Novel nucleic acid sequences and their use in methods for achieving pathogen resistance in plants

This application is a divisional of U.S. application Ser. No. 10/488,222, filed Mar. 2, 2004 which is a 35 U.S.C. 371 National stage filing of International Application No. PCT/EP2002/009719, filed Aug. 3, 2002, which claims priority to German Application No. 10142579.1, filed Sep. 3, 2001 and German Application No. 10229729.0, filed Jul. 2, 2002. The entire contents of each of these applications are hereby incorporated by reference herein.

The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is Revised_Sequence_List_—12810_—00810_US. The size of the text file is 171 KB, and the text file was created on Feb. 18, 2009.

The invention relates to novel RacB cDNA sequences from barley and to expression cassettes and vectors comprising these sequences. The invention furthermore relates to transgenic plants transformed with these expression cassettes or vectors, to cultures, parts or transgenic propagation material derived from them, and to their use for the production of foodstuffs, feeding stuffs, seed, pharmaceuticals or fine chemicals. The invention furthermore relates to methods of generating or increasing a pathogen resistance in plants by reducing the expression of an RacB protein or of a functional equivalent thereof.

The aim of plant biotechnology work is the generation of plants with advantageous novel properties, for example for increasing agricultural productivity, increasing the quality in the case of foodstuffs, or for producing specific chemicals or pharmaceuticals (Dunwell J M (2000) J Exp Bot 51 Spec No: 487-96). The plant\'s natural defense mechanisms against pathogens are frequently insufficient. Fungal diseases alone result in annual yield losses of many billions of US$. The introduction of foreign genes from plants, animals or microbial sources can increase the defenses. Examples are the protection of tobacco against feeding damage by insects by expressing Bacillus thuringiensis endotoxins under the control of the 35S CaMV promoter (Vaeck et al. (1987) Nature 328:33-37) or the protection of tobacco against fungal infection by expressing a bean chitinase under the control of the CaMV promoter (Broglie et al. (1991) Science 254:1194-1197). However, most of the approaches described only offer resistance to a single pathogen or a narrow spectrum of pathogens.

Only a few approaches exist which impart a resistance to a broader spectrum of pathogens, in particular fungal pathogens, to plants. Systemic acquired resistance (SAR)—a defense mechanism in a variety of plant/pathogen interactions—can be mediated by the application of endogenous messenger substances such as jasmonate (JA) or salicylic acid (SA) (Ward, et al. (1991) Plant Cell 3:1085-1094; Uknes, et al. (1992) Plant Cell 4(6):645-656). Similar effects can also be achieved by synthetic compounds such as 2,6-dichloroisonicotinic acid (INA) or S-methyl benzo(1,2,3)thiadiazole-7-thiocarboxylate (BTH; Bion®) (Friedrich et al. (1996) Plant J 10(1):61-70; Lawton et al. (1996) Plant J. 10:71-82). The expression of pathogenesis-related (PR) proteins, which are highly regulated in the case of an SAR, may also cause pathogen resistance in some cases.

In barley, the Mlo locus has been described for some time as a negative regulator of plant defense. The loss, or loss of function, of the Mlo gene causes an increased and, above all, race-unspecific resistance for example against a large number of mildews (Büschges R et al. (1997) Cell 88:695-705; Jorgensen J H (1977) Euphytica 26:55-62; Lyngkjaer M F et al. (1995) Plant Pathol 44:786-790). The Mlo phenotype is inherited recessively, which also suggests a function as a susceptibility gene. Mlo-deficient barley varieties obtained by traditional breeding are already being widely used in agriculture. Although these varieties are being grown intensively, this resistance has proved to be extraordinarily durable, probably owing to the recessivity. Resistance breakdown has not been observed as yet. Mlo-like resistances in other plants, especially in cereal species, have not been described even though wheat, rye and other cereals are also attacked by comparable mildew pathogens. The reason in the case of wheat may be, for example, the existence of a hexaploid genome, which makes the identification of mutants in which each of the six copies of the gene has been inactivated extremely difficult.

The Mlo gene has only recently been cloned (Büschges R et al. (1997) Cell 88:695-705; WO 98/04586; Schulze-Lefert P, Vogel J (2000) Trends Plant Sci. 5:343-348). As a consequence, various homologs have been isolated from other cereal species. Various methods for obtaining pathogen resistance using these genes have been described (WO 98/04586; WO 00/01722; WO 99/47552).

Mlo resistance of a plant to mildew pathogens manifests itself in two important events, both of which bring about resistance to penetration: cell wall apposition (CWA) underneath the penetration site of the pathogen in the epidermal cell wall. Spreading of this fungal pathogen is almost exclusively restricted to this subcellular structure (Jorgensen J H and Mortensen K (1977) Phytopathology 67:678-685; Freialdenhoven A et al. (1996) Plant Cell 8:5-14). This reaction is caused by the genes Ror1 and Ror2, which are required for the effect of Mlo (Peterhänsel C et al. (1997) 9:1397-1409).

The disadvantage in Mlo pathogen resistance is that Mlo-deficient plants—even in the absence of a pathogen—initiate a defense mechanism which manifests itself for example in the spontaneous death of leaf cells (Wolter M et al. (1993) Mol Gen Genet 239:122-128). A further disadvantage is that the Mlo-deficient genotypes are hypersusceptible to hemibiotrophic pathogens such as Magnaporte grisea (M. grisea) and Cochliobolus sativus (Bipolaris sorokiniana) (Jarosch B et al. (1999) Mol Plant Microbe Interact 12:508-514; Kumar J et al. (2001) Phytopathology 91:127-133). The Mlo gene therefore appears to be a negative regulator of cell death. Again, the cause is probably the induction of cell death in the absence of the Mlo gene, which increases the susceptibility to these fairly necrotrophic pathogens. This ambivalent effect, which limits the biotechnological use of Mlo, is probably due to the fact that necrotrophic fungi are capable of exploiting the more pronounced HR of the Mlo-deficient host plant for their infection process. A resistance comparable to Mlo deficiency, but without the characteristic of inducing cell death, would be desirable.

The proteins Rho, Rac and Cdc42 are members of the small GTP (guanosine triphosphate) binding protein family and regulate a large number of intracellular processes as “molecular switches”, both in plant and animal organisms. As elements of signal transduction, they play an important role in the conversion of extracellular stimuli. For example, they regulate NADPH oxidase and thus the release of reactive oxygen molecules (“oxidative burst”). Animal or human Rac1 is essential for the formation of the active NADPH oxidase complex which, in turn, is important for the formation of superoxide, thus contributing to plant defense (Irani K and Goldschmidt-Clermont P J (1998) Biochem Pharmacol 55: 1339-1346). The function in plant defense in plants and animals is largely analogous (Kwong et al. (1995) J Biol Chem 270(34): 19868-19872; Dusi et al. (1996) Biochem J 314:409-412; Diekmann et al. (1994) Science 265:531-533; Purgin et al. (1997) The Plant Cell 9:2077-2091; Kleinberg et al. (1994) Biochemistry 33:2490-2495; Prigmore et al. (1995) Journal of Biol Chem 27(18): 10717-10722; Irani et al. (1997) Science 275:1649-1652; Low et al. (1994) Advances in Molecular Genetics of Plant-Microbe Interactions 3:361-369 (1994) eds. M J Daniels, Kluwer Acadmic Publishers, Netherlands; Mehdy et al. (1994) Plant Physiol 105: 467-472; Sundaresan et al. (1996) Biochem J 318:379-382). Moreover, GTP binding proteins function in restructuring the cytoskeleton and in cell transformation (Symon M. (1996) TIBS 21: 178-181), and also in the activation of transcription (Hill et al. (1995) Cell 81:1159-1170; Chandra et al. (1996) Proc Natl Acad Sci USA 93:13393-13397).

In plants, there exists a substantial family of Rac-like proteins (Winge et al. (1997) Plant Mol Biol 35:483-495), which is also termed Rop family (Lin et al. (1997) The Plant Cell 9:1647-1659). In plants, the Rac proteins appear to have a function in the release of reactive oxygen molecules as the consequence of pathogen infection (Groom Q J et al. (1996) Plant J 10: 515-522; Hassanain H H et al. (2000) Biochem Biophys Res Commun 272(3):783-788; Ono E et al. (2001) Proc Natl Acad Sci USA 98: 759-764). Rac modulates, inter alia, cell wall architecture, signal transduction in the meristem and the defense against pathogens (Valster A H et al. (2000) Trends Cell Biol 10(4):141-146). When the constitutively active form is overexpressed, Rac1 from rice is capable of inducing a hypersensitive response (HR) at the sites of M. grisea attack, thus causing pathogen resistance. Analogously, the expression of a negative dominant form of Rac1 brings about an increased susceptibility to M. grisea (Kawasaki T et al. (1999) Proc Natl Acad Sci USA 96:10922-10926; Ono E et al. (2001) Proc Natl Acad Sci USA 98: 759-764). These findings suggest that an overexpression of Rac proteins in the plant can bring about advantageous effects with regard to plant defense.

WO 00/15815 describes five Rac genes from maize. Although methods for both an up regulation and a down regulation of Rac proteins are described and speculatively discussed in connection with obtaining a resistance to pathogens (p. 55/line 25 et seq.), the only technical teaching, which describes this use in real terms, concerns merely an overexpression of the claimed Rac genes for obtaining pathogen resistance (p. 60/line 21 et seq.). The author postulates quite unambiguously and in agreement with the situation described in the prior art (p. 60/line 31 et seq.): “Thus the present invention is useful in protecting plants from pathogens. Once a plant is transformed with a polynucleotide sequence encoding an Rac polypeptide, expression of the polypeptide in the plant confers resistance to infection by plant pathogens.” The rationale behind this hypothesis (plants defense via reactive oxygen molecules) is explained hereinbelow and supported by a large number of references. Beyond this, no differentiation is being made between the five claimed Rac genes.

It is an object of the present invention to provide novel methods of plant defense against pathogens which bring about an effective defense against as broad a spectrum of pathogens as possible in as large a number of plant species as possible, preferably the crop plants used in agriculture. We have found that this object is achieved by the method according to the invention.

The invention firstly comprises a method of generating or increasing the resistance to at least one pathogen in plants, which comprises the following steps

a) reducing the amount, activity or function of an RacB protein in a plant or a tissue, organ, part or cell thereof, and

b) selecting those plants in which, as opposed or as compared to the original plant, the resistance to at least one pathogen exists or is increased.