US 2004OO67506A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0067506 A1 Scheres et al. (43) Pub. Date: Apr. 8, 2004 (54) NOVEL ROOT SPECIFIC PROMOTER Publication Classification DRIVING THE EXPRESSION OF ANOVEL LRR RECEPTOR-LIKE KINASE 51) Int.nt. Cl.C.7 ............................ C12O 1/68; CO7H 21/04 C07K 14/705; C12P 21/02 (76) Inventors: Ben Scheres, Ac Utrecht (NL); Renze (52) U.S. Cl. ......................... 435/6; 435/69.1; 435/320.1; Heidstra, Lh Utrecht (NL) 435/325; 530/350, 536/23.5 Correspondence Address: (57) ABSTRACT Ann R Pokalsky The present invention relates to the field of plant molecular Dilworth & Barrese biology, more particularly to the root-specific gene expres 333 Earle Ovington Boulevard Sion in plants. The invention provides nucleic acids for a Uniondale, NY 11553 (US) novel transcriptional regulatory root-specific promoter and nucleic acid and protein Sequences coding for a new LRR (21) Appl. No.: 10/433,731 receptor-kinase protein, further Specified as a root clavata 1 homolog (RCH1). Further provided are compositions com (22) PCT Filed: Dec. 4, 2001 prising nucleic acids, polypeptides, antibodies and vectors. The invention further provides for methods for modifying (86) PCT No.: PCT/EPO1/14154 cell fate and/or plant development and/or plant morphology Related U.S. Application Data and/or plant biochemistry and/or plant physiology compris ing the modification of expression in particular cells, tissues (60) Provisional application No. 60/256,204, filed on Dec. or organs of a plant of the novel LRR receptor-like kinase or 15, 2000. comprising the expressing of a gene of interest under the control of the novel transcriptional regulatory root-specific (30) Foreign Application Priority Data promoter. Further are provided compounds interacting with the new polypeptides for use as herbicides or growth regu Dec. 4, 2000 (EP)........................................ OO87O288.8 lators. Patent Application Publication Apr. 8, 2004 Sheet 1 of 40 US 2004/0067506 A1 Figure 1 Carrant opinion in Part slokogy Patent Application Publication Apr. 8, 2004 Sheet 2 of 40 US 2004/0067506 A1 Primers used in experiments described SECR D No. Primer Sequence (5'-3' Details SEC ID No. 6 LRR1 AAYAAYTTMASYGGTKMDWTHCC Degenerate LRR primer SEC ID No. 7 LRR2 CARYTHAMCGGWGAAAHCC Degenerate LRR primer SEQID No. 8 LRR3 MAYYTSAYGGAHCTATHCC Degenerate LRR primer SEOD No. 9 KIN1 THCCWTTWGSCATGTANTCATA Degenerate kinase primer SECR D No. 10 KN2 AYTCDGGOGCHAHGTADCC Degenerate kinase primer SEQD No. 11 KN3 MAYHCCRAARCTRTAVACATC Degenerate kinase primer SEO D No. 12 RCHF CGATCAGACACAAGAACAT RCH1 specific primer SEQ ID No. 13 RCHR AGCAATGGTGTTGGAAGAA RCH1 specific primer SECD No. 14 UBF TGCAGATCTTYGGAAGAC Compl. to 5' end of repeating ubiquitin unit SEQD No. 15 UBIR GACTCCTTCTGGATGTTG Compl. to 3' end of repeating ubiquitin unit SEQID No. 16 M13F TGTAAAACGACGGCCAGT Compl. to p3S sequence SEQ ID No. 17 PRCH1R CACACAGGATCCAATCGGCATTT Compl. to RCH1 promoter, includes small GCAAAGACATA Coding sequence and BamH site Patent Application Publication Apr. 8, 2004 Sheet 3 of 40 US 2004/0067506 A1 Figure 3 Patent Application Publication Apr. 8, 2004 Sheet 5 of 40 US 2004/0067506 A1 GTCTAGCGGTATTAGGCGTGGGCGGATACGAGCAAACAAATGATTCGAGATGATAACGACAGAGACTGGAGA AAAICTAGGACATGGCAATTCACACCTTTTCAGAAACCAACTTCACAGTCGAACATGTACCAAGTGTTTGGTAGAAG GTAATGITATAGGGAAAGGTTGCTCCGGGATAGGTACAAAGCTGAAAGCCTAACAGAGAAGTCATCGCGGTGAAAAAG Figure 5-2 CTCTGGCCAGTGACAGACCTAATCGAATGAGAAAACTAAGTCATCAGGAGTTCGAGACTCATTCTCAGCTGAAGAAA AACACTGGACGACAGACACAAGAACATGTACGCTCTTGGGATGTGTGGAACAAGAACACTAGACTTCTTAGT AGATTATATGTCAAATGGGAGTTTGGGAAGTIGCTTCACGAAAGGAGCGGTGTATGTAGCTTAGGATGGGAAGTGAGG TACAAGATTATACTTGGTGCAGCTCAGGGTTTGGCTTACTTGCACCATGACTGTGTTCCTCCCATTGTTCATAGAGACAT CAAGGCAAATAATATTCTGATTGGCCCGATTTTGAACCTTACATTGGAGATTTCGGACTCGCTAAGCTTGTTGAGATG GCGACTITGCTCGTTCTTCCAACACCATTGCTGGTTCCTATGGTTACATAGCTCCAGGTACTTCCCAAATTTCTGTTTTG CTGCTAATTAGTTAACTTGATACACATAAGTACTAGAGCAGCCTAGTAATGGTAGAAATTCAGGTTTAAGT GGTTTGGTTTGGCATTTTGAAAAGAATAGTATTGGTTCAGTGATTTCGATTTGGTTAAAATTTTAGAAAAAA AATGGTAGAAGAAATAATTTTTGATTAATATATACACTACTGIATACGATTTAAAAAAAAAATTGATTGGTTTGGT CTCGATATTTTGCTTGTAGCTTGGTTCAATATTTACTATATATATGATTCATGACTTTCATACTAATCAATATTTGTT CGGTTTCGTTTTGATTAGCTGAAAGTGAATCATATAGAAAAATGACCAACTATCATATACTTACACGCTACAAG ATTTTCGATGTAATTTTGGTGTAAGCATTTIGGTTTTATATGTTCATCCTTAGTAACTAACAACAAATATCAATTGGGA AGCATAACGTTATATATTCCCATAAGGCTATAACACTATAATTATGCAGAATACGGATACTCAATGAAGATAACAGAGAA AAGCGACGTGTACAGCTATGGAGTCGAGTGCTCGAGGACTAACGGGTAAGCAACCAATCGACCAACGATACCAGATG GACTCCACATAGTGGACTGGGTCAAGAAAATCAGAGACATACAAGTAATCGACCAAGGACTACAAGCAAGACCAGAGTCA GAGGTTGAAGAGATGATGCAAACGCTAGGAGTCGCGCTTTTAGCATTAATCCAATACCCGAAGACAGGCCTACAATGAA AGATGTGGCTGCTATGCTAGGAGATATGTCAAGAGAGAGAGGAATCGATGAAAGTTGATGGTTGCTCGGGAAGTGTA ACAATGGAAGAGAACGTGGCAAAGATGATTCGACTTCATCGGTTATGCAACAAACGGCTAAGTATTGAGAAGTAGTAGC ACGAGTTCTCTGCGTCTTCTTTGCTTTACTCTTCTTCTTCTTCTGCTACTTCTAATGTTAGACCAAATCTTAAATAA Patent Application Publication Apr. 8, 2004 Sheet 7 of 40 US 2004/0067506 A1 Figure 7 Patent Application Publication Apr. 8, 2004 Sheet 10 of 40 US 2004/0067506A1 Figure 10-1 CLV1 1 . mamrillkthl. ... lflhlyliffspcfayt. dimevillnlkssm 38 . : . : RCH1 1 MSLQMPIPRKKALTVSHFSITLSLFLAFFISSTSASTNEVSALISWLHSS 50 39 igpkghglhdwihss spdahcsfsgvscdddarvislinv. 77 : . : 51 NSPPPSVFSGWNPSDSDPCQWPYITCSSPDNKLVTEINVVSVQLALPFPP 100 78 . Sftp. lfgtispeigmilthlvinlitlaannftgelplem 114 . : : . : : 101 NISSFTSLOKLVISNTNLTGAISSEIGDCSELIVIDLSSNSLVGEIPSSL 150 115 ksiltslkvlinisnngnlitgtfpgeilkamvdlevliditynnnfngklippem 164 . : : . : 151 GKLKNLQELCINSNG. LTGKIPPE. LGDCVSLKNLEIFDNYLSENLPLET 1.98 165 selkklkylsfggnf.fsgeipesygdiqsleylglngaglisgkSpafls 213 I. : . : 199 GKISTLESIRAGGNSELSGKIPEEIGNCRNLKVLGLAATKISGSLPWSLG 248 214 rlknlremyigyynsytggvprefggltkleildmaSctltgeiptslisn 263 . : : : . : : . : . : 249 QLSKLQSLFV. YSTMLSGEIPKELGNCSELINTFLYDNDLSGTLPKELGK 297 264 lkhlhtliflhinnlitghippelsgilvs.lksldlisingltgeipos finlg 313 . 298 LONLEKMLLWQNNLHGPIPEEIGFMKSLNAIDLSMNYFSGTIPKSFGNLS 347 314 nitlinlfrnnlygqipe . aigellipkl 339 - : : : . 348 NLQELMLSSNNITGSIPSILSDCTKLVOFQIDANQISGLIPPEIGLLKEL 397 340 evfevwennftlqlpanlgringnlikldvisonhltglipkdlcrgeklem 389 : : : II. : : . 398 NIFLGWONKLEGNIPDELAGCONLQALDLSQNYLTGSLPAGLFQLRNLTK 447 390 lilsnniff fgpipeelgkcksltkirivknllingtvpaglfnlplvtiie 439 : . ::: : : : 448 LLLISNAISGVIPLETGNCTSLVRLRLVNNRITGEIPKGIGFLONLSFLD 497 440 ltdniffsgelpvtmsg. divldqiylisnnwfsgeippaignfpnligtliflcd 488 I. : . : : . 498 LSENNLSGPVPLEISNCRQLQMLNLSNNTLQGYLPLSLSSLTKLQVLDVS 547 489 rnrfirginipreifelkhlsrints annitggipdsisrcstlisvdlsrn 538 : . : . 548 SNDLTGKIPDSLGHLISLNRLILSKNSFNGEIPSSLGHCTNLQLLDLSSN 597 539 ringeipkginnvknlg. tilnisgnqltgsiptgignmtslittldlsfnd 587 O 0. d O o h d to e s a e p d 598 NISGTIPEELFDIQDLDIALNLSWNSLDGFIPERISALNRLSVLDISHNM 647 Patent Application Publication Apr. 8, 2004 Sheet 12 of 40 US 2004/0067506A1 Figur 11 AB019227.pro ACOO2334 pro ACOO7651 pro RCH1 pro TO5050.pro CLV1 pro ER.pro BRI1 pro 110.0 100 80 60 40 20 O Patent Application Publication Apr. 8, 2004 Sheet 13 of 40 US 2004/0067506A1 Figure 12 AC002328.pro APO00603.pro BRI pro AC006532.pro AB007644.pro T48499 pro AB010698.pro AC005967.pro AC002339.pro ABO 19235pro T48489-pro ER.pro TO4587 pro AB016874.pro TO5322.pro ACO34257.pro ACO08017.pro ACO21198.pro AL161513.pro AC005395.pro RCH1 pro AB028621 pro AB011476.pro TO5050 pro ACO 15446.pro AB019227.pro AC002334 pro ACOO7651 pro TO5898.pro T46033pro CLV1 pro TO5335.pro TOO712pro T10659.pro AB016887 pro ABO25613.pro AB025627 pro ACOOO 132.pro APOOO417 pro ACO67754.pro AF1494.13.pro TO5897 pro HAEpro ACO101.55pro RPS5pro RPS2pro RPP8.pro RPM1.pro RPS4pro N.pro RPP5pro 329.6 ---- - - - - 300 250 200 150 OO 5O O Patent Application Publication Apr. 8, 2004 Sheet 14 of 40 US 2004/0067506 A1 Figure 13 SEQID NO 18: 842 bp AAGCTTAAATGACAATTTAGTACCTTGGGTTGGTCATGATTTAGAGCGGAACAAATATACCAT ACATCAAACGAGGATATACAGAGAAAATTCATGGAAGTATGGAATTTAGAGGACAATTTCTC TTCTGGGCTACAACGGACCGGCCCATTCGCTCATTTACCCAGAGGTATCGAGTTTGTGGACTTT TGATGCCGCTAGAGACTATTGGCATCGGATTGAAAAAAATGTTTACTTCGTTGTTAACAATTTT CTGAATGCAATATTTTCCTTGTCATGAATATTTAAACTTGTTATTACTTTCTTTTAGCTTAGGTG TGGACAATTATGGAGTTTACTTCAAACGAGGAAGAATCTTAAACGCTCGGTTCAGGTCTCGAA AACAAACCAACTCACAATCCTGACTTAATTGAGGAAAACAATGCAAAACCACATGCATGCTT CCATATTTCTATCATAATCTTATAAGAAAAAACACTACTAAGTGAAATGATTCTGTATATATAT AACCAATGCCTTTTGTTTTGTGATATTTTATGTATATATAACTATTGACTTTTGTCATCTATGGA TAGTGTCTCGGGCTCTTGGCAAACATATTTCAAAGAAAAGTTAATGACTGTAATTAATTAATC TGAAGCTAGAAACAGAACCCCGAGGTAAAAGAAAAAGACAGAGCACATGAAGTTTAGTACTT TTATATATTTAATATATCATTCTTTCTTATTGCTTATCTCTAAAGCAAAAACTTCCCTAAACCCT AAGCCAAAGGACTCAGATCGATGCAGAACCAAGAAGGCTTGTTTTGGATTTGAGAGCCAAAT GCAAAGAAAAAAACTCTT Patent Application Publication Apr. 8, 2004 Sheet 15 of 40 US 2004/0067506A1 NCBI Sequence Viewer Figure 14 NCB Nucleotide banner PubMed Nucleotide search Nucleotide 1: G '3510337" (GenBank Arabidopsis thaliana genomi... PubMed, Protein, Related Sequences, 1 LOCUS AB017061 61712 bp DNA PN 09-AUG-2000 DEFINITION Arabidopsis thaliana genomic DNA, chromosome 5, TAC clone:K19 E20. ACCESSION AB017061 WERSION ABO1761.. G:35.1337 SOURCEKEYWORDS Arabidopsis thaliana (strain: Columbia) DNA, clone lib: Mitsui TAC clone K9E20. ORGANISM ArabidopsisEukaryota; Wiridiplantae; thaliana Embryophyta; Tracheophyta; Spermatophyta Magnoliophyta eudicotyledons; core eudicots; Rosidae; eurosids II Brassicallies; Brassicaceae; Arabidopsis. REFERENCE 1 (sites) AUTHORS Kaneko, T., Katoh, T., Sato, S., Nakamura, Y., Asamizu, R., Kotani, H., Miyajima, N. and Tabata, S. TITLE Structural analysis of