AU9270898A

AU9270898A - Petal-specific promoter and method for obtaining plants having flowers with no petals

Info

Publication number: AU9270898A
Application number: AU92708/98A
Authority: AU
Inventors: Ines Brocard; Florence Charlot; Philippe Guerche; Evelyne Teoule
Original assignee: Institut National de la Recherche Agronomique INRA
Current assignee: Institut National de la Recherche Agronomique INRA
Priority date: 1997-09-23
Filing date: 1998-09-23
Publication date: 1999-04-12
Anticipated expiration: 2018-09-23
Also published as: FR2768746B1; WO1999015679A1; EP1017833A1; FR2768746A1; JP2001517450A; CA2304569A1; AU740911C; AU740911B2

Description

WO 99/15679 - 1 - PCT/FR98/02043 PETAL-SPECIFIC PROMOTER AND METHOD FOR PRODUCING PLANTS HAVING FLOWERS WITH NO PETALS The present invention concerns, in particular, a petal-specific promoter and a method for producing 5 plants having flowers with no petals. The advantage of producing plants lacking petals came from the observation that senescent petals, by falling onto the leaves, might provide preferred seats of infection for the spores of certain pathogenic 10 fungi. In the case of rape, for example, the mode of infection of Sclerotinia sclerotiorum follows principally this route. This fungus is indeed responsible for important damage in cultures of rape (Lamarque, 1983), and no genetic resistance is known to 15 this fungus, either in rape or in the neighboring species. Thus, at the current time, only preventive chemical treatments are used. Sclerotinia sclerotiorum control via plants whose flowers would have no petals would make it 20 possible to diminish the use of fungicide, and thus to limit the subsequent pollution of the soils. It involves, therefore, producing plants having flowers with no petals, and in this way testing a strategy of control of the abovementioned fungus, based 25 on a "physical" resistance and not on the use of resistance genes in the conventional sense. The present invention proposes, therefore, to produce plants whose flowers would be lacking in petals. It consists in using a promoter region which 30 controls the expression, specifically in the petals, of a sequence (orf) encoding a molecule which is capable of modifying the natural properties of the petal, or of inhibiting the formation thereof. In this way, modifying the structure, the 35 shape, the coloration and/or the petal structure of flowers may be envisaged, by placing, downstream of the above-described promoter region, genes which are RA4/ involved in the biosynthesis of pigments, or regulatory genes such as the MYB proteins (Noda et al. 1994). This WO 99/15679 - 2 - PCT/FR98/02043 type of experiment has already been carried out (Elomma et al., 1996; Gutterson, 1995) . However, the promoters used are rather of constitutive type, such as the 35S of CaMV, whereas it would be advantageous to confine 5 the expression of the transgene to the targeted organ. The creation of original ornamental plants may thus, in the context of the present invention, be envisaged. A subject of the present invention is, therefore, a nucleotide sequence for which it has been 10 demonstrated that the corresponding gene is expressed specifically in the petal, this nucleotide sequence corresponds to SEQ ID No. 5. Consequently, a subject of the present invention is a nucleotide sequence which corresponds to 15 all or part: a) of the sequence according to SEQ ID No. 5, or b) of a sequence which hybridized to the sequence according to a), or 20 c) of a sequence which has at least 80% homology with a) or b). In the context of the present invention, the most valuable part of this nucleotide sequence is the promoter region, which is defined as being the sequence 25 preceding (on the 5'side) the translation start codon (ATG) . Stricto sensu, this promoter region stretches from nucleotide 1 to nucleotide 3265 (i.e. to the last nucleotide immediately preceding the ATG codon) , but, taking into account the restriction sites, this region 30 preferably stretches from nucleotide 1 to nucleotide 3233 (corresponding to the site AvaI), and even more preferably from nucleotide 2911 to nucleotide 3233 of SEQ ID No. 5. This promoter region precedes, therefore, in 35 the natural state, an orf which is expressed specifically in the petals, and when this orf is replaced (by genetic manipulation) by another orf, whose product is a cytotoxic molecule, the latter is 40 X)c WO 99/15679 - 3 - PCT/FR98/02043 capable of destroying only said petals. The replacement may also be carried out by a gene part which is capable, during its specific expression in the petal, of modifying the properties of origin thereof. 5 A subject of the present invention is, therefore, also cell-expression vectors comprising a promoter region as described above, placed upstream of a DNA sequence encoding a product which is capable of modifying the structure, the shape, the coloration 10 and/or the petal texture of flowers, and a method for producing ornamental plants, which comprises the insertion into said plants of one of these vectors.' The invention also comprises the case where said DNA sequence encodes a cytotoxic product. 15 Advantageously, the cytotoxic product in question is a ribonuclease. Specifically, when this RNAse is expressed specifically in the petals, it will destroy all the RNAs thereof, as a result of which the petal will not be able to survive. Preferably, the 20 RNAse is barnase, whose corresponding orf has been isolated from Bacillus amyloliquefasciens (Hartley RW, 1988). It involves, therefore, introducing a vector in accordance with the invention into a bacterial strain 25 which is capable of carrying out the transformation of plant cells, such as Agrobacterium tumefaciens. This may, in particular, be carried out by the method of infiltration of Arabidopsis thaliana plants, described by Bechtold et al., 1993. This technique consists in 30 introducing the bacterium into the cells of the floral scapes, by infiltration under vacuum. The plants are then planted out under glass, and their seeds harvested. About one seed in a thousand gives rise to plants of which all the cells carry the transgene. The 35 transformation of other plants, and in particular of rape, may be carried out through Agrobacterium tumefaciens and/or Agrobacterium rhizogenes, with the aid of various techniques which are now conventional WO 99/15679 - 4 - PCT/FR98/02043 (transformation of foliar disks, of hypocotyls, of floral scapes, etc.), combining a phase of coculture of the bacterium with plant tissues, followed by the selection and regeneration of the transformed cells 5 into whole plants. Other transformation techniques do not use this bacterium, but make it possible to transfer the cloned gene directly into cells or tissues (electroporation, particle gun, etc.) and to select and obtain transformed plants (technique reviewed by 10 Siemens and Schieder). A subject of the present invention is also plant cells transformed with a vector in accordance with the invention, and plants comprising said cells. The subject of the invention is also plants whose 15 flowers have no petals. As indicated above, the present invention thus makes it possible to produce plants whose flowers have no petals; the method in accordance with the invention comprising the insertion into the plants of a vector as 20 described above and comprising a DNA sequence encoding a cytotoxic product. In the context of the present invention, it may also be envisaged to produce hybrid plants by crossing two lines whose combined agronomic qualities would be 25 sought. However, in order for the entomophilous pollination to operate optimally, it is necessary for the parents of the hybrid in question to carry petals. Such a cross is, therefore, only possible by means of a two-component system of activation of the toxic gene. 30 The principle of such a system consists in having two lines, each carrying a constituent which has no cytotoxic activity. The specific toxic activity is then restored in the hybrids of these two lines by combination of the two constituents. 35 A possible example of such a system consists in inactivating the expression product whose control is desired by insertion of at least one stop codon at the start of the corresponding coding sequence, then adding WO 99/15679 - 5 - PCT/FR98/02043 into the system, in trans, a tRNA, termed "suppressor", which will recognize the stop codon(s) and supply the amino acid it is carrying, instead of terminating the translation. The protein will thus be able to be 5 translated in full, and its activity restored. Such a system has already been tried out regarding the sequence encoding the GUS gene into which the amber stop codon was inserted, the suppressor tRNA used being a leucine carrier. In addition, the functionality of 10 such a system of transactivation using a tRNALeu suppressor has been verified in planta in Arabidopsis thaliana and Nicotiana tabacum. This model was then applied to the case of barnase. Mutated genes (i.e. genes into which a stop codon has been inserted) 15 encoding barnase, and which are dependent upon the expression of the tRNALeu gene, have been obtained and tested in transient expression in tobacco protoplasts (Choisne Nathalie, 1997). The present invention thus also concerns a 20 method for producing hybrid plants whose flowers have no petals, and comprising the steps of: a) transformation of plants of a line A with a vector in accordance with the invention, and comprising a DNA sequence encoding a 25 cytotoxic sequence modified by the insertion of at least one stop codon, b) crossing of the plants of line A thus obtained with plants of line B expressing the gene of a tRNA suppressor, 30 c) selection of the hybrid plants having flowers with no petals. In the context of the present invention, the plants of line A are transformed with a construct similar to pIB352, as represented in Figure 7. 35 Advantageously, the plants in accordance with the invention belong to the Brassicacea family; preferably, the plant is rape.

WO 99/15679 - 6 - PCT/FR98/02043 Figure 1 illustrates the analysis by Northern hybridization of polyA+ RNA (2 pg) and total RNAs (10 pg) from rape. The membrane is hybridized with the P-labeled whole cDNA 9.2. Revelation is carried out 5 after 24 hours of exposure at -80*C with a screen. The mRNAs identified have an approximate size of 800 bp. Plantule 1: plantule of one week; Plantule 2: plantule of two weeks. Figure 2 illustrates the comparison of the 10 protein sequences from Arabidopsis thaliana (above) and from rape (below) deduced, respectively, from cDNA X74360 (SEQ ID No. 1) and 9.2 (SEQ ID No. 2). The protein from Arabidopsis thaliana has a length of 140 aa, while the protein from rape has a length of 15 147 aa, the homology between the two being 74.6%. The stars mark the amino acids which are common to the two sequences, and the dots appearing in the cDNA from Arabidopsis thaliana have been indicated only to enable the sequences which are common to the two plants to be 20 placed opposite one another, the Arabidopsis thaliana sequence having to be read continuously, i.e. disregarding said dots. Figure 3 represents the alignment of the nucleotide sequences of the cDNAs 9.2 from rape (below) 25 and X74360 from Arabidopsis thaliana (above) , the two sequences having a total homology of 83%. Figure 4 represents the partial restriction maps of the genomic clones (A: Aval, B: BamHl, EI: EcoR1, EV: EcoRV, H: HindIII, Hc: HincII, P: PstI, S: 30 Sacl, Sl: Sall, Xb: Xbal, Xh: Xhol). Figure 5 represents the 5'-3' sequence of the genomic clone 4.1.1 (SEQ ID No. 5). The palindromic sequence has been underlined twice, the coding sequence has been underlined once. The following restriction 35 sites have been marked: BamHI (at position 1): GGATCC; SalI (at position 2911) : GTCGAC and AvaI (at position 3229) : CCCGAG.

WO 99/15679 - 7 - PCT/FR98/02043 Figure 6 represents the constructions carried out with the promoters of the genomic clones 4.1.1 and 8.1.1. distal promoter region of the genomic 5 clone 4.1.1 palindromic sequence proximal promoter region of the genomic clone 4.1.1 322 bp promoter region of the genomic 10 clone 4.1.1 322 bp promoter region of the genomic clone 8.1.1 terminator of the nopaline synthase gene coding sequence of the gus reporter gene 15 coding sequence of the gene 4.1.1 3' untranslated region of the gene 4.1.1 Figure 7 illustrates the constructs prepared with the 322 bp promoter of the genomic clone 4.1.1. 322 bp promoter of the genomic clone 20 4.1.1 coding sequence of the gus reporter gene coding sequence of the gene for wild type barnase coding sequence of the gene for mutated 25 barnase terminator of the nopaline synthase gene terminator 19S of CaMV The invention is not limited to sole description above, it will be better understood in the 30 light of the examples below, which are, however, given only as illustrations. EXAMPLE 1: Demonstration of a petal-specific promoter The first step consists in obtaining 35 complementary DNA (cDNA) clones which are expressed specifically in the petal. For this, the cDNAs were synthesized from petal messenger RNA (mRNA) from rape. In parallel, cDNAs were synthesized from mRNA from WO 99/15679 - 8 - PCT/FR98/02043 leaves, from floral buds whose petals have been removed and from stamens. The cDNAs from said organs or tissues were subtracted from the cDNAs derived from the mRNAs which 5 were expressed in the rape petal. The molecules resulting from this subtraction were used in an experiment of differential hybridization of a petal cDNA library, according to a technique similar to that presented by Atanassov et al., 1996. 10 Several rape DNA clones were isolated at the conclusion of this experiment. Their expression profile was studied by the technique of Northern molecular hybridization. In the absence of clones which are strictly specific for the petal (at the detection 15 threshold of the technique), the most relevant candidate was retained for the rest of the studies; it is clone 9.2. This clone is strongly expressed in the petal at the young stage (bud of about 3 mm) and very weakly in the stamens (Figure 1). 20 Homology searches of sequences in the databanks show a strong similarity between the protein deduced from the open reading frame (orf) of clone 9.2 and the coding sequence of an Arabidopsis thaliana gene (X74360) which encodes a putative wall protein, whose 25 expression is regulated by the gibberellins (Phillips and Huttly, 1994) (Figure 2). The degree of homology shown by the corresponding respective cDNA sequences is greater than 80% in the first 500 bases, then disappears totally over the remaining 220 (Figure 3). 30 The rape cDNA clone 9.2 was used as a probe to screen a rape genomic library. Seven genomic clones were isolated. On the basis of the restriction maps and the sequences, these seven clones divide up into two groups, suggesting the existence in rape of a family of 35 at least two genes, named, in the remainder of the text, 4.1.1 and 8.1.1 (Figure 4) . The cDNA 9.2 is derived from the gene corresponding to the genomic clone 4.1.1.

WO 99/15679 - 9 - PCT/FR98/02043 A preliminary study by PCR amplification was carried out on the clone 9.4.1 which belongs to the group of 4.1.1. Specifically, the structure of the genomic clone made it possible to amplify an upstream 5 region of 3233 bp, using techniques of amplification of large DNA fragments, and of progressive sequencing by PCR. This 3233 bp region stretches from nucleotide 1 to nucleotide 3233 of the sequence represented in 10 Figure 5, and it ends at the level of the AvaI site, at the level of which the cleavage was carried out, as well as the cloning, to obtain "blunt ends". Then, the upstream regions possibly containing the regulatory sequences were subcloned from the two 15 genomic clones (4.1.1 and 8.1.1) into cloning vectors. Currently, more than 4 kb of sequence corresponding, in the majority, to the orf and to the upstream regions (Figure 5) are thus available for the clone 4.1.1. EXAMPLE 2: Verification of the specificity of 20 the promoter region Different constructs comprising the GUS reporter gene placed under the control of certain of these sequences were prepared in order to study the expression of these chimeric genes (i.e. consisting of 25 the coding sequence of a known gene, preceded by the promoter region in accordance with the invention) in transformed plants from Arabidopsis thaliana and from rape. These constructs fall into two categories, as a 30 function of the orf which is placed under the control of the regulatory sequences: - the GUS reporter gene, to study the expression profiles and verify the specificity conferred by the promoter, 35 - the gene for wild-type or inactivated barnase, to prevent the formation of the petal by expression, in this organ, of this f~E FP WO 99/15679 - 10 - PCT/FR98/02043 toxic gene (Figures 6 and 7 detail the composition of each construct). The expression profiles of the GUS reporter gene, in the Arabidopsis transformants obtained in the 5 case of the pIB100, show a certain variability over the plants as a whole (see Table 1 below, which enumerates the parts of the transformed plants in which a blue coloration was observed). However, in nearly half the plants having a blue coloration (13/30), the reporter 10 gene is expressed only in the petals (at the detection threshold of the technique). In certain plants, a weak expression in the stamens, which is relatively unsurprising on account of the results of the Northern hybridizations, but also sometimes an expression in 15 other floral organs, is found, which might suggest the influence of positional effects of the transgene, due to its small size. However, the existence of a significant proportion of plants having the expected profile leads to the thought that the 322 bp proximal 20 fragment is capable of conferring an expression which is specific to the petal. The stability of this expression was tested in the descendants on the self fertilization of these plants. For most, the "petal" specificity was indeed found (data not shown). 25 Longer promoter sequences were also used via the constructs pIB102 and pIB105, and the transformed plants from Arabidopsis thaliana were observed (Table 2 enumerates the parts of the plants which are transformed by pIB102 and have a blue coloration, Table 30 3 enumerates the parts of the plants which are transformed by pIB105 and have a blue coloration). The petal specificity is not again found in the proportion previously observed, because in almost all cases the reporter gene is effectively expressed in the petal, 35 but also in other organs of the flower. Similarly, transformed rape plants were obtained with a construct comprising, as a regulatory sequence, the 3233 bp upstream fragment of the gene -I!4 Cu WO 99/15679 - 11 - PCT/FR98/02043 4.1.1, which was cloned after PCR amplification. In the nine rape plants which could already be observed, the reporter gene is expressed in the petal, but also in other organs of the flower (data not shown), as is 5 observed in Arabidopsis with these large promoter regions. These results suggest that these fragments are too long, whereas it is thought that the preceding one (322 bp) might be a little short and, therefore, 10 amplify the possible positional effects. The latter, however, gives rise to the most promising results. The promoters pIB351 and pIB352 (Figure 7), which are analogous to the pIB100, but comprise, respectively, the coding sequence of the gene for wild 15 type barnase, and this same sequence inactivated by insertion of a stop codon (then named mutated barnase), instead of the coding sequence of the reporter gene, have been introduced into Arabidopsis thaliana (results not yet available). TR4,

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110 99/15679 - 12 - PCT/FR9B/02043 -Y, oz 04 04 o 4.~) t R 0 0 4-) vi +4 044 04J'HJ'H04 U)0~ -H 04 H~ 40 I q0 4-) 4-H ) - H a) 0 m 00 H- 00 100 WQ ) 00>E )- 10 a)W ~ ~ t~ 0. 4 00 4 .0i 0.004 uQ tv0o4- 04 mU 0 0 04 0 ~ 04 Q4 3 04~ (L 0 C) O 04r 4-o *H4 0- 4 ' 0 - ) 4-J >, rg 4- 1)4) 1 to 0 ST -H 4-J r; WO0 99/15679 -13 - PCT/FR98/02043 W w 0 0 .4 ii 04 CO) 04) o 4-) Ho 0a r 0 r 0 1 _ El4 I 01 04 I4 a) .I Q u CL a) rt4 Co -H -H -H -0C 04 0 0 0 0 0) c Q) 2 )U) 0)u P : Co 4-4 H 4-40 414 - 4 Co I : -I Iv - T + CO) -70 WO 99/15679 - 14 -PCT/FR98/02043 cc %0 W4 4 4, 0 0, 0- 4- 'D -4 ( -) -4- (1)0 a) 0 0 04 -0 1 4 4 o. )(a 4- () -4 0 0. 0.0404 Q H I I I I ID 0W 0 a) Q4 04 0 0)) W 4- 0 4-) -4 -H -4 -4 E I -H r H (a 41 0.- 4)ra ( CoA 0. r.. r W -1 0H a) H -- 0 4 0H -4 -4 -4 a 4 04 a a) O 0 a ) ) r -4 4-4 4U4 A ' I~ WO 99/15679 - 15 - PCT/FR98/02043 REFERENCES Atanassov I et al. (1996) Plant Science 118, 185-194 5 Bechtold N. et al (1993) Comptes-Rendus de l'Acad6mie des Sciences 316, 1194-1199 Choisne Nathalie (1997). Etude de l'expression in vivo d'une gene d'ARNt leu de Phaseolus vulgaris et 10 l'utilisation de ce gane dans un systeme de suppression [Study of the expression in vivo of a leu tRNA gene from Phaseolus vulgaris, and use of this gene in a suppression system]. Doctoral thesis from the University of Paris XI (Order 15 No. 4691). Elomaa P. et al. (1996). Molecular Breeding 2 : 41-50. Gutterson N. (1995). HortScience, Vol. 30(5), August 20 1995. Hartley RW, 1988. Barnase and barstar : expression of its cloned inhibitor permits expression of a cloned ribonuclease. J. Mol. Biol, 202, 913-915. 25 Lamarque C. (1983) Proc. 6th int. Rapeseed Cong. 1983, Paris, France, pp 903-907 Noda K-I, et al. (1994). Nature. Vol 369. 23 June 1994. 30 Phillips A.L. and Huttly A.K. (1994). Plant Mol Biol. 24 : 603-615 Siemens and Schieder 1996. Plant Tissue Culture and 35 Biotechnology, 2, 66-75

Claims

1- Nucleotide sequence corresponding to all or part: a) of the sequence according to SEQ ID No. 5, 5 or b) of a sequence which hybridizes to the sequence according to a), or c) of a sequence which has at least 80% homology with a) or b). 10

2. Nucleotide sequence according to Claim 1, corresponding to all or part: a) of the sequence which stretches from nucleotide 1 to nucleotide 3233 and preferably from nucleotide 2911 to 15 nucleotide 3233 of SEQ ID No. 5, or b) of a sequence which hybridizes to the sequence according to a), or c) of a sequence which has at least 80% homology with a) or b). 20

3. Cell-expression vector comprising a sequence according to Claim 2, placed upstream of a DNA sequence encoding a product which is capable of modifying the structure, the shape, the coloration and/or the petal texture of flowers. 25

4. Cell-expression vector comprising a sequence according to Claim 2, placed upstream of a DNA sequence encoding a cytotoxic product.

5. Vector according to Claim 4, characterized in that the cytotoxic product is a ribonuclease and 30 preferably barnase.

6. Plant cells transformed with a vector according to one of Claims 3 to 5.

7. Plants comprising cells according to Claim 6.

8. Plants whose flowers have no petals. 35

9. Method for producing ornamental plants, comprising the insertion into said plants of a vector according to Claim 3. WO 99/15679 _ 17 _ PCT/FR98/02043

10. Method for producing plants whose flowers have no petals, comprising the insertion into said plants of a vector according-to Claim 4 or 5.

11. Method for producing hybrid plants whose 5 flowers have no petals, comprising the steps of: a) transformation of plants of a line A with a vector according to Claim 4 or 5,. modified by insertion of at least one stop codon into the coding sequence of the DNA, 10 b) crossing of the plants of line A obtained -in a) with plants of line B expressing the gene of a tRNA suppressor, c) selection of the hybrid plants having flowers with no petals. 15

12. Plants according to Claim 7 or 8, or obtained by the use of the method according to Claim 10 or 11, characterized in that they belong to the Brassicacea family, preferably in that the plant is rape.