either theuidA gene or a synthetic Bacillus thuringiensis cry1B gene. Although uidA and cry1B encode very different proteins consistent results were obtained from their respective histochemical and fluorometric and immunoblot detections in T3 transgenic rice lines. In response to mechanical wounding, a 4–5 fold increase in GUS activity and a Cry1B accumulation reaching 0.1–0.2% of total soluble proteins were observed from basal and undetectable levels respectively in leaf tissue. The establishment of the time-course of wound response in both systems revealed a maximum induction level 12–16 h after treatment. From both systems we also deduced that the C1 region is not active in pollen and seed endosperm. Three independent transformation events expressing cry1B under the control of the C1 region exhibited protection against striped stem borer damage and showed 100% mortality of second instar larvae 8 days after release. These results illustrate the first evidence that wound-inducible expression of a Bacillus thuringiensis endotoxin gene affords full protection to transgenic rice plants.

Regulatory roles of benzyl adenine and sucrose during wound response of the ribosomal protein gene, rpL34

We previously reported that a large subunit ribosomal protein gene, rpL34, was wound-inducible in tobacco leaves, and that benzyl adenine (BA) enhanced the wound-inducible expression of the gene. Here we report that the wound-inducible expression of the rpL34 gene is enhanced by sucrose. The regulatory roles of BA and sucrose in wound-signalling pathways were investigated using transgenic plants expressing a fusion molecule between the rpL34 promoter and the chloramphenicol acetyltransferase (cat) gene. Wounding caused a progressive increase in CAT activity; BA and sucrose enhanced the response. This indicates that regulation occurs at the transcriptional level. Diethyldithiocarbamic acid (DIECA) and other potent inhibitors of the octadecanoid pathway inhibited the rpL34 promoter, and also reduced BA inducibility of that wound response. However, inhibitors of the octadecanoid pathway did not affect the sucrose response. Protein kinase inhibitors increased BA enhancement while decreasing the sucrose effect, suggesting that protein kinases differentially regulate BA- and sucrose-signalling in the promoter's wound response. Taken together, BA and sucrose enhanced the wound response of the rpL34 promoter via different signalling pathways, although they exerted overlapping effects on wound-induction of the promoter.

Tacrolimus impairs wound healing: a possible role of decreased nitric oxide synthesis.

The effect of the immunosuppressant tacrolimus on wound healing is not known. Tacrolimus has been shown to decrease nitric oxide synthesis. The systemic inhibition of wound nitric oxide synthesis leads to impaired healing. We studied the effect of systemic tacrolimus treatment on wound-breaking strength and collagen deposition 10 days after wounding in rats and to correlate the outcome of healing with wound nitric oxide synthesis. Beginning at the day of wounding, rats were treated once daily by intraperitoneal injections with 0.5, 1.0, or 2.0 mg tacrolimus/kg body weight. Nitrite and nitrate were measured in wound fluid as an index of wound nitric oxide synthesis. Expression of inducible nitric oxide synthase in the wound was investigated by immunohistochemistry. Splenic lymphocytes were tested for proliferative activity. Tacrolimus levels in blood and wound fluid were measured by enzyme-linked immunosorbent assay. Systemic tacrolimus treatment was well tolerated by all rats. Tacrolimus accumulated in wound fluid. Tacrolimus levels in wound fluid were found to be approximately 10-fold higher than blood levels (P < 0.001). Tacrolimus (2.0 mg/kg/day) reduced wound-breaking strength (P < 0.01) and collagen deposition (P < 0.05). This was paralleled by decreased wound nitrite + nitrate levels (P < 0.001) and wound-inducible nitric oxide synthase expression. Splenic lymphocyte proliferative activity was significantly decreased by 1.0 and 2.0 mg tacrolimus/kg body weight/day (P < 0.05), indicating that the tacrolimus doses used were immunosuppressive. Our data show for the first time that tacrolimus impairs wound healing, and this is reflected by diminished wound nitric oxide synthesis.

A 42 bp fragment of the pmas1' promoter containing an ocs-like element confers a developmental, wound- and chemically inducible expression pattern.

Synthesis of mannopine in plant tissues infected with Agrobacterium tumefaciens is controlled by a divergent promoter (pmas2' and pmas1') that in 479 bp contains all the cis-acting elements necessary to direct tissue-specific and wound-inducible expression. In this report, using transgenic tobacco plants harboring a pmas1'-beta-glucuronidase (GUS) gene fusion, we investigated the developmental expression pattern directed by pmas1' in the early stages of development and the responses of pmas1' to different chemical inducers. It was found that this promoter can respond to auxins, cytokinins, methyl jasmonate (MJ), salicylic acid (SA) and its analogue 2,6-dichloroisonicotinic acid (iNA). Treatment with chemical inducers also showed that the effects of iNA are organ-dependent, that wound-induction is a complex response mediated by at least two different chemical signals, and that MJ stimulates changes in the tissue-specific and developmental expression pattern directed by the ptmas1' promoter. Using chimeric promoters we demonstrate that an ocs-like element (ocs+1) directs MJ responses in an orientation-dependent manner and that sequences around the ocs+1 are important to maintain the inducible and developmental properties of this cis-regulatory element.

Cloning of the wound-inducible protein CBP20 and expression in suspension cultures of tobacco

One of the proteins secreted from suspension-cultured tobacco cells was identified as the wound-inducible protein CBP20. Four cDNA clones, CBP20/1-CBP20/4, containing fragments of different lengths of the corresponding gene were isolated by immunoscreening two libraries prepared from tobacco leaves (CBP20/1-3) and the suspension culture line S-2 (CBP20/4). The clones, CBP20/1 and CBP20/4, with the largest inserts are identical and homologous to the clone cbp20-44 isolated by Ponstein et al. [1]from a cDNA library prepared from tobacco ( Nicotiana tabacum Samsun NN) leaves inoculated with tobacco mosaic virus. The protein CBP20 is constantly present in suspension cultures of tobacco during subculturing in the presence of different combinations of plant growth regulators. However, accumulation of CBP20 in the culture medium is stimulated by 2,4 dichlorophenoxyacetic acid (2,4 D). The auxin induces a slight transient increase of the mRNA level indicating that the synthesis of CBP20 is at least partly regulated by the mRNA level. CBP20 is synthesized as a precursor of approximately 21 kDa. Maturation occurs over 3 h in a similar temporal manner to that of the vacuolar β-1,3-glucanase resulting in a protein of approximately 19 kDa.