WO2002078427A2

WO2002078427A2 - Specification of meiocyte and tapetal cell layers in plants

Info

Publication number: WO2002078427A2
Application number: PCT/GB2002/001478
Authority: WO
Inventors: Hugh Dickinson; Roderick Scott; Claudia Canales
Original assignee: Isis Innovation Limited
Priority date: 2001-03-30
Filing date: 2002-03-28
Publication date: 2002-10-10
Also published as: GB0108048D0; WO2002078427A3; AU2002241163A1

Abstract

The invention provides a plant which is, or is capable of being, male-sterile by virtue of modulation of the expression of an EXTRASPOROGENOUS CELL (ESP )gene, or of the activity of the encoded ESP protein.

Description

SPECIFICATION OF MEIOCYTE AND TAPETAL CELL LAYERS IN PLANTS

FIELD OF THE INVENTION

The present invention is in the field of plant biotechnology. It relates more particularly to the provision of male-sterile plants, especially for use in breeding programmes and agriculture. This is based on the cloning and characterisation of a new gene, EXTRASPOROGENOUS CELLS (ESP), whose sequence is provided herein.

BACKGROUND OF THE INVENTION

Cell specification in plants

Plants differ from other multicellular organisms in that they develop from meristems. Embryonic morphology develops exclusively from the shoot and root apical meristems (SAM and RAM) and the fate of cells generated at these meristems is largely dependent upon their position. Genes involved in the specification of cell fates at the RAM and SAM have been identified and events at the SAM during flowering have been well characterised. Apart from genes directing the transition to flowering, the "mapping out" - in space and time - of the floral apex into domains subsequently to be occupied by the floral organs is achieved by the combinatorial action of a range of known homeotic genes. However, even in the well-characterised model plant Arabidopsis, while many of the sequences controlling development of the floral organs are known, little information is available on the genes directing development of the male and female germlines within them. Certainly SPOROCYTELESS/NOZZLE is expressed in the very young anther, and may play a part in the differentiation of the archesporeal tissue, but this gene seems to play little, if any, role in the specification of key cell types. AGAMOUS is transcribed in the anther, but its expression pattern suggests that it also is uninvolved in the determination of cell fate. Importantly, the readily-understandable combination of meristem and developing files of cells that characterise the SAM and RAM does not pertain in the anther, as a relatively small number of divisions of the L2 layer (which even differ between species) within a single sheet of epidermal cells gives rise to the meiocytes, the surrounding tapetal layer, a middle layer and the endothecium. Logic dictates that cell type within each anther loculus is specified in a radial fashion, but there is currently no knowledge as to how it is regulated at a molecular level. The many male sterile mutants of Arabidopsis do little to elucidate these events, for they mostly take effect well after cell fate has been determined. Further, the paucity of mutations acting at early developmental stages frustrates any effort to study the specification of cell fate and interrelations between cell types during development.

Male sterility

Male-sterile plants are extremely useful in plant breeding programmes. In crops such as maize, the majority of seed sold to farmers is so-called FI hybrid seed. An FI hybrid is a first-generation cross between two lines, generally inbred or elite inbred lines, these inbred lines being largely homozygous. A cross between the two lines therefore has a very well-defined genotype, which is advantageous to the farmer. Moreover, FI hybrid plants generally benefit from "hybrid vigour", i.e. improved agronomic properties compared to the parent lines or progeny of subsequent generations. However, an FI hybrid of known genetic composition can only be created if it can be assured that there is a genuine out-cross between the two parent lines. Often, the parent lines are capable of self-pollination and, if that occurs, not all of the seed produced is FI hybrid seed. To overcome this, plant breeders have traditionally removed the male reproductive parts of the female parent lines by hand, a procedure known as "de-tasselling" in maize. With the advent of biotechnology, various methods become available to render the plants automatically, or inducibly, male-sterile instead. Various male sterility systems are thus available. Some rely on so-called cytopiasmic male sterility, based in the mitochondria. Others rely on, for example, the expression of diphtheria toxin or RNA enzymes as in the Barnase/Barstar system. Current male sterility technologies do however suffer from several drawbacks. Cytopiasmic male sterility is technically difficult to engineer and also "leaky" in the sense that it is not fully effective and some plants remain male-fertile. Diphtheria toxin has the disadvantage that transgenic crops containing toxic products are unattractive to the consumer. The Barnase/Barstar system has the disadvantage that any problems with the localisation of the expression of the RNA gene to the anther leads to a great deal of damage in other cells of the plant. Another disadvantage of some currently available systems is that they also compromise female fertility, which is undesirable.

There is therefore a need for the development of improved male sterility systems. Since an understanding of the processes which generate genetic variation in crops is of key importance to the plant breeding industry, the molecular analysis of the mechanisms central to the origin and specification of plant germlines must remain a high priority.

An increasing number of male-sterility mutations have been identified in Arabidopsis, which fall naturally into three classes: (1) structural, resulting from morphological anomalies (e.g. stamenless); (2) functional, where viable pollen is produced but is prevented from affecting fertilisation; (3) and sporogenous where the lesion affects germ line development. Significantly, despite the large number of sporogenous mutants described, very few mutants have been reported which affect the specification, organisation or development of the microsporangial cell layers. Important in this context has been the recent cloning of SPOROCYTELESS/NOZZLE which appears to be involved in the specification of sporogenous cell fate and in the development of the anther wall. This gene, which may encode a MADS-box transcription factor, has also a dramatic effect on female phenotype. SUMMARY OF THE INVENTION

The present invention is based on the cloning and characterisation of a new gene, EXTRASPOROGENOUS CELLS (ESP), whose sequence is provided herein. Importantly, esp mutants fail to form fertile pollen; although ESP anthers commence early development they fail to form the key nutritive tapetal layer - with the result that the cell line destined to become pollen grains (the sporogenous cells or "germ line") fails immediately after meiosis. Very significantly, the failure of the tapetum to form is accompanied by the initial generation of a larger number of germ line cells, even though these then die.

Molecular analysis has identified the ESP gene as encoding a leucine-repeat receptor kinase and it is reasonable to assume that ESP forms part of a signalling system involved in cell type specification early in anther development.

We have now cloned the gene encoding ESP in Arabidopsis, and its sequence is provided herein. In addition, we have confirmed that it is expressed specifically in the young, developing anther, and in the ovule. In fact, our studies point to the transcript being present in very young reproductive apices, effectively delimiting those regions which will develop into anthers and ovules. No other gene with this type of expression has been discovered.

These are significant findings, and the ESP gene forms the basis of novel and improved male sterility strategies.

Accordingly, the invention provides:

a plant which is, or is capable of being, male-sterile by virtue of modulation of the expression of an EXTRASPOROGENOUS CELLS (ESP) gene, or of the activity of the encoded ESP protein. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: C24 Arabidopsis genomic sequence of ESP.

Figure 2: Amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

Vectors and chimeric genes

Genes and polynucleotides according to the invention

The ESP gene whose genomic sequence is provided herein was cloned from Arabidopsis. However, the skilled person will appreciate that homologous genes will exist in other species. In particular, it is envisaged that homologous genes will exist the plants mentioned in the section below entitled "Plants of the invention". Based on the genomic DNA sequence and the amino acid sequence of ESP provided herein, a skilled person would readily be able to design probes and primers to identify and obtain homologues in other plant species. Such homologues form part of the invention.

A preferred homologue of the invention or polynucleotide of the invention, which may be isolated form, generally:

(a) encodes a polypeptide sequence as set out in SEQ ID NO: 1 ; or

(b) has a cDNA sequence having 60% or more homology to a sequence of (a); or

(c) has a cDNA sequence capable of hybridising selectively to the complement of a sequence of (a); and when functional, encodes a leucine-repeat receptor kinase; and, when non-functional, causes the plant to fail to produce fertile pollen.

Hybridisable sequences

A polynucleotide of the invention may hybridise selectively to the coding sequence of (a) at a level significantly above background. Background hybridisation may occur, for example because of other cDNAs present in a cDNA library. The signal level generated is typically at least 10 fold, preferably at least 100 fold, as that generated by background hybridisation. The intensity of interaction may be measured, for example by radiolabelling the probe, e.g. with ³²P. Selective hybridisation is typically achieved using conditions of medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C, for example 45 to 50, 50 to 55 or 55 to 60°C, e.g. at 50 or 60°C.

However, such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual). For example, if high stringency is required, suitable conditions include 0.2 x SSX at around 60°C, for example 40 to 50°C, 50 to 60°C or 60 to 70°C, e.g. at 50 or 60°C. If lower stringency is required, suitable conditions include 2 x SSC at around 60°C, for example 40 to 50°C, 50 to 60°C or 60 to 70°C, e.g. at 50 or 60°C.

Stringency typically occurs in a range from about Tm-5°C (5°C below the melting temperature (Tm) of the two sequences hybridising to each other in a duplex) to about 20°C to 25°C below Tm. Thus, according to the invention, a hybridisable sequence may be one which hybridises at a temperature of from Tm to Tm-25°C, e.g. Tm to Tm-5°C, Tm-5 to Tm-10°C, Tm-10 to Tm-20°C or Tm-20 to Tm-25°C. Homologous sequences

A polynucleotide sequence of the invention, will typically comprise a coding sequence at least 60% or 70%, preferably at least 80 or 90% and more preferably at least 95, 98 or 99%, homologous to the coding sequence of (a) above.

Such homology will preferably apply over a region of at least 20, preferably at least 50, for instance 100 to 500 or more, contiguous nucleotides.

Methods of measuring nucleic acid and polypeptides homology are well known in the art. These methods can be applied to measurement of homology for both polypeptides and nucleic acids of the invention. For example, the UWGCG Package provides the BESTFIT program which can be used to calculate homology (Devereux et al, 1984, Nucleic Acids Research 12, p.387-395).

Similarly, the PILEUP and BLAST algorithms can be used to line up sequences (for example as described in Altschul, S.F., 1993, J. Mol. Evol. 30:290-300; Altschul, S.F. et al, 1990) J Mol. Biol. 215:403-410).

Many different settings are possible for such programs. According to the invention, the default settings may be used.

In more detail, the BLAST algorithm is suitable for determining sequence similarity and it is described in Altschul et al (1990) J Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi/nlm.hih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11 , the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Nad. Acad. Sci. USA 89: 10915- 10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g. Karlin and Altschul (1993) Proc. Nad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a fused gene or cDNA if the smallest sum probability in comparison of the test nucleic acid to a fused nucleic acid is less than about 1 , preferably less than about 0.1 , more preferably less than about 0.01 , and most preferably less than about 0.001.

Fragments

Also included within the scope of the invention are sequences which are fragments of the sequences of (a) to (c) above but have the properties of the polypeptides of invention as defined aboved. Degenerate sequences

Also included within the scope of the invention are sequences that differ from those of (b) and (c) above but which, because of the degeneracy of the genetic code, encode the same polypeptides.

Complementary sequences

In addition, the invention provides polynucleotides having sequences complementary to any of the above-mentioned sequences. Such polynucleotides may be useful in antisense and/or RNAi applications as discussed herein.

Further properties

Polynucleotides of the invention may comprise DNA or RNA. They may also be polynucleotides which include within them synthetic or modified nucleotides.

Polynucleotides of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe, e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will preferably be at least 10, preferably at least 15 or 20, for example at least 25, 30 or 40 nucleotides in length. These will be useful in identifying species homologues and allelic variants as discussed above.

Polynucleotides such as a DNA polynucleotides and primers according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. The polynucleotides are typically provided in isolated and/or purified form. In general, primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.

Genomic clones corresponding to the homologues of the invention and containing, for example introns and promoter regions are also aspects of the invention and may also be produced using recombinant means, for example using PCR (polymerase chain reaction) cloning techniques.

Polynucleotides which are not 100%) homologous to the sequences of the present invention but fall within the scope of the invention, as described above, can be obtained in a number of ways, for example by probing cDNA or genomic libraries from other plant species with probes derived from SEQ ID NO: 1, 3 or 5. Degenerate probes can be prepared by means known in the art to take into account the possibility of degenerate variation between the DNA sequences of SEQ ID NO: 1, 3 or 5 and the sequences being probed for under conditions of medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C), or other suitable conditions (e.g. as described above).

Allelic variants and species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding likely conserved amino acid sequences. Likely conserved sequences can be predicted from aligning the amino acid sequences of the invention with each other and/or with those of any homologous sequences known in the art. The primers will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences. Alternatively, such polynucleotides may be obtained by site-directed mutagenesis. This may be useful where, for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequences may be desired in order to introduce restriction enzyme recognition sites, or to alter the properties or function of the polypeptides encoded by the polynucleotides.

The invention further provides double stranded polynucleotides comprising a polynucleotide of the invention and its complement.

Polynucleotides, probes or primers of the invention may carry a revealing label. Suitable labels include radiosotopes such as ³²P or ³⁵S, enzyme labels, or other protein labels such as biotin. Such labels may be added to polynucleotides, probes or primers of the invention and may be detected using techniques known per se.

Polypeptides of the invention

Polypeptides of the invention may be encoded by polypeptides as described above. A polypeptide of the invention may consist essentially of the amino acid sequence set out in SEQ ID NO: 1 or a substantially homologous sequence, or of a fragment of either of these sequences, as long as the properties of the invention are maintained.

In particular, a polypeptide of the invention may comprise:

(a) the polypeptide sequence of SEQ ID NO: 1 ;

(b) a polypeptide sequence at least 60, 70, 80, 90, 95, 98 or 99% homologous to, a polypeptide of (a); or

(c) an allelic variant or species homologue of a sequence of (a). Allelic variants

An allelic variant will be a variant which occurs naturally and which will function in a substantially similar manner to the protein of SEQ ID NO: 1. Similarly, a species homologue of the protein will be the equivalent protein which occurs naturally in another species.

Homologues

A polypeptide of the invention is preferably at least 60% homologous to the protein of SEQ ID NO: 1 more preferably at least 80 or 90% and more preferably still at least 95, 97 or 99% homologous thereto over a region of at least 20, preferably at least 30, for instance at least 40, 60 or 100 or more contiguous amino acids. Methods of measuring protein homology are well known in the art and it will be understood by those of skill in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as "hard homology").

Degrees of homology can be measured by well-known methods, as discussed herein for polynucleotide sequences.

The sequence of the polypeptides of SEQ ID NOs: 2, 4 and 6 and of the allelic variants and species homologues can be modified to provide further polypeptides of the invention.

Substitutions

Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions. For example, a total of up to 1, 2, 5, 10 or 20 amino acids may be substituted over a length of 50, 100 or 200 amino acids in the polypeptides. For example, up to 20 amino acids substituted over any length of 50 amino acids. The modified polypeptide generally retains the neurological properties of the invention, as defined herein. Conservative substitutions may be made, for example according to the following table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

Fragments

Polypeptides of the invention also include fragments of the above-mentioned full length polypeptides and variants thereof, including fragments of the sequence set out in SEQ ID NO1 : 1. Such fragments typically retain the properties of the invention.

Suitable fragments will generally be at least about 20, e.g. at least 20, 50 or 100 amino acids in size. Polypeptide fragments of the polypeptides of SEQ ID No:l and allelic and species variants thereof may contain one or more (e.g. 2, 3, 5, 5 to 10 or more) substitutions, deletions or insertions, including conservative substitutions. Each substitution, insertion or deletion may be of any length, e.g. 1, 2, 3, 4, 5, 5 to 10 or 10 to 20 amino acids in length. Isolation and purification

Polypeptides and polynucleotides of the invention may be in a substantially isolated form. It will be understood that they may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A polypeptide or polynucleotide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide or polynucleotide, as the case may be, in a preparation in which more than 70%), e.g. more than 80, 90, 95, 98 or 99% of the polypeptide in the preparation is a polypeptide of the invention.

Production of polypeptides

Polypeptides of the invention may be produced in any suitable manner. It is preferred that they be produced recombinantly from polynucleotides of the invention by expression in a suitable host cell, e.g. a bacterial, yeast or plant cell, preferably a plant cell, e.g. of a species mentioned herein, either in culture or in planta. Polypeptides may be recovered and, optionally, purified by techniques known in the art. This can be done using known techniques.

Replicable vectors

Polynucleotides of the invention can be incorporated into recombinant replicable vectors. Such vectors may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and cultivating the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below in connection with expression vectors. Bacterial cells, especially E. coli are preferred. Chimeric genes and expression vectors

In particular, the invention provides a chimeric gene comprising, operably linked to one or more regulatory sequences capable of securing its expression in a cell, a coding sequence encoding a polypeptide of the invention.

Such chimeric genes can, in turn, be incorporated into expression vectors. Thus, preferably, a polynucleotide of the invention is operably linked to regulatory sequences capable of effecting the expression of the coding sequence by a host cell. Such chimeric genes and expression vectors can be used to express the polypeptides of the invention.

The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequences.

Such chimeric genes and expression vectors may be introduced into a suitable host cell to provide for expression of a polypeptide or polypeptide fragment of the invention, as described below.

The vectors may be for example, plasmid, cosmid, virus or phage vectors provided with an origin of replication, preferably a promoter for the expression of the said polynucleotide and optionally an enhancer and/or a regulator of the promoter. A terminator sequence may also be present, as may a polyadenylation sequence. The vectors may contain one or more selectable marker genes, for example antibiotic ampicillin resistance genes. These will generally be operably linked to regulatory sequences capable of securing their expression in the host cell, as described herein for the coding sequences of the invention. Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell. The vector may also be adapted to be used in vivo, for example for generation of transgenic plants of the invention.

So far as plasmid vectors are concerned, plasmids derived from the Ti plasmid of Agrobacterium tumefaciens are especially preferred, as are plasmids derived from the Ri plasmid of Agrobacterium rhizogenes.

A further embodiment of the invention provides host cells transformed or transfected with the vectors for the replication and expression of polynucleotides of the invention. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (bacterial), plant, yeast, insect or mammalian cells, bacterial and plant cells being preferred.

Polynucleotides according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides may also be produced by synthetic means. Such antisense polynucleotides may be used in a method of reducing the levels of expression of polypeptides having the sequence of SEQ ID NO: 1, or variants or species homologues thereof in planta.

An antisense polynucleotide of the invention may be capable of hybridising to mRNA of a gene of the invention, or a variant or species homologue thereof, as defined herein (a "target" mRNA) and may thus inhibit expression by interfering with one or more aspects of mRNA metabolism including transcription, mRNA processing, mRNA transport from the nucleus, translation or mRNA degradation .

The antisense polynucleotide may be DNA, but is typically RNA. The antisense polynucleotide may be provided as single or double stranded polynucleotide. The antisense polynucleotide typically hybridises to the target mRNA to form a duplex (typically an RNA-RNA duplex) which can cause direct inhibition of translation and/or destabilisation of the mRNA. Such a duplex may be susceptible to degradation by nucleases.

The antisense polynucleotide may hybridise to all or part of the target mRNA. Typically the antisense polynucleotide hybridises to the ribosome binding region or the coding region of the target mRNA. The polynucleotide may be complementary to all of or a region of the target mRNA. For example, the polynucleotide may be the exact complement of all or a part of target mRNA. However, absolute complementary is not required and polynucleotides which have sufficient complementarity to form a duplex having a melting temperature of greater than 20°C, 30°C, or 40°C under physiological conditions are particularly suitable for use in the present invention. The polynucleotide may be a polynucleotide which hybridises to the target mRNA under conditions of medium to high stringency such as 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C.

In one preferred embodiment the antisense polynucleotide sequence is complementary to the entire coding sequence of the target mRNA and to the nucleotides of the mRNA immediately 5' of the coding sequence. However, the polynucleotide may hybridise to all or part of the 5'- or 3'- untranslated region of the mRNA. The antisense polynucleotide may be of any length but will typically be from 6 to 40 nucleotides in length. More preferably it will be from 12 to 20 nucleotides in length. The polynucleotide may be at least 40, for example at least 60 or at least 80, nucleotides in length and up to 100, 200, 300, 400, 500, 1000 or more nucleotides in length. In one embodiment the length of the antisense oligonucleotide is the same as that of the target mRNA or up to a few nucleotides, such as 5 or 10 nucleotides, shorter than SEQ ID NO: 1.

Promoters and other regulatory elements may be selected to be compatible with the host cell for which the expression vector is designed. Promoters suitable for use in plant cells may be derived, for example, from plants or from bacteria that associate with plants or from plant viruses. Thus, promoters from Agrobacterium spp. including the nopaline synthase (nos), octopine synthase (ocs) and mannopine synthase (mas) promoters are preferred. Also preferred are plant promoters such as the ribulose bisphosphate small subunit promoter (rubisco ssu), histone promoters (EP-A-0 507,698), the rice actin promoter (US Patent No. 5,641,876) and the phaseolin promoter. Also preferred are plant viral promoters such as the cauliflower mosaic virus (CAMV) 35S and 19S promoters, and the circovirus promoter (AU-A-689,311).

Depending on the pattern of expression desired, promoters may be constitutive, tissue- or stage-specific; and/or inducible. For example, strong constitutive expression in plants can be obtained with the CAMV 35S, Rubisco ssu, or histone promoters mentioned above. Also, tissue-specific or stage-specific promoters may be used to target expression of polypeptides of the invention to particular tissues in a transgenic plant or to particular stages in its development. Promoters specific to the anther, or to early anther development are particularly advantageous.

Inducible promoters are particularly preferred. Alcohol-inducible and herbicide- inducible promoters are available. Chemically inducible promoters such as those activated by herbicide safeners may also be used, for example the maize GST 27 promoter (WO97/11189), the maize In2-1 promoter (WO90/11361), the maize In2-2 promoter (De Veylder et al, Plant Cell Physiology, Vol. 38, pp568-577 (1997).

Especially where expression in plant cells is desired, other regulatory signals may also be incorporated in the vector, for example a terminator and/or polyadenylation site. Preferred terminators include the nos terminator and the histone terminator of EP-A-0 633,317 although other terminators functional in plant cells may also be used. Additionally, sequences encoding secretory signals or transit peptides may be included. On expression, these elements direct secretion from the cell or target the polypeptide of the invention to a particular location within the cell. For example, sequences may be added to target the expressed polypeptide to the nucleus or plastids (e.g. chloroplasts) of a plant cell.

Some examples are signal-peptide encoding DNA/RNA sequences which target proteins to the extracellular matrix of the plant cell, such as the signal sequence of the Nicoliana plumb aginifolia extension gene; signal peptides which target proteins to the vacuole, like those of the sweet potato sporamin gene and the barley lectin gene; signal peptides which cause proteins to be secreted such as that of PRIb; or the barley α-amylase leader sequence; and signal peptides which target proteins to the plastids such as that of rapeseed enoyl-Acp reductase.

Typically, therefore, a chimeric gene comprises the following elements in 5' to 3' orientation: a promoter functional in a host (preferably plant) cell, as defined above, a polynucleotide of the invention and a terminator functional in said cell, as defined above.

Other elements, for example enhancers, may also be present in a vector of the invention. Enhancers include the tobacco etch virus (TEV) enhancer and the tobacco mosaic virus (TMV) enhancer (WO87/07644).

Similarly, an origin of replication may be present. Sequences capable of securing integration into a cells genome, e.g. Agrobacterium tumefaciens T-DNA sequences may be present.

Further, selectable marker genes, under control of their own regulatory sequences may be included. These include antibiotic resistance genes. Examples include genes that confer resistance to the antibiotics kanamycin and/or neomycin (e.g. the nptl and nptll genes) or chloramphenicol (e.g. the CAT gene). Herbicide resistance genes may also be used as selectable markers. Notably, genes conferring resistance to herbicides such as bialaphos, glyphosate or an isoxazole herbicide may be used. Particular examples are described in EP-A-0 242,236, EP-A-0 242,246, GB-A-2, 197,653, WO91/02701, WO95/06128, WO96/38567 and WO97/04103. Likewise, scorable marker genes may be present. Some examples are the β-glucuronidase (GUS) β- galactosidase luciferase and green fluorescent protein (gfp) genes.

Expression in host cells

Expression in the host cell may be transient although, preferably, integration of the polynucleotide or chimeric gene of the invention into the cell's genome is achieved.

Cell culture will take place under standard conditions. Commercially available cultural media for cell culture are widely available and can be used in accordance with manufacturers' instructions.

Plants of the invention

The present invention is in principle applicable to any plant species, notably arable crop species, tree species and species used in horticulture, especially ornamentals. Preferred dicotyledonous crop plants include tomato; potato; sugarbeet cassava; cruciferous crops, including oilseed rape; linseed; tobacco; sunflower; fibre crops such as cotton; and leguminous crops such as peas, beans, especially soybean, and alfalfa. Brassicas are particularly preferred. Preferred monocotyledonous plants include graminaceous plants such as wheat, maize, rice, oats, barley, rye, sorghum, triticale and sugar cane. Maize is particularly preferred.

The invention is also particularly useful in the context of transgenic trees, where male sterility is of benefit because it prevents dispersal of transgenic pollen. Transformation/regeneration techniques

The cell used for transformation may be from any suitable organism, preferably a plant as defined herein and may be in any form. For example, it may be an isolated cell, e.g. a protoplast, or it may be part of a plant tissue, e.g. a callus, for example a solid or liquid callus culture, or a tissue excised from a plant, or it may be part of a whole plant. It may, for example, be part of an embryo, or a meristem, e.g. an apical meristem of a shoot. Transformation may thus give rise to a chimeric tissue or plant in which some cells are transgenic and some are not.

Transformation techniques

Cell transformation may be achieved by any suitable transformation method. Preferred transformation techniques include electroporation of plant protoplasts (Taylor and Walbot, 1985), PEG-based procedures (Golds et al, 1993), microinjection (Neuhas et al, 1987; Potrykus et al, 1985), injection by galinstan expansion femtosyringe (Knoblauch et al, 1999), Agrobacrerium-based transformation and particle bombardment. Particle bombardment is particularly preferred.

Selection of transformed cells

Cells generated by the transformation techniques discussed above will typically be present in chimeric tissues, and thus will be surrounded by other non-transformed cells. Standard selection techniques using co-transforming selectable and/or scorible markers can then be used to identify and obtain transformed cells. Regeneration and breeding

Transformed cells may be regenerated into a transgenic plant by techniques known in the art. These may involve the use of plant growth substances such as auxins, giberellins and/or cytokinins to stimulate the growth and/or division of the transgenic cell. Similarly, techniques such as somatic embryogenesis and meristem culture may be used. Regeneration techniques are well known in the art and examples can be found in, e.g. US 4,459,355, US 4,536,475, US 5,464,763, US 5, 177,010, US 5, 187,073, EP 267,159, EP 604, 662, EP 672, 752, US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,253, US 5,405,765, EP 442,174, EP 486,233, EP 486,234, EP 539,563, EP 674,725, WO91/02071, WO 95/06128 and WO 97/32977.

In many such techniques, one step is the formation of a callus, i.e. a plant tissue comprising expanding and/or dividing cells. Such calli are a further aspect of the invention as are other types of plant cell cultures and plant parts. Thus, for example, the invention provides transgenic plant tissues and parts, including embryos, meristems, seeds, shoots, roots, stems, leaves and flower parts. These may be chimeric in the sense that some of their cells are transgenic and some are not.

Regeneration procedures will typically involve the selection of transgenic cells by means of marker genes. The regeneration step gives rise to a first generation transgenic plant. The invention also provides methods of obtaining transgenic plants of further generations from this first generation plant. These are known as progeny plants. Progeny plants of second, third, fourth, fifth, sixth and further generations may be obtained from the first generation progeny plant by any means known in the art. Thus, the invention provides a method of obtaining a transgenic plant of the invention comprising obtaining a second-generation transgenic progeny plant from a first- generation progeny plant of the invention, and optionally obtaining transgenic plants of one or more further generations from the second-generation progeny plant thus obtained.

Such progeny plants are desirable because the first generation plant may not have all the characteristics required for cultivation. For example, for the production of first generation transgenic plants, a plant of a taxon that is easy to transform and regenerate may be chosen. It may therefore be necessary to introduce further characteristics in one or more subsequent generations of progeny plants before a plant more suitable for cultivation is produced.

Progeny plants may be produced from their predecessors of earlier generations by any known technique. In particular, progeny plants may be produced by:

obtaining a transgenic seed from a transgenic plant of the invention belonging to a previous generation, then obtaining a transgenic progeny plant of the invention belonging to a new generation by growing up the seed; and/or

propagating clonally a transgenic plant of the invention belonging to a previous generation to give a transgenic progeny plant of the invention belonging to a new generation; and/or

crossing a first-generation transgenic plant of the invention belonging to a previous generation with another compatible plant to give a transgenic progeny plant of the invention belonging to a new generation; and optionally

obtaining transgenic progeny plants of one or more further generations from the progeny plant thus obtained. These techniques may be used in any combination. For example, clonal propagation and sexual propagation may be used at different points in a process that gives rise to a plant suitable for cultivation. In particular, repetitive back-crossing with a plant taxon with agronomically desirable characteristics may be undertaken. Further steps of removing cells from a plant and regenerating new plants therefrom may also be carried out.

Also, further desirable characteristics may be introduced by transforming the cells, plant tissues, plants or seeds, at any suitable stage in the above process, to introduce desirable coding sequences other than the polynucleotides of the invention. This may be carried out by conventional breeding techniques, e.g. fertilizing a plant of the invention with pollen from a plant with the desired additional characteristic. Alternatively, the characteristic can be added by further transformation of the plant obtained by the method of the invention, using the techniques described herein for transformation. Preferably, different transgenes are linked to different selectable of scorable markers to allow selection for both the presence of further transgenes. Selection, regeneration and breeding techniques for transformed plants are known in the art.

Uses of plants of the invention

The invention also provides methods of obtaining crop products by harvesting, and optionally processing further, cells, calli, plants or seeds of the invention. By crop product is meant any useful product obtainable from a crop plant.

Such a product may be obtainable directly by harvesting or indirectly, by harvesting and further processing. Directly obtainable products include: grains, e.g. grains of monocotyledonous species, preferably graminaceous species, for example wheat, oats, rye, rice, maize, sorghum, triticale, especially wheat; other seeds; shoots, especially tubers, such as potato tubers; fruit; and other plant parts, for example as defined herein. Alternatively, such a product may be obtainable indirectly, by harvesting and further processing. Examples of products obtainable by further processing are: flour; oil; rubber; beverages such as juices and fermented and/or distilled alcoholic beverages; food products made from directly obtained or further processed material, e.g. bread made from flour or margarine made from oil; tobacco and tobacco products such as cigarettes and cigars; fibres, e.g. cotton, linen, flax and hemp fibres and textile items made therefrom; paper or timber derived from woody plants.

Additional features of plants of the invention

In addition to the transgenes of the invention, plants of the invention may be transgenic in other respects. For example, they may be transformed such that they comprise genes for herbicide, insecticide or disease resistance. Preferred herbicide resistance genes may be responsible for, for example, tolerance to: Glyphosate (e.g. using an EPSP synthase gene (e.g. EP-A-0 293,358) or a glyphosate oxidoreductase (WO 92/000377) gene); or tolerance to fosametin; a dihalobenzonitrile; glufosinate, e.g. using a phosphinothrycin acetyl transferase (PAT) or glutamine synthase gene (cf. EP-A-0 242,236); asulam, e.g. using a dihydropteroate synthase gene (EP-A-0 369,367); or a sulphonylurea, e.g. using an ALS gene); diphenyl ethers such as acifluorfen or oxyfluorfen, e.g. using a protoporphyrogen oxidase gene); an oxadiazole such as oxadiazon; a cyclic imide such as chlorophthalim; a phenyl pyrazole such as TNP, or a phenopylate or carbamate analogue thereof; spectinomycin e.g. using the aadA gene, as exemplified below.

Insect resistance may be introduced, for example using genes encoding Bacillus thuringiensis (Bt) toxins. Likewise, genes for disease resistance may be introduced, e.g. as in WO91/02701 or WO95/06128. Transformation may also lead to the introduction of a selectable marker gene i.e. marker genes that allow transformed cells to survive in the presence of agents that kill non-transformed cells. Any selectable marker gene may be used in the transforming polynucleotide of the invention. Some examples have already been given above. Typically, herbicide resistance genes, e.g. as defined above, may be used as selectable markers. Alternatively, coding regions that encode products which provide resistance to aminoglycoside antibiotics may be used as selectable marker, for example, encoded products that provide resistance to kanomycin, neomycin or chloramphenicol. The encoded polypeptide may cause morphological alterations to cultured transformed cells, such as isopentyltransferase (Kunkel et al, 1999). The encoded polypeptide may be a scorable marker, which allows transformed cells to be distinguished from non-transformed cells, generally by alteration of the transformed cell's optical properties. Any scorable marker may be used. Preferred scorable markers include, polypeptides which are able to alter the appearance or optical properties of transformed cells, for example: β-glucoronidase (i.e. the uidA:G\ ^> gene); fluorescent proteins such as green fluorescent protein (GFP), yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP); or luminescent proteins such as luciferase or aequorin. Cells with scorable optical differences can be sorted using techniques such as fluorescence activated cell sorting (FACS). In a preferred embodiment, the polynucleotide of the invention comprises a selectable marker and a scorable marker, for example, the FLARE-S marker genes which comprise aadA and GFP (Khan and Maliga, 1999).

Similarly, plants of the invention may be transformed such that they express polypeptides whose mass production is desirable, e.g. components of antibodies, or pharmaceutically active polypeptides such as interferon-gamma. Male sterility and breeding strategies

Male sterility may be achieved by modulating the expression of an ESP gene, or the activity of an ESP protein, in any suitable way.

Modulation of the ESP gene according to the invention will have the effect of inducing sterility solely in the male germline, leaving the female ovules unaffected. The ability to induce solely male sterility in crop plants without the use of aggressive (e.g. diphtheria toxin, Barnase) genes transferred from other kingdoms, and through the simple down-regulation of an endogenous sequence is highly advantageous. Further, since down-regulation of ESP will eliminate the germline at a very early stage, the likelihood of developmental leakiness is small, as virtually none of the anther tissues will have had the opportunity to develop. The most preferred technologies for the down-regulation of ESP are antisense or RNAi. Both produce species of RNA which silence the endogenous gene, and rarely interfer with the expression of other sequences. Strategies involving the mutation of the ESP gene in crops are less preferred.

In particular, male sterility may be achieved by antisense inhibition of an ESP gene. Alternatively, it may be achieved by RNA Interference (RNAi) techniques. Antisense or RNAi techniques are known in the art. RNAi inhibition may, in particular, be achieved by transformation with a construct comprising back-to-back sense and antisense ESP DNA sequences. This gives rise to sense and antisense RNA, which associates to give double-stranded RNA, which is broken up into short segments of dsRNA. The antisense strands of those segments then combine to achieve inhibition of ESP mRNA expressed from the endogenous gene by an antisense mechanism.

Alternatively, male sterility may be achieved by mutation of an ESP gene. Such a mutation may be in the regulatory regions of the gene, e.g. in the promoter, or it may be in the coding sequence. The mutation may completely prevent, or diminish, expression, e.g. if the mutation is in the regulatory regions. Alternatively, it may permit expression but lead to the production of a non-functional ESP protein, e.g. by the introduction of a stop codon that terminates translation prematurely, or by a deletion, substitution or insertion that renders the protein non-functional.

In principle, it may also be possible to inhibit the activity of ESP protein by means of antibodies to ESP or by applying inhibitors of ESP to plants. For example, antibodies could be generated in situ by transforming the plant cells of the invention with DNA encoding them. Similarly, transformation could be carried out with a gene encoding an inhibitor of ESP.

Antisense and RNAi approaches are preferred. For antisense inhibition, a construct comprising a polynucleotide capable of selectively hybridising to all or part of the ESP gene sequence is introduced into the cell. Desirably, its expression is under the control of an inducible promoter so that the transgenic plant of the invention can be rendered male sterile on demand.

The antisense sequence may extend over the whole ESP genomic sequence, or may extend over only part of it, as long as it is of sufficient length to block expression. For example, the antisense sequence may be directed to the promoter or to part of the coding region of the ESP gene. Multiple antisense sequences to different parts of the gene may be used. Suitable antisense sequences may be 10 or more, 20 or more, 50 or more, 100 or more, 200 or more, 500 or more, 1000 or more, 2000 or more or 3000 or more nucleotides in length.

The above applies, mutatis mutandis, to RNAi-based male sterility strategies of the invention. Another preferred technique is the use of recombinase-encoding constructs to excise, for example, antisense or RNAi constructs according to the invention. Recombinase systems to do this are available in the art. In outline, a construct, for example, an antisense construct of the invention, is flanked by sequences that are recognised by the recombinase enzyme. The recombinase enzyme is then capable of splicing the flanking sequences together, thus excising the construct of the invention. In a particularly preferred embodiment, male sterility is achieved by means of an introduced antisense or RNAi construct. Of course, this plant is generally the female parent plant in any cross as it is male-sterile (though it may be a male parent, e.g. for purposes of propagation, when an inducible male-seterility characteristic is not switched on). The male parent plant which supplies the pollen in the cross is transformed with a construct encoding a recombinase enzyme, and the antisense or RNAi construct in the female parent plant is flanked by the sequences recognised by the recombinase. Thus, when the two plants are crossed, they recombinase is expressed in the progeny and splices out the antisense or RNAi construct. The use of recombinases in this manner is particularly advantageous because it eliminates the transgenic construct of the invention, which is beneficial from the point of view of public perception. Even more desirably, the recombinase construct may be self- excising, in that it is itself flanked by sequences which the enzyme it encodes can excise.

An example of a commercial breeding strategy in a crop such as Brassica would involve transformation of elite Brassica lines with either antisense or RNAi constructs driven by an inducible promoter. There are a number of such promoters available activated by agents ranging from herbicides to steroids. To generate F, seed, the potential seed proteins would be grown to near maturity and then the ESP down-regulating sequence activated by the appropriate agent. The flowers formed would contain no pollen but fertile ovules. These ovules would then be pollinated by the pollen donor of choice, which could be grown close by, and all the seed produced would be by the transgenic line as fertile F, hybrids. An alternative approach would be to excise the transgene in the F, using a recombinase strategy. Thus, the seed parent would contain the antisense/RNAi construct bordered by recombinase target sequences. The pollen parent would contain a recombinase construct which would be expressed in the zygote and excise the antisense/RNAi transgene. The seed produced by the F, would then be fully fertile. Other more complex strategies would involve the recombinase itself being bordered by target sequences as well as the antisense RNAi construct. If the female parent contained both the antisense/RNAi construct and a factor (encoding by a transgene or otherwise) which activated the recombinase from the male parent, one could envisage a situation where, on pollination, the factor from the female activated the male recombinase which excised both itself and the antisense RNAi transgene. There are also circumstances in crop plants where the harvested produce is not the seed, and here the antisense RNAi sequence could be constitutively expressed.

Antibodies

The invention also provides monoclonal or polyclonal antibodies which specifically recognise polypeptides of the invention, and methods of making such antibodies. Antibodies of the invention bind specifically to the polypeptides of the invention.

Monoclonal antibodies may be prepared by conventional hybridoma technology using polypeptides of the invention as immunogens. Polyclonal antibodies may also be prepared by conventional means which comprise inoculating a host animal, for example a rat or a rabbit, with a polypeptide of the invention, or a fragment thereof comprising an epitope, and recovering immune serum. In order that such antibodies may be made, polypeptides may be haptenised to another polypeptide for use as immunogens in animals or humans. For the purposes of this invention, the term "antibody" includes antibody fragments such as Fv, F(ab) and F(ab)₂ fragments, as well as single-chain antibodies. Antibodies to the polypeptides of the invention can be produced by use of the following methods. An antibody to the substance may be produced by raising antibody in a host animal against the whole substance or an antigenic epitope thereof (hereinafter "the immunogen"). Methods of producing monoclonal and polyclonal antibodies are well-known.

A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG faction purified.

A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein, Nature (1975) 256, 495-497).

An immortalised cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

For production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat or mouse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier. The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified. Thus, the invention provides the use of a polypeptide of the invention, or a fragment thereof comprising an epitope, in the production of antibodies that specifically recognise a polypeptide of the invention. For these purposes, it should be noted that the fragments may not function as plant-protective polypeptides, because an epitope may be contained within a region too small to retain function as a plant-protective polypeptide. Similarly, the invention provides methods of producing antibodies by inoculating animals with a polypeptide of the invention or a fragment thereof containing an epitope and recovering immune serum. This will generate polyclonal antibodies.

Antibodies may also be generated using β-cells in vitro instead of in vivo.

Antibodies to polypeptides of the invention may also be identified by phage display techniques.

Antibodies of the invention can be used to identify compounds whose structural properties (e.g. shape, charge) correspond to those of the polypeptides of the invention. Thus, they may be used to screen for compounds that mimic the functional properties of the polypeptides of the invention.

The invention is illustrated below by means of the following Examples.

EXAMPLES

Identification of ES mutants

Plants with mutations in ESP {esplesp plants) fail to form an organised tapetum, developing instead a larger number of sporogenous cells (meiocytes). 3 alleles of ESP have been generated by fast neutron radiation, all of which fail to develop complete tapetal layers. Microscopic analysis reveals meiosis to initiate and progress normally to the tetrad stage in all the sporogenous cells of esp/esp plants, although callose deposition is clearly aberrant, failing to completely invest the developing meiocytes.

Neither electron nor confocal microscopy can positively identify the developmental fates of the cell layers immediately surrounding the meiocytes in esp mutants, however, all studies suggest a normal endothecium is always formed.

Fine mapping of ESP

ESP has been fine mapped in C24 plants. Primary linkage analysis placed ESP close to CIC12 on the top arm of chromosome 5, and subsequent mapping has positioned the ESP locus within a 250 kb interval spanned by 4 PI clones. Southern blot analysis of 3 ESP alleles have detected allele-specific polymorphisms on a single 80kb PI clone (K2N18), and analysis of sub-clones using 4 restriction enzymes has identified polymoφhisms for the different alleles. Sequence is now available for a BAC clone containing K2N18, and the restriction sites responsible for the polymoφhisms have been detected by Southern blot analysis. Two regions have been identified as possessing the sites necessary to give the observed polymoφhisms, each between 4- 8kb in length.

Cloning of ESP

ESP was cloned by positional cloning. Provided below is the ESP genomic sequence of C24 Arabidopsis (Figure 1). Also provided is the ESP amino acid sequence (SΕQ ID NO: 1; Figure 2).

Molecular analysis has identified the ESP gene as encoding a leucine-repeat receptor kinase and it is reasonable to assume that ESP forms part of a signalling system involved in cell type specification early in anther development. Expression studies

RT-PCR studies have demonstrated that ESP's expression is specific in time and place. It is expressed early on in plant development, in the young, developing anther.

Our initial experiments suggested that ESP was expressed quite early in the anther and ovule, but not in other parts of the plant. Further, and importantly, the gene remains highly expressed though male and female development, with some expression in the silique. This late development is not associated with any clear phenotype except on rare occasions ESP lines may have lowered seed fertility.

The female expression pattern of ESP is very striking, being first centred in the region where the megasporocyte is specified and thereafter transcripts being strikingly present in a band corresponding with the developing integuments. Despite this dramatic expression pattern we have as yet detected no female esp phenotype.

This reinforces the view that ESP acts to regulate the specification of germline cells from a pool of undifferentiated reproductive cells and serves to confirm that ESP could participate in a highly effective male sterility system for application in crop improvement strategies.

Claims

1. A plant which is, or is capable of being, male-sterile by virtue of modulation of the expression of an EXTRASPOROGENOUS CELLS (ESP) gene, or of the activity of the encoded ESP protein.

2. A plant according to claim 1 which is, or is capable of being, male-sterile by virtue of a transgenic construct which provides for expression of antisense RNA or of RNA Interference (RNAi), which inhibits the expression of the ESP gene.

3. A plant according to claim 1 which is male-sterile by virtue of a mutation in the ESP gene.

4. A plant according to claim 1 or 2 which is male fertile but inducibly male- sterile, or male-sterile but inducibly male-fertile.

5. A plant according to claim 4 which is inducibly male-sterile by virtue of a transgenic construct as defined in claim 2 wherein the expression of the antisense RNA or RNAi is under the control of an inducible promoter.

A plant according to any one of the preceding claims wherein the ESP gene:

(a) encodes a polypeptide sequence as set out in SEQ ID NO: 1; or (c) has a cDNA sequence having 60%) or more homology to a sequence of (a); or

(c) has a cDNA sequence capable of hybridising selectively to the complement of a sequence of (a); and

when functional, encodes a leucine-repeat receptor; and, when non-functional, causes the plant to fail to produce fertile pollen.

7. A plant according to any one of the preceding claims which is of a monocot or dicot arable crop species, optionally a Brassica or maize plant; or a tree species.

8. A progeny plant of a plant according to any one of the preceding claims.

9. A progeny plant according to claim 8 which is an FI hybrid plant.

10. A seed of a plant according to any one of the preceding claims.

11. A seed of an F 1 hybrid plant according to claim 9.

12. A progeny plant according to claim 8 or 9 or a seed according to claim 10 or 11 which is male-fertile.

13. A plant or seed according to any one of the preceding claims which is a maize or Brassica plant or seed.

14. A method of producing a plant according to any one of claims 1 to 9 or 12 or 13 or a seed according to any one of claims 10 to 12 or 13 comprising: (a) transforming a plant cell with a transgenic construct that renders a plant regenerated from the cell male-sterile, or capable of being male-sterile, by virtue of inactivation or suppression of an ESP gene; or (a') introducing an inactivating mutation of the ESP gene; and (b) regenerating an F0 generation plant from the transformed cell; and optionally (c) producing plants of an FI, F2 or subsequent generations from said F0 plant; and optionally (d) harvesting seed from said plants of any of the F0, FI, F2 or subsequent generations.

15. A method according to claim 14 wherein the transgenic construct is as defined in any one of claims 2, 5 or 6.

16. A method according to claim 14 or 15 wherein a male-sterile plant obtained from step (b) or (c) in combination with step (a) is crossed with a male-fertile plant containing a transgenic construct comprising a sequence coding for a recombinase enzyme which, when expressed, generates a recombinase that excises the transgenic construct that renders the regenerated plant male-sterile to the extent that suppression of ESP is removed, and optionally also excises the recombinase 's own coding sequence, thereby generating a male-fertile progeny plant according to claim 12.

17. Use of a plant according to any one of claims 2 to 9 or 12 or 13 or obtained by a method according to any one of claims 14 to 16, or of a seed according to any one of claims 10, 11 or 13 or of a construct as defined in any one of claims 2, 5 or 6, in a plant breeding programme; or for the production of seed for planting; or for the production of seed for consumption or processing into food; or for use in an industrial process.

18. A crop product obtained or obtainable from a plant according to any one of claims 2 to 9 or 12 or 13 or a seed according to any one of claims 10, 11 or 13.

19. An ESP polynucleotide which:

(a) encodes a polypeptide sequence as set out in SΕQ ID NO: 1 ; or

(b) has a cDNA sequence having 60%> or more homology to a sequence of (a); or

encodes a leucine-repeat receptor kinase.

20. A polynucleotide sequence capable of hybridising selectively to all or part of the mRNA expressed from a gene as defined in claim 7 and thus achieving antisense inhibition of said gene.

21. A vector comprising a polynucleotide according to claim 19 to 20 operably linked to a promoter.

22. A vector according to claim 21 wherein the promoter is stage-specific and/or tissue specific and/or inducible.

23. A vector according to claim 21 or 22 wherein the polynucleotide is an antisense polynucleotide according to claim 20 and the promoter is an inducible promoter.

24. A cell transformed with a polynucleotide sequence according to claim 19 or 20 or a vector according to claim 21, 22 or 23.

25. A cell according to claim 24 which is a plant cell.

26. A method of making a cell according to claim 24 comprising transforming a cell with a polynucleotide sequence according to claim 19 or 20 or a vector according to claim 21, 22 or 23.

27. A method according to claim 25 further comprising regenerating a plant from said transformed cell, and optionally further propagating said plant to give progeny plants of first, second or subsequent generations.

28. A polypeptide encoded by a polynucleotide according to claim 19.

29. A polypeptide according to claim 27 having the sequence of SEQ ID NO: 1.

30. An antibody which specifically recognises a polypeptide according to claim 27 or 28.

31. A primer, or pair of primers, capable of amplifying a polynucleotide as defined in claim 19 or 20 in a Polymerase Chain Reaction.

32. A labelled probe capable of hybridising to and thus detecting a polynucleotide as defined in claim 19 or 20.