Transgenic pathogen-resistant organism

Abstract

transgenic pathogen-resistant organism whose genome contains at least two different genes under the control of active promoters with pathogen-inhibiting action. This organism is distinguished by a synergistic pathogen-inhibiting action. This action is evident particularly when the genes code for the gene products chitinase (chis, chig), glucanase (glug), protein synthesis inhibitor (psi) and antifungal protein (afp).

Description

where W1 and W2 indicate the efficacies of the individual proteins, which is defined as that percentage deviation of the growth plot (in the presence of the protein) from the untreated control. The efficacy for a protein (at a defined time in the growth plot) is given by:
W1=(OD(K)−OD(P))/OD(K)×100 (percent)
In this, OD(K) is the optical density of the untreated control and OD(P) is the optical density of the culture treated with the protein.

Thus, on combined use of two proteins, the following statements were possible: if the efficacy G measured in the experiment is identical to the expected value E, the behavior is additive. If, on the other hand, G is greater than E, the behavior is synergistic.

Using this test model, it emerged that the proteins ChiS, PSI, AFP, ChiG and GluG used in the Examples surprisingly have synergistic inhibitory effects on various fungi, and these effects were achieved both by the combination of two types of proteins and by multiple combination of the above-mentioned proteins.

For example, the following values were determined from the combination of ChiS and PSI protein and from the combination of AFP protein and PSI protein on the fungus Rhizoctonia solani (in each case two different ChiS and AFP concentrations with a constant RIP concentration):

ChiS+PSI:

The expected values were: E1=29.9% and E2=44.5%

The measured values were: G1=60.4% and G2=64.1%

The proteins ChiS and PSI therefore act together in a synergistic manner in the inhibition of the growth of R. solani.

FIG. 1 shows the results obtained with the combination of the proteins and with the individual substances. According to the Figure, various ChiS concentrations (0.5 μg/ml and 0.05 μg/ml) are combined with PSi protein (1.0 μg/ml.

AFP+PSI:

The expected values were: E1=39.9% and E2=41.9%

The measured values were: G1=57.7% and G2=65.4%

The AFP and PSI combination also according to this shows a synergistic inhibition of growth of the fungus R. solani. FIG. 2 indicates the test results with various AFP concentrations (0.4 μg/ml and 0.04 μg/ml) combined with PSI protein (1.0 μg/ml).

EXAMPLE 2:

Transgenic plants

In order to obtain the organisms according to the invention with DNA sequences which act together synergistically, initially transgenic plants which contained at least one of the genes which act together synergistically were generated.

ChiS in transgenic slants

Initially a ChiS gene was fused to plant regulatory sequences.

A ChiS gene 1.8 Kb in size was sequenced by using synthetic oligonucleotides in the dideoxy sequencing method of Sanger et al. in Proc. Natl. Acad. Sci. USA, 74 (1977), 5463-5467.

The 35S promoter originating from cauliflower mosaic virus (CamV) (400 bp (according to Töpfer et al. in Nucl. Acid. Res., 15 (1987), 5890)) underwent transcriptional fusion to the ChiS gene. The termination signal, which is 0.2 Kb in size, of the 35S gene of CamV, whose functionality in dicotyledonous plants is known, was used 3′ from the ChiS gene. The chimeric gene 35S-ChiS was cloned into the pLS034 vector by means of the Agrobacterium tumefaciens transformation system in tobacco and potato plants, and kanamycin-resistant plants were regenerated.

It was possible to detect both the ChiS gene and the corresponding mRNA as well as the gene product protein in the resulting plants.

PSI in transgenic plants

PolyA RNA was initially isolated from ripe barley seeds (Hordeum vulgare L. cv. Piggy) and deposited in a cDNA gene bank λ-gt-11-phages. The details of the process are to be found in R. Lea in Plant. Biol., 12 (1989), 673-682. Monospecific PSI antibodies were then used to identify CDNA clones.

Subsequently, the PSI-positive λ-gt-11-phages were isolated, cloned further and sequenced by the dideoxy sequencing method of Sanger et al. indicated above. The DNA cloned into E. coli was then transferred in the manner described above by conjugation into Agrobacterium LBA4404.

Both the transferred gene and mRNA and gene product were detectable in corresponding transgenic tobacco, potato, rape, strawberry and tomato plants.

AFP in transgenic plants

For the cloning in the vector, the cDNA sequence of the antifungal peptide is provided with ends which can be ligated into BamH1 and Sal1 restriction cleavage sites. The cloning vector used was pDH51 (Pietrzak et al. in Nucl. Acids Res. 14 (1986), 5857). The vector pDH51 was opened with the restriction enzymes BamH1 and Sal1 between promoter and terminator. The vector pDH51 is a pUC18 derivative which contains promoter and terminator sequences of the 35S transcript from cauliflower mosaic virus. These sequences are recognized by the plant's transcription apparatus and lead to strong constitutive expression of the gene associated with them in plants. The DNA of the antifungal peptide is then cloned via the BamH1 and Sal1 cleavage site into the vector. Finally, the transcription unit—promoter, gene and terminator—is cut out of the vector using the restriction enzyme EcoRI and cloned into a plant transformation vector. The following vectors and their derivatives can, for example, be used as plant transformation vector:

• • pOCA18 (Olszewski et al. in Nucl. Acids Res., 16 (1988), 10765) pPCV310 (Koncz and Shell in MGG 204 (1986), 383) and pBin19 (Bevan et al. Nucl. Acids. Res. 12 (1984), 8711)

After the transcription unit and the vector had been ligated via the EcoRI cleavage site, the construct was conjugated into the Agrobacterium strain MP90RK (Koncz and Shell (see above)) or IHA101 (Hood et al. in J. Bacteriol. 168 (1986), 1291).

Transgenic tobacco, potato, strawberry, rape and tomato plants were then transformed by the method described above. Transformed shoots are selected on the basis of the cotransferred resistance to the antibiotic kanamycin. Expression of the antifungal protein in the transformed crop plants was checked and confirmed by DNA analysis (Southern blotting), RNA analysis (Northern blotting) and protein analysis with specific antibodies (Western blotting).

ChiG and GluG in transgenic plants

ChiG- and GluG-transgenic plants which were both Southern-, Northern- and Western-positive were obtainable in analogy to the plants described above.

ChiS, PSI, AFP, ChiG, GluG in transgenic monocotyledonous plants.

It was possible by means of direct gene transfer to integrate the abovementioned genes into the genome of monocotyledonous plants such as, for example, corn. This resulted in transgenic plants which were Southern- and Northern- and Western-positive.

Combination of various fungus-resistance genes in transgenic plants

The previously obtained tobacco, corn, rape, strawberry, potato and tomato plants were crossed together and selected for plants containing in each case the fungus-resistant genes of both parents. In addition, transgenic plants were obtained by transforming them initially with one and then with one or more other gene. Finally, plants were also transformed with vectors which contained various resistance genes. Fungus-resistance tests were done with this plant material. Surprisingly, in all cases synergistic effects, not just additive effects, in respect of fungus resistance are observed.

For example, a tobacco plant which expresses ChiS and PSI shows a considerably greater resistance to Rhizoctonia infestation than the plants which expressed only ChiS or PSI or which would result from the additive resistance.

A synergistic inhibitory effect on infestation with Rhizoctonia solani also results from combined expressed of PSI- and AFP-transgenic tobacco. Combination of two or more different genes (ChiS, RIP, AFP, ChiG and GluG) in a wide variety of transgenic plants also led to synergistic inhibitory effects on various fungi.

Whereas wild-type plants have index values from 38 to 46 in tests on 20 seedlings, it emerges with transgenic tobacco according to the invention that the latter grows as well in the presence of the fungus Rhizoctonia solani as do control plants (index value 10-12) cultivated on Rhizoctonia-free soil.

Claims

1. A process for producing a plant having increased resistance to fungal attack, comprising topically applying, to a transgenic plant, a first gene product of a gene selected from the group consisting of a chig gene from barley, a glug gene from barley, a psi gene from barley, and an afp gene from Aspergillus giganteus, wherein the transgenic plant carries at least two transgenes, each operably linked to a plant-functional promoter, wherein one transgene is a chis gene from serratia marcescens and a second transgene is a gene selected from the group consisting of a chig gene from barley, a glug gene from barley, a psi gene from barley, and an afp gene from Aspergillus giganteus, provided that the second transgene does not encode the first gene product.

2. A process for producing a plant having increased resistance to fungal attack, comprising topically applying, to a transgenic plant, a first gene product of a gene selected from the group consisting of a chig gene from barley, a glug gene from barley, a psi gene from barley, and a chis gene from serratia marcescens, wherein the transgenic plant carries at least two transgenes, each operably linked to a plant-functional promoter, wherein one transgene is an afp gene from Aspergillus giganteus and a second transgene is a gene selected from the group consisting of a chig gene from barley, a glug gene from barley, a psi gene from barley, and a chis gene from serratia marcescens, provided that the second transgene does not encode the first gene product.