Site-specific binding of a nuclear factor to the carrot extensin gene is influenced by both ethylene and wounding

Experiments conducted in vitro using the electrophoretic mobility shift assay have shown that a single region of the extensin gene of carrot (Daucus carota L.) interacts with a protein factor designated Extensin Gene Binding Factor-1 (EGBF-1) present in nuclear extracts obtained from carrot roots. This interaction is sequence-specific as judged by the failure of other plant gene sequences to compete with the extensin gene for EGBF-1 binding. The EGBF-1 activity is organspecific, not being expressed in nuclear extracts obtained from carrot leaves or stems. Both ethylene treatment and wounding of roots are shown to have a controlling influence on the expression of EGBF-1 activity in nuclear extracts. These results demonstrate that at least three distinct signals: ethylene treatment, wounding, and development, are important in determining the activity of EGBF-1 in nuclear extracts, and indicate a role for EGBF-1 in stress-related signal transduction and the regulation of extensin-gene expression.Abbreviations bp base pair(s) - EGBF extensin-gene binding factor - EMSA electrophoretic mobility shift assay - HRGP hydroxyproline-rich glycoprotein - kb kilobase     

Purification of extensin from cell walls of tomato (hybrid of Lycopersicon esculentum and L. peruvianum) cells in suspension culture

Extensin, a hydroxyproline-rich glycoprotein comprising substantial amounts of -l-arabinose-hydroxyproline glycosidic linkages is believed to be insolubilized in the cell wall during host-pathogen interaction by a peroxidase/hydroperoxide-mediated cross-linking process. Both extensin precursor and extensin peroxidase were ionically eluted from intact water-washed tomato (hybrid) of Lycopersicon esculentum Mill. and L. peruvianum L. (Mill.) cells in suspension cultures and purified to homogeneity by a rapid and simple procedure under mild and non-destructive experimental conditions. The molecular weight of native extensin precursor was estimated to be greater than 240–300 kDa by Superose-12 gel-filtration chromatography. Extensin monomers have previously been designated a molecular weight of approximately 80 kDa. Our results indicate that salt-eluted extensin precursor is not monomeric. Agarose-gel electrophoresis, Superose-12-gel-filtration, extensin-peroxidase-catalysed cross-linking, Mono-S ion-exchange fast protein liquid chromatography (FPLC), and peptide-sequencing data confirmed the homogeneity of the extensin preparation. Evidence that the purified protein was extensin is attributed to the presence of the putative sequence motif — Ser (Hyp)₄ — within the N-terminal end of the protein. Treatment of extensin with trifluoroacetic acid demonstrated that arabinose was the principal carbohydrate. The amino-acid composition of the purified extensin was similar to those reported in the literature. The cross-linking of extensin in vitro upon incubation with extensin peroxidase and exogenous H₂O₂ was characteristic of other reported extensins. Furthermore, Mono-S ion-exchange FPLC of native extensin precursor resolved it into two isoforms, A (90%) and B (10%). The amino-acid compositions of extensin A and extensin B were found to be similar to each other and both extensins were cross-linked in vitro by extensin peroxidase.Abbreviations CM-cellulose carboxymethyl-cellulose - FPLC fast protein liquid chromatography - HF hydrogen fluoride - HRGP hydroxyproline-rich glycoprotein - Hyp hydroxyproline - V_c retention volume - TCA trichloroacetic acid - TFA tri-fluoroacetic acid This work was supported by a A.F.R.C. postdoctoral assistantship to Michael D. Brownleader. We thank Dr. Anthony K. Allen (Department of Biochemistry, Charing Cross and Westminster Hospital, London, UK) for performing the amino-acid analysis and Mrs. Margaret Pickering (Department of Biochemistry, Royal Holloway) for performing the peptide-sequence analysis of extensin. We also express our gratitide to Dr. A. Mort (Oklahoma State University) for performing the HF-deglycosylation of extensin.     

A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris

A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)₄ repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.     

Extensin from suspension-cultured potato cells: a hydroxyproline-rich glycoprotein, devoid of agglutinin activity

Enhanced deposition and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in the plant cell wall is acknowledged to contribute to the formation of a resistant barrier against pathogen infection. We have isolated, from suspension-cultured potato (Solanum tuberosum L. cv. Desiree) cells, two forms of soluble HRGP, a cross-linked and a monomeric form; the latter can be converted to the cross-linked form by incubation with tomato extensin peroxidase and H₂O₂. The monomeric form was purified by Sephacryl S-200 gel-filtration, reverse-phase high-performance liquid chromatography and Mono-S cation-exchange chromatography into two isoforms (A, a minor form; B, a major form). The properties of the B isoform were further investigated. A quantitative enzyme-linked immuno-sorbent assay of the B isoform, using tomato extensin antiserum, showed a titration curve at a high antibody-dilution range comparable to that of purified tomato extensin monomer (M.D. Brownleader and P.M. Dey, 1993, Planta 191: 457–469). The amino acid and carbohydrate compositions were similar to those of tomato extensin, but did not match well with the other two HRGPs from potato, potato lectin and potato bacterial agglutinin. These observations demonstrate the similarities of the B isoform to extensin. The homogeneity of the B isoform was demonstrated by its ability to be fully cross-linked in vitro, leaving no residual protein, into a high-molecular-weight form by the action of extensin peroxidase. The trifluoroacetic acid-deglycosylated sample migrated as a single protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, SDS-PAGE and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry indicated a molecular weight of approximately 67 kDa. Circular-dichroism spectroscopy demonstrated that the molecule possesses an extended polyproline II helix conformation with no evidence of α- helix or β- sheet secondary structure. In conclusion, we refer to this HRGP as potato extensin. As proposed for other extensins, potato extensin is likely to play a role in cell wall architecture and plant disease resistance. Received: 25 November 1996 / Accepted 13 January 1997     

Induced resistance against Alternaria brassicae blight of mustard through plant extracts

An experiment was conducted to study the effect of plant extracts on soluble sugar, soluble phenol and defence-related enzymes response against Alternaria blight in mustard crop. The efficacy of six selected plant extracts (5 and 10%) used as foliar sprays at 60 and 70 days after sowing and mustard leaves was used for investigation. The results indicate that soluble phenol and sugar content in mustard leaves significantly increases in response to spraying of Azadirachta indica seed kernel, Calotropis procera and A. indica leaf extracts. The soluble protein, viz. peroxidase, polyphenol oxidase and phenylalanine ammonia lyase content, was higher in mustard leaves sprayed with C. procera leaves extract, A. indica seed kernel and Allium sativum bulb extract.     

Enzymic cross-linkage of monomeric extensin precursors in vitro

Rapidly growing tomato (Lycopersicon esculentum) cell suspension cultures contain transiently high levels of cell surface, salt-elutable, monomeric precursors to the covalently cross-linked extensin network of the primary cell wall. Thus, we purified a highly soluble monomeric extensin substrate from rapidly growing cells, and devised a soluble in vitro cross-linking assay based on Superose-6 fast protein liquid chromatography separation, which resolved extensin monomers from the newly formed oligomers within 25 minutes. Salt elution of slowly growing (early stationary phase) cells yielded little or no extensin monomers but did give a highly active enzymic preparation that specifically cross-linked extensin monomers in the presence of hydrogen peroxide, judging from: (a) a decrease in the extensin monomer peak on fast protein liquid chromatography gel filtration, (b) appearance of oligomeric peaks, and (c) direct electron microscopical observation of the cross-linked oligomers. The cross-linking reaction had a broad pH optimum between 5.5 and 6.5. An approach to substrate saturation of the enzyme required extensin monomer concentrations of 20 to 40 milligrams per milliliter. Preincubation with catalase completely inhibited the cross-linking reaction, which was highly dependent on hydrogen peroxide and optimal at 15 to 50 micromolar. We therefore identified the cross-linking activity as extensin peroxidase.     

Levels of 4-hydroxystilbene-oxidizing isoperoxidases related to constitutive disease resistance in in vitro-cultured grapevine

A zymographic screening of peroxidases (EC 1.11.1.7) capable of oxidizing 4-hydroxystilbene was carried out by means of the peroxidase-catalyzed oxidative coupling of 4-hydroxystilbene and 4-aminoantipyrine. This screening reveals that only a few isoperoxidases are active in oxidizing 4-hydroxystilbene to viniferin-type compounds in in vitro cultures of grapevine. Unlike total peroxidase activity measured with 4-methoxy--naphthol, the levels of total peroxidase activity measured using 4-hydroxystilbene are related to disease resistance against downy mildew in axillary bud cultures of Vitis vinifera and (Vitis vinifera x Vitis rupestris) x Vitis riparia. From this observation, and using the above zymographic assay, it was found that a 4-hydroxystilbene-oxidizing isoperoxidase was overexpressed in both leaves and stems of the hybrid in relation to the increase in disease resistance of this species. These results suggest that constitutive 4-hydroxystilbene-oxidizing isoperoxidases may participate through their role in viniferin synthesis in the constitutive resistance mechanism that grapevines show against downy mildew.Abbreviations 4-AAP 4-aminoantipyrine - HRP horseradish peroxidase - 4-HS hydroxystilbene - HSPrx 4-hydroxystilbene-oxidizing peroxidase - 4-MN 4-methoxy--naphthol     

Identification of a wound-induced inhibitor of a nuclear factor that binds the carrot extensin gene

Following wounding of carrot (Daucus carota L.) roots, the activity of a nuclear factor (EGBF-1) that binds a 5′-region of the carrot extensin gene declines to undetectable levels within 48 h. Mixing of nuclear extracts from wounded roots with nuclear extracts from unwounded roots has demonstrated the existence of a wound-induced inhibitor of EGBF-1. Inhibition of EGBF-1 DNA-binding activity by nuclear extracts from wounded roots is shown to be specific for EGBF-1, and to be destroyed by heat treatment. In addition, inhibition is saturable and occurs rapidly. Active EGBF-1 can be reconstituted from its inhibited state by renaturation of proteins from mixed extracts following denaturation by boiling in sodium dodecyl sulfate and 2-mercaptoethanol, and electrophoretic separation, indicating that inhibition is dependent upon the reversible interaction of EGBF-1 with a titratable factor. However, EGBF-1 activity could not be detected in nuclear extracts from wounded roots following denaturation and electrophoretic separation. Inhibitory activity was not detectable in nuclear extracts from roots that had been trated with ethylene. The action of the inhibitor indicates one possible mechanism for the control of EGBF-1 activity in carrot roots following wounding.     

Cross-linking of soluble extensin in isolated cell walls

The extensin component of primary cell walls has generally been considered to be an intrinsically insoluble cell wall glycoprotein. Recent data have established that cell wall extensin is in fact secreted in a soluble monomeric form which slowly becomes insolubilized in the cell wall probably through the oxidative formation of isodityrosine cross-links. We now show that isolated cell walls from aerated root slices of Daucus carota have the capacity to insolubilize extensin through the formation of isodityrosine. This in vitro cross-linking is specific for the extensin glycoprotein, as other wall proteins are not cross-linked by the isolated wall system. Although extensin can be cross-linked in solution by peroxidase and H₂O₂, dityrosine and not isodityrosine is the phenolic cross-link formed. Wall-catalyzed cross-linking of soluble extensin is inhibited by l-ascorbate, and both the initial rate and total extent of cross-linking are inhibited by acidic pH in the physiological range (pH 4 to 6). We suggest several mechanisms by which acid might inhibit cross-linking and propose that cytoplasmic factors (ascorbate and/or hydrogen ions) may regulate the solubility of extensin in vivo.     

Formation of Isodityrosine by Peroxidase Isozymes

Fry, S. C. 1987. Formation of isodityrosine by peroxidase isozymes.—J.exp. Bot. 38: 853–862. Tyrosine residues of extensin are oxidatively coupled in vivoto form isodityrosine bridges, whereas treatment of purifiedextensin with H₂O₂+ peroxidase in vitro yields only dityrosine.Two explanations for the correct mode of coupling in vivo weretested. The first, that the pH of the cell wall is lower thanthat (pH 9-0) at which in vitro experiments have been conducted,provided part of the answer since treatment of L-tyrosine withH₂O₂+peroxidase in vitro at pH 37–5 yielded some isodityrosine.The second, that the wall contains other isozymes of peroxidasethan the basic isozyme usually studied in vitro, appeared unlikelybecause several sharply contrasting isozymes yielded similarisodityrosine: dityrosine ratios from L-tyrosine+ H₂O₂ at anygiven pH. The isozymes were also similar in their ability tooxidize tyrosine-dimers further to higher polymers. It is concludedthat the formation of isodityrosine in vivo is dictated by neighbouringwall molecules, possibly ionically-bound pectins, which modifythe local environment of the tyrosine residues of extensin. Key words: Isodityrosine, peroxidase isozymes, extensin     

Ethylene effect on extensin and peroxidase distribution in the subapical region of pea epicotyls

In dark grown pea (Pisum sativum) seedlings ethylene causes the triple response in which elongation growth is inhibited, radial growth is promoted, and orientation of shoots to gravity is altered. The distribution of extensin and peroxidase activity in pea epicotyls upon ethylene treatment was studied by tissue printing on nitrocellulose paper. It was found that the localization of extensin and peroxidase activity changes after 72 and 96 hours of ethylene treatment. In untreated plants, peroxidase activity is detected only in the vascular bundles. Nonetheless, after 72 and 96 hours of ethylene treatment peroxidase activity is hardly detected in the vascular system but present in the epidermal and cortical cells. Extensin increases in the epidermal and cortical cells upon ethylene treatment but it also appears in the vascular system when peroxidase activity is no longer detected.     

Isoperoxidase Activity and Induction in Cultured Tissues of Wild Carrot: a Comparison of Proembryos and Embryos

Several anodic isoperoxidases were found in embryonic tissues of cultured wild carrot, Daucus carota L., which were not present in the proembryo masses from which they originate. This difference is further reflected in the higher specific activity of peroxidase in embryo extracts as compared to proembryonic tissues. The absence of anodic isoperoxidases and depressed peroxidase activity in carrot tissue cultures in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) suggests a regulatory role for this plant growth regulator in controlling peroxidase activity.     

Antibody localization of extensin in cell walls of carrot storage roots

The accumulation and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in cell walls of dicotyledonous plants has been correlated with a number of wall-strengthening phenomena. Polyclonal antibodies raised against glycosylated extensin-1, the most abundant HRGP in carrot (Daucus carota L.) cell walls, recognize this antigen on gel and dot blots and on thin sections of epoxy-embedded carrot-root cell walls. Since wall labeling can be largely reduced by preincubating the antibodies with purified extensin-1, most labeling can be attributed to recognition of this antigen. The remaining label may be the result of recognition of extensin-2, a second carrot HRGP, or other wall components (cellulose, hemicellulose and pectin are not recognized). Extensin-1 label was distributed quite uniformly across the cell wall but was absent from the expanded middle lamella at the intersection of three or more cells and was reduced in the narrow middle lamella between two cells. This distribution is essentially the same as that of cellulose. Because of limitations of this labeling technique, it is not possible to construct a complete model of the structure of the cross-linked extensin matrix. Nonetheless, short, linear arrays of gold particles may represent small portions of the extensin matrix or of individual extensin molecules as they are exposed on the surface of sections. These and other results presented here indicate that: a) newly synthesized extensin is added to the wall by intussusception; b) extensin cannot cross the middle lamella separating the walls of adjacent cells; and c) incorporation of extensin is a late event in the development of phloem-parenchyma cell walls in carrot.Abbreviations dE-1 antibodies antibodies raised against deglycosylated extensin 1 - ELISA enzyme-linked immunosorbant assay - gE-1 antibodies antibodies raised against glycosylated extensin 1 - HRGP hydroxyproline-rich glycoprotein - PAGE polyacrylamide gel electrophoresis - RG-1 rhamnogalacturonan I - SDS sodium dodecyl sulfate     

胡萝卜愈伤组织伸展蛋白纯化及某些生化特性的研究

本文报道了胡萝卜愈伤组织伸展蛋白的柱色谱纯化,电泳性质,氨基酸组成及其电镜观察结果。用CM-cellulose柱色谱纯化胡萝卜愈伤组织伸展蛋白时,仅发现有一个组分;它的电泳性质与胡萝卜根产生的第一种类型伸展蛋白相同;它的羟脯氨酸/絲氨酸克分子数比例大约为4/1(羟脯氨酸,42.1mol％;丝氨酸,12.8mol％);此外,在电子显微镜下观察,伸展蛋白具有典型棒状分子结构。     

Evidence for Compartmentation of Tryptophan in Cultured Plant Tissues: Free Tryptophan Levels and Inhibition of Anthranilate Synthetase

1. Anthranilate synthetase activity in crude extracts from tissue cultures of Daucus carota L. (carrot), Nicotiana tabacum L. (tobacco; cv. Wisconsin 38 and xanthi), Glycine max Merr. (soybean) and Oryza sativa L. (rice) was completely inhibited by l -tryptophan (5 to 50 μM). Mutant carrot and tobacco lines, capable of growth in the presence of 5-methyltryptophan, required 500 to more than 1000 μM tryptophan for complete inhibition of enzyme activity, respectively. 2. Except for the mutant tobacco line, the concentrations of free tryptophan in all tissue cultures tested were greater than the levels necessary to completely inhibit the respective anthranilate synthetase activities in vitro. These findings would indicate that much of the free tryptophan is compartmentalized away from the regulatory enzyme, anthranilate synthetase. This could implicate compartmentalization of the inhibitor as a biosynthetic control mechanism. 3. During the growth of normal and mutant carrot tissues the anthranilate synthetase enzyme must be at least 7.8 and 10.8% active, respectively, in order to accumulate the amount of tryptophan found in the tissues. 4. Of the substrates and cofactors required for anthranilate synthetase activity in vitro, Mg²⁺ and glutamine were present at near optimal levels in the carrot and tobacco tissues, but chorismate was found to be significantly below the optimal concentrations.     

Antifungal and enzymatic evaluation of plant crude extracts derived from cinnamon and rosemary against Sclerotinia carrot rot

The objective of this study was to assess the antifungal potential of plant crude extracts derived from cinnamon and rosemary against three isolates of Sclerotinia sclerotiorum under in vitro and in vivo conditions. The crude extracts were obtained using two different solvents including ethyl acetate (EA) and ethanol. The results showed that crude extracts of cinnamon are able to reduce mycelial growth of isolate 2 at volatile and contact phase by 35.4% and 78.2%, respectively. Furthermore, sclerotial myceliogenic at contact phase and carpogenic germination of isolate 2 were inhibited by 94.3% and 68.1%, respectively. In general, rosemary extracts showed less inhibitory efficacy than cinnamon. As the most effective treatment, EA extract of cinnamon was analysed using Gas Chromatography/Mass Spectrometry. The results showed the presence of 33 components and the major constituents were E‐cinnamaldehyde (66.4%) followed by Alpha‐Muurolene (4.86%), Alpha‐Copaene (4.73%) and 2H‐1‐Benzopyran‐2‐one (3.72%). The enzyme analysis showed that the activity of phenylalanine ammonia lyase, polyphenoloxidase and peroxidase decrease in the inoculated carrots after application of plant crude extracts indicating that they cannot be considered as resistance inducers against Sclerotinia carrot rot. In conclusion, cinnamon extract was found to be more effective against the pathogen. Although crude extracts of cinnamon and rosemary were able to reduce severity of carrot rot during storage, EA extract of cinnamon (2 g L^?1) was found to have practically significant effect against the disease.     

UHPLC/HR‐ESI‐MS/MS Profiling of Phenolics from Tunisian Lycium arabicum Boiss. Antioxidant and Anti‐lipase Activities’ Evaluation

This study was performed in the aim to evaluate nine different extracts from Tunisian Lycium arabicum for their total phenolic and total flavonoid contents, phytochemical analyses as well as their antioxidant and anti‐lipase activities. The in vitro antioxidant property was investigated using three complementary methods (DPPH, ferric reducing antioxidant power (FRAP), and β‐carotene‐linoleic acid bleaching assays) while anti‐lipase activity was evaluated using 4‐methylumbelliferyl oleate method. From all of the tested extracts the most potent found to be the polar MeOH extracts especially those of stems and leaves. In order to investigate the chemical composition of these extracts and possible correlation of their constituents with the observed activities, an UHPLC/HR‐ESI‐MS/MS analysis was performed. Several compounds belonging to different chemical classes were tentatively identified such as rutin and kampferol rutinoside, the major constituents of the leaves, and N‐caffeoyltyramine, lyciumide A, N‐dihydrocaffeoyltyramine as well as fatty acids: trihydroxyoctadecadienoic acid and hydroxyoctadecadienoic acid isomers were detected abundantly in the stems. These results showed that the MeOH extracts of stems and leaves of L. arabicum can be considered as a potential source of biological active compounds.     

Changes in Peroxidases and Catalase Activity During in vitro Rooting

Enzyme changes in non-rooted (treated with Fe-EDTA) and rooted (treated with Fe-EDDHA) stems of rootstock GF-677 (Prunus amygdalus×P. persica) during adventitious root formation in vitro have been recorded. The first roots appeared approximately after 12 d on the rooting medium. By contrast to non-rooted stems, rooted stems showed a maximum of soluble peroxidase activity on the 9^th day, of ionically bound peroxidase to cell wall on the 6^th and 12^th day and of catalase on the 6^th and the 15^th day. A time course study of changes of soluble peroxidases isoenzymes showed that there was a band visible only in the rooted stems and also a new band appeared three days before the emergence of roots.     

Characterization of a hydroxyproline-rich glycoprotein in pearl millet and its differential expression in response to the downy mildew pathogen <Emphasis Type="Italic">Sclerospora graminicola</Emphasis>

A monoclonal antibody, JIM 20, derived against an extensin type of hydroxyproline-rich glycoprotein (HRGP) from pea, showed high affinity for HRGP in pearl millet [Pennisetum glaucum (L.) R. Br.]. Electrophoretic separation of Tris–SDS extracted proteins from suspension cells of pearl millet revealed a range of PM-HRGP polypeptides having a glycan epitope, which reacted with JIM 20. A high molecular mass band, probably an HRGP aggregate or polymer, and a few low molecular mass polypeptides were recognized by JIM 20 during Western blot analysis. Treatment of pearl millet suspension cells with hydrogen peroxide in the presence of an endogenous peroxidase resulted in insolubilization of HRGP polypeptides with molecular weights between 45 and 33 kDa. To investigate the gene coding for an extensin type of HRGP, a fosmid-based genomic library of pearl millet having a fourfold genome coverage was constructed. A partial sequence of 378 bp of an HRGP gene was obtained by PCR amplification of pearl millet DNA with a primer pair designed from the conserved regions of monocotyledon extensin type of HRGPs. Screening the genomic library using the homologous probe developed from the 378-bp PCR product resulted in the isolation of five fosmid clones. Restriction mapping of these fosmids resulted in an 11.8-kb region around an HRGP gene in pearl millet. The newly characterized gene, PM-HRGP, had all the characteristic features of a monocotyledon extensin type of HRGP. An intron at the 3′ untranslated region of the gene was identified by cDNA cloning. Differential expression of the PM-HRGP gene was observed during compatible and incompatible interactions of pearl millet with the downy mildew pathogen Sclerospora graminicola (Sacc) Schroet. Induced expression of the gene was observed only in case of an incompatible interaction.