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WO 2010/037714 Al (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENTCOOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 8 April 2010 (08.04.2010) WO 2010/037714 Al (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 15/82 (2006.01) C12N 15/31 (2006.01) kind of national protection available): AE, AG, AL, AM, AOlH 5/00 (2006.01) AOlN 63/00 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, C12N 15/52 (2006.01) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, PCT/EP2009/062533 KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (22) International Filing Date: ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 28 September 2009 (28.09.2009) NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, (25) Filing Language: English TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 081655 13.6 30 September 2008 (30.09.2008) EP GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (71) Applicant (for all designated States except US): BASF TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, PLANT SCIENCE GMBH [DE/DE]; 67056 Lud- ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, wigshafen (DE). MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, (72) Inventors; and ML, MR, NE, SN, TD, TG). (75) Inventors/ Applicants (for US only): PLESCH, Gunnar [DE/DE]; Plantagenhof 1, 14482 Potsdam (DE). PUZIO, Published: Piotr [DE/BE]; Rene v. d. Puttestraat 1, B-9030 Mariak- — with international search report (Art. 21(3)) erke (BE). FRANK, Markus [DE/DE]; Weinstrasse 107, 67434 Neustadt (DE). — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of (74) Agent: FITZNER, Uwe; Hauser Ring 10, 40878 Ratin- amendments (Rule 48.2(h)) gen (DE). — with sequence listing part of description (Rule 5.2(a)) (54) Title: METHOD FOR PRODUCING A TRANSGENIC PLANT CELL, A PLANT OR A PART THEREOF WITH IN- CREASED RESISTANCE BIOTIC STRESS (57) Abstract: The invention relates to the control of pathogens. Disclosed herein are methods of producing transgenic plants with increased pathogen resistance, expression vectors comprising polynucleotides encoding for functional proteins, and trans genie plants and seeds generated thereof. Method for producing a transgenic plant cell, a plant or a part thereof with increased resistance biotic stress [0001.1.1.1] The invention relates to the control of pathogens. Disclosed herein are methods of producing transgenic plants with increased pathogen resistance, ex pression vectors comprising polynucleotides encoding for functional proteins, and transgenic plants and seeds generated thereof. [0002.1.1.1] In particular, this invention relates to a transgenic plant cell, a plant or a part thereof with increased resistance to biotic stress, preferably pathogenic fungi as compared to a corresponding non-transformed wild type control, by increasing or gen erating one or more activities of biotic stress related protein (BSRP) and to a method for producing said plant cell, a plant or a part thereof. [0003.1.1.1] The invention also deals with methods of producing and screening for and breeding such plant cells or plants. [0004.1.1.1] Population increases and climate change have brought the possibility of global food, feed, and fuel shortages into sharp focus in recent years. Additionally, plant performance in terms of growth, development, biomass accumulation and yield depends under field conditions on acclimation ability to the environmental changes and tolerance to plant diseases. Stresses, biotic and abiotic, exert a critical influence on crop yields. Pathogen attacks are sometimes the most devastating biotic stresses. The enhancement of disease re sistance in crops can contributed significantly to increasing the productivity of crops and decreasing the application of pesticides, which can adversely affect human health and the environment when applicated in excess. Plant diseases are infectious diseases which are caused by biotic stress and noninfec tious diseases caused by abiotic stress. Biotic stress is caused by phytopathogenes respective bacteria, fungi, nematodes, v i ruses, mollicutes (mycoplasmas, spiroplasmas), protozoa, phanerogams; rickettsias, and viroids, insects and parasitic plants. Abiotic stress means "sub-optimal growing condition", referring to environmental stress, damages by weather and other environmental factors, limited water and nutrient avail ability and sub-optimal disposability. Limited water availability can induce drought, heat, cold or salt stress. [0005.1.1.1] Plants infectious diseases, in other words biotic stress, are responsible for significant crop losses worldwide, resulting from both infection of growing plants and destruction of harvested crops. Crop losses and crop yield losses of major crops such as rice, maize (corn) and wheat caused by these stresses represent a significant eco nomic and political factor and contribute to food shortages worldwide. [0006.1.1.1] Agricultural biotechnology has attempted to meet humanity's growing needs through genetic modifications of plants that could increase crop yield, for exam ple, by conferring better tolerance to biotic and abiotic stress or by increasing biomass. The plants' natural defense mechanisms against pathogens are frequently insufficient. At the moment many genetical and biotechnological approaches are known in order to obtain plants growing under conditions of biotic stress. [0007.1.1.1] The introduction of foreign genes from plants, animals or microbial sources can increase the defenses. Examples are the protection against feeding dam age by insects by expressing Bacillus thuringiensis endotoxins (Vaeck et al. (1987) Nature 328:33-37) or the protection against fungal infection by expressing a bean chiti- nase (Broglie et al. (1991) Science 254:1 194-1 197). [0008.1 . 1 .1] Most pathogens are host-specific to a particular plant species, genus or family. For instance, blackspot of rose will not attack marigolds or lettuce. Therefore, most of the approaches described only offer resistance to a single pathogen or a na r row spectrum of pathogens. Plant-pathogen interactions are sometimes controlled by specific interactions between avirulence genes of pathogens and gene-for-gene disease resistance (R) genes of plants. Many authors have reported the enhancement of disease resistance by trans genic approaches, as increasing the expression of the disease resistance (R) genes of plants. The characteristic defence reaction of R-gene mediated resistance is the hyper sensitive response (HR), a strong resistance reaction comprising a programmed cell death of the attacked plant cell. Small antimicrobial peptides play an important role as part of the natural defense systems of plants against infectious microorganisms. A ntim icrobial peptides are usually small, cationic, and amphipathic and have open-chain forms. Various types of antimicrobial peptides have been identified in plants, including thionins, maize zeamatin, coffee circulin, and wheat puroindoline and plant defensins (Kawata et. al, JARQ 37 (2), 7 1 - 76 (2003)). [0009.1 . 1 .1] Only a few approaches exist which impart a resistance to a broader spectrum of pathogens to plants. Systemic acquired resistance (SAR)-a defense mechanism in a variety of plant/pathogen interactions -can be conferred by the appli cation of endogenous messenger substances such as jasmonic acid (JA) or salicylic acid (SA) (Ward, et al. (1991) Plant Cell 3:1085-1694; 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; Bion200 ) (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 upregulated in the case of SAR, may also cause pathogen resistance in some cases. [0010.1 . 1 .1] In barley, the loss of the MIo gene causes an increased and, above all, race-unspecific resistance against a large number of mildew species (Buschges 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). mlo-Wke resistances in other plants, in particular in cereal species, are not described. Various methods using the MIo gene and ho- mologs from other cereal species for obtaining a pathogen resistance are described (WO 98/04586; WO 00/01722; WO 99/47552). The disadvantage is that the mlo- mediated defense mechanism comprises a spontaneous die-off of leaf cells (Wolter M et al. (1993) MoI Gen Genet 239: 122-128). Another disadvantage is that the MIo- deficient genotypes show hypersensitivity to hemibiotrophic pathogens such as Mag- naporte grisea (M. grisea) and Cochliobolus sativus (Bipolaris sorokiniana) (Jarosch B et al. (1999) MoI Plant Microbe Interact 12:508-514; Kumar J et al. (2001 ) Phytopathol ogy 9 1:127-133). [001 1. 1 . 1 .1] The liberation of reactive oxygen species (ROS) is ascribed an impor tant protection function in the reaction on plant pathogens (Wojtaszek P (1997). It has been shown that mutations in the catalytic subunit of NADPH oxidase in Arabidopsis thaliana show a reduced accumulation of reactive oxygen intermediates (ROI). With regard to the hypersensitive reaction (HR), the results were heterogeneous: while in- fection with the aviralulent and bactrium Pseudomonas syringae showed a reduced HR in a double mutant, the virulent oomycete Peronospora parasitica showed an increased HR.
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