PLANTS HAVING INCREASED RESISTANCE TO PLANT PATHOGENS, AND METHOD FOR CREATING INCREASED PATHOGEN RESISTANCE IN PLANTS

Abstract

The invention relates to plants with increased resistance to plant pathogens, wherein the intracellular concentration of inositol pyrophosphate InsP.sub.7 and/or InsP.sub.8 in said plants is increased in comparison to the wild-type plant. In particular, the invention involves plants with increased expression of at least one protein involved in the synthesis of inositol pyrophosphates InsP.sub.7 and/or InsP.sub.8, such as, for example, proteins VIH2 and VIH1. The plants according to the invention are particularly resistant to the following plant pathogens: herbivore insects, for example larvae of agriculturally relevant pests, pathogenic fungi, such as necrotrophic fungi, or other plant pests, such as biotrophic pathogens. The invention further relates to the method for increasing plant resistance to plant pathogens, wherein the intracellular concentration of inositol pyrophosphates InsP.sub.7 and/or InsP.sub.8 is increased in comparison to the wild-type plant.

Claims

1. A plant with increased resistance to plant pathogens, in which the intracellular concentration of inositol pyrophosphates InsP.sub.7 and/or InsP.sub.8 is increased in comparison with the wildtype plant.

2. The plant as claimed in claim 1, having inducible or increased expression of at least one protein involved in the synthesis of inositol pyrophosphates InsP.sub.7 and/or InsP.sub.8.

3. The plant as claimed in claim 1, wherein the expression and/or the activity of a protein selected from the group consisting of VIH2 encoded by a nucleotide sequence 2 (GenBank Accession: At3g01310), VIH1 encoded by a nucleotide sequence 1 (GenBank Accession: At5g15070), and a homologous protein capable of synthesizing inositol pyrophosphates InsP.sub.7 and/or InsP.sub.8, is in the whole plant or in specific tissues inducible, or is increased in comparison with the wildtypes.

4. (canceled)

5. The plant according to claim 3, in which the nucleotide sequence 1 or the nucleotide sequence 2 originates from the same plant species or from a different organism.

6. The plant according to claim 3, wherein the nucleotide sequence 1 or the nucleotide sequence 2 is under the control of a promoter that is selected from the group consisting of an inducible promoter and a constitutive promoter.

7. (canceled)

8. The plant according to claim 6, wherein the promoter is tissue-specific, for example leaf-, fruit- or seed-specific.

9. The plant according to claim 1, wherein the plant pathogens are herbivorous insects, for example larvae of agriculturally relevant pests, such as the small cabbage white or the owlet moth, or pathogenic fungi, such as necrotrophic fungi, for example, representatives of the genera Alternaria or Botrytis, or other plant pests, including biotrophic pathogens.

10. A method for increasing plant resistance against plant pathogens, wherein the intracellular concentration of inositol pyrophosphates InsP.sub.7 and/or InsP.sub.8 is increased in comparison to the wildtype plant.

11. The method according to claim 10, wherein the plants are treated with InsP.sub.7, with InsP.sub.8 and/or with InsP.sub.7 or InsP.sub.8 derivatives, for example in form of sprinkling, spraying or the like.

12. The method according to claim 11, wherein the derivatives are membrane permeable esters.

13. The plant as claimed in claim 2, wherein the expression and/or the activity of a protein selected from the group consisting of VIH2 encoded by a nucleotide sequence 2 (GenBank Accession: At3g01310), VIH1 encoded by a nucleotide sequence 1 (GenBank Accession: At5g15070), and a homologous protein capable of synthesizing inositol pyrophosphates InsP.sub.7 and/or InsP.sub.8, is in the whole plant or in specific tissues inducible, or is increased in comparison with the wildtypes.

14. The plant according to claim 13, in which the nucleotide sequence 1 or the nucleotide sequence 2 originates from the same plant species or from a different organism.

15. The plant according to claim 13, wherein the nucleotide sequence 1 or the nucleotide sequence 2 is under the control of a promoter that is selected from the group consisting of an inducible promoter and a constitutive promoter.

16. The plant according to claim 14, wherein the nucleotide sequence 1 or the nucleotide sequence 2 is under the control of a promoter that is selected from the group consisting of an inducible promoter and a constitutive promoter.

17. The plant according to claim 15, wherein the promoter is tissue-specific, for example leaf-, fruit- or seed-specific.

18. The plant according to claim 16, wherein the promoter is tissue-specific, for example leaf-, fruit- or seed-specific.

19. The plant according to claim 2, wherein the plant pathogens are herbivorous insects, for example larvae of agriculturally relevant pests, such as the small cabbage white or the owlet moth, or pathogenic fungi, such as necrotrophic fungi, for example, representatives of the genera Alternaria or Botrytis, or other plant pests, including biotrophic pathogens.

20. The plant according to claim 3, wherein the plant pathogens are herbivorous insects, for example larvae of agriculturally relevant pests, such as the small cabbage white or the owlet moth, or pathogenic fungi, such as necrotrophic fungi, for example, representatives of the genera Alternaria or Botrytis, or other plant pests, including biotrophic pathogens.

Description

[0026] Further advantages, features and possible applications of the invention are described in the following with reference to the below described exemplary embodiment referring to the figures.

[0027] FIG. 1: Arabidopsis VIH2 loss of function mutants show a reduced resistance to larvae of the small cabbage white (Pieris rapae). The larvae development was investigated in a so-called ‘no choice’ assay. In this assay, one single caterpillar in larvae stage L1 is placed on a single 5-week-old plant of the indicated genotype, and prevented from escaping using gauze. Col-0 denotes the used wildtype Arabidopsis thaliana (thale cress) ecotype, vih2-3 and vih2-4 are VIH2 loss of function mutants in Col-0 background. The fresh weight of the caterpillars was determined after 7 days. The values indicate the mean value (±standard error) (n=20). Statistically significant differences are indicated by an asterisk (t-test; *p<0.02).

[0028] FIG. 2: Arabidopsis VIH2 loss of function mutant shows a reduced resistance to larvae of the owlet moth Mamestra brassicae (cabbage moth). The Larvae development was investigated in a so-called ‘no choice’ assay. In this assay, one single caterpillar in larvae stage L1 is placed on a single 5-week-old plant of the indicated genotype, and prevented from escaping using gauze. Col-0 denotes the used wildtype Arabidopsis thaliana (thale cress) ecotype, vih2-4 is a VIH2 loss of function mutant in Col-0 background. The fresh weight of the caterpillars was determined after 8 days. The values indicate the mean value (±standard error) (n=20). Statistically significant differences are indicated by an asterisk (t-test; *p<0.02).

[0029] FIG. 3: Arabidopsis VIH2 overexpressing lines show increased resistance to larvae of the owlet moth Mamestra brassicae (cabbage moth). The Larvae development was investigated in a so-called ‘no choice’ assay. In this assay, one single caterpillar in larvae stage L1 is placed on a single 5-week-old plant of the indicated genotype, and prevented from escaping using gauze. Col-0 denotes the used wildtype Arabidopsis thaliana (thale cress) ecotype, “CaMV 35S: VIH2” are transgenic plants in which the kinase domain of the wild-type VIH2 gene is overexpressed under the control of the strong viral CaMV 35S promoter. The fresh weight of the caterpillars was determined after 8 days. The values indicate the mean value (±standard error) (n=20). Statistically significant differences are indicated by an asterisk (t-test; *p<0.05). These experiments show that an increase in the expression of VIH2 (which is linked to an increase in the inositol pyrophosphate InsP.sub.8) leads to an increased resistance of the plants to herbivorous insect pests. The growth of pests on the transgenic plants (and in approximation of pest-induced damage) is reduced by approx. 30%. There are no “undesirable” side effects of the inositol pyrophosphate increase: the plants are healthy and develop and reproduce normally.

[0030] FIG. 4: In Arabidopsis, over-expression of VIH2 leads to increased resistance to the necrotrophic ascomycete Alternaria brassicicola, whereas VIH2 loss of function mutants show reduced resistance. Infection experiments with Alternaria brassicicola (isolate MUCL 20297) were carried out as described previously.sup.3. For this purpose, spores were adjusted to a density of 5×10.sup.5 spores/ml, four to six 5 μl drops of the spore suspension were applied to the leaf surface, and the plants were incubated at 100% humidity, at 22° C. and an 8-hour/16 hours light/darkness-rhythm for 7-10 days (number of plants≧15). Subsequently the disease symptoms were documented and divided into different classes for each phenotype of leaf damage. The classes were as follows. Class I (cross-striped): light brown spots on the infection site; class II (oblique striped): dark brown spots on the infection site and first signs of necrosis; class III (consistently black): progressive necrosis and leaf maceration. The distributions of the data were evaluated with a Chi-square test and showed that the differences between Col-0 and vih2-3, between Col-0 and vih2-4, as well as between Col-0 and CaMV 35S: VIH2 are significant (p<0.05). Vih2-3 and vih2-4 are VIH2 loss of function mutants, CaMV 35S: VIH2 are transgenic plants in which the kinase domain of the wild-type VIH2 gene is overexpressed under the control of the strong, viral CaMV 35S promoter. These experiments show that an increase in the expression of VIH2 (which is associated with an increase in the inositol pyrophosphate InsP.sub.8) leads to an increased resistance of the plants against nectrotrophic fungi, since the fungus-induced damage on such plants is significantly reduced.

[0031] FIG. 5: Arabidopsis VIH2 loss of function mutants show a reduced resistance to the necrotrophic fungus and causative agent of the grey mould Botrytis cinerea. A 5 μL large drop of a conidia (spore) suspension of Botrytis cinerea was pipetted onto the leaf surface of a 5-week-old plant. This was done for 5 grown leaves per plant and a total of 20 plants of the indicated genotype. Only leaves younger than the 4.sup.th leaf of the respective plant were used. Thereafter, the inoculated plants were incubated at 100% humidity for 3 days at 21° C. in a climatic chamber under a 10-hour/14-hour light/dark-rhythm. Subsequently, the disease symptoms were documented and divided into different classes according to the size and the time of occurrence of lesions. The classes were as follows: class I (cross-striped): lesions of 2 mm diameter; class II (continuous black): lesions of 2 mm diameter with chlorosis; class III (oblique striped): lesions of 2-4 mm diameter with chlorosis; class IV (longitudinally striped): lesions with a diameter >4 mm and chlorosis. The distributions of the data was done with a chi-square test and show that the differences between Col-0 and vih2-3 as well as between Col-0 and vih2-4 are significant (p<0.001). The conidial suspensions for these experiments were prepared as described previously.sup.4. For this purpose, conidia from Botrytis cinerea were inoculated from a glycerol stock to semi-concentrated potato dextrose broth solid medium (PDB, Difco™) and incubated for 2 weeks at 22° C. under a 10-hour/14-hour light/dark-rhythm. Subsequently, conidia were washed from the surface of the solid medium with semi-concentrated PDB liquid medium, filtered through glass wool, and the conidia-density was determined with a counting chamber. The suspension was adjusted in semi-concentrated PDB-liquid medium to a density of 5×10.sup.5 conidia/ml, incubated at room temperature for 2 hours and used for leaf inoculation. The experiments were repeated 3 times with similar results.

EXEMPLARY EMBODIMENTS

[0032] The experiments were performed with isogenic lines of the same ecotype (Arabiopsis thaliana, Col-0), which are characterized by presence (Col-0) and absence (vih2-3 and vih2-4) of an intact VIH2 gene (and thus VIH2 protein), or by increased expression of the VIH2 kinase domain (CaMV 35S: VIH2). In the latter plants (CaMV 35S: VIH2), the kinase domain of the wild-type VIH2 gene was under the control of the strong viral CaMV 35S promoter. For this purpose, the VIH2 kinase sequence was amplified by an Arabidopsis cDNA and inserted into the vector pENTR™/D-TOPO® (Invitrogen Life Technologies). From there the VIH2 kinase domain sequence was transferred by Gateway® LR Clonase™ II (Invitrogen Life Technologies) into the binary plant transformation vector pGWB441 (Nakagawa et al., 2007, Biosci. Biotechnol. Biochem, 71, 2095-2100). The vector produced thereby “pGWB441-VIH2 KD” was used for the transformation of Arabidopsis plants. Several independent transformants were selected on kanamycin, no longer segregating CaMV 35S: VIH2 T3 plants were established and an increased InsP.sub.8 biosynthesis was confirmed in these plants. FIGS. 3 and 4 show examples of experiments with one of these lines demonstrating that the increased expression of the VIH2 kinase domain (and accompanying increased production of InsP.sub.8) leads to increased resistance to both larvae of the herbivorous insect Mamestra brassicae (cabbage moth, FIG. 3) as well as against the necrotrophic fungus Alternaria brassicicola (FIG. 4). The used VIH2 loss of function plants (vih2-3 and vih2-4) originate from publically accessible seed repositories as follows: Arabidopsis thaliana Col-0 (Columbia-0, CS60000 whose genome is completely sequenced) from the ‘Salk Institute Genomic Analysis Laboratory’ (USA); Vih2-3 (SAIL_165_F12) from the Syngenta Arabidopsis Insertion Library (SAIL) collection. This line was made available to us through the ‘Arabidopsis Biological Research Center’ (ABRC) of Ohio State University’ (USA); Vih2-4 (GK-080A07) originates from the collection “Genomanalyse im biologischen System Pflanze (GABI-KAT)” of the university Bielefeld. In both cases (SAIL and GABI-KAT), Arabidopsis thaliana Col-0 plants were transformed by transformation with specific T-DNA-containing vectors with the aid of agrobacteria. The T-DNAs integrate largely un-directed into the genome and, depending on the genomic locus, can lead to the destruction of the affected gene (and thus the loss of the protein encoded by this gene). A plurality of such plants are sequenced in the corresponding repositories with the aid of T-DNA-specific oligonucleotides in order to determine the insertion site and thus the identity of the affected gene in a specific plant. Corresponding data are made available online in order to enable the identification of a potential loss of function mutant of the desired protein. This detour (non-directed insertion and subsequent genotyping) is necessary, since in a higher plant a targeted generation of ‘knockout’ plants by the principle of homologous recombination (in contrast to, for example, baker's yeast or mice) is very ineffective.

[0033] The exemplary embodiments indicate that VIH2 loss of function mutants have reduced resistance to herbivorous insects and nectrotrophic fungi (FIGS. 1, 2, 4 and 5), whereas the increased expression of VIH2 kinase domain (and thus the increased production of InsP.sub.8) leads to an increased resistance to herbivorous insects and nectrotrophic fungi (FIGS. 3 and 4).

REFERENCES

[0034] 1. Dean R, Van Kan J A, Pretorius Z A, Hammond-Kosack K E, Di Pietro A, Spanu P D, et al. The top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 2012, 13 (4): 414-430. [0035] 2. Dakin K, Li W H. Cell membrane permeable esters of D-myo-inositol 1, 4,5-trisphosphates. Cell calcium 2007, 42 (3): 291-301. [0036] 3. Kemmerling B, Schwedt A, Rodriguez P, Mazzotta S, Frank M, Abu Qamar S, et al. The BRIT-associated kinase 1, BAK1, has a Brassinoli-independent role in plant cell death control. Current Biology, 2007, 17 (13): 1116-1122. [0037] 4. Submit Corrections Close Van Wees S C, Van Pelt J A, Bakker P A, Pieterse C M. Bioassays for assessing jasmonate-dependent defenses triggered by pathogens, herbivorous insects, or beneficial rhizobacteria. Methods Mol Biol 2013, 1011: 35-49.