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(12) United States Patent (10) Patent No.: US 8,373,022 B2 Schultheifs Et Al

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(12) United States Patent (10) Patent No.: US 8,373,022 B2 Schultheifs Et Al US008373022B2 (12) United States Patent (10) Patent No.: US 8,373,022 B2 Schultheifs et al. (45) Date of Patent: Feb. 12, 2013 (54) METHODS FOR INCREASING THE “Oryza sativa BI-1 mRNA for Bax Inhibitor-1, Complete cds.”. RESISTANCE IN PLANTS TO BIOTROPIC EMBL Database, Accession No. AB025926, Jan. 19, 2000. FUNG Imani J., et al., “Expression of Barley BAX Inhibitor-1 in Carrots Confers Resistance to Botrytis cinerea'. Molecular Plant Pathology, 2006, vol. 7, No. 4, pp. 279-284. (75) Inventors: Holger Schulthei?B, Neustadt (DE): Eichmann, R., et al., The Barley Apoptosis Suppressor Homologue Markus Frank, Neustadt (DE); Bax Inhibitor-1 Compromises Nonhost Penetration Resistance of Caroline Höfle, Wolfersdorf (DE) Barley to the Inappropriate Pathogen Blumeria graminis f.sp. tritici. Molecular Plant Microbe Interactions, 2004, vol. 17. No. 5, pp. (73) Assignee: BASF Plant Science GmbH, 484-490. Ludwigshafen (DE) Hickelhoven, R., et al., “Overexpression of Barley BAX Inhibitor 1 Induces Breakdown of mlo-Mediated Penetration Resistance to (*) Notice: Subject to any disclaimer, the term of this Blumeria graminis”. Proc. Natl. Acad. Sci. U.S.A., 2003, vol. 100, patent is extended or adjusted under 35 No. 9, pp. 5555-5560. U.S.C. 154(b) by 552 days. Hickelhoven, R., et al., “Differential Expression of Putative Cell Death Regulator Genes in Near-Isogenic, Resistant and Susceptible (21) Appl. No.: 12/446,641 Barley Lines during Interaction with the Powdery Mildew Fungus'. Plant Molecular Biology, 2001, vol. 47, pp. 739-748. (22) PCT Filed: Oct. 24, 2007 Sanchez, P, et al., “AtBI-1, a Plant Homologue of Bax Inhibitor-1, Suppresses Bax-Induced Cell Death in Yeast and is Rapidly (86). PCT No.: PCT/EP2007/061436 Upregulated during Wounding and Pathogen Challenge”. The Plant Journal, 2000, vol. 21, No. 4, pp. 393-399. S371 (c)(1), Adendorff, R., et al., “Scanning Electron Microscopy of Direct Host (2), (4) Date: Apr. 22, 2009 Leaf Penetration by Urediospore-Derived Infection Structures of Phakopsora apoda'. Mycological Research, 2000, vol. 104, No. 3, (87) PCT Pub. No.: WO2008/0498.65 pp. 317-324. Chae, H.-J., et al., “Evolutionary Conserved Cytoprotection Provided PCT Pub. Date: May 2, 2008 by Bax Inhibitor-1 Homologs from Animals, Plants, and Yeast”, Gene, 2003, vol. 323, pp. 101-113. (65) Prior Publication Data Lacomme, C., et al., “Bax-Induced Cell Death in Tobacco is Similar US 2010/0088777 A1 Apr. 8, 2010 to the Hypersensitive Response'. Proc. Natl. Acad. Sci. U.S.A., 1999, vol. 96, pp. 7956-7961. (30) Foreign Application Priority Data Kawal-Yamada, M., et al., “Dissection of Arabidopsis Bax Inhibi tor-1 Suppressing Bax-, Hydrogen Peroxide-, and Salicylic Acid Oct. 24, 2006 (EP) ..................................... O6122870 Induced Cell Death'. The Plant Cell, 2004, vol. 16, pp. 21-32. Hickelhoven, R., “BAX Inhibitor-1, an Ancient Cell Death Suppres (51) Int. Cl. sor in Animals and Plants with Prokaryotic Relatives'. Apoptosis, CI2N 15/09 (2006.01) 2004, vol. 9, pp. 299-307. CI2N 15/82 (2006.01) Bolduc, N., et al., “Molecular Characterization of Two Plant BI-1 (52) U.S. Cl. ........ 800/279: 800/278; 800/312:800/298; Homologues which Suppress Bax-Induced Apoptosis in Human 293 Cells”. Planta, 2003, vol. 216, pp. 377-386. 435/320.1; 435/69.1 Xu, Q., et al., “Bax inhibitor-1, a Mammalian Apoptosis Suppressor (58) Field of Classification Search ........................ None Identified by Functional Screening in Yeast'. Molecular Cell, 1998, See application file for complete search history. vol. 1, pp. 337-346. Matsumura, H., et al., “Overexpression of Bax Inhibitor Suppresses (56) References Cited the Fungal Elicitor-Induced Cell Death in Rice (Oryza saliva L.) U.S. PATENT DOCUMENTS Cells”. The Plant Journal, 2003, vol. 33, pp. 425-434. 6,229,067 B1 5, 2001 Sonnewald et al. (Continued) 6,451,604 B1* 9/2002 Flinn et al. .................... 435/468 2003/OOO9785 A1 1/2003 Reed Primary Examiner — Medina AIbrahim 2006, OO64775 A1 3, 2006 Frank et al. (74) Attorney, Agent, or Firm — Novak Druce Connolly FOREIGN PATENT DOCUMENTS Bove + Quigg LLP EP O864.650 A2 9, 1998 WO WO-OO,26391 5, 2000 (57) ABSTRACT WO WO-O2/10.1079 A2 12/2002 The present invention relates to methods of generating or WO WO-2004/081217 A1 9, 2004 increasing resistance to at least one biotrophic pathogen in a WO WO-2007/080143 A1 7/2007 plant or a part of a plant by increasing the protein quantity or OTHER PUBLICATIONS function of at least one Bax Inhibitor-1 (BI-1) protein in at Mittler et al. Plant Cell (1996) 8:1991-2001.* least one part of the plant. Moreover, the invention relates to "Brassica napus Bax Inhibitor 1 (BI-1) mRNA. Complete cds.”. polypeptide sequences and nucleic acid sequences which EMBL Database, Accession No. AF390555, Jul. 17, 2001. code for a BI-1 protein, and to expression cassettes, vectors “Arabidopsis thaliana At3I-1 mRNA for Bax Inhibitor-1, Complete and organisms which comprise Such sequences or such a cds, EMBL Database. Accession No. AB025927, Jan. 19, 2000. protein. "Nicotiana tabacum Bax Inhibitor 1 (B1-1) mRNA. Complete cds.”. EMBL Database, Accession No. AF390556, Jul. 17, 2001. 16 Claims, 15 Drawing Sheets US 8,373,022 B2 Page 2 OTHER PUBLICATIONS Bay Inhibitor-1 (AtEI-1), PNAS, 2001, vol. 98, No. 21, pp. 12295 Baek. D., et al., “Bax-induced CellDeath of Arabidopsis is Meditated 12300. through Reactive Oxygen-Dependent and -Independent Processes'. Lincoln, J., et al., “Expression of the Antiapoptotic Baculovirus p35 Plant Molecular Biology, 2004, vol. 56, pp. 15-27. Gene in Tomato Blocks Programmed Cell Death and Provides Broad Bolduc, N., et al., “Antisense Down Regulation of NtEI-1 in Tobacco Spectrum Resistance to Disease'. Proceedings of the National Acad BY-2Cells Induces Accelerated CellDeath upon Carbon Starvation'. emy of Sciences of the U.S., vol.99, No. 23 (2002), pp. 15217-15221. FEBS Letters, 2002, vol. 532, pp. 111-114. Shirasu, K., et al., “Regulators of Cell Death in Disease Resistance'. Watanabe, N., et al., “Arabidopsis Sax Inhibitor-1 Functions as an Plant Molecular Biology, vol. 44 (2000), pp. 371-385. Attenuator of Biotic and Abiotic Types of Cell Death'. The Plant "Hordeum vulgare mRNA for BAX Inhibitor-1 (pb.I-1 Gene)”. Journal, 2006, vol. 45, pp. 884-894. GenBank Accession No. AJ290421, Jan. 18, 2002. Roth, W., et al., “Apoptosis and Cancer. When BAX is Trailing Nelson, H., et al. “Respective roles of the epidermis and mesophyll in Away”. Nature Medicine, 2002, vol. 8, No. 3, pp. 216-218. the resistance of barley to powdery mildew', Ann. Phytopath. Soc. Kawai, M., et al., “Evolutionary Conserved Plant Homologue of the Japan (1989) 55: 156-160. Bax Inhibitor-1 (BI-1) Gene Capable of Suppressing Bax-Induced Ryals, J.A., et al., “Systemic Acquired Resistance'.The Plant Cell Cell Death in Yeast”, FEBS Letters, 1999, vol. 464, pp. 143-147. (1996) vol. 8: 1809-1819. Kawai-Yamada, M., et al., “Mammalian Bax-Induced Plant Cell Death Can Be Down-Regulated by Overexpression of Arabidopsis * cited by examiner U.S. Patent Feb. 12, 2013 Sheet 1 of 15 US 8,373,022 B2 Figure 1a (Page 1 of 3) 50 AtBI-1 () --------- MDAFSSFFDSQPGs---RSWSYDSLKNFRQISPAVONHLKR BnBI (l) --------- MDSFSSFFDSQPGS---RSWSYDSLKNLRQISPSVONHLKR GmB2 (1) ----- RLQAMDAFNSFFDS------ RNRWNYDTLKNFRQISPVVQNHLKQ Grabi3 (1) ITKTIRFDSLFSMDTFFKSPSSSSSRSRWSYDTLKNFREISPLVONHIKI, HVB (l) -----------MDAFYSTS---SAAASGWGHDSLKNFRQISPAVQSHLKL NtEI (1) --------- MESCTSFFNSQSASS-RNRWSYDSLKNFRQISPFWOTHLKK OSBI (1) -----------MDAFYSTSSAYGAAASGWGYDSLKNFRQISPAVOSHLK, TaBI.i. (l) ---------------- TaBI.8 (1) - - - - - - - - - - - - - - - - - - FSGTFRNSRSDDFVLCELQRELPRCRDATLTV TaB5 new (l) --------------------------------------- - - - - - WAMPGR ZInBI.4 (1) ---------------- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2n36 (1) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ZInBI33 (1) ------- aws m m -- - ----- -- a-- w w nom ww m www- w w w m wwn wa w man w awm ana, mamm - w - - a we w ZimBI8 (1) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Consensus (1) F S W YDSLKN R ISP WQ HLK 51 OO At Ben (39) VYLTLCCALVASAFGAYLHVLWNIGGILTTIGCEGTMIWLISCRPYEHOK BInBI (39) VYLTLCCALVASAFGAYLHVLWNIGGILTTIGCFGSMIWLLSCPPYEQQK GraBI2 (40) VYFTLCFAVVAAAVGAYLHVLLNIGGFLTTVACMGSSFWLLSTPPFEERK GmBI3 (51) WYFTLCCAVVAAAVGAFLHVLWNIGGFLTTLASIGSMFWLLSTPPFEEOK HVB (37) VYLTLCFALASSAVGAYLHIALNIGGMLTMLACVGTIAWMFSVPVYEERK Nts (41) VYLSLCCALVASAAGAYLHILWNIGGLLTTLGCVGSIVWLMATPLYEEQK OSBI-1 (40) VYLTLCVALAASAVGAYLHVALNIGGMLTMLGCVGSIAWLFSVPVFEERK TaBI 11 (1) ---------------------------------------------------- TaB 8 (33) VYVIPIVGRIKSAAGAYLHIALNIGGMLTMLACIGTIAWMFSVPVYEERK TaBI5 new (7) RFRLTYALPGLICRGCLPAHCPEHWRDADNARVYRNHRLDVLGASLRGEE ZInB4 (1) - - - - - - - ------ www.worm or w w w w w - - - - - - or or GSAWLFSVPWYEERK 2fB 6 (1) ---------------------WNIGWRLTMLGCGSIDWLFSVPVYEERK Z333 (1) ---------------------WNIGGTLTMLGCVGSIAWLFSVPVYEERK ZInBI8 (l) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ----------------- CorSei SS 51) WY TLC AL ASA GAYLHV NIGG LT LGCIGSI WL, S PVYEERK O 15 O AtBI-1 89) RLSLLFVSAVLEGASVGPLIKVAIDVDPSILITAFVGTAIAFVCFSAAAM BB - i. (89) RLSLL FLSAVLEGASVGPLIKVAVDFDPSILITAFVGTAIAFICFSGAAM GmBI2 30) RVTTLLMAASLFOGSSIGPLIDLAIHIDPSLIFSAFVGTALAFACFSGAAL GmBI3 (101) RLSLIMASALFQGASIGPLIDLAFAIDPGI, IIGAFVATSLAFACFSAVAL HVBI-1 87) RFGLIMGAALLEGASVGPLIEAIDFDPSILVTGFVGTAIAFGCFSGAA. NtE3 - 1 91) RIALLMAAALFKGASIGPLIELAIDFDPSIVIGAFVGCAVAFGCFSAAAM OSBI, 90)
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