FORM 2
THE PATENTS ACT, 1970 [39 OF 1970]
COMPLETE SPECIFICATION [See Section 10; rule 13]
"POLLEN SPECIFIC PROMOTER'
SYNGENTA LIMITED {formerly ZENECA LIMITED], of European Regional Centre, Priestley Road, Surrey Research Park, Guildford, Surrey GU2 7YH, United Kingdom,
The following specification particularly describes the nature of the invention and the manner in which it is to be performed:-

POLLEN SPECIFIC PROMOTER
7 The present invention relates to a promoter sequence which is specific tor pollen, to constructs and transgemc plant ceils and plants comprising the promoter as well as to methods onransforming pollen and controlling fertility in plants using this promoter.
in order to introduce desirable genetic traits from two plants into a single plant, such as a variety or hybrid, cross-breeding represents the traditional approach. In order to reliably obtain consistent hybrids, it is necessary to ensure that the self-pollination of the parent plants does not take place.
This can be achieved by ensuring thai one of the parent lines is male sterile. Various techniques tor producing male sterility are known and have been proposed in the an. One method involves removal of the anthers or tassels of the female parent plant, either manually or mechanically. This plant may then ottiy be tenilised'by pollen from the male parent and therefore its progeny will be hybrid. However, such 2 process is labour intensive and not altogether reliable as it is possible ih7420 (1996V HJ Rogers et c/.., Plant J. 4: 875-882 (1993) and CJ Staiger etal.., Plant J. 4: 631-641 (1993)), putative ascorbate oxidasss, 2 Kunitz protein inhibitor and many others whose function cannot be inferred by homology to known genes. The temporal expression of such genes has been srudied and many are found to be expressed late in microsporogenesis reaching a maximum in mature microsporocytes, In some cases continued expression in the pollen tube has also been demonstrated [AK Kononowicz si ai.. Plant Cell 4; 513-524 (1992)). These genes are referred to as "late genes". The majority of expression at this stage is from the vegetative

cell rather than from the generative cell and ii is likely that the majority of these laic' £enes are transcribed from the vegetative nucleus: although (his has only been demonstrated tor oni! "taie^ne (D. TwelL Plant J!>: SS7-S9^ (1992). A distinct class of genes expressed in ambers is found 10 have a different expression programme, being first detectable soon after the tetrad stage and declining in expression well before pollen maturity. It is likely that the major role of these 'early' genes may be during microspore differentiation and development rather than.pollen tube growth. In addition. US Patent No. 5.0S6.169 fMascarenhas) describes the isolation of the first pollen-specific promoter from corn.
The applicants have isolawd a further promoter which is specifically expressed only in pollen tissue. The promoter is derived from a 'late' pollen expressed gene isolated from maize. ZmC5.
Thus, according to a first aspect of the present invention there is provided a rccombinam nucleic acid sequence which comprises a promoter sequence of the ZmCS gene in maize, or a variant or fragment thereof, which acts as a promoter in pollen.
A? used herein, the term "fragment" includes.one or more regions of the basic sequence which rstnin promoter na;'.'i;y. \Vhcr-.; ihf frngiTierus comprise one or more regions, they may be joined together dir^aiy or they may be spaced apart by additional bases.
The expression "variant" with reference to the present invention means any subiiiitution of, variation of, modification of, replacement of, deletion of or the addition of one or more nucleotides from or to the nucleic acid sequence providing the resultant sequence exhibits pollen promoter expression. The term also includes sequence that can substantially hybridise to the nucleic acid sequence.
As used herein, the expression "ZmCS gene7' refers to the gene of maize which encodes a 563 amino acid sequence as described herein, A cDNA sequence encoding this sequence is defined in EMBL YI32S5
The promoter sequence of the present inversion is comprised within the clone deposited National Collection of Industrial and Marine Bacteria as NCIMB 40915 on 26 Jan I99S. This is a Sail fragment derived as described hereinafter. The promoter region ties within a region which consists of approximately 2kb of sequence upstream of the transcription start site of the ZmC5 gene of maize as shown in Figure I hereinafter.

According to a preferred embodiment of the present invention, there is provided a recomb'inant nucleic acid sequence which comprises a promoter sequence comprising at least part "of the DNA sequence as shown in Figure 5 oral least part of a sequence encoding a promoter which has substantially similar activity to the promoter encoded by Figure 5 or a variant or iragmem thereof.
The term "substantially similar ac;ivi;y" includes DMA sequences which are complementary to and hybridise to the DNA of the present invention and which code for a promoter which acts in pollen. Preferably, such hybridisation occurs at, or between, low and high stringency conditions. In genera! terms, low stringency conditions can be defined as ?> \ SCC at ambient temperature of between about 60flC to about 65°C. and high stringency conditions as 0.1 x SSC at about 65°C. SSC is the name of a buffer of 0.15M NaG. 0.0 ISM irisodium curate. 3 >: SSC is three times as sirong as SSC and so on.
The pollen specific promoter of the present invention may be used to engineer male sterility by driving genes capable of interfering with pollen production or viability, or to express genes of intercut specifically in pollen grains.
According to a second aspect of the present inversion, ihe promoter sequence may form pan of an expression cassenc m combination with g;:v,'S whose expression in pollen, and particularly in lote polien production, may be desirable. These include genes which have iin impact on pollen or pollen production. Such acnes may be those involved in the control of male-fertility, genes which encode insecdcida! toxins (which would then be targeted 10 insect species which feed on pollen), or genes which would enhance or modify the nutritional value of the pollen. In addition, the promoter sequence could be used to drive expression of a selectable marker for use in pollen transformation, Examples of suitable selectable marker series include antibiotic resistance genes such os Icariamycin resistance gene, hygromycm resistance gene ajid the PAT resistance gene so as to enable stable transfonnants to be identified depending on the species e.g. com, rice, wheat.
The term "expression cassette" - which is synonymous with terms such as tlDNA construe:", '"hybrid" and "conjugate" - includes an effect gene directly or indirectly attached to the regulator promoter, such as to form a cassene. An example of an indirect attachment is the provision of a suitable spacer group such as an iruron sequence intermediate the promoter and the target gene. The DNA sequences may furthermore be on different vectors and are

therefore not necessarily located on the same vector. The same is true for the term "rused" \n relation to the present invention which includes direct or indirect attachment. Such consfoxis aiso include plasmids and pha^e which are suitable for transforming a eel! of
interest.
According to a preferred embodiment, expression cassettes of the present invention comprise a promoter sequence as described above which is arranged to control expression of a gene which is deleterious to pollen development, such as genes encoding barnase. adenine audeoude iranslocator. mutant tubuims, T-urf (as claimed in WO 97/04116) or tretulose phosphate phosphatase (TPP).
For instance. W093/25695 describes the use of the gene bamase which encodes a cytoioxic protein . which is under the control of a tape turn specific promoter. Expression of barnase in me tapeial cells disrupts these cells and leads to disruption of pollen production.
Rjbozymes are RNA molecules capable of catalysing endonucleolyiic cleavage reactions. They can catalyse reactions in trans and can be targeted to different sequences. They aiv [herefore potential alternatives to antisense as a means of modulating gene expression. (Hasseihof and Gerhch (1938) Nature V'ol 334. 5S5-:-9n or V-'e^ncr ,^ a', f i '^M'i Mo! Gen C-enc; 2-45; (465-470) have demonstrated {{\z gt-ncraiion of a ;raiu -ciominant mutation by expression of a ribozyme gene in planes. If required, the pollen specific promoter of the present invention may be used to control expression of the ribo'^ymes such ihai they are specifically expressed in pollen.
Baulcombe (1997) describes a method of gene silencing in iransgenic plants via the use of replicable viral RNA vectors (Amp!icons7U) which may also be useful as a means of knocking out expression of endogenous genes, This method has the advantage that it produces a dominant mutation i.e. is scorable in the heierozygous stale and knocks out alt copies of a targeied gene and may also knock out isoforms. This is a clear advantage in wheat which is hexaploid. Fertility could then be restored by using'an tnducible promoter to drive the expression of a functional copy of the knocked out gene. By including the pollen specific promoter as the elements of the A/nplicon™ vector, expression of the gene would then take place specifically in the pollen.
The use of cyioto.xic or disrupter genes as means of disrupting pollen production requires the expression of restorer genes to regain fertility. Suitably the construct further

comprises a casseue comprising ;i nuclcotide sequence which is able to overcome the effcci of said deleterious gene, such us a restorer ^eno such as barstur in ihc case ofbamase orTPS in the case of TPP. or 3 sequence which encodes a construct which is sense or antisense to a deleterious eene.
An alternative means of controlling expression of deleterious genes is to use operator sequences. Operator sequences such as lac, tei, 434 etc. may be inserted into promoter regions us described in Vv'O 90/08830. Represser molecules can then bind to these operator sequences and prevent transcription of the downstream gene, for example a gene delecerious to pollen development (Wilde et ul. (1992) £MBO L U. 1551). Furthermore. \\ is possible to engineer operator sequences with enhanced binding capacity such as the Lac l£ His muiam as described in ('Lehjnmg tff a/. (19S7) E.MBQ . 6. 3145-3153). This has a change of amino acid from tyrosine to hisiidine at position 17 thus giving tight control of expression. Used in combination with inducible expression of the represser this then allows expression of an inactivating gene to be cumed off
In this way, the expression cassettes may be incorporated into expression systems which may be used in the control of fertility of a p!am as described above
""• The icr.ii "expression system" means thai the system defir.erf above c;in be expressed in an appropriate organism, tissue, cell or medium. The system may comprise one or more expression cassettes and may also comprise additional component irni ensure 10 increase expression of the targsi gene by use of the regulator promoter.
According 10 a third aspect of the present invention, there is provided an expression system comprising
(a) a fust promoter sequence which is expressed specifically in pollen;
(b) a first gene which, when expressed, disrupts pollen, biogenesis, under the control of said
pollen specific promoter:
(c) a-second promoter sequence responsive 10 the presence or absence of an exogenous
chemical inducer; and
(d) a second gene encoding an element which can inhibit either expression of said first gene,
or can inhibit the protein coded for by said first gene, operably linked to and under the
control of said second promoter sequence.

Elements (a') and (b) and (c) and (d) above may be provided by one or iwo individual vectors, but preferably are contained in the same vector to ensure co-seg re Cation. These can
-4
be used to transform or co-transform plant ceils so as to allow the appropriate interaction between the elements to taker place.
The second promoter sequence and the second gene provide for chemical "switching" on and off of the first gene, Where the second promoter sequence is responsive to the presence of the exogenous chemical inducer. application of the chemical inducer to pollen or to a plant will have ihc effect of switching on the second gene which thereby counteracts the effect of the first gene. The absence of the chemical inducer will have n similar effect where the second promoter sequence is active only in the absence of the chemical inducer.
Elements (c) and (d) are suitably in the form of an expression cassette comprising a nucleotide sequence which is able 10 overcome the effect of said deleterious gene. such as a restorer gene such as barstar in the case of b amass or TPS in the case of TPP, or a sequence which encodes a construct which is sense or amistnse to a deleterious gene, or a gene encoding a represser molecule in inc c;ise 0:'operator sequences being used opcraiiveiy interconnected with an inuucibie pro;noier.
The expression system of the present invention may further comprise a selectable marker, such as herbicide resistance genes or antibiotic resistance genes so as 10 allow stable transformants to be identified depending on the species eg corn, rice, wheat. The presence of a herbicide resistance gene also allows selection of male sterile progeny in a segregating population.
Transformation of a plant with such an expression system will result in the production of male sterile plants and methods of producing such a plant form a fourth aspect of the present invention-Expression systems in accordance with this embodiment of the present invention wherein the gene is deleterious to viable pollen production, are useful in the production of hybrids but are especially useful when the male sterile line can be made homozygou's. When "late" promoters, such as the ZmC5 promoter described above, are used, because the (Straiagene) was replaced with a Mr! recognition site by insertion of the an oiigonucleonde linker MKLJNK.4 (5^-TCGATTCGGCCGCCGCCGAA-31) imo the digested San she. A 0.9kb, flamHI-rYindlll fraement carrying the coding region ot'bamase followed by a bacterial-promoter-driven barstar coding region, was inserted imo the corresponding fragment of the modified pBluescript. The nos terminator on a Hwdlli* 'Ieted with slmmp alkaline phophaiasc C3-7°C, i hour). A 1.9kb 5amHl fragment of the C5 5'flanking region was ligated into this, followed by digestion with 5amHI and Psti to check for presence and orientation of the ifiser:. rcb'pcctively. The resulting plasmid was nanied pSK-C5-BBN (Figure 6). The entire cassep.e is then removed as an £roRl- Not\ fragment to a binar/ plant transformation vsaor pVD6. The constrjct is then introduced into Agrobacterium Tumefaciens by the freese-ihaw method- Standard techniques are used to introduce the DNA inro tobacco.
EXAMPLES
Analysis of sterile transscnic plants
Primary transformancs are seiecteci by gro^vth on kanamyctn in cissue cuinue and this confirmed by PCR analysis. The plants are grown to maturity in the glass house . Pollen is collected from anthers after dehiscence and a vital stain is used to establish whether the pollen is fertile or sterile. 50% of the pollen is expected to be sterile. Backcrossing these plants with wild type plants (after anther removal) ot alio%ving self pollination to occur results in progeny in which 50% of pollen is sterile.

Other modifications to the present invention will be apparent to those skilled in the art without departing from the scope ofihc invention.

WE CLAIMS

1. A recombinant nucleic acid sequence which comprises a promoter sequence of the ZmC5 gene in maize, or a variant or fragment thereof, which acts as a promoter in pollen.

2. A recombinant nucleic acid sequence according to claim I which comprises
approximately 2kb upstream of the transcriptions! start site of [he ZmC5 gene of maize as shown in Figure 1.

3. A recombinant nucleic acid sequence according to claim I or claim 2 which
comprises a promoter sequence comprising at least pan of the DMA sequence as shown in Figure 5 or at least pan of a sequence which encodes a promoter which has substantially similar activity to that shown in Figure \ or 2. Variant or fragment thereof.

4. An expression cascara comprising a nucleic acid sequence according any one ai
claims I to 3 wherein the scissile vs managed Vo control a aeni -vh;ch ii is desired 10 be expressed in pollen and which encodes 0 produce capable of having an impact on pollen production, insecticidal coxias, or enhances or modifies the nutritional value of pollen.

5. An expression cassette according to claim 4 wherein said gene comprises a gene
which is deleterious to pollen development.

6. An expression cassette according to claim 5 wherein said gene comprises a gene
encoding either bamase, adenine nucleotide translocaior, mutant mbuiins, T-urf or
trehalose phosphate phosphatase (TPP) or a nbozyme.

7. An expression cassette according to claim 4 wherein the said gene comprises a
selectable marker gene.

8. An expression casscnc according to claim 7 wherein the selectable marker gene
comprises and antibiotic resistance gene.

9. An expression system comprising an expression cassette according to any one of
claims 4 to 8.

10. An expression system according to claim 9 which comprises a gene which is
deleterious to pollen, and which further comprises an expression cassette comprising a second nucleic acid sequence which encodes a peptide or protein able to overcome the effect of sajd deleterious gene, operativcSy interconnected with a chemical inducible promoter.

11. An expression system according to clairn 10 wherein the said nucleic acid sequence comprises a restorer gene.

12. An expression system according 10 claim 1J wherein the said restorer gene is bars tar or TPS.

13. An expression system according to claim 10 wherein said nucleic acid sequence
encodes a construct which is sense or amisense to said deleterious gene so as to
suppress expression thereof.

14. An expression system according to claim 10 wherein said second nucleic acid
sequence encodes a represser protein to lac, tec, 434, tac-His, which interacts with an operator sequence which is operably linked to the nucleic acid sequence of any one of claims I to 3, or an Amplicon™, so as to prevent expression of the first gene which it is desired to be expressed in pollen.

15. An expression system according to any one of claims 10 to 14 wherein the inducible promoter is the AlcA/R, GST or Ecdysone inducible promoter.

16. An expression system according to any one of claims 9 :o 13 which further comprises a selection marker.

17. An expression system according to anyone of claims 9 to 16 wherein the gene whichii is desired to be expressed in pollen is linked to a herbicide resistance gene,

18. An expression system comprising
(a) a first promoter sequence which is expressed specifically in pollen;
(b) a first gene which when expressed, disrupts pollen biogenesis, under the control of said pollen specific promoter;
(c) a second promoter sequence responsive to the presence or absence of an
exogenous chemical mducer; and
(d) a second gene encoding an elernsm which^can inhibit either expression of said first gsnc; or can inhibit, the s^id first protein, operably linked to and under the control nf said second promoter sequence.

19. A method of producing a plar.c which method comprises transforming a plan: eel!
with an expression system according to any one of claims 9 to IS.

20. A plant cell which comprises an expression system according to any one of claims 9 to 18.

21. A plant which comprises cells according to claim 20.

22. A method of inducing male sterility in plants which method comprises transforming a plant with an expression system according to any one of claims 10 to 18.

23. A method of controlling chc fertility of a plant which comprises transforming said plant with an expression system according to claim 11 or claim IS, and when fertility is required to be restored, activating the inducible promoter,

24. A method according to claim 23 therein the inducible promoter is activated by
application of a chemical to (he plant.

25. A repSicable viral RNA vector (Amplicon™) which comprises a recombinani nucleic
acid according to any one of claims 1 10 3.

26. A method of transforming pollen which comprises transforming pollen cells with an
expression system according to claim 9 or claim 18.

27. A rtcombinant nucleic acid: art expression cassette, an expression system or a method substantially as hereinbefore described w\ih reference to any one of [lie accompanying Figures.

28. A process for preparing an expression,system, for use in the control of
fertility of a plant or plant cell or for use in expression in an
appropriate organism, tissue, cell or medium, wherein the process
comprises the step of mixing the following :
(a) a first pollen specific promoter sequence which is expressed
specifically in pollen;
(b) a first gene which when expressed, disrupts pollen biogenesis,
under the control of said pollen specific promoter sequence;
(c) a second promoter sequence responsive to the presence or
absence of an exogenous chemical inducer; and
(d) a second gene encoding an element which can inhibit either
expression of said first gene, or can inhibit the said first pollen
specific promoter sequence, operable linked to and under the
control of said second promoter sequence; and
(e) optionally one or more expression cassettes of the kind such as
herein described;
in the conventional manner to result in the expression system,

29. A process as claimed in claim 28, wherein optionally a gene
deleterious to pollen and an expression cassette comprising a second
nucleic acid sequence capable of encoding a peptide or protein able to
overcome the effect of said deleterious gene, operatively
interconnected with a chemical inducible promoter are incorporated.

30. A process as claimed in claim 29, wherein the said nucleic acid
sequence comprises a restorer gene.

31. A process as claimed in claim 30, wherein the said restorer gene is
barstar or TPS.

32. A process as claimed in claim 29, wherein the said nucleic acid
sequence is capable of encoding a construct which is sense or
antisense to said deleterious gene so as to suppress expression
thereof.

33. A process as claimed in claim 29, wherein the said second nucleic
acid sequence is capable of encoding a represser protein to lac, tet,
434, lac-His, which interacts -with an operator sequence which is operably linked to the nucleic acid sequence of any one of claims 1 to 3, or an Amplicon™, so as to prevent expression of the first gene which is desired to be expressed in pollen.

34. A process as claimed in any of the preceding claims, wherein the said
inducible promoter is the AlcA/R, GST or Ecdysone inducible
promoter.

35. A process as claimed in any of the preceding claims, wherein
optionally a selection marker is also incorporated.

36. A process as claimed in any of the preceding claims, wherein the gene
which is desired to be expressed in pollen is linked to a herbicide
resistance gene.

37. A process as claimed in claim 28, wherein the said expression
cassette comprises a nucleic acid sequence, preferably a recombinant
nucleic acid sequence,

38. A process as claimed in claim 37, wherein the said nucleic acid
sequence comprises a promoter sequence of the ZmCS gene in maize,
or a variant or fragment thereof, which acts as a promoter in pollen.

39. A process as claimed in claim 38, wherein the said nucleic acid
sequence comprises approximately 2kb upstream of the
transcriptional start site of the ZmCS gene of maize.

40. A process as claimed in claim 37 or 38, wherein the said nucleic acid
sequence comprises a promoter sequence comprising at least part of
the DNA sequence or at least part of a sequence which is capable of
encoding a promoter which has substantially similar activity, or a
variant or fragment thereof,

41. A process as claimed in claim 28 or 37, wherein the said expression
cassette is arranged to control a gene which is desired to be
expressed in pollen and which encodes a product capable of having an
impact on pollen production, insecticidal toxins, or enhances or
modifies the nutritional value of pollen,

42. A process as claimed in claim 41, wherein the said gene comprises a
gene which is deleterious to pollen development.

43. A process as claimed in claim 42, wherein the said gene comprises a
gene encoding either bamase, adenine nucleotide translocate*,
mutant tubulins, T-urf or trehalose phosphate phosphatase (TPP) or a
nbozyme.

44. A process as claimed in claim 411 wherein the said gene comprises a
selectable marker gene.

45. A process as claimed in claim 44, wherein the said selectable marker
gene comprises an antibiotic resistance gene.

46. A method of inducing male sterility in plants comprising the step of
transforming a plant with an expression system prepared by any one
of the preceding claims 28 to 45.

47. A method of controlling the fertility of a plant comprising the step of transforming said plant with an expression system prepared by any one of the preceding claims 28 to 45 and activating the inducible promoter for restoring the fertility.

48. A method as claimed in claim 47r wherein the said inducible promoter
is activated by application of a chemical, such as herbicide safener to
the plant.

49. A method of transforming pollen comprising the step of transforming
pollen cells with an expression system prepared by any one of the
preceding claims 28 to 45.

50. A method as claimed in claims 46, 47, 48 or 49, wherein the said
expression cassette is introduced in the plant or plant cell via
infection by Agro bacterium tumefactions.