DE4117026A1 - Prodn. of pathogen-resistant organisms, esp. plants - by introduction of chitinase gene giving plants with increased resistance to attack by pathogenic fungi - Google Patents

Abstract

Prodn. of pathogen-resistant organisms is effected by introducing a chitinase gene under the control of a transcriptionally active promoter into the genetic material of an organism. An exogenous (esp. bacterial) chitinase gene is pref. used, esp. a Serratia marcescens gene coding for coupled exofendochitinase activity. The S. marcescens ChiS gene has been isolated (from an unspecified source) as a 1-8 Kb DNA fragment. The ChiS gene is present as a pLChiA insert in E. coli A5178 (Phytopathol., 79, 1246 1989). Fusion of the ChiS DNA fragment with the cauliflower mosaic virus 35S promoter yields a chimeric 35S-ChiS gene, which is cloned in pLS034 and used to transform tobacco and potatoe plants via Agrobacterium tumefaciens LBA 4404. Similarly, fusion of ChiS with the wound- and pathogen-inducible wunl promoter yields a chimeric wunl-ChiS gene for transformation of tobacco and potato plants. USE - The process may be used to produce transgenic plants with increased resistance to attack by pathogenic fungi, e.g. Rhizoctonia solani in the case of tobacco plants

Description

Process for the production of pathogen-resistant eukaryotes

The invention relates to a method for generating pathogen-resistant organisms, generated by the method Organisms as well as new DNA transfer vectors and DNA expression vectors.

State of the art

The infestation of a plant by pathogens calls a number different reactions on the plant side. These include e.g. B. changes in cell wall structure, the synthesis of antimicrobial phytoalexins, the Accumulation of so-called PR proteins (PR = "Pathogenesis-related"), protease inhibitors and Enzymes with hydrolytic functions (Hahlbrock and Grisebach, 1979; Darvill and Albersheim 1984; van Loon, 1985).

Enzymes with hydrolytic functions also include chitin-degrading enzymes, so-called chitinases. Chitin is a component of the cell wall of many phytopathogenic fungi and insects, while plants do not have chitin. In Several cases have shown that plants Chitinases increased after a pathogenic attack produce (Joosten and de Wit, 1989; Legrand et al., 1987, Nasser et al., 1988). In no case, however, so far shown that plants are produced by the production of Chitinases have increased resistance to pathogens receive.

The invention described here is based on the hypothesis that that the attacking fungus adheres to the plant's own Chitinase has "gotten used" to the extent that it has none Impairment to the fungus. This results the idea of "foreign" chitinase genes in the genome of Insert plants so that the attacking pathogen Chitinases found in the plant to which it was previously concerned  could not adapt. This way the pathogen no longer infect the plant. The origin of the "alien" chitinase blessing can be an alien plant, but it can also be chitinase genes from mushrooms and Bacteria act. In this invention is representative for a "foreign" chitinase gene a gene from the bacterium Serratia marcescens has been used.

invention

From the soil bacterium Serratia marcescens a 1.8 Kb large DNA fragment isolated that for a chitinase, called ChiS. In vitro investigations with purified ChiS protein have been shown to be low in Concentrations can inhibit the growth of fungi. The cause of the inhibition is that the ChiS protein surprisingly about a combined Endo / exo-chitinase activity is associated with the Hyphal peaks of the fungus can be destroyed. To this In this way the fungus cannot continue to grow and is inhibited. The pathogen-inhibiting property of the ChiS protein is on Plants transferable if the ChiS gene is located under the Control of a transcriptionally active promoter in the Genome of plants incorporated. Using transformed Tobacco plants could be shown to be plants resistant to ChiS protein production different plant pathogens have become, such as B. against the fungus Rhizoctonia solani. Thus are newly acquired Pathogen resistance properties of transgenic plants correlated directly with the expression of ChiS genes.

Summary

The invention describes the discovery, characterization and application of a bacterial chitinase, ChiS. ChiS is characterized by its high stability and  their ability to reduce the cell wall of pathogens due to a to destroy combined endo / exo-chitinase activity and thus to be able to inhibit the pathogen. These Properties of the ChiS protein are on plants transferable. By introducing the ChiS gene into one Plant heritage and its expression, the plant becomes made resistant to pathogenic infestation.  

material media

Bacterial media:
The media used to grow bacteria were given the information by Maniatis et al. (1982).

Plant media:
The media used are derived from the media (MS) given by Murashige and Skoog (1962).

3MS: MS + 3% sucrose
3MSC: MS + 3% sucrose, 500 µg / ml Claforan
MSC15: MS + 2% sucrose, 500 µg / ml Claforan,
+ 100 µg / ml kanamycin sulfate
MSC16: MS + 0.5 µg / ml BAP + 0.1 µg / ml NAA
+ 100 µg / ml kanamycin sulfate
+ 500 µg / ml Claforan

An additional 6.5 g / l Bacto agar was used for solid medium admitted.

Tribes and vectors

E. coli strains:
BMH 71-18:
delta (lac-proAB), thi, supE;
F '(laci ^q , ZdeltaM15, proA⁺B⁺)
(Messing et al. 1977)

Agrobacterial strains:
LBA 4404: (Hoekema et al., 1983)

Plasmids:
pUC8 (Vieira and Messing, 1982) pPR69 (a derivative of bin 19, Bevan 1984)

Plants:
Nicotiana tabacum SRI
Solanum tuberosum Desiree

Mushrooms:
Rhizoctonia solani, Basidiomycet, (gift from Prof. I. Chet, Hebrew University, Israel)

Methods used

All standard molecular biological methods, such as B. Restriction analysis, plasmid isolation, mini preparation of Plasmid DNA, transformation of bacteria, etc. unless otherwise stated, as in Maniatis et al., (1982).

In vitro fungal growth test with ChiS protein

Based on the work of Broekaert et al., 1990 with a defined amount of Rhizoctonia solani mushroom mycelium 100 μl of potato dextrose solution are added and added Microtiter plates at 25 ° C. incubated. It is Growth of the fungus with the increase in optical density 495 nm linearly correlated. By adding proteins with potentially fungal growth inhibitory activity their properties due to a lower increase in optical density can be demonstrated.

Analysis of the ChiS nucleotide sequence

The 1.8 Kb ChiS gene was created using synthetic oligonucleotides according to the Dideoxy sequencing method of Sanger et al., 1977 sequenced.

DNA transfer in agrobacteria transformation

The DNA cloned in E. coli was according to that of Van Haute et al., (1983) described method by conjugation  A.tumefaciens LBA4404 (Hoekema et al., 1983).

DNA analysis

Checking the DNA transfer into the Agrobacterium was done by isolating Agrobacteria DNA after the by Ebert et al. (1987) method described. Restriction cleavage of the DNA, the transfer on Nitrocellulose and hybridization against radioactive marked ChiS probe gave information about one successful DNA transfer into the Agrobacterium.

Transformation of tobacco / potato plants with agrobacteria Cultivation of agrobacteria

The agrobacteria LBA4404 required for infection were grown in selective antibiotic medium (Zambrisky et al., 1983), sedimented by centrifugation and washed in YEB medium without antibiotics (YEB = 0.5% meat extract; 0.2% yeast Extract; 0.5% peptone; 0.5% sucrose; 2 mM MgSO ₄ ). After renewed sedimentation and absorption in MgSO ₄ , the bacteria could be used for infection.

Leaf disk infection

Sterile tobacco or Potato leaves used. Pieces of leaf about 1cm in the agrobacterial suspension as described above submerged with subsequent transfer to 3MS medium. After 2 days of incubation in 16 hours of light and 25 ° C-27 ° C the leaf pieces were transferred to MSC16 medium. After 4-6 Weekly appearing shoots were cut and opened MSC15 medium implemented. Roots were formed further analyzed.

DNA analysis of plants

1-2 g of leaf tissue are mortared in an Eppendorf vessel with sea sand, with 1 vol. Extraction buffer (2% CTAB (cetyl-trimetylammonium bromide); 1.4 M NaCL; 100 mM Tris / HCL pH 8.0; 20 mM EDTA) added and 10 minutes at 64 ° C. incubated. Coarse fragments are separated by centrifugation (5 minutes at 13,000 rpm). The extract is shaken with 1 volume of chloroform / isoamyl alcohol and freed of protein. CTAB-nucleic acid complexes are then precipitated (30 minutes at room temperature) by adding 1 volume of precipitation buffer (1% CTAB; 50 mM Tris / HCL pH 8.0; 10 mM EDTA) and pelleted by centrifugation. To separate the CTAB, the pellet is dissolved in 1 M CsCl. The DNA is then precipitated by adding 2 vol. ETOH at room temperature, centrifuged for 5 minutes, washed with 70% ETOH and finally taken up in H ₂ O. The restriction digestion of the isolated DNA, the gel electrophoretic separation of the DNA with agarose, the transfer of the DNA to a nylon membrane is described in Maniatis et al., 1982. Hybridization against radioactively labeled DNA samples was carried out according to Logemann et al., 1987.

RNA analysis of plants

The isolation of total RNA from different organs, the Transfer to nylon membranes as well as hybridization against radioactively labeled DNA samples were carried out according to Logemann et al., 1987.

Protein analysis of transgenic plants

5 g of sheet material are mixed with liquid nitrogen and mortar. The shredded material is then 1-2 ml extraction buffer / g leaf and added to Homogeneity further mortarized (extraction buffer: 50 mM Tris / HCL pH 7.5; 5mM EDTA, 10mM mercaptoethanol; 5 mM PMSF, 25% glycerin, 1 µg / µl pepstatin, 1 µg / µl leupeptin). Rough Parts are removed by centrifugation (10 minutes at 9000 rpm, at 4 ° C.) Low molecular weight components by dialysis against 20 mM Tris / HCL pH 7.5; 10 mM NaCL  separated. 1 mg each of the protein extracts are by SDS-PAGE separated by electrophoresis and then by Electroblotting transferred to nitrocellulose. The on Proteins bound to the filter are de specific antibody analyzed.

Infection of transgenic plants with Rhizoctonia solani

The fungus Rhizoctonia solani is in a liquid medium (Potato dextrose agar from Difco) at 25 ° C. dressed and harvested after 5-6 days. By means of a Büchner funnel and connected feeding bottle will be Medium pulled off. The remaining mushroom mycelium is included a scalpel broken into the smallest possible fragments. The The desired amount of mushroom mycelium is weighed in and with 5 liters sterile uniform earth (ED73) thoroughly mixed. These Soil is distributed in a bowl and with those to be tested Plants planted. Under constant environmental conditions (Temp: 20-24 ° C .; 80-85% rel.humidity) it will Plant growth every 24 hours by determination the shoot length (distance between soil and vegetation point).

Results Isolation and purification of ChiS protein

As described in Shapira et al., 1989, the E. coli contains Strain A5178 the plasmid pLChiA. Located on this plasmid the ChiS gene is under the control of the heat-inducible Promotors PL (PL-Chis). For the production of ChiS protein A5178 bacteria were heat induced, and then the Bacteria separated by centrifugation. The Media excess was one fractional Ammonium sulfate precipitated. The fractions with 30-60% ammonium sulfate saturation was several hours dialyzed (against 20 mM Tris / HCL pH 7.5) and then on a preparative polyacrylamide gel. The dominant ChiS protein band was excised from the gel and the protein elutes from the gel. That way purified native ChiS protein (95% purity) was used for the Determination of the enzymatic properties as well as for the in Vitro growth tests with the fungus Rhizoctonia solani used.

Production of ChiS antibodies

For the production of ChiS antibodies this was from the heat-induced A5178 bacterial supernatant isolated ChiS protein selectively precipitated with the help of ammonium sulfate and through a preparative denaturing Polyacrylamide gel electrophoresis separated and purified. Most recently, the ChiS protein was used in rabbits for harvesting injected with ChiS antibodies. The antibodies obtained turned out to be highly specific against ChiS protein targeted antibodies.

Analysis of the enzymatic properties of the ChiS protein Exo-chitinase activity

The substance p-nitrophenyl-N, N ′ -diacetyl-chitobiose is known as a substrate for exo-chitinases. Cleavage of the para-nitrophenyl residue (see FIG. 2C) leads to a yellowing which can be analyzed photometrically (Roberts et al., 1988). As shown in Figure 2A, purified ChiS protein is able to effectively convert the substrate. As a result, the ChiS protein has exo-chitinase activity.

Endo-chitinase activity

As described by Wolf et al., 1991, the substance CM-chitin-RBV can be digested by chitinases. The substrate is a long chain of sugar molecules to which the dye RBV (Remazol-Brilliant-Violett) is bound. A cleavage can only take place between the sugar molecules (see FIG. 2C), so that only endochitinases can be considered for this process. Figure 2B shows that ChiS protein is able to digest CM-chitin-RBV. As a result, ChiS protein has endo-chitinase activity.

pH optimum

Purified native ChiS protein was mixed with the substrate CM-chitin-RBV at different pH values in order to be able to determine the pH optimum. ChiS shows a broad pH spectrum with optimal activity at pH 7.5 to 9.5 ( Fig. 3).

Optimal temperature

The temperature optimum of purified ChiS protein was analyzed using the substrate CM-chitin-RBV. The high temperature stability of ChiS is remarkable; the temperature optimum is 62 ° C., the activity at room temperature is 20% of the optimum ( FIG. 4).

Lysozyme activity

Some vegetable chitinases are known to:  additionally have lysozyme activity. Investigations with the ChiS protein, however, have shown that none Lysozyme activity is present.

Mushroom growth test with purified ChiS protein

In order to be able to test the influence of ChiS protein on the growth of phytopathogenic fungi, in vitro tests were carried out with the basidiomycete Rhizoctonia solani containing chitin. As described in methods, the growth of Rhizoctonia solani in microtiter plates was analyzed photometrically. Fig. 5 shows a typical growth curve of the fungus. However, the addition of 0.05 µg of purified native ChiS protein completely stops the growth of the fungus. The test with the cooked ChiS sample shows that the ChiS protein is the cause of the fungus inhibition. If the ChiS sample is boiled before the start of the experiment (15 minutes at 100 ° C.) And the ChiS protein is denatured, the fungal growth can no longer be inhibited.

Fusion of the ChiS gene with plant regulatory sequences

The 35S promoter (400 bp) from the cauliflower mosaic virus (CamV) (Töpfer et al., 1987) was transcriptionally fused to the ChiS gene. 3 'of the ChiS gene, the 0.2 Kb termination signal of the 35S gene of the CamV was used, the functionality of which is known in dicotyledonous plants ( FIG. 6). The chimeric gene 35S-ChiS was cloned into the vector pLSO34, transformed into tobacco and potato plants by means of the Agrobacterium tumefaciens transformation system, and kanamycin-resistant plants were regenerated.

Detection of the 35S-ChiS gene in transgenic plants

With the help of Southern blot analysis, transgenic tobacco and Potato plants regarding correct integration  of 35S- examined. In 80% of the plants analyzed a correct integration could be proven.

Detection of ChiS mRNA in transgenic plants

50 µg of total RNA each were isolated from 35S-ChiS transgenic tobacco leaves and examined for the presence of ChiS mRNA ( FIG. 7). An RNA band (2.0 Kb) hybridizing with ChiS is recognizable in 35S-ChiS transgenic plants. The size of the RNA corresponds to the expected size (1.8 Kb ChiS gene plus 0.2 Kb polyA-tail) No ChiS mRNA was detected in untransformed tobacco leaves (K).

Detection of ChiS protein in transgenic plants

Whole protein was isolated from 35S-ChiS transgenic tobacco and potato leaves in order to be able to detect the presence of ChiS protein with the help of ChiS antibodies. As shown in FIG. 8, ChiS proteins with different sizes (58 Kd, 61 Kd, 64 Kd, 66 Kd) can be detected. Further analyzes have shown that the 64 Kd and 66 Kd ChiS proteins are located in the intercellular space of the plant cells. From these observations it can be concluded that the ChiS protein after its synthesis in the cytoplasm (61 Kd) enters the endoplasmic reticulum (58 Kd after cleavage of the leader peptide), where it is glycosylated (bringing the molecular weight to 64 Kd or 66 Kd is increased) and is finally secreted into the intercellular space.

Detection of ChiS protein activity in transgenic plants

In order to be able to demonstrate the enzymatic activity of ChiS protein in transgenic plants, the entire protein was isolated from the intercellular spaces 35S-ChiS of transgenic tobacco and potato plants, separated by gel electrophoresis and blotted onto a substrate gel (contains CM-chitin-RBV). Proteins with chitinase activity can be recognized as bright bands in this way. FIG. 9 shows that the ChiS transgenic plants have an additional chitinase activity compared to untransformed plants, which is attributable to the ChiS protein on account of the running behavior. It could thus be shown that the 35S-ChiS transgenic tobacco and potato plants have an additional, ChiS-related, chitinase activity. This additional ChiS activity could also be demonstrated in other organs (root, stem, etc.) in further experiments.

Establishment of an in vivo "Rhizoctonia tobacco infection system

The Basidiomycet Rhizoctonia solani is a fungal pathogen that also affects tobacco plants. If tobacco plants are cultivated on soil containing Rhizoctonia, the roots, the leaves lying on the ground and the lower stem area are infested within a short time. The destruction of the tissue there results in a slowdown in growth, which can be followed by continuous measurements of the distance between the plant vegetation point and the shoot base. As shown in FIG. 10, a direct correlation between fungal attack and the growth rate of the plant can be demonstrated. The more the soil is infected with Rhizoctonia solani, the more infested the tobacco plants and the less the plant growth.

Infection of 35S-ChiS transgenic tobacco plants with Rhizoctonia solani

10 independent 35S-ChiS transgenic tobacco plants and 10 untransformed tobacco plants (SRI) were planted as 5 cm plants in soil containing various concentrations of Rhizoctonia solani fungus (096, 0.0896; 0.16%; 0.24%) had been inoculated. The occurrence of infestation features (destruction of the lower stem area, maceration of the lower leaf area) was examined daily. After 10 days, the percentage of infected plants depending on the mushroom oculum was created in the form of a graph. It can be seen that in all cases the 35S-ChiS transgenic plants are less affected ( FIG. 11).

In another experiment, the average growth of 10 independently transformed 35S-ChiS transgenic tobacco plants and 10 untransformed SRI tobacco plants in Rhizoctonia solani-infected soil (0.2%) was analyzed. As FIG. 12 shows, untransformed SRI tobacco plants grow significantly less than 35S-ChiS transgenic tobacco plants in the given 9-day period. As a result, the 35S-ChiS transgenic tobacco plants are more resistant to fungal attack.

Examinations with subsequent 35S-ChiS tobacco generations confirm the examinations from the parental generation. A direct correlation between the expression of ChiS protein and the acquired resistance to Rhizoctonia solani was demonstrated. A representative picture of the size difference between 35S-ChiS transgenic tobacco plants and untransformed SRI tobacco plants after growing on fungus-infected soil is shown in FIG. 13.

Use of a wun1-ChiS chimeric gene to generate Fungal resistance in transgenic plants

Analogous to the experiments with the chimeric gene 35S-ChiS, experiments with the chimeric gene wun1-ChiS were also undertaken. It is a transcriptional fusion of the wound and pathogen-inducible plant promoter wun1 (Logemann et al., 1989) with the ChiS gene. 3 ′ of the ChiS gene, the polyA signal from the 35S promoter was used as a termination signal.

This chimeric gene, called wun1-ChiS, has been found in tobacco and Potato plants transformed. Analogous to the expression data  with 35S-ChiS transgenic plants produce wun1-ChiS transgenic plants ChiS-RNA and enzymatically active ChiS protein, provided that the appropriate tissue previously was wounded. Even the wun1-ChiS transgenic plants have a resistance to Rhizoctonia infestation the expression of the ChiS gene is due.

literature

Bevan, M. (1984). Binary Agrobacterium vectors for plant transformation. Nucl. Acids. Res. 12: 8711-8721
Broekaert, WF, Cammue, BPA, and Vanderleyden, J. (1990). An automated quantitative assay for fungal growth inhibition. FEMS Microbiology Letters, in press.
Darvill, AG, and Albersheim, P. (1984). Phytoalexins and their elicitors - A defense against microbial infection in plants. Ann. Rev. Plant Physiol. 35: 34-275.
Ebert, PR, SB, An, G. (1987). Identification of an essential upstream element in the nopalin synthase promoter by stable and transient assays. Proc. of course. Acad. Sci USA 84: 5745-5749
Hahlbrock, K., and Grisebach, H. (1979). Enzymatic controls in the biosynthesis of lignin and flavonoids. Ann. Rev. Plant Physiol. 30: 105-130.
Hoekema, A., Hirsch; P., Hoykaas, P., Schilperoort, R. (1983). A binary plant vector strategy based on separation of vir- and T-region of A. tumefaciens. Nature 303: 179-180
Joosten, MHAJ, de Wit, PJGM, (1989). Identification of several pathogenesis related proteins in tomato leaves inoculated with Cladosporium fluvum (syn. Fulvia fulva) as 1,3-beta-glucanases and chitinases. Plant Physiol., 89: 945-951.
Legrand, M., Kauffmann, S., Geoffroy, P., Fritig, B., (1987). Biological function of pathogenesis related proteins: four tobacco pathogenesis related proteins are chitinases. Proc. Natl. Acad. Sci. USA 84: 6750-6754.
Logemann, J., Schell, J. and Willmitzer, L. (1987). Improved method for the isolation of RNA from plant tissues. Anal. Biochem., 163: 16-20.
Logemann, J., Lipphardt, S., Lörz, H., Häuser, I., Willmitzer, L., and Schell, J., (1989). 5 ′ upstream sequences from the wun1 gene are responsible for gene activation by wounding in transgenic plants. The Plant Cell 1: 151-158.
Maniatis, T., Fritsch, EF, Sambrook, J. (1982). Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Messing, J., Geraghty, D., Hu, NT, Kridl, J. and Rubenstein, I. (1983). Plant Gene Structure. In: Kosuge, F., Meredith, CP, Hollaender, A. (eds.). Genetic engineering of plants. Plenum Press, New York: 211-227.
Murashige, T., Skoog, F. (1962). A rapid method for rapid growth and bioassays wit tobacco tissue cultures. Physiol. Plant., 15: 473-497.
Nasser, W., de Tapia, M., Kauffmann, S., Montasser-Kouhsari, S., Burkard, G. (1988). Identification and characterization of maize pathogenesis related proteins. Four maize PR proteins are chitinases. Plant Mol. Biol. 11: 529-538.
Roberts, WK, Selitrennikoff, CP, (1988). Plant and bacterial chitinases differ in antifungal activity. J. Gen. Microbiol., 134: 169-176.
Sanger, F., Nicklen, S., Coulson, AR. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74: 5463-5467.
Shapira, R., Ordiell, A., Chet, I., Oppenheim, AB, (1989). Control of plant diseases by chitinase expressed from cloned DNA in E. coli. Phytopathology, 79: 1246-1249.
Töpfer, R., Matzeit, V., Gronenborn, B., Schell, J., and Steinbiß, HH, (1987). A set of plant expression vectors for transcriptional and translational fusions. Nucl. Acid. Res., 15: 5890.
Van Haute, E., Joos, H., Maes, M., Warren, G., Van Montagu, M., Schell, J. (1983). Intergenic transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for reversed genetics of Ti-plasmids of Agrobacterium tumefaciens. EMBO J., 2: 411-418.
Van Loon, LC (1985). Pathogenesis-related proteins. Plant Mol. Biol. 4: 111-116.
Vieira, J., Messing, J. (1982). The puc plasmid, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene, 19: 259-268.
Zambrisky, P., Joos, H., Genetello, C., Leemans, J., Van Montagu, M., Schell, J. (1983). Ti-plasmid vector for the introduction of DNA into plant cells without alteration of theit normal capacity. EMBO J., 1: 147-152.

Figure description

Fig. 1 Isolation and purification of ChiS protein from bacteria: E.coli strain A5187 contains the 1.8 kb ChiS gene under the control of a heat-inducible PL promoter. After heat induction, the total protein of the bacterial supernatant was isolated, precipitated with 80% ammonium sulfate and separated on a 10% SDS polyacrylamide gel. Row 1: Total protein from heat-induced A5187 bacteria. Row 2 : total protein from non-heat-induced A5187 bacteria. Row 3 : protein size standard. The ChiS protein (58Kd) is marked with an arrow.

Fig. 2 Detection of Endo / Exo-chitinase activity of ChiS:

Fig. 2A Detection of exochitinase activity: Different amounts of purified ChiS protein were mixed with the substrate p-nitrophenyl-N, N'-diacetyl-chitobiose and the increase in absorbance at OD = measured over time.

FIG. 2B Detection of endochitinase activity: The substrate CMV-chitin-RBV was added to various amounts of purified ChiS protein and the increase in absorbance over time at OD = 550 was measured.

Fig. 2C model of the exo / endochitinase activity of ChiS: Due to the molecular structure of the substrates p-nitrophenyl-N, N'-diacetylchitobiose and CM-chitin-RBV, chitinolytic digestion is only from the end of the molecule (exochitinase) or only from the middle of the molecule (endochitinase) possible. The target of the respective chitinase is marked with an arrow.

Fig. 3 Analysis of the optimum pH for the ChiS protein: 20 ng of purified ChiS protein was at 37 ° C. incubated with the substrate CMV-chitin-RBV (2mg / ml) using different pH concentrations. The activity of the ChiS protein was calculated from three independent absorbance measurements at the times 0, 20, 40, 60 minutes. 1 mUnit is defined as the change in absorbance of 0.001 units per minute.

FIG. 4 Analysis of the optimum temperature for the ChiS protein: 20 ng of purified ChiS protein was incubated at pH 7.5 with the substrate CMV-chitin-RBV (2 mg / ml), using different temperatures. The activity of the ChiS protein was calculated from three independent absorbance measurements at the times 0, 20, 40, 60 minutes. 1 mUnit is defined as the change in absorbance of 0.001 units per minute.

Fig. 5 fungal growth assay with purified protein ChiS: mycelium of the phytopathogenic fungus Rhizoctonia solani was grown in a total volume of 100 ul medium / well of a microtiter plate and various ChiS protein concentrations added (see Methods). The growth of the fungus was monitored at an OD of 495 nm over a period of 55 hours. A measuring point is composed of the average of 12 parallel approaches. Batches without protein (R.solani pur) or batches with heat-denatured (10 minutes at 100 ° C.) ChiS protein (ChiS denat) served as controls.

Fig. 6 Construction of the chimeric gene 35S-ChiS: 35S-ChiS: 400 base pairs of the 35S promoter (p35S) from the cauliflower mosaic virus were transcriptionally fused with the 1.8 Kb ChiS gene. In order to achieve a correct termination of the transcription, the termination signal of the 35S gene was used 3 ′ from the ChiS gene (35SpA). The chimeric 35S-ChiS gene was cloned into the pLSO34 vector. pLSO34 contains a GUS gene under the control of the wound-inducible promoter wun1. In this way, a preliminary examination of transgenic plants with regard to the correct integration of the DNA into the plant genome can be analyzed with the aid of GUS tests. Furthermore, an NPTII gene is located in the vector pLSO34 under the control of the NOS promoter. In this way, a selection of regenerated plant material with the help of kanamycin is made possible. The DNA that is within the T-DNA boundaries (RB, LB) is integrated into the plant genome.

Fig. 7: Northern blot analysis of wun1-ChiS transgenic tobacco plants
50 µg total leaf RNA from tobacco plants was separated by gel electrophoresis, transferred to nylon membranes and hybridized against radioactive ChiS-DNA. No ChiS-RNA is detectable in untransformed tobacco plants (K).

Fig. 8: Western blot analysis of 35S-ChiS transgenic tobacco plants
A total protein extract was prepared from 5 g leaf material from a 35S-ChiS transgenic tobacco plant. The protein solution obtained was divided and subjected to fractional acetone precipitation with different saturation percentages on the one hand, and fractional ammonium sulfate precipitation with different saturation percentages on the other hand. The protein fractions obtained were separated on an 8% denaturing polyacrylamide gel, transferred to nitrocellulose and treated with the ChiS antibody (see methods). 200 ng of purified bacterial ChiS protein was used as positive control (K).

Fig. 9: Detection of ChiS-dependent chitinase activity in transgenic plants:
100 ug total protein from intercellular spaces of different 35S-ChiS transgenic (35S-ChiS) and untransformed (under) tobacco and potato leaves were separated on a native protein gel, transferred to a second protein gel (contains 0.1% CM-chitin-RBV) and then developed for 24 hours at room temperature. The bands document proteins with chitinase activity. ChiS: 2 µg of purified bacterial ChiS protein.

FIG. 10 is a function of the growth rate of untransformed SRI tobacco plants by the fungus amount: 10 The untransformed SRI tobacco plants of the same size were grown in soil which had been treated with various fungal concentrations. The mean values of the increase in length for the measured times are plotted in the diagram.

Fig. 11 Incidence of Rhizoctonia solani infected ChiS transgenic and untransformed tobacco plants. The diagram shows the proportions of the infected 35S-ChiS transgenic tobacco plants or untransformed SRI plants depending on the respective mushroom inoculum for the ninth day after the start of the test. The occurrence of leaf and / or stem rot was rated as infestation.

Fig. 12 Average shoot length increase of Rhizoctonia solani infected tobacco plants: 10 independent 35S-ChiS transgenic tobacco plants and 10 untransformed SRI control plants were infected with Rhizoctonia solani (2 g fungus / 1 liter soil) and with regard to the shoot length increase (given by the shoot length at the time of measurement minus the initial length). The average shoot length increase over a period of 9 days is plotted (calculated error probability: less than 0.5%).

(0.2%) were cultured Representative difference in size between 35S ChiS transgenic tobacco plants and SRI control plants inoculated for 10 days at a Rhizoctonia solani floor: Fig. 13 size difference between fungus infected 35S ChiS transgenic plants and control plants.

Claims (5)

1. Process for the production of pathogen-resistant organisms, characterized in that a chitinase gene is introduced into the genetic makeup of organisms under the control of a transcriptionally active pro motor. 2. The method according to claim 1, characterized, that you ver a chitinase gene of alien origin turns. 3. The method according to claim 1 or 2, characterized, that one uses the gene of a bacterial chitinase. 4. The method according to claim 3, characterized, that one can use a chitinase from Serratia marcescens det, which has a high protein stability and has a coupled exo / endochitinase activity. 5. DNA transfer vectors and DNA expression vectors with inserted gene according to claim 4.