EP1078089A2

EP1078089A2 - Method for producing plants having an increased tolerance against drought and/or fungal attack and/or increased salt concentrations and/or extreme temperature by the expression of plasmodesmata-localized proteins

Info

Publication number: EP1078089A2
Application number: EP99950352A
Authority: EP
Inventors: Wolfgang Rohde; Dirk PRÜFER; Eckhard Tacke; Peter Pasemann; Francesco Salamini
Original assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Current assignee: Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV; Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority date: 1998-05-13
Filing date: 1999-05-12
Publication date: 2001-02-28
Also published as: CA2328131A1; WO1999058653A3; WO1999058653A2; AU4260999A

Abstract

The invention relates to the use of nucleic acids which code a (poly)peptide with an intrinsic affinity to plasmodesmata, to the production of plants or parts thereof having an increased tolerance against drought and/or fungal infections and/or increased salt concentrations and/or extreme temperature (heat, cold), and to corresponding methods. According to the invention, a plant, a plant tissue or a plant cell is advantageously transfected with the nucleic acid. The nucleic acid preferably codes a virus-coded transport protein which, in an especially preferred embodiment, is a derivative of the pr17 protein with a hydrophilic N-terminal extension.

Description

Process for the production of plants with increased tolerance to

Dryness and / or fungal attack and / or increased salt concentrations and / or extreme temperature due to the expression of plasma-localized proteins

The invention relates to the use of nucleic acids encoding a (poly) peptide with an intrinsic affinity for plasmodesms, for the production of plants or parts thereof with increased tolerance to drought and / or fungal infections and / or increased salt concentrations and / or extreme temperatures (cold , Heat) and corresponding procedures. A plant, a plant tissue or a plant cell is advantageously transfected with the nucleic acid. The nucleic acid preferably encodes a virus-encoded transport protein, which in a particularly preferred embodiment is a derivative of the pr17 protein with a hydrophilic N-terminal extension.

Several documents are cited below, the disclosure content of which is hereby incorporated by reference and the technical teaching of which can be applied within the meaning of the present invention.

A goal of classic plant breeding is the creation of high-yielding varieties that have an increased tolerance to environmental factors or are resistant to stress factors. These stress factors can be both biotic (insects, viruses, fungi, etc.) and abiotic (extreme temperatures, salt, drought). While wild plants in stress-dominated locations have adapted to the extreme living conditions, drought, heat or salinity of the soil limit the possibilities for cultivating crops in such areas. On the other hand, agriculture in other locations also suffers major losses due to abiotic stress, as the example of the drought in 1983 in the USA has shown: Almost half of the total corn harvest and one third of the expected soybean yield were destroyed by the continuing drought. All abiotic stress conditions mentioned have in common that they are intracellular

Disrupt water balance. Plants can, however, within certain limits

Set stress conditions (Bohnert, (1995) Plant Cell 7: 1099-1011). For example,

Proteins and metabolites of plant metabolism such as sugar alcohols,

Proline or glycine betaine as accumulating as a result of abiotic stress

Osmoregulators have been identified. Various were based on this

Strategies developed to produce transgenic plants with increased tolerance to such factors or increased stress resistance through genetic engineering changes (review by Holmberg and Bülow (1998) Trends Plant

Science 3: 61-66). An example of plant proteins as anti-stress factors are the so-called LEA (late embryogenesis abundant) proteins, their increased

Expression correlated with physiological and environmental stress and the one

Represent protective function for the plant under extreme stress conditions (see e.g. Chandler (1994) Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 113-141). The genetic modification of rice with the help of the barley LEA gene HVA1 actually resulted in an increased tolerance to drought and salt

(Xu (1996) Plant Physiology 110: 249-257). Other work on expressing a

LEA genes in a heterologous system could not support these findings

(Iturriaga (1992) Plant Mol. Biol. 20: 555-558).

Among the plant metabolites identified as anti-stress factors or osmoprotectors is glycine betaine, the effectiveness of which has been demonstrated, for example, in maize (Saneoka, (1995) Plant Physiol. 107: 631-638). Betaine aldehyde dehydrogenase (BADH) is involved in the synthesis of glycine betaine in plant chloroplasts (Rhodes, (1993) Annu. Rev. Plant Physiol. Plant Mol. Biol. 44: 357-384). Transgenic tobacco plants expressing a bacterial BADH (Holmstrom, (1994) Plant. J. 6: 749-758) or a plant BADH (Rathinasabapathi, (1994) Planta 193: 155-162) showed the expected resistance to betaine aldehyde by conversion to glycine betaine in amounts measured in stressed plants. However, an increased stress tolerance of these plants has not been reported. Sugar derivatives such as sugar alcohols or fructans are obviously also formed and accumulated to an increased extent in the stress response of the plant. The

Increasing the level of mannitol in transgenic tobacco plants by expressing a bacterial mannitol-1-phosphate dehydrogenase caused an increase

Salt tolerance (Tarczynski, (1993) Science 259: 508-510). Similarly, transgenic tobacco plants with an increased fructan content compared with

Control plants have increased drought resistance (Pilon-Smith,

(1995) Plant Physiol. 107: 125-130).

All these genetic engineering processes mentioned so far have in common that they result in an increased synthesis of osmoprotectors in the plant, which means e.g. the normal growth of the plant can be impaired (see Rathinasabapathi, op. cit). In other cases, bacterial genes are expressed in the plant, which is not necessarily associated with optimal expression and thus suboptimal stress management. The object underlying the present invention was therefore to provide a method for providing plants with increased stress resistance, which nevertheless have essentially normal growth. This stress resistance (or tolerance) should preferably relate to both biotic and abiotic stress factors. This object is achieved by the embodiments characterized in the claims.

The invention thus relates to the use of a nucleic acid which encodes a (poly) peptide with an intrinsic affinity for plasmodesms for the production of plants or parts thereof with increased tolerance to drought and / or fungal infections and / or increased salt concentrations and / or extreme temperatures; i.e. Cold and / or heat. According to the invention, a plant, a plant tissue or a plant cell is usually transfected with such a nucleic acid by conventional methods.

All higher plants are characterized by the fact that they are capable of photosynthesizing sugars and their derivatives, which, as mentioned above, can serve as osmoprotectors under stress conditions. The intracellular Concentration of the sugar and sugar derivatives - especially in the leaves to protect the photosynthesis-active chloroplasts - was solved according to the invention in a surprisingly simple manner by the measure identified above. In addition to the fact that this measure is technically simple for the person skilled in the art, it is particularly advantageous that, according to the invention, the stress resistance can be increased with a mechanism which is valid in many and possibly even all plants. Surprisingly, the plant tolerance to both abiotic and biotic factors could be increased with the method according to the invention.

The term "increased salt concentration" refers to salt concentrations in the soil, which lead to an increased ion concentration in the plant, which then leads to reduced growth. The absolute salt concentrations in the soil, which are to be regarded as increased, are different for different plants, but can be determined by a person skilled in the art using conventional methods, e.g. based on the disclosure content of Greenway and Munns (1980) Ann. Rev. Plant Physiol. 31: 149-190.

For the expression of the nucleic acid encoding a (poly) peptide with an intrinsic affinity for plasmodesms in plant cells, this is linked to regulatory sequences which ensure transcription in plant cells. These include promoters in particular. In general, any promoter active in plant cells can be used for the expression.

The promoter can be selected so that the expression is constitutive or only in a certain tissue, at a certain time in plant development or at a time determined by external influences. The promoter can be homologous or heterologous to the plant. Useful promoters are, for example, the 35S RNA promoter from the Cauliflower Mosaic Virus (CaMV) and the ubiquitin promoter from maize for constitutive expression. Furthermore, a transcription termination sequence is usually present which serves to correctly terminate the transcription and can also be used to add a poly-A tail to the transcript which has a function in the stabilization the transcripts are attached. Such elements, such as the terminator of the

Octopin synthase genes from agrobacteria have been described in the literature (cf. Gielen, EMBO J. 8 (1989), 23-29) and can be exchanged as desired. In a preferred one

In one embodiment, the promoter is the 35S CaMV promoter.

In addition to the nucleic acid which encodes a (poly) peptide with an intrinsic affinity for plasmodesms, the plant which is used or produced in one of the methods or uses according to the invention can contain further recombinant DNA molecules which, for. B. can be used for crop protection or quality improvements of the plant or its harvest products. Examples of plant protection measures are: (i) herbicide tolerance (DE-A-3701623; Stalker (1988) Science 242, 419), (ii) insect resistance (Vaek (1987) Plant Cell 5, 159-169), (iii) virus resistance (Powell (1986) Science 232, 738-743) and (vi) ozone resistance (Van Camp (1994) BioTech. 12, 165-168). Examples of quality increases are: (i) increase in the shelf life of fruit (Oeller (1991) Science 254, 437-439), (ii) increase in starch production in potato tubers (Stark (1992) Science 242, 419), (iii) change in Starch (Visser (1991) Mol. Gen. Genet. 225, 289-296) and lipid composition (Voelker (1992) Science 257, 72-74) and (iv) production of non-plant polymers (Porier (1992) Science 256, 520- 523).

The basic requirement for the production of transgenic plants is the availability of suitable transformation systems. Several methods are currently available for transformation. The most frequently used method for the transformation of dicotyledonous plants is the Agrobacterium -mediated gene transfer. Here, the natural ability of the soil bacteria to exploit genetic material in the plant genome is used. Other suitable methods are, for example, protoplast transformation by polyethylene glycol-induced DNA uptake, electroporation, sonication or microinjection, and the transformation of intact cells or tissue by micro- or macroinjection into tissue or embryos, tissue electroporation, incubation of dry embryos in DNA-containing solution, vacuum infiltration of seeds and organic Gene transfer (for an overview: see, for example, Potrykus, Physiol. Plant (1990), 269-273 and Christou (1996) Trends in Plant Science 1, 423-431).

During the transformation of dicotyledonous plants using Ti plasmid vector systems

With the help of Agrobacterium tumefaciens is well established, recent work indicates that monocotyledonous plants are very well amenable to transformation using Agrobacterium-based vectors (Chan, Plant Mol. Biol. 22 (1993),

491-506; Hiei, Plant J. 6 (1994), 271-282; Bytebier, Proc. Natl. Acad. Be. USA 84

(1987), 5345-5349; Raineri, Bio / Technology 8 (1990), 33-38; Gould, Plant Physiol.

95: 426-434 (1991); Mooney, Plant, Cell Tiss. & Org. Cult. 25: 209-218 (1991); Li,

Plant Mol. Biol. 20 (1992), 1037-1048). The methods for introducing foreign DNA using the biolistic method or through

Protoplast transformation is known (see e.g. Christou (1996) Trends in Plant

Science 1, 423-431; Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A

Multi-Volume Comprehensive Treatise (H. Rehm, G. Reed, A. Pühler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge). Three of the above transformation systems have been used for different in the past

Cereals are established: the electroporation of tissue, the transformation of

Protoplasts and DNA transfer by particle bombardment into regenerable ones

Tissues and cells (for overview: Jahne, Euphytica 85 (1995), 35 - 44). The

Transformation of wheat has been described in various ways in the literature (for

Overview: Maheshwari, Critical Reviews in Plant Science 14 (2) (1995), 149 - 178).

The person skilled in the art can refer to the markers for the selection of transformed plant cells,

Use plant tissue and plants. The transgenic plant cells can be regenerated into whole plants using techniques known to those skilled in the art.

The person skilled in the art can e.g. also use molecular biological methods such as PCR to identify these plants. On the other hand, the

Of course, a specialist, e.g. after selfing or backcrossing against the parent,

Plant the seeds of such plants on selective media and use the

Germination of these seeds or survival of the plants in a later one

Stage of development (depending on the chosen promoter) conclude whether the

Plants are transgenic or not. The transgenic plants can act principally plants of any plant species, ie both monocot and dicot plants.

Because only a few cells have the desired cells regardless of the transformation process

In addition to the target gene, a selectable marker is integrated into the plant genome in a conventional manner, which enables the identification of transgenic cells. Currently, selections are being transformed

Plant cells primarily used genes that have a herbicide or

Communicate antibiotic tolerance. Suitable resistance genes are, for example, the bar-

Gene from Streptomyces hygroscopicus, the resistance to the total herbicide

Phosphinothricin mediates (De Block (1987) EMBO J. 6, 2513-2518) or the nptll-

Gene from the transposon Tn5 from Escherichia coli, which is resistant to the

Antibiotic Kanamycin effects (Herrera-Estrella (1983) EMBO J. 2, 987-995).

Other selection systems are e.g. B. Expression of a Mannose 6 Phosphate

Isomerase and positive selection on mannose-containing nutrient media (WO

94/20627). Another method takes advantage of the ability of a deaminase

Aspergillus terreus from detoxifying the insecticide Blasticidin S (Tamura (1995)

Biosci. Biotechnol. Biochem. 59, 2336-2338). Of course, the expert knows that the

Selection markers do not necessarily have to be present in the vector which contains the recombinant DNA molecule, but can also be co-transformed with them

(Lyznik (1989) Plant Mol. Biol. 13, 151-161; Peng (1995) Plant Mol. Biol. 27, 91-104).

This option is useful, for example, if no physical coupling of the marker gene and the information to be transmitted is desired.

In a preferred embodiment of the use according to the invention, a plant is also regenerated from the transfected plant cell. The plants can be regenerated by customary methods which are familiar to the person skilled in the art.

In a particularly preferred embodiment of the use according to the invention, further plants or plant cells are further produced from the regenerated plant following the regeneration step. In a further preferred embodiment of the invention

Use is the (poly) peptide a virus-encoded transport protein.

The expression of plant viral proteins which are involved in the transport of viral information from cell to cell (transport proteins) also influence the transport or metabolism of starch, sugars and sugar derivatives in such a way that they are present in the photosynthesis-active leaves of the plant during the light period accumulate higher than normal values (see e.g. Lucas (1993) Planta 190: 88-

96; Olesinski (1995), Planta 197: 118-126; Olesinski, (1996) Plant Physiol. 111: 541-

550; Herbers (1997) Plant J. 12: 1045-1056; Almon (1997) Plant Physiol. 115:

1599-1607). The increase in plant tolerance to the above-mentioned environmental factors by the expression of such proteins with an intrinsic affinity for plasmodesms, the connecting channels adjacent

Cells in plants according to this preferred embodiment of the invention were not readily derived from the prior art. The actual function of virus-coded transport proteins (TP) is to ensure that the genetic information of a virus is transported from cell to cell, thus allowing a virus to spread from the original site of infection to the entire plant.

In a particularly preferred embodiment of the use according to the invention, the virus-encoded transport protein is the potato leaf roll virus (PLRV) transport protein pr17 or a derivative thereof.

As explained in the examples, the TP of the potato leaf roll virus (potato leafroll virus, PLRV) and the crop plant potato were chosen as the model system. The PLRV-TP, which is a feature of a particularly preferred embodiment of the method according to the invention, is a 17 kDa protein (pr17) which is responsible for the transfer of the genomic RNA from cell to cell. It is encoded by the open reading frame (ORF) ORF4; this gene is located within ORF3, the gene for the viral capsid protein CP, but in a different reading frame. The protein has an amino-terminal domain for the formation of homopolymers (Tacke (1993), Virology 197: 274-282.) And a carboxy-terminal domain for binding single-stranded nucleic acids (Tacke (1991), J. Gen. Virol. 72: 2035-2038). This protein, which is phosphorylated in planta (Tacke (1993), op. Cit; Sokolova (1997), FEBS Lett. 400: 201-205), is expressed seven times more strongly than the viral coat protein gene (Tacke (1990), J. Gen Virol. 71: 2265-2272).

In PLRV-infected and in pr17-transgenic potato plants, the pr17 is predominantly due to the plasmodesms between the sieve element and the escort cell of the

Phloems localized (Schmitz (1997), Virology 235: 311-322), to which the virus is limited during its propagation in the plant. Expression of a mutated pr17 protein in transgenic potato plants causes broad-spectrum resistance to the most important potato viruses (Tacke (1996), Nature Biotechnology 14: 1597-1601).

At the same time, however, it was observed in the course of this invention that the expression of WT and mutant PLRV-TPs in transgenic potato and tobacco plants led to an increase in the intracellular concentrations of sugars (sucrose, fructose,

Glucose) and sugar derivatives such as starch.

In a further preferred embodiment of the use according to the invention, the derivative is a pr17 protein with a hydrophilic N-terminal extension.

Particularly good results in the sense of this invention were found when a variant of the pr17 gene was used which carries an N-terminal extension (pr17-N). In one exemplary embodiment, the polylinker (multiple cloning site; MCS) of the bluescript vector was fused translationally to the δ ′ end of the pr17-WT gene, and both the first two AUG translation codons of pr17 were mutated into ACG codons by targeted mutagenesis AUG translation start codon inserted into the polylinker sequence (Figure 1). This change results in the expression of a derivative (pr17-N) of the pr17-WT protein with a hydrophilic extension by the sequence MAELGSGSELHRGGGRSRTS (SEQ ID NO: 1) at the amino terminus (Tacke et al., 1996, op. Cit .; Figure 2) . Such transgenic potato plants show broad-spectrum resistance to the potato viruses PLRV, PVY and PVX as well as an increased concentration of sugars and sugar derivatives in the greenhouse test.

For expression in plants, this gene was under the transcriptional control of the 35S promoter and terminator of the cauliflower mosaic virus (CaMV) in the vector pRT103 (Töpfer (1987), Nucleic Acids Res. 15: 5890) and these

Transcription unit (Figure 1) then integrated into the binary plant transformation vector pBIN19 (Bevan (1984), Nucleic Acids Res. 12: 8711-8721). This vector was transferred into the Agrobacterium tumefaciens-Siamm LBA4404 (pAL4404) (Hoekema (1983), Nature 303: 179-180), which was used to transform Solanum tuberosum Var. Linda was deployed. Four (L4, L6, L7 and L8; see also Tacke (1996), op. Cit.) Of the independent transgenic potato lines as well as the original potato variety Linda were selected for the further experiments on induced tolerance.

As already mentioned above, in a particularly preferred embodiment of the use according to the invention, the hydrophilic extension comprises the amino acid sequence MAELGSGSELHRGGGRSRTS.

In another embodiment of the use according to the invention, the plant, the plant tissue or the plant cells come from the potato, from tobacco, from cereals or vegetables or are potatoes, tobacco plants, cereal plants or vegetable plants.

In a further preferred embodiment of the use according to the invention, increasing the tolerance of the plants to fungal infections is one of the tolerance to infections with Phytophtora infestans.

As a surprising finding according to this preferred embodiment, it was found that transgenic lines obtained with the method according to the invention are also distinguished by a statistically significant tolerance to Phytophthora infestans, the causative agent of late blight.

The invention further relates to methods for producing plants or parts thereof with increased tolerance to drought and / or fungal infections and / or increased salt concentrations and / or extreme temperature (heat, cold), wherein (a) a plant, plant tissue or plant cell with one

Transfected nucleic acid encoding a (poly) peptide with an intrinsic affinity for plasmodesms.

In a preferred embodiment of the method according to the invention, furthermore

(b) regenerates a plant from the transfected plant cell.

In a particularly preferred embodiment of the method according to the invention, one produces

(c) following step (b), further plants or plant cells from the plant obtained in (b).

In a further preferred embodiment of the method according to the invention, the (poly) peptide is a virus-encoded transport protein.

In a particularly preferred embodiment of the method according to the invention, the virus-encoded transport protein is the potato leaf roll virus (PLRV) transport protein pr17 or a derivative thereof.

In a further preferred embodiment of the method according to the invention, the derivative is a pr17 protein with a hydrophilic N-terminal extension.

As already mentioned above, in a particularly preferred embodiment of the method according to the invention, the hydrophilic extension comprises the amino acid sequence MAELGSGSELHRGGGRSRTS.

In another embodiment of the method according to the invention, the plant, the plant tissue or the plant cells come from the potato, from tobacco, from cereals or vegetables or are potatoes, tobacco plants, cereal plants or vegetable plants. In a further preferred embodiment of the method according to the invention, increasing the tolerance of the plants to fungal infections is one of the tolerance to infections with Phytophtora infestans.

The figures show:

Figure 1

Preparation of plasmid p17N. The two AUG codons of the wild-type pr17 gene were mutated into ACG by specific mutagenesis and a translation initiation codon was introduced into the polylinker sequence.

Figure 2

Nucleotide and amino acid sequence of the mutated pr17N gene or protein.

Figure 3

Result of resistance test # 1 (in the greenhouse) with 5 plants each from the starting variety Linda (L) and the pr17N transgenic lines L4, L6, L7 and L8. A. Overall view of the experiment. B. View of one plant per line.

Figure 4

Result of resistance test # 2 (in the phytochamber) with 6 plants each from the starting variety Linda (L) and the pr17N transgenic lines L4, L6, L7 and L8. A. Partial view of the overall experiment. B. View selected plants

Figure 5

Evaluation of infestation of leaf disks in the laboratory test with P. infestans race 1-11 (experiment # 2) after 9, 10 and 13 days after infection (dpi).

Figure 6

Cumulative representation of two attempts to infect potato leaf slices with P. infestans breed 1-11. Figure 7

Habitus of the non-transgenic potato variety Linda (L) and the 4 transgenic lines

L4, L6, L7 and L8 after 5 weeks at 100 mM NaCl. Individual plants of the

Partial experiments (A) were shown in (B) for better illustration. In the

Source variety Linda, the lower leaves have died.

Figure 8

Habitus of individual plants of the non-transgenic potato variety Linda (L) and the 4 transgenic lines L4, L6, L7 and L8 after 5 weeks at 100 mM NaCI. The Linda variety clearly shows the remains of the dead lower leaves and the changes in the stem.

The examples illustrate the invention.

Example 1: Preparation of plasmid p17N

A modification at the 5 'end of the pr17 gene (ORF4) was achieved by translational fusion of the multiple cloning site of the Bluescript vector, introduction of an optimized translation initiation codon and mutation of the two pr17-WT AUG initiation codons to ACG (FIG. 1). This change results in the expression of a derivative (pr17-N) of the pr17-WT protein with a hydrophilic extension by the sequence MAELGSGSELHRGGGRSRTS at the amino terminus (Tacke (1996), op. Cit; Figure 2). The preparation of the plasmid p17N is described in Schmitz (1996), Nucleic Acids Res. 24: 257-263 (referred to there as p17 / NIII).

Example 2: Introduction of T-DNA into the recipient organism

After transformation of E. coli S17-1 cells and mating with the A. tumefaciens strain LBA 4404 (pAL 4404) (Hoekema er al., 1983), agrobacteria which carried the plasmid p17N were used for plant transformation. Leaves of S. tuberosum var. Linda from the sterile culture were cut off at the base and 10 min in liquid MS medium with an agrobacteria grown overnight. Culture incubated. After two days of incubation on solid MS medium, the

Leaves washed and laid out on selection, regeneration medium. This consisted of MS medium complemented with 0.02 mg / 1 naphthylacetic acid, 0.02 mg / 1

Giberellic acid, 2 mg / 1 zeatin riboside, 500 mg / 1 claforan and 100 mg / l

Kanamycin sulfate. Shoots formed after 6-8 weeks and became

Root formation on MS medium with 250 mg / l claforan and 150 mg / l kanamycin sulfate implemented.

Example 3: Characterization of the transgenic lines obtained

Expression of the N-terminally modified PLRV 17K TP was detected in the Western blot with the help of 17K-specific antisera as described in Tacke et al., 1996 (op. Cit.). For this purpose, protein extracts were made from leaf material, the proteins were separated on a 12.5% polyacrylamide gel, transferred to nitrocellulose membranes and incubated with a 17K-specific antibody. Bound antibodies were detected using proven methods by incubation with sheep anti-rabbit IgG / peroxidase conjugate and the peroxidase substrate of the ECL kit (Amersham).

Example 4: Resistance tests with Phytophthora infestans

In order to determine the quantitative resistance, infestation tests with the

Herb and tuber pathogen, Phytophthora infestans. As

Race 1-11 of the pathogen was used in the inoculum (this breed becomes the

Conservation cultivated on leaf material of the Desiree potato variety). To do this

Potato flakes in an irrigation box (Gieffers (1989), J. Phytopathology

126: 115-132), which enables a permanent water supply, inoculated with the pathogen and designed below 10 ° C. The light was supplied with 10 tubes of the

Type Osram, L16, W / 25 White Universal for 16 hours a day with a medium one

-1 -2 photoperiodically active radiation of approx. 100 μmol sm. The sporangia formed after 8-10 days were rinsed off with water from the leaflets. The proportion of living sporangia was determined using the Behr staining method (Behr (1955), Zentralblatt für Bakt. Etc. II 108: 23/24, 641-656). For infestation tests

4 -1 a suspension with a sporangia density of 10 ml was used.

Based on the Hodgson test (Hodgson (1961), American Potato Journal

38: 259-264) a new test method was developed. Leaf disks with a diameter of 20 mm are produced using a punch developed for the test. The

Punching process works with low pressure, so that the sheet edges are not crushed and

Necrosis and bacterial rot can be avoided.

The leaf disks are placed in irrigation boxes (Gieffers et al., Op. Cit.)

Filter paper laid out with the bottom up. The constant water film on the filter paper provides the leaf disks with sufficient water. For inoculation, a drop with a sporangia suspension (200 sporangia / 20μl) is placed on the

Pipette in the center of the disc.

The inoculated leaf disks are kept under a permanent temperature of 10 ° C and the above. Incubated light conditions. The infectious zoospores hatch under these conditions. After about 6 days, the first sporangia are formed, the

Infestation is assessed after 8 to 10 days.

The leaf disk infestation, visible by sporangia and

Leaf tissue decay is estimated as a percentage.

An important prerequisite for the test is that the ones to be tested

Potato plants cultivated under the same conditions and the leaf slices of

Papers of the same leaf days and the same leaf position can be obtained.

In this way, the quantitative infestation of greenhouse and field material can be checked. The level of infestation determines the quantitative level of resistance.

Example 5: Induced tolerance to drought as an example of increased tolerance to abiotic stress

In each case 5 or 6 plants of the transgenic lines L4, L6, L7 and L8 and the starting variety Linda were kept under dry stress in the 6-8-leaf stage for 8 weeks, the plants being watered once after 3 and after 6 weeks. The transgenic plants were shown in two independent experiments a significantly increased tolerance to water stress, as in Table 1 and

Figures 3 and 4 emerges. Table 1: Evaluation of the potato lines L4, L6, L7 and

L8 and the starting variety Linda after 8 weeks of water stress

Line / variety # surviving plants / # of the examined plants

Exp. 1 ^* * Exp. 2 * ^**

Linda 1 ^* / 5 1 ^* / 6

L4 4/5 6/6

L6 5/5 6/6

L7 5/5 6/6

L8 5/5 5/6

* the surviving plant develops a new sprout from the tuber; all original plant parts are dead in contrast to the surviving plants of the transgenic lines L4, L6, L7 and L8.

** This experiment was carried out under controlled conditions in the greenhouse.

*** This experiment was carried out under controlled conditions in a phyto chamber.

Example 6: Induced tolerance to Phytophthora infestans as an example of increased tolerance to fungal attack

Two independent infestation tests with P. infestans were carried out with breeds 1-11 on leaf disks of greenhouse material. 8 leaf disks from 8 plants per transgenic line and starting variety Linda were infected for infection in the Laboratory test used. The inoculum contained 200 sporangia per 20 ul water; the

The leaf disks were incubated at 10 ° C. and ratings were given after 9, 10 and

Made 13 days after infection (dpi). All 4 transgenic lines showed significantly less infection compared to P. infestans up to 10 dpi. Then the

Infestation increased rapidly in L7 and L8, while L6 and especially L4 continued to maintain their relatively lower infestation level (FIG. 5). The mean of both experiments showed a significant difference in the resistance behavior of the 4 tested transgenes

Linda lines compared to the non-transgenic starting variety Linda (Figure 6).

Example 7: Induced tolerance to salt as another example of increased tolerance to abiotic stress

In a test series of 4 plants each from lines L4, L6, L7 and L8 and the non-transgenic starting variety Linda (L), the potato plants in granules were kept under salt stress for 5 weeks, salt in the form of NaCl in concentrations of 10.25 , 50, 75, 100, 200, 400 and 1000 mM was contained in the water. The highest concentration of 1000 mM caused severe damage to all plants after 2 weeks and these plants were dead after 5 weeks, while the transgenic lines, but not the Linda variety, survived a salt concentration of 400 mM despite severe damage. As shown in FIGS. 7 and 8 for irrigation with 100 mM NaCl after 5 weeks, all transgenic and all non-transgenic plants had a similar phenotype (the somewhat lower growth height of the transgenic lines, as can be seen in FIG. 7B, results from the genetic modification) from the salt stress): In the non-transgenic variety Linda, the lower leaves have died and the stalk shows characteristic, pathological changes (severe narrowing, brown color), as can be seen in Figure 8, while the stems of all transgenic lines remain green ( Figures 7, 8) and all leaves are preserved (Figure 7A, B). At most there are isolated necroses on the lower leaves (Figure 8). Despite the salt stress, flowers form on all plants and tuber formation occurs (Figure 8).

Claims

PATENT CLAIMS

1. Use of a nucleic acid encoding a (poly) peptide with an intrinsic affinity for plasmodesms for the production of plants or parts thereof with increased tolerance to drought and / or fungal infections and / or increased salt concentrations and / or extreme temperature (heat, cold ).

2. Use according to claim 1, further comprising regenerating a plant from the transfected plant cell.

3. Use according to claim 2, wherein, following the regeneration, further plants or plant cells are also generated from the regenerated plant.

4. Use according to any one of claims 1 to 3, wherein the (poly) peptide is a virus-encoded transport protein.

5. Use according to claim 4, wherein the virus-encoded transport protein is the potato leaf trivivirus (PLRV) transport protein pr17 or a derivative thereof.

6. Use according to claim 5, wherein the derivative is a pr17 protein with a hydrophilic N-terminal extension.

7. Use according to claim 6, wherein the hydrophilic extension is the amino acid MAELSGSGSELHRGGGRSRTS.

8. Use according to one of claims 1 to 7, wherein the plant, the plant tissue or the plant cells originate from the potato, from tobacco, from cereals or vegetables or are potatoes, tobacco plants, cereal plants or vegetable plants.

9. Use according to one of claims 1 to 8, wherein the increase in the tolerance of plants to fungal infections is an increase in the tolerance to infections with Phytophtora infestans.

10. Process for the production of plants or parts thereof with increased tolerance to drought and / or fungal infections and / or increased salt concentrations and / or extreme temperature (heat, cold), wherein

(a) transfecting a plant, plant tissue or plant cell with a nucleic acid encoding a (poly) peptide with an intrinsic affinity for plasmodesms.

11. The method of claim 10, further comprising

(b) regenerates a plant from the transfected plant cell.

12. The method of claim 11, further comprising, following step (b)

(c) other plants or plant cells are produced from the plant obtained in (b).

13. The method according to any one of claims 10 to 12, wherein the (poly) peptide is a virus-encoded transport protein.

14. The method of claim 13, wherein the virus-encoded transport protein is the potato leaf roll virus (PLRV) transport protein pr17 or a derivative thereof.

15. The method of claim 14, wherein the derivative is a pr17 protein with a hydrophilic N-terminal extension.

16. The method of claim 15, wherein the hydrophilic extension is the amino acid MAELSGSGSELHRGGGRSRTS.

17. The method according to any one of claims 10 to 16, wherein the plant, the

Plant tissue or the plant cells originate from the potato, tobacco, cereals or vegetables or are potatoes, tobacco plants, cereal plants or vegetable plants.

18. The method according to any one of claims 10 to 17, wherein the increase in the tolerance of plants to fungal infections is an increase in the tolerance to infections with Phytophtora infestans.