WO2011154385A1

WO2011154385A1 - Plant promoters and uses thereof

Info

Publication number: WO2011154385A1
Application number: PCT/EP2011/059355
Authority: WO
Inventors: Alain Tissier; Christophe Sallaud
Original assignee: Philip Morris Products, S.A.
Priority date: 2010-06-07
Filing date: 2011-06-07
Publication date: 2011-12-15

Abstract

The invention encompasses transcriptional promoters active in the trichomes, nucleic acid constructs comprising said promoters, and their uses for genetically modifying cells, seeds or plants. The invention also concerns methods of using the promoters for expressing proteins or metabolites of interest.

Description

PLANT PROMOTERS AND USES THEREOF FIELD OF THE INVENTION

The invention relates to methods and compositions for modifying plants and protein expression in plants. The invention encompasses transcriptional promoters active in the trichomes, nucleic acid constructs comprising said promoters, and their uses for genetically modifying cells, seeds or plants. The invention also concerns methods of using the promoters for expressing proteins or metabolites of interest. The invention is generally applicable to any plant having glandular trichomes, and to the expression of any compound or protein of interest.

INTRODUCTION

The surface of the leaves of Angiosperms contains organs called trichomes which, depending on their anatomy, are divided into two main types: non-secreting trichomes and secreting trichomes (also termed glandular or glandular secreting trichomes). The main function of glandular secreting trichomes is to secrete resins and essential oils at the leaf surface. Glandular trichomes exhibit a wide diversity in their shape and the molecules they produce (Fahn, A. (2000). Advances in Botanical Research Incorporating Advances in Plant Pathology, Vol 31, 37-75; Wagner, G.J. (1991) Plant Physiol, 96, 675-679; Wagner G J, et al. (2004) Ann. Botany 93:3-1 1). They are sites of intense metabolic activity, compartmentalized, and in some species their secretion may amount to 30% of the leaf dry weight (Dell, B. and McComb, A.J. (1978). Advances in Botanical Research, 6, 227-316). In the tobacco plant, the leaf surface is covered by a resinous material secreted by the trichomes and which is composed of diterpenoids and sucrose esters. The glandular secreting trichomes consist of a base of 3 to 5 linearly arranged cells and a head containing 2 to 20 secretory cells. The secretory cells primarily secrete sucrose esters and diterpenes. Diterpenes may account for up to 60% of the exudate and 10% of the dry weight of an adult plant (Wagner et al., 2004, supra). In cultivated tobacco species ( icotiana tabacum), the diterpenes produced belong to two separate families, the labdanes and the cembranes. The tobacco diterpenoids include macrocyclic cembranoids (alpha- and beta- cembratrien-diols being the major compounds) and bicyclic labdanes (czs-abienol and labdane- diol). The relative levels of production of the two families depend on the cultivar, although cembrane production is generally four to ten times higher than labdane production. In N. sylvestris, a progenitor of N. tabacum, labdanes are absent but a compound from the cembrane family, cembratriene-diol (CBT-diol), by itself accounts for more than 60% of the terpenes produced by the plant. Multiple enzymes are involved in the pathway for the biosynthesis of such diterpenoids in tobacco trichomes and some of the individual steps appear to be catalyzed by more than one functionally related enzymes. For example, more than one cembratriene-ol (CBT-ol) cyclases may be responsible for CBT-ol synthesis, and several CBT-ol hydroxylases may convert CBT-ol to CBT- diol.

The expression of proteins in plants and/or the genetic modification of plants in order to confer them with particular properties are of major interest. The availability of tools which can regulate the expression of genes in plants, tissue-specific expression in particular, is therefore important in the exploitation and development of these systems. In this regard, the present invention, which provides promoters enabling expression in trichomes, would offer many uses and advantages.

SUMMAR Y OF THE INVENTION The present invention provides the identification and characterization of regulatory nucleic acid sequences of plant origin, which make it possible to specifically direct the expression of a nucleic acid of interest in the secretory cells of plant trichomes. These promoters are easy to manipulate as modules, of reasonable size, can be adapted to the expression of heterologous genes, and can drive expression specifically in certain plant tissues. The invention concerns an isolated nucleic acid comprising a nucleotide sequence (i) that is at least 80% identical to SEQ ID NO: 1 or 2, (ii) that is identical to at least 100 consecutive nucleotides of SEQ ID NO: 1 or 2; or (iii) that hybridizes under stringent conditions to a nucleic acid probe that comprises the nucleotide sequence of SEQ ID NO: 1 or 2, or the complement of the nucleotide sequence of SEQ ID NO: 1 or 2, wherein said stringent conditions comprise hybridizing the nucleic acid probe to filter-bound nucleic acid in 6x SSC at about 45°C followed by one or more washes in O.lx SSC at about 68°C. Preferably, the nucleotide sequence exhibits transcriptional promoter activity in glandular trichome cells, preferably specific transcriptional promoter activity in glandular trichome cells. Optionally, the nucleic acid is a nucleic acid vector capable of propagation in a plant cell, transformation of a plant cell, or both. In a preferred embodiment, the nucleic acid further comprises an expressible sequence that is operatively linked to the nucleotide sequence to enable transcription of said expressible sequence. Said expressible sequence can encode a polypeptide of interest, in particular an enzyme that modifies an exudate molecule in a trichome; a bacterial protein, an animal protein, a mammalian protein, a plant protein, a non-tobacco protein, or a viral protein; an activator of transcription, or a suppressor of transcription; or production of said polypeptide reduces infection or infestation by a pathogen, a microorganism, a bacterium, a virus, a fungi, or an insect. Alternatively, said expressible sequence can encode an antisense nucleic acid or a R Ai.

The invention also concerns a modified plant cell comprising the nucleic acid according to the present invention. Preferably, the cell is of a plant from the family of Solanaceae, Asteraceae, Cannabaceae or Lamiaceae.

The invention further concerns a method for making of a polypeptide or protein, comprising introducing into a plant cell the nucleic acid according to the present invention, optionally generating a plant from the plant cell, and expressing an expressible sequence that encodes said polypeptide or protein. Preferably, the plant produces the polypeptide in a trichome. More preferably, the method further comprises collecting exudate at the leaf surface, and optionally recovering said polypeptide from said exudate.

One of the advantages of the invention is that the plant secretes the protein in the glandular trichome, and can be recovered from the exudate at the leaf surface.

The invention concerns the use of a nucleic acid according to the present invention or of a modified plant cell according to the present invention for the expression of a protein, polypeptide or nucleic acid in the trichomes of a plant, in particular for the specific expression of a protein in the trichomes of a plant. It also concerns the use of a nucleic acid according to the present invention or of a modified plant cell according to the present invention for the making of a molecule of interest in the trichomes of a plant, wherein the molecule of interest is a secondary metabolite, a biosynthetic product, a biosynthetic intermediate, an alkaloid, or a flavour.

As described in detail hereinbelow, the invention is applicable to the expression of any gene or expressible sequence of interest in any plant or plant tissue, preferably higher plants comprising glandular trichomes, for different applications, such as for the production of pharmaceutical or other products of interest, or for the production of plants having improved or adapted properties.

BRIEF DESCRIPTION OF THE DRA WING

FIGURE 1 : Map of the pLIBRO-66 plasmid derived from the pCAMBIA 1391Z T-DNA plasmid with the 1.4 kb fragment of the NtCPS2 gene upstream of the uidA gene.

FIGURE 2: GUS staining of plants carrying the pLIBRO-66 construct. Leaf from line number 3843 showing the highly specific localization of GUS activity in the trichome secretory cells. The cells of the trichome stalk are not stained. The black horizontal bar indicates.

DETAIIED DESCRIPTION OF THE INVENTION The present application provides nucleic acids with transcriptional promoter activity in trichomes and their uses which include the production of ribonucleic acids, polypeptides and metabolites of interest in trichomes.

The nucleic acids of the invention are derived from promoters of CBT-ol synthase genes of tobacco. Using the sequence of a cDNA coding for cembratriene-ol cyclase (CBT-ol cyclase), a terpene synthase, genes displaying strong sequence similarity were identified from genomic DNA from the species Nicotiana sylvestris, using PCR methods. An analysis of the expression of such genes showed that they are expressed in tobacco trichomes. Promoter sequences have been identified and characterized, and shown to be located upstream of the ATG of the CBT-ol cyclase gene (SEQ ID No 3). The sequences are given in SEQ ID Nos. 1 and 2. Construction of expression cassettes using said promoters has demonstrated an expression profile which is specific of trichome secretory cells.

The present application therefore provides promoters, particularly advantageous for the expression of proteins, polypeptides and nucleic acids of interest in plants or plant tissues, in particular for specific expression in trichomes, and more particularly, in the secretory cells of glandular trichomes. These promoters are particularly advantageous since they enable the collection of produced recombinant polypeptides directly in the exudate secreted by leaves of the plant. The invention concerns an isolated nucleic acid having functional transcriptional promoter activity in glandular trichomes, comprising the sequence set forth in SEQ ID No. 1 or 2 or the complementary sequence thereof, a fragment of the nucleic acid having at least 100 consecutive bases or a functional variant thereof, a sequence displaying at least 80% identity with one of said sequences. Preferably, the activity is specific for glandular trichomes, in particular that of the secretory cells of glandular trichomes.

In one embodiment of the invention, isolated nucleic acids comprising the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 are provided. In another embodiment, the invention provides isolated nucleic acids comprising a nucleotide sequence that is at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98% or 99% identical to SEQ ID NO: 1 or 2. In yet another embodiment, the invention provides isolated nucleic acids comprising a fragment or a functional variant of the promoters of the invention. In yet another embodiment, the invention provides isolated nucleic acids comprising a nucleotide sequence that is identical to at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive nucleotides of SEQ ID NO: 1 or 2. In yet another embodiment, the invention provides isolated nucleic acids that hybridizes under stringent conditions to a nucleic acid probe that comprises the nucleotide sequence of SEQ ID NO: 1 or 2, or the complement of the nucleotide sequence of SEQ ID NO: 1 or 2. Stringent hybridization conditions include without limitations medium stringency conditions, for example, hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2x SSC containing optionally 0.2% SDS at about 50°C to 65°C, or high stringency conditions, for example, hybridization to filter-bound nucleic acid in 6xSSC at about 45°C followed by one or more washes in O. lxSSC containing optionally 0.2% SDS at about 68°C, or under other stringent hybridization conditions which are apparent to those of skill in the art (see, for example, Ausubel, F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, at pages 6.3.1-6.3.6 and 2.10.3).

In another embodiment, the nucleic acids of the invention can comprise an expressible sequence that is operatively linked to the nucleotide sequence of the invention so as to enable transcription of the expressible sequence. Such a nucleic acid is also referred to herein as an expression cassette. In yet another embodiment, the nucleic acids comprising the nucleotide sequence of the invention is a vector capable of propagation in a plant cell, transformation of a plant cell, or both. Such nucleic acids comprise one or more cloning sites for insertion of an expressible sequence, preferably at a distance suitable for initiation of transcription. Therefore, the invention also concerns an expression construct comprising an expressible sequence operatively linked to a nucleic acid such as defined hereinabove; and any vector or modified plant cell comprising a construct such as defined hereinabove.

Preferably, the isolated nucleic acids described hereinabove exhibit transcriptional promoter activity in a trichome or in glandular trichome cells. The transcriptional promoter activity of the isolated nucleic acids can be specific of a trichome or of glandular trichome cells. More specifically, the fragments of sequence of SEQ ID NO: 1 or 2, the nucleic acids displaying identity with sequence of SEQ ID NO: 1 or 2 or the nucleic acids hybridizing with sequence of SEQ ID NO: 1 or 2 preferably conserve the transcriptional activity of the nucleic acid of SEQ ID NO: 1 or 2 or the signal of expression specificity.

As illustrated in the examples, the 1460 base pair sequence of the NtCPS2 gene promoter (SEQ ID No. 2) was cloned upstream of a uidA reporter gene. This reporter gene construct was inserted into a T-DNA based vector (pBI121) comprising a kanamycin resistance gene under the control of the nopaline synthase promoter of Agrobacterium tumefasciens (nos). The construct was introduced into N. sylvestris by genetic transformation using the Agrobacterium tumefaciens LBA4404 (Hoekema et al., 1993, Nature 303: 179-180). UidA gene expression was detected in several transformants. The results in FIG. 2 show that expression was present in secretory cells, but not in the cells constituting the base of the trichomes. Furthermore, no uidA expression was detected in any of the other organs of the plant. These data indicate that the sequence under study can direct the expression of a heterologous gene in the secretory cells of glandular trichomes in a highly specific manner.

In the spirit of the invention, the term "nucleic acid" shall be understood to mean a polymer of nucleotides, including DNA or RNA, either single- or double-stranded. These can be molecules that are synthetic or semi-synthetic, recombinant, optionally amplified or cloned, chemically modified or containing non-natural bases. The phrase "nucleic acid sequence" or "nucleotide sequence" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. The term "isolated" when referring to nucleic acids, refer to those that have been purified away from other cellular components and contaminants, i.e., other cellular nucleic acids and/or proteins, by standard techniques, including, for example, alkaline/SDS treatment, CsCl banding, column chromatography, and others purification techniques well known in the art. See, e.g., Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques (Berger and Kimmel (eds.), San Diego: Academic Press, Inc. (1987)), and Current Protocols in Molecular Biology (Ausubel, et al., (ed.), Greene Publishing and Wiley-Interscience, New York (1987)), both of which are incorporated herein by reference.

The term "specific" used herein in connection with a promoter shall be understood to mean a promoter that is mainly active in a given tissue or recognized group of cells. It shall be understood that a residual expression, generally lower, in other tissues or cells cannot be entirely excluded. For example, residual expression in other tissues or cells does not exceed 30% of the expression observed in glandular trichomes, preferably it does not exceed 20%, 15%, 10%, 5%, 2% or 1%. A feature of the invention is the provision of nucleic acids that exhibit transcriptional promoter activity only in or that are specific of trichomes, glandular trichome cells, and preferably secretory cells of trichome.

A nucleic acid "fragment" according to the invention refers to a fragment comprising at least 10 consecutive bases, more generally at least 20, 30, 40, 50, 60, 70, 80, 90 or 100. Nucleic acid "fragments" are typically fragments containing 100, 200, 300, 400 or 500 consecutive nucleotides of the sequence or more. Nucleic acid "fragments" are preferably fragments containing at least 100, 200, 300, 400 or 500 consecutive nucleotides of the sequence. Nucleic acid "fragments" can be used alone, or combined with other transcriptional regions, in order to construct chimeric promoters having a specificity for glandular trichomes, in particular for secretory cells of glandular trichomes.

The term "functional variant" designates any nucleic acid bearing one or more modifications (that is to say for example a mutation, deletion, or addition of one or more bases) with respect to the parent sequences described herein, and conserving an activity, either of a transcriptional promoter, or a signal of expression specificity, or of transit. For example the functional variants can correspond to promoters derived from the corresponding genes of other plant species. For instance, the studies described in the examples herein concern sequences from the species Nicotiana sylvestris, one of the presumed diploid parents of cultivated tobacco, N. tabacum. It is known that the sequences of N. sylvestris and N. tabacum are extremely similar (up to 99% sequence identity between N. tabacum and N. sylvestris sequences), such that the promoters of the corresponding genes of N. tabacum constitute functional variants in the context of the invention, and can be easily prepared by conventional methods (hybridization, amplification, and the like). Other functional variants are nucleic acids, synthetic, recombinant or natural, whose sequence hybridizes, under stringent conditions, with one of the sequences SEQ ID No. 1 or 2 or a fragment having at least 100 consecutive nucleotides and which has transcriptional promoter activity, in particular specific of glandular trichome cells.

The skilled artisan can test the transcriptional activity of fragments and variants by routine experiments by introducing the nucleic acid fragments and variants into a vector in such a way that it is operatively linked to a reporter gene, as described above and in the example section.

The percentage of sequence identity between two biological sequences can be determined by comparing two optimally aligned sequences over a window of comparison of, for example, at least 20 positions. Preferably, the percent identity is determined over two sequences of identical size. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.), 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection. These references are incorporated herein by reference. The term "expressible sequence" refers to a nucleotide sequence that can be transcribed to produce a functional product, including but not limited to a messenger RNA, a RNAi, or an antisense nucleic acid. Non-limiting examples of expressible sequence includes cDNA, genomic DNA, genomic DNA that comprises one or more exons that are transcribed, a gene sequence that contains one or more mutant nucleotide(s) which is not present in a naturally occurring sequence of the same gene. The term "expression cassette" refers to a nucleic acid construct comprising a regulatory region that is operatively linked to an expressible sequence. The term "operatively linked" indicates that the regulatory region and the expressible sequence are joined in the expression cassette such that transcription of the expressible sequence is regulated by the regulatory region. The regulatory region typically comprising a transcriptional promoter that is placed upstream at the 5' end of the expressible sequence of interest at a distance which is effective for regulation of expression. A spacer may be present between the regulatory region and the expressible sequence. The sequence and length of the spacer can be determined by methods well known in the art.

In various embodiments of the invention, the expressible sequence encodes a polypeptide or protein of interest, which can be a bacterial protein, an animal protein, a mammalian protein, a plant protein, a tobacco protein, a non-tobacco protein, or a viral protein. The polypeptide can be an enzyme, preferably an enzyme that modifies an exudate molecule in a trichome, resulting in a change in the composition of the exudate collected at leaf surface. The polypeptide can be a transcription factor, an activator of transcription, or a suppressor of transcription. In other embodiments of the invention, the expression of the polypeptide or protein of interest in a plant can reduces infection or infestation by a pathogen, a microorganism, a bacterium, a virus, a fungus, or an insect.

Alternatively, the expressible sequence encodes a nucleic acid of interest such as an antisense nucleic acid, a ribozyme or a RNAi.

As used herein, an RNAi molecule is a double-stranded or single-stranded polynucleotide that initiates RNA interference in a cell, resulting in a decrease in gene expression of a target gene. Preferably, the decrease in expression is specific for the target gene. As used herein, the term short, interfering RNA molecule (siRNA) re feres to a double-stranded RNA molecule that forms a RNA- induced silencing complex (RISC) in a cell. In various embodiment, a RNAi molecule of the invention is a nucleic acid molecule that comprises a portion of the nucleic acid sequence of the gene of interest, and can be a double-stranded DNA molecule, a double-stranded RNA molecule, a single-stranded DNA molecule, a single-stranded RNA molecule, or a double-stranded DNA-RNA hybrid. In one embodiment, the RNAi molecule is a double-stranded RNA molecule that comprises, independently in each strand, at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ribonucleotides. In a specific embodiment, the RNAi molecule is a siRNA. In another embodiment, the RNAi molecule is a polynucleotide which can serve as a template to produce, by the action of a DNA-dependent RNA polymerase or a RNA-dependent RNA polymerase, a RNA molecule a portion of which forms a portion of a siR A. In a specific embodiment, the RNAi molecule features regulatory elements that enable inducible action of a RNA polymerase. In a specific embodiment, the RNAi molecule comprises a hairpin structure. In a specific embodiment, the bases on each strand of a double-stranded RNAi molecule are perfectly complementary.

The vectors of the invention can be DNA or RNA, circular or linear, single- or double-stranded. Typically, it is a plasmid, phage, phagemid, virus, cosmid, artificial chromosome, etc. Examples of plant vectors are described in the literature, among which one can cite in particular the A. tumefaciens T-DNA plasmids pBIN19 (Bevan, 1984, Nucl. Acids Res. 12:8711 -8721), pPZPlOO (Hajdukewicz et al, 1994), pCAMBIA series (R. Jefferson, CAMBIA, Australia). The vectors of the invention can additionally comprise an origin of replication, a selection gene, a plant recombination sequence, or a combination of two or more of the foregoing. The vectors can be constructed by conventional molecular biology methods, well known to those skilled in the art, using for example restriction enzymes, ligation, cloning, replication, etc. Specific examples of vectors according to the invention are provided in the experimental section, and include in particular pLIBRO-66 and pLIBRO-68.

The expressed polypeptide or protein encoded by the expressible sequence is recombinant. Indeed, the polypeptide or protein of interest is either a partly or entirely heterologous (i.e., foreign) to the genetically modified plant or plant cell into which it is introduced; or, is homologous to an endogenous gene of the genetically modified plant or plant cell into which it is introduced, but it is either inserted into the plant's genome or chromosome at a location which differs from that of the natural gene; or operably linked to a regulatory region which differs from the natural regulatory region of the natural gene. In addition, the expression cassette of the invention may be recombinant and not naturally found because the sequence having a transcriptional activity of the invention is not operative ly linked to the natural coding sequence associated therewith (in particular NtCPS2 coding sequence), but is operatively linked to a distinct expressible sequence. Therefore, such an expression cassette will be referred herein as "recombinant expression cassette". One aspect of the invention concerns a method for producing a recombinant protein, a recombinant polypeptide, or a nucleic acid (e.g., an antisense nucleic acid or RNAi) in a plant cell, preferably a cell of a trichome, comprising introducing into said plant cell a construct such as defined hereinabove, comprising a gene or sequence coding for said protein, polypeptide, or nucleic acid, and optionally regenerating a plant from said cell. Another aspect of the invention concerns a method for making a plant that produces a recombinant protein, a recombinant polypeptide, or a nucleic acid (e.g., an antisense nucleic acid or RNAi), comprising introducing into a plant cell or seed a construct such as defined hereinabove, comprising a gene or sequence coding for said protein, polypeptide, or nucleic acid, and regenerating a plant from said cell. The nucleic acid to be produced is preferably a single stranded or double stranded RNA. It is preferably an antisense nucleic acid or a RNAi. An additional aspect of the invention concerns a method for producing a metabolite of interest in a plant cell, preferably a cell of a trichome, comprising introducing into said plant cell a construct such as defined hereinabove, comprising a gene or sequence that encodes for a protein functionally associated with the production of said metabolite in said plant cell, and optionally regenerating a plant from said cell.

The nucleic acid constructs of the invention can be used to genetically modify plants, and in particular to introduce and express proteins, polypeptides, nucleic acids in plant cells, plant parts, as well as whole plants. Introduction of the nucleic acid constructs into a plant cell or tissue, including a seed or a plant part, can be carried out by any method known to those skilled in the art. Many methods are well known in the field which include but are not limited to the use of Agrobacterium tumefaciens, electroporation, conjugative transfer, ballistic methods, and the like.

A commonly used method is based on Agrobacterium tumefaciens which involves introducing a genetic construct of the invention into A. tumefaciens, then contacting plant cells or plant parts with said bacterium. A. tumefaciens-based transformation methods are described in Horsch et al., 1985 (Science 227: 1229-1231) or Hooykaas and Schilperoort, 1992 (Plant Mol. Biol. 19:15-38). Another method of plant transformation is based on projecting microparticles (typically microbeads) to which gene constructs are attached, directly on plant cells, then culturing said cells in order to regenerate a transgenic plant. The particles which are used are typically gold particles, which are typically projected by means of a particle gun (see in particular Russell et al., In Vitro Cell. Dev. Biol., 1992, 28P, p. 97-105). The microinjection method is based primarily on injecting the gene constructs into plant protoplasts or embryos, then cultivating said tissues so as to regenerate whole plants. Many other well known methods and protocols implementing the above methods are described in Siemens, J and Schieder, 1996 (Plant Tiss. Cult. Biotechnol. 2:66-75) and can be employed in the invention. The invention also provides a plant cell modified by the introduction of a nucleic acid of the invention. In particular, the plant cell is a cell of a plant from the Solanaceae, Asteraceae, Cannabaceae or Lamiaceae family. The cell can be cultured in vitro, or it can be used to regenerate tissues or whole plants, in order to produce polypeptides in culture, in a greenhouse, or in the field.

Once regenerated, the transgenic plants can be tested for expression of the product of interest in the trichomes, in particular in the trichome secretory cells. This can be done by collecting the leaf exudate and testing for the presence of the product in said exudate. This can also be done by analyzing the presence of the expression product of the gene of interest in the leaves and, more particular, in the trichome cells (for example by analyzing mRNA or genomic DNA with specific primers or probes). Optionally the plants can be selected, crossed, treated, etc. in order to obtain plants displaying improved levels of expression.

The present invention also concerns a modified plant or plant cell, said plant or plant cell comprises: a recombinant expression cassette of the invention;

more than one copy of expression cassette of the invention; indeed, the copy number of an expressible sequence of interest is increased compared to the wildtype plant or plant cell in order to increase the production of the expressible sequence; for instance, 2, 3, 4, 5 or 10 copies are contemplated;

an expression cassette of the invention, which can be either stably integrated into chromosome of the plant or plant cell genome at a location which differs from that of the natural gene, or maintain in an epigenic (i.e., extrachromosomal) form;

wherein the expression cassette comprises a nucleic acid comprising a nucleotide sequence (i) that is at least 80% identical to SEQ ID NO: 1 or 2, (ii) that is identical to at least 100 consecutive nucleotides of SEQ ID NO: 1 or 2; or (iii) that hybridizes under stringent conditions to a nucleic acid probe that comprises the nucleotide sequence of SEQ ID NO: 1 or 2, or the complement of the nucleotide sequence of SEQ ID NO: 1 or 2, operably linked to an expressible sequence. In various embodiments, the invention provides methods for making a polypeptide, in particular in the glandular trichome of a plant, comprising introducing into a plant cell a nucleic acid according to the invention, such as but not limited to the expression cassette described hereinabove, which comprises a sequence encoding for the polypeptide. The methods can independently comprise the steps of culturing the plant cell, regenerating a plant from the plant cell, growing the regenerated plant, and harvesting the leaves of the regenerated plant. The invention encompasses methods comprising providing or growing a plant comprising a nucleic acid according to the invention, such as but not limited to the expression cassette described hereinabove, which comprises a sequence encoding for the polypeptide. The invention can thus be used to modify plants, plant cells or tissues, in order to cause them to make certain products of interest. Accordingly, the uses of the nucleic acids of the invention to modify a plant, a plant cell, or plant tissue, to make a molecule of interest, such as but limited to a polypeptide, a secondary metabolite, a biosynthetic product, a biosynthetic intermediate, an alkaloid, a terpenoid, or a flavour, are contemplated. The invention can be used to express products of interest specifically in the secretory cells of glandular trichomes of higher plants, in particular, Angiosperms. The invention can be applied to any plants from families having glandular trichomes, for example Asteraceae, Solanaceae, Cannabaceae and Lamiaceae. The invention is particularly adapted to plants from the Solanaceae family, such as for example the genuses Solanum, Lycopersicon, Capsicum, Petunia, Datura, Atropa, etc., and to Nicotianae, for example Nicotiana sylvestris and Nicotiana tabacum.

In one embodiment, the recombinant polypeptide is expressed in the trichome which leads to production by the secretory cells of a molecule secreted in the leaf surface exudate and/or to a modification of the exudate composition. The molecule so produced can be recovered in the exudate. In another embodiment, the plant comprising the nucleic acid of the invention secretes the polypeptide in the glandular trichome, and the polypeptide is recovered in the exudate at the leaf surface.

The molecule or product of interest can be a recombinant polypeptide, including peptides, proteins, enzymes, antibodies, and the like. In particular it can be a protein having a biological activity of industrial interest, for example medical or phytosanitary. It can also be a protein intended to confer particular properties to the plant (in particular a modification of the composition of the leaf exudate, such as resistance to pathogens (insects, fungi, bacteria, viruses, etc.), improved growth, a modified metabolite content, or a modified synthetic pathway, etc.

The invention can also be used in order to express, in a plant cell or a plant, a transcriptional activator. In this case, the transcriptional activator produced will enable control of the expression of a protein of interest placed under control of a promoter responding to the activator. Such a binary system, using two cassettes (present on a same vector or on different vectors) makes it possible to amplify the specific expression obtained with the aid of the invention. One such example is a system comprising a first cassette containing the gene coding for a transcriptional activator (for example GAL4) under the control of the trichome-specific promoter, and another cassette comprising the gene coding for the protein of interest under the control of elements known to be regulated by the transcription factor (Gal4).

Another amplification system consists in the use of a viral RNA vector. In fact, some plant RNA viruses code for an RNA-dependent RNA polymerase which allows amplification of the transcripts of a given gene. In this embodiment, a gene encoding the product of interest is cloned downstream from the promoter of said RNA polymerase, in place of the open reading frame coding for the viral coat protein. The virus is itself placed under the control of the specific promoter. Expression of the virus is therefore restricted to trichome secretory cells, thereby allowing selective amplification of expression of the gene in said cells. Other aspects and advantages of the invention will become apparent in the following examples, which are given for purposes of illustration and not by way of limitation.

EXAMPLES

IDENTIFICATION OF THE NTCPS2 PROMOTER SEQUENCE

Using a technique called adapter anchor PCR (Siebert P D, et al. (1995) Nucl. Acids Res. 23:1087- 1088), DNA fragments of unknown sequence flanking a known sequence can be amplified and sequenced. This technique is used to determine the sequence of fragments of genomic DNA located upstream of the coding region of NtCPS2 from Nicotiana tabacum var. Basma Drama, an oriental variety which produces high levels of cis-abienol. Genomic DNA is digested following standard molecular biological methods with restriction enzymes producing blunt ends (e.g. EcoRV, Dral, Sspl, Nael) and ligated to adapters composed of the following oligonucleotides:

ADPR1 5'-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGGAGGT (SEQ ID No 4) and ADPR2 5 '-ACCTCCCC (SEQ ID No 5).

Flanking regions are then amplified after two PCR with primers complementary to the known sequence and to the adapter. The second PCR is done with nested primers to ensure specificity of the amplification. The amplifications are performed with Taq polymerase (Eppendorf) and 25 ng of genomic DNA in 25 μL under the following cycling conditions: 94°C for 2 min, then 35 cycles of 94°C for 30 sec, 68°C for 30 sec, and then 1 cycle at 72°C for 5 min. PCR fragments are then purified (QiaQuick, Qiagen) and directly sequenced. Thus, after two rounds of adapter anchor PCR, the sequence of a fragment of 1447 bp from the translation initiation codon could be determined (see SEQ ID No 3). Rapid Amplification of cDNA ends (RACE) with a commercial kit (AMBION™) is used to determine the site of transcription initiation which was found to be located 119 bp upstream of the ATG translation initiation codon. The sequence of the promoter fragment together with the 5' untranslated leader and the beginning of the open reading frame are shown in SEQ ID No 3.

AMPLIFICATION OF THE NTCPS2 FRAGMENT AND CLONING INTO A T-DNA BINARY VECTOR

Two lengths (1.4 and 0.7 kb) of the promoter are tested for their trichome specific transcription activity. Both fragments are amplified by PCR with primers carrying an EcoRI site for the upstream (or forward) primer and a BamHI site for the downstream (or reverse) primer. Thus, for the 0.7 kb fragment the sequences of the primers are 5'-gaattcgaggtcttaatatgtgttcaatccaga (SEQ ID No 6) for the upstream primer and 5'ggatcctttctaatttaattttgttttattcttc (SEQ ID No 7) for the downstream primer (SEQ ID No 1). For the 1.4 kb fragment the sequence of the upstream primer was 5'- ggaattctgcaaatctcccaacattatcacctt (SEQ ID No 8) and the same reverse primer as for the 0.7 kb fragment are used (SEQ ID No 2). The amplifications are carried out on tobacco genomic DNA from the variety Basma Drama with Taq polymerase (Eppendorf) under the following conditions: 1 cycle at 94°C for 2 min, 35 cycles at 94°C for 30 s, 57°C for 30 s, and 72°C for 1 min, followed by a single extension cycle at 72°C for 5 min. The PCR products are purified on a Qiaquick column (Qiagen) and cloned into the the pGEM-T plasmid (Promega). The plasmids with the fragments are then checked by sequencing and named pCPS2-0.7 and pCPS2-1.4 for the 0.7 kb and 1.4 kb fragments respectively. Both plasmids are then digested with the restriction enzymes EcoRI and BamHI to release the promoter fragments which are purified after gel electrophoresis (Qiaquick, Qiagen). In parallel, the T-DNA binary vector pCAMBIA 1391Z, which carries a uidA reporter gene with an intron, is also digested with EcoRI and BamHI and purified on Qiaquick (Qiagen). The promoter fragments are then ligated to the linearized pCAMBIA 1391Z to afford plasmids pLIBRO- 68 and pLIBRO-66 (Figure 1). These binary T-DNA plasmids are then introduced into Agrobacterium tumefaciens strain LBA 4404 by electroporation. GENETIC TRANSFORMATION

Transgenic lines of Nicotiana sylvestris carrying the T-DNA fragment of plasmids pLIBRO-66 and pLIBRO-68 are recovered upon co-cultivation of Nicotiana sylvestris leaf discs with A. tumefaciens LBA4404 strains harboring the pLIBRO-66 and pLIBRO-68 plasmids, according to the method of Horsch et al. (Horsch R B, et al. (1985) Science 227:1229-1231). Transgenic calli and plants are selected on 50 mg/L hygromycin and the bacteria are eliminated by 250 mg/L carbenicillin. Transgenic lines carrying a single copy of the T-DNA are selected by real time quantitative PCR.

GUS HISTOCHEMISTRY OF TRANSGENIC LINES

The visualization of the expression of the uidA gene which encodes the glucuronidase (GUS) activity is carried out by staining the plant tissue with X-glucuronide as per the protocol described in Jefferson et al. (Jefferson R A. (1987) Plant Mol. Biol. Rep. 5:387-405; Jefferson, R.A., et al. (1987). EMBO Journal 6, 3901-3907). Stained tissues are observed under a binocular microscope and photographs were taken. Thus, the expression profile in several lines carrying either pLIBRO-66 or pLIBRO-68 is determined. Typical images of these observations are shown in Figure 2. With both constructs, the staining is highly specific and strictly localized to the secretory cells of the glandular trichomes.

Claims

1. An isolated nucleic acid comprising a nucleotide sequence (i) that is at least 80% identical to SEQ ID NO: 1 or 2, (ii) that is identical to at least 100 consecutive nucleotides of SEQ ID NO: 1 or 2; or (iii) that hybridizes under stringent conditions to a nucleic acid probe that comprises the nucleotide sequence of SEQ ID NO: 1 or 2, or the complement of the nucleotide sequence of SEQ ID NO: 1 or 2, wherein said stringent conditions comprise hybridizing the nucleic acid probe to filter-bound nucleic acid in 6x SSC at about 45°C followed by one or more washes in O.lx SSC at about 68°C

2. The nucleic acid according to claim 1 , characterized in that the nucleotide sequence exhibits transcriptional promoter activity in glandular trichome cells, preferably specific transcriptional promoter activity in glandular trichome cells.

3. The nucleic acid according to claim 1 or 2, wherein the nucleic acid is a nucleic acid vector capable of propagation in a plant cell, transformation of a plant cell, or both.

4. The nucleic acid according to any one of claims 1 to 3, further comprising an expressible sequence that is operatively linked to the nucleotide sequence to enable transcription of said expressible sequence.

5. The nucleic acid according to claim 4, wherein said expressible sequence encodes a polypeptide of interest.

6. The nucleic acid according to claim 5, wherein said polypeptide is an enzyme that modifies an exudate molecule in a trichome.

7. The nucleic acid according to claim 5, wherein said polypeptide is a bacterial protein, an animal protein, a mammalian protein, a plant protein, a non-tobacco protein, or a viral protein.

8. The nucleic acid according to claim 5, wherein said polypeptide is an activator of transcription, or a suppressor of transcription.

9. The nucleic acid according to claim 5, wherein production of said polypeptide reduces infection or infestation by a pathogen, a microorganism, a bacterium, a virus, a fungi, or an insect.

10. The nucleic acid according to claim 4, wherein said expressible sequence encodes an antisense nucleic acid or a R Ai.

11. A modified plant cell comprising the nucleic acid according to one of claims 1 to 10.

12. The modified plant cell according to claim 11 , characterized in that the cell is of a plant from the family of Solanaceae, Asteraceae, Cannabaceae or Lamiaceae.

13. A method for making of a polypeptide or protein, comprising introducing into a plant cell the nucleic acid according to any one of claims 1 to 9 , optionally generating a plant from the plant cell, and expressing an expressible sequence that encodes said polypeptide or protein.

14. The method according to any of claim 13, characterized in that the plant produces the polypeptide or protein in a trichome.

15. The method according to any of claim 13 to 14, further comprising collecting exudate at the leaf surface, and optionally recovering said polypeptide from said exudate.

16. Use of a nucleic acid according to any one of claims 1 to 10 or of a modified plant cell according to claim 1 1 or 12 for the expression of a protein, polypeptide or nucleic acid in the trichomes of a plant.

17. Use of a nucleic acid according to one of claims 1 to 10 or of a modified plant cell according to claim 1 1 or 12 for the making of a molecule of interest in the trichomes of a plant, wherein the molecule of interest is a secondary metabolite, a biosynthetic product, a biosynthetic intermediate, an alkaloid, or a flavour.