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United States Patent (19) 11 Patent Number: 5,254,801 Dotson Et Al

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United States Patent (19) 11 Patent Number: 5,254,801 Dotson Et Al US00525480A United States Patent (19) 11 Patent Number: 5,254,801 Dotson et al. 45 Date of Patent: Oct. 19, 1993 54) HETEROLOGOUS DOMINANT 56) References Cited CONDITIONAL LETHAL GENE WHICH ISA PUBLICATIONS PHOSPHONATE MONOESTER HYDROLASE AND USE THEREOF IN Fitzgibbon (Dec. 1988) University Microfilms Interna PLANTS tional 1989, Abstract. Fitzgibbon et al. (Nov. 1990) Applied and Environmen tal Microbiology 56(11): 3382-3388. (75) Inventors: Stanton B. Dotson, Fenton; Ganesh Moore, et al. (Aug. 1983) Applied and Environmental M. Kishore, Chesterfield, both of Microbiology 46: 316-320. Mo. Lee, et al. (1988) Science 239: 1288-1291. Walden, et al. (1990) Eur. J. Biochem 192: 563-564. Mariani, et al. (Oct. 1990) Nature 347: 737-741. 73) Assignee: Monsanto Company, St. Louis, Mo. Shinabarger, et al. (Nov. 1984) Applied and En vironmenal Microbiology 48(5): 1049-1050. (21) Appl. No.: 621,670 Primary Examiner-Che S. Chereskin Attorney, Agent, or Firm-Dennis R. Hoerner, Jr.; 22) Filed: Dec. 3, 1990 Howard C. Stanley 57 ABSTRACT (51) Int. Cl. ......................... A01H 4/00; C12N 5/14; A class of heterologous dominant conditional lethal C12N 15/82 genes is disclosed. The gene is useful in genetically (52) U.S. Cl. ................................. 800/205; 435/240.4; modifying plant cells and plants to selectively induce 435/1723; 435/320.1; 435/69.1; 935/64; cellular lethality for purposes such as inducing male 935/67; 800/DIG. 15 sterility for hybrid seed production, cell ablation and (58) Field of Search ....................... 800/205, DIG. 15; counter-negative selection. 435/320.1, 172.3, 240.4, 69.1; 536/27; 935/64, 67 20 Claims, 10 Drawing Sheets U.S. Patent Oct. 19, 1993 Sheet 2 of 10 5,254,801 ATGACCAGAA AAAATGTCC GCTTATCGTC GGATCAAT 40 4. GGCGAGCAGA TTTTATCCCT CACCGAGC GGGCGGAGGG 80 81 GCGCGAACC TCCAAAA CTCCCAATCT TGATCGTCTT 120 121 GCCGGGAAG GCTTGACCTT CCGCAA CA GTCACGACGT 60 61 GCGTGCCGTG TGGTCCGGCA AGGGCAAGCC TGCGACGGG 200 201 CCTCTACCTé AGAACCACC GGGCGGGCA GAACACG 240 241 CCGCTTGACC AGCGCCAC AAACCGGC AAGGCCCGC 280 281 GCGCCATTGG CTACGATCCC GCGC CATG GACACCAC 320 321 CACGACACC GATCCGCGCA CAACCTCTGC AAGGGATCCG 360 361 CGTTTCACGG TCCTGGGCGA CATCATGGAC GGCTTTCGTT 400 40 CGGCGGCGC ACGAGCCC AATAGGAGG GGTATTTTGG 440 441 CGGGGGCG CAGAACGGC TCGAACTGCC AGAGAACCGC 480 481 GAAGAACT GGCTGCCGGA AGGGAACA TCCGTTCCCG 520 521 GTGCACCGA CAAACCGTCG CGCACCGA AGGAATTTTC 560 56. GGA CGACA TTCTTCACGG AGCGCGCCCT GACAATCG 600 601 AAGGGCAGGG ACGGCAAGCC TTTCTTCCTG CATCTTGGCT 640 641 ATTATCGCCC GCATCCGCCT TTCGTAGCCT CCGCGCCCA 680 581 CCATGCGATG TACAAAGCCG AAGATAGCC TGCGCCTATA 720 721 CGGCGGAGA ATCCGGAGC CGAAGCGGCA CAGCACCGC 760 761. TCAGAAGCA CTATATCGAC CACATCAGAC GCGGCTCGTT 800 801 CTTCCATGGC GCGGAAGGCT CGGGAGCAAC GCTGATGAA 840 841 GGCGAAATTC GCCAGATGCG CGCTACATA GCGGACTGA 880 88. CACCGAGAT CGACGATTGT CTGGGGAGGG TCTTTGCCTA 920 921 CCGAGAA ACCGGTCAGT GGGACGACAC GCTGATTATC 960 961. TTCACGAGCG ATCATGGCGA ACAACGGGC GACACACC OOO 1001 GCTCGGCAA GATCGGTTAC AAGCCGAAA GCTTCCGTAT 1040 104 TCCCTTGGTC AAAAGGATG CGGGACAGAA CCGGCACGCC 080 1081 GGCCAGACG AAGAAGGC CCCGAAAGC ATCGACGTCA 120 1121 GCCGACCA CCCGAAGG CTGGGCGGGG AAACGCCTCG 1160 1161 CGCCTGCGAC GGCCGTTCGC TGTTGCCGTT TCTGGCTGAG 200 201 GGAAAGCCCT CCGACTGGCG CACGGAACTA CATACGAGT 1240 1241 TCGATTTTCG CGATGTCTTC TACGACAGC CGCAGAACTC 1280 28 GGTCCAGCTT TCCCAGGATG AGCAGCC CTGTGTGATC 1320 1321 GAGGACGAAA ACACAAGTA CGTGCATTTT GCCGCCCTGC 1360 1361 CGCCGCTGTT CTTCGATCTG AAGGCAGACC CGCAGAA 400 1401 CAGCAATCTG GCGGCGATC CTGCTTATGC GGCCCTCGTT 1440 1441 CGIGACTATG CCCAGAAGGC ATGTCGGG CGACTGTCTC 480 1481 ATGCCGACCG GACACTCACC CATACAGA CCAGCCCGCA 520 1521 AGGGCTGACA ACGCGCAACC ATTGA 1545 Figure 2. U.S. Patent Oct. 19, 1993 Sheet 4 of 10 5,254,801 /199108S 0/9\\ON 689|HUu8€. Z01l'HooE 0188NOWd U.S. Patent Oct. 19, 1993 Sheet 5 of 10 5,254,801 40 41 80 81 120 121 160 161 200 201 240 241. 280 281 320 321 360 361 400 401 440 441 480 481 520 521. 560 56. 600 60. NGACCTCATT CGTTCTGATA TATAACA GT AATAACGA 640 641 AATTGTGTGT AACTATTCA CAGAAA AAG ACTG 680 681 GAAGAGA GAAAACAGTA AGAAGG AAAGAAAA 720 721 AGAAAGA CTTATTATAA ATTAATTATA TTGAATCTTA 760 761 AAAAAGT TATGCAATTT GAGTTGTTTG ATGTGTGTTA 800 801 AGACTTAGGT TGATCATGTA GTCGAGAAGG TGTTAATAA 840 841 AGATGCTA TGGAAAGATG TAGTTGGCTT CTTTTTCTTT 880 881 TTTCCCTCTC TATATAGTAC AGAGACA TGTTACACAC 920 92 AAAGATG AGCTAGGA ATTTCTACCT CAAATTCT 960 961 TCATTATTGT ACCAATTTTT GGACACCA ATAACGAG 1000 1001 CTTGCAAATC CAAATATCT CACACCAAA ACATCATACA 1040 1041 AAAATA CTCTTTTCTT CTACCTAGGC AAAATGGCAA 1080 108 CCACGATTCT TGTTGTTATT CTTATTACA CTAGTGTTCT 20 ill21 TACTTACCCT AAAAIGCGA GGACGCAA GGCAATGAAG 1160 161 GAAAAACCAA AAGCATCAGC TGAGAACAG AAGAAA 1200 120 TCCAGCACCC TTTATCGCCT TTTTTTGGTG GTTTTGGTGG 1240 1241 GAGAGG GCAATTAGGC CTCCATTTGG TTTAGGAGCC 1280 1281 GGCTTTGGTG GATTTGGTGG TAGTATTGGA GGTGCTTTTG 1320 1321 GAAGGG TGGTCCTTTC GCCGGAAAG GTGGAACAAG 1360 1361 TAGGCTAGA AGTGGAAGTG GAAGTGGAAG TGGATTTGGT 1400 1401 TCTGGTATTA AGAAGGG TGGAAAAA GGTGGCAATA 1440 441 ACCAATGA TAAAATTGAA GGTGAACTG AACAGGGA 480 48 CCTAGGGAG GGAGGGAG CAACAATAA AAATGATATG 1520 1521 CACCACCA GAACTTAAAC TCACTAATTA TAATAAAAG 1560 1.56 ATTGCTAAAT CATAGCATCT AGTAGATNN NNNNNNNNNN 1600 160 NNGAATTC 1608 Figure 5. U.S. Patent Oct. 19, 1993 Sheet 6 of 10 5,254,801 Cv91.III68 886],100N /f792A'HOOEY 8.//ZAHOOEY 668|OBS 0/188|Hul08. 5988|OBS U.S. Patent Oct. 19, 1993 Sheet 7 of 10 5,254,801 000€IHuuB8| 60ye100N. "Zeun61-I U.S. Patent Oct. 19, 1993 Sheet 9 of 10 5,254,801 ·6eunfil U.S. Patent Oct. 19, 1993 Sheet 10 of 10 5,254,801 000€|Hudb8 6078||OON ºo!,eunfil-l 38/9IOON 5,254,801 1. 2 expressed by wild-type cells limiting their use to cells HETEROLOGOUS DOMINANT CONDITIONAL which carry null mutations in the wild-type genes. Het LETHAL GENE WHICH IS A PHOSPHONATE erologous dominant conditional lethal genes have the MONOESTER HYDROLASE AND USE THEREOF greatest potential for controlled cell lethality. The N PLANTS genes are by definition, non-lethal in the absence of the controlled application of a heterologous protoxin and This invention relates in general to plant molecular utilize protoxins which are not substrates for normal biology and, more particularly, to a class of phospho cellular enzymes. In plants, the only example of a heter nate monoester hydrolases which are useful as heterolo ologous conditional lethal gene is theiaah gene encod gous dominant conditional lethal genes. 10 ing indoleacetamide hydrolase which can convert non toxic levels of naphthalene acetamide into toxic levels BACKGROUND OF THE INVENTION of the auxin, naphthalene acetic acid (Klee et al., 1987). Recent advances in genetic engineering have pro Since naphthalene acetamide is itself toxic at high lev vided the requisite tools to transform plants to contain els, the conditional lethal phenotype is difficult to con foreign genes. It is now possible to genetically improve 15 trol. Viral thymidine kinase is an example of a heterolo plants to have unique characteristics of agronomic im gous dominant conditional lethal gene in mammalian portance. Certainly, one such advantageous trait is con cells. Unlike the cellular thymidine kinase, the viral trolled plant cell lethality. Expression of a lethal gene in thymidine kinase protein is able to activate pyrimidine a cell can be used to specifically kill or prevent develop analogs such as acycolvir and gancyclovia into toxic ment of that targeted cell type from within an organism 20 products (Elion, 1978 and Mansour et al., 1988). As or from within a population of cells. A lethal gene has described below, a novel conditional lethal gene is potential applications for hybrid seed production, cell needed in plant biology which, has no affect on cellular ablation, and negative (counter) selection. metabolism in the absence of protoxin, utilizes a pro To be generally useful, lethal genes must be express toxin which is several orders of magnitude less toxic ible behind a variety of promoters, be effective in a 25 than the activated toxin, has a unique substrate specific variety of cell types and be cell autonomous. Both dom ity distinct from plant cellular enzymes and which uti inant and conditional (inducible) lethal genes have been lizes a protoxin inexpensive to synthesize on a large described in the literature. Dominant lethal genes in scale. clude the expression of a protein which is directly toxic HYBRID SEED PRODUCTION such as ricin which inhibits ribosomes (Landel et al., 30 1988), diphtheria toxin A (Dip-A gene) which ADP Plant breeders can design a cross between two par ribosylates elongation factor 2 (Palmiter et al., 1987) ents such that the progeny outperform both of the par and RNAses and DNAses which degrade critical ge ents. This increased performance is from heterosis or netic molecules (Mariani et al., 1990). Dominant lethal hybrid vigour. The increased yield associated with hy genes may also include antisense and ribozyme genes 35 brid vigour makes hybrid seed profitable for farmer to which target RNA molecules for critical cellular pro purchase seed each year rather than saving seed from cesses. The advantage of such dominant lethal genes is the previous year and profitable for a plant breeding good efficacy and strict cell autonomous expression. company to sell hybrid seed. Heterosis has been identi The use of dominant lethal genes is severely restricted fied in all major crop species, however most crops are when expression of the lethal gene is critical for the not currently amenable to commercial hybrid seed pro survival or reproduction of the organism preventing the duction which requires three
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