AU705759B2 - Inhibition of cell respiration and production of male sterile plants - Google Patents

Description

WO 97/04116 PCT/GB96/01675 -1- INHIBITION OF CELL RESPIRATION AND PRODUCTION OF MALE STERILE PLANTS The present invention relates to a method of producing male sterile plants by use of a gene, which is expressible in plants and inhibits an essential cell function, hence disrupting full expression of a selected plant characteristic.

Our International Patent Application No. WO 90/08831 describes and claims the disruption of respiration using a variety of disrupter genes which we refer to also as "pollen-inactivating genes".

The ability of such inactivating genes to function in this way varies and, therefore, there is a need for further improved gene sequences so that appropriate selection for specific applications may be made.

An object of this invention is to provide genes for use in inhibiting gene expression.

According to the present invention there is provided a method of inhibiting gene expression in a target plant tissue comprising stably transforming a plant cell of a type from which a whole plant may be regenerated with a gene construct carrying a tissue-specific or a development-specific promoter which operates in the cells of the target plant tissue and a disrupter gene encoding a protein which is capable, when expressed, of inhibiting respiration in the cells of the said target tissue resulting in death of the cells characterised in that the said disrupter gene is selected from the group consisting of the T-urfl3 gene, genes encoding an at or P-tubulin, short sense co-suppression of two essential maize cell cycle genes. cdc25 and replication origin activator (ROA) and a short sense construct to the adenine nucleotide translocator (ANT) of the inner mitochondrial membrane.

Down regulation of gene activity due to short sense co-suppression is described in our International Patent Application No.WO 90/08299.

The ct- or P-tubulin genes act as disrupters by de-stabilizing microtubule arrays in plant cells, hence inhibiting essential microtubule function in the said target tissue resulting in death of the cells.

The use of short sense co-suppression of two essential maize cell cycle genes, and replication origin activator (ROA) disrupts cell division and hence provide a growth defect in the targeted organ or tissue.

Preferably the promoter is an anther- and/or tapetum-specific promoter or a pollen-specific promoter, so that on expression of the said disrupter protein therein the regenerated plant is in male sterile. More preferably the said anther and/or tapetum-specific WO 97/04116 PCT/GB96/01675 -2promoter was isolated using the cDNA sequences shown in Figure 1 or 2 or 3 of the accompanying drawings and using the techniques described in our International Patent Application No. WO 90/08826.

Plasmids containing the DNA sequences shown in Figures 1, 2 and 3 have been deposited under the terms of the Budapest Treaty, details being as follows: Plasmid pMS10 in an Escherichia coli strain RR1 host, containing the gene sequence shown in Figure 1 herewith, and deposited with the National Collection of Industrial Marine Bacteria on 9th January 1989 under the Accession Number NCIB 40090.

Plasmid pMS14 in an Escherichia coli strain DH5c( host, containing the gene o1 sequence shown in Figure 2 herewith, and deposited with the National Collection of Industrial Marine Bacteria on 9th January 1989 under the Accession Number NCIB 40099.

Plasmid pMS 18 in an Escherichia coli strain RR1 host, containing the gene sequence shown in Figure 3 herewith, and deposited with the National Collection of Industrial Marine Bacteria on 9th January 1989 under the Accession Number NCIB 40100.

The isolation and characterisation of these gene sequences of this invention are described in full in WO 93/01294.

Other promoters may also be used, for example a promoter such as the tapetum specific MFS 14 promoter.

The present invention also provides a plant having stably incorporated in its genome by transformation a gene construct carrying a gene construct carrying a tissue-specific or a development-specific promoter which operates in the cells of the target plant tissue and a disrupter gene encoding a protein which is capable, when expressed, of inhibiting an essential cell function such as respiration, microtubule arrays or cell division in the cells of the said target tissue resulting in death of the cells.

The invention also provides a plant, particularly a monocotyledonous plant, and more particularly a corn plant, having stably incorporated within its genome a gene construct carrying a tissue-specific promoter which operates in the cells of the said target tissue and a disrupter gene encoding a protein which is capable of inhibiting an essential cell function such as respiration or microtubules in the said cells of the said target tissue resulting in death of the cells characterised in that the said disrupter gene is selected from the T-urfl3 gene, a short sense construct of the adenine nucleotide translocator, genes encoding an c- or 0-tubulin and short sense down-regulation of the essential cell cycle genes, cdc25 and ROA.

WO 97/04116 PCT/GB96/01675 -3 These gene constructs may be used as a means of inhibiting cell growth in a range of organisms from simple unicells to complex multicellular organisms such as plants and animals.

By the use of tissue- or cell-specific promoters, particular cells or tissue may be targeted and destroyed within complex organisms. One particular application intended for this invention is in the destruction of cells essential for male flower development, leading to male sterility.

The invention therefore provides a method of preventing or inhibiting growth and development of plant cells based on gene constructs which inhibit an essential cell function such as respiration or microtubules. The technique has wide application in a number of crops where inhibition of particular cells or tissue is required.

Of particular interest is the inhibition of male fertility in maize for the production of F hybrids in situ. The concept of inhibition of mitochondrial function as a mechanism for male sterility arises from some previous research on T-type cytoplasmic male sterility in maize (cms-T) which has shown an association between the male sterile phenotype and mitochondrial dysfuction. Although a direct causal relationship has yet to be established between mitochondrial dysfunction and cms-T, an increasing body of evidence suggests that fully functional mitochondria, particularly in the tapetal cells, are essential. This is particularly critical during microsporogenesis since the metabolic demands placed on the tapetal cells results in a 40-fold increase in mitochondrial number.

Thus we provide a number of negative mutations which act upon mitochondria to inhibit functional respiration. When specifically expressed in maize anther tissue these mutations will result in a male sterile phenotype.

We also use expression of oc- or P-tubulin genes to disrupt cell function. During normal cell life, expression oftubulin genes is closely regulated by their endogenous promoters and closely matches the requirements of cells for these proteins which are polymerised and assembled into microtubules during growth and development of the plant. By expressing tubulin genes in an unregulated fashion using non-tubulin promoters in a particular tissue or stage of development, the equilibrium between free tubulin monomers and those polmerised in microtubules is disrupted resulting in instability of the microtubule complex and cellular dysfuntion. When expressed in the tapetum or other cells of the anther this latter effect will cause the plants to be sterile.

WO 97/04116 PCT/GB96/01675 -4- We also propose the use of short sense down-regulation of essential cell cycle genes, eg cdc25 and ROA. When expressed in the tapetum or other cells of the anther this latter effect will cause the plants to be sterile.

The method employed for transformation of the plant cells is not especially germane to this invention and any method suitable for the target plant may be employed. Transgenic plants are obtained by regeneration from the transformed cells. Numerous transformation procedures are known from the literature such as agroinfection using Agrobacterium tumefjciens or its Ti plasmid, electroporation, microinjection of plant cells and protoplasts, microprojectile transformation and pollen tube transformation, to mention but a few.

Reference may be made to the literature for full details of the known methods.

The development and testing of these gene constructs as disrupters of mitochondrial function in the unicellular organism, yeast, will now be described. A mechanism by which these gene constructs may be used to inhibit plant cell growth and differentiation in transformed plants will also be described. The object of these procedures is to use yeast as a model system for the identification and optimisation of gene constructs for expressing proteins which disrupt mitochondrial function. Plant cells will then be transformed with the selected constructs and whole plants regenerated therefrom.

The accompanying drawings are as follows: Figure 1 shows the DNA sequence of an anther-specific cDNA, carried by plasmid Figure 2 shows the DNA sequence of a tapetum-specific cDNA, carried by plasmid pMS14; Figure 3 shows the DNA sequence of an anther-specific cDNA, carried by plasmid pMS18; Figure 4 shows the sequence of the T-urfl3 gene (SEQ ID NO 1) with the primers Turf-I (SEQ ID NO 2) and Turf-2R (SEQ ID NO 3) underlined; Figure 5 shows DNA encoding the 59 amino acid region from the ATP-2 gene of Nicotinia plumbaginifolia ((SEQ ID NOS 4 and 5) with primers PREB-IB (SEQ ID NO 6) and PREB-R (SEQ ID NO 7) shown; Figure 6 shows the cleavage site of the pre-P sequence; Figure 7 is a map of vector pCaMVIiN; Figure 8 is a map of vector RMS17; WO 97/04116 PCT/GB96/01675 Figure 9 is a map of vector pIE109; Figure 10 shows the MFS14 promoter sequence (SEQ ID NO 8) with the following features: position 2198 transcription start CCT"A"CAA (concensus CTC"A"TCA) position 2167 ATCCATT (possible TATA box motif) position 2141 CCAT (possible CAAT box motif) position 2233 cdna start CAC"A"CAG position 2295 translation start GCAACAATGGCG (concensus

TAAACAATGGCT);

Figure 11 is a map of vector RMS 11; 1 0 Figure 12 is a map of vector pMANT3 Figure 13 illustrates the contruction of vectors for maize cell line transformation: and Figure 14 is a plot showing numbers of transformants produced in the various different experiments.

The invention will now be illustrated by the following Examples.

Example 1 Construction of the maize transformation vector. RMS17.

We used the PCR to amplify the T-urfl3 gene from cms-T maize (line RW33:TMS) and the mitochondrial targeting sequence, pre-P, from Nicotiana plumhbaginolia

DNA

samples from plant material were prepared using the method described by Edwards et al (Nucliec Acids Research 1991, 19, 1349).

The complete T-urfl3 gene was amplified in the PCR using primers turf-i (5'ATCGGATCCATGATCACTACTTTCTTAAACCTTCCT-3', SEQ ID NO 2) and turf- 2R (5'TAGTCTAGATCACGGTACTTGTACGCTATCGGT-3', SEQ ID NO 3) designed from sequence information provided by Dewey et al (1986, Cell, 44, 429-449). The PCR conditions were 35 cycles of denaturing at 94 0 C for 0.8 min, annealing at 65 0 C for 1 min and extension at 72 0 C for 2.5 mins. To aid subsequent cloning the PCR primers were designed such that they introduce unique BamHI and XbaI restriction sites at the 5' and 3' ends of the gene respectively. The position of these primers relative to the T-urfl3 gene sequence is shown in Figure 4.

Similarly the 59 amino region from the ATP2 gene ofNicotiana plumbaginifolia which encodes the functional pre-P mitrochondrial targeting sequence was amplified in the PCR using the primers PREB-IB (5'ATCGGTACCGCCATGGCTTCTCGGAGGCTTCTCGCCT-3, SEQ ID NO 6) and PREB-R (5'ATCGGATCCCGCTGCGGAGGTAGCGTA-3', SEQ ID NO 7) designed using sequence information provided from Boutry et al (1987, Nature, 328, 341). The PCR WO 97/04116 PCT/GB96/01675 -6conditions were as described above except that the annealing temperature was reduced to 0 C. To aid subsequent cloning PBEB-IB and PREB-2R were designed such that they introduce unique KpnI and BamHI restriction sites at the 5' and 3' ends of the amplified fragment respectively. The position of these primers relative to the ATP2 gene is shown in Figure Following amplification the pre-B PCR fragment was digested with KpnI and BamHI to generate cohesive ends and cloned into the corresponding sites of the vector pUC18 to give plasmid pPB The TURF-13 PCR product was then digested with BamHI and XbaI and cloned into the corresponding sites in pPB 1 to give plasmid pPB2. In pPB2 the pre-0 0o sequence is fused in frame with the T-urfl3 gene so that following expression in a plant cell the full product will be transported to mitochondria. Cleavage of the pre-P sequence at the predicted site between residues 55-56 will release the T-urfl3 protein which includes at its NH2-terminus an additional 4 residues from the pre-P sequence (Figure 6).

The pre-P/T-urfl3 gene fusion in pBB2 removed by digestion with the enzymes Kpn I and Sal I, blunted-ended and cloned into the plasmid pCAMVI 1 N (Figure 7) which was digested with BamHI and blunt-ended to give pPB3. This cloning step places the pre-P/Turfl3 gene fusion under transcriptional control of the CAMV 35S promoter. TheAdh/I intron is present in this construct to boost expression levels in corn cells (Mascarenhas et al., 1990.

Plant Mol. Biol., 15, 913-920) and the nos 3' sequence provides a polyA addition site. To produce the final vector, RMS17 (Figure 8) the PAT selection cassette from plE109 (Figure 9) which allows in vitro selection of transformed corn cells on bialaphos was introduced as an EcoRI fragment into the unique EcoRI site of pPB3.

Example 2 Transformation of BMS corn cells with RMS 17.

The objective of this experiment was to show that expression of the prep/TURF-13 gene construct in cultured BMS corn cells results in a reduction in cell viability as measured by the establishment of transgenic calli following transformation. The RMS17 vector (Figure 8) was introduced into cultured BMS cells using a silcon carbide fibre-mediated transformation technique as follows: Preparation of silicon carbide whiskers Dry whiskers were always handled in a fume cabinet, to prevent inhalation and possible lung damage. These whiskers may be carcinogenic as they have similar properties to WO 97/04116 PCT/GB96/01675 -7asbestos. The Silar SC-9 whiskers were provided by the Advanced Composite Material Corporation Greer, South Carolina,USA. The sterile whisker suspensions were prepared in advance as follows. Approximately 50mg of whiskers were deposited into a pre-weighed ml Eppendorf tube, which was capped and reweighed to determine the weight of the whiskers. The cap of the tube was perforated with a syringe needle and covered with a double layer of aluminium foil. The tube was autoclaved (121 0 C, 15psi, for 20 minutes) and dried. Fresh whisker suspensions were prepared for each experiment, as it had been reported that the level of DNA transformation when using fresh suspensions was higher than that of older suspensions. A 5% (weight/volume) whisker suspension was prepared using sterile deionised water. This was vortexed for a few seconds to suspend the whiskers immediately before use.

DNA transformation into cells All procedures were carried out in a laminar air flow cabinet under aseptic conditions. The DNA was transformed into the cells using the following approach. Specific modifications to this method are indicated in the text.

Cell and whisker suspensions were pipetted using cut down Gilson pipette tips. 100l of fresh BMS medium (see appendix 1) was measured into a sterile Eppendorftube. To this was added 40Vl of the 5% whisker suspension and 25pl (lmgy/ml) of the plasmid DNA, which was vortexed at top speed for 60 seconds using a desktop vortex unit (Vortex Genie 2 Scientific industries, Inc). Immediately after this period of vortexing, 500pl of the cell suspension was added ie 250pl of packed cells. The Eppendorf tube was then capped and vortexed at top speed for 60 seconds in an upright position. The same procedure was used to transform the other cell lines.

Three controls were included in this experiment. Two positive control vectors were pPG3 which contains the PAT selection cassette alone and RMS 15, which is identical to RMS 17 except that the T-urfl3 gene is replaced by the mitochondrial uncoupling protein gene, UCP, which jhas no effect on cultured BMS cells. The pre-0 targeting sequence is present in both constructs. A negative control, which should completely prevent establishment of transgenic calli, was provided by RMS 13. which is identical to RMS17 except that the prep/T-urfl3 gene fusion is replaced by the cytotoxic ribonuclease gene, barnase.

The mean numbers of transgenic calli established in this experiment are shown in Table 1.

WO 97/04116 -8- TABLE 1 PCT/GR96/01675 No. oftransgenic calli per replicate Vector Mean 1 2 3 pPG3 44 33 39 RMS13 0 0 0 0 40 30 43 38 RMS17 12 13 23 16 These data show that relative to the two positive controls, pPG3 and RMS15, expression of the preB/T-urfl3 gene fusion results in a signficant decrease or better) in the establishment oftransgenic calli. This suggests that targeting T-urfl3 protein to mitochondria has a deleterious effect on these cells, presumably due impartment of mitochondrial function.

Expression of the cytotoxic ribonuclease, barnase, completely abolishes the establishment of transformed calli.

Example 3 Construction of the maize transformation vector. RMS 11 RMS 11 is a transformation vector in which expression of the pre-P/T-urfl3 gene fusion is controlled by the maize tapteum promoter, MFS14. The sequence of the MFS14 promoter and untranslated leader region from position -2198 to +97 is shown in Figure 10. In this way, expression of the T-urfl3 protein is limited to the cells producing pollen and not throughout the whole plant.

To construct RMS11, the Kpn I SalI fragment from pPB2 containing the pre-P/Turfl3 gene fusion was blunt-ended and ligated into the blunt ended BamHI site of plasmid pSC9 to yield pPB4. In pPB4 the pre-P/T-urfl3 gene fusion is now positioned between the 152 to +97 MFS14 promoter fragment and the nos 3' polyadenylation sequence. This complete cassette was removed from pPB4 by digestion with SacI and EcoRI and cloned into the corresponding sites of pSC7 to give plasmid pB5. pSC7 contains the -153 to -5800 region of the MFS14 promoter so that the introduction of the Sad EcoRI fragment from pPB4 recreates the full 5.8 kb MFS14 promoter.

RMS1 I (Figure 11) was completed by introduction of the PAT in vitro selection cassette from plE109 into the unique EcoRI site of WO 97/04116 PCT/GB96/01675 -9- Example 4 Transformation of maize cells with RMS 11 by particle bombardment to provide stably transformed, male sterile plants.

The maize transformation vector, RMS 11 was used to transform regenerable maize cell cultures by particle bombardment.

Culture Material Friable embryogenic Type II callus was initiated from immature zygotic embryos excised from either greenhouse or filed grown A188 plants 10-12 days after pollination with pollen from the inbred B73. The medium used for callus initiateion and maintenance was based onN6 medium as modified by Armstrong and Green. Specifically, the medium contained 6mM L-proline, 2% sucrose, 2 mg/l 2,4-dichlorophenoxy- acetic acid (2,4-D) and 3% Gelrite (Trade Mark, Caroline Biological Supply) at pH 6.0. Callus was grown for 4-4 weeks prior to suspension culture initiation. Suspension cultures were initiated in a MS-based liquid medium containing 100 mg/l myo-inositol, 2 mg/l 2,4-D, 2 mg/l 1-naphthaleneacetic acid (NAA), 6mM proline, 200 mg/1 casein hydrolysate (Difco Laboratories), 35 (w/v sucrose and 5% coconut water (Difco Laboratories) at pH Cell suspensions were maintained in theis medium in 125 ml Erlenmeyer flasks at 28 0 C in the dark on a gyrating shaker at 125 rpm. Suspension were subcultured every 3.5 days by addition of 3ml packed volume of cells and 10 ml culture medium to 20 ml of fresh culture medium. The suspension cultures were typically 6 months to one year old at the time of bombardment. Suspension cultures recovered from cryopreservation were used in some transformations.

Microproiectile Bombardment Cell suspensions were sieved through a 1.0 mm and then a 0.5mm screen. A packed volume of 0.2 ml of the cells which passed through the sieves was then suspended in 5 ml of suspension medium and evenly distributed on to a Whatman No.4 filter paper disc via vacuum filtration using a 4.7 cm microanalysis holder. Precipitation of supercoiled plasmid DNA on to tungsten particles and bombardment using the DuPont PDS-1000 Biolistics (Trade Mark) apparatus were essentialy as described by the manufacturers. Target plates were bombarded once.

WO 97/04116 PCT/GB96/01675 Transformant selection and plant regeneration Following bombardment, each filter disc (with cells) was transferred to N6 based medium containing 100 mg/l myo-inositol, 2 mg/1 2,4-D, 3% sucrose, and 0.3% (w/v) Gelrite at pH 6.0. For selection using the NPTII gene, this medium was supplemented with 200 mg/l kanamycin sulphate. The filter discs were transferred to fresh medium containing the selection agent after seven and again after 14 days. The suspension was divided into two equal aliquots and each was evenly plated over 20 ml of solidified media containing the selective agent and 3% Gelrite in 100 x 20 mm Petri dishes. After 2-5 weeks, rapidly growing, putatively transformed calli were removed and transferred to the surface of fresh selection medium. Plants were renerated by transferring tissue to MS based medium containing 1 g/l myo-inositol, 1 mg/l NAA, 6% sucrose, and 3% Gelrite at pH After 2-3 weeks, the tissue was transferred to MS media containing 0.25 mg/l NAA, and 3% sucrose and placed in the light, where embryo germination occurred. Plants were then grown in half-strength MS based medium containing 500 mg/l myo-inositol, 3% (w/v) sucrose and 0.3% Gelrite at pH 6.0 for approximately 1-2 weeks priro to transfer to the greenhouse.

After transfer to the glasshouse, plants within each independently transformed clone were tested for the presence of the MFS 14/pre-B/T-urfl3 gene construct using the PCR.

DNA was extracted from small leaf samples using the technique described by Edwards et al (1991, Nucleic Acids Research, 19, 1349) and used in the PCR with the primers, 14-SA AGACGCTGAGCTCAAGGACGTGA-3' SEQ ID NO 9)and turf-2R (see Example 1 for the sequence of this primer).

At flowering the plants within each independently transformed clone were assessed in the glasshouse using a visual scale developed for CMS lines and described in Table 2. Plants scoring 4 and below are functionally sterile. The accumulated sterility scores for each of the independent PCR positive clones is shown in Table 3 and compared to a maize line which was generated by bombardment with RMS 11 but which is PCR negative for the MFS 14/pre-B/Turfl3 gene construct.

Sterile plants were backcrossed with pollen from fertile, non-transgenic BE70 plants.

the progeny seeds arising from one of these crosses (clone YK23, plant 5 x BE70) were planted in the glasshouse and allowed to flower. The presence of the MFS 4/pre-B/T-urfl3 gene construct as assessed by PCR and PAT test (the latter determines whether the selectable WO 97/04116 PCT/GB96/01675 11 marker used inthe transformation process is present) and the fertility scores of the plants are shown in Table 4. Plants which were PCR negative were rogued from the glasshouse prior to flowering. Plants 1 and 2 had an inconclusive PCR test and were kept until flowering. Plant 12 was kept as a control. As can be seen in Table 4, 6 of the progeny plants were sterile and this sterility correlated with the presence of the transgene as assessed either by PCR or PAT testing. This is consistent the the presence of a single transgenic locus imparting sterility.

TABLE 2 Anther Classification (After L.M. Josephson) Class 0 No anthers exerted Class 1 Less than half of the anthers exerted and all were small, dry and hard with no pollen shed.

Class 2 Most of anthers exerted but all were small, dary and hard with no pollen shed.

Class 3 Partially fertile anthers exerted with some pollen shed, proportion of anthers exerted was highly variable.

Class 4 Slightly abnormal anthers with approximately 75 to 100 percent exertion.

Class 5 Normal anthers and fully fertile.

Class 4 through 5 tassels were considered fertile.

TABLE 3 CLONE NO. OF PLANTS FERTILITY SCORE

P.C.R.

WK 23 2 0 3 3 WK 23 2 0 YK 23 3 0 1 4 YE23 4 WO 97/04116 PCT/GB96/01675 12- TABLE 4

NT

YK23/5/1 YK23/5/2 YK23/5/3 YK23/5/4 YK23/5/5 YK23/5/6 YK23/5/7 YK23/5/8 YK23/5/9 YK23/5/10 YK23/5/11 YK23/5/12 PCR TEST PAT TEST FERTILITY

SCORE

0

ROGUED

ROGUED

NO GERMINATION 1 0

ROGUED

0

ROGUED

0 Example Construction of the maize transformation vector pRMS-23 We have tested whether expression of a short-sense construct from the maize adenine nucleotide translocator (ANT) gene will give rise to a defect in the growth of maize cells. A fragment of the maize ANT was isolated using the PCR and primers designed from the sequence of the maize gene published by Bathgate et al (1989, Eur. J. Biochem., 83, 303- 310).

The fragment of theANT gene was amplified in the PCR using primers ATGCCCGGGCTTGCAATGTCTGTTAGCGGTGGCATCA-3', SEQ ID NO 10) and MANT-2RB (5-ATGCCCGGGCGATGGGGTAAGATGCAAGACCA-3', SEQ ID NO 11).

The PCR conditions were 35 cycles of denaturing at 94oC for 0.8 min, annealing at 65°C for 1 min and extension at 72°C for 2.5 mins. To aid subsequent cloning the PCR primers were designed such that they introduce unique Smal restriction sites at the 5' and 3' ends of the gene. The sequence of the maize ANT gene was published in Eur. J. Biochem. (1989) 183, 303-310. The MANT-1 primer sequence appears at the beginning of the coding sequence of the gene and the MANT-2R primer sequence is near the end of the gene.

WO 97/04116 PCT/GB96/01675 13 Following PCR which produced a DNA fragment of the predicted size of 1050 bp, the DNA was digested with Smal and subcloned into the Sinal site ofpUC8 to give pMANT1.

Subsequently the nos 3' polyadenylation signal sequence was introduced 3' to the ANT gene as a SacI- EcoRI fragment into the corresponding sites in pMANTI to yield pMANT2.

A

IindIII BanHI fragment carrying the CaMV 35s promoter and ADH lintron from pCaMVI 1 N (Figure 5) was introduced into the corresponding sites of pMANT2 to yield pMANT 3. pRMS-23 (Figure 12) was completed by introduction of the PAT in vitro selection cassette from pIE109 (Figure 7) into the unique EcoRI site of pMANT3.

Example 6 Transformation of BMS corn cells with pRMS-23 The objective of this experiment was to show that expression of pRMS-23 in cultured BMS corn cells results in a reduction in cell viability as measured by the establishment of transgenic calli following transformation in two separate experiments. The vector DNAs were introduced into cultured BMS cells using the silcon carbide fibre transformation techniqiue as described in Example 2.

Following transformation with pRMS23 the mean numbers of transgenic calli established were determined relative a positive control pPG3 wihich contains the in vitro selection cassette alone (Table These data show that expression of a short sense adeneine nucleotide translocator gene results in a significant decrease in the establishment of transgenic calli.

Experiment 1 pPG3 pRMS-23 Experiment 2 pPG3 pRMS-23 TABLE No of transgenic calli per replicate 1 2 3 16 20 22 3 4 3 49 48 40 10 15 23 Mean 19 3 46 16 WO 97/04116 PCT/GB96/01675 -14- Example 7 Construction of the maize transformation vectors. pTBR and pTBS We tested whether un-regulated expression of a-tubulin genes will give rise to a defect in the growth of maize cells. Two constructs containing the coding sequence from atubulin cDNAs isolated from two biotypes ofEleusine indica were prepared. pTBR (Figure 13) contains the cc-tubulin cDNA from a dinitroaniline resistant biotype ofEleusine indica cloned as a blunt-ended Hinf I fragment into the blunt-ended BamHI site of pCaMVIIN (Figure pTBS (Figure 13) contains the a-tubulin cDNA from a dinitroaniline sensitive biotype cloned exactly as described for pTBR.

Example 8 Transformation of BMS corn cells with pTBR and pTBS.

The objective of this experiment was to show that expression of pTBR and pTBS in cultured BMS corn cells results in a reduction in cell viability as measured by the establishment of transgenic calli following transformation. The vector DNAs were introduced into cultured BMS cells using the silcon carbide fibre transformation techniqiue as described in Example 2.

Following transformation with pTBR and pTBS the mean numbers of transgenic calli established were determined relative a positive control pPG3 wihich contains the in vitro selection cassette alone (Figure 14). These data show that non-regulated expression of an atubulin gene from either biotype ofEleusine indica results in a significant decrease in the establishment of transgenic calli.

WO 97/04116 PCT/GB96/01675 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: ZENECA LIMITED STREET: 15 Stanhope Gate CITY: London COUNTRY: UK POSTAL CODE (ZIP): W1Y 6LN (ii) TITLE OF INVENTION: Production of Male Sterile Plants (iii) NUMBER OF SEQUENCES: 11 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 357 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: T-urfl3 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATCGGATCCA TGATCACTAC TTTCTTAAAC CTTCCTCCCT TTGATCAAGG TTTGGTATTT TTCGGTTCTA TTTTTATTTT TTTTTTGTGC ATATTATTGA TAAAGGGATA TCTCCGTAAA 120 ATGGATGATT CCTATTTGGC TCAACTCTCC GAGTTAGCCA ACCACAATAG AGTGGAAGCG 180 GCAAAAGCGG GCCACGTGGC CCTGCATGAG CTATCCTTCT CGTGGTTGAG GGGGGTTCAA 240 ATTAGGGTGA GGACCTTACC TATACAACGG AATGAAGGAG GGGGTCGAAG CAACGACCAA 300 TCCACTCTCT CTAAGCCTAA GTATTCCTCA ATGACCGATA GCGTACAAGT ACCGTGA 357 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (vi) ORIGINAL SOURCE: ORGANISM: Turf-i primer WO 97/04116 PCT/GB96/01675 16- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATCGGATCCA TGATCACTAC TTTCTTAAAC CTTCCT 36 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (Vi) ORIGINAL SOURCE: ORGANISM: Turf-2R primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TAGTCTAGAT CACGGTACTT GTACGCTATC

GGT

33 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 269 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: ATP-2 gene of Nicotinia plumbaginifolia (ix) FEATURE: NAME/KEY:

CDS

LOCATION:1..267 OTHER INFORMATION:/codon start= 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ATG GCT TCT CGG AGG CTT CTC GCC TCT CTC CTC CGT CAA TCG GCT CAA 48 Met Ala Ser Arg Arg Leu Leu Ala Ser Leu Leu Arg Gln Ser Ala Gin 1 5 10 CGT GGC GGC GGT CTA ATT TCC CGA TCG TCA GGA AAC TCC ATC CCT AAA 96 Arg Gly Gly Gly Leu Ile Ser Arg Ser Ser Gly Asn Ser Ile Pro Lys 25 TCC GCT TCA CGC GCC TCT TCA CGC GCA TCC CCT AAG GGA TTC CTC TTA 144 Ser Ala Ser Arg Ala Ser Ser Arg Ala Ser Pro Lys Gly Phe Leu Leu 40 AAC CGC GCC GTA CAG TAC GCT ACC TCC GCA GCG GCA CCG GCA TCT CAG 192 Asn Arg Ala Val Gin Tyr Ala Thr Ser Ala Ala Ala Pro Ala Ser Gln 55 CCA TCA ACA CCA CCA AAG TCC GCC AGT GAA CCG TCC GGA AAA ATT ACC 240 WO 97/04116 PCT/GB96/01675 17- Pro Ser Thr Pro Pro Lys Ser Ala Ser Glu Pro Ser Gly Lys Ile Thr 70 75 GAT GAG TTC ACC GGC GCT GGT TCG ATC GG Asp Glu Phe Thr Gly Ala Gly Ser Ile 269 INFORMATION FOR SEQ ID NO: I0 SEQUENCE

CHARACTERISTICS:

LENGTH: 89 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Ala Ser Arg Arg Leu Leu Ala Ser Leu Leu Arg Gin Ser Ala Gin 1 5 10 Arg Gly Gly Gly Leu Ile Ser Arg Ser Ser Gly Asn Ser Ile Pro Lys 25 Ser Ala Ser Arg Ala Ser Ser Arg Ala Ser Pro Lys Gly Phe Leu Leu 40 Asn Arg Ala Val Gin Tyr Ala Thr Ser Ala Ala Ala Pro Ala Ser Gin 55 Pro Ser Thr Pro Pro Lys Ser Ala Ser Glu Pro Ser Gly Lys Ile Thr 70 75 Asp Glu Phe Thr Gly Ala Gly Ser Ile INFORMATION FOR SEQ ID NO: 6: SEQUENCE

CHARACTERISTICS:

LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (vi) ORIGINAL

SOURCE:

ORGANISM: PREB-IB primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ATCGGTACCG CCATGGCTTC TCGGAGGCTT CTCGCCT 37 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA WO 97/04116 PCT/GB96/01675 18ivi) ORIGINAL SOURCE: ORGANISM: PREB-2R primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 7: ATCGGATCCC GCTGCGGAGG

TAGCGTA

2) INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 2285 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL

SOURCE:

ORGANISM: MFS14 Promoter (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: AAAAGCGTAC CAGTAAGGGA TAAAGAAAAT AAACAAACAC GAAATGCTTC

CCCATCGGCC

AATTCGCCTA

GGTGTGCTAG

CCAGAAAACT

TAGAAAACTC

CGGTGATCAT

ATGGCTTCTG

TCTCAAAATA

GTTTAGCCAT

CCGGACCCTA

GGCTGTGGGG

GACCGATTGA

TAATGTTGAT

GGGGGCAGAA

CATACTGCCG

GGTTCTGTGT

ATGTGTATCC

GCTATTATTG

GGGCGCCACG

CGACGGTCTG

TGTAAGGGCC

AGGGCCGTCG ATCTGCCAAG ATCAACATAC

CATATAATGG

ATAAATATCA

TGTTACTAGT

ATACACGTTA

ATTTTCTAAT

TGCAAATGGT

CTACATACTG

ACTTGAAGCG

AAGATAGAAG

GACGATCCAC CATGTATTGT ATTGTTGTTG

CTCATCGGTC

GAACTGGCCT

TGGTATCCTT

ATATGATTGC

CTCTTCCCTA

TTCATTCGGT

CGGCCTGTGA

TTGTGGTATG

GATATGTCCG

CGTAGCCCTG

GAACTATCCA

CAAGGACGAT

ATGGGGCTGC

TTCATATGAT

TGGTTCTTCT

TTTAATAGAT

AGATGTGACG

GACAAGAATT

GTCGGACTCT

ATATGTTCGT

GCCCCTTGTG

CCCACTCTAT

ATTTTCTACA

CGGGCATTGT

TCATATGGCG

TAATAATTTG

GTGGTACATC

GTGCGGCCCG

GACAAAAGCT

GGTGATGGTA

AAATCCCCAT

GGAAAACTAG

AACCCTCTAA

TGGATGTCGT

TCGGTATCGC

TCTCCTTCGT

TAATAGCCGG

GTGTGCTTCT

GATATGGGAC

CCACACCAAC

TTGCACTCGG

TATGCCGACC

TGTTGTGGGT

GTGCATAGTG

TGAACTGGCC

GACTATCCGG

CGGACGGTCC

TTTGCCCTGG

TTGTCGCCGA

GAGCAACAGA

TCTAATGCTT

CGGGTTTACT

ATGTTTGACA

TTGACACATG

CTTTAGGATA

CCTATTTTCA

AATGTCAAAC

TCCGAGTCTA

TGGCAGACCA

TTCTTGCCAT

TAAATATGTA

TGCGACAGTA

GGTTGTGTTA

CAGAGAAAGC

GACCGTGTAG

ATATGAGTT C

TGTATTAGAC

CTTCTCCAAC

CATTTCATTA

TACGTTAGGG

ACTTTCTGGT

TAGTCCTCGT

TTGTCCGGGG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 AGATATCGGT GTGATCTTTA GAACCGCCAT TTGATGGCCT GAGTTTTAGT

AGATCTAGAC

WO 97/04116 PCT/GB96/01675 19 ACATTTCCCC AACGGAGTCG CCAAkA.AGTG TGTTGGCGCC GATCCAGGCG

CGAAACACTG

GAGATGGACC

GTTTGGCGGI

CGACTCTCCT

CTACGTGTGT

CGCAGAGGGT

CTTCTTCTTC

GGAGGAGAGA

TTGTAGGGTG

ACGTAGAGCC

GAAGAGAGGT

AGAACTACTG

CTAATGCATA

AAGTAATCTG

ACTGTAGCCC

GTTTTCCGAG

CTAATCTCAC

ACTGATTCTA

CTCATGCGCG

AGTTAACCCT

CTGTTGTGCG

ATTTGTAGGG

ATTTAACATA

TTAAAGAGGC

AGATGCTAGA

CGTCCACGCC

GGCGGAGATG

GGACGTGACA

CCGCGCGTAC

CCAAGCTCCC

GCGTCCTGAT

CATTGCACCC

TTCAAAGCTC

GTTCTCCGGG TGAGGACGGT

CCGCGACCTG

CCGGACGGTC

GCAGCCGACC

TGTCTTGGCG

GAAGGATTGA

AGGTAAAAAC

TTTATCTAT.

GGTTTTAGTT

AACCATTCGT

CTGTGATTTG

CCAAGTGCTG

GCTCGTCCAG

AACGGCAACA

CTCGCGTTCA

CCGTAGGTGA

GAACCTACpA,

CGTCGTCTGC

TAGATCTCGC

TCGACAGGCC

GGTAGAAGGA

GAGAAGTGGA

TAAAGGGGAG

AATAAATC CT GC GC CAC CGCC

GTGGCATATA

CGAAAGGAAT

CTCAGCGCTG

AAGGCGGAAA

GTTGGCTCAC

GCCATGCAAA

ATAGCGTGCA

GGCTCGGACG

CTCCCGGGAG

ACACAATACG

GGCTAAACTT

CTTCATTTGA

GTATGGACCC

GCGAGAAACT

T GCC CGT CCC

AAACCACATT

CGTTTTCGGA

AGCACCTGTG

GGCCGAGACG

ACAACAGCAG

GGTCCAGCAG

GTCGCGATGG

GGACCGTCGG

CCTCTAGTCG

GGGCTAAACT

TCGATTGTGG

GTTACAAGC

C

CGGAACTCTA

G CGAT CGCT C T GCAATAAAA

GGACCCAAAA

TTGTCTTCCT

CTGAGCTCAA

CTCGCTCGCC

1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2285 23 GGTCGCCGCG

CGCCCTGATC

CACACAGCAA GCCAGCAGAG CAGAAAGCAG CCGCAGCCCC AGCCCCCACA

AAGACGAAGG

CAACA

INFORMATION FOR SEQ ID NO: 9: SEQUENCE

CHARACTERISTICS:

LENGTH: 23 base pairs 4i TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (vi) ORIGINAL

SOURCE:

ORGANISM: 14-SA Primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AGACGCTGAG CTCAAGGACG

TGA

INFORMATION FOR SEQ ID NO: SEQUENCE

CHARACTERISTICS:

LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/04116 PCT/GB96/01675 (ii) MOLECULE TYPE: DNA (vi) ORIGINAL SOURCE: ORGANISM: MANT-1 Primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATGCCCGGGC TTGCAATGTC TGTTAGCGGT GGCATCA 37 INFORMATION FOR SEQ ID NO: 11: SEQUENCE

CHARACTERISTICS:

LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (vi) ORIGINAL

SOURCE:

A) ORGANISM: MANT-2RB Primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATGCCCGGGC GATGGGGTAA GATGCAAGAC

CA

Claims (14)

1. A method of inhibiting gene expression in a target plant tissue which comprises stably transforming a plant cell of a type from which a Whole plant may be regenerated with a gene construct carrying a tissue-spedfic or a development-specific promoter which operates in the cells of the target plant issue and a sequence which encodes a fusion of a mitochondrial targeting sequence and a T-urfl3 disrupter gene product, which fusion protein is capable, when expressed, of inhibiting respiration in the cells of the said target tissue.

2. A method according to claim 1 wherein the mitochondrial targeting sequence is the functional pre-P mitochondrial targeting sequence from Nicotianaplumbaginolia.

3. A method according to claim 1 or claim 2 wherein the tissue-specific is an anther or 15 tapetum specific promoter.

4. A method according to claim 3 wherein the tissue specific promoter is a tapetum specific promoter.

5. A plant having stably incorporated in its genome by trasformation a gene construct carrying a gene construct carrying a tissue-specific or a development-specific promoter which operates in the cells of the target plant tissue and a disrupter gene encoding a fusion of a mitochondrial targeting sequence and a T-urfl3 disrupter gene product, which fusion protein is capable, when expressed, of inhibiting an essential cell function in the cells of the said target tissue.

6. A plant having stably incorporated within its genomc a gene construct carrying a tissue-specific promoter which operates in the cells of the said target tissue and a disrupter gene encoding a fusion of a mitochondrial targeting sequence and a T-urfl 3 disrupter gene product, which fusion protein is capable of inhibiting an essential cell Sfunction in the said cells of the said target tissue.

7. A plant as claimed in claim 5 or claim 6 which is a inorlocotyledonous plant S. A plant as claimed in claim 7 which is a corn plant.

9. A plant as claimed in any one of claims 5 to 8, wherein'fthe promoter is an anther- and/or tapeturn-specific promoter. A plant as claimed in claim 9 wherein the promoter is a tapetum-specific prOrnoter.

11. Amtethod as claimed in claim 4 or aplant as claimed izn claim 10 wherein the promoter may be isolated using the cDNA sequences of any of Figures I to 3. S12. A method or a plant as claimed in claim 1ll, wherein tho promoter is the promoter o 15 from the MffS14 gene (SEQ ID NO 8) 1 13. A method of inhibiting gene expression in a target plant tissue which comprises stably transforming a plant cell of a type from which a whole plant may be regeneratd wtageecnrutarying a tapetum-specific promoter which operates in tapetal cells of the target plant and a T-urfl 3 disrupter gene, which is capable, when expressed, of inibiting essential cell. function in the cells of the S* tapetum.

14. A mnale sterile corn plant having stably incorporated within its genome a gene construct carrying a tapetum-specific promoter which operates in the cells of tapeturn '.:and a T-urfl 3 disrupter gene encoding a protein which is capable of inhibiting an essential cell function in the cells of the tapetum. A method as defined in claim I of inhibiting gene expressionl in a target plant tissue substantially as herein described with reference to any, onl of Exaffplcs I to 4 herein. I/Q 23

16. A plant as defined in claim 5 substantially as herein des~ribed %iffi reference to any one of Examples 1 to 4 herein.

17. A plant as defined in claim 6 substantially as herein described with reference to any one of Examplesi to 4 herein

18. A male sterile corn plant as defined in claim~ 1.4 sub stan~ially as herein described with reference to any one of Examples I to 4 herein.

19. A method according to claim 1 or claim 13 substantially as hereinbefore( described with reference to any of the examples. DATED: 11 February, 1999 PHILLIPS ORMONDE FITZPATRICK Attor ne ys for: ZENECA LIMITED