US008373022B2

(12) 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 , 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. " 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 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) RFGILLAAALLEGASVGPLIKLAVIDFDSSILVTAFVGTAIAFGCFTCAAI TaBI.1. (1) ------AAI TaBI 8 (83) RFGLTMGAALLEGASVGPLIELAIDFDPSILVTGFVGTAIAFGCFSGAAJ TaB45 new 57) EVWAADGCSI, LEGASVGPLIELAIDFDPSILVTGFVGTAIAFGCFSGAAI Zrin Bll 4 (17) RYWLLMAAALLEGASVGPLIKLAVEFDPSILVTAFVGTAIAFACFSCAAM ZimBI 6 30) RYGLLMAAALLEGASVGPLVKLAVEFDPSILVTAFVGTAIAFACFSGAAM Zrins 33 30) RYGLLMAAALLEGASVGPLVKLAVEFDPSII.VTAFVGTAIAFACFSGAPW Zm EI 8 (1) ------WID, DSRLWTAFWG AWAFACFSGAA Consensus (101) R LLMAAALLEGASVGPLI LAIDFDPSILVTAFVGTAIAFACFSGAAI U.S. Patent Feb. 12, 2013 Sheet 2 of 15 US 8,373,022 B2

Figure 1b (Page 2 of 3)

151 200 AtBI- (39) LARRREYLYIGGLESSGLSMLMWLOFASSIFG-GSASI FKFEI.YFGLLF BnBI-1 (139) LARRREYLYLGGLLSSGLSMLMWLOFASSIFG-GSASI FKFELYFGLLIF GraB2 (140) VARRREYLYIGGLVSSGLSILLWLHFASSIFG-GSTALFKFELYFGLLVF GmB3 (151) WARRREYTYLGGLLSSWLSILMWLHSDSSLEG-GSIALFKFELYFGLLVF HVBI-1 (137) IAKRREYLYLGGILLSSGLSILLWLQFVTSIFGHSS-GSFMFEVYFGLLEF NtBI-1 (141) VARRREYLYLGGLLSSGLSILFWLHFASSIFG-GSMALFKFEVYFGLLVF OSBI-1 (140) VAKRREYLYLGGLLSSGLSILLWLQFAASIFGHST-GSFMFEVYFGLLIF TaBI 11 (4) IAKRREYLYIGGLLSSGISILL WI.QFATSIFGHSS-GSFMFEVYFGI.L.I.F TaBI 8 (33) IAKRREYLYLGGILLSSG------LTT, TaBI5 new (107) IAKRREYLYLGGLISSGLSILLWLQFATSIFGHSS-GSFMFEVYFGLLIF Zrin B1 4 (67) VAKRREYLYLGGLLSSGLSILLWI.QFAASIFGHQSTSSFMFEVYFGLI.I.F ZInBI 6 (80). WARRREYLYLGGLLSSGLSILLWLQLAASI F-GHSATSFMFEVYFGLLIF ZmB33 (80), WQAR-EYLYLGGCSRRGSPSCSGCSSPPPSS--ALRNSFMFEVYFGLLII, Zia BI8 (29) IAKRREYLYLGGLLSSGLSILLWLQEATSIFGHTS-ATFMFELYFGLLVF Consensus (151) VAKRREYLYLGGLLSSGISILLWLQFASSIFG S ASFMFEVYFGLLIF

201 25 O At Bi- (188) VGYMVVDTOEITEKAHLGDMDYVKHSLTLFTDFVAVFVRILITMLKNSAD F3B-1. (188) VGYMVVDTODIIEKAHLGDMDYVKHSLTLFTDFVAVFVRVLIIMLKNSAD GmBI2 (189) VGYIVVDTQEIVERAHLGDLDYVKHALTLFTDLVAVFVRILVIMLKNSTE GmBT3 (200) WGYVIVDTQEIIERAHFGDLDYVKHALTLFTDLAAIFVRILIIMLKNSSE HVB- (186) LGYMVYDTQEIIERAHHGDMDYIKHAILTLFTDFVAVLVRVLTIMLKNAGD Nt B- (190) WGYIIFDTODIIEKAHIGDI.DYVKHALTLFTDFVAVFVRILIIMLKNASD Os B- (189) LGYMVYDTOEIIERAHHGDMDYIKHALTLFTDFVAVLVRILVIMLKNASD TaBI 11 (53) LGYMVYDTOELIERAHHGDMDYIKHALTLFTDFVAVLVRELIIMLKNAGD TaBI, 8 (154) ------TaBI5 new (156) IGYMVYDTQEIIERAHHGDMDYIKHALTLFTDFWAVLVRVLII LLKNAAD 2IBI 4 (117). LGYMVYDTQEVIERAHHG------ZimBI16 (129) LGYVVYDT------a m ------2IB33 (127) LG - - - - m ZBI 8 (78) LGYMVFDTOEIIERAHRGDMDYIKHALTIFTDFWAVLVRILVIMMKNAQE Consen Sls (201) IGYMVYDTOEIIERAH GDMDYIKHALTLFTDFVAV VRILIIMLKNA D

251. 300 - Atisi-in-(238)-KEEKKKKRRN------GBVK-E-Y&e YRVW-PI-RYYLEASEGEQFeF. BnBI-1 (238) KEDKKKRRRN----- D-KVRKKAK--SGCYWCFKK ------KRWG GmBI2 (239) RNEKKKKRRD------GmBI3 (250) RNEKKKKRRD--ADRPTRAQASLQ-FSLWRIHN------FR-CWSW HVBI-1 (236) KSEDKKKRKRG------S------w -- www.m. new NtBI-1 (240) KEEKKKKRRN- - - -CISGYSKTL-L-NLAFSCS---TSVDLRQVCC--FG OsBI-1 (239) KSEEKKRKKRS-ELLFPLCT-EKTTAAIASTYYDRAALQLGFMVNTSSFA TaBI11 (103) KSEDKKKRKRRS------TaBE18 (155) ------TaBI5 new (206) KVGGQEEEEEKS------ZimBI14 (135) ------ZnaBI16 (137) ------2IaBI.33 (129) ------ZInBT8 (128) KSQDEKKRK------Consensus (251) K E KKKRR U.S. Patent Feb. 12, 2013 Sheet 3 of 15 US 8,373,022 B2

Figure 1c (Page 3 of 3)

301 350 AtBI-1 (278) H-KG-SACFTSAQVPSSDCK------LECCSSFHKLLFFKSL BnBI-1 (269) VISTDMIALVFFTCLEQFW------QHTLRICVFLLVTPDCEWI GmBI2 (249) ------w m. m. wwn mw wax r m me m. m. m. m. mm M- w w -m we ---, -w w ---w war am wa waw as aw GmBI3 (288) LWSYVFAVMVNVRISFKHLHMYLPIS-CVV-HHTLV-KKKKKKKKKKKKK HVBI-1 ((248) ww. --- -- m --- ... we ... --- w w w w -- www.m. - . . . . . m ------w w - - NtBI-1 (279) NASD-AARLCYAACQCGYGGT-MVLF----PKHTIK-HACLHYIDNLRVY OsBI-1 (287) FC-YGVNLLRFVVVVALOILACYMTRIFL-WWSR-SKRENTSSFATNLFA Consensus (301)

351. 4 O O AtBI-1 (312) VLLIASYOAKNNVGK------SC, NF, KCVHERKKKKKKKKK - w - - - BnBI-1 (307) SILKLC-KLSVGS------GmBI2 (249) ------GmBI3 (335) KKKKKXXXXXXXXXXXX-XXXXXGVCGLRYSRHSSNH-EGSLW-PGLC HVBI-1 (248) ------NtBI-1 (322) YLFLLPFAVLGCS-LYS-FSVMLDHLLS-RLISHIDGRNENSHRRPNLFK OsBI-1 (334) FW-LMMILSPKKKK------Consensus (35)

AtBI-1 (348) ------BnBI-1 (319) ------GinBI2 (249) ------GmBI3 (380) ACIDTVH-FGCNLCANS-YNVE-FI-EK-EEEEERLIG-PIAMCRVIWFV HVBT-1 (248) ------NtBI-1 (369) TEAQL------Consensus (401)

451. 5 OO GmBI3 (424) ENT - LAV-KLLVPICS.--LAMCLL-W-MSGFL. NIFICIC-S-YIV-TS Consensus (451)

5 O. 52 GmBI3 (464) FLGLKKEKKKKK Consensus (50)

U.S. Patent Feb. 12, 2013 Sheet 8 of 15 US 8,373,022 B2

A. Y., : : 3. S O. Sait. (...: 4. tha. YSTSSFrps-QPGsrss. BYG ASGGrisk E. sap.

H. Wri. Li-::$ 33: TMLAggTI3.- : : Hrs EE--- ESEFE... 113

O. Sait. LR-IgG. inicsvgs. LFSwwFEE- . Livir. 6 A. tha. JK-EGILITIGg:IGTMI.E.L.SCPPYEEI-8 : i. & S2's ity is 5 A. sap. THFIQaletts LGSLILtiti.MathSHETECRLG GLEP:LEFCW, it A. V. , SGS 3 O. salt. SFGHSTGS 176 A. tha. tow: assif58 GSI 1.75 H. Sap, LLS3 (FFG-SEF 69.

A. vi. 3. O. sat. 1. tha. isfs.III.iii. H. sap.

H. wui. O. sat. 4. tha. H. sap. U.S. Patent Feb. 12, 2013 Sheet 9 of 15 US 8,373,022 B2

Figure 7

is

as . . . . E E. 2.

1100 - 1350 Pressure psi U.S. Patent Feb. 12, 2013 Sheet 10 of 15 US 8,373,022 B2

Figure 8

. Control . . . . a Hvb-l.

Experiments U.S. Patent Feb. 12, 2013 Sheet 11 of 15 US 8,373,022 B2

Figure 9

#6(1)E81 (T1) bp NTCNTCPKPK 8 910. 111

30 are: 630 a.

Occasion U.S. Patent Feb. 12, 2013 Sheet 12 of 15 US 8,373,022 B2

Figure 10 Transformed cells Transformed cells Penetration rate which interact with which are penetrated of the transformed Experiment Constructs used soybean rust by soybean rust cells (% pg.Y 1-GFP+pgYl if 43 pgYa-GFF+ pGY-HwBE 3. 3. 23,08 2 p(Yi-GFPipGY S 35 55. p(SY-GFP+ gGYi-HwBf 36 2. d53

3. pcyl-GFP-pgYi. 5 g 82.9 pGYi-GFP+ ------...--29-...--its Cgntrol w8. CYeral mean averaged ower the experiments 53.3 3,83 standard deviation 0.38 2.8 t-test 3

U.S. Patent Feb. 12, 2013 Sheet 15 of 15 US 8,373,022 B2

Figure 12

is (JESL1 1#6 (2) (T) (T2) E15L7P (T2) US 8,373,022 B2 1. 2 METHODS FOR INCREASING THE have a firm cell wall during at least one stage in their life RESISTANCE IN PLANTS TO BIOTROPIC history. This cell wall usually consists of chitin or, in some FUNG cases such as the Oomycota, of cellulose. The vegetative part of the fungus (thallus) is usually haploid, in rare cases diploid. RELATED APPLICATIONS The thallus of lower fungi (Myxomycota, interalia) consists of ameboidal cells or plasmodia (naked, polynuclear proto This application is a national stage application (under 35 plasma). Eumycota have budding cells, as in the case of yeasts U.S.C. S371) of PCT/EP2007/061436, filed Oct. 24, 2007, (for example Saccharomyces cerevisiae), or form a mycelium which claims benefit of European application 06122870.6, which consists of threadlike hyphae. As the result of hyphal filed Oct. 24, 2006. 10 aggregation, specific organs for propagation (fruiting bodies) SUBMISSION OF SEQUENCE LISTING or for Surviving unfavorable environmental conditions (scle The Sequence Listing associated with this application is rotia) may be formed. Propagation and multiplication is usu filed in electronic format via EFS-Web and hereby incorpo ally by way of spores; asexually by means of conidia, ure rated by reference into the specification in its entirety. The dospores, sporarigiospores, chlamydospores and Zoospores, name of the text file containing the Sequence Listing is 15 and sexually with oospores, ascospores, Zygospores and Sequence Listing 13987 00101. The size of the text file is basidiospores. 161 KB, and the text file was created on Feb. 27, 2012. Approximately 100 000 different fungal species are known to date. Among these, however, only 5% are plant pathogens. FIELD OF THE INVENTION The Basidiomycota are a division of the true fungi which are characterized by the development of aparticular structure, the The present invention relates to methods for generating or basidium, on which the basidiospores mature. The Basidi increasing resistance to at least one biotrophic pathogen in a omycota also include the generally known mushrooms. The plant or a part of a plant by increasing the protein quantity or Basidiomycota are predominantly heterothallic and self-ster function of at least one Bax inhibitor-1 (BI-1) protein in at ille. Mating occurs by Somatogamy. During somatogamy, two least one part of the plant. Moreover, the invention relates to 25 compatible, haploid, mononuclear mycelia or sporidia coa polypeptide sequences and nucleic acid sequences which lesce. The resulting dikaryotic mycelium constitutes the code for a BI-1 protein, and to expression cassettes, vectors dominant phase of the life cycle over a prolonged period. The and organisms which comprise Such sequences or such a only two phytopathogenic genera of the Basidiomycota are protein, in particular to recombinant plants, to cultures, parts the Smuts (Ustilages) and the rusts (Uredinales). Smuts only or recombinant propagation material derived there from, and 30 attack angiosperms and use to be of great economical impor to the use of same for the production of foodstuffs, feeding tance. Nowadays they are controlled successfully by suitable stuffs, seed, pharmaceuticals or fine chemicals. active substances and tight seed control. The rusts are still Somewhat more important nowadays. They are biotrophic BACKGROUND OF THE INVENTION and can have a complicated development cycle with up to five 35 different spores stages (spermatium, aecidiospore, ure The cultivation of agricultural crop plants serves mainly dospore, teleutospore and basidiospore). Rusts which for the production of foodstuffs for humans and feedingstuffs develop all spore stages are referred to as macrocyctic rusts. If for animals. The last 25 years have seen pronounced yield Some stages are absent, these rusts are referred to as being increases in crop production. This was the result of a good macrocyclic. “Imperfect rusts' lack the basidiospores. Some combination of altered production techniques, newly devel 40 rusts change their hosts during their development. These are oped varieties, fertilization and, last but not least, increased referred to as heteroecious. Host alternation can be linked to crop protection. In the light of an ever increasing world popu nuclear-phase alternation. In contrast, autoecious rusts com lation, safeguarding food production gains increasingly in plete all of their development on one host. A traditional importance. It has been estimated that 7 billion people will example of a macrocyclic heteroecious rust is black rust of inhabit Earth in 2010. To feed all these people, without the 45 cereals, Puccinia graminis. P. graminis, in its dikaryotic proportion of malnourished people increasing, food produc stage, attacks predominantly wheat. The haplont is patho tion would have to be increased by 60% (Entrup N. L. et al., genic to barberry (Börner H. Pflanzenkrankheiten and Pflan Lehrbuch des Pflanzenbaues Textbook of crop production. Zenschutz Plant disease and plant protection, Ulmer Verlag Thomas Mann Verlag, Gelsenkirchen, 2000). Efficient crop Stuttgart, 1997: Sitte P. et al., Strasburger Lehrbuch der protection is a decisive factor in this context. Monocultures in 50 Botanik Textbook of Botany, Gustav Fischer Verlag, Stut particular, which are the rule nowadays, are highly suscep tgart, 1998; Entrup N. L. et al., Lehrbuch des Pflanzenbaues tible to an epidemic-like spreading of diseases. The result are Textbook of crop production, Thomas Mann Verlag, markedly reduced yields. To date, the pathogenic organisms Gelsenkirchen, 2000). have been controlled mainly by using pesticides. Nowadays, During the infection of plants by pathogenic fungi, differ in contrast, the possibility of directly modifying the genetic 55 ent phases are usually observed. The first phases of the inter disposition of a plant or pathogen is open to man. action between phytopathogenic fungi and their potential Fungi are distributed worldwide so they may form a het host plants are decisive for the colonization of the plant by the erogeneous group with a range of species. They are eukary fungus. During the first stage of the infection, the spores otes, do not contain chlorophyll and are therefore het become attached to the Surface of the plants, germinate, and erotrophic. Hence, they rely on external carbon sources which 60 the fungus penetrates the plant. The attachment of the spores they tap as parasites, saprophytes or symbionts. Saprophytes requires either an active metabolism, which is the case in live exclusively on dead plant material. Parasitic fungi feed on Colletotrichum graminicola, or it is passive, as is the case live tissue and must have concluded their development before with Magnaporthe grisea. In the latter case, moisture leads to the plant has died. Facultative parasites can feed both on live the secretion of a "mucilage adhesive', by means of which the and on dead tissue. Symbionts, such as mycorrhiza, live in 65 spore attaches (Howard R.J. et al., Annu. Rev. Microbiol. 50. close association with the plants. Fungi have one or more 491 (1996)). Spore germination is induced either by unspe nuclei per cell and are homokaryotic or heterokaryotic. Fungi cific inductors such as water, nutrients, ethylene or, more US 8,373,022 B2 3 4 rarely, as is the case in Phyllosticta ampelicida, by hydropho Interaktionen von Pflanzen und phytopathogenen Pilzen In bic surfaces (Kuo K. et al., Fungal Genet. Biol. 20, 18 (1996)). teractions between plants and phytopathogenic fungi. Some fungi develop two germ tubes, as is the case in powdery Gustav Fischer Verlag, Jena, 1996). In the case of compatible mildew cereals, Blumeria graminis, while other fungi interaction, the pathogen develops at the expense of the plant, develop only one germ tube (Green J. R. et al., The powdery thus causing the formation of disease symptoms such as wilt mildews, a comprehensive treatise; The formation and func ing, necroses and chloroses. However, if basic compatibility tion of infection and feeding structures, APS Press, 2002). exists, the plant can still defend itself against the pathogen Fungi may penetrate the plant via existing ports such as when a resistance mutation has taken place in the plant. The stomata, lenticels, hydatodes and wounds, or else they pen resistance of the plant, thus acquired, is referred to as host etrate the plant epidermis directly as the result of the mechani 10 cal force and with the aid of cell-wall-digesting enzymes. resistance. It is only directed against a certain individual Specific infection structures are developed for penetration of pathogen and can be overcome readily by the latter. The the plant. These pressure organs are referred to as appressoria pathogens in question are mostly, but not always, biotrophic and allow the fungus to build up a high pressure above a pathogens. Host resistance can be subdivided into non-race discrete point. It is estimated that appressorium of M. grisea 15 specific horizontal resistances which, in most cases, involves reaches a pressure of 80 bar (Howard R. J. et al., Annu. Rev. several genes, and race-specific vertical resistance. The latter Microbial. 50, 491 (1996)). Most rusts, in contrast, penetrate is only effective against certain, individual races of a patho the plant via the stomata. The Soya rust Phakopsora gen, while the plant defends itself a priori against most patho pachyrhizi directly penetrates the plant epidermis and there gens. This phenomenon is referred to as basic incompatibility fore resembles powdery mildew of cereals, B. graminis, in its or non-host resistance. In contrast to host resistance, non-host penetration behavior (Koch E. et al., Phytopath. p. 106, 302 resistance is based on a series of causes and not on individual (1983); Tucker S. L. et al., Annu. Rev. Phytopathol. 39,385 genes. Firstly, the pathogen may lack the necessary pathoge (2001); Green J. R. et al., The powdery mildews, a compre nicity factors, or else the plant is capable of recognizing, and hensive treatise; The formation and function of infection and Successfully defending itself, against the pathogen. Another feeding structures, APS Press, 2002). 25 term which is important in particular for agriculture is toler Phytopathogenic fungi do not always colonize all of the ance. A plant is tolerant to a pathogen when it can be attacked, plant; in contrast, sometimes it is only specific areas or tissues but the attack does not lead to the development of disease which are colonized. Following the successful invasion of the systems and yield reduction (Prell H. H., Interaktionen von plant, phytopathogenic fungi follow different nutritional Pflanzen und phytopathogenen Pilzen Interactions between strategies. Pertotrophic or necrotrophic pathogens kill the 30 plants and phytopathogenic fungi, Gustav Fischer Verlag, host cells by means of extracellular enzymes or toxins and Jena, 1996). For the purposes of the description of the present feed by degrading the dead cells. Some genera with per invention, generating, or increasing, a resistance is to com totrophic nutrition are the Fusaria sp., Alternaria sp. and prise an increase or generation of any type of resistance, but Cochliobolus. Most fungi use this feeding strategy. The also of tolerance, i.e. in particular all cases of an increased biotrophic phytopathogenic fungi. Such as mildew and many 35 tolerance or resistance which lead to reduced yield losses as rusts, depend, for their nutrition, on the metabolism of live caused by the pathogen. cells. An intermediate position is occupied by the hemi In connection with resistance responses of plants, the term biotrophic pathogenic fungi, which include the genera Phy resistance factors includes structures, Substances and pro thophtora and Peronospora. Most of these are biotrophs at the cesses which prevent or inhibit attack of the plant by potential beginning of their development and only change over to a 40 pathogens. If the resistance factors are already constitutively pertotrophic lifestyle during the later stages of their develop present in the plant, they are referred to as pre-formed resis ment (Prell H. H., Interaktionen von Pflanzen and phyto tance factors. Induced resistance factors are only formed pathogenen Pilzen Interactions between plants and phyto when a recognition response between the plant and the poten pathogenic fungi, Gustav Fischer Verlag, Jena, 1996). The tial pathogen has taken place. Recognition can be described plants have developed defense mechanisms to avoid infec 45 as a signal/sensor response (elicitor/receptor model, Keen N. tion. Another intermediate position is occupied for example T. et al., Phytopathology 62,768 (1972)). The signal are plant by soybean rust, which penetrates the epidermis directly, Substances or Substances produced by the pathogen, known as whereupon the penetrated cell becomes necrotic; after the elicitors, which bind to a sensor or receptor which is specific penetration, the fungus changes over to an obligatory for the elicitor in question. This binding triggers one or more biotrophic lifestyle. The subgroup of the biotrophic fungal 50 effectors, which may be, for example, signal transduction pathogens which follows essentially Such an infection strat chains and induce the resistance response. A whole series of egy will, for the purposes of the present description, be Substances act as elicitors. These include proteins, glycopro referred to as being “heminecrotrophic''. teins, glucans and lipids (Garcia Bruggeretal. MPMI 19, 711 It must be emphasized that plants, during their develop (2006)). Plant cell wall degradation products which are ment, are exposed to constant attack by a large number of 55 released by enzymes of the pathogen or else by wounding of phytopathogenic organisms. Nevertheless, colonization of the plant may also induce a resistance response. In this con the plant by phytopathogenic organisms is the exception text, an avirulence factor of the pathogen and the correspond rather than the rule. Before a pathogen can attack the plant, it ing resistance gene of the plant is frequently mentioned has to overcome a series of barriers. Frequently, the pathogen (“Gene-for-gene hypothesis', nor, J. Agric. Res. 74, 241 has developed specific pathogenicity factors which are 60 (1947)). The pathogen can prevent recognition by the plant by adapted to the plant, known as virulence factors, in order to means of structural modifications of the elicitor, masking of overcome these barriers. In such a case, the plant becomes a the recognition sequence or by competition with another Sub host plant for the pathogen, i.e. the latter is virulent on the stance for the binding sites on the elicitor or receptor. plant. There is a basic compatibility between the plant and the Preformed resistance factors form the first defense against pathogen. This means that the physiological and biochemical 65 colonization by pathogenic organisms. These factors can be prerequisites which are required for the colonization are morphological factors or else Substances of the secondary already in existence or have been produced (Prell H. H., plant metabolism (phytoanticipins). Morphological factors US 8,373,022 B2 5 6 which prevent colonization are hairy leaves, stomatal density reacts with a non-host-plant. Decisive for pathogen defense is and shape, and the nature of the cuticle and of the cell wall. the quality of the recognition and the quantity and speed of the Recognition, of the pathogen, by the plant may also lead to resistance response (Thordal-Christensen H. Current Opin the induction of resistance factors, i.e. morphological and ion in Plant Biology 6,351. (2003)). physiological resistance responses. Many of these responses 5 A plant disease which has become increasingly important are the result of a signal cascade. Signal molecules such as in recent times is soybean rust. The disease is caused by the Ca", NO, reactive oxygen compounds and phytohormones pathogenic rusts Phakopsora pachyrhizi (Sydow) and Pha Such as ethylene and jasmonate are involved in the cascades kopsora meibomiae (Arthur). They belong to the class Basidi and contribute to the crosslinking of the signal pathways. omycota, order Uredinales, family Phakopsoraceae. The two Resistance responses in which morphological structures in 10 species are very closely related with one another. The inter the plant cell are modified are the formation of cell wall genic sequences of their rRNA genes show 80% similarity appositions (papillae), cork and abscission layers, thylae and (Frederick R. D. et al., Phytopathology 92,217 (2002)). The the impregnation of the cell wall. The beginning of penetra species are distinguished by morphological characteristics of tion by a pathogenic fungus can trigger the formation of the teliospores (Ono Y. et al., Mycol. Res. 96, 825 (1992)). papillae. Lignin, callose, Suberin and hydroxyproline-rich 15 Bothrusts infect a widespectrum of host plants. P. pachyrhizi, proteins are deposited at the inner cell wall opposite the also referred to as Asian soybean rust, is the more aggressive potential penetration site and are crosslinked with one pathogen on Soybeans (Glycine max), and is therefore, at least another. Callose can be stained by the intercalation of aniline currently, of great importance for agriculture. P. pachyrhizi is blue. In addition, the papilla formed accumulates phenols, capable of infecting 31 species from 17 families of the Legu reactive oxygen species and hydrolases (Hickelhoven R. et minosae under natural conditions and is capable of growing al., Plant Physiol. 119, 1251 (1999); Assaad F. F. et al., Mol. on further 60 species under controlled conditions (Sinclair et Biol. of the Cell, 15, 5118 (2004)). The development of papil al. (eds.), Proceedings of the soybean rust workshop (1995), lae leads to a Substantially thickened cell wall and may pre National Soybean Research Laboratory, Publication No. 1 vent penetration of the pathogenic fungus. Physiological pro (1996); Rytter J. L. et al., Plant Dis. 87, 818 (1984)). P. cesses which contribute to induced resistance are 25 meibomiae has been found in the Caribbean Basin and in depolarization of the cell membrane, the oxidative burst, the Puerto Rico, and has not caused substantial damage as yet. hypersensitive reaction, the formation of phytoalexins and P. pachyrhizi was originally discovered in Japan in 1902. the expression of pathogenesis-related proteins (PR pro From there.P pachyrhizi spread over large parts of Asia and teins). One of the first responses to contact with an elicitor is over India and Australia and, finally, reached Africa in 1996. the depolarization of the cell membrane. This results in a 30 In 2001, the fungus arrived in South America and reached pronounced efflux of Cl and K" ions, linked with pro America for the first time in 2004 (Sconyers E.L. et al., at nounced water loss. It is assumed that depolarization triggers ers.usda.gov/Features/SoyBeanRust? (2005)). In South an increased Ca" concentration, which is an important signal America in particular, P. pachyrhizi caused big yield losses of molecule (Ward J. M. et al., Plant Cell 7, 833 (1995)) and up to 80%. It is estimated that 1.5 million tons of the Brazilian plays a role in the hypersensitive reaction (HR) (Wendehenne 35 soybean harvest 2005/2006 alone have succumbed to infec a et al., Plant Cell 14, 1937 (2002)). HR in plants is a form of tion with soybean rust. P pachyrhizi is a hemicyclic rust programmed cell death. It allows the plant to stop the fungus which forms three types of spores. The formation of even after penetration of the latter by denying it a source of teliospores was observed in Asia towards the end of the veg nutrients. The course of HR appears to depend on the com etation period (Yeh C.C. et al., Phytopathology 71, 1111 bination of plant and pathogen. However, protein biosynthe 40 (1981)). The formation of basidiospores, in contrast, is only sis, an intact cytoskeleton and salicylic acid appear to be known under laboratory conditions. The most important necessary for inducing HR (Heath M., Plant Mol. Biol. 44. spore form are the uredospores, which are formed over the 321 (2000)). entire vegetation period and which serve to spread the dis A very rapid response to the pathogen is the oxidative burst, ease. These spores are formed in large amounts and are the formation of reactive oxygen species, such as the Super 45 capable of spreading over wide distances with the aid of wind oxide anion O, the hydroxyl radical OH and hydrogen and rain. P. pachyrhizi is an obligate biotroph. If a uredospore peroxide. These compounds are formed by various oxidases. arrives on a Suitable host, it germinates with a single germ The hydroxyl radical acts locally, while HO can diffuse via tube. At the end of this germ tube, an appressorium develops the membranes. Both oxidize polyunsaturated fatty acids and and rapidly reaches the size of the spore. With the appresso can thus destroy membranes (Grant J. J. et al., Plant Physiol. 50 rium, the fungus is capable of building up a large pressure and 124, 21 (2000)). HO is also suspected of performing a of penetrating the epidermal cells directly with the aid of a function in gene regulation. In addition, the compounds Sup penetration hypha. The penetration hypha of P. pachyrhizi port the defense responses by crosslinking the cell wall com grows through the epidermal cell and, once it reaches the ponents, by increasing lignification and by exerting a toxic intercellular space in the leaf, forms the first septum. It now effect on pathogens (Garcia-Brugger A. et al., MPMI 19, 711 55 continues to grow in the leaf as a primary hypha. As early as (2006)). Last but not least, the pathogen attack leads to the 24-48 h after the infection, the firsthaustorial mother cell is expression of genes which code for PR proteins and for phy divided by a septum, and a sacciform haustorium is formed in toalexins. PR proteins are a heterogeneous group of proteins a mesophyll cell of the leaf. The cell wall of the mesophyll cell which have a toxic effect on penetrating fungi. The term is penetrated, but the plasmallemma is only folded, so that the phytoalexins refers to low-molecular-weight antimicrobially 60 cell remains alive and can act as a nutrient source. The nutri active Substances whose synthesis is triggered by biotic or ents travel from the membrane of the live host cell via the abiotic stress (Prell H. H., Interaktionen von Pflanzen and extrahaustorial matrix to the haustorium. The epidermal cell phytopathogenen Pilzen Interactions between plants and which has been penetrated at the beginning turns necrotic phytopathogenic fungi, Gustav Fischer Verlag, Jena, 1996; shortly after penetration. This manner of infection of a van Loon L. C. et al., Physiol. Mol. Plant Physiol. 55, 85 65 biotrophic pathogen, as is used by P pachyrhizi, will there (1999)). The responses described proceed partly not only fore be referred to as “heminecrotrophic' for the purposes of when the pathogen interact with a host plant, but also when it the description of the present invention. The firsturedospores US 8,373,022 B2 7 8 are found only 11-12 days after the infection, and the cycle Genetics and Biology 42, 949 (2005). The utilization of can start afresh (Koch E. et al., Phytopath, p. 106, 302 potential resistance sources from representatives of the peren (1983)). nial Subgenus Soja is limited (Hartman G. I. et al., Plant The crop plant soybean Glycine max (L.) Merr. belongs to Disease 76,396 (1992)). So far, all crosses have only led to the family Leguminosae, subfamily Papilionoideae, tribe 5 sterile progeny (Singh R. et al., Wendl. Theor. Appl. Genet 74, Phaseoleae, genus Glycine Willd. and Subgenus soja 391 (1987). (Moench). Soybean is planted in more than 35 countries. The efficient control of soybean rust with fungicides Some of the most important production areas are located in requires low application into the foliage of the plants, since the United States, China, Korea, Argentina and Brazil. It is infection occurs first on the lower leaves. A double treatment considered to be one of the oldest crop plants and was domes 10 has proved to be effective. A disadvantage of the fungicides ticated for the first time in China between the 11" and 17" used is that, as the result of their specific mechanism of action, century (Hymowitz T. Econ. Bot. 24, 408 (1970)). It was resistances may develop readily. A potential alternative to the introduced into the United States in 1765; the United States use of fungicides is the use of glyphosate-resistant soybean are currently one of the largest Soya production areas. Wild plants. In a greenhouse experiment, soybean plants were Soybean species can be found in China, Korea, Japan, Taiwan 15 treated with the herbicide three days before inoculation, and and the former USSR. Morphological, cytological and a reduction of rust-caused lesions by 46-70% was observed molecular evidence Suggests that G. Soia is the ancestor of the (Feng C. C. P. et al., PNAS 102, 17290 (2005)). Whether this cultivated form G. max. Being a subtropical plant, soybeans effect will also be retained in field trials remains to be seen. prefer a mean annual temperature of 5.9-27°C.; they are not In recent years, P pachyrhizi has gained in importance as frost resistant (OECD, Consensus document on the biology of pest in Soybean production. There was therefore a demand in Gycinemax (L.) Merr. (Soybean); Series on harmonization of the prior art for developing methods of controlling the fungus. regulatory oversight in biotechnology No. 15, ENV/JM/ Right now, plant breeding cannot be expected to contribute MONO(2000)9). Soybeans are currently an important oil and since the available resistance sources are not accessible. protein source. This extensive use of soya in food production Treatment with fungicides has a limited efficiency and is only underlines the importance of efficient control of soybean rust. 25 effective when the disease is yet to break out. This is why in Soybean plants are infected by P. pachyrhizi by windborne particular the pathosystem Soya/P pachyrhizi is the method uredospores. The first discernible symptoms are small yellow of choice for a recombinant approach. For an approach to be to reddish-brown lesions on the upper surface of the leaf, Successful, it is initially important to have detailed knowledge which later spread further until all of the leaf finally turns of the course of infection and the response of the plant, in chlorotic and dies. Upon advanced infection, the lesions are 30 particular during the first stages of the infection. Potential found on all of the plant. The first uredia have a diameter of candidate genes which confer resistance must be identified, 100-200 um and are found on the underside of the leaf 10-14 and their effect in the interaction between plant and pathogen days after the infection; they can produce spores for three to must be characterized. The stable transformation of plants is six weeks. Telia are formed subepidermally and mostly occur very time-consuming and costly, which is why transient on the periphery of the lesions. The spores are first yellow to 35 transformation, which makes possible a characterization of brown and later turn black. The first symptoms are frequently the plant/pathogen interaction at the cell level, is preferred. first observed on the older leaves. The rapid development of The method of choice here is the transient transformation of the disease correlates with the beginning of flowering (R1+) leaves with the gene gun. In plants, non-host-resistance is and finally destroys all of the foliage. The fact that most of the particularly effective and durable and based on the fact that photosynthetically active area is destroyed and that water and 40 quantity and speed of the resistance response is increased in nutrients are extracted by the fungus leads to reduced produc comparison with the compatibility response. Thus, the char tivity of the plant (Sconyers E.L. et al., at ers.usda.gov/Fea acterization of decisive genes in non-host-resistance can tures/SoyBeanRust/ (2005)). serve to identify potential candidate genes for the generation In order to germinate, P pachyrhizi requires moisture in the of resistant plants. Thus, to also study the response of a form of dew or the like on the upper surface of the leaf. The 45 non-host-plant to Soya rust, the system barley (Hordeum Vul fungus is encouraged in particular by frequent rain and tem gare)/P pachyrhizi was chosen since barley is a model plant peratures of between 15 and 29° C. (Sconyers E.L. et al., at which has been described in detail. Moreover, the pathosys ers.usda.gov/Features/SoyBeanRust/ (2005)). Frequently, tem barley/with Blumeria graminis f.sp. hordei (Bgh) and the disease starts at discrete locations and Subsequently with Blumeria graminis f.sp. tritici (Bgt), which is incompat spreads rapidly over the entire field. The fungus is autoecious, 50 ible, has been studied in great detail. Although Bgh and P i.e. it requires no host alternation for its development, and it pachyrhizi differ in Some respects, they share at least pheno can persist readily on its numerous alternative host plants. In typically some steps at the beginning of the infection process. the United States, kudzu vine (Pueraria lobata), which origi Thus, the analysis in barley can provide information on the nates in Japan, is considered to be a potential host plant on mechanisms of the non-host-resistance to P. pachyrhizi in which P pachyrhizi can overwinter and provide fresh inocu 55 comparison with the host resistance of barley. Thus, candi lum in the next spring. date genes were tested in transiently transformed barley P pachyrhizi can currently be controlled in the field only by leaves for their effect in the resistance to P. pachyrhizi. The means of fungicides. Soybean plants with resistance to the transient transformation with the gene gun provides a method entire spectrum of the isolates are not available. When search of studying, in the pathosystem barley/P pachyrhizi, a series ing for resistant plants, four dominant genes Rippl-4, which 60 of genes which may be involved in the resistance response. mediate resistance of soya to P pachyrhizi, were discovered; Programmed cell death (PCD) is an important process in however, this resistance is only isolate-specific (Hartwig E. E. the development and stress response of plants and animals. et al., CropScience, 23, 237 (1983); Hartwig E. E., Crop Some morphological and biochemical changes in the cell, Science 26, 1135 (1986)). Since the resistance was only based Such as chromatin condensation, shrinking of the cytoplasm on individual genes, it was lost rapidly. Only the Rpp4-me 65 and DNA fragmentation, appear to be shared by plants and diated resistance has as yet only been broken down under animals (Lam E. et al., Nature 411, 848 (2001)). A clear greenhouse conditions (Posada-Buitrago M. L. et al., Fungal distinction between PCD and HR, which is caused by biotic US 8,373,022 B2 10 or abiotic stress, is not possible (Heath M., Plant Mol. Biol. Plant J. 45,884 (2006)), and the reduction of the BI-1 expres 44, 321 (2000)). One activator of PCD in animals is BAX. sion after the treatment of rice cells with M. grisea elicitor BAX develops channels in the outer mitochondrial mem extract, demonstrates that the expression patterns can differ brane and causes the release of cytochrome c. This triggers a greatly, depending on the inducing factor and on the plant. caspase cascade, and thus the proteolysis of proteins which The expression patterns suggest a role of BI-1 in the regula the cell needs to survive (Green D. R. etal, Science 281, 1309 tion of the stress-induced PCD or HR and in the resistance (1998). The overexpression of BAX in tobacco (N. tabacum, response to pathogens. The influence on the HR can increase Lacomme C. et al., Proc. Nat. Acad. Sci. USA 96, 7956 the resistance of a plant, especially if the pathogens are per (1999)) and Arabidopsis thaliana (Kawai-Yamada M. et al., totrophic or hemibiotrophic fungi. Overexpression of BI-1 in Plant Cell 16, 21 (2004)) causes PCD and thus suggests 10 carrots (Daucus carota Ssp. sativa) leads to resistance of the similar mechanisms in plants and animals. However, no BAX plants to Botrytis cinerea Omani J. et al., Mol. Plant Physiol. homologs have been identified in plants. However, a BAX in press). In tomatoes, the expression of the PCD inhibitor antagonistic regulator of PCD, the Bax inhibitor-1 (BI-1), is p35 protects against Alternaria alternata, Colletotrichum conserved in plants, animals and other organisms such as coccodes and Pseudomonas Syringae (Lincoln J. E. et al., yeast. Similar proteins have been identified since in A. 15 Proc. Nat. Acad. Sci. USA99, 15217 (2002)). thaliana, H. vulgare, Brassica napus, , Against this background, there was a continuous demand Oryza sativa and N. tabacum (Hickelhoven R., Apoptosis 9. in the prior art for crop plants with an increased resistance to 299 (2004)). In experiments with BI-1/GFP fusion proteins, a pathogens. Only few approaches exist which confer, to plants, localization of BI-1 in the membrane of the endoplasmic a resistance to a broader spectrum of pathogens, especially reticulum (ER) and the nuclear membrane has been observed fungal pathogens. Systemic acquired resistance (SAR)—a (Eichmann R. et al., Mol. Plant Microbe Interact. 17, 484 defense mechanism in various plant/pathogen interactions— (2004)). The protein has a size of 25-27 kDa and has 6-7 can be conferred by application of endogenous messenger transmembrane domains. The C-terminal end, which is prob Substances such as jasmonate (JA) or salicylic acid (SA) ably located in the cytoplasm (Bolduc N. et al., Planta 216, (Ward J. M., et al., Plant Cell 3, 1085 (1991); Uknes et al., 377 (2003)), is essential for the function of BI-1 (Kawai 25 4.(6), 645 (1992)). Similar effects can also be brought about Yamada et al., Plant Cell 16, 21 (2004)). It is possible that the by synthetic compounds such as 2,6-dichloroisonicotinic transmembrane domains form an ion channel (Bolduc N. et acid (DCINA) or benzo(1.2.3)thiadiazole-7-thiocarboxylic al., Planta 216,377 (2003)). Thus, BI-1 might have a function acid S-methyl ester (BTH: Bion.R.) (Friedrich et al., Plant J. in regulating the cytosolic Ca" leveland/or the redox state of 10(1), 61 (1996); Lawton et al., Plant J. 10, 71 (1996)). Also, the cell as the result of the ER's storage function for Ca" (Xu 30 expression of “pathogenesis-related’ (PR) proteins, which Q. et al., Mol. Cell 18, 1084 (1998); Balduc N. et al., FEBS has been upregulated in the context of SAR, may partly bring Lett532, 111 (2003); Hickelhoven R. et al., Proc. Natl. Aced about resistance to pathogens. Sci. USA 29, 5555 (2003); Matsumura H. et al., Plant J.33, In barley, the Milo locus has been described as a negative 425 (2003)). In cells, there is no direct physical inter regulator of pathogen defense. The loss, or loss of function, of action between BI-1 and Bax. However, BIO0-1 interacts with 35 the Mlogene brings about an increased, race-unspecific resis other PCD regulators (Xu Q. et al., Mol. Cell 18, 1084 tance to a large number of mildew isolates (Biischges R. et al., (1998)). It is probable that BI-1, in plants, also interacts with Cell 88,695 1997); Jorgensen J. H., Euphytica 26, 55 (1997); other PCD regulators, thus influencing the resistance Lyngkjaer M. F. et al., Plant Pathol 44, 786 (1995)). responses. In Arabidopsis, BI-1 is capable of Suppressing The Mlo gene has been described (Bischges R. et al., Cell BAX and the HO, have induced PCD (Baeket al., Plant Mol. 40 88, 695 (1997); WO 98/04586: Schulze-Lefert P. et al., Biol. 56, 15 (2004); Kawai-Yamada M. et al., Plant Cell 16, Trends Plant Sci. 5,343 (2000)). Various Mlo homologs from 21 (2004)). Therefore, it probably regulates the processes at a other cereal species have been isolated. Methods using these level lower than the oxidative stress response (Kawai-Ya genes for obtaining pathogen resistance have been described mada M. et al., Plant Cell 16, 21 (2004)). (WO 98/04586: WO 00/01722; WO99/47552). The disad The expression of BI-1 is induced by biotic and abiotic 45 vantage is that Mlo-deficient plants also initiate the above stress such as attack by pathogens or wounding, but also in mentioned defense mechanism in the absence of a pathogen, aging tissues (Balduc N. et al., FEBS Lett. 532, 111 (2003); which manifests itself in the spontaneous dying of plant cells Hickelhoven R., Apoptosis 9, 299 (2004)). In Arabidopsis, (Wolter M. et al., Mol. Gen. Genet. 239, 122 (1993)). As the the mRNA levels of BI-1 are increased after heat shock (Wa result, mlo-resistant plants suffer a yield loss of up to 5% tanabe N. et al., Plant J. 45,884 (2006)). BI-1 expression is 50 (Jörgensen J.H. Euphytica 63, 141 (1992)). The spontaneous induced in tomato (Lycopersicum esculentum) by H2O, and dying of the leaf cells furthermore brings about a disadvan in Arabidopsis by HO and salicylic acid (Hickelhoven R. tageous hypersusceptibility to necrotrophic and hemi Apoptosis 9, 299 (2004); Kawai-Yamada M. et al., Plant Cell biotrophic pathogens such as Magnaporthe grisea (M. 16, 21 (2004)). This suggests that BI-1 has a function in grisea) or Cochliobolus sativus (Bipolaris Sorokiniana) pathogen defense, because this is where both Substances play 55 (Jarosch B. et al., Mol Plant Microbe Interact. 12,508 (1999); an important role (Prell H. H., Interaktionen von Pflanzen and Kumar J. et al., Phytopathology 91, 127 (2001)). phytopathogenen Pilzen Interactions between plants and Apoptosis, also referred to as programmed cell death, is an phytopathogenic fungi, Gustav Fischer Verlag, Jena, 1996). essential mechanism for maintaining tissue homoeostasis, Accordingly, the infection of barley with Bgh or Bgt trig and, as such, counteracts cell division as a negatively-regu gers an increased expression of BI-1 (Hickelhoven R. et al., 60 lating mechanism. In the multi-celled organism, apoptosis is Plant Mol. Biol. 47, 739 (2001); Eichmann R. et al., Mol. a natural component of ontogenesis, and involved, interalia, Plant Microbe Interact. 17, 484 (2004)). In rice, the expres in organ development and the removal of senescent, infected sion is biphasic after infection with M. grisea. It is first or mutated cells. As the result of apoptosis, undesired cells are slightly increased, but is reduced 12 hours after the infection eliminated in an efficient manner. Interference with, or inhi only to rise again (Matsumura H. et al., Plant J. 33, 425 65 bition of apoptosis contributes to the pathogenesis of a vari (2003)). The increased expression of BI-1 in Arabidopsis ety of diseases, among which carcinogenesis. The main effec cells after treatment with fumonisin B1 (Watanabe N. et al., tors of apoptosis are aspartate-specific cysteine proteases, US 8,373,022 B2 11 12 which are known by the name of caspases. They can generally The roll of BI-1 has been tested in three independent be activated by at least two apoptotic signal pathways: firstly experiments in the transient transformation system. In the by the activation of the control. 53% (averaged over the experiments) of the trans TNF (tumor necrosis factor) receptor family; secondly, the formed cells which interacted with P. pachyrhizi were pen mitochondria play a central role. Activation of the mitochon etrated, while only 37% of the BI-1 transformed cells were drial apoptosis signal pathway is regulated by proteins of the penetrated (FIG. 8: Table FIG. 10). The data are based on Bcl-2 family. This protein family consists of antiapoptotic three independent experiments. Barley leaves were trans and proapoptotic proteins such as, for example, Bax. In the formed with the reporter gene construct pGY1-GFP and the case of an apoptotic stimulus, the Bax protein undergoes an blank vector in order to act as controls. The penetration rate in 10 the BI-1 transformed cells differs significantly from the WT allosteric conformation change, which leads to the anchoring (P<0.05). After the evaluation, the cells which have been of the protein in the external mitochondrial membrane, and to transiently transformed with BI-1 therefore surprisingly its oligomerization. As the result of these oligomers, proapo show a significantly increased penetration resistance to P ptotic molecules are released from the mitochondria into the pachyrhizi (P<0.05; FIG. 8: Table FIG. 10). A noteworthy cytosol and bring about an apoptotic signal cascade and, 15 aspect of the observation under the microscope was that the ultimately, the degradation of specific cellular substrates, BI-1-forming cells had a markedly more vital appearance resulting in cell death. The Bax inhibitor-1 (BI1) was isolated than cells, which expressed GFP or ADF3. via its property of inhibiting the proapoptotic effect of BAX Thus, even the transient overexpression of the cell death (Xu Q. et al., Mol Cell 1(3), 337 (1998)). BI1 is a highly inhibitor BI-1 in barley revealed, surprisingly, the trend to an conserved protein. It is found predominantly as an integral increased resistance of the plant cells to penetration by Soy constituent of intracellular membranes. BI1 interacts with bean rust. To confirm this, the interaction between transgenic bcl-2 and bcl-X1. The overexpression of BI1 in mammalian barley plants cv. 'Golden Promise' (GP) which contain a cells suppresses the proapoptotic effect of BAX, etoposid and GFP-BI-1 overexpression construct was studied under the staurosporin, but not of Fasantigen (Roth W. et al., Nat. Med. microscope in comparison with the wild type (WT) of this 8, 216 (2002)). The inhibition of BI1 by antisense RNA, in 25 variety. To generate the transgenic plants, a GFP-BI-1 fusion contrast, induces apoptosis (Xu Q. et al., Mol Cell 1(3), 337 under the control of the constitutive CaMV 35S promoter was (1998)). The first plant homologs of BI1 have been isolated used, thus ensuring sufficient expression of the protein. from rice and Arabidopsis (Kawai et al., FEBS Lett 464, 143 In preliminary experiments, leaves of the WT cv. 'Golden (1999); Sanchez et al., Plant J. 21, 393 (2000)). These plant Promise' and of the transgenic barley line (cv. “Golden proteins suppress the BAX-induced cell death in yeast. The 30 Promise') #6(1)E8L1(T1)" were inoculated with P. amino acid sequence homology with human BI1 is approxi pachyrhizi and, 24 hours after the inoculation, fixed in mately 45%. In recombinant plants, the Arabidopsis homolog destaining solution. After destaining was complete, the leaves AtBI1 is capable of suppressing the proapoptotic effect of were stained with aniline blue. Aniline blue intercalates into murine BAX (Kawai-Yamada M. et al., Proc. Natl. Acad. Sci. the structure of callose and thus preferentially stains papillae USA 98(21), 12295 (2001)). The rice (Oryza sativa) BI1 35 where callose undergoes accumulation and crosslinking with homolog OSBI1 is expressed in all plant tissues (Kawai et al., other polymeric substances. Cells which, as the result of a FEBS Lett 464, 143(1999)). Furthermore described are BI1 hypersensitive response (HR), have undergone a similar pro genes from barley (Hordeum vulgare; GenBank Acc.-No.: cess as apoptosis in mammaliancells, will, after staining with AJ290421), rice (GenBank Acc.-No.: AB025926), Arabidop aniline blue, also show a light fluorescence. The number of sis (GenBank Acc.-No.: AB025927), tobacco (GenBank 40 spores, the germinated spores with germ tubes which had Acc.-No.: AF390556) and oilseed rape (GenBank Acc.-No.: already formed an appressorium and, as cell response, the AF390555, Bolduc N. et al., Planta 216, 377-386 (2003)). appressoria with underlying papillae and the HR were The expression of BI1 in barley is upregulated as the result of counted on the inoculated barley leaves. Larger spore infection with mildew (Hickelhoven R. et al., Plant Mol. agglomerations where an assignation of the appressoria to the Biol. 47(6), 739 (2001)). 45 spores was no longer possible were not included. As far as WO 00/26391 describes the overexpression, in plants, of possible, at least 100 spores with appressoria were counted the antiapoptotic genes Ced-9 from C. elegans, sf IAP from per leaf. Even in this experiment, a significantly increased Spodoptera frugiperda, bcl-2 from humans and bcl-Xl from formation of papillae and a significantly decreased HR of the chicken for increasing the resistance to necrotrophic or hemi cells was observed in the transgenic line (P<0.01: Table FIG. biotrophic fungi. Plant BI1 homologs are not disclosed. 50 11A/B, FIG. 12). Expression is under the control of constitutive promoters. Both Bgh (Hickelhoven R., FEMS Microbiol. Letters 245, Furthermore described is the expression of a BI1 protein from 9(2005)) and P. pachyrhizi (Koch E. et al., Phytopath, p. 106, Arabidopsis under the strong constitutive 35S CaMV pro 302 (1983)) are biotrophic pathogens. An HR of the infected moter in rice cells and a hereby-induced resistance to cell cells can therefore stop the development of the fungi since it death-inducing Substances from Magnaporthe grisea (Mat 55 deprives them from their food source. If the HR is prevented, sumura H. et al., Plant J. 33,425 (2003)). the cells may become more sensitive to infection by Originally, the prior art described that constitutive expres biotrophic pathogens. Despite this, barley cells which have sion of an inhibitor of the programmed cell death in plants can been transiently transformed with a BI-1 overexpression con bring about resistance to necrotrophic fungi. struct Surprisingly show an increased resistance to penetra However, the person skilled in the art was faced in particu 60 tion by P. pachyrhizi. This is Surprising because an increased lar with the problem of providing methods for the pathogen sensitivity to the biotrophic fungus would have been expected defense in plants, in particular against biotrophic pathogens. However, the HR is not the only resistance reaction in the Surprisingly, the problem is solved by the inventive meth resistance of barley and wheat to the incompatible pathogen ods, peptide sequences, nucleic acid sequences, expression P. pachyrhizi, and perhaps not the decisive one Thus, the cassettes, vectors and organisms defined in the main claims, 65 defense of wheat against P pachyrhizi is papille formation using a BI1 protein. The dependent claims define specific, (Hoppe H. H. et al., Pro. Intern. Congress of SABRAO especially preferred use forms of the present invention. (Bangkok 1985) 1986). Barley responds with papilla forma US 8,373,022 B2 13 14 tion and with HR of the infected cells, depending on the other genes. Since the identification was performed at the variety. Moreover, barley with the mlo5 allele responds with DNA level, no comments can be made on the expression of papilla increased papilla formation. The defense of barley to BI-1. It must also be borne in mind that even minor damage to Bgt is also affected by papilla formation, but mostly by an HR the seeds or the leaves can have a decisive effect, or prevent, of infected cells (Hickelhoven R. et al., Mol. Plant Pathol. 2, the development of the plant. Thus, some seeds of the line 199 (2001). In contrast to P. pachyrhizi, the transient overex #6(2)E15L7P2 (T2) did not germinate. pression of BI-1 in barley leads to an increased penetration rate of the cells by Bgt (Eichmann R. et al., Mol. Plant SUMMARY OF THE INVENTION Microbe Interact. 17, 484 (2004)). Likewise, barley plants with the mlo5 allele, which confers broad resistance to Bgh 10 Consequently, a first Subject matter of the invention relates demonstrate, in the case of transient overexpression of BI-1, to a method of generating or increasing a resistance in the greater sensitivity of the cells to penetration by Bgh (Hick plant, or a part of a plant, to a pathogen which is preferably a elhoven R. et al., Proc. Natl. Acad. Sci. USA,29,5555 (2003). biotroph. The method comprises a step in which the amount Although the resistance of barley with themlo5 allele is based of a Bax inhibitor-1 (BI-1) protein or its function in the plant not on an HR of the infected cells, but on a more efficient 15 or at least a part of a plant is increased. A part of a plant is accumulation of antimicrobial compounds, HO and an understood as meaning the entire plant, one or more of its increased formation of papillae, the inhibition of the HR also organs, a tissue or at least a cell. The method furthermore appears to affect the other resistance responses (Hickelhoven comprises the step of selecting a plant or a part of a plant in R. et al., Plant Physiol. 119, 1251 (1999)). Without causing which the BI1 protein or its function has been increased in limitation by theory, these observations of a reduced penetra Such a way that it shows increased resistance in comparison tion resistance as the result of inhibition of HR suggests that with a starting plant, in which the increase in the amount of crosslinked regulation of the resistance responses. The Sus function of the BI1 protein in comparison with the starting picion that the resistance responses are subject to crosslinked plant or its part is increased. regulation is Supported by the microscopic analysis of trans genic barley plants which have been inoculated with P. 25 BRIEF DESCRIPTION OF THE DRAWINGS pachyrhizi. These barley plants contain a BI-1 overexpression construct and respond to infection with increased papilla FIG. 1a-1C: Alignment of protein sequences of various formation. While only 16-26% of the infectcd cells show HR BI-1 proteins from plants. AtBI-1 (SEQ ID NO: 4): Arabi on the leaves of the transgenic plants, HR was observed in dopsis: BnBI-1 (SEQID NO: 10): Brassica napus (oil seed approximately 50% of the infected cells on the WT leaves. 30 rape); GmBI2 (SEQ ID NO: 12): Glycine max (soybean: Consequently, in the barley variety “Golden Promise', the variant 1); GmBI3 (SEQID NO: 14): Glycinemax (soybean: HR of the cells also appears to be an important resistance variant 2): HVBI-1 (SEQID NO: 2): Hordeum vulgare (bar mechanism against P. pachyrhizi, which is a biotroph, ley); NtEI-1 (SEQID NO: 6): Nicotiana tabacum (tobacco); although P pachyrhizi does not utilize the epidermal cells as OsBI-1 (SEQID NO:8): Oryza sativa (rice); TaBI11 (SEQID a food source, at least not in soybean, but only forms haustoria 35 NO: 20): Triticum aestivum (wheat, variant 1); TaBI18 (SEQ in the mesophyll (Koch E. et al., Phytopath, p. 106, 302 ID NO: 26): Triticum aestivum (wheat, variant 2); TaBI5 new (1983)). In general, however, the fungus did not reach this (SEQ ID NO: 16): Triticum aestivum (wheat, variant 3): stage on the non-host plant barley. Inhibition of the HR makes ZmBI14 (SEQ ID NO: 22): Zea mays (: variant 1); it possible for Bgt to penetrate the cells, and the fungus can ZmBI16 (SEQ ID NO: 24): Zea mays (maize: variant 2): establish itself successfully (Eichmann R. et al., Mol. Plant 40 ZmBI33 (SEQ ID NO: 28): Zea mays (maize: variant 3): Microbe Interact. 17, 484 (2004)). Further fungus-specific ZmBI8 (SEQ ID NO: 18): Zea mays (maize: variant 4): factors appear to be necessary for this process. It appears that Consensus (SEQ ID NO: 61): consensus sequence derived these specific factors of Bgt are capable of suppressing either from the alignment. the alternative resistance response of the plant cell or the FIG. 2:Vector map for the vectorpUbiBI-1 (Ubi: ubiquitin recognition by the latter. These factors are probably absent in 45 promoter; BI-1 nucleic acid sequence coding for barley BI1 P. pachyrhizi. Thus, the plant cell is perhaps capable of rec protein; ter: transcription terminator). Also indicated is the ognizing P pachyrhizi and, since BI-1 Suppresses the HR, localization of the cleavage sites of various restriction capable of inducing an alternative resistance response, papilla enzymes. formation. Recognition of P pachyrhizi by the plant is Sup FIG. 3: Vector map for the vector plO114UbiBI-1 (Ubi: ported by the observation of increased papilla formation in 50 ubiquitin promoter, BI-1 nucleic acid sequence coding for barley with the mlo5 allele. However, the reason why the barley BI1 protein; ter: transcription terminator). Also indi plant is capable of recognizing the pathogen P. pachyrhizi, cated is the localization of the cleavage sites of various which is specialized to host plants of a completely different restriction enzymes. order, remains unexplained (Sinclair J. B. et al. (eds.), Pro FIG. 4: Vector map for the vector pCxoBI-1 (Oxo: TaGer ceedings of the soybean rust workshop (1995), National Soy 55 min 9f-2.8 promoter; BI-1 nucleic acid sequence coding for bean Research Laboratory, Publication No. 1 (1996)). The barley BI1 protein; ter: transcription terminator). Also indi “recognition feature' of P. pachyrhizi might be an unspecific cated is the localization of the cleavage sites of various elicitor (pathogenesis-associated molecular patterns restriction enzymes. PAMP) such as the INF 1 from P infestans, which triggers an FIG. 5: Vector map for the vector plO114OxoBI-1 (Oxo: HR in Nicotiana sp. (Kamoun S. et al., Plant Cell 10, 1413 60 TaGermin 9f2.8 promoter; BI-1 nucleic acid sequence cod (1998)). PAMPs are recognized by the plant as foreign mol ing for barley BI1 protein; ter: transcription terminator). Also ecules and trigger a resistance response. Not all leaves of the indicated is the localization of the cleavage sites of various transgenic plant show increased papilla formation, although restriction enzymes. the BI-1 gene construct has been identified in them by means FIG. 6: Alignment of the protein sequences of BI-1 pro of PCR. This might be the consequence of an unfavorable 65 teins from barley (Hordeum Vulgare, GenBank Acc.-No.: insertion type of the construct, which prevents an effective CAC37797), rice (Oryza sativa, GenBank Acc.-No.: expression of BI-1 and/or leads to antagonistic effects by Q9MBD8), Arabidopsis thaliana (GenBank Acc.-No.: US 8,373,022 B2 15 16 Q9LD45) and humans (Homo sapiens, GenBank Acc.-No.: Phacopsora. In especially preferred embodiments, the patho AAB87479). Amino acids shown against the black back gen is Phacopsora pachyrhizi and/or P. meibomiae (together ground are identical in all species. Amino acids shown against also referred to as “soybean rust” or “soya rust'). Preference a gray background are only identical in plants. Bars indicate being given to the former. When the pathogen is selected from the predicted seven transmembrane domains in HvBI 1. “H. 5 the group of the biotrophic pathogens or fungi, it is preferred vul.”: SEQID NO: 2: “O. sat.”: SEQIDNO:8: “A. tha.”: SEQ in some embodiments that the pathogenis other than powdery ID NO: 4; “H. sap.”: SEQID NO: 60. mildew or downy mildew. FIG. 7: Diagram of the transformation rate as a function of In preferred embodiments, the step of increasing the the conditions during the transformation of soybean leaves amount or function of the BI1 protein is accomplished by (A) and barley leaves (B) with the gene gun. For the transfor 10 biotechnological methods. The term “biotechnological mation, in each case 1.6 g of DNA were used per bombard methods comprises, interalia, the recombinant expression of ment; particles of diameter 0.6 um were used for barley leaves the protein in a cell, preferably in operable linkage with, and and with lum diameter for soybean. The number of trans driven by, a heterologous promoter. The abovementioned formed cells was determined 24 hours after the transforma increase, however, can also be accomplished for example by tion. 15 Substituting only the endogenous promoter or, for example, FIG. 8: Diagram of the penetration rate of P pachyrhizi in the silencing of factors which generally inhibit the expression barley cells which have been transformed transiently with a of the endogenous BI1 protein. Accordingly, the term “bio BI 1 overexpression construct in comparison with the control. logical method’ comprises all methods which are open to The data are based on three independent experiments. By way modern molecular biology, in particular the methods of of control, barley leaves were transformed with the reporter recombinant gene technology, which are known to the skilled gene construct pGY 1 GFP and with the blank vector. In the BI worker and which will be discussed in greater detail in the 1 transformed cells, the penetration rate differs significantly course of the description. from the WT (P<0.05). The plant in which the resistance is generated is preferably FIG. 9: PCR for detecting the GFP BI 1 construct in the selected from the group of the monocotyledonous plants, in transgenic barley lines #6(1)E4L3P5 (T2), 1#6(2)E15L7P1 25 particular comprising wheat, oats, millet, barley, rye, maize, (T2), #6(2)E15L7P2 (T2) and #6(1)E8L1(T1) (“Golden rice, Sorghum, triticale, spelt or Sugarcane; or from the group Promise). The PCR was carried out with genomic DNA of of the dicotyledonous plants, in particular comprising Arabi the plants with the GFP-specific primers. In positive trans dopsis, cotton, buckwheat, potato, cabbages, cress, linseed, genic plants, the entire GFP, which is 740 bp in size is ampli oil seed rape, tomato, aubergine, bell peppers, Sunflower, fied. The negative control used was genomic DNA from the 30 tobacco, Tagetes, lettuce, Calendula, melon, pumpkin/ WT “Golden Promise (WT) or water (NTC). The GFP of squash, courgette, Sugarbeet, ornamentals, trees and legumes. plasmid pGY 1 GFP was amplified as the positive control Since soybean rust attacks more than 70 leguminous species, (PC). the plant is especially preferably selected, among the FIG. 10: Data obtained for the penetration rate of P legumes, at least in cases where the pathogen is a hemine pachyrhizi in barley cells which have been transformed tran 35 crotrophic pathogen or a rust or soybean rust pathogen. siently with a BI 1 overexpression construct. The data shown Consequently, the plant is selected especially preferably are those of FIG. 8 (see also examples hereinbelow). among the legumes, comprising plants of the genus Phaseo FIG. 11 A/B: Counted spores and cell responses in the lus (comprising French bean, dwarf bean, climbing bean interaction between soybean rust and transgenic barley lines (Phaseolus. vulgaris), Lima bean (Phaseolus lunatus L.), with the GFP BI 1 overexpression construct #6(1)E4L3P5 40 Tepary bean (Phaseolus acutifolius A. Gray), runner bean (T2), 1 #6(2)E15L7P1(T2), #6(2)E15L7P2(T2) and #6(1) (Phaseolus coccineus)); the genus Glycine (comprising Gly E8L1(T1) CV. ‘Golden Promise. The WT acted as the con cine soia, soybeans (Glycine max (L.) Merill)); pea (Pisum) trol. Leaves of 7-day old plants were inoculated with soybean (comprising shelling peas (Pisum sativum L. convar. Sati rust, fixed after 24 hours and stained with aniline blue for vum), also called Smooth or round-seeded peas; marrowfat counting. 45 pea (Pisum sativum L. convar. medullare Alef. emend. C.O. FIG. 12: Relative fraction of papillae formation and HR Lehm), Sugar pea (Pisum sativum L. convar. axiphium Alef following inoculation with P pachyrhizi in barley leaves of emend. C.O. Lehm), also called Snow pea, edible-podded pea the transgenic lines (CV. ‘Golden Promise') #6(1)E4L3P5 or mangetout, (Pisum granda Sneida L. convar. Sneidulo p. (T2), 1#6(2)E15L7P1 (T2), #6(2)E15L7P2 (T2) and #6(1) shneiderium)); peanut (Arachis hypogaea), (Trifolium E8L 1 (T1) in comparison with the WT. The primary leaves 50 spec.), medick (Medicago), kudzu vine (Pueraria lobata), were removed seven days after Sowing the barley seeds inocu common lucerne, (M. sativa L.), chickpea (Cicer), lated with P. pachyrhizi and evaluated 24 hours after the lentils (Lens) (Lens culinaris Medik.), lupins (Lupinus); inoculation. The data shown are the mean values of the WT vetches (Vicia), field bean, broadbean (Vicia faba), vetchling and of the individual lines. The error bars represent the stan (Lathyrus) (comprising chickling pea (Lathyrus sativus), dard deviation. A Student's t-test reveals that the relative 55 heath pea (Lathyrus tuberosus)); genus Vigna (comprising papillae formation and HR in the transgenic lines are signifi bean (Vigna aconitifolia (Jacq.) Maréchal), adzuki bean cantly different from the WT, P-0.001. (Vigna angularis (Willd.) Ohwi & H. Ohashi), urd bean (Vi gna mungo (L.) Hepper), mung bean (Vigna radiata (L.) R. DETAILED DESCRIPTION OF THE INVENTION Wilczek), bambara groundnut (Vigna subterrane (L.) Verdc.), 60 rice bean (Vigna umbellata (Thunb.) Ohwi & H. Ohashi), In a preferred embodiment, the resistance is a resistance to Vigna vexillata (L.) A. Rich., Vigna unguiculata (L.) Walp., in at least one biotrophic pathogen, preferably a biotrophic fun the three Subspecies asparagus bean, cowpea, catang bean)); gus, and especially preferably a heminecrotrophic fungus as pigeonpea (Caianus Cajan (L) Millsp.), the genus Macroty defined herein. In preferred embodiments of the present loma (comprising geocarpa groundnut (Macrotyloma geo invention, the biotrophic fungus is selected from the group 65 carpum (Harms) Maréchal &. Baudet), horse bean (Macro Basidiomycota, preferably the Uredinales (rusts), especially tyloma unifiorum (Lam.) Verdc.)); goa bean (Psophocarpus preferably the Melompsoraceae, and in particular the genus tetragonolobus (L.) DC.), African yam bean (Sphenostylis US 8,373,022 B2 17 18 Stenocarpa (Hochst. ex A. Rich.) Harms), Egyptian black mal appendages of various form, structure and function are bean, dolichos bean, lablab bean (Lablab purpureus (L.) referred to as trichomes and, in the present context, likewise Sweet), yam bean (Pachyrhizus), guar bean (Cyanopsis tet come under the term “epidermis’. They occur in the form of ragonolobus (L.) Taub.); and the genus Canavalia (compris protective hairs, Supportive hairs and gland hairs in the form ing jack bean (Canavalia ensiformis (L.) DC.), Sword bean 5 of scales, different papillae and, in the case of roots, as absor (Canavalia gladiata (Jacq.) DC.)). bent hairs. They are formed exclusively by epidermal cells. In any of the embodiments of the present invention which Frequently, a trichome is formed by only one Such a cell, are disclosed herein, it is especially preferred that the amount however, occasionally, more than one cell is involved in its or function of the BI1 protein is increased at least in the formation. epidermis, preferably essentially in a tissue-specific manner 10 The term "epidermis' likewise comprises papillae. Papil in the epidermis; in particular, it is preferred that the amount lae are bulges of the epidermal surface. The textbook example or function of the BI1 protein is specifically increased in the are the papillae on flower surfaces of pansy (Viola tricolor) epidermis and/or essentially not increased in the mesophyll. and the upper Surfaces of the leaves of many species from By epidermis, the skilled worker means the predominant tropical rain forests. They impart a velvet-like consistency to epidermal tissue of primary aerial plant parts, for example of 15 the surface. Some epidermal cells can form water stores. A the shoot, the leaves, flowers, fruits and seeds. The epidermal typical example are the water vesicles at the Surfaces of many cells secrete outwardly a water-repellent layer, the cuticle. Mesembryanthemum species and other Succulents. In some The roots are surrounded by the rhizodermis, which, in many plants, for example in the case of campanula (Campanula ways, resembles the epidermis, but also shows pronounced persicifolia), the outer walls of the epidermis are thickened differences. While the outermost layer of the apical meristem 20 like a lens. gives rise to the epidermis, the formation of the rhizodermis is The bulk of all tissues is the parenchyma. The parenchy much less clear. Depending on the species, it can be consid matic tissues include the mesophyll which, in leaves, can be ered, in phylogenetic terms, either as part of the calyptra or as differentiated into palisade parenchyma and Spongy paren part of the primary cortex. The epidermis has a number of chyma. functions: it protects the plant against desiccation and regu- 25 Accordingly the skilled worker understands, by meso lates the transpiration rate. It protects the plant against a wide phyll, a parenchymatic tissue. Parenchymatic cells are always range of chemical and physical external influences, against alive, in most cases isodiametric, rarely elongated. The pith of being fed upon by animals and against attack by parasites. It the shoots, the storage tissues of the fruits, seeds, the root and is involved in gas exchange, in the secretion of certain other underground organs are: also parenchymas, as is the metabolites and in the absorption of water. It comprises 30 mesophyll. receptors for light and mechanical stimuli. it thus acts as a In the leaves of most ferms and phanerogams, especially in signal transformer between the environment and the plant. In the case of the dicots and many monocots, the mesophyll is accordance with its various functions, the epidermis com Subdivided into palisade parenchyma and spongy paren prises a number of differently differentiated cells. To this chyma. A “typical leaf is of dorsiventral organization. In must be added species-specific variants and different organi- 35 most cases, the palisade parenchyma is at the upper Surface of zations of the epidermides in the individual parts of a plant. the leaf immediately underneath the epidermis. The sponge Essentially, it consists of three categories of cells: the “actual parenchyma fills the underlying space. It is interspersed by a epidermal cells, the cells of the stomata and of the trichomes Voluminous intercellular system whose gas space is in direct (Greek: trichoma, hair), epidermal appendages of varying contact with the external space via the stomata. shape, structure and function. 40 The palisade parenchyma consists of elongated cylindrical The “actual', i.e. the least specialized, epidermal cells cells. In some species, the cells are irregular, occasionally account for the bulk of the cells of the epidermal tissue. In bifurcate (Y-shaped: arm palisade parenchyma). Such vari topview, they appear either polygonal (slab or plate shaped) ants are found in ferns, conifers and a few angiosperms (for or elongated. The walls between them are often wavy or example in Ranunculaceae and Caprifoliaceae species ex sinuate. It is not known what induces this shape during devel- 45 ample: elder). Besides the widest-spread organization form opment; existing hypotheses only offer unsatisfactory expla which has just been described, the following variants have nations herefor. Elongated epidermal cells can be found in been found: organs or parts of organs that are elongated themselves, thus, palisade parenchyma on the abaxial leaf Surface. Particu for example, in stems, petioles, leaf veins and on the leaves of larly noticeable in scaly leaves. Example: arbor vitae most monocots. The upper Surface and underSurface of lami- 50 (Thuja), and on the leaves of wild garlic ( ursi nae can be covered in epidermides with different structures, it being possible for the shape of the cells, the wall thickness palisade parenchyma on both leaf surfaces (adaxial and and the distribution and number of specialized cells (stomata abaxial). Frequently in plants which grow in dry habitats and/or trichomes) per unit area to vary. A high degree of (xerophytes). Example: prickly lettuce (Lactuca ser variation is also found within individual families, for example 55 riola); in the Crassulaceae. In most cases, the epidermis consists of ring-shaped closed palisade parenchyma. In cylindrically a single layer, though multi-layered water-storing epider organized leaves and in conifers needles. mides have been found among species from a plurality of The variability of the cells of the spongy parenchyma, and families (Moraceae: most Ficus species; Piperaceae. Pepero the organization of the Spongy parenchyma itself, are even nia, Begoniaceae, Malvaceae and the like). Epidermal cells 60 more varied than that of the palisade parenchyma. It is most however secrete a cuticle on the outside which covers all frequently referred to as aerenchyma since it comprises a epidermal Surfaces as an uninterrupted film. It may either be multiplicity of interconnected intercellular spaces. smooth or structured by bulges, rods, folds and furrows. How The mesophyll may comprise what is known as the assimi ever, the folding of the cuticle, which can be observed when lation tissue, but the terms mesophyll and assimilation tissue viewing the Surface, is not always caused by cuticular rods. 65 are not to be used synonymously. There are chloroplast-free Indeed, there are cases where cuticular folding is merely the leaves whose organization differs only to a minor extent from expression of the underlying bulges of the cell wall. Epider comparable green leaves. As a consequence, they comprise US 8,373,022 B2 19 20 mesophyll, but assimilation does not take place; conversely, HvPAL, acc. X97313; Wei Y., Zhang Z., Andersen C. H., assimilation also takes place in, for example, sections of the Schmelzer E., Gregersen Collinge D. B. Smedegaard shoot. Petersen V. and Thordal-Christensen H. Plant Molecular In the present description, the epidermis is characterized in Biology 36,101 (1998). biochemical terms. In a preferred embodiment, the epidermis 5 In preferred embodiments, the mesophyll is characterized can be characterized by the activity of one or more of the by the fact that all the abovementioned promoters are active in following promoters: the tissue or the cell. In another embodiment, the mesophyll WIR5 (=GstA1); acc. X56012: Dudler & Schweizer, comprises the fact that only some of the promoters are active, GLP4, acc. AJ310534; Wei Y., Zhang Z. Andersen C. H., for example preferably 2, 3, 5 or especially preferably 7 or Schmelzer E. Gregersen Collinge D. B. Smedegaard 10 more, but at least from only one of those detailed above. In preferred embodiments, all of the abovementioned pro Petersen V. and Thordal-Christensen H., Plant Molecu moters are active in a plant used or produced in accordance lar Biology 36, 101 (1998), with the invention or in the epidermis and in the mesophyll in GLP2a, acc. AJ237942, Schweizer P., Christoffel A. and a plant according to the invention. In one embodiment, only Dudler R., Plant J. 20,541 (1999); Prx7, acc. AJO03141, 15 Some of the abovementioned promoters are active, for Kristensen B. K., Ammitzböll H., Rasmussen S. K. and example preferably 2, 5, or especially preferably 7 or more; Nielsen K. A., Molecular Plant Pathology, 206), 311 however, at least one of the promoters detailed above is active (2001); in each case. GerA, acc. AF250933; Wu S., Druka A., Horvath H., In preferred embodiments, the increase in the protein quan Kleinhofs. A., Kannangara G. and von Wettstein D., tity or function of the BI1 protein takes place in a constitutive Plant Phys Biochem 38,685 (2000); or tissue-specific manner. In especially preferred embodi OsROC1, acc. AP004656 ments, an essentially tissue-specific increase in the protein RTBV, acc. AAV62708, AAV62707; Klöti.A., Henrich C., quantity or protein function takes place in an essentially epi Bieri S., He X., Chen G., Burkhardt P. K., Warm J., dermis-specific manner, for example by recombinant expres Lucca P. Hahn T. Potrykus I. and Fitterer J., PMB 40, 25 sion of a nucleic acid. Sequence coding for said BI1 protein 249 (1999); under the control of an epidermis-specific promoter. In par Chitinase ChtC2-Promotor from potato (Ancillo et al., ticular, the increase in the expression or function of the BI1 Planta. 217(4), 566, (2003)); protein takes place in the epidermis, where, however, the AtProT3 Promotor (Grallath et al., Plant Physiology. 137 expression of the BI-1 protein in the mesophyll remains 30 essentially unchanged, or it is reduced, and where other tis (1), 117 (2005)) Sues are unaffected. SHN-Promotors from Arabidopsis (AP2/EREBP tran As described in the present text, in one embodiment, the Scription factors involved in cutin and wax production) expression or function of the protein according to the inven (Aarón et al., Plant Cell. 16(9), 2463 (2004)); tion or of the BI-1 characterized in the present text is GSTA1 from wheat (Dudler et al., WP2005306368 and 35 increased at least in the epidermis of a plant An increase in Altpeter at al., Plant Molecular Biology. 57(2), 271 expression can be achieved as described hereinbelow. By (2005)). increased expression or function, the present text means both In preferred embodiments, the epidermis is characterized the activation or enhancement of the expression or function of by the fact that all the abovementioned promoters are active in the endogenous protein including a de novo expression, but the tissue or the cell. In other preferred embodiments, the 40 also an increase in or enhancement as the result of the expres epidermis is characterized by the fact that only some of the sion of a transgenic protein or factor. promoters are active, for example preferably 2, 3, 5 or most In an especially preferred embodiment, the increase in the preferably 7 or more, but at least from only one of those protein quantity or function of at least one plant BI1 protein detailed above. can be combined with an mlo-resistant phenotype or with the In a preferred embodiment, the mesophyll is characterized 45 inhibition or reduction, in comparison with a control plant, of in biochemical terms. The mesophyll can be characterized by the expression of MLO, RacB and/or NaOX in the plant or a the activity of one or more of the following promoters: part thereof, for example in a tissue, but especially advanta PPCZm1 (=PEPC): Kausch A. P. Owen T. P. Zachwieja S. geously at least in the epidermis or a considerable number of J., Flynn A. R. and Sheen J., Plant Mol. Biol. 45, 1 the epidermal cells, and/or with the increase in the expression (2001); 50 or function of PEN2 and/or PEN1 in the plant, for example OsrbcS, Kyozuka at al., PlaNT Phys 102, 991 (1993); constitutively, or a part thereof, for example in a tissue, but Kyozuka J. McElroy D., Hayakawa T., Xie Y., Wu R. especially advantageously at least in the epidermis or in a and Shimamoto K., Plant Phys. 102,991 (1993); considerable number of epidermal cells, with the proviso that OsPPDK, acc. ACO99041: the expression of a plant BI1 protein in the leaf epidermis TaGF-2.8, acc. M63223; Schweizer P., Christoffel A and 55 remains essentially unchanged or is reduced. Dudler R., Plant J. 20, 541 (1999); The Milo locus has been described in barley as negative TaFBPase, acc. X53957: regulator of pathogen defense. The loss, or loss of function, of TaWIS1, acc. AF467542; US 200220115849; the Mlogene brings about an increased, race-unspecific resis HvBIS1, acc. AF467539; US 200220115849; tance to a number of mildew isolates (Bischges R. et al., Cell ZmMIS1, acc. AF467514; US 200220115849; 60 88, 695 (1997); Jorgensen J. H., Euphytica 26, 55 (1977); HvPR1a, acc. X74939; Bryngelsson et al., Mol. Plant Lyngkjaer M. F. et al., Plant Pathol. 44, 786 (1995)). An Microbe Interacti. 7 (2), 267 (1994): mlo-resistant phenotype can be obtained as described in the HvPR1 b, acc. X74940; Bryngelsson et al., Mol. Plant prior art. Methods using these genes for obtaining a pathogen Microbe Interact. 7(2), 267 (1994); HvB1.3gluc; acc. resistance are described, inter alia, in WO 98/04586: WO AF479647; 65 00/01722; WO99/47552. HvPrXS, acc. AJ276227; Kristensen et al., Molecular Plant In one embodiment of the present invention, the activity, Pathology, 206), 311 (2001): expression or function of MLO, RacB and/or NaOX in the US 8,373,022 B2 21 22 plant or a part thereof, for example in a tissue, but especially and/or SNAP34 in the plant, for example constitutively, or in advantageously at least in the epidermis or a substantial num a part thereof, for example in a tissue, but especially advan ber of epidermal cells, can advantageously be inhibited or tageously at least in the epidermis or a substantial number of reduced in comparison with a control plant or a part thereof. epidermal cells will preferably increase the resistance or By reducing the activity or function of MLO, RacB and/or 5 withstanding power to biotrophic pathogens in the plants NaOX in the plant or a part thereof, for example in a tissue, but produced in accordance with the invention. The increase in especially advantageously at least in the epidermis or a Sub the activity or function, if appropriate the expression, of stantial number of epidermal cells, it is preferred to increase PEN2 is described in detail in WO 03/074688, which is here the resistance, or withstanding power, to biotrophic patho with expressly incorporated into the present description. The gens in plants produced in accordance with the invention. The 10 description of the abovementioned publication describes pro activity or function of MLO, Rach3 and/or NaOX can be cesses and methods for reducing or inhibiting the activity or reduced or inhibited analogously to what has been described function of PEN2; the examples detail specifically how this for MLO in WO98/04586. WO 00/01722; WO99/47552 and can be performed. The reduction or inhibition of the activity the other publications mentioned hereinbelow, whose content or function of PEN2 is especially preferably carried out in is herewith expressly incorporated into the present descrip 15 accordance with the embodiments and examples which are tion, in particular for describing the activity and inhibition of especially preferred in WO 03/074688 and in the organisms MLO. The description of the abovementioned publications detailed therein as being especially preferred, in particular in describes processes, methods and especially preferred plants, for example constitutively, or in a part thereof, for embodiments for reducing or inhibiting the activity or func example in a tissue, but especially advantageously at least in tion of MLO; the examples detail specifically how this can be the epidermis or a considerable part of the epidermal cells. In performed. WO 03074688, the skilled worker will find the sequences The reduction of the activity or function, if appropriate the which code for PEN2 proteins and can also identify PEN2 by expression, of RacB is described in detail in WO 2003/20939, means of the method provided in WO 03/074688. which is herewith expressly incorporated into the present The expression of PEN1 and SNAP34 can be increased description. 25 analogously to the methods described in WO 03/074688. The description of the abovementioned publication Owing to his general expert knowledge and the prior art with describes processes and methods for reducing or inhibiting which he is familiar, the skilled worker can isolate and over the activity or function of proteins; the examples detail spe express PEN1 and SNAP34 nucleic acid sequences and pro cifically how this can be performed. It is especially preferred tein sequences. SEQ ID No. 39 describes the nucleic acid to carry out the reduction or inhibition of the activity or 30 sequence which codes for PEN1 from barley; the protein function of RacB as described in the embodiments and the sequence is described in SEQ ID No. 40. SEQ ID No.: 41 examples which are especially preferred in WO 2003/20939 describes the nucleic acid sequences which codes for PEN1 and in the organisms specified therein as being especially from Arabidopsis thaliana, the protein sequence is described preferred, in particular in a plant or a part thereof, for example in SEQ ID No:42. PEN1 from Arabidopis thaliana is pub in a tissue, but especially advantageously at least in the epi 35 lished under the accession numbers NM 202559 and NM dermis or a substantial number of epidermal cells. The reduc 112015. The homolog from barley is disclosed in accession tion of the activity or function, if appropriate the expression, numbers AY246907 and AY246906 as ROR2. They are mem of RacB is described in detail in WO 2003/20939. In WO bers of the fairly large family of the syntaxin proteins. Thus, 2003/20939, the skilled worker can find the sequences which the skilled worker can use simple homology comparisons for code for RacB proteins and can also identify Rach3 by means 40 identifying further syntaxin proteins which are expressed as of the method provided in WO 2003/20939. potential resistance genes in the method according to the The reduction of the activity or function, if appropriate of invention. the expression, of NaOX is described in detail in WO 2004/ SEQID No. 43 describes the nucleic acid sequence which 09820 (=PCT/EP/03/07589) which is herewith expressly codes for SNAP34 from barley; the protein sequence is incorporated into the present description. The description of 45 described in SEQ ID No. 44. The SNAP-34 homolog from the abovementioned publication describes processes and barley is also published as AY 247208 (SNAP-34). Homologs methods for reducing or inhibiting the activity or function of whose function is unknown and which might play a role in the NaOx; the examples detail specifically how this can be per resistance are published as AY 247209. (SNAP-28) and AY formed. It is especially preferred to carry out the reduction or 247210 (SNAP-25). The following Arabidopsis genes show a inhibition of the activity or function of NaOx as described in 50 higher degree of homology with barley SNAP34 than barley the embodiments and the examples which are especially pre SNAP-28 or SNAP-25 to SNAP-34 and can thus advanta ferred in WO 2004/09820 (=PCT/EP/03/07589) and in the geously be co-overexpressed as potential resistance-mediat organisms specified therein as being especially preferred, in ing genes: particular in a plant or a part thereof, for example in a tissue, AAM 62553 Arabidopsis SNAP25a but especially advantageously at least in the epidermis or a 55 NP 200929 Arabidopsis. SNAP33b substantial number of epidermal cells. In WO 2004/09820 NP 172842 Arabidopsis SNAP30 (PCT/EP/03/07589), the skilled worker can find the NP 196405 Arabidopsis SNAP29 sequences which code for NaOX proteins and can also iden Accordingly, the invention also relates to a plant in which tify NaOx by means of the method provided in WO 2004/ a polypeptide wheh is encoded by a nucleic acid molecule 09820 (=PCT/EP/03/07589). 60 comprising the sequences shown in SEQID No. 39, 41 or 43 In one embodiment of the present invention, the activity, or one of the sequences shown in the abovementioned data expression or function of PEN1, PEN2 and/or SNAP34 can base publications or which comprises one of the amino acid advantageously be increased in the plant, for example consti sequences shown in the abovementioned database publica tutively, or in a part thereof, for example in a tissue, but tions or in SEQID No. 40, 42 or 44, or which is a functional especially advantageously at least in the epidermis or a Sub 65 equivalent thereofor which has at least 50%, preferably 70%, stantial number of epidermal cells. The increase in activity, more preferably 80%, even more preferably 90%. 95% or which also comprises a de novo expression, of PEN1, PEN2 more homology with the abovementioned sequences at the US 8,373,022 B2 23 24 coding nucleic acid molecule level or, preferably, at the amino Operable linkage is understood as meaning, for example, acid level is overexpressed at least furthermore in the epider the sequential arrangement of a promoter and the nucleic acid mis, or relates to a plant in which the above-characterized sequence to be expressed and, if appropriate, further regula polypeptide is activated, or its activity or function increased, tory elements such as, for example, a terminator, in Such a constitutively or in a part, for example in a tissue, but espe 5 way that each of the regulatory elements can fulfil its function cially advantageously at least in the epidermis or a substantial in the recombinant expression of the nucleic acid sequence, number of epidermal cells. depending on the arrangement of the nucleic acid sequences A reduction of the expression or activity of a protein can be to make sense RNA or antisense RNA. This does not neces brought about by the methods with which the skilled worker sarily require direct linkage in the chemical sense. Genetic is familiar, for example mutagenesis, for example EMS, if 10 control sequences such as, for example, enhancer sequences, appropriate TILLING, iRNA; ribozyme, silencing, knockout, can also exert their function on the target sequence from and the like. Reduction methods are described in particular in positions which are somewhat distant, or indeed from other WO 2003/20939, whose methods can readily be adapted to DNA molecules (cis or trans localization). Preferred arrange the sequences described herein, which is why the content of ments are those in which the nucleic acid sequence to be WO 2003/20939 is explicitly incorporated herein. 15 expressed recombinantly is positioned downstream of the The lowering or reduction of the expression of a protein, sequence which acts as promoter, so that the two sequences the activity or the function can be performed in many ways. are covalently bonded with one another. The distance “Lowering”, “to lower”, “reduction” or “to reduce” is to be between the promoter sequence and the nucleic acid sequence understood in the broad sense in connection with the present to be expressed recombinantly is preferably less than 200 invention and comprises the partial or essentially complete base pairs, especially preferably less than 100 base pairs, very prevention or blocking of the functionality or a protein, as the especially preferably less than 50 base pairs. result of different cell-biological mechanisms. The generation of an operable linkage can be accomplished A reduction for the purposes of the invention also comprise by means of current recombination and cloning techniques, as a quantitative reduction of a protein down to an essentially is the generation of an expression cassette. Such techniques complete absence of the protein (i.e. lacking detectability of 25 are described, for example, in Maniatis T., Fritsch E. F. and activity or function or lacking immunological detectability of Sambrook J., Molecular Cloning: A Laboratory Manual, the protein). In this context, the expression of a certain protein Cold Spring Harbor Laboratory, Cold. Spring Harbor (N.Y.) or the activity, or function, in a cell or an organism is prefer (1989), in Silhavy T. J., Berman M. L. and Enquist L. W., ably reduced by more than 50%, especially preferably by Experiments with Gene Fusions, Cold Spring Harbor Labo more than 80%, very especially. preferably by more than 30 ratory, Cold Spring Harbor (N.Y.) (1984), in Ausubel F. M. et 90%. al., Current Protocols in Molecular Biology, Greene Publish The methods of dsRNAi, cosuppression by means of sense ing Assoc. and Wiley Interscience (1987) and in Gelvin et al. RNA and “VIGS (“virus induced gene silencing') are also in Plant Molecular Biology Manual (1990). However, it is referred to as “post-transcriptional gene silencing (PTGS). also possible to position, between the two sequences, further PTGS methods, like the reduction of the function or activity 35 sequences which have, for example, the function of a linker with dominant-negative variants, are especially advantageous with certain restriction enzyme cleavage sites, or of a signal because the requirements to homology between the endog peptide. The insertion of sequences may also lead to the enous gene to be Suppressed and the recombinantly expressed expression of fusion proteins. Preferably, the expression cas sense or dsRNA nucleic acid sequence (or between the sette, consisting of a linkage of promoter and nucleic acid endogenous gene and its dominant-negative variant, respec 40 sequence to be expressed, can exist as integrated into a vector tively) are lower than, for example in the case of a traditional and can be inserted into a plant genome by, for example, antisense approach. Such homology criteria are mentioned in transformation. The control elements preferably mediate an the description of the dsRNAi method and can generally be epidermis-specific expression. applied to PIGS methods or dominant-negative approaches. For the purposes of the present invention, “approximately “Introduction' comprises, within the context of the present 45 in connection with numbers or sizes means a range of num invention, all methods which are capable of introducing a bers or sizes around the numerical value or the size. In gen compound, directly or indirectly, into the epidermis or a Sub eral, the term “approximately means a range of in each case stantial part of the epidermal cells, compartment or tissues of 10% above and below the value detailed. same, or which are Suitable for generating it therein. This For the purposes of the present invention, “plant’ means all comprises direct and indirect methods. The introduction can 50 genera and species of higher and lower plants of the plant lead to a transient presence of a compound (for example a kingdom. The term includes the mature plants, seed, fruits dsRNA) or else to a stable presence. Introducing, comprises, and seedlings, and parts, propagation material, plant organs, for example, methods such as transfection, transduction or tissue, protoplasts, callus and other cultures, for example cell transformation. cultures, derived therefrom, and any other types of associa In expression constructs of the present invention, a nucleic 55 tions of plants cells to the functional or structural units. acid molecule disclosed herein, whose expression (transcrip Mature plants means plants at any developmental stage tion and, if appropriate, translation) generates a correspond beyond the seedling stage. Seedling means a young, imma ing amino acid molecule, is preferably in operation linkage ture plant in an early developmental stage. with at least one genetic controller (for example a promoter) "Plant comprises all annual and perennial, monocotyle which ensures expression in an organism, preferably in 60 donous and dicotyledonous plants and includes by way of plants, preferably an epidermis-specific expression. If the example, but not by limitation, those of the genera Cucurbita, expression construct is to be introduced directly into the Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, plant, plant-specific genetic control elements (for example Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, promoters) are preferred, where, as can be seen from what has Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, been said above, the epidermis-specific activity of the pro 65 Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nic moter is mandatory in most use forms, as described herein Otiana, Solarium, Petunia, Digitalis, Majorana, Ciahorium, above. Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Het US 8,373,022 B2 25 26 erocallis, Nemesis, Pelargonium, Panieum, Pennisetum, tance to at least one pathogen exists or is increased, all those Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, methods which are suitable for recognizing an existing or Glycine, Pisum, Phaseolus, Lawn, Oryza, Zea, , Hor increasing pathogen resistance. This can be for example deum, Secale, Triticum, Sorghum, Picea and Populus. symptoms of the pathogen infection (for example develop The term “plant preferably comprises the monocotyle ment of necroses in the case of fungal infection), but may also comprise the above-described symptoms, which affect the donous and dicotyledonous crop plants. Preferred within the quality of the plant, the quantity of the yield, the suitability for Scope of the invention are plants which are employed as use as feedstuff or foodstuff, and the like. foodstuffs or feedingstuffs, very especially preferred are agri For the purposes of the invention, “pathogen' means by culturally important monocotyledonous and dicotyledonous way of example, but not by limitation, viruses or viroids, genera and species, as detailed in the claims. 10 bacteria, fungi, animal pests such as, for example, or "Pathogen resistance” means the reduction or diminishing nematodes. Fungi, in particular biotrophic or hemine of disease symptoms of a plant as the result of attack by at crotrophic fungi as defined herein, are especially preferred. least one pathogen. The symptoms can be manifold in nature, However, it can be assumed that the mesophyll-specific but preferably comprise those which directly or indirectly expression of a BI1 protein also brings about a resistance to lead to a negative effect on plant quality, yield quantity, the 15 other pathogens since a resistance to stress factors in total is suitability for use as foodstuff or feedingstuff, or else which being generated. make Sowing, planting, harvesting or processing of the crop Pathogens which may be mentioned by way of example, more difficult. For the purposes of the present invention, but not by limitation, are the following: "pathogen tolerance' is, in particular, to be considered as 1. Fungal Pathogens of Fungus-Like Pathogens: being comprised by "pathogen resistance'. Fungal pathogens or fungus-like pathogens (such as, for “Confering”, “existing”, “generating or “increasing (of) example, Chromista) preferably belong to the group compris a resistance means that the defense mechanisms of a particu ing Plasmodiophoramycota, Oomycota, Ascomycota, lar plant species or variety displays increased resistance to Chytridiomycetes, Zygomycetes, Basidiomycota and Deu one or more pathogens, as the result of the application of the teromycetes (Fungi imperfecti). Pathogens which may be method according to the invention, in comparison with the 25 mentioned by way of example, but not by limitation, are those wild type of the plant (“starting plant”) to which this method detailed in Tables 1 to 4, and the diseases which areassociated according to the invention has not been applied, under other with them. wise essentially identical conditions (such as, for example, climatic conditions, culture conditions, type of stress, patho TABLE 1 gen species and the like). in this context, the increased resis 30 tance preferably manifests itselfin a reduced manifestation of Diseases caused by biotrophic phytopathogenic fungi the disease symptoms, where disease symptoms—in addition Disease Pathogen to the abovementioned adverse effects—also comprises for example the penetration efficiency of a pathogen into the Leafrust Puccinia recondita 35 Yellow rust Pstriiformis plant or plant cells, or the proliferation efficiency in or on Powdery mildew Erysiphe graminist Blumenia graminis same. in this context, the disease symptoms are preferably Rust (common corn) Puccinia sorghi reduced by at least 5%, 10% or at least 20%, especially Rust (Southern corn) Puccinia polysora preferably by at least 40% or 60%, very especially preferably Tobacco leaf spot Cercospora nicotianae Rust (soybean) Phakopsora pachyrhizi, P. meibomiae by at least 70% or 80%, most preferably by at least 90% or 40 Rust (tropical corn) Physopella pallescens, P Zeae = 95%. Angiopsora zeae “Selection' means, with regard to plants where—as opposed to, or in comparison with, the starting plant—resis TABLE 2 Diseases caused by necrotrophic and/or hemibiotrophic fungi and Oomycetes Disease Pathogen Plume blotch Septoria (Stagonospora) nodorum Leaf blotch Septoria tritici Earfusarioses Fusarium spp. Eyespot Pseudocercosporella herpotrichoides Smut Ustilago spp. Late blight Phytophthora infestans Bunt Tilletia caries Take-all Gaettmannomyces graminis Anthrocnose leafblight Colletotrichum graminicola (teleomorph: Glomerella Anthracnose stalk rot graminicola Politis); Glomerella tucumanensis (anamorph: Glomerella falcatum Went) Aspergilius ear and Aspergiiitisfiavits kernel rot Banded leaf and sheath spot Rhizoctonia Soiani Kuhn = Rhizoctonia (“Wurzeltöter) microsclerotia J. Matz (telomorph: Thanatephorus citchineris) Blackbundle disease Acremonium stricium W. Gams = alosporium acremonium Auct. non Corda Black kernel rot Lasiodiplodia theobromae = Botryodiplodia theobromae Borde blanco Marasnielius sp. Brown spot (black spot, stalk rot) Physoderma maydis US 8,373,022 B2 27 28 TABLE 2-continued Diseases caused by necrotrophic and/or hemibiotrophic fungi and Oomycetes Disease Pathogen Cephalosporium kernel rot Acremonium strictum = Cephalosporium acremonium Charcoal rot Macrophomina phaseolina Coricitin ear rot Thanatephorus cucumenis = Corticium Sasaki Curvularia leaf spot Curvatiaria Clavata, C. eragrostidis, = C. machians (teleomorph: Cochliobolus eragirostidis), Curvularia inaequalis, C. intermedia (teleomorph: Cochliobolus intermedius), Curvularia lunata (teleomorph: Cochliobolus lunatus), Curvularia pallescens (teleomorph: Cochliobolus pallescens), Curvularia Senegalensis, C. tuberculata (teleomorph: Cochliobolus tuberculatus) Didymella leaf spot Didymelia exitalis Diplodia ear and stalk rot Diplodia frumenti (teleomorph: Botryosphaeria festicae) Diplodia ear and stalk rot, seed rot and Diplodia maydis = Seedling blight Stenocarpella maydis Diplodia leaf spot or streak Stenocarpella macrospora = Diplodialeaf macrospora Brown stripe downy Sclerophthora rayssiae var. zeae mildew Crazy top downy mildew Sclerophthora macrospora = Sclerospora macrospora Green ear downy mildew (graminicola Sclerospora graminicola downy mildew) Dry ear rot (cob, Nigrospora oryzae kernel and stalk rot) (teleomorph: Khuskia oryzae) Earrots (minor) Alternaria alternata = A. tentiis, Aspergillus glaucus, A. niger, Aspergillus spp., Botrytis cinerea (teleomorph: Botryotinia fickeiana), Cunninghamelia sp., Curvularia pallescens, Doratomyces Stemonitis = Cephalotrichum stemonitis, Fusarium cultimorum, Gonatobotry's simplex, Pithomyces maydicus, Rhizopus microsports Tiegh., R. Stoionifer = R. nigricans, Scopulariopsis briumpti Ergot (horse's tooth) Claviceps gigantea (anamorph: Sphacelia sp.) Eyespot Aureobasidium zeae = Kabatiella zeae Fusarium ear and stalk rot Fusarium subgiitiinans = F moniiforme var. subglutinians Fusarium kernel, root and stalk rot, seed Fusarium moniiforme rot and seedling blight (teleomorph: Gibberella filiikuroi) Fusarium stalk rot, Fusaritin avenacetin Seedling root rot (teleomorph: Gibberella avenacea) Gibbereia ear and stalk rot Gibbereia zeae (anamorph: Fusaritingraminearlin) Gray ear rot Botryosphaeria zeae = Physalospora zeae (anamorph: Macrophoma zeae) Gray leaf spot Cercospora Sorghi = C. Sorghi var. maydis, C. zeae (Cercospora leaf spot) maydis Helminthosporium root rot Exserohilum pedicellatum = Heiminthosporium pedicellatum (teleomorph: Setosphaeria pedicellata) Hormodendrum ear rot Cladosporium cladosporioides = Hormodendrum (Cladosporium rot) cladosporioides, C. herbarum (teleomorph: Mycosphaerelia tassiana) Leaf spots, minor Alternaria alternata, Ascochyta maydis, A. tritici, A. Zeicola, Bipolaris victoriae = Heiminthosporium victoriae (teleomorph: Cochliobolus victoriae), C. sativus (anamorph: Bipolaris Sorokiniana = H. Sorokinianum = H. sativum), Epicoccim nigrum, Exserohilum prolatum = Drechslera prolata (teleomorph: Setosphaeria prolata) Graphium penicillioides, Leptosphaeria maydis, Leptothyrium zeae, Ophiosphaerelia herpoiricha, (anamorph: Scolecosporiella sp.), US 8,373,022 B2 29 30 TABLE 2-continued Diseases caused by necrotrophic and/or hemibiotrophic fungi and Oomycetes Disease Pathogen Paraphaeosphaeria michotii, Phoma sp., Septoria zeae, S. Zeicola, S. Zeina Northern corn leafblight (white blast, Setosphaeria turcica (anamorph: Exserohilum crown stalk rot, stripe) turcicum = Heiminthosporium turcicum) Northern corn leaf spot Helminthosporium Cochliobolus carbonum (anamorph: Bipolaris zeicola = ear rot (race 1) Helminthosporium carbonum) Penicilium ear rot (blue eye, blue mold) Penicilitim spp., P. Chrysogeniim, P. expanslim, P. Oxalictim Phaeocytostroma stalk and rootrot Phaeocytostrona ambiguttin, = Phaeocytosporelia 288 Phaeosphaeria leaf spot Phaeosphaeria maydis = Sphaerulina maydis Physalospora ear rot (Botryosphaeria ear Botryosphaeria festicae = Physalospora zeicola rot) (anamorph: Diplodiafitinenii) Purple leaf sheath Hemiparasitic bacteria and fungi Pyrenochaeta stalk and rootrot Phoma terrestris = Pyrenochaeta terrestris Pythium root rot Pythium spp., P arrhenomanes, Pgraminicola Pythium stalk rot Pythium aphanidermatum = P buileri L. Red kernel disease (ear mold, leaf and Epicocci in nigrin seed rot) Rhizoctonia ear rot (sclerotial rot) Rhizoctonia zeae (teleomorph: Waitea circinata) Rhizoctonia root and stalk rot Rhizoctonia Soiani, Rhizoctonia zeae Rootrots (minor) Alternaria alternata, Cercospora Sorghi, DictoChaeta fertilis, Fusarium acuminatum (teleomorph: Gibberella acuminata), F equiseti (teleomorph: G. intricans), Foxysportin, F. pallidoroseum, F. poae, F roseum, G. cyanogena, (anamorph: F. Sulphureum), Microdochium boileyi, Mucor sp., Periconia circinata, Phytophthora cactorum, P drechsleri, P. nicotianae var. parasitica, Rhizop its arrhizus Rostratum leaf spot (Helminthosporium Setosphaeria rostrata, (anamorph: leaf disease, ear and stalk rot) xserohilum rostratum = Heiminthosporium rostratin) Java downy mildew Peronosclerospora maydis = Sclerospora maydis Philippine downy mildew Peronosclerospora philippinensis = Sclerospora philippinensis Sorghum downy mildew Peronosclerospora sorghi = Sclerospora Sorghi Spontaneim downy mildew Peronosclerospora spontanea = Sclerospora spontanea Sugarcane downy mildew Peronosclerospora sacchari = Sclerospora sacchari Sclerotium ear rot (southern blight) Sclerotium rolfsii Sacc. (teleomorph: Athelia rolfsii) Seed rot-seedling blight Bipolaris Sorokiniana, B. Zeicola = Heiminthosporium carbonum, Diplodia maydis, Exserohilum pediciliatum, Exserohilum turcicum = Helminthosporium turcicum, Fusarium avenaceum, F. culmorum, F moniiforme, Gibberella zeae (anamorph: F. graminearlin), Macrophonina phaseolina, Penicilium spp., Phomopsis sp., Pythium spp., Rhizoctonia Solani, R. Zeae, Sclerotium rolfsii, Spicaria sp. Selenophoma leaf spot Selenophoma sp. Sheath rot Gaeli mannomyces graminis Shuck rot Myrothecium gramineum Silage mold Monascus purpureus, M rather Smut, common Ustilago zeae = U. maydis Smut, false Ustilaginoidea virens Smut, head Sphacelotheca reiliana = Sporisorium holicisorghi Southern corn leafblight and stalk rot Cochliobolus heterostrophus (anamorph: Bipolaris maydis = Heiminthosporium maydis) Southern leaf spot Stenocarpella macrospora = Diplodia macrospora Stalk rots (minor) Cercospora Sorghi, Fusarium episphaeria, F. merismoides, Foxysporum Schlechtend, F. poae, F. roseum, F. Solani (teleomorph: Nectria haematococca), Firicinctum, Mariannaea elegans, Mucor sp., Rhopographits zeae, Spicaria sp. Storage rots Aspergillus spp., Penicillium spp. und weitere Pilze Tar spot Phyllachora maydis Trichoderma ear rot and rootrot Trichoderma viride = T, ignorum teleomorph: Hypocreasp. White ear rot, root and stalk rot Stenocarpella maydis = Diplodia zeae US 8,373,022 B2 31 32 TABLE 2-continued Diseases caused by necrotrophic and/or hemibiotrophic fungi and Oomycetes Disease Pathogen Yellow leafblight Ascochyta ischaemi, Phyllosticta maydis (teleomorph: Mycosphaerella zeae-navalis) Zonate leaf spot Gloeocercospora Sorghi

10 TABLE 4 grasses), Uromyces appendiculatus (brown rust of bean), Sclerotium rolfsii (root and stem rots of many Diseases caused by fungi and Oomycetes with unclear classification plants). regarding biotrophic, hemibiotrophic or necrotrophic behavior Deuteromycetes (Fungi imperfecti) Such as Septoria (Stagonospora) nodorum (glume blotch) of wheat (Sep Disease Pathogen 15 toria tritici), Pseudocercosporella herpotrichoides Hyalothyridium leaf spot Hyalothyridium maydis (eyespot of wheat, barley, rye), Rynchosporium secalis Late wilt Cephalosporium maydis (leaf spot on rye and barley). Alternaria Solani (early blight of potato, tomato), Phoma betae (blackleg on The following are especially preferred: Beta beet), Cercospora beticola (leaf spot on Beta beet), Plasmodiophoromycota Such as Plasmodiophora brassi Alternaria brassicae (black spot on oilseed rape, cab bage and other crucifers), Verticillium dahliae (verticil cae (clubroot of crucifers), Spongospora subterranea, lium wilt), Colletotrichum lindemuthianum (bean Polymyxa graminis, anthracnose), Phoma lingam (blackleg of cabbage and Oomycota Such as Bremia lactucae (downy mildew of oilseed rape). Botrytis cinerea (grey mold of grapevine, lettuce), Peronospora (downy mildew) in Snapdragon (P 25 strawberry, tomato, hops and the like). antirrhini), onion (P destructor), spinach (P effusa), Especially preferred are biotrophic pathogens, among soybean (P. manchurica), tobacco (“blue mold': P taba which in particular heminecrotrophic pathogens, i.e. Phako cina) alfalfa and clover (P trifolium), Pseudoperono psora pachyrhizi and/or those pathogens which have essen spora hamuli (downy mildew of hops), Plasmopara tially a similar infection mechanism as Phakopsora (downy mildew in grapevines) (P. viticola) and Sun 30 pachyrhizi, as described herein. Particularly preferred are flower (Phalstedli), Sclerophthora macrospora (downy pathogens from the group Uredinales (rusts), among which in mildew in cereals and grasses), Pythium (for example particular the Melompsoraceae. Especially preferred are damping-off of Beta beet caused by P. debaryanum), Phakopsora pachyrhizi and/or Phakopsora meibomiae. Phytophthora infestans (late blight in potato and in 2. Bacterial Pathogens: tomato and the like), Albugo spec. 35 The pathogens and, the diseases associated with them Ascomycota Such as Microdochium nivale (Snow mold of which are mentioned in Table 5 may be mentioned by way of rye and wheat), Fusarium graminearum, Fusarium cul example but not by limitation. morum (partial ear sterility mainly in wheat), Fusarium TABLE 5 Oxysporum (Fusarium wilt of tomato), Blumeria grami 40 nis (powdery mildew of barley (f.sp. hordei) and wheat (f.sp. tritici)), Erysiphe pisi (powdery mildew of pea), Bacterial diseases Nectria galligena (Nectria canker of fruit trees), Disease Pathogen Uncinula necator (powdery mildew of grapevine), Bacterial leafblight and stalk rot Pseudomonas a venae Subsp. avenae Pseudopeziza tracheiphila (red fire disease of grape 45 Bacterial leaf spot Xanthomonas campestris pv. holicicola Vine), Claviceps purpurea (ergot on, for example, rye Bacterial stalk rot Enterobacter dissolvens = Erwinia dissolvens and grasses), Gaeumannomyces graminis (take-all on Bacterial stalk and top rot Erwinia carotovora Subsp. carotovora, wheat, rye and other grasses), Magnaporthe grisea, Erwinia chrysanthemipv. zeae Pyrenophora graminea (leaf stripe of barley), Pyreno Bacterial stripe Pseudomonas andropogonis 50 Chocolate spot Pseudomonas Syringae pv. phora teres (net blotch of barley), Pyrenophora tritici corona faciens repentis (leaf blight of wheat), Venturia inaequalis Goss's bacterial wilt and blight Clavibacter michiganensis subsp. (apple scab), Sclerotinia Sclerotium (stalk break, stem (leaf freckles and wilt) nebraskensis = Corynebacterium michiganense pv. andnebraskense rot), Pseudopeziza medicaginis (leaf spot of alfalfa, Holcus spot Pseudomonas Syringae pv. Syringae white and red clover). Purple leaf sheath Hemiparasitic bacteria Basidiomycetes Such as Tiphula incarnate (typhula blight 55 Seed rot-seedling blight Bacilius subtiis Stewart's disease (bacterial wilt) Panioea Stewari = Erwinia Stewari on barley, rye, wheat), Ustilago maydis (blister Smut on Corn stunt (achapparramiento, Spiroplasma kuinkei maize.), Ustilago nuda (loose Smut on barley), Ustilago maize stunt, Mesa Central or Rio tritici (loose Smut on wheat, spelt), Ustilago avenae Grande maize stunt) (loose Smut on oats), Rhizoctonia Solani (rhizoctonia 60 root rot of potato), Sphacelotheca spp. (head Smut of 3. Viral Pathogens: Sorghum), Melampsora lini (rust of flax), Puccinia “Viral pathogen includes all plant viruses such as, for graminis (stem rust of wheat, barley, rye, oats), Puccinia example, tobacco or cucumber mosaic virus, ringspot virus, recondite (leafrust on wheat), Puccinia disperse (brown necrosis virus, maize dwarf mosaic virus and the like. rust on rye), Puccinia hordei (leaf rust of barley), Puc 65 The pathogens and diseases associated with them which cinia coronata (crown rust of oats), Puccinia Striiformis are mentioned in Table 6 may be mentioned by way of (yellow rust of wheat, barley, rye and a large number of example, but not by limitation. US 8,373,022 B2 33 34 TABLE 6

Viral diseases Disease Pathogen American wheat striate American wheat striate mosaic virus (AWSMV) (wheat striate mosaic) Barley Stripe mosaic Barley stripe mosaic virus (BSMV) Barley yellow dwarf Barley yellow dwarf virus (BYDV) Brome mosaic Brome mosaic virus (BMV) Cereal chlorotic mottle Cereal chlorotic mottle virus (CCMV) Corn chlorotic vein banding Corn chlorotic vein banding virus (CCVBV) (Brazilian maize mosaic) Corn lethal necrosis Virus complex of Maize chlorotic mottle virus (MCMV) and Maize dwarf mosaic virus (MDMV). A or B or Wheat streak mosaic virus (WSMV) Cucumber mosaic Cucumber mosaic virus (CMV) Cynodon chlorotic streak Cynodon chlorotic streak virus (CCSV) Johnsongrass mosaic Johnsongrass mosaic virus (JGMV) Maize bushy stunt Mycoplasma-like organism (MLO) associated Maize chlorotic dwarf Maize chlorotic dwarf virus (MCDV) Maize chlorotic mottle Maize chlorotic mottle virus (MCMV) Maize dwarf mosaic Maize dwarf mosaic virus (MDMV) strains A, D, E and F Maize leaf fleck Maize leaf fleck virus (MLFV) Maize line Maize line virus (MLV) Maize mosaic (corn leaf stripe, Maize mosaic virus (MMV) enanismo rayado) Maize mottle and chlorotic stunt Maize mottle and chlorotic stunt virus Maize pellucid ringspot Maize pellucid ringspot virus (MPRV) Maize raya gruesa Maize raya gruesa virus (MRGV) Maize rayado fino (fine striping Maize rayado fino virus (MRFV) disease) Maize red leaf and red stripe Mollicute Maize red stripe Maize red stripe virus (MRSV) Maize ring mottle Maize ring mottle virus (MRMV) Maize rio IV Maize rio cuarto virus (MRCV) Maize rough dwarf Maize rough dwarf virus (MRDV) (nanismo ruvido) (Cereal tillering disease virus) Maize sterile stunt Maize sterile stunt virus (strains of barley yellow striate virus) Maize streak Maize streak virus (MSV) Maize stripe (maize chlorotic Maize stripe virus stripe, maize hoja blanca) Maize stunting Maize stunting virus Maize tasselabortion Maize tassel abortion virus (MTAV) Maize vein enation Maize vein enation virus (MVEV) Maize wallaby ear Maize wallaby ear virus (MWEV) Maize white leaf Maize white leaf virus Maize white line mosaic Maize white line mosaic virus (MWLMV) Millet red leaf Millet red leaf virus (MRLV) Northern cereal mosaic Northern cereal mosaic virus (NCMV) Oat pseudorosette (Zakuklivanie) Oat pseudorosette virus Oat sterile dwarf Oat sterile dwarf virus (OSDV) Rice black-streaked dwarf Rice black-streaked dwarf virus (RBSDV) Rice stripe Rice stripe virus (RSV) Sorghum mosaic Sorghum mosaic virus (SrMV) (auch: Sugarcane mosaic virus (SCMV) Stämme H, I and M) Sugarcane Fiji disease Sugarcane Fiji disease virus (FDV) Sugarcane mosaic Sugarcane mosaic virus (SCMV) strains A, B, D, E, SC, BC, Sabi and MB (formerly MDMV-B) Wheat spot mosaic Wheat spot mosaic virus (WSMV)

4. Animal Pests lillus, Aeolus mancus, Horistonotus uhlerii, Sphenophorus 4.1 Pathogenic Insects: 55 maidis, Sphenophorus zeae, Sphenophorus parvulus, Sphe The follovving may be mentioned by way of example, but nophorus callosus, Phyllogphaga spp., Anuraphis maidiradi not by limitation: insects such as, for example, beetles, cat cis, Delia platura, Colaspis brunnea, Stenolophus lecontei erpillars, lice or mites. and Clivinia impressifions. Preferred insects are those of the genera Coleoptera, Other examples are: barley leaf beetle (Oulema melano Diptera, Hymenoptera, , Mallophaga, 60 Homoptera, Hemiptera, Orthoptera, Thysanoptera, Der pus), frit fly (Oscinella frit), wireworms ( lineatus) maptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc. and aphids (such as, for example, the oat grain aphid Rho Especially preferred are coleopteran and lepidopte an insects palosiphum padi, the blackberry aphid Sitobion avenae). such as, for example, the European corn borer (ECB), 4.2 Nematodes: Diabrotica barberi, Diabrotica undecimpunctata, 65 The pathogens and the diseases associated with them men Diabrotica virgifera, Agrotis ipsilon, Crymodes devastator; tioned in Table 7 may be mentioned by way of example, but Feltia ducens, Agrotis gladiaria, Melanotus spp., Aeolus mel not by limitation. US 8,373,022 B2 35 36 TABLE 7 Empoasca fabae, Acrosternum hilare, Melanoplus femurru brum, Melanoplus differentialis, Hylenya platura, Serico Parasitic nematodes thrips variabilis, Thrips tabaci, Tetranychus turkestani, Tet Damage Pathogenic nematode ranychus urticae, 3. Oil Seed Rape: Awl Dolichodorus spp., D. heterocephalus Bulb and stem nematode Dity lenchus dipsaci Fungal, bacterial or viral pathogens: Albugo candida, disease: bulb eelworm Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia Burrowing Radopholus similis Solani, Sclerotinia Sclerotiorum, Mycosphaerella brassic Cyst nematode disease Heterodera avenae, H. zeae, Punctodera cola, Pythium ultinum, Peronospora parasitica, Fusarium chaicoensis 10 Dagger Xiphinema spp., X. americantin, roseum, Alternaria alternate. X. mediterraneum 4. Alfalfa: False root-knot Nacobbus dorsais Fungal, bacterial or viral pathogens: Clavibater michi Lance, Columbia Hoplolaimus columbus 8CC. Hoplolaint is spp., H. galeathis ganese subsp. insidiosum, Pythium ultimum, Pythium irregu Lesion Pratylenchus spp., P. brachyurus, P. crenatus, lare, Pythium splendens, Pythium debaryanum, Pythium aph P hexincisus, P. neglectiis, Ppenetrans, 15 anidermatum, Phytophthora megasperma, Peronospora P scribneri, Pihornei, P zeae Needle Longidorus spp., L. breviannitiatus trifoliorum, Phoma medicaginis var. medicaginis, Cer Ring Criconemella spp., C. ornata cospora medicaginis, Pseudopeziza medicaginis, Leptotro Root-knot disease Meloidogyne spp., M. Chitwoodi, M. incognita, chila medicaginis, Fusarium, Xanthomonas campestris p.V. M.javanica Spiral Helicotylenchus spp. alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Sting Belonoiaint is spp., B. longicatidatus Stemphylium alfalfae. Stubby-root Paratrichodorus spp., P christiei, P. minor, 5. Wheat: Putinisticit is actitis, Trichodorus spp. Fungal, bacterial or viral pathogens: Pseudomonas Syrin Stunt Tvlenchorhynchus dubius gae p.V. atrofaciens, Urocystis agropyri, Xanthomonas 25 campestris p.V. translucens, Pseudomonas Syringae p.V. Very especially preferred are Globodera rostochiensis and syringae, Alternaria alternate, Cladosporium herbarium, G. pallida (cysteelworm on potato, tomato and other Solan Fusarium graminearum, Fusarium avenaceum, Fusarium aceae), Heterodera Schachtii (beet eelVVorm on Sugar and culmorum, Ustilago tritici, Ascochyta tritici, Cephalospo fodder beet, oilseed rape, cabbage and the like), Heterodera rium gramineum, Collotetrichum graminicola, Erysiphe avenae (oat cyst nematode on oat and other cereal species), 30 graminis f.sp. tritici, Puccinia graminis f.sp. tritici, Puccinia. Dity lenchus dipsaci (stem or bulb eelworm, stem eelworm of recondita f.sp. tritici, Puccinia striiformis, Pyrenophora rye, oats, maize, clover, tobacco, beet), Anguilla tritici (grain tritici-repentis, Septoria (Stagonospora) nodorum, Septoria nematode, cockle disease of wheat (spelt, rye), Meloidogyne tritici, Septoria avenae, Pseudocercosporella herpotri hapla (root-knot nematode of carrot, cucumber, lettuce, choides, Rhizoctonia Solani, Rhizoctonia cerealis, Gaeuman tomato, potato, Sugar beet, lucerne). 35 nomyces graminis var. tritici, Pythium aphanidermatum, Examples of preferred fungal or viral pathogens for the Pythium arrhenomanes, Pythium ultimum, Bipolaris Soro individual varieties are: Kiniana, barley tellow dwarf virus, brome mosaic virus, soil 1. Barley: borne wheat mosaic virus, wheat streak mosaic virus, wheat Fungal, bacterial and viral pathogens: Puccinia graminis spindle streak virus, American wheat striate virus, Claviceps f.sp. hordei barley yellow dwarf virus (BYDV), 40 purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tille Pathogenic insects/nematodes: Ostrinia nubilalis (Euro tia indica, Rhizoctonia Solani, Pythium arrhenomannes, pean corn borer); Agrotis Ipsilon, Schizaphis graminum, Pythium gramicola, Pythium aphanidermatum, high plains Blissus leucopterus leucopterus, Acrosternum hilare, Eus virus, European wheat striate virus, Puccinia graminis f.sp. chistus servus, Deliaplatura, Mayetiola destructor, Petrobia tritici (wheat stem rust), Blumeria (Erysiphe) graminis f.sp. latens. 45 tritici (wheat powdery mildew). 2. Soybean: Pathogenic insects/nematodes: Pseudaletia unipunctata, Fungal, bacterial or viral pathogens: Phytophthora Spodoptera, frugiperda, Elasmopalpus lignosellus, Agrotis megasperma fisp. glycinea, Macrophomina phaseolina, Orthogonia, Elasmopalpus Zignosellus, Oulema melanopus, Rhizoctonia Solani, Sclerotinia Sclerotiorum, Fusarium Hypera punctata, Diabrotica undecimpunctata howardi: Oxysporum, Diaporthe phaseolorum var. Sojae (Phomopsis 50 Russian wheat aphid; Schizaphis graminum, Macrosiphun soiae), Diaporthe phaseolorum var. caulivora, Sclerotium avenae, Melanoplus femurrubrum, Melanoplus differentia rolfsii, Cercospora kikuchii, Cercospora Sojina, Peronospora lis, Melanoplus sanguinipes, Mayetiola destructor, Sito manshurica, Colletotrichum dematium (Colletotrichum trun diplosis mosellana, Meromyza americana, Hvlemya coarc catum), Corynespora cassicola, Septoria glycines, Phyllos tata, Frankliniella fusca, Cephus cinctus, Aceria tulipae, ticta sojicola, Alternaria alternata, Pseudomonas syringae 55 6. Sunflower: p.V. glycinea, Xanthomonas campestris p.V. phaseoli, Fungal, bacterial or viral pathogens: Plasmophora halste Microsphaera diffussa, Fusarium semitectum, Phialophora dii, Sclerotinia sclerotiorum, Aster Yellows, Septoria gregata, Soybean mosaic virus, soybean rust, Glomerella gly helianthi, Phomopsis helianthi, Alternaria helianthi, Alter cines, tobacco ring spot virus, tobacco streak virus, Phakop naria Zinniae, Botrytis cinerea, Phoma macdonaldii, Macro Sorapachyrhizi, Pythium aphanidermatum, Pythium ulti 60 phomina phaseolina, Erysiphe cichoracearum, Rhizopus mum, Pythium debaryanum, tomato spotted wilt virus, Oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia Heterodera glycines Fusarium Solani; particularly soybean helianthi, Verticillium dahliae, Erwinia carotovorum p.V. ruSt. Carotovora, Cephalosporium acremonium, Phytophthora Pathogenic insects/nematodes: Pseudoplusia includens, Cryptogea, Albugo tragopogonis. Anticarsia gemmatalis, Plathylpena scabra, Ostrinia nubila 65 Pathogenic insects/nematodes: Suleima helianthana, lis, Agrotis ipsilon, Spodoptera exigua, Heliothis virescens, Homoeosoma electellum; Zygogramma exclamationis. Both Helicoverpa zea, Epillachna varivestis, Myzus persicae, yrus gibbosus, Neolasioptera murtfeldtiana, US 8,373,022 B2 37 38 7. Maize: 9. Cotton: Fungal, bacterial or viral pathogens: Fusarium monili Pathogenic insects/nematodes: Heliothis virescens, Heli forme var. subglutinans, Erwinia Stewartii, Fusarium monili coverpa zea, Spodoptera exigua, Pectinophora gossypiella, forme, Gibberella zeae (Fusarium graminearum), Stenocar Anthonomus grandis grandis, Aphis gossypii. Pseudato pella maydi (Diplodia maydis), Pythium irregulare, Pythium moscelis seriatus, Trialeurodes abutilonea, Lygus lineolaris, debaryanum, Pythium graminicola, Pythium splendens, Melanoplus femurrubrum, Melanoplus differentialis, Thrips Pythium ultimum, Pythium aphanidermatum, Aspergillus fia tabaci (onion thrips); Franklinkiella fisca, Tetranychus cin vus, Bipolaris maydis 0, T (Cochliobolus heterostrophus), nabarinus, Tetranychus urticae. Helminthosporium carbonum I, II & Ill (Cochliobolus car 10. Rice: bonum), Exserohilum turcicum I, II & Ill, Helminthosporium 10 pedicellatum, Physoderma maydis, Phyllosticta maydis, Pathogenic insects/nematodes: Diatraea saccharalis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Spodoptera frugiperda, Helicoverpa zea, Colaspis brunnea, Puccinia sorghi, Puccinia polysora, Macrophomina phaseo Lissorhoptrus Oryzophilus, Sitophilus Oryzae, Nephotettix lina, Penicillium oxalicum, Nigrospora Oryzae, Cladospo nigropictus, Blissus leucopterus leucopterus, Acrosternum hilare. rium herbarum, Curvularia lunata, Curvularia inaequalis, 15 Curvularia pallescens, Clavibacter michiganese Subsp. 11. Oilseed rape: nebraskense, Trichoderma viride, maize dwarf mosaic virus Pathogenic insects/nematodes: Brevicoryne brassicae, A & B, wheat streak mosaic virus, maize chlorotic dwarf Phyilotrea cruciferae, Mamestra Conigurata, Plutella xylos virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chry tella, Delia ssp. Santherni p.V. Zea, Erwinia corotovora, Cornstunt spiro The processes and methods according to the invention plasma, Diplodia macrospora, Sclerophthora macrospora, firstly relate by preference to Soya, plant parts, cells and/or Peronosclerospora sorghi, Peronosclerospora philippinesis, seed thereof. Thereof. Equally, the processes and methods Peronosclerospora maydis, Peronosclerospora sacchari, according to the invention relate by preference to Soybean Spacelotheca reiliana, Physopella zeae, Cephalosporium ruSt. maydis, Caphalosporium acremonium, maize chlorotic 25 For the purposes of the invention, “BI1 protein’ means mottle virus, high plains virus, maize mosaic virus, maize polypeptides which have at least one sequence with at least rayado fino virus, maize streak virus (MSV), maize stripe 50%, preferably at least 80%, especially preferably at least virus, maize rough dwarf virus. 90%, very especially preferably at least 95% and especially Pathogenic insects/nematodes: Ostrinia nubilalis, Agrotis preferably 100% homology with a BI1 consensus motif ipsilon, Helicoverpa zea, Spodoptera frugiperda, Diatraea 30 grandiosella, Elasmopalpus lignosellus, Diatraea sacchara selected from the group consisting of lis, Diabrotica virgifera, Diabrotica, longicornis barberi; Diabrotica undecimpunctata howardi; Melanotus spp.; (SEQ ID NO: 49) Cyclocephala borealis, Cyclocephala immaculate, Popillia a) H (L/I). KXVY japonica, Chaetocnema pullicaria, Sphenophorus maidis, 35 Rhopalosiphum maidis, Anuraphis maidiradicis, Blissus leu (SEO ID NO : 50) copterus leucopterus, Melanoplus femurrubrum, Melano b) AXGA (Y/F) XH plus sanguinipes, Hvlemva platura, Agromyza. parvicornis, (SEQ ID NO: 51) Anaphothrips obscrurus, Solenopsis milesta, Tetranychus c) NIGG urticae. 40 (SEQ ID NO: 52) 8. Sorghum: d) P (V/P) (Y/F) E (E/O) (R/Q) KR Fungal, bacterial or viral pathogens: Exserohilum turci cum, Colletotrichum graminicola (Glomerella graminicola), (SEO ID NO : 53) Cercospora sorghi, Gloeocercospora sorghi, Ascochyta e) (EAQ) G (A/S) S (WAI) GPL Sorghina, Pseudomonas Syringae p.V. syringae, Xanthomo 45 (SEQ ID NO: 54) nas Campestris p.V. holcicola, Pseudomonas andropogonis, f) DP (SAG) (LAI) (IAL) Puccinia purpurea, Macrophomina phaseolina, Perconia cir cinata, Fusarium monilifonne, Alternaria alternate, Bipo (SEO ID NO : 55) laris sorghicola, Helminthosporium sorghicola, Curvularia g) V (G/A) T (A/S) (L/I)AF (A/G) CF (S/T) lunata, Phoma insidiosa, Pseudomonas a venae (Pseudomo 50 (SEO ID NO. 56) nas alboprecipitans), Ramulispora sorghi, Ramulispora h) YL (YAF) LGG, Sorghicola, Phyllachara sacchari, Sporisorium reilianum (SEO ID NO : 57) (Sphacelotheca reiliana), Sphacelotheca Cruenta, Sporiso preferably EYLYLGG rium sorghi, Sugarcane mosaic H. maize dwarf mosaic virus A & 8, Claviceps sorghi, Rhizoctonia Solani, Acremonium 55 (SEO ID NO. 58) strictum, Sclerophthona macrospora, Peronosclerospora i) L (LAW) SS (GAW) L (SAT) (IAM) L (LAM)W Sorghi, Peronosclerospora philippinensis, Sclerospora (SEO ID NO. 59) graminicola, Fusarium graminearum, Fusarium oxysporum, j) DTGX (I/V) (I/V) E. Pythium arrhenomanes, Pythium graminicola. Pathogenic insects/nematodes: Chilo partellus, 60 Especially preferred in this context is the BI consensus Spodoptera frugiperda, Helicoverpa zea, Elasmopalpus motif f) YL(Y/F)LGG (SEQ ID NO: 56), very especially lignosellus, Feltia subterranea, Phyllophaga crimita, Ele preferred is (EYLYLGG, SEQ ID NO: 57). This motif is Odes, Conoderus und Aeolus spp., Oulema melanopus, Cha characteristic for plant BI1 proteins. Sequences with homol etocnema pulicaria, Sphenophorus maidis, Rhopalosiphun ogy to at least 2 or 3 of these motifs (a to ) are especially maidis, Siphaflava, Blissus leucopterus eucopterus, Con 65 preferably found in a BI1 protein, very especially preferably tarinia sorghicola, Tetranychus cinnabarinus, Tetranychus at least 4 or 5, most preferably all motifs a to j. Further urticae. BI1-typical sequence motifs can be derived by the skilled US 8,373,022 B2 39 40 worker without difficulty from the sequence alignment of BI1 nucleic acid sequence over in each case the entire sequence proteins as shown in FIG. 1 or 6. length which, in turn, is calculated by alignment with the aid Especially preferred for the use in the methods disclosed of the program algorithm GAP ( Package Version herein are BI1 proteins which are encoded by a polypeptide 10.0, University of Wisconsin, Genetics Computer Group which comprises at least one sequence selected from the (GCG), Madison, USA; Altschul at al., Nucleic Acids Res. group consisting of 25, 3389 (1997)) setting the following parameters: a) the sequences as shown in SEQID NO: 2, 4, 6, 8, 10.12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 38 or 46; b) sequences with at least 50%, more preferably 60%, 70%, 80%, 85% or 90%, especially preferably 95, 97 or 99% or 10 Gap weight:50 Length weight: 3 more identity with one of the sequences as shown in SEQ Average match: 10 Average mismatch: O ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 38 or 46; and For example a sequence which has at least 80% homology c) sequences which comprise at least one part-sequence of at with sequence SEQ ID NO: 1 at the nucleic acid level is least 10 contiguous amino acid residues of one of the 15 understood as mening a sequence which, upon alignment sequences as shown in SEQID NO: 2, 4, 6, 8, 10, 12, 14, with the sequence SEQ ID NO: 1 by the above program 16, 18, 20, 22, 24, 26, 28, 30, 32, 38 or 46, and/or which algorithm with the above parameter set, has at least 80% comprise at least a part-sequence of at least 20 contiguous homology. amino acid residues, where the part-sequence has at 80%, Homology between two polypeptides is understood as preferably 85% or 90%, especially preferably 95, 97 or meaning the identity of the amino acid sequence over in each 99% or more identity with the corresponding part-se case the entire sequence length which is calculated by align quence from one of the sequences as shown in SEQID NO: ment with the aid of the program algorithm GAP (Wisconsin 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 38 Package Version 10.0, University of Wisconsin, Genetics or 46. Computer Group (GCG), Madison, USA), setting the follow Comprised in accordance with the invention by the term BI 25 ing parameters: protein are in particular natural or artificial mutations of the BI1 polypeptides as shown in SEQID NO: 2, 4, 6, 8, 10, 38 and in particular 46, and homologous or similar polypeptides from other organisms, preferably plants, which furthermore Gap weight: 8 Length weight: 2 Average match: 2.912 have essentially identical properties. Mutations comprise 30 Average mismatch: -2003 Substitutions, additions, deletions, inversions or insertions of one or more amino acid residues. For example a sequence which has at least 80% homology Also comprised are thus use forms utilizing BI1 proteins with sequence SEQ ID NO: 2 at the protein level is under from nonplant organisms such as for example humans (Gen stood as meaning a sequence which, upon comparison with BankAcc.-No.: P55061), rat (GenBankAcc.-No.: P55062) or 35 the sequence SEQID NO: 2 by the above program algorithm Drosophila (GenBank Acc.-No.: Q9VSH3). Motifs which with the above parameter set, has at least 80% homology. are conserved between plant and nonplant BI1 proteins can be BI1 proteins also comprise those polypeptides which are identified readily by sequence alignments (cf. alignment in encoded by nucleic acid sequences which hybridize under Bolduc N. et al., Planta 216,377 (2003); FIGS. 1 and 6), standard conditions with one of the BI1 nucleic acid Thus, the present invention also comprises for example 40 sequences described by SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, those polypeptides which are obtained by modification of a 17, 19, 21, 23, 25, 27, 29, 31, 37 and 45, the nucleic acid polypeptideas shown in SEQID NO: 2, 4, 6, 8, 10,38 and 46. sequence which is complementary thereto or parts of the The sequences from other plants which are homologous to above, and which have essentially identical properties as the the BI1 sequences disclosed within the scope of the present proteins described under SEQID NO:2, 4, 6, 8, 10,38 and 46. invention can be found for example by 45 “Standard hybridization conditions” is to be understood in a) database search in libraries of organisms whose genomic or the broad sense and means stringent or else less stringent cDNA sequence is known in full or in part, using the BI1 hybridization conditions. Such hybridization conditions are sequences provided as search sequence, or described, interalia, by Sambrook.J., Fritsch E. F., Maniatis T. b) screening gene libraries or cDNA libraries using the BI1 et al., in Molecular Cloning (A Laboratory Manual), 2nd sequences provided as probe. 50 Edition, Cold Spring Harbor Laboratory Press, 1989, pages Screening cDNA libraries or genomic libraries (for 9.31-9.57) or in Current Protocols in Molecular Biology, John example using one of the nucleic acid sequences described Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the under SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, conditions during the wash step can be selected from the 27, 29, 31, 37 and 45 or parts of these as probe) is a method of range of conditions delimited by low-stringency conditions identifying homologous or similar or identical sequences, 55 (approximately 2xSSC at 50° C.) and high-stringency condi which method is known to the skilled worker. In this context, tions (approximately 0.2xSSC at 50° C., preferably at 65° C.) the probes derived from the nucleic acid sequences as shown (20xSSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). In addi in SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, tion, the temperature during the wash step can be raised from 29, 31, 37 and 45 have a length of at least 20 bp, preferably at low-stringency conditions at room temperature, approxi least 50 bp, especially preferably at least 100 bp, very espe 60 mately 22°C., to higher-stringency conditions at approxi cially preferably at least 200 bp, most preferably at least 400 mately 65° C. Both of the parameters salt concentration and bp. A DNA strand which is complementary to the sequences temperature can be varied simultaneously, or else one of the described under SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, two parameters can be kept constant while only the other is 21, 23, 25, 27, 29, 31, 37 and 45 may also be employed for varied. Denaturants, for example formamide or SDS, may screening the libraries. 65 also be employed during the hybridization. In the presence of Homology between two nucleic acid sequences is under 50% formamide, hybridization is preferably effected at 42 stood as meaning, in the present context, the identity of the C. US 8,373,022 B2 41 42 “Essential properties' means, with regard to a B1 protein, context, the increase amounts to at least 10%, preferably at one or more of the following properties: least 30% or at least 50%, especially preferably at least 70% a) Conferring or increasing the pathogen resistance to at least or 100%, very especially preferably at least 200% or 500%. one pathogen while increasing the protein quantity or func most preferably at least 1000%. Methods of increasing the tion of said BI protein in at least one tissue of the plant, function comprise, beside the above-described method of preferably at least in the epidermis of the plant. increasing the protein quantity (which, as a rule, also b) Nonappearance of a spontaneously induced cell death increases the function) furthermore—by way of example, but when increasing the protein quantity or the function of said not by limitation in particular the introduction of mutations BI protein. into a BI1 protein or the inhibition of a putative BI1 inhibitor, c) The property of significantly inhibiting the BAX-induced 10 and the like. apoptosis in the case of transient cotransfection of Bax The BI1 protein quantity can be increased for example, but with said BI1 protein, for example in HEK293 cells. Suit not by limitation, by one of the following methods: able methods have been described (Bolduc N. et al. Planta a) recombinant expression or overexpression of a BI1 protein 216,377 (2003)). by introducing a recombinant expression cassette compris d) The presence of five to seven putative transmembrane 15 ing a nucleic acid sequence coding for a BI1 protein under domains within said BI1 protein. the control of a tissue-specific promoter, where said pro e) Preferential localization in cell membranes, in particular moter has activity preferably essentially specifically in the the nuclear membrane, the ER membrane and/or the thy leaf epidermis and/or no activity in the mesophyll. lakoid membrane. b) modification (for example substitution) of the regulatory In this context, the quantitative manifestation of said prop regions (for example the promoter region) of an endog erties of a BI1 protein may deviate up or down in comparison enous BI1 gene, for example Substitution for a tissue-spe with the value obtained for the BI1 protein as shown in SEQ cific promoter by means of homologous recombination, ID NO: 2, 4, 6, 8, 10, 38 or 46. where said promoter has activity preferably essentially For the purposes of the present invention, the term specifically in the leaf epidermis and/or no activity in the “increasing the BI1 protein quantity or function' is to be 25 mesophyll. understood in the broad sense and may be based on different c) insertion of a nucleic acid sequence, coding for a BI1 cell-biological mechanisms. protein, into the plant genome downstream of a tissue “Protein quantity” means the amount of a BI1 protein in the specific promoter by means of homologous recombination, organism, tissue, cell or cell compartment detailed. where said promoter has activity preferably essentially “Increasing the protein quantity” means the quantitative 30 specifically in the leaf epidermis and/or no activity in the increase of the amount of a BI1 protein in the organism, mesophyll. tissue, cell or cell compartment detailed, for example by d) increasing the expression of an endogenous BI1 protein by means of one of the methods described hereinbelow, in com introducing a transcription factor (for example artificial parison with the wild type of the same genus and species, to transcription factor from the class of the Zinc finger pro which this method has not been applied, but on the otherwise 35 teins) which is suitable for inducing the expression of said identical conditions (such as, for example, culture conditions, BI1 protein. Preferred is the introduction of a recombinant age of the plants and the like). In this context, the increase expression cassette comprising a nucleic acid sequence amounts to at least 10%, preferably at least 30% or at least coding for said transcription factor under the control of a 50%, especially preferably at least 70% or 100%, very espe tissue-specific promoter, where said promoter has activity cially preferably at least 200% or 500%, most preferably at 40 preferably essentially specifically in the leaf epidermis least 1000%. The protein quantity can be determined by and/or no activity in the mesophyll. means of a variety of methods with which the skilled worker For the purposes of the present invention, the term “to is familiar. Examples which may be mentioned, but not by introduce/introduction' generally comprises all methods limitation, are the micro-biuret method (Goa.J., Scand.J. Clin. which are suitable for transferring the compound to be intro Lab. Invest. 5, 218 (1953)), the Folin-Ciocalteu method 45 duced, either directly or indirectly, into a plant or into a cell, (Lowry O. H. et al., J Biol Chem 193, 265 (1951)) or the compartment, tissue, organ or seed thereof, or generating it measurement of the adsorption of CBB G-250 (Bradford M. therein. This comprises direct and indirect methods. The M., Analyt. Biochem. 72,248 (1976)). Furthermore, a quan introduction can lead to a transient presence of said com tification can be accomplished via immunological methods pound or else to a stable or inducible presence. “Introducing such as, for example, Western blot The generation of suitable 50 comprises for example methods such as transfection, trans BI1 antibodies and the procedure of BI1 Western blots is duction or transformation. described (Bolduc N. et al., FEBS Lett532, 111 (2002)). An In the recombinant expression cassettes which are indirect quantification can be accomplished via Northern employed within the scope of the present invention, a nucleic blots, where, as a rule, the mRNA quantity correlates well acid molecule (for example coding for a BI1 protein) is in with the resulting protein quantity. Suitable methods have 55 operable linkage with at least one tissue-specific promoter, been described (Bolduc N. et al., Planta 216, 377 (2003); where said promoter has activity preferably essentially spe Matsumura H. et al., Plant J. 33,425 (2003)). cifically in the leaf epidermis and/or no activity in the meso “Function' preferably means the property of a BI1 protein phyll, and where the promoter is heterologous with regard to of reducing the spontaneously induced cell death and/or the the nucleic acid sequence to be expressed, i.e. does not natu property of inhibiting the apoptosis-inducing effect of Bax. 60 rally occur in combination with same. The recombinant Such functions are among the essential properties of a BI1 expression cassettes according to the invention may option protein. ally comprise further genetic control elements. Within the scope of the present invention, “increasing the Operable linkage is to be understood as meaning, for function means, for example, the quantitative increase of the example, the sequential arrangement of said promoter with inhibitor effect on the Bax-induced apoptotic cell death, 65 the nucleic acid sequence to be expressed and, if appropriate, which can be determined quantitatively by methods with further regulatory elements such as, for example, a terminator which the skilled worker is familiar (see herein above). In this in Such away that each of the regulatory elements can fulfil its US 8,373,022 B2 43 44 function when the nucleic acid sequence is expressed recom sible an increased recombinant expression of the nucleic acid binantly. To this end, direct linkage in the chemical sense is sequence. Additional advantageous sequences, such as fur not necessarily required. Genetic control sequences such as, ther regulatory elements or terminators, may also be inserted for example, enhancer sequences, can also exert their func at the 3' end of the nucleic acid sequences to be expressed tion on the target sequence from positions which are further recombinantly. One or more copies of the nucleic acid away, or indeed from other DNA molecules. sequences to be expressed recombinantly may be present in Preferred arrangements are those in which the nucleic acid the gene construct. sequence to be expressed recombinantly is positioned down Polyadenylation signals which are suitable as control stream of the sequence acting as promoter, so that the two sequences are plant polyadenylation signals, preferably those sequences are linked covalently to each other. The distance 10 which essentially correspond to T-DNA polyadenylation sig between the promoter sequence and the nucleic acid sequence nals from Agrobacterium tumefaciens, in particular the OCS to be expressed recombinantly is preferably less than 200 (octopin synthase) terminator and the NOS (nopalin syn base pairs, especially preferably less than 100 base pairs, very thase) terminator. especially preferably less than 50 base pairs. Operable link Control sequences are furthermore to be understood as age, and a recombinant expression cassette, can be generated 15 those which make possible homologous recombination or by means of customary recombination and cloning tech insertion into the genome of a host organism or which permit niques as are described above. However, further sequences removal from the genome. In the case of homologous recom which, for example, act as a linker with specific cleavage sites bination, for example the natural promoter of a BI1 gene may for restriction enzymes, or as a signal peptide, may also be be exchanged for one of the preferred tissue-specific promot positioned between the two sequences. The insertion of ers. Methods such as the cre/lox technology permit a tissue sequences may also lead to the expression of fusion proteins. specific, if appropriate inducible, removal of the recombinant Preferably, the expression cassette, consisting of a linkage expression cassette from the genome of the host organism of promoter and nucleic acid sequence to be expressed, can (Sauer B. Methods. 14(4), 381 (1998)). In this method, spe exist in a vector-integrated form and be inserted into a plant cific flanking sequences (lox sequences), which later allow genome, for example by transformation. 25 removal by means of cre recombinase, are attached to the However, a recombinant expression cassette also denotes target gene. those constructions in which the promoter is positioned A recombinant expression cassette and the vectors derived upstream of an endogenous BI1 gene, for example by means from it may comprise further functional elements. The term of homologous recombination, thus controlling the expres functional element is to be understood in the broad sense and sion of the BI1 protein. Analogously, the nucleic acid 30 refers to all those elements which have an effect on the gen sequence to be expressed (for example coding for a BI1 eration, amplification or function of the recombinant expres protein) can be placed downstream of an endogenous pro sion cassettes, vectors or recombinant organisms according to moter in such a way that the same effect is manifested. Both the invention. The following may be mentioned by way of approaches lead to inventive recombinant expression cas example, but not by limitation: SetteS. 35 a) Selection markers which confer a resistance to a metabo By “tissue-specific promoter with activity essentially spe lism inhibitor such as 2-deoxyglucose-6-phosphate (WO cifically in the leaf epidermis' there are generally to be under 98/45456), antibiotics or biocides, preferably herbicides, stood, for the purposes of the present invention, those pro Such as, for example, kanamycin, G 418, bleomycin or moters which are Suitable of ensuring or increasing hygromycin, or else phosphinothricin and the like. Espe recombinant expression of a nucleic acid sequence in at least 40 cially preferred selection markers are those which confer one plant tissue with the proviso that resistance to herbicides. Examples which may be men a) the expression is manifested at least in the epidermis and tioned are DNA sequences which code for phosphinothri preferably not in the mesophyll, or remains essentially cin acetyltransferases (PAT) and which inactivate glutamin unchanged in the mesophyll, where tissues other than the synthase inhibitors (bar and pat genes), 5-enolpyru two tissues mentioned are not been taken into consider 45 vylshikimate-3-phosphate synthase genes (EPSP synthase ation, and genes), which confer resistance to GlyphosatR) (N- b) the recombinant expression under the control of said pro (phosphonomethyl)glycine), the goX gene, which encodes moter in said plant tissue amounts to at least five times, GlyphosatR)-degrading enzymes (Glyphosate oxidoreduc preferably at least ten times, especially preferably at least tase), the deh gene (encoding a dehalogenase which inac hundred times, the expression of a comparative plant. 50 tivates Dalapon(R), and bXn genes, which encode bro Genetic control sequences furthermore also encompass the moxynil-degrading nitrilase enzymes, the ansa gene, 5'-untranslated regions, introns or noncoding 3'-region of which confers resistance to the antibiotic spectinomycin, genes, such as, for example, the actin-1 intron, or the Adhl-S the streptomycin phosphotransferase (SPT) gene, which introns 1, 2 and 6 (general reference: The Maize Handbook, allows resistance to streptomycin, the neomycin phospho Chapter 116, Freeling and Walbot, Eds. Springer, N.Y. 55 transferase (NPTII) gene, which confers resistance to (1994)). It has been demonstrated that they may play a sig kanamycin or geneticidin, the hygromycin phosphotrans nificant role in the regulation of gene expression. Thus, it has ferase (HPT) gene, which mediates resistance to hygromy been demonstrated that 5'-untranslated sequences can cin, the acetolactate synthase gene (ALS), which confers enhance the transient expression of heterologous genes. resistance to sulfonylurea herbicides (for example mutated Examples of translation enhancers which may be mentioned 60 ALS variants with, for example, the S4 and/or Hra muta are the tobacco mosaic virus 5' leader sequence (Gallie et al., tion), and the acetolactate synthase gene (ALS), which Nucl. Acids Res. 15,8693 (1987)) and the like. Furthermore confers resistance to imidazolinone herbicides. they may promote tissue specificity (Raster J. et al., Plant J 15, b) Reporter genes which code for readily quantifiable pro 435 (1998)). teins and, via their color or enzyme. activity, make possible The recombinant expression cassette may advantageously 65 an assessment of the transformation efficacy, the site of comprise one or more of what. are known as enhancer expression or the time of expression. Very especially pre sequences, linked operably to the promoter, which make pos ferred in this context are genes coding for reporter proteins US 8,373,022 B2 45 46 (Schenborn E. Groskreutz D., Mol. Biotechnol. 13(1), 29 by Kung S.D. and Wu R, Academic Press, p. 128-143 (1993), (1999)) such as the green fluorescent protein (GFP) (Sheen and in Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. et al. Plant Journal 8(5), 777 (1995); Heseloff et al., Proc. 42, 205 (1991)). Natl. Acad. Sci. USA 94(6), 2122 (1997); Reichel et al., For example, the DNA or RNA can be introduced directly Proc. Natl. Acad. Sci. USA93(12),5888 (1996); Tianet al., by microinjection or by bombardment with DNA-coated Plant Cell Rep. 16, 267 (1997); WO 97/41228; Chui W. L. microparticles. Also, the cell can be permeabilized chemi et al., Curr. Biol. 6, 325 (1996); Leffel S. M. et al., Bio cally, for example using polyethylene glycol, so that DNA can techniques 23(5), 912 (1997)), chloramphenicol trans enter the cell by diffusion. The DNA can also be introduced ferase, a luciferase (Ow et al., Science 234,856 (1986); by protoplast fusion with other DNA-containing units such as Millar et al., Plant Mol. Biol. Rep. 10, 324 (1992)), the 10 aequorin gene (Prasher et al., Biochem. BiophyS. Res. minicells, cells, lysosomes or liposomes. Another Suitable Commun. 126(3), 1259 (1985)), B-galactosidase, R locus method of introducing DNA is electroporation, where the gene (encoding a protein which regulates the production of cells are permeabilized reversibly by an electrical pulse. Suit anthocyanin pigments (red coloring) in plant tissue and able methods have been described (for example by Bilang et thus makes possible the direct analysis of the promoter 15 al., Gene 100,247 (1991); Scheidetal, Mol. Gen. Genet. 228, activity without addition of further auxiliary substances or 104 (1991); Guerche et al., Plant Science 52, 111 (1987); chromogenic Substrates; Dellaporta et al. in Chromosome Neuhause et al., Theor. Appl. Genet. 75, 30 (1987); Kleinet Structure and Function: Impact of New Concepts, 18th al., Nature 327, 70 (1987); Howell et al., Science 208, 1265 Stadler Genetics Symposium, 11 (1988)), with B-glucu (1980); Horsch et al., Science 227, 1229 (1985): DeBlocket ronidase being very especially preferred (Jefferson et al., al., Plant Physiol 91,694 (1989)). EMBO.J. 6,3901 (1987)). In plants, the above-described methods of transforming c) Origins of replication, which ensure amplification of the and regenerating plants from plant tissues or plant cells are recombinant expression cassettes or vectors according to exploited for transient or stable transformation. Suitable the invention in for example, E. coli. Examples which may methods are especially protoplast transformation by polyeth be mentioned are ORI (origin of DNA replication), the 25 ylene-glycol-induced DNA uptake, the biolistic method with pBR322 ori or the P15A ori (Sambrook et al., Molecular the gene gun, what is known as the particle bombardment Cloning. A Laboratory Manual, 2nd ed. Cold Spring Har method, electroporation, incubation of dry embryos in DNA bor Laboratory Press, Cold Spring Harbor, N.Y., 1989). containing solution, and microinjection. d) Elements which are necessary for Agrobacterium-medi In addition to these “direct transformation techniques, ated plant transformation, Such as, for example, the right or 30 transformation can also be effected by bacterial infection by left border of the T-DNA or the vir region. means of Agrobacterium tumefaciens or Agrobacterium To select cells which have successfully undergone homolo rhizogenes. The Agrobacterium-mediated transformation is gous recombination, or else to select transformed cells, it is, best suited to dicotyledonous plant cells. The methods are as a rule, necessary additionally to introduce a selectable described, for example, by Horsch R. B. et al., Science 225, marker which confers resistance to a biocide (for example 35 1229 (1985). herbicide), a metabolism inhibitor Such as 2-deoxyglucose When agrobacteria are used, the expression cassette must 6-phosphate (WO 98/45456) or an antibiotic to the cells be integrated into specific plasmids, either into a shuttle or which have Successfully undergone recombination. The intermediate vector, or into a binary vector. If a Ti or Ri selection marker permits the selection of the transformed plasmid is to be used for the transformation, at least the right cells from untransformed ones (McCormicket al., Plant Cell 40 border, but in most cases the right and left border, of the Tior Reports 5, 81 (1986)). Ri plasmid T-DNA is linked to the expression cassette to be The introduction of a recombinant expression cassette introduced in the form of a flanking region. according to the invention into an organism or cells, tissues, Binary vectors are preferably used. Binary vectors are organs, parts or seeds thereof (preferably into plants or plant capable of replication both in E. coli and in Agrobacterium. cells, tissue, organs, parts or seeds) can be effected advanta 45 As a rule, they comprise a selection marker gene and a linker geously using vectors which comprise the recombinant or polylinker flanked by the right and left T-DNA border expression cassettes. The recombinant expression cassette sequence. They can be transferred directly into Agrobacte can be introduced into the, vector (for example a plasmid) via rium (Holsters et al., Mol. Gen. Genet. 163, 181 (1978)). The a suitable restriction cleavage site. The plasmid formed is first selection marker gene permits a selection of transformed introduced into E. coli. Correctly transformed E. coli are 50 agrobacteria and is, for example, the nptII gene, which con selected, grown, and the recombinant plasmid is obtained by fers resistance to kanamycin. The agrobacterium which acts the methods familiar to the skilled worker. Restriction analy as host organism in this case should already contain a plasmid sis and sequencing may serve to verify the cloning step. with the vir region. The latter is required for transferring the Examples of vectors may be plasmids, cosmids, phages, T-DNA to the plant cell. An agrobacterium transformed in viruses or else agrobacteria. In an advantageous embodiment, 55 this way can be used for transforming plant cells. The use of the expression cassette is introduced by means of plasmid T-DNA for transforming plant cells has been studied and vectors. Preferred vectors are those which make possible described intensively (EP 120516; Hoekema in The Binary stable integration of the recombinant expression cassette into Plant Vector System, Offsetdrukkerij Kanters B. V., the host genome. Alblasserdam, Chapter V: An et al., EMBO.J. 4, 277 (1985)). The generation of a transformed organism (or of a trans 60 Various binary vectors are known, some of which are com formed cell or tissue) requires introducing the DNA, RNA or mercially available such as, for example, pBI101.2 or protein in question into the relevant host cell. pBIN19 (Bevan et al., Nucl. Acids Res. 12, 8711 (1984): A plurality of methods are available for this procedure, Clontech Laboratories, Inc. USA). Further promoters which which is referred to as transformation (or transduction or are suitable for expression in plants have been described transfection) (Keown et al., Methods Enzymol. 185, 527 65 (Rogers et al., Methods Enzymol. 153,253 (1987); Schardlet (1990); Jenes B. et al., Techniques for Gene Transfer, in: al., Gene 61, 1 (1987); Berger et al., Proc. Natl. Acad. Sci. Transgenic Plants, Vol. 1, Engineering and Utilization, edited USA 86, 8402 (1989)). US 8,373,022 B2 47 48 Direct transformation techniques are suitable for any “artificial methods such as, for example, mutagenization. organism and cell type. The plasmid used need not meet any Such methods have been described (U.S. Pat. No. 5,565,350; particular requirements in the case of the injection or elec WO 00/15815; also see above). troporation of DNA or RNA into plant cells. Simple plasmids The invention also relates to recombinant organisms trans such as those of the puC series may be used. If complete formed with at least one of the nucleic acid sequences accord plants are to be regenerated from the transformed cells, it is ing to the invention, expression cassette according to the necessary for an additional selectable marker gene to be invention or vector according to the invention, and to cells, located on the plasmid. cell cultures, tissues, parts—such as, for example, leaves, Stably transformed cells, i.e. those which comprise the roots and the like in the case of plant organisms—or propa introduced DNA integrated into the DNA of the host cell, can 10 gation material derived from Such organisms. The term organ be selected from untransformed cells when a selectable ism is to be understood in the broad sense and refers to marker is part of the DNA introduced. Examples of genes prokaiyotic and eukaryotic organisms, preferably bacteria, which can act as markers are all those which are capable of yeasts, fungi, animal organisms and plant organisms. Host or conferring resistance to antibiotics or herbicides (such as starting organisms which are preferred as recombinant organ kanamycin, G 418, bleomycin, hygromycin or phosphino 15 isms are mainly plants in accordance with the above defini thricin) (see above). Transformed cells which express such tion. marker genes are capable of Surviving in the presence of The invention furthermore relates to the use of the recom concentrations of a corresponding antibiotic or herbicide binant organisms according to the invention and of the cells, which kill an untransformed wild type. Examples of suitable cell cultures, parts—such as for example, roots, leaves and the selection markers are mentioned above. As soon as a trans like in the case of recombinant plant organisms—derived formed plant cell has been generated, a complete plant can be from them, and to recombinant propagation material Such as obtained by using methods known to the skilled worker. Start seeds or fruits, for the production of foodstuffs or feeding ing material in this context is, for example, callus cultures. stuffs, pharmaceuticals or fine chemicals. The development of shoot and root and can be induced in the Furthermore is a nucleic acid molecule which is antisense known manner in these as yet undifferentiated cell biomasses. 25 to the nucleic acid according to the invention, a monoclonal The plantlets obtained can be grown on and bred. The skilled antibody which binds specifically to the polypeptide accord worker is familiar with methods of regenerating plant parts of ing to the invention, and a fungicide which comprises the entire plants starting from plant cells. For example, methods nucleic acid according to the invention, the vector according described by Fennell et al., Plant Cell Rep. 11, 567 (1992); to the invention, in particular an infectious, for example viral, Stoeger et al., Plant Cell Rep. 14, 273 (1995); Jahne et al., 30 vector according to the invention, the polypeptide according Theor. Appl. Genet. 89, 525 (1994) are used in this context. to the invention in a form which is suitable for application to The plants obtained can be bred and/or hybridized in the plants, for example in encapsulated form or in an infectious customary manner. Two or more generations should be grown organism which is preferably suitable for transferring nucleic in order to ensure that the genomic integration is stable and acids or for expressing genes in a cell. Such as an agrobacte hereditary. 35 rium or a virus. The method according to the invention can advantageously In one embodiment, the invention relates to the use of a be combined with further methods which bring about patho BI-1 encoding nucleic acid molecule or of a BI-1 protein for gen resistance (for example to insects, fungi, bacteria, nema the generation of a pathogen-resistant plant, preferably for the todes and the like), stress resistance or another improvement generation of a fungus-resistant plant or for the generation of of the plant properties. Examples are mentioned, inter &la, by 40 a fungicide which brings this about, or for controlling or Dunwell J. M., J. Exp. Bot. 51 (Spec No), 487 (2000). treating plants which are attacked, or liable to be attacked, by With regard to, for example a nucleic acid sequence, an pathogens. expression cassette or a vector comprising said nucleic acid Sequences sequence or an organism transformed with said nucleic acid 1. SEQ ID NO: 1: Nucleic acid sequence coding for a BI1 sequence, expression cassette or vector, the term “recombi 45 protein from barley (Hordeum vulgare). nant’ means all those constructs which are the result of 2. SEQ ID NO: 2: Amino acid sequence coding for a BI1 recombinant methods and in which either protein from barley (Hordeum vulgare). a) the BI1 nucleic acid sequence or 3. SEQ ID NO:3: Nucleic acid sequence coding for a BI1 b) a genetic control sequence, for example promoter, which is protein from Arabidopsis thaliana. operably linked with the BI1 nucleic acid sequence, or 50 4. SEQ ID NO: 4: Amino acid sequence coding for a BI1 c) (a) and (b) protein from Arabidopsis thaliana. are not located in their natural genetic environment or have 5. SEQ ID NO 5: Nucleic acid sequence coding for a BI1 been modified by recombinant methods, an example of the protein from tobacco. modification being a substitution, addition, deletion, inver 6. SEQ ID NO: 6: Amino acid sequence coding for a BI1 sion or insertion of one or more nucleotide residues. Natural 55 protein from tobacco. genetic environment refers to the natural chromosomal locus 7. Nucleic acid sequence coding for a BI1 protein from rice. in the organism of origin, or to the presence in a genomic 8. Amino acid sequence coding for a BI1 protein from rice. library. In the case of a genomic library, the natural genetic 9. SEQ ID NO: 9: Nucleic acid sequence coding for a BI1 environment of the nucleic acid sequence is preferably protein from oil seed rape. retained, at least in part. The environment flanks the nucleic 60 10. SEQID NO: 10: Amino acid sequence coding for a BI1 acid sequence at least at one side and has a sequence of at least protein from oil seed rape. 50 bp, preferably at least 500 bp, especially preferably at least 11. SEQID NO: 11: Nucleic acid sequence coding for part of 1000 bp, very especially preferably at least 5000 bp, in length. a BI1 protein from soybean. A naturally occurring expression cassette—for example the 12. SEQID NO: 12: Amino acid sequence coding for part of naturally occurring combination of the BI1 promoter with the 65 a BI1 protein from soybean. corresponding BI1 gene—becomes a recombinant expres 13. SEQID NO: 13: Nucleic acid sequence coding for part of sion cassette when it is modified by non-natural, synthetic a BI1 protein from soybean. US 8,373,022 B2 49 50 14. SEQID NO: 14: Amino acid sequence coding for part of 47. SEQID NO: 47: GFP primer 1 (see herein below) a BI1 protein from soybean. 48. SEQID NO: 48: GFP primer 2 (see herein below) 15. SEQID NO: 15: Nucleic acid sequence coding for part of a BI1 protein from wheat. EXAMPLES 16. SEQID NO: 16: Amino acid sequence coding for part of 5 a BI1 protein from wheat. General Methods: 17. SEQID NO: 17: Nucleic acid sequence coding for part of The chemical synthesis of oligonucleotides can be a BI1 protein from maize. effected, for example, in the known fashion using the phos 18. SEQID NO: 18: Amino acid sequence coding for part of phoamidite method (Voet, Voet, 2nd Edition, Wiley Press 10 New York, pages 896-897). The cloning steps carried out for a BI1 protein from maize. the purposes of the present invention Such as, for example, 19. SEQID NO: 19: Nucleic acid sequence coding for part of restriction cleavages, agarose gel electrophoresis, purifica a BI1 protein from wheat. tion of DNA fragments, transfer of nucleic acids to nitrocel 20. SEQID NO:20: Amino acid sequence coding for part of lulose and nylon membranes, linking DNA fragments, trans a BI1 protein from wheat. 15 formation of E. coli cells, bacterial cultures, phage 21. SEQID NO: 21: Nucleic acid sequence coding for part of multiplication and sequence analysis of recombinant DNA, a BI1 protein from maize. are carried out as described by Sambrook et al. Cold Spring 22. SEQID NO: 22: Amino acid sequence coding for part of Harbor Laboratory Press (1989), ISBN 0-87969-309-6. The a BI1 protein from maize. sequencing of recombinant DNA molecules is carried out 23. SEQID NO: 23: Nucleic acid sequence coding for part of with an MWG-Licor laser fluorescence DNA sequencer fol a BI1 protein from maize. lowing the method of Sanger (Sanger et al., Proc. Natl. Acad. 24. SEQID NO: 24: Amino acid sequence coding for part of Sci. USA 74,5463 (1977)). a BI1 protein from maize. 25. SEQID NO: 25: Nucleic acid sequence coding for part of Example 1 a BI1 protein from wheat. 25 26. SEQID NO: 26: Amino acid sequence coding for part of Inoculation with P. pachyrhizi a BI1 protein from wheat. 27. SEQID NO: 27: Nucleic acid sequence coding for part of Suitable spore material (Uredospores) of the pathogen a BI1 protein from maize. Phakopsora pachyrhizi was obtained from BASF Aktieng 28. SEQID NO: 28: Amino acid sequence coding for part of 30 esellschaft. To this end, spores of soybean plants which had a BI1 protein from maize. been inoculated 2-3 weeks earlier were used directly or 29. SEQ ID NO 29: Nucleic acid sequence coding for the shaken onto aluminum foil and stored in the dark at room patatin promote from potato. temperature on a desiccant (TROPAGel, Tropack, Lahnau). 30. SEQ ID NO:30: Nucleic acid sequence coding for the To prepare a spore suspension, the spores were washed Germin 9f-3.8 promoter from wheat. 35 from the leaves with Tween/HO (0.1%). The spores were 31. SEQ ID NO:31: Nucleic acid sequence coding for the counted using a Thoma hematocytometer under the light Arabidopsis CAB-2 promoter microscope. To carry out the inoculation, the leaves were 32. SEQ ID NO:32: Nucleic acid sequence coding for the fixed on 1% H2O agar using a metal ring. Various methods PPCZm1 promoter from maize. were finally used for the inoculation. For the spray inocula 33. SEQ ID NO:33: Nucleic acid sequence coding for the 40 tion, the spore suspension was placed into a spray bottle recombinant expression vector pubiBI-1 operated with pressurized air and were distributed uniformly 34. SEQ ID NO:34: Nucleic acid sequence coding for the on the plants until the upper surface of the leaf was thoroughly recombinant expression vector plo114UbiBI-1 moistened. To obtain a higher spore density, leaves which had 35. SEQ ID NO:35: Nucleic acid sequence coding for the previously been sprayed with Tween/HO were additionally recombinant expression vector pCXoBI-1 45 dry-inoculated in a precipitation column. To this end, the 36. SEQ ID NO:36: Nucleic acid sequence coding for the column is placed over the plates with the moistened leaves. recombinant expression vector plo114OXoBI-1 The young spores of infected Soybean plants are blown into 37. SEQID NO:37: Nucleic acid sequence coding for BI-1 the column via an inlet flap. As the result of the draft, the protein from wheat spores are fluidized, finely distributed, and fall down onto the 38. SEQ ID NO:38: Amino acid sequence coding for BI-1 50 leaves. protein from wheat To obtain higherinoculation densities without spore aggre 39. SEQ ID NO: 39: Nucleic acid sequence for PEN1 gation, the leaves were, as an alternative, covered with a layer (—ROR2) from barley of a spore Suspension and, after 15 minutes, removed from the 40. SEQID NO: 40: Amino acid sequence coding for PEN1 Suspension (layering method). After this time, Sufficient (—ROR2) from barley 55 adhesion of the precipitated spores was, already ensured. The 41. SEQ ID NO: 41: Nucleic acid sequence for PEN1 inoculated leaves were incubated in a chamber with on aver (—ROR2) from Arabidopsis thaliana age 25°C. and an atmospheric humidity of 71.2%. 42. SEQID NO: 42: Amino acid sequence coding for PEN 1 To assess the course of the infection under the microscope, (—ROR2) from Arabidopsis thaliana leaf samples were taken 0 hours post inoculation (hpi), 6 hpi, 43. SEQ ID NO: 43: Nucleic acid sequence coding for 60 24 hpi and 48 hpi, and stained by different methods. SNAP34 from barley a) Coomassie Staining 44. SEQ ID NO: 44: Amino acid sequence coding for To carry out the Coomassie staining, the infected leaf mate SNAP34 from barley rial was harvested and destained overnight at room tempera 45. SEQID NO. 45: Nucleic acid sequence coding for. BI-1 ture. For the microscopy, the leaf material was covered in from Soya 65 Coomassie solution, and the solution was rinsed off after 5 46. SEQ ID NO: 46: Amino acid sequence coding for BI-1 minutes using a little water. The sample was viewed under the from Soya microscope immediately thereafter. US 8,373,022 B2 51 52 b) Calcofluor Staining nol and dried, within a radius of approx. 1 cm. The macro To prepare the Calcofluor staining solution, 0.03% of Cal carriers were then incubated at room temperature until all of cofluor White (Sigma-Aldrich) was dissolved in 50 mM Tris/ the ethanol had evaporated. HCl pH 8.0 nd 0.01% Tween 20. To stain the extracellular The transient biolistic transformation of barley and soya fungal structures, the infected leaf was immersed in the stain leaves was performed using the Biolistic Particle Delivery ing solution for 30 seconds and then washed with water for 10 System PDS-1000/He system (Bio-Rad, Munich). The mate seconds. The stained fungal structures fluoresce pale blue rial used for the transformation was preferably barley leaves under UV excitation. of 7-day old barley plants (cv. Hanna) or the first two leaves c) Aniline Blue Staining (two-leaf stage) of soybean plants (cv. 'Oxford). The leaves 10 to be transformed were placed on 1% (wlv) water agar and The leaf material was transferred into Falcon tubes or fixed using a metal ring. dishes with destaining Solution and incubated overnight at In order to determine the optimum ratio between pressure RT. Thereafter, the destaining solution was removed and the applied and residual pressure in the vacuum chamber for the leaves were washed 2x with water. For the staining, the leaves transformation, various pressure/residual-pressure combina were covered for 1.5-2 h in aniline blue staining solution and 15 tions were tested. Residual pressures of 15-27 inches Hg and Subsequently viewed directly under the microscope. rupture disks rated at 650-1800 psi were used After the bom d) Wheat Germ. Agglutinin Alexa Fluor 488 Staining (WGA bardment, the chamber was ventilated, the carrier together Staining) with the leaves was removed, and the leaves were incubated For the WGA staining of P. pachyrhizi, inoculated barley for at least 24 hours at room temperature before being viewed leaves were placed into 10% (wlv) KOH for 30–45 minutes at under the microscope or stained. RT. Inoculated soybean leaves were dissected into 1 cm Gold particles were used for the ballistic transformation of sections and boiled for 5 minutes in 10% (wlv) KOH. There the leaves. It emerged that bombardment with particles 0.6 after, the barley and the soybean leaves were washed 5 times um in diameter was best Suited to barley since they caused less for 3-5 minutes with 1x PBS buffer. For the staining, the leaf tissue injury. In the case of the transient transformation of material was placed into WGA staining solution and allowed 25 Soybeans, the highest transformation rate was obtained with to infiltrate for 10 minutes under a residual pressure of 100 the 1 um particles. mbar. Thereafter, the material was viewed directly under the For the transient transformation to be successful, the ratios microscope or stored in the staining Solution at 4°C. in the between the pressure for accelerating the particles, the dark. residual pressure in the vacuum chamber and the distance 30 between sample and particles must be adapted to each other. Example 2 To achieve this, various conditions were tested for barley leaves and soybean leaves. In the case of soybean, a pressure Transient Biolistic Transformation of Plant Cells of 900 psi with a residual pressure in the vacuum chamber of 25 inches Hg and a sample distance of 9 cm has proven 35 successful (FIG. 7A). In the case of barley leaves, the highest For the transient overexpresssion of the Bax inhibitor (for cell numbers were obtained with a pressure of 1100 psi, a constructs and other methods, see patent WO 2004/081217) residual pressure in the vacuum chamber of 25 inches Hg and by biolistic transformation, 30-100 mg of gold particles were a distance between leaves and particles of 9 cm (FIG. 7B). weighed, resuspended in 1 ml of 70% strength EtOH and To study the effects of BI-1 on the interaction between P shaken for 3-5 minutes. After incubation for 1 hour at room 40 pachyrhizi and barley, use was made of the transient biolistic temperature, pelletization was effected by centrifugation (1 transformation. The primary leaves of seven-day old barley min, 10000 rpm, Eppendorfmicrocentrifuge). Thereafter, the plants (cv. Hanna) were bombarded with overexpression particles were washed 3x with sterile water and subsequently constructs of BI-1 (pGY1-BI-1, Hickelhoven R. et al., 2001). taken up in sterile 50% (v/v) glycerol and stored at 4° C. These plasmids contain a Camv 35S promoter, which ensures When 30 mg of gold particles were weighed in, the solution 45 constitutive expression of the genes. A CaMV 35S/GFP con contained approximately 25 mg/ml gold. Prior to use the struct as reporter gene was bombarded into the cells together particles were again shaken in order to distribute them thor with the expression plasmids (Schweizer P. et al., MPMI 12, oughly and Sonicated for 15 seconds in a Sonicator. 647 (1999)). The blank vector pGY-1 together with the GFP/ In general, enough gold particles for at least three bom reporter gene construct was used as the control. The inocula bardments were prepared. Up to the precipitation step, the 50 tion with P pachyrhizi was effected 24 hours after the trans mixture was shaken vigorously for a few seconds on a Vortex formation by precipitation from a spore suspension (layering mixer after each step. For the DNA precipitation into gold method, 2-3x10 spores/ml). After incubation for 18 hours, particles for 3 bombardments, one 12.5 ul of the thoroughly the interactions were evaluated under the microscope follow shaken gold particle glycerol solution were removed and ing Calcofluor staining of the extracellular fungal structures. treated with the desired DNA (pGY1-BI-1, pGY1-GFP. 55 The structures counted were GFP-forming cells which had pGY1, see WO 2004/081217). 1.6 ug/ul DNA were employed been penetrated by the fungus and GFP-forming cells which per bombardment on the plasmid. Thereafter, 12.5ul of 2.5M had been attacked by the fungus, but could either successfully CaCl, solution and 5 ul of 0.1 M spermidine solution were defend themselves against it (or where the fungus has died added. The mixture was shaken for 3 minutes, treated with 70 prior to penetration). In addition, the total number of GFP ul of 70% (v/v) ethanol and carefully inverted. After addition 60 forming cells was counted. To carry out the evaluation, the of 70 ul of 100% ethanol, the mixture was again mixed thor relative penetration rates were calculated on the basis of the oughly by inverting and incubated at -20°C. for up to 1 hour. total number of transformed cells which interacted with soy The particles were pelleted by centrifugation (1 min, 11000 bean rust. rpm, Eppendorf microcentrifuge. Weaseling-Berzdorf), In the control, 53% of the transformed cells which inter resuspended in 18 ul of 100% ethanol and distributed as 65 acted with P. pachyrhizi were penetrated (averaged over the uniformly as possible on the macrocarriers (Bio-Rad, experiments), while only 37% of the BI-1-transformed cells Munich), which had previously been washed with 100% etha were penetrated (see Table FIG. 10 and FIG. 8). According to US 8,373,022 B2 53 the evaluation, the penetration resistance of the cells which -continued had been transformed transiently with BI-1 is significantly 5. Elongation 72° C. 5 min increased over P pachyrhizi. Viewing under the microscope 6. Storage 4° C. revealed that the BI-1-forming cells had a markedly more vital appearance than cells which only expressed GFP. Using standard methods, the DNA was separated and ana Example 3 lyzed by gel electrophoresis in a 1% agarose gel (w/v. INVITROGEN, Karlsruhe; in 1x TAE buffer) (Sambrook et Stable Transformation of Barley, and Detection of al., Molecular Cloning: A Laboratory Manual, Cold Spring 10 Harbor Laboratory, Cold Spring Harbor (N.Y.) 1989). the GFP:BI-1 Transgene In a total of six plants out of the transgenic lines #6(2) E15L7P2 (T2) and #6(1)E8L1(T1), it was not possible to To study the effect of BI-1 on interaction of barley with detect the 740 by GFP fragment, and thus the construct. The Phakopsora pachyrhizi in greater detail, stably transformed data of these plants plants 5, 10 and 19 of line #6(1)E8L 1 (T1) barley plants which overexpressed a GFP:BI-1 fusion protein and plant 3, 11 and 12 of line #6(2) E15L7P2 (T2) were were generated. The agrobacterium-mediated stable trans 15 therefore not taken into consideration in the calculations formation used for this purpose is a technique which has been established for years and whose methods have already been which follow (see FIG.9). published in a number of review articles (Cheng Z. et al., Example 4 Plant Mol. Biol. 60, 583 (2004); Taylor et al., DNA & Cell Biology. 21 (12), 963 (2002); Rakoczy-Trojanowska, Cellu Interaction of P. pachyrhizi with Transgenic Bax lar & Molecular Biology Letters. 7(3), 849 (2002); Inhibitor-1 Overexpressing Barley Grabowska A., Acta Physiologiae Plantarum. 26(4), 451 (2004); Chesnokov V. Selskokhozyalistvennaya Biologiya. To verify the resistance-increasing effect as the result of 1, 26 (2004). Even the transformation of monocotyledonous transient overexpression of BI-1, the interaction between P species such as, for example, barley, which was still difficult 25 in the 1990s, is now a standard technique (Travella S. et al., pachyrhizi and transgenic barley plants cv. 'Golden Promise Plant Cell Reports 23(12), 780 (2005); Murray F. et at, Plant (GP), which contain a GFP-BI-1 overexpression construct Cell Reports 22(6), 397 (2004)). was studied under the microscope in comparison with the Since it was not known of the resulting transgenic plants wild type (WT) of this variety. To generate the transgenic whether they were homozygous lines, the presence of the plants, a GFP-BI-1 fusion under the control of the constitutive overexpression construct had first to be detected. To this end, 30 CaMV 35S promoter was used, thus ensuring a sufficient the genomic DNA was isolated from one leaf of all of the expression of the protein. plants. This was done using the DNeasy 96 Plant Kit (Qui In each case 20 plants of the wild type and of four trans agen, Hilden; following the manufacturers instructions). The genic lines #6(1)E4L3P5 (T2), 1#6(2) E15L7P1 (T2), #6(2) overexpression construct GFP-BI-1 was detected with GFP E15L7P2 (T2) and #6(1)E8L1(T1) were grown. Only 14 35 plants germinated from the seeds of line #6(2)E15L7P2 (T2). specific primers by means of PCR for the presence of the Since there was only a limited amount of seed available, the construct. The Phusion Hot Start (Finnzymes, Espoo) was experiments were carried out with a smaller number of plants used for the in-vitro amplification of DNA by PCR (Mullis & of this line. Seven days after Sowing, the primary leaf was Faloona, 1987). removed, inoculated with the P pachyrhizi with the aid of the precipitation column and fixed in destaining Solution 24 40 Primer 1: hours after the inoculation. After destaining of the leaves was (SEO ID NO: 47) complete, they were stained with aniline blue. Aniline blue 5'-ATGGTGAGCAAGGGCGAGGA-3' intercollates into the structure of callose and thus preferen tially stains papillae where callose accumulates and Primer 2: crosslinks with other polymeric substances. Cells which (SEQ ID NO: 48) 45 have, as the result of a hypersensitivity response (HR), under 5'-TTGAACAACGATGTGCAAGACTCCTTGTACAGCTCGTCCATGC-3') gone a process which resembles apoptosis in mammalian PCR approach for detecting the GFP:BI-1 transgene: cells, also show a light fluorescence after staining with aniline blue. The number of spores, the germinated spores with germ tube which had already formed an appressorium and, as cell 50 response, the appressoria with the papillae which lie under DMSO 0.6 ul neath, and the HR, were counted on the inoculated barley Buffer (5x) (F-519 GC) 4 Il DNA 3 ul leaves. Larger spore accumulations where an allocation of the dNTP mix (10 mM each) 0.4 Il appressoria to the spores was no longer possible, were not Primer 1 (20 pmol) 1 Jul included in the count. As far as possible, at least 100 spores Primer 2 (20 pmol) 1 Jul 55 with appressoria were counted per leaf (see Table in FIG. 11). Hot Start Phusion F 540L 0.2 pil The analysis of the leaves under the microscope revealed Water 9.8 ul the formation of papillae, which was frequently markedly increased in the transgenic lines. In contrast, an appearance of The PCR was carried out a therrnocycler with heatable lid HR in the infected cells was observed less frequently in the (Tgradient; Biometra, Göttingen transgenic plants. In the wild type, an average of 36% of the PCR Programme: 60 infected cells developed papillae as defence response, 45% of the cells responded with an HR. In contrast, the formation of papillae in the transgenic lines amounted to between 50 and 1. Initial denaturation 98°C. 30 sec 60%. The HR, which amounted to 16-26%, was also mark 2. Denaturation 98 C. 10 Sec 35 cycles of steps 2-4 edly less pronounced in comparison with the wild type (FIG. 3. Annealing 58° C. 30 Sec 65 12). According to examination with the so-called “Student 4. Elongation 72° C. 30 Sec test” (t-Test), the relative formation of papillae in the trans genic lines is significantly increased over the WT and the HR US 8,373,022 B2 55 56 is significantly reduced (P<0.001). According to the observa as described above. The NtEI-1-transformed soya plants tions in these experiments, BI-1 prevents the programmed showed a markedly reduced soya rust infection in comparison cell death and promotes papillae formation, by the cells, as with the wild-type Soya plants—the reduction amounted on alternative defence. average to in the region of over 30%. Example 5 5 Likewise, soya plants transformed with NTBI-1 and with SELDA were generated and inoculated with P. pachyrhizi as Interaction of P. pachyrhizi with Transgenic described above. The NtEBI-1+SELDA-transformed soya Bax-Inhibitor-1-Overexpressing Soya plants likewise showed a markedly reduced soya rust infec 10 tion in comparison with the wild-type Soya plants—the Soya plants which overexpress NtEI-1 were generated by reduction here amounted on average to in the region of over methods known perse and were inoculated with P. pachyrhizi 15%.

SEQUENCE LISTING

NUMBER OF SEO ID NOS: 61

SEO ID NO 1 LENGTH: 744 TYPE: DNA ORGANISM: Hordeum vulgare FEATURE; NAME/KEY: CDS LOCATION: (1) . . (741) OTHER INFORMATION: coding for BI1-protein

< 4 OOs SEQUENCE: 1.

atg gac gcc ttic tog acc tcg tcg gcg gcg gcg agc ggc tigg gC 48 Met Asp Ala Phe Ser Thir Ser Ser Ala Ala Ala Ser Gly Trp Gly 1. 10 15

CaC gac t cc citc. aag aac titc cgc Cag atc. to c cc c gcc gtg cag ticc 96 His Asp Ser Luell Lys Asn Phe Arg Glin Ile Ser Pro Ala Val Glin Ser 2O 25 3O

CaC citc. aag citc. gtt tac Ctg act Cta tgc titt gca ct g gcc tica tot 144 His Lell Lys Luell Wall Luell Thir Lell Cys Phe Ala Lieu Ala Ser Ser 35 4 O 45

gcc gtg ggit tac Cta CaC att gcc Ctg aac at C ggc ggg atg Ctg 192 Ala Wall Gly Luell His Ile Ala Lell Asn Ile Gly Gly Met Leu SO 55 6 O

aca atg citc. tgt gt C gga act atc. gcc atg ttic ticg gtg cca 24 O Thir Met Lell Cys Wall Gly Thir Ile Ala Met Phe Ser Wall Pro 65 70 8O

gtc tat gag agg aag agg titt 999 Ctg atg ggit gca gcc ctic 288 Wall Glu Arg Lys Arg Phe Gly Lell Luell Met Gly Ala Ala Lieu 85 9 O 95

Ctg gaa 999 tog gtt gga cott Ctg att gag citt gcc at a gac titt 336 Luell Glu Gly Ser Wall Gly Pro Lell Ile Glu Luell Ala Ile Asp Phe 105 110

gac C Ca agc citc. gtg aca 999 titt gtc gga acc gcc atc gcc titt 384 Asp Pro Ser Luell Wall Thir Gly Phe Wall Gly Thir Ala Ile Ala Phe 115 12O 125

999 tgc tto tot ggc gcc gcc atc. atc. gcc aag cgc agg gag tac Ctg 432 Gly Cys Phe Ser Gly Ala Ala Ile Ile Ala Lys Arg Arg Glu Tyr Lieu. 13 O 135 14 O

tac citc. ggit ggc Ctg citc. tog tot ggc Ctg tog at C ctg. Ct c tig citg 48O Tyr Lell Gly Gly Luell Luell Ser Ser Gly Lell Ser Ile Lieu Lleu Trp Lieu. 145 15 O 155 16 O

cag titt gtc acg to c at C titt ggc CaC t cc tot ggc agc titc atgttt 528 Glin Phe Wall Thir Ser Ile Phe Gly His Ser Ser Gly Ser Phe Met Phe 1.65 17 O 17s

gag gtt tac titt ggc Ctg ttg atc. tto Ctg 999 tac at g g to tac gac 576 Glu Wall Phe Gly Luell Luell Ile Phe Lell Gly Met Val Tyr Asp 18O 185 190

acg Cag gag at C at C gag agg CaC Cat ggc gac atg gac tac atc 624 US 8,373,022 B2 57 58 - Continued

Thir Glin Glu Ile Ile Glu Arg Ala His His Gly Asp Met Asp Tyr Ile 195 2OO 2O5 aag CaC gcc citc. a CC citc. tto acc gac titt gtt gcc gtc citc. gt C cga 672 Lys His Ala Luell Thir Lell Phe Thir Asp Phe Wall Ala Wall Luell Wall Arg 21 O 215 22O gtc citc. at C at C atg citc. aag aac gca ggc gac aag tcg gag gac aag 72 O Wall Luell Ile Ile Met Lell Asn Ala Gly Asp Ser Glu Asp Lys 225 23 O 235 24 O aag aag agg aag agg 999 t cc tga 744 Lys Arg Lys Arg Gly Ser 245

SEQ ID NO 2 LENGTH: 247 TYPE : PRT ORGANISM: Hordeum vulgare

< 4 OOs SEQUENCE: 2

Met Asp Ala Phe Ser Thir Ser Ser Ala Ala Ala Ser Gly Trp Gly 1. 1O 15

His Asp Ser Luell Asn Phe Arg Glin Ile Ser Pro Ala Wall Glin Ser 25 3 O

His Luell Lys Luell Wall Lell Thir Luell Phe Ala Lell Ala Ser Ser 35 4 O 45

Ala Wall Gly Ala Tyr Lell His Ile Ala Luell ASn Ile Gly Gly Met Lel SO 55 60

Thir Met Luell Ala Wall Gly Thir Ile Ala Trp Met Phe Ser Wall 65 70

Wall Glu Glu Arg Arg Phe Gly Luell Luell Met Gly Ala Ala Lel 85 90 95

Lell Glu Gly Ala Ser Wall Gly Pro Luell Ile Glu Lell Ala Ile Asp Phe 105 11 O

Asp Pro Ser Ile Lell Wall Thir Gly Phe Wall Gly Thir Ala Ile Ala Phe 115 12 O 125

Gly Cys Phe Ser Gly Ala Ala Ile Ile Ala Arg Arg Glu Lel 13 O 135 14 O

Tyr Luell Gly Gly Lell Lell Ser Ser Gly Luell Ser Ile Lell Luell Trp Lel 145 150 155 160

Glin Phe Wall Thir Ser Ile Phe Gly His Ser Ser Gly Ser Phe Met Phe 1.65 17O 17s

Glu Wall Phe Gly Lell Lell Ile Phe Luell Gly Tyr Met Wall Asp 18O 185 19 O

Thir Glin Glu Ile Ile Glu Arg Ala His His Gly Asp Met Asp Ile 195

His Ala Luell Thir Lell Phe Thir Asp Phe Wall Ala Wall Luell Wall Arg 21 O 215 22O

Wall Luell Ile Ile Met Lell Asn Ala Gly Asp Ser Glu Asp Lys 225 23 O 235 24 O

Arg Arg Gly Ser 245

SEQ ID NO 3 LENGTH: 1067 TYPE: DNA ORGANISM: Arabidopsis thaliana FEATURE: NAME/KEY: CDS LOCATION: (1) . . (741) OTHER INFORMATION: coding for BI1-protein US 8,373,022 B2 59 60 - Continued

<4 OOs, SEQUENCE: 3 atg gat gcg ttic tict tcc titc titc. gat tot caa cott ggit agc aga agc 48 Met Asp Ala Phe Ser Ser Phe Phe Asp Ser Glin Pro Gly Ser Arg Ser 1. 5 1O 15 tgg agc tat gat t ct citt aaa aac ttic cqt cag att tot CC a gcc gtt 96 Trp Ser Asp Ser Lieu Lys Asn Phe Arg Glin Ile Ser Pro Ala Wall 25 3 O

Cag aat Cat citt aaa cqg gtt tat ttg acc tita tgt gct Ctt gtg 144 Glin Asn His Lieu Lys Arg Val Tyr Lieu. Thir Lieu Cys Ala Lieu Wall 35 4 O 45 gcg tot gcc titt gga gct tac ct c cat gtg ctic tgg aat at C ggc ggit 192 Ala Ser Ala Phe Gly Ala Tyr Lieu His Val Lieu. Trp Asn Ile Gly Gly SO 55 60 att citt aca acg att gga tigt att gga act atg att tgg citc. ct t t ca. 24 O Ile Luell Thir Thir Ile Gly Cys Ile Gly Thr Met Ile Trp Luell Luell Ser 65 70 7s 8O

cott cott tat gaa cac caa aaa agg citt tot citt Ctg titt gtg tot 288 Pro Pro Tyr Glu. His Gln Lys Arg Lieu. Ser Lell Lell Phe Wall Ser 85 90 95 gct gtt citt gala ggit gct tct gtt ggc ccc ttg atc. a.a.a. gtg gca att 336 Ala Wall Luell Glu Gly Ala Ser Val Gly Pro Leu Ile Wall Ala Ile 105 11 O gat gtt gac cca agc atc citt atc act gca titt gtt gga act gcg at a 384 Asp Wall Asp Pro Ser Ile Lieu. Ile Thir Ala Phe Wall Gly Thir Ala Ile 115 12 O 125 gcg titt gt C tgt titc. tca gca gca gca atgtta gca aga cgc agg gag 432 Ala Phe Wall Cys Phe Ser Ala Ala Ala Met Lieu. Ala Arg Arg Arg Glu. 13 O 135 14 O tat citc. tac Ctt gga gga citg Ctt to a tot ggc ttg tot atg cta atg Tyr Luell Lieu. Gly Gly Lieu. Lieu Ser Ser Gly Lell Ser Met Luell Met 145 150 155 160 tgg citc. cag titt gcc tict tca atc titt ggt ggc tot gca tot at c titt 528 Trp Luell Glin Phe Ala Ser Ser Ile Phe Gly Gly Ser Ala Ser Ile Phe 1.65 17O 17s aag titt gag ttg tac titt gga citt ttg atc titt gtg gga tac atg gtg 576 Lys Phe Glu Leu Tyr Phe Gly Lieu eu. Ile Phe Wall Gly Tyr Met Wall 18O 185 19 O gtg gac aca caa gag att at a gala aag gca cac citc. ggit gac atg gac 624 Wall Asp Thir Glin Glu Ile Ile Glu Lys Ala His Lell Gly Asp Met Asp 195 2O5 tat gta a.a.a. cat tcc titg acc citt ttic act gac titt gta gct gtg titt 672 Tyr Wall His Ser Lieu. Thir Lieu. Phe Thr Asp Phe Wall Ala Wall Phe 21 O 215 22O gtt cgg att ctic atc ata atgttg aag aac toa gca gat a.a.a. gala gag 72 O Wall Arg Ile Lieu. Ile Ile Met Lieu. Lys Asn. Ser Ala Asp Lys Glu Glu 225 23 O 235 24 O aag aag aag aaa agg aga aac taggggatg taaagtaa at tta actitt at 771 Lys Lys Lys Lys Arg Arg Asn 245 ggttgttatc gtgttgttggCC actittgaaga tatt acttgt tag cact ct c tattggtgac 831 caga catgtt to Cactaaaa. aggatctgct tgttt cactt ctgcacaagt acCat Cttca 891. gattgtaaat gacticgagtg ttgttcttct titt Catalaac ttttgttctt talagagtttg 951 gttctactga ttgcatctta cCaagctaag aataatgtag gaaaatgata atcctgttta 1011 aattitt Ctaa aatgtgtgca titt cagaaaa aaaaaaaaaa. aaaaaaaaaa. aaaaaa. 1067

<210s, SEQ ID NO 4 &211s LENGTH: 247 US 8,373,022 B2 61 - Continued

212. TYPE: PRT <213> ORGANISM: Arabidopsis thaliana <4 OOs, SEQUENCE: 4 Met Asp Ala Phe Ser Ser Phe Phe Asp Ser Gln Pro Gly Ser Arg Ser 1. 5 1O 15 Trp Ser Tyr Asp Ser Lieu Lys Asn. Phe Arg Glin Ile Ser Pro Ala Val 2O 25 3 O Glin Asn His Lieu Lys Arg Val Tyr Lieu. Thir Lieu. Cys Cys Ala Lieu Val 35 4 O 45 Ala Ser Ala Phe Gly Ala Tyr Lieu. His Val Lieu. Trp Asn. Ile Gly Gly SO 55 60 Ile Lieu. Thir Thr Ile Gly Cys Ile Gly Thr Met Ile Trp Lieu. Leu Ser 65 70 7s 8O Cys Pro Pro Tyr Glu. His Gln Lys Arg Leu Ser Lieu Lleu Phe Val Ser 85 90 95 Ala Val Lieu. Glu Gly Ala Ser Val Gly Pro Lieu. Ile Llys Val Ala Ile 1OO 105 11 O Asp Val Asp Pro Ser Ile Lieu. Ile Thr Ala Phe Val Gly Thr Ala Ile 115 12 O 125 Ala Phe Val Cys Phe Ser Ala Ala Ala Met Lieu Ala Arg Arg Arg Glu 13 O 135 14 O Tyr Lieu. Tyr Lieu. Gly Gly Lieu. Lieu. Ser Ser Gly Lieu. Ser Met Lieu Met 145 150 155 160 Trp Leu Glin Phe Ala Ser Ser Ile Phe Gly Gly Ser Ala Ser Ile Phe 1.65 17O 17s Llys Phe Glu Lieu. Tyr Phe Gly Lieu. Lieu. Ile Phe Val Gly Tyr Met Val 18O 185 19 O Val Asp Thr Glin Glu Ile Ile Glu Lys Ala His Lieu. Gly Asp Met Asp 195 2OO 2O5 Tyr Val Llys His Ser Lieu. Thir Lieu Phe Thr Asp Phe Val Ala Val Phe 21 O 215 22O Val Arg Ile Lieu. Ile Ile Met Lieu Lys Asn. Ser Ala Asp Llys Glu Glu 225 23 O 235 24 O Llys Llys Llys Lys Arg Arg Asn 245

<210s, SEQ ID NO 5 &211s LENGTH: 116 O &212s. TYPE: DNA <213> ORGANISM: Nicotiana tabacum 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (747) <223> OTHER INFORMATION: coding for BI1-protein

<4 OOs, SEQUENCE: 5 atg gag tot togc aca tog titc ttic aat to a cag tog gcg tog tot cqc 48 Met Glu Ser Cys Thr Ser Phe Phe Asin Ser Glin Ser Ala Ser Ser Arg 1. 5 1O 15 aat cqc togg agt tac gat t ct citt aag aac titc cqc cag atc. tct coc 96 Asn Arg Trp Ser Tyr Asp Ser Lieu Lys Asn. Phe Arg Glin Ile Ser Pro 2O 25 3 O titt gtt caa act cat citc aaa aag git c tac citt to a tta tigt togt gct 144 Phe Val Glin Thir His Lieu Lys Llys Val Tyr Lieu. Ser Lieu. Cys Cys Ala 35 4 O 45 tta gtt gct tcg gct gct gga got tac citt cac att citt togg aac att 192 Lieu Val Ala Ser Ala Ala Gly Ala Tyr Lieu. His Ile Lieu. Trp Asn. Ile SO 55 60

US 8,373,022 B2 65 66 - Continued Lieu Val Ala Ser Ala Ala Gly Ala Lieu. His Ile Lell Trp Asn Ile SO 55 60

Gly G ly Lieu. Lieu. Thir Thr Lieu. Gly Val Gly Ser Ile Wall Trp Luell 65 70 7s 8O

Met A. la Thr Pro Leu Tyr Glu Glu Glin Lys Arg Ile Ala Luell Luell Met 85 90 95

Ala Ala Ala Lieu. Phe Lys Gly Ala Ser Ile Gly Pro Lell Ile Glu Luell 1OO 105 11 O

Ala I le Asp Phe Asp Pro Ser Ile Wall Ile Gly Ala Phe Wall Gly 115 12 O 125

Ala Val Ala Phe Gly Cys Phe Ser Ala Ala Ala Met Wall Ala Arg Arg 13 O 135 14 O

Arg G lu Tyr Lieu. Tyr Lieu. Gly Gly Luell Luell Ser Ser Gly Luell Ser Ile 145 150 155 160

Lieu. Phe Trp Lieu. His Phe Ala Ser Ser Ile Phe Gly Gly Ser Met Ala 1.65 17O 17s

Lieu. Phe Llys Phe Glu Val Tyr Phe Gly Lieu. Luell Wall Phe Wall Gly Tyr 18O 185 19 O

Ile I le Phe Asp Thr Glin Asp Ile Ile Glu Lys Ala His Luell Gly Asp 195 2OO

Lieu. Asp Tyr Val Llys His Ala Lieu Thir Lieu. Phe Thir Asp Phe Wall Ala 2 10 215 22O

Val Phe Val Arg Ile Lieu. Ile Ile Met Lieu Lys Asn Ala Ser Asp Lys 225 23 O 235 24 O

Glu G lu Lys Llys Llys Lys Arg Arg Asn 245

SEO ID NO 7 LENGTH: 1056 TYPE: DNA ORGANISM: Oryza sativa FEATURE: NAME/KEY: CDS LOCATION: (1) . . (747) OTHER INFORMATION: coding for BI1-protein SEQUENCE: 7 atg 9 ac goc titc tact cq acc ticg tog gcg tac gga gcg gcg gcg agc 48 Met Asp Ala Phe Tyr Ser Thr Ser Ser Ala Tyr Gly Ala Ala Ala Ser 1. 5 1O 15 ggc tigg ggc tac gac tog Citg aag aac ttic cqc Cag atc. to c cc c gcc 96 Gly. T rp Gly Tyr Asp Ser Lieu Lys Asn Phe Arg Glin Ile Ser Pro Ala 2O 25 3 O gtc c ag to c cac ct c aag ctic gtt tac ct g aca Cta tgc gtC gcc Ctg 144 Wall G lin Ser His Lieu Lys Lieu Val Lieu. Thir Lell Cys Wall Ala Luell 35 4 O 45

Ctg cac gtc gcc ttg aac at C ggc 192 Ala Ala Ser Ala Val Gly Ala Tyr Luell His Wall Ala Lell Asn Ile Gly SO 55 60 ggg a tg ttg act atg Ctic ggg tec gtg ggg agc atc. gcc tgg ttg ttic 24 O Gly M et Lieu. Thir Met Leu Gly Cys Wall Gly Ser Ile Ala Trp Luell Phe 65 70 7s 8O tCg g tg cct gt C titt gag gag agg aag agg ttt 999 att citc. ttg gcc 288 Ser Wal Pro Val Phe Glu Glu Arg Lys Arg Phe Gly Ile Luell Luell Ala 85 90 95

cc ctg. Ctg gaa ggg gct tca gtt ggg cct Ctg atc. aag citt gct 336 Ala Ala Lieu. Lieu. Glu Gly Ala Ser Wall Gly Pro Lell Ile Lys Luell Ala 105 11 O gta g ac titt gac toa agc att ct c gta aca gca titt gtt gga act gcc 384 US 8,373,022 B2 67 68 - Continued Val Asp Phe Asp Ser Ser Ile Lieu. Wall. Thir Ala Phe Val Gly Thr Ala 115 12 O 125 att go a titt ggg togc titc act togc gct gcc atc gtt gcc aag Cdt agg 432 Ile Ala Phe Gly Cys Phe Thr Cys Ala Ala Ile Val Ala Lys Arg Arg 13 O 135 14 O gag tac Ct c tac Ctt ggt ggt ttg ctic tot tot ggc ctic toc atc ct g Glu Tyr Lieu. Tyr Lieu. Gly Gly Lieu. Luell Ser Ser Gly Lieu. Ser Ile Lieu. 145 150 155 160 ctic togg citg cag titt gcc gca to c atc titt ggc cac toc acc ggc agc 528 Lieu. Trp Lieu. Glin Phe Ala Ala Ser Ile Phe Gly His Ser Thr Gly Ser 1.65 17O 17s titc atg titt gag gtt tac titt ggc ctg ttg atc ttic Ctgggg tac atg 576 Phe Met Phe Glu Val Tyr Phe Gly Lieu. Lieu. Ile Phe Leu Gly Tyr Met 18O 185 19 O gtg tat gac acg cag gag atc at C gag agg gct cac cac ggit gac atg 624 Val Tyr Asp Thr Glin Glu Ile Ile Glu Arg Ala His His Gly Asp Met 195 2O5 gac tac atc aag cac goa citc acc ct c titc act gac titc gtg gcc gtC 672 Asp Tyr Ile Llys His Ala Lieu. Thr Lieu. Phe Thr Asp Phe Val Ala Val 21 O 215 22O citt gtc cqg at c ctic gtc atc atg ctic aag aac gcg tct gac aag tog 72 O Lieu Val Arg Ile Lieu Val Ile Met Lieu Lys Asn Ala Ser Asp Llys Ser 225 23 O 235 24 O gag gag aag aag agg aag aag agg tot tdagagcttic tott cocqct 767 Glu Glu Lys Lys Arg Llys Lys Arg Ser 245 ttgcacataa gaaaaaacca cc.gcggct at tgcct citacg tattatgaca gagcc.gcact 827 t caactgggt titt atggtga atacaagttc ttttgcattt tgttgatacg gtgtgaatct 887 tcticaggttt gtcgt.cgtag tagctittgca aatactagoa tgctacatga cacggat citt 947 tctgtaatgg tdtcgcgtt gat.cgaaacg tgaaaacaca tott catttg cqactaattit 1 OOf gtttgc ctitt ttgattga tgatgatcct titcCC Caaaa. aaaaaaaaa. 1056

<210s, SEQ ID NO 8 &211s LENGTH: 249 212. TYPE: PRT <213> ORGANISM: Oryza sativa

<4 OOs, SEQUENCE: 8 Met Asp Ala Phe Tyr Ser Thr Ser Ser Ala Tyr Gly Ala Ala Ala Ser 1. 5 1O 15

Gly Trp Gly Tyr Asp Ser Lieu Lys Asin Phe Arg Glin Ile Ser Pro Ala 25 3 O Val Glin Ser His Lieu Lys Lieu Val Tyr Lieu. Thr Lieu. CyS Val Ala Lieu. 35 45

Ala Ala Ser Ala Val Gly Ala Tyr Lieu. His Wall Ala Lieu. Asn. Ile Gly SO 55 60 Gly Met Lieu. Thr Met Leu Gly Cys Val Gly Ser Ile Ala Trp Lieu. Phe 65 70 7s 8O Ser Val Pro Val Phe Glu Glu Arg Lys Arg Phe Gly Ile Lieu. Lieu Ala 85 90 95 Ala Ala Lieu. Lieu. Glu Gly Ala Ser Val Gly Pro Lieu. Ile Llys Lieu Ala 105 11 O

Val Asp Phe Asp Ser Ser Ile Lieu. Wall. Thir Ala Phe Val Gly Thr Ala 115 12 O 125

Ile Ala Phe Gly Cys Phe Thr Cys Ala Ala Ile Val Ala Lys Arg Arg 13 O 135 14 O US 8,373,022 B2 69 70 - Continued

Glu Tyr Lieu. Tyr Lieu. Gly Gly Lieu. Luell Ser Ser Gly Lell Ser Ile Luell 145 150 155 160

Leu. T rp Lieu. Glin Phe Ala Ala Ser Ile Phe Gly His Ser Thir Gly Ser 1.65 17O 17s

Phe Met Phe Glu Val Tyr Phe Gly Luell Lieu. Ile Phe Lell Gly Tyr Met 18O 185 19 O

Val Tyr Asp Thr Glin Glu Ile Ile Glu Arg Ala His His Gly Asp Met 195 2OO

Asp Tyr Ile Llys His Ala Lieu. Thr Luell Phe Thir Asp Phe Wall Ala Wall 2 10 215 22O

Luell Wal Arg Ile Lieu Val Ile Met Luell Lys Asn Ala Ser Asp Ser 225 23 O 235 24 O

Glu G lu Lys Lys Arg Llys Lys Arg Ser 245

SEO ID NO 9 LENGTH: 973 TYPE: DNA ORGANISM: Brassica napus FEATURE: NAME/KEY: CDS LOCATION: (1) . . (741) OTHER INFORMATION: coding for BI1-protein SEQUENCE: 9 atg 9 at tca ttc. tcg toc titc titc gat tot Caa cott ggit agc aga agc 48 Met Asp Ser Phe Ser Ser Phe Phe Asp Ser Glin Pro Gly Ser Arg Ser 1. 5 1O 15 tgg agc tat gat t ct citc aaa aac citc. cgt cag att tot cc c to c gt C 96 Trp S er Tyr Asp Ser Lieu Lys Asn Luell Arg Glin Ile Ser Pro Ser Wall 2O 25 3 O

Cag a at cat ct c aag agg gtt tat citc. act ctd tgt tgt gct citc. gtt 144 Glin Asn His Lieu Lys Arg Val Tyr Luell Thir Lieu. Cys Cys Ala Luell Wall 35 4 O 45 gcg t cit gcg titt gga gct tac ct c CaC gtg ctic tgg aac at a ggt ggt 192 Ala S er Ala Phe Gly Ala Tyr Lieu. His Wall Lieu. Trp Asn Ile Gly Gly SO 55 60 att C to act acc att gga tigc titt gga agc atg att tgg Ctg citc. to c 24 O Ile L. eu. Thir Thr Ile Gly Cys Phe Gly Ser Met Ile Trp Luell Luell Ser 65 7s 8O tgt c ct cot tat gaa caa caa aag agg citt too citt Ctg titt Ctg tot 288 Cys P ro Pro Tyr Glu Gln Gln Lys Arg Luell Ser Lell Lell Phe Luell Ser 85 90 95 gct g tt ct c gaa got gct tca gtt ggt cc c ttg atc. a.a.a. gtg gca gtt 336 Ala Wall Lieu. Glu Gly Ala Ser Val Gly Pro Leu. Ile Wall Ala Wall 1OO 105 11 O gat t tt gac cca agc atc ctic atc act gcg titt gtc gga act gcg at a 384 Asp Phe Asp Pro Ser Ile Lieu. Ile Thir Ala Phe Wall Gly Thir Ala Ile 115 12 O 125 gcc t tt at c tdt titc. tca ggg gca gcg atgttg gca aga cgc aga gag 432 Ala Phe Ile Cys Phe Ser Gly Ala Ala Met Leu Ala Arg Arg Arg Glu 1. 3 O 135 14 O tac C. tc tac Ct c ga gga citg citt to a tot ggc ttg t cc atg citt atg Tyr L eu Tyr Lieu. Gly Gly Lieu. Lieu. Ser Ser Gly Lell Ser Met Luell Met 145 150 155 160 tgg C tt cag titt gcc tict tcc atc titt ggit gc tot gca to c at C titt 528 Trp L. eul Glin Phe Ala Ser Ser Ile Phe Gly Gly Ser Ala Ser Ile Phe 1.65 17O 17s aag t tt gag ct c tac titt gga ct c ttg at c titt gtg gga tac atg gtg 576 Llys Phe Glu Lieu. Tyr Phe Gly Lieu. Luell Ile Phe Wall Gly Tyr Met Wall 18O 185 19 O US 8,373,022 B2 71 - Continued gtg gaC act caa gat att at a gag aag gcc cac ctic ggit gaC atg gat 624 Val Asp Thr Glin Asp Ile Ile Glu Lys Ala His Lieu. Gly Asp Met Asp 195 2OO 2O5 tac gitg aaa cat tcc titg acc citt titc acc gat titt gta gct gtg titt 672 Tyr Val Llys His Ser Lieu. Thir Lieu Phe Thr Asp Phe Val Ala Val Phe 21 O 215 22O gtt cqt gtt Ct c atc att atg ctg aag aac tog gca gat aaa gala gat 72 O Val Arg Val Lieu. Ile Ile Met Lieu Lys Asn. Ser Ala Asp Llys Glu Asp 225 23 O 235 24 O aaa aag aag agg agg agg aac tagactaaa aagtgaga aa gaaagctaaa 771 Llys Llys Lys Arg Arg Arg Asn 245 tagagtgggt gttatgtgtg tttcaaaaaa taaaaaagag tiggtgttat aagtacagac 831 atgatagogt toggtgtttitt tacttgtttg gaacagttitt ggtaacaa.ca cacgttacgt. 891. atttgttgt at tcc tottagt gactic cagat tdtgaatgga t cagtat citt gaaactgtgt 951 tgaaaattat cagttgggag ct 973

<210s, SEQ ID NO 10 &211s LENGTH: 247 212. TYPE: PRT <213> ORGANISM: Brassica napus <4 OOs, SEQUENCE: 10 Met Asp Ser Phe Ser Ser Phe Phe Asp Ser Gln Pro Gly Ser Arg Ser 1. 5 1O 15 Trp Ser Tyr Asp Ser Lieu Lys Asn Lieu. Arg Glin Ile Ser Pro Ser Val 2O 25 3 O Glin Asn His Lieu Lys Arg Val Tyr Lieu. Thir Lieu. Cys Cys Ala Lieu Val 35 4 O 45 Ala Ser Ala Phe Gly Ala Tyr Lieu. His Val Lieu. Trp Asn. Ile Gly Gly SO 55 60 Ile Lieu. Thir Thr Ile Gly Cys Phe Gly Ser Met Ile Trp Lieu. Leu Ser 65 70 7s 8O Cys Pro Pro Tyr Glu Glin Gln Lys Arg Lieu. Ser Lieu Lleu Phe Lieu. Ser 85 90 95 Ala Val Lieu. Glu Gly Ala Ser Val Gly Pro Lieu. Ile Llys Val Ala Val 1OO 105 11 O Asp Phe Asp Pro Ser Ile Lieu. Ile Thr Ala Phe Val Gly Thr Ala Ile 115 12 O 125 Ala Phe Ile Cys Phe Ser Gly Ala Ala Met Lieu Ala Arg Arg Arg Glu 13 O 135 14 O Tyr Lieu. Tyr Lieu. Gly Gly Lieu. Lieu. Ser Ser Gly Lieu. Ser Met Lieu Met 145 150 155 160 Trp Leu Glin Phe Ala Ser Ser Ile Phe Gly Gly Ser Ala Ser Ile Phe 1.65 17O 17s Llys Phe Glu Lieu. Tyr Phe Gly Lieu. Lieu. Ile Phe Val Gly Tyr Met Val 18O 185 19 O Val Asp Thr Glin Asp Ile Ile Glu Lys Ala His Lieu. Gly Asp Met Asp 195 2OO 2O5 Tyr Val Llys His Ser Lieu. Thir Lieu Phe Thr Asp Phe Val Ala Val Phe 21 O 215 22O Val Arg Val Lieu. Ile Ile Met Lieu Lys Asn. Ser Ala Asp Llys Glu Asp 225 23 O 235 24 O

Llys Llys Lys Arg Arg Arg Asn 245

US 8,373,022 B2 75 76 - Continued

TYPE PRT ORGANISM: Glycine max

< 4 OOs SEQUENCE: 12

Arg Lieu. Glin Ala Met Asp Ala Phe Asn Ser Phe Phe Asp Ser Arg Asn 1. 15

Arg Trp Asn Tyr Asp Thr Lieu Lys Asn Phe Arg Glin Ile Ser Pro Wall 25 3 O

Val Glin Asn His Lieu Lys Glin Val Tyr Phe Thir Lieu. Cys Phe Ala Val 35 4 O 45

Val Ala Ala Ala Val Gly Ala Tyr Luell His Wall Lieu. Lieu. Asn. Ile Gly SO 55 60

Gly Phe Lieu. Thir Thr Val Ala Cys Met Gly Ser Ser Phe Trp Lieu. Leu 65 70 8O

Ser Thr Pro Pro Phe Glu Glu Arg Arg Wall Thir Lieu. Lieu Met Ala 85 90 95

Ala Ser Leu Phe Glin Gly Ser Ser Ile Gly Pro Lieu. Ile Asp Lieu Ala 105 11 O

Ile His Ile Asp Pro Ser Lieu. Ile Phe Ser Ala Phe Val Gly Thr Ala 115 12 O 125

Lieu Ala Phe Ala Cys Phe Ser Gly Ala Ala Luell Val Ala Arg Arg Arg 13 O 135 14 O

Glu Tyr Lieu. Tyr Lieu. Gly Gly Lieu. Wall Ser Ser Gly Lieu. Ser Ile Lieu. 145 150 155 160

Lieu. Trp Lieu. His Phe Ala Ser Ser Ile Phe Gly Gly Ser Thr Ala Lieu. 1.65 17O 17s

Phe Llys Phe Glu Lieu. Tyr Phe Gly Luell Luell Wall Phe Val Gly Tyr Ile 18O 185 19 O

Val Val Asp Thr Glin Glu Ile Val Glu Arg Ala His Lieu. Gly Asp Lieu. 195 2OO 2O5

Asp Tyr Val Llys His Ala Lieu. Thr Luell Phe Thir Asp Lieu Val Ala Val 21 O 215 22O

Phe Val Arg Ile Lieu Val Ile Met Luell ASn Ser Thr Glu Arg Asn 225 23 O 235 24 O Glu Lys Llys Llys Lys Arg Arg Asp 245

SEQ ID NO 13 LENGTH: 1510 TYPE: DNA ORGANISM: Glycine max FEATURE: NAME/KEY: CDS LOCATION: (1) . . (777) OTHER INFORMATION: coding for BI-1 protein FEATURE: NAMEAKEY: misc feature LOCATION: (1046) ... (1095) OTHER INFORMATION: n is a

SEQUENCE: 13 atc acg aaa act at a cqa titc gat to c ttg titt tcq atg gac act titc 48 Ile Thr Lys Thr Ile Arg Phe Asp Ser Luell Phe Ser Met Asp Thr Phe 1. 5 1O 15 ttcaag toc coa tot tot tot tot tog aga agc cgc tigg agt tac gat 96 Phe Llys Ser Pro Ser Ser Ser Ser Ser Arg Ser Arg Trp Ser Tyr Asp 25 3 O act ct c aag aat titc cqc gag atc tot cc.g citc. gtt cag aat cac atc 144 Thir Lieu Lys Asn. Phe Arg Glu Ile Ser Pro Luell Wall Glin Asn His Ile 35 4 O 45

US 8,373,022 B2 79 80 - Continued

<210s, SEQ ID NO 14 &211s LENGTH: 259 212. TYPE : PRT <213> ORGANISM: Glycine ax

<4 OOs, SEQUENCE: 14

Ile Thir Lys Thr Ile Arg Phe Asp Ser Luell Phe Ser Met Asp Thir Phe 1. 5 15

Phe Lys Ser Pro Ser Ser Ser Ser Ser Arg Ser Arg Trp Ser Asp 2O 25 3 O

Thir Luell Lys Asn Phe Arg Glu Ile Ser Pro Luell Wall Glin Asn His Ile 35 4 O 45

Luell Wall Phe Thir Lell Ala Wall Wall Ala Ala Ala Wall SO 55 60

Gly Ala Phe Luell His Wall Lell Trp Asn Ile Gly Gly Phe Luell Thir Thir 65 70

Lell Ala Ser Ile Gly Ser Met Phe Trp Luell Luell Ser Thir Pro Pro Phe 85 90 95

Glu Glu Glin Lys Arg Lell Ser Luell Luell Met Ala Ser Ala Luell Phe Glin 105 11 O

Gly Ala Ser Ile Gly Pro Lell Ile Asp Luell Ala Phe Ala Ile Asp Pro 115 12 O 125

Gly Luell Ile Ile Gly Ala Phe Wall Ala Thir Ser Lell Ala Phe Ala 13 O 135 14 O

Phe Ser Ala Wall Ala Lell Wall Ala Arg Arg Arg Glu Luell Tyr Luell 145 150 155 160

Gly Gly Luell Luell Ser Ser Trp Luell Ser Ile Luell Met Luell His Ser 1.65 17O 17s

Asp Ser Ser Luell Phe Gly Gly Ser Ile Ala Luell Phe Phe Glu Luell 18O 185 19 O

Phe Gly Luell Lell Wall Phe Wall Gly Tyr Wall Ile Wall Asp Thir Glin 195 2O5

Glu Ile Ile Glu Arg Ala His Phe Gly Asp Luell Asp Wall His 21 O 215

Ala Luell Thir Luell Phe Thir Asp Luell Ala Ala Ile Phe Wall Arg Ile Luell 225 23 O 235 24 O

Ile Ile Met Luell Lys Asn Ser Ser Glu Arg ASn Glu Lys 245 250 255 Arg Arg Asp

SEO ID NO 15 LENGTH: 651 TYPE: DNA ORGANISM: Triticum aestivum FEATURE: NAME/KEY: CDS LOCATION: (1) . . (651) OTHER INFORMATION: coding for BI-1 protein

SEQUENCE: 15 gtc gca atg cc.g ggit cga cga titt cgt Ctg acc tat gct ttg cott ggc 48 Wall Ala Met Pro Gly Arg Arg Phe Arg Luell Thir Tyr Ala Luell Pro Gly 1. 5 1O 15 citc. at C tgc cgt 999 tgc tta cott gca Cat tgc cott gaa Cat tgg cgg 96 Lell Ile Cys Arg Gly Cys Lell Pro Ala His Cys Pro Glu His Trp Arg 2O 25 3 O gat gct gac aat gct cgc gtg tat cgg aac Cat cgc Ctg gat gtt citc. 144 Asp Ala Asp Asn Ala Arg Wall Arg Asn His Arg Lell Asp Wall Luell US 8,373,022 B2 81 82 - Continued

35 4 O 45 ggit gcc agt Cta cga gga gag gala gag gtt tgg gct gct gat 999 tgc 192 Gly Ala Ser Luell Arg Gly Glu Glu Glu Wall Trp Ala Ala Asp Gly Cys SO 55 60 agc citc. Ctg gala 999 gct toa gtt gga cott Ctg att gag citt gcc at a 24 O Ser Lel Luell Glu Gly Ala Ser Wall Gly Pro Luell Ile Glu Luell Ala Ile 65 70 7s 8O gac titt gac CC a agt atc. citc. gtg aca 999 titt gtc gga acc gcc at C 288 Asp Phe Asp Pro Ser Ile Lell Wall Thir Gly Phe Wall Gly Thir Ala Ile 85 90 95 gcc ttic 999 tto tot ggc gcc gcc at C atc. gcc aag cgc agg gag 336 Ala Phe Gly Phe Ser Gly Ala Ala Ile Ile Ala Lys Arg Arg Glu 105 11 O tac Ctg tac citc. ggit ggit Ctg citc. to c to c ggc Ctg tcg at C Ctg citc. 384 Lel Tyr Luell Gly Gly Lell Luell Ser Ser Gly Lell Ser Ile Luell Luell 115 12 O 125 tgg Ctg cag titt gcc acg t cc at C titt ggc CaC t cc tot ggc agc ttic 432 Trp Lel Glin Phe Ala Thir Ser Ile Phe Gly His Ser Ser Gly Ser Phe 13 O 135 14 O atg titt gag gtt tac titt ggc Ctg ttg at C titc gga tac atg gtg Met Phe Glu Wall Tyr Phe Gly Luell Luell Ile Phe Lell Gly Met Wall 145 150 155 160

gac acg cag gag atc. atc. gag agg gcg CaC CaC ggc gac atg gat 528 Asp Thir Glin Glu Ile Ile Glu Arg Ala His His Gly Asp Met Asp 1.65 17O 17s

at C aag CaC gcg citc. a CC citc. ttic acc gac tto gtc gcc gtt citc. 576 Ile Lys His Ala Lell Thir Luell Phe Thir Asp Phe Wall Ala Wall Luell 18O 185 19 O gtc cgc gt C citc. atc. atc. ttg citc. aag aac gca gcg gac aag gt C gga 624 Wall Arg Wall Luell Ile Ile Lell Luell Lys Asn Ala Ala Asp Lys Wall Gly 195 2OO 2O5 ggc Cala gala gag gag gaa gag aag to c 651 Gly Glin Glu Glu Glu Glu Glu Lys Ser 21 O 215

<210s, SEQ ID NO 16 &211s LENGTH: 217 212. TYPE : PRT &213s ORGANISM: Triticum aestivum

<4 OOs, SEQUENCE: 16

Wall Ala Met Pro Gly Arg Arg Phe Arg Luell Thir Ala Luell Pro Gly 1. 5 15

Lell Ile Cys Arg Gly Lell Pro Ala His Pro Glu His Trp Arg 25 3 O

Asp Ala Asp Asn Ala Arg Wall Tyr Arg Asn His Arg Lell Asp Wall Luell 35 4 O 45

Gly Ala Ser Luell Arg Gly Glu Glu Glu Wall Trp Ala Ala Asp Gly SO 55 60

Ser Luell Luell Glu Gly Ala Ser Wall Gly Pro Luell Ile Glu Luell Ala Ile 65 70

Asp Phe Asp Pro Ser Ile Lell Wall Thir Gly Phe Wall Gly Thir Ala Ile 85 90 95

Ala Phe Gly Cys Phe Ser Gly Ala Ala Ile Ile Ala Arg Arg Glu 105 11 O

Luell Tyr Luell Gly Gly Lell Luell Ser Ser Gly Lell Ser Ile Luell Luell 115 12 O 125

Trp Luell Glin Phe Ala Thir Ser Ile Phe Gly His Ser Ser Gly Ser Phe 13 O 135 14 O US 8,373,022 B2 83 - Continued

Met Phe Glu Val Tyr Phe Gly Lieu. Lieu. Ile Phe Leu Gly Tyr Met Val 145 150 155 160 Tyr Asp Thr Glin Glu Ile Ile Glu Arg Ala His His Gly Asp Met Asp 1.65 17O 17s Tyr Ile Llys His Ala Lieu. Thir Lieu. Phe Thr Asp Phe Val Ala Val Lieu. 18O 185 19 O Val Arg Val Lieu. Ile Ile Lieu. Lieu Lys Asn Ala Ala Asp Llys Val Gly 195 2OO 2O5 Gly Glin Glu Glu Glu Glu Glu Lys Ser 21 O 215

<210s, SEQ ID NO 17 &211s LENGTH: 412 &212s. TYPE: DNA <213> ORGANISM: Zea mays 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (3) ... (410) <223> OTHER INFORMATION: coding for BI1-protein <4 OOs, SEQUENCE: 17 tt gtt att gac ttg gat tcg agg att Ctc gtc act gcg ttic gtc ggg 47 Val Ile Asp Lieu. Asp Ser Arg Ile Lieu Val Thir Ala Phe Val Gly 1. 5 10 15 acc goa gtt got titt gca togc titc. tct ggc gct gcc atc atc gcc aag 95 Thir Ala Val Ala Phe Ala Cys Phe Ser Gly Ala Ala Ile Ile Ala Lys 2O 25 3 O cgc agg gaa tac Ctg tac Ct c ggc ggit ctg. Ctt t catct ggc ct c to c 143 Arg Arg Glu Tyr Lieu. Tyr Lieu. Gly Gly Lieu. Lieu. Ser Ser Gly Lieu. Ser 35 4 O 45 att citt ct c togg citg cag titt got act tca atc titt ggc cac acc agc 191 Ile Leu Lleu Trp Leu Glin Phe Ala Thr Ser Ile Phe Gly His Thr Ser SO 55 6 O gcc acc titc atgttt gag ctic tac titt ggc ctic ctd gtt titc ct g gga 239 Ala Thr Phe Met Phe Glu Lieu. Tyr Phe Gly Lieu. Leu Val Phe Leu Gly 65 70 7s tat atg gtg titt gac acc cag gag at C at C gag agg gcg cac cqt ggg 287 Tyr Met Val Phe Asp Thr Glin Glu Ile Ile Glu Arg Ala His Arg Gly 8O 85 90 95 gac atg gac tac atc aag cac gog citg act ct c titc acc gac titt gtt 335 Asp Met Asp Tyr Ile Llys His Ala Lieu. Thir Lieu. Phe Thr Asp Phe Val 1 OO 105 11 O gcg gtt Ctt gtt ca atc Ctt gt C at C atg atg aag aat gca cag gag 383 Ala Val Lieu Val Arg Ile Lieu Val Ile Met Met Lys Asn Ala Glin Glu 115 12 O 125 aaa to C caa gac gag aag aag agg aag aa 412 Llys Ser Glin Asp Glu Lys Lys Arg Llys 13 O 135

<210s, SEQ ID NO 18 &211s LENGTH: 136 212. TYPE: PRT <213> ORGANISM: Zea mays <4 OOs, SEQUENCE: 18 Val Ile Asp Lieu. Asp Ser Arg Ile Lieu Val Thr Ala Phe Val Gly Thr 1. 5 1O 15 Ala Val Ala Phe Ala Cys Phe Ser Gly Ala Ala Ile Ile Ala Lys Arg 2O 25 3 O

Arg Glu Tyr Lieu. Tyr Lieu. Gly Gly Lieu. Lieu. Ser Ser Gly Lieu. Ser Ile 35 4 O 45 US 8,373,022 B2 85 86 - Continued

Lell Luell Trp Luell Glin Phe Ala Thir Ser Ile Phe Gly His Thir Ser Ala SO 55 60

Thir Phe Met Phe Glu Lell Phe Gly Luell Luell Wall Phe Luell Gly Tyr 65 70

Met Wall Phe Asp Thir Glin Glu Ile Ile Glu Arg Ala His Arg Gly Asp 85 90 95

Met Asp Ile Lys His Ala Luell Thir Luell Phe Thir Asp Phe Wall Ala 1OO 105 11 O

Wall Luell Wall Arg Ile Lell Wall Ile Met Met Lys Asn Ala Glin Glu Lys 115 12 O 125

Ser Glin Asp Glu Lys Arg 13 O 135

SEQ ID NO 19 LENGTH: 345 TYPE: DNA ORGANISM: Triticum aestivum FEATURE: NAME/KEY: CDS LOCATION: (1) . . (342)

<4 OOs, SEQUENCE: 19 gcc gcc at C at C gcc aag cgc agg gag tac Ctg tac citc. ggt ggc Ctg 48 Ala Ala Ile Ile Ala Arg Arg Glu Tyr Luell Lell Gly Gly Luell 1. 5 1O 15 citc. to c to c ggc Ctg tcg atc. Ctg citc. tgg Ctg Cag titt gcc acg to c 96 Lell Ser Ser Gly Lell Ser Ile Luell Luell Trp Luell Glin Phe Ala Thir Ser 2O 25 3 O atc. titt ggc CaC t cc tot ggc agc ttic atg titt gag gtt tac titt ggc 144 Ile Phe Gly His Ser Ser Gly Ser Phe Met Phe Glu Wall Phe Gly 35 4 O 45

Ctg ttg at C titt Ctg gga tac atg gtg gac acg Cag gag at C at C 192 Lell Luell Ile Phe Lell Gly Tyr Met Wall Asp Thir Glin Glu Ile Ile SO 55 60 gag agg gcg CaC CaC ggc gac atg gac atc. aag CaC gcg citc. acc 24 O Glu Arg Ala His His Gly Asp Met Asp Ile His Ala Luell Thir 65 70 7s 8O citc. ttic acc gac titt gtc gcc gt C citc. gt C cgg atc. citc. at C at C atg 288 Lell Phe Thir Asp Phe Wall Ala Wall Luell Wall Arg Ile Lell Ile Ile Met 85 90 95 citc. aag aac gca ggc gac aag tog gag gac aag aag aag agg aag agg 336 Lell Lys Asn Ala Gly Asp Ser Glu Asp Arg Lys Arg 1OO 105 11 O agg to c tga 345 Arg Ser

<210s, SEQ ID NO 2 O &211s LENGTH: 114 212. TYPE : PRT &213s ORGANISM: Triticum aestivum

<4 OOs, SEQUENCE:

Ala Ala Ile Ile Ala Arg Arg Glu Tyr Luell Lell Gly Gly Luell 1. 5 1O 15

Lell Ser Ser Gly Lell Ser Ile Luell Luell Trp Luell Glin Phe Ala Thir Ser 2O 25 3 O

Ile Phe Gly His Ser Ser Gly Ser Phe Met Phe Glu Wall Phe Gly 35 4 O 45

Lell Luell Ile Phe Lell Gly Tyr Met Wall Tyr Asp Thir Glin Glu Ile Ile SO 55 60

US 8,373,022 B2 93 94 - Continued

Glu Luell Glin Arg Glu Lell Pro Arg Cys Arg Asp Ala Thir Luell Thir Wall 25 3 O

Wall Wall Ile Pro Ile Wall Gly Arg Ile Lys Ser Ala Ala Gly Ala 35 4 O 45

Luell His Ile Ala Lell Asn Ile Gly Gly Met Lel Thir Met Luell Ala SO 55 60

Cys Ile Gly Thir Ile Ala Trp Met Phe Ser Wall Wall Glu Glu 65 70

Arg Arg Phe Gly Lell Lell Met Gly Ala Ala Lel Lell Glu Gly Ala 85 90 95

Ser Wall Gly Pro Lell Ile Glu Luell Ala Ile Asp Phe Asp Pro Ser Ile 105 11 O

Lell Wall Thir Gly Phe Wall Gly Thir Ala Ile Ala Phe Gly Phe Ser 115 12 O 125

Gly Ala Ala Ile Ile Ala Lys Arg Arg Glu Tyr Lel Luell Gly Gly 13 O 135 14 O

Lell Luell Ser Ser Gly Lell Thir Ile Luell Luell 145 150

SEO ID NO 27 LENGTH: 388 TYPE: DNA ORGANISM: Zea mays FEATURE: NAME/KEY: CDS LOCATION: (3) ... (386) OTHER INFORMATION: coding for BI1-protein

SEQUENCE: 27 tc tig aac atc ggc ggg acg ctg aca atg Ctic ggit to gtc. g.gc agc 47 Trp Asn Ile Gly Gly Thr Lieu. Thr Met Leu Gly Cys Val Gly Ser 1. 5 10 15 atc. gcc tgg citc. tto tcg gtg cc c gt C tac gag gag agg aag agg tat 95 Ile Ala Trp Luell Phe Ser Wall Pro Wall Tyr Glu Glu Arg Lys Arg Tyr 2O 25 3 O

999 Ctg Ctg atg gcg gct gcc citc. Ctg gala ggc gct tcg gtC gga cc c 143 Gly Luell Luell Met Ala Ala Ala Luell Luell Glu Gly Ala Ser Wall Gly Pro 35 4 O 45 citc. gt C aag citc. gcc gtg gaa titt gac CC a agc atc. Ctg gtg acg gcg 191 Lell Wall Lys Luell Ala Wall Glu Phe Asp Pro Ser Ile Lell Wall Thir Ala SO 55 6 O tto gtg 999 act gcc atc. gcg ttic gcg tgc titc t cc ggc gcg CC a tgg 239 Phe Wall Gly Thir Ala Ile Ala Phe Ala Cys Phe Ser Gly Ala Pro Trp 65 70 7s tgg cag gcc agg gag tac citc. tac Ctg ggc ggc tot cgt. cga ggc 287 Trp Glin Ala Arg Glu Tyr Lell Luell Gly Gly Cys Ser Arg Arg Gly 8O 85 90 95 tot CC a to c tgc tot ggc tgc agc tog cc.g cott C Ca tot tog gca citc. 335 Ser Pro Ser Cys Ser Gly Cys Ser Ser Pro Pro Pro Ser Ser Ala Luell 1 OO 105 11 O cgc aac agc ttic atg tto gag gt C tac ttic 999 Ctg citc. att citt Ctg 383 Arg Asn Ser Phe Met Phe Glu Wall Tyr Phe Gly Lell Lell Ile Luell Luell 115 12 O 125 ggc ta 388 Gly

<210s, SEQ ID NO 28 &211s LENGTH: 128 212. TYPE : PRT <213> ORGANISM: Zea mays US 8,373,022 B2 95 - Continued

<4 OOs, SEQUENCE: 28 Trp Asn. Ile Gly Gly Thr Lieu. Thr Met Leu Gly Cys Val Gly Ser Ile 1. 1O 15

Ala Trp Lieu. Phe Ser Wall Pro Wall Tyr Glu Glu Arg Lys Arg 25 3 O

Lieu. Luell Met Ala Ala Ala Lieu. Lieu. Glu Gly Ala Ser Val Gly Pro Leu 35 4 O 45

Val Llys Lieu. Ala Val Glu Phe Asp Pro Ser Ile Leul Wall. Thir Ala Phe SO 55 60

Val Gly Thr Ala Ile Ala Phe Ala Cys Phe Ser Gly Ala Pro Trp Trp 65 70 7s Glin Ala Arg Glu Tyr Lieu. Tyr Lieu. Gly Gly Cys Ser Arg Arg Gly Ser 85 90 95

Pro Ser Cys Ser Gly Cys Ser Ser Pro Pro Pro Ser Ser Ala Lieu. Arg 105 11 O

Asn. Ser Phe Met Phe Glu Val Tyr Phe Gly Lieu. Lieu. Ile Lieu. Lieu. Gly 115 12 O 125

<210s, SEQ I D NO 29 &211s LENGT H: 1737 212. TYPE : DNA <213s ORGANISM: Solanum tuberosum 22 Os. FEATU RE: <221> NAME/KEY: promoter &222s. LOCAT ION: (1) . . (1737) <223> OTHER INFORMATION: patatin promoter <4 OOs, SEQUENCE: 29 aagct tatgt tgc catatag agtagtttgt gatggtatac ttcatalaact ttaact tatg 6 O ttaaatttgt aatgataaaa tttittattgt aaattaaaaa. ttact tataa. aattgggcat 12 O tatalacatat gaaaga caaa ttgttgttaca tattt tact t ttgactittaa tatgaatatt 18O toaatttaala toattgttitt at t t t c tott to t t t t taca ggtataaaag gtgaaaattg 24 O alagcaagatt gattgcaa.gc tatgtgtcac cacgittattg at actittgga agaaatttitt 3OO actitatatgt Ctttgtttag gagtaatatt tgatatgttt tagttagatt ttcttgt cat 360 titatgctitta gtataattitt agittatttitt attatatgat Catgggtgaa ttittgataca aatatttittg toattaaata aattaattta t cacaacttg attactitt.ca gtgacaaaaa atgt attgtc gtag tacc ct ttitttgttga atatgaataa ttitt t t t tatt tttgttgacaa 54 O ttgtaattgt Cactactitat gataatattt agtgacatat atgtcgt.cgg taaaa.gcaaa cactitt cagt gacaaaataa tagatttaat Cacaaaatta tta acct titt ttataataat 660 aaattitat CC Ctaatttata catttaagga caaagtattt tttittatata taaaaaatag 72 O t ctittagtga cgatcgtagt gttgagticta gaaat cataa tgttgaatct agaaaaatct

Catgcagtgt aaaataalacc tcaaaaagga cgttcagt cc atagaggggg tgtatgtgac 84 O acco Caac Ct Cagcaaaaga aaacCtcCCt t caacaagga catttgcggit gctaaacaat 9 OO ttcaagttctic at Cacacata tatttatt at ataatactaa taaagaatag aaaaggaaag 96.O gtaalacat ca ttaaatcgt.c tttgtatatt tittagtgaca actgattgac gaaatcttitt tcqt cacaca aaatttittag tgacgaaa.ca tgatttatag atgatgaaat tatttgtc.cc 108 O toataatcta atttgttgta gtgat catta citcctttgtt tgttt tattt gtcatgttag 114 O to Cattaaaa. aaaaat at Ct citott citt at gtacgtgaat ggttggalacg gatct attat 12 OO ataatactaa taaagaatag aaaaaggaaa gtgagtgagg titcgagggag agaatctgtt 126 O

US 8,373,022 B2 99 100 - Continued <222s. LOCATION: (1) ... (959) <223> OTHER INFORMATION: CAB-2 promoter <4 OOs, SEQUENCE: 31 gaatt catgt gtgagggcaa ttagtgattg taaaaataaa attgttgttitt gtaaaaaact 6 O tttactgtcg aaattattta gggtgatgaaaaaat cagta aactacgaat gatagcttaa 12 O agagtttcta t caaagtgat tdaggaatag tttgttgcaa attaa acct c taacaaaatg 18O ttittctgttg toggtttitt catct ctacaaa ttittgaattt tatgatgaat tagaaagata 24 O gaatgagtta ctittagattt taaaaggttgttcaagttta caaaacagat tact agaatc 3OO atgattaaaa atttacaa.gc tacatattgt ctaaaccaat gatgttgaac ataccagatg 360 at agtttitt.c agtgtttgaa caatcaattig gatagitttitt atgtttctgc aaaatatgca 42O aataat cagt gtttittgagt ctittgcattt tdatttaaaa gcaaaaacaa citgagtttca 48O aggittaaatt aattacatta t t catgagat titat caggitt agtggataaa citgacaatgg 54 O aatcaatgtt attgtaaatt ggtag tigatgttggacittct aatgttactic tictatgatgt 6OO titcggit catc ggitat cacac tat citt tact tittatttaaa gogaaagat.ca cacaaataag 660 titat ct citat t cagaact at taagctgctt coaaaag act togcaa.catgt gigt ct cqaaa 72 O tgctttggct gcaatgaaaa aat catagca aaa.gctagtg gactagagac to Cacatala 78O gaatagtaaa cqttaaaacc aaaatctoaa aaatccaatgagtaaagaga tatagattac 84 O tt catagata acaaacgtta citcgcaattt toctatataa tocaa.ccct a cct aaccatt 9 OO ttcaat cact ct cact caca agittagt cac caaaaaaaaa aaaaacacaa aaagttt ca 959

<210s, SEQ ID NO 32 &211s LENGTH: 445 &212s. TYPE: DNA <213> ORGANISM: Zea mays 22 Os. FEATURE: <221> NAME/KEY: promoter <222s. LOCATION: (1) ... (445) <223> OTHER INFORMATION: PPCZmil promoter <4 OOs, SEQUENCE: 32 gaattic caaa aatagacacg gcaattittct tatt cacaga aaaaatataa citacaactaa 6 O t ccc caagtic cacagggatt agggat caat citgcaaaact aaaagtact t t tacagttgt 12 O acttgg catg agt catgtga C catgagaga ggcgcacggit to agcaaagc alacataaaat 18O tctic caaacg ggc.ccc.gc.ca cacacgatca ccatcacccc cqggctic ccg acc cagtaca 24 O aataga cacg cacact coca act coccacc catct cogcc gcgcacaccg cccaatcagc 3OO caat ct cotc ctic ctic ct co got ct cagac gag cagoggit togc catcact citccact tcc 360 cacgc.ccgct gcgggctcgc aggcggcaga gaattgttctg. tcc.gc.cggg toggaatttg 42O attcggtcgg attic.cgtgcg cc.gcg 445

<210s, SEQ ID NO 33 &211s LENGTH: 5455 &212s. TYPE: DNA <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of the artificial sequence: recombinant expression vector pubiBI-1

<4 OOs, SEQUENCE: 33 ggggat.cctic tagagt cac Ctgcaggcgg cc.gcact agt gattaggatt C caacgc.gag 6 O cCagga caag Cagga acct tcgtgcgag gcgaggcc.gc ccc.gctc.cga titcgatt.cga 12 O

US 8,373,022 B2 133 134 - Continued

Asp Ser Luell Asn Phe Arg Glu Ile Ser Pro Ala Wall Glin Ser 2O 25 3 O

CaC citc. aag ct c gtt tac Ctg acc Cta tgc titt gcc Ctg gcc to a tot 144 His Luell Llys Lieu. Wall Lell Thir Luell Cys Phe Ala Lell Ala Ser Ser 35 4 O 45 gcc gtg ggit gct tac Ctg CaC att gcc Ctg aac atc. ggit 999 atg Ctg 192 Ala Wall Gly Ala Tyr Lell His Ile Ala Luell ASn Ile Gly Gly Met Lel SO 55 60 a Ca atg Ct c gog tgt gtt gga acc at C gcc atg tto tot gtg CC a 24 O Thir Met Luell Ala Cys Wall Gly Thir Ile Ala Met Phe Ser Wall 65 70 8O gtc tat gag gag agg aag agg titt 999 Ctg Ctg atg ggit gca gcc citc. 288 Wall Glu Glu Arg Arg Phe Gly Luell Luell Met Gly Ala Ala Lel 85 90 95

Ctg gala ggg gct tcg gtt gga cott Ctg att gag citt gcc at a gac titt 336 Lell Glu Gly Ala Ser Wall Gly Pro Luell Ile Glu Lell Ala Ile Asp Phe 105 11 O gac CC a agt atc citc. gtg a Ca 999 titt gt C gga a CC gcc at C gcc ttic 384 Asp Pro Ser Ie Lell Wall Thir Gly Phe Wall Gly Thir Ala Ile Ala Phe 115 12 O 125

999 tgc ttic tot ggc gcc gcc at C at C gcc aag cgc agg gag tac Ctg 432 Gly Cys Phe Ser Gly Ala Ala Ile Ile Ala Arg Arg Glu Tyr Lel 13 O 135 14 O tac citc. ggt get Ctg citc. t cc to c ggc Ctg tog atc. Ctg citc. tgg Ctg Tyr Luell Gly Gly Lell Lell Ser Ser Gly Luell Ser Ile Lell Luell Trp Lel 145 150 155 160

Cag titt gcc acg t cc atc. titt ggc CaC to c tot ggc agc ttic atg titt 528 Glin Phe Ala Thr Ser Ile Phe Gly His Ser Ser Gly Ser Phe Met Phe 1.65 17O 17s gag gtt tact titt ggc Ctg ttg at C ttic Ctg gga tac atg gtg gac 576 Glu Wall Tyr Phe Gly Lell Lell Ile Phe Luell Gly Tyr Met Wall Asp 18O 185 19 O acg cag gag at C atc. gag agg gcg CaC CaC ggc gac atg gat at C 624 Thir Glin Glu Ile Ile Glu Arg Ala His His Gly Asp Met Asp Ile 195 2OO 2O5 aag CaC gcg ct c a CC citc. tto acc gac ttic gtc gcc gtt citc. gt C cgc 672 Lys His Ala Lieu Thir Lell Phe Thir Asp Phe Wall Ala Wall Luell Wall Arg 21 O 215 22O gtc citc. at C at C atg citc. aag aac gca ggc gac aag tcg gag gac aag 72 O Wall Luell Ile Ile Met Lell Lys Asn Ala Gly Asp Lys Ser Glu Asp Lys 225 23 O 235 24 O aag aag agg aag agg 999 t cc tga 744 Lys Lys Arg Llys Arg Gly Ser 245

<210s, SEQ ID NO 38 &211s LENGTH: 247 212. TYPE: PRT &213s ORGANISM: Triticum aestivum

<4 OOs, SEQUENCE: 38

Asp Ala Phe Tyr Ser Thir Ser Ser Ala Ala Ala Ser Gly Trp Gly 5 1O 15

Asp Ser Luell Lys Asn Phe Arg Glu Ile Ser Pro Ala Wall Glin Ser 2O 25 3 O

His Luell Llys Lieu. Wall Lell Thir Luell Phe Ala Lell Ala Ser Ser 35 4 O 45

Ala Wall Gly Ala Tyr Lell His Ile Ala Luell ASn Ile Gly Gly Met Luell SO 55 60

Thir Met Lieu Ala Cys Wall Gly Thir Ile Ala Trp Met Phe Ser Wall Pro US 8,373,022 B2 135 136 - Continued

65 70

Wall Glu Glu Arg Arg Phe Gly Luell Luell Met Gly Ala Ala Lieu. 85 90 95

Lell Glu Gly Ala Ser Wall Gly Pro Luell Ile Glu Lieu Ala Ile Asp Phe 105 11 O

Asp Pro Ser Ile Lell Wall Thir Gly Phe Wall Gly Thir Ala Ile Ala Phe 115 12 O 125

Gly Cys Phe Ser Gly Ala Ala Ile Ile Ala Lys Arg Arg Glu Tyr Lieu. 13 O 135 14 O

Tyr Luell Gly Gly Lell Lell Ser Ser Gly Luell Ser Ile Lieu. Lieu. Trp Lieu. 145 150 155 160

Glin Phe Ala Thir Ser Ile Phe Gly His Ser Ser Gly Ser Phe Met Phe 1.65 17O 17s

Glu Wall Phe Gly Lell Lell Ile Phe Luell Gly Tyr Met Val Tyr Asp 18O 185 19 O

Thir Glin Glu Ile Ile Glu Arg Ala His His Gly Asp Met Asp Tyr Ile 195 2O5

His Ala Luell Thir Lell Phe Thir Asp Phe Wall Ala Val Lieu Val Arg 21 O 215 22O

Wall Luell Ile Ile Met Lell Asn Ala Gly Asp Llys Ser Glu Asp Llys 225 23 O 235 24 O

Arg Arg Gly Ser 245

SEO ID NO 39 LENGTH: 1293 TYPE: DNA ORGANISM: Hordeum vulgare FEATURE: NAME/KEY: CDS LOCATION: (173) . . (1126) OTHER INFORMATION: coding for Hordeum vulgare subsp. vulgare syntaxin (Ror2)

<4 OOs, SEQUENCE: 39 gtaact aacc cct tct tcct c ccttgtc.ca citcc.gctitct c cc catccaa gaalacagcgc 6 O

Caac agct Co acc catcgag gagaatcaag aaaccgc.gc.c ggcgtggtga t Caagga cat 12 O c catcgat.cg atcgaccgac cct gccttgc ctdag to aac ccggcggcag cc atgaac 178 Met Asn 1. aac citc. ttic tog agc tcg tgg aag cgg gcg ggc gcg gig gc gaC ggg 226 Asn Luell Phe Ser Ser Ser Trp Lys Arg Ala Gly Ala Gly Gly Asp Gly 10 15 gac Ctg gag tog ggc ggc ggc ggc gtg gag atg acg gC9 cc.g. CC9 gC 274 Asp Luell Glu Ser Gly Gly Gly Gly Wall Glu Met Thr Ala Pro Pro Gly 2O 25 3O gcc gcg gcg 999 gcg agc Ctg gac cgc ttic titc gag gac gtg gag ticg 322 Ala Ala Ala Gly Ala Ser Lell Asp Arg Phe Phe Glu Asp Val Glu Ser 35 4 O 45 SO atc. aag gac gac Ctg cgg gag Ctg gag cgg atc. cag cqc toc ct c cac 37 O Ile Lys Asp Asp Lell Arg Glu Luell Glu Arg Ile Glin Arg Ser Lieu. His 55 60 65 gac ggc aac gag tcg ggc aag tog citc. CaC gac gcg tcg gC9 gtg C9C 418 Asp Gly Asn Glu Ser Gly Lys Ser Luell His Asp Ala Ser Ala Val Arg 70 7s 8O gcg citc. cgc to c cgc atg gac gcc gac gtg gcc gcc gcc at C aag aag 466 Ala Luell Arg Ser Arg Met Asp Ala Asp Wall Ala Ala Ala Ile Llys Llys 85 9 O 95

US 8,373,022 B2 139 140 - Continued

SO 55 60

Lell His Asp Gly Asn Glu Ser Gly Ser Luell His Asp Ala Ser Ala 65 70

Wall Arg Ala Luell Arg Ser Arg Met Asp Ala Asp Wall Ala Ala Ala Ile 85 90 95

Ala Lys Wall Wall Luell Arg Luell Glu Ser Lell Asp Arg Ala 105 11 O

Asn Ala Ala Asn Arg Ser Wall Ala Gly Gly Pro Gly Ser Ser Thir 115 12 O 125

Asp Arg Thir Arg Thir Ser Wall Wall Ala Gly Luell Arg Luell Arg 13 O 135 14 O

Asp Ala Met Glu Ser Phe Ser Ser Luell Arg Ser Arg Ile Thir Ser Glu 145 150 155 160

Arg Glu Thir Wall Ala Arg Arg Phe Thir Wall Thir Gly Ser Glin 1.65 17O 17s

Pro Asp Glu Ala Thir Lell Asp Thir Luell Ala Glu Thir Gly Glu Gly Glu 18O 185 19 O

Arg Luell Luell Glin Arg Ala Ile Ala Glu Glin Glin Gly Arg Gly Glu Wall 195

Lell Gly Wall Wall Ala Glu Ile Glin Glu Arg His Gly Ala Wall Ala Asp 21 O 215 22O

Lell Glu Arg Ser Lell Lell Glu Luell Glin Glin Wall Phe Asn Asp Met Ala 225 23 O 235 24 O

Wall Luell Wall Ala Ala Glin Gly Glu Glin Luell Asp Asp Ile Glu Gly His 245 250 255

Wall Gly Arg Ala Arg Ser Phe Wall Asp Arg Gly Arg Glu Glin Luell Glin 26 O 265 27 O

Wall Ala Arg His Glin Ser Ser Arg Trp Thir Phe Ile Gly 28O 285

Ile Gly Ile Luell Lell Wall Wall Ile Luell Ile Ile Wall Ile Pro Ile Wall 29 O 295 3 OO

Lell Asn Thir Asn Lys Ser Asn Asn Asn ASn Ser Glin Glin 3. OS 310 315

SEQ ID NO 41 LENGTH: 948 TYPE: DNA ORGANISM: Arabidopsis thaliana FEATURE: NAME/KEY: CDS LOCATION: (1) ... (945) OTHER INFORMATION: coding for Arabidopsis thaliana syntaxin 121 (SYP121) / syntaxin-related protein (SYR1) (At3 g1182O)

SEQUENCE: 41 atg gcg aat cc c gcg gga toa acc ggt ggt gtg aac citc. gac aag ttic 48 Met Ala Asn Pro Ala Gly Ser Thir Gly Gly Wall Asn Lell Asp Lys Phe 1. 5 15 tto gala gat gtt gaa tot gtg a.a.a. gala gag Cta aag gag Cta gat cgg 96 Phe Glu Asp Wall Glu Ser Wall Glu Glu Luell Lys Glu Luell Asp Arg 2O 25 3 O citc. aac gala aca citc. tot toa tgt CaC gag cag agc aag acg citt CaC 144 Lell Asn Glu Thir Lell Ser Ser Cys His Glu Glin Ser Lys Thir Luell His 35 4 O 45 aat gct a.a.a. gcc gtt a.a.a. gat citc. cgg tot a.a.a. atg gac ggt gac gtt 192 Asn Ala Ala Wall Asp Luell Arg Ser Lys Met Asp Gly Asp Wall SO 55 60

US 8,373,022 B2 143 144 - Continued

Asn Ala Ala Wall Asp Luell Arg Ser Lys Met Asp Gly Asp Wall SO 55 60

Gly Wall Ala Luell Lys Lys Ala Met Ile Lys Wall Lys Luell Glu Ala 65 70

Lell Asp Arg Ala Asn Ala Ala Asn Arg Ser Luell Pro Gly Gly Pro 85 90 95

Gly Ser Ser Ser Asp Arg Thir Arg Thir Ser Wall Lell Asn Gly Luell Arg 105 11 O

Luell Met Asp Ser Met Asp Ser Phe ASn Arg Lell Arg Glu Luell 115 12 O 125

Ile Ser Ser Glu Tyr Arg Glu Thir Wall Glin Arg Arg Phe Thir Wall 13 O 135 14 O

Thir Gly Glu Asn Pro Asp Glu Arg Thir Luell Asp Arg Lell Ile Ser Thir 145 150 155 160

Gly Glu Ser Glu Arg Phe Lell Glin Ala Ile Glin Glu Glin Gly Arg 1.65 17O 17s

Gly Arg Wall Luell Asp Thir Ile Asn Glu Ile Glin Glu Arg His Asp Ala 18O 185 19 O

Wall Asp Ile Glu Asn Luell Arg Glu Luell His Glin Wall Phe Luell 195

Asp Met Ala Wall Lell Wall Glu His Glin Gly Ala Glin Lell Asp Asp Ile 21 O 215

Glu Ser His Wall Gly Arg Ala Ser Ser Phe Ile Arg Gly Gly Thir Asp 225 23 O 235 24 O

Glin Luell Glin Thir Ala Arg Wall Glin Lys ASn Thir Arg Trp Thir 245 250 255

Ile Ala Ile Ile Ile Lell Ile Ile Ile Ile Thir Wall Wall Wall Luell 26 O 265 27 O

Ala Wall Luell Pro Trp Asn Asn Ser Ser Gly Gly Gly Gly Gly Gly 28O 285

Gly Gly Gly Gly Thir Thir Gly Gly Ser Glin Pro Asn Ser Gly Thir Pro 29 O 295 3 OO

Pro Asn Pro Pro Glin Ala Arg Arg Luell Luell Arg 3. OS 310 315

SEQ ID NO 43 LENGTH: 1275 TYPE: DNA ORGANISM: Hordeum vulgare FEATURE: NAME/KEY: CDS LOCATION: (80) ... (1006) OTHER INFORMATION: coding for Hordeum vulgare subsp. vulgare SNAP-34

<4 OOs, SEQUENCE: 43 ggcc cct coa ccccaccc.ca cccagt cqct gcggatactt gattctgcta citcggc.cagc 6 O gatcgatcto goctocqcc atg agc gcc acc agg ccc toc ttic titc ccc ticc 112 Met Ser Ala Thr Arg Pro Ser Phe Phe Pro Ser 1. 1. O aac aac aac agg aac aag c cc gcc acc cgg aac c cc tto gac to c gac 16 O Asn Asn Asn Arg Asn Lys Pro Ala Thir Arg ASn Pro Phe Asp Ser Asp 15 2O 25 tcg gac gac gac ggc ggc atg gcc cgg cgc ggc cc.g gcg cgg gcc tog Ser Asp Asp Asp Gly Gly Met Ala Arg Arg Gly Pro Ala Arg Ala Ser 3 O 35 4 O t cc gt C acc c cc gcc 999 gcc agg gcc t cc tog gcc 256

US 8,373,022 B2 147 148 - Continued

212. TYPE: PRT <213> ORGANISM: Hordeum vulgare <4 OOs, SEQUENCE: 44

Met Ser Ala Thr Arg Pro Ser Phe Phe Pro Ser Asn Asn Asn Arg Asn 1. 5 1O 15 Llys Pro Ala Thir Arg ASn Pro Phe Asp Ser Asp Ser Asp Asp Asp Gly 2O 25 3 O

Gly Met Ala Arg Arg Gly Pro Ala Arg Ala Ser Ser Val Pro Thr Pro 35 4 O 45

Ala Ala Gly Pro Ala Arg Ala Ser Ser Ala Pro Ile Pro Ala Asp Glu SO 55 60 Ala Asp Glin Arg Gly Ala Lieu. Phe Gly Ala Gly Pro Ala Pro Ser Gly 65 70 7s Phe Ala Ser Ser Ser Ser Ala Ala Ala Arg Gly Arg Tyr Arg Asn Asp 85 90 95

Phe Arg Asp Ser Gly Gly Val Glu Ala Glin Ser Val Glin Glu Lieu. Glu 1OO 105 11 O Gly Tyr Ala Ala Tyr Lys Ala Glu Glu Thir Thr Arg Arg Val Asp Gly 115 12 O 125

Cys Lieu. Arg Val Ala Glu Glu Met Arg Asp Thr Ala Ser Lys Thr Luell 13 O 135 14 O

Lieu. Glin Val His Glin Glin Gly Glin Glin Ile Arg Arg Thr His Ala Met 145 150 155 160 Ala Val Asp Ile Asp Glin Asp Lieu. Ser Arg Gly Glu Lys Lieu. Lieu. Gly 1.65 17O 17s Asp Lieu. Gly Gly Lieu. Phe Ser Lys Llys Trp Llys Pro Llys Lys Asn Gly 18O 185 19 O Ala Ile Arg Gly Pro Met Lieu. Thir Arg Asp Asp Ser Phe Ile Arg 195 2OO 2O5

Gly Ser His Met Glu Glin Arg His Llys Lieu. Gly Lieu. Ser Asp Arg Pro 21 O 215 22O

His Arg Ser Asn Ala Arg Glin Phe Leu Ser Glu Pro Thr Ser Gly Luell 225 23 O 235 24 O Glu Lys Val Glu Val Glu Lys Ala Lys Glin Asp Asp Gly Lieu. Ser Asp 245 250 255 Lieu. Ser Asp Ile Lieu. Thr Glu Lieu Lys Gly Met Ala Ile Asp Met Gly 26 O 265 27 O Thr Glu Ile Glu Gly Glin Thr Lys Asp Lieu. Gly His Ala Glu Lys Asp 27s 28O 285 Phe Asp Glu Lieu. Asn Tyr Arg Val Lys Gly Ala Asn Ala Arg Thr Arg 29 O 295 3 OO Arg Lieu. Lieu. Gly Arg 3. OS

<210s, SEQ ID NO 45 &211s LENGTH: 1398 &212s. TYPE: DNA <213> ORGANISM: Glycine max 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (212) ... (946) 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1367) . . (1367) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1368) . . (1368) US 8,373,022 B2 149 150 - Continued <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1369) . . (1369 <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1370) . . (1370) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1371) . . (1371 <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1372) . . (1372) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1373) . . (1373) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1374) . . (1374) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1375) . . (1375) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1376) (1376) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1377) . . (1377) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1378) . . (1378) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1379) . . (1379) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1380) . . (1380) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1381) . . (1381 <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1382) . . (1382) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1383) . . (1383) <223> OTHER INFORMATION: n is a, c, g, or t 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1384) . . (1384) <223> OTHER INFORMATION: n is a, c, g, or t <4 OOs, SEQUENCE: 45 ggaattitc.cg tdgcgaccca toggacgctic gtc.cgacgca toaac actac ggc.cgactgg 6 O aggcgacttgaatgaaaggg aggtaatctt aagtggalacc gtttgcaaaa ttaccc.gttc 12 O citcgtacctic gaatgcagtic taaacttggg gtc.gctgaag to acago cat atacgacact 18O agacat attt gtgttgtct co gattgcaa.gc a atg gac toc titc aat tcc titc 232 Met Asp Ser Phe Asn Ser Phe 1. 5 titc gat tca aca aac cqa togg aat tac gat act citc aaa aac titc cqt 28O Phe Asp Ser Thir Asn Arg Trp Asn Tyr Asp Thir Lieu Lys Asn. Phe Arg

US 8,373,022 B2 153 154 - Continued

<4 OOs, SEQUENCE: 46

Met Asp Ser Phe Asn Ser Phe Phe Asp Ser Thir Asn Arg Trp Asn Tyr 1. 15

Asp Thir Luell Lys Asn Phe Arg Glin Ile Ser Pro Wall Wall Glin Asn His 2O 25 3 O

Lell Wall Tyr Phe Thir Luell Phe Ala Wall Wall Ala Ala Ala 4 O 45

Wall Gly Lell His Wall Luell Luell Asn Ile Gly Gly Phe Luell Thir SO 55 60

Thir Wall Wall Gly Ser Ser Wall Trp Luell Luell Ser Thir Pro Pro 65 70 7s

Phe Glu Arg Lys Arg Val Thir Luell Luell Met Ala Ala Ser Luell Phe 85 90 95

Glin Gly Ser Ile Gly Pro Luell Ile Asp Lieu. Ala Ile Glin Ile Asp 105 11 O

Pro Ser Ile Phe Ser Ala Phe Wall Gly Thir Ser Lell Ala Phe Ala 12 O 125

Phe Ser Gly Ala Ala Lieu Wall Ala Arg Arg Arg Glu Luell Tyr 13 O 135 14 O

Lell Gly Gly Luell Wall Ser Ser Gly Luell Ser Ile Lieu. Lell Luell His 145 150 155 160

Phe Ala Ser Ser Ile Phe Gly Gly Ser Thir Ala Lieu. Phe Phe Glu 1.65 17s

Lell Tyr Phe Gly Lell Lieu Wall Phe Wall Gly Tyr Ile Wall Wall Asp Thir 18O 185 19 O

Glin Glu Ile Wall Glu Arg Ala His Luell Gly Asp Lieu. Asp Wall 195

His Ala Luell Thir Lell Phe Thir Asp Luell Wall Ala Wall Phe Wall Arg Ile 21 O 215 22O

Lell Wall Ile Met Lell Lys Asn Ser Thir Glu Arg Asn Glu Lys 225 23 O 235 24 O Lys Arg Arg Asp

SEO ID NO 47 LENGTH: TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: GFP-Primer 1

<4 OOs, SEQUENCE: 47 atggtgagca agggcgagga

SEQ ID NO 48 LENGTH: 43 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: GFP-Primer 2

<4 OOs, SEQUENCE: 48 ttgaacaacg atgtgcaaga citccttgtac agct cqtcca togc

SEQ ID NO 49 LENGTH: 6 TYPE : PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: BI1 consensus motif US 8,373,022 B2 155 156 - Continued

22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Xaa is Lieu or Ile 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Xaa is any amino acid <4 OOs, SEQUENCE: 49 His Xaa Lys Xaa Val Tyr 1. 5

<210s, SEQ ID NO 50 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: BI1 consensus motif 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Xaa is any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa is Tyr or Phe 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Xaa is any amino acid <4 OOs, SEQUENCE: 50 Ala Xaa Gly Ala Xaa Xaa His 1. 5

<210s, SEQ ID NO 51 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: BI1 consensus motif

<4 OOs, SEQUENCE: 51 Asn. Ile Gly Gly 1.

<210s, SEQ ID NO 52 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: BI1 consensus motif 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Xaa is Wall or Pro 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Xaa is Tyr or Phe 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa is Glu or Glin 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Xaa is Arg or Glin <4 OOs, SEQUENCE: 52 Pro Xaa Xala Glu Xaa Xaa Lys Arg 1. 5 US 8,373,022 B2 157 158 - Continued

SEO ID NO 53 LENGTH: 8 TYPE PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: BI1 consensus motif FEATURE: NAME/KEY: MISC FEATURE LOCATION: (1) ... (1) OTHER INFORMATION: Xaa is Glu or Glin FEATURE: NAME/KEY: MISC FEATURE LOCATION: (3) ... (3) OTHER INFORMATION: Xaa is Ala or Ser FEATURE: NAME/KEY: MISC FEATURE LOCATION: (5) . . (5) OTHER INFORMATION: Xala is Wall or Ile

<4 OOs, SEQUENCE: 53 Xaa Gly Xaa Ser Xaa Gly Pro Lieu. 1. 5

SEO ID NO 54 LENGTH: 5 TYPE PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: BI1 consensus motif FEATURE: NAME/KEY: MISC FEATURE LOCATION: (3) ... (3) OTHER INFORMATION: Xala is Ser or Gly FEATURE: NAME/KEY: MISC FEATURE LOCATION: (4) ... (4) OTHER INFORMATION: Xaa is Lieu or Ile FEATURE: NAME/KEY: MISC FEATURE LOCATION: (5) . . (5) OTHER INFORMATION: Xaa is Ile or Lell

<4 OOs, SEQUENCE: 54 Asp Pro Xaa Xala Xaa 1. 5

SEO ID NO 55 LENGTH: 11 TYPE PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: BI1 CCS eSS motif FEATURE: NAME/KEY: SC FEATURE LOCATION: (2) ... (2) OTHER INFORMATION: Xaa is Gly o Ala FEATURE: NAME/KEY: SC FEATURE LOCATION: (4) ... (4) OTHER INFORMATION: Xaa is Ala o Ser FEATURE: NAME/KEY: SC FEATURE LOCATION: (5) . . (5) OTHER INFORMATION: Xaa is Luell o Ile FEATURE: NAME/KEY: SC FEATURE LOCATION: (8) ... (8) OTHER INFORMATION: Xaa is Ala o Gly FEATURE: NAME/KEY: SC FEATURE LOCATION: (11) . . (11) OTHER INFORMATION: Xaa is Ser o Thir

<4 OO > SEQUENCE: 55 US 8,373,022 B2 159 160 - Continued

Val Xala Thr Xaa Xaa Ala Phe Xaa Cys Phe Xaa 1. 5 1O

<210s, SEQ ID NO 56 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: BI1 consensus motif 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: Xaa is Tyr or Phe <4 OOs, SEQUENCE: 56 Tyr Lieu. Xaa Lieu. Gly Gly 1. 5

<210s, SEQ ID NO 57 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: BI1 consensus motif

<4 OO > SEQUENCE: 57 Glu Tyr Lieu. Tyr Lieu. Gly Gly 1. 5

<210s, SEQ ID NO 58 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: BI1 consensus motif 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Xaa is Lieu or Wall 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa is Gly or Trp 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (7) . . (7) <223> OTHER INFORMATION: Xaa is Ser or Thr 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: Xaa is Ile or Met 22 Os. FEATURE: <221 > NAMEAKEY: SC FEATURE <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: Xaa is Lieu or Met

<4 OOs, SEQUENCE: 58 Lieu. Xaa Ser Ser Xaa Lieu. Xaa Xala Lieu. Xaa Trp 1. 5 1O

<210s, SEQ ID NO 59 &211s LENGTH: 7 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: BI1 consensus motif 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Xaa is any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE US 8,373,022 B2 161 162 - Continued <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa is Ile or Wall 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (6) . . (6) <223> OTHER INFORMATION: Xaa is Ile or Wall

<4 OO > SEQUENCE: 59 Asp Thr Gly Xaa Xaa Xaa Glu 1. 5

<210s, SEQ ID NO 60 &211s LENGTH: 237 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 60 Met Asn. Ile Phe Asp Arg Lys Ile Asin Phe Asp Ala Lieu. Lieu Lys Phe 1. 5 1O 15 Ser His Ile Thr Pro Ser Thr Glin Gln His Leu Lys Llys Val Tyr Ala 2O 25 3 O Ser Phe Ala Lieu. Cys Met Phe Val Ala Ala Ala Gly Ala Tyr Val His 35 4 O 45 Met Val Thr His Phe Ile Glin Ala Gly Lieu. Leu Ser Ala Leu Gly Ser SO 55 60 Lieu. Ile Leu Met Ile Trp Leu Met Ala Thr Pro His Ser His Glu Thr 65 70 7s 8O Glu Gln Lys Arg Lieu. Gly Lieu. Lieu Ala Gly Phe Ala Phe Lieu. Thr Gly 85 90 95 Val Gly Lieu. Gly Pro Ala Lieu. Glu Phe Cys Ile Ala Val Asn Pro Ser 1OO 105 11 O Ile Leu Pro Thr Ala Phe Met Gly Thr Ala Met Ile Phe Thr Cys Phe 115 12 O 125 Thir Lieu. Ser Ala Lieu. Tyr Ala Arg Arg Arg Ser Tyr Lieu. Phe Lieu. Gly 13 O 135 14 O Gly Ile Lieu Met Ser Ala Lieu. Ser Lieu. Lieu. Lieu Lleu Ser Ser Lieu. Gly 145 150 155 160 Asn Val Phe Phe Gly Ser Ile Trp Leu Phe Glin Ala Asn Lieu. Tyr Val 1.65 17O 17s Gly Lieu Val Val Met Cys Gly Phe Val Lieu. Phe Asp Thr Glin Lieu. Ile 18O 185 19 O Ile Glu Lys Ala Glu. His Gly Asp Glin Asp Tyr Ile Trp His Cys Ile 195 2OO 2O5 Asp Lieu. Phe Lieu. Asp Phe Ile Thr Val Phe Arg Llys Lieu Met Met Ile 21 O 215 22O Lieu Ala Met Asn. Glu Lys Asp Llys Llys Lys Glu Lys Llys 225 23 O 235

<210s, SEQ ID NO 61 &211s LENGTH: 244 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Consensus sequence 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (239) <223> OTHER INFORMATION: Xaa at positions 2, 3, 5-13, 15, 22, 24, 28, 31, 35, 38, 42, 45, 49, 56, 57, 62, 65. 73, 76, 78. 87, 88, 106, 168, 169, 171, 2O2, 222, 234, 237 and 239 can be any amino acid

<4 OOs, SEQUENCE: 61 US 8,373,022 B2 163 164 - Continued

Phe Xaa Xala Ser Xaa Xala Xala Xala Xala Xaa Xala Xala Xala Trp Xaa 15

Asp Ser Lieu Lys Asn. Xaa Arg Xaa Ile Ser Pro Xala Wall Glin Xaa His 25 3 O

Lell Xaa Wall Tyr Xaa Thir Luell Xaa Ala Lell Xaa Ala Ser Ala 35 4 O 45

Xaa Gly Ala Tyr Lieu. His Wall Xaa Xala Asn. Ile Gly Gly Xaa eu. Thir SO 55 60

Xala Luell Gly Ile Gly Ser Ile Xaa Trp Luell Xala Ser Xaa Wall 65 70 8O

Glu Glu Arg Llys Arg Xaa Xala Luell Lieu Met Ala Ala Ala Cell Luell 85 90 95

Glu Gly Ala Ser Wall Gly Pro Leu Ile Xaa Lieu Ala Ile Asp Phe Asp 105 11 O

Pro Ser Ile Luell Wall. Thir Ala Phe Wall Gly Thir Ala Ile Ala Phe Ala 115 12 O 125

Phe Ser Gly Ala Ala Ile Wall Ala Arg Arg Glu Lieu. Tyr 13 O 135 14 O

Lell Gly Gly Lieu. Luell Ser Ser Gly Luell Ser Ile Lieu. Luell Trp Lel Glin 145 150 155 160

Phe Ala Ser Ser Ile Phe Gly Xaa Xala Ser Xaa Ala Ser Phe Met Phe 1.65 17O 17s

Glu Wall Phe Gly Lieu. Lieu. Ile Phe Leu Gly Tyr Met Wall Asp 18O 185 19 O

Thir Glin Glu Ile Ile Glu Arg Ala His Xaa Gly Asp Met Asp Ile 195 2O5

His Ala Luell Thir Lell Phe Thir Asp Phe Wall Ala Wall Xaa Val Arg 21 O 215

Ile Luell Ile Ile Met Leu Asn Ala Xala Asp Xaa Glu Xaa Lys 225 23 O 235 24 O Arg Arg

We claim: 3. The method of claim 1, wherein the BI1 protein com 1. A method of generating or increasing resistance to at prises at least one sequence which has at least 98% sequence least one biotrophic fungus of the genus Phakopsora in a 45 homology with at least one BI1 consensus motif selected from plant, plant part, plant tissue, or plant cell, comprising: the group consisting of a) increasing protein quantity or function of at least one Bax inhibitor-1 (BI1) protein in a plant or part thereof in comparison with a control plant or part thereof by: (SEQ ID NO: 49) i) stably transforming a plant cell with a recombinant 50 a) H (LAI) KXVY, expression cassette comprising a nucleic acid (SEO ID NO : 50) sequence encoding the BI1 protein operably linked to b) AXGA (YAF) XH, a heterologous tissue-specific promoter, ii) regenerating a plant from said plant cell, and (SEQ ID NO: 51) iii) expressing the nucleic acid sequence encoding the 55 c) NIGG, BI1 protein in at least one part or tissue of said plant, b) selecting a plant or part thereof having resistance to at (SEQ ID NO: 52) least one biotrophic fungus of the genus Phakopsora d) P (V/P) (Y/F) E (E/O) (R/Q) KR, generated or increased in comparison with a control (SEO ID NO : 53) plant or part thereof, 60 e) (EAQ) G (AAS) S (VAI) GPL, wherein the plant is a soybean plant and the BI1 protein comprises an amino acid sequence having at least 90% (SEQ ID NO: 54) sequence identity with the amino acid sequence of SEQ f) DP (SAG) (LAI) (IAL), ID NO: 6. (SEO ID NO : 55) 2. The method of claim 1, wherein the at least one 65 g) V (G/A) T (A/S) (L/I) AF (A/G) CF (S/T), biotrophic fungus is Phakopsora pachyrhizi or Phakopsora meibomiae. US 8,373,022 B2 165 166 - Continued 8. A recombinant soybean plant comprising at least one (SEO ID NO. 56) expression cassette of claim 6 or at least one vector compris h) YL (YAF) LGG, ing said expression cassette. (SEO ID NO. 58) 9. The recombinant soybean plant of claim 8, wherein the i) L (LAW) SS (G/W) L (SAT) (IAM) L (LAM)W, 5 Soybean plant has an mlo-resistant phenotype. and 10. The method of claim 1, wherein the tissue-specific promoter is an epidermis-specific promoter. (SEO ID NO. 59) 11. The method of claim 1, wherein the BI1 protein com j) DTGX (I/V) (I/V) E. prises an amino acid sequence having at least 98% sequence 4. The method of claim 1, wherein expression of the BI1 10 identity with the amino acid sequence of SEQID NO: 6. protein is increased at least in epidermis, essentially tissue 12. The method of claim 1, wherein the BI1 protein com specifically in epidermis, and/or essentially not increased in prises an amino acid sequence having at least 95% sequence mesophyll. identity with the amino acid sequence of SEQID NO: 6. 5. The method of claim 1, wherein the plant additionally 13. The method of claim 1, wherein the BI1 protein com has: 15 prises the amino acid sequence of SEQ ID NO: 6 or is a) an mlo-resistant phenotype, encoded by the polynucleotide sequence of SEQID NO: 5. b) inhibited or reduced expression or function of MLO, 14. The recombinant expression cassette of claim 6. RacB and/or NaOx at least in epidermis in comparison wherein the BI1 protein comprises an amino acid sequence with a control plant, and/or having at least 98% sequence identity with the amino acid c) increased expression or function of PEN2, SNAP34 sequence of SEQID NO: 6. and/or PEN1 at least in epidermis in comparison with a 15. The recombinant expression cassette of claim 6, control plant. wherein the BI1 protein comprises an amino acid sequence 6. A recombinant expression cassette comprising a nucleic having at least 95% sequence identity with the amino acid acid sequence encoding a BI1 protein operably linked to a sequence of SEQID NO: 6. heterologous epidermis-specific promoter, wherein the BI1 25 16. The recombinant expression cassette of claim 6, protein comprises an amino acid sequence having at least wherein the BI1 protein comprises the amino acid sequence of 90% sequence identity with the amino acid sequence of SEQ SEQID NO: 6 or is encoded by the polynucleotide sequence ID NO: 6. of SEQ ID NO: 5. 7. A recombinant vector comprising the expression cas sette of claim 6.