CA2350186A1 - Promoters for gene expression in the roots of plants - Google Patents

Abstract

The present invention relates to promoters effecting root-specific expressio n of coding nucleotide sequences controlled thereby and expression cassettes, recombinant vectors and microorganisms that contain such promoters. The invention also relates to transformed transgenic plants, in addition to a method for the production and a method for the isolation of root-specific promoters.

Description

VOSSI US & PARTN ER
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+49-89-41 30 44 00 PCT application ETH Zurich Our ref.: C 2645 PCT
Promoters for gene expression in roots of plants Description The present invention relates to promoters causing a root-specific expression of coding nucleotide sequences which are controlled by the promoters for tissue-specific gene expression in plants, expression cassettes, recombinant vectors and microorganisms comprising such promoters, transgenic plants transformed with them, a method for the production of transgenic plants and a method for the isolation of root-specific promoters.
In the following, documents from the state of the art are cited the disclosure content of which is incorporated in this application by reference.
The use of plants modified in their genotype by means of genetic engineering has proven to be advantageous in many areas of agriculture and often to be the only way to transmit certain properties to useful plants. The principal aims are plant protection as well as an increase of quality of the harvestable products.
Various methods for the genetic modification of dicotyledonous and monocotyledonous plants are known (cf., amongst others, Gasser and Fraley, Science 244 (1989), 1293-1299; Potrykus, Ann. Rev. Plant Mol. Biol. Plant Physiol.
42 (1991 ), 205-225). All the methods are based on the transmission of gene constructs which, in most cases, are new combinations of specific coding regions of structural genes with promoter regions of other structural genes as well as transcription terminators.
The provision of promoters plays a great role for the production of transgenic plants since the specificity of a promoter is crucial for the point in time, the type of tissue and the intensity in which a structural gene transmitted by means of genetic engineering is expressed.
A multitude of promoters controlling the expression of foreign genes in plants is known. The promoter most commonly used is the 35S CaMV promoter (Franck et al., Cell 1 (1980), 285-294) leading to a constitutive expression of the gene introduced.
Often, however, inducible promoters are used, for example for wound induction (DE-\\Ntvossiusl\Allgemein\Daten-1\ast\translations\Eng\C\C2645PCT.doc V
A-3843628), chemical induction (Ward et al., Plant Molec. Biol. 22 (1993), 361-366) or light induction (Fluhr et al., Science 232 (1986), 1106-1112).
Also, the use of cell- and tissue-specific promoters was described: guard cell-specific gene expression (DE-A-4207358), seed-, tuber- and fruit-specific gene expression (summarized in Edwards and Coruzzi, Annu. Rev. Genet. 24 (1990), 275-303; DE-A-3843627), phloem-specific gene expression (Schmulling et al., Plant Cell 1 (1989), 665-670), root nodule-specific gene expression (DE-A-3702497) or meristem-specific gene expression (Ito et al., Plant Mol. Biol. 24 (1994), 863-878).
Promoters mediating the gene expression in roots include, for example, the class II
patatin promoter (Koster-Topfer et al., Mol. Gen. Genet. 219 (1989), 390-396) exhibiting a high expression in potato tubers and in specific cell layers of root tips after fusion with the reporter gene of the f3-glucuronidase (GUS) gene. GUS
fusion experiments with the agropine synthase promoter (ags) (Inoguchi et al., Plant Phys.
149 (1996), 73-78) exhibited a high GUS activity primarily in roots.
Transgenic Arabidopsis plants containing an AKT1 (= potassium channel)-GUS-construct (Lagarde et al., Plant J. 9 (1996), 195-203) particularly led to an expression in the outer cell layers of mature root segments. A portion with a length of 636 by of the 5' region of the TobRB7 (= water channel) gene (Yamamoto et al., Plant Cell 3 (1991 ), 371-382) mediated a root-specific GUS expression in transgenic tobacco plants.
Furthermore, GUS fusion experiments with the promoter of an extensin gene were described wherein GUS activity in soy bean seedlings could be determined depending on the developmental stage in the hypocotyl and in the root and/or specific zones of the root (Ahn et al., Plant Cell 8 (1996), 1477-1490).
The use of the promoters described is often problematic. Promoters causing a constitutive expression of the genes which are controlled by said promoters can be used, for example, for the production of plants which are tolerant to herbicides and resistant to pathogens. The disadvantage of these promoters, however, is that the products of the genes which are controlled by them are present in all parts of the plant, including the harvested parts of the plants, which can be undesirable in some cases. Inducible promoters are not unproblematic neither since the induction conditions are typically difficult to control in plants which are used agriculturally outdoors.
If the various approaches of genetic modification in plants are to be carried out successfully, it is furthermore necessary that the genes which are to be regulated in different ways are subjected to the control of different promoters. It is, thus, necessary to provide different promoter systems with different specificities.
Only a limited number of promoters regulating the gene expression in roots has been known so far. if specific approaches of genetic modification in plants are to be carried out successfully, it is necessary to provide further alternative promoter systems for the gene expression in roots, which, in comparison to known systems, are regulated differently.
Therefore, the technical problem underlying the present invention is to provide means for an organ-specific, preferably root-specific gene expression of plants.
These means should be suitable, for example, for the expression of genes influencing the uptake of nutrients from the soil and for the expression of genes modifying the growth of roots.
This technical problem has been solved by the provision of the embodiments characterized in the claims.
Thus, the present invention relates to a promoter selected from the group consisting of a) promoters comprising the nucleic acid sequence indicated under SEQ ID No.
1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6;
b) promoters comprising a functional part of the nucleic acid sequence indicated under SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No.
or SEQ ID No. 6 and causing a root-specific expression of a coding nucleotide sequence controlled by them in plants;
c) promoters having a sequence which hybridizes with one of the sequences shown under SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ
ID No. 5 or SEQ ID No. 6 and which causes a root-specific expression of a coding nucleotide sequence controlled by them in plants; and d) promoters of genes encoding proteins the amino acid sequence of which exhibits a homology of at least 60% to the amino acid sequence indicated under SEQ ID No. 8, wherein these promoters cause a root-specific expression of a coding nucleotide sequence controlled by them in plants.
In a preferred embodiment, the promoters of the invention are promoters from plant genes or derived therefrom.
Within the context of the present invention, a "promoter" is a DNA sequence comprising the regulatory portion of a gene, preferably of a structural gene.
The "regulatory portion" of a gene is understood to be the part which determines the expression of the gene. A regulatory portion has a sequence motif where transcription factors and RNA polymerise assemble and induce the transcription of the coding portion of the gene. Furthermore, the regulatory portion may comprise one t v or more positive regulatory elements, so-called enhancers. In addition or instead, it may also contain negative regulatory elements, so-called silencers. A
"structural gene" is a genetic unit of regulatory and coding portion whose gene product is a protein. The information for the primary amino acid sequence of the protein is contained in the coding portion of the structural gene, while the regulatory part determines when and in which tissues and in which amounts the transcript of the coding portion according to which the gene product is synthesized is formed.
The promoters according to the invention may originate from, for example, plant genes, be modified by recombinant DNA techniques and/or be produced synthetically.
Within the meaning of the present invention, the term "root-specific" means that a foreign gene which is under the control of a promoter according to the invention is expressed in the roots. Within the meaning of the present invention, root specificity is particularly given when the promoter according to the invention favours the expression of a foreign gene in the roots in comparison to mature leaves and causes a significantly, for example at least 2- to 5-fold, preferably 5- to 10-fold, particularly preferred 10- to 100-fold increased expression.
In the context of the present invention, root specificity can be analysed via reporter gene experiments. For testing an isolated promoter sequence for promoter activity in roots, the promoter can, for example, be operatively linked to a reporter gene, such as the f3-glucuronidase gene from E. coli, in an expression cassette or in a vector for plant transformation. This construct is used for the transformation of plants.
Subsequently, the expression of the f3-glucuronidase in roots is determined in comparison to mature leaves, as has been described, for example, by Martin et al.
(The GUS Reporter System as a Tool to Study Plant Gene Expression, In: GUS
protocols: Using the GUS Gene as a Reporter of Gene Expression, Academic Press (1992), 23-43).
The term "root" is known to the person skilled in the art. In addition, it is referred to Strasburger (Lehrbuch der Botanik fur Hochschulen: Begr. by Eduard Strasburger u.a., new edition by Peter Sitte, Hubert Ziegler u.a., 34. Aufl., 1998, Fischer (Gustav)-Verlag, Stuttgart).
Surprisingly, it was found that a promoter with the nucleotide sequence shown under SEQ ID No. 1, SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID
No. 6 causes a root-specific expression of a coding nucleotide sequence which is controlled by said promoters in plants.

This observation is particularly surprising since a genomic fragment (SEQ ID
No. 13) of the promoter region (cf. Example 2) which has an extension of approximately by in comparison to SEQ ID No. 1 cannot mediate a root-specific GUS
expression.
Only shortened promoter fragments (SEQ ID No. 1 to 6) - in comparison to the large fragment (SEQ ID No. 13) - which were produced as described in Example 3C lead to a root-specific gene expression of a coding nucleotide sequence controlled by the promoter.
During the analysis of the cDNA sequence (SEQ ID No. 7), three further ATG
translation initiation codons (positions 42-44, 65-67 and 142-144 of SEQ ID
No. 7) are found in the 5' region upstream of the actual ATG (pos. 256-258, SEQ ID
No. 7), which lead to several upstream open reading frames (uORFs). The presence of such uORFs can have a negative influence on the translation rate of specific mRNAs.
The various promoter fragments are preferably constructed in a way so that they do not comprise this 5' upstream region (pos. 1-255, SEQ ID No. 7).
The promoter according to the invention allows for a root-specific gene expression of a coding nucleotide sequence controlled by the promoter. It represents an interesting alternative to other root-specific promoters since it can also mediate the gene expression in root hairs. Due to the bigger surface of the root hairs in comparison to the root, there is the possibility that the expression of such genes can be manipulated more effectively by means of the promoter according to the invention whose gene products, for example, mediate the transport of nutrients and metabolites via the root hair cells.
Various possible applications for the promoter according to the invention are at disposal. One possibility, for example, is the production of transgenic plants which, due to a modified root-hair metabolism that has a positive influence on the root surroundings (rhizosphere), show an increased yield. Furthermore, the promoters according to the invention can be used for the root-specific expression of genes mediating, for example, the absorption of heavy metal ions from contaminated soil.
By means of the promoters according to the invention it would, thus, be possible to produce plants which can be used in phytoremediation.
In another embodiment of the invention, the promoters according to the invention allow for the expression of a coding nucleotide sequence controlled by said promoters to be caused in specific root cells as, for example, in root cells of the primary root, in root hairs of the primary root, in root cells of the root tip or in root hairs of the primary root below the hypocotyl.
s Apart from a promoter exhibiting the complete sequence shown under SEQ ID No.
1, SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6, the present invention also relates to promoters exhibiting a functional part of this sequence and causing a root-specific expression of a coding nucleotide sequence controlled by these promoters in plants.
In this context, a "functional part" is to be understood as sequences which, despite deviating nucleotide sequence, still have the desired functions, for example promoter activity and tissue or organ specificity. A way of measuring promoter activity is, for example, the expression rate determined for a specific marker gene which is subjected under the regulatory control of the promoter according to the invention.
Suitable marker genes are, for example, the 13-glucuronidase (GUS) gene from E.
coli or the green fluorescence protein (GFP) gene (Baulcombe et al., Plant J.
7 (16) (1993), 1045-1053). The organ and tissue specificity can easily be established by comparing the expression rates determined for the individual tissues or organs of the plant for the above marker genes. Within the meaning of the present invention, functional parts of the promoter sequences comprise naturally-occurring variants of the sequences described herein as well as artificial nucleotide sequences, for example, obtained by chemical synthesis.
In particular, a functional part is also the natural or artificial mutation of an originally isolated promoter sequence which further exhibits the desired functions.
Mutations comprise substitutions, additions, deletions, exchanges and/or insertions of one or more nucleotide residues. Thus, the present invention also comprises nucleotide sequences which can be obtained by modifications of the nucleotide sequence depicted under SEQ ID No. 1, SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID
No. 5 or SEQ ID No. 6. The aim of such a modification can be, for example, to further delimit the promoter sequence contained therein or, for example, to introduce further restriction sites.
Functional parts of a promoter sequence also comprise promoter variants whose promoter activity, compared to the wild type, is decreased or increased.
In principle, the activity of a eukaryotic RNA polymerase II promoter is caused by the synergetic interaction of various trans-active factors (DNA binding proteins) which bind to the various cis-regulatory DNA elements present in the promoter. These factors interact directly or indirectly with single or several factors of the basal transcription machinery which finally leads to the formation of a pre-initiation complex near the transcription start site (Drapkin et al., Current Opinion in Cell Biology 5 (1993), 469-476). It could be called a modular set-up of eukaryotic RNA
polymerase II promoters, wherein various cis-elements (modules) as partial components each determine the total activity of the promoter (Tjian and Maniatis, Cell 77 (1994), 5-8).

This modular set-up was elucidated, for example, by promoter studies with the cauliflower mosaic virus (CaMV) 35S promoter (Benfey and Chua, Science 250 (1990), 959-966; Benfey et al., EMBO J. 9 (1990), 1677-1684; Benfey et al., EMBO
J. 9 (1990), 1685-1696). Due to the different tissue specificities that different restriction sub-fragments of the -343 to +8 (relative to the transcription start site) promoter mediate in transgenic tobacco plants, the promoter was divided into 6 sub-domains. The strong, constitutive expression which the complete promoter mediates can, thus, be sectioned into tissue-specific partial activities.
Single sub-domains of the promoter according to the invention which mediate potential tissue specificity can be identified, for example, by fusion with a minimal promoter reporter gene cassette. A minimal promoter is a DNA sequence comprising a TATA box, which is located about 20 to 30 base pairs upstream of the transcription start site, or an initiator sequence (Smale and Baltimore, Cell 57 (1989), 103-113;
Zawel and Reinberg, Proc. Natl. Acad. Sci. 44 (1993), 67-108; Conaway and Conaway, Annu. Rev. Biochem. 62 (1993), 161-190). Minimal promoters are, for example, the -63 to +8 035S promoter (Frohberg, Dissertation at the FU Berlin FB
Biologie (1994)), the -332 to +14 minimal patatin class I promoter as well as the -176 to +4 minimal PetE promoter (Pwee et al., Plant J. 3 (1993), 437-449).
Furthermore, such sub-domains or cis-elements of the promoter according to the invention can also be identified via deletion analyses and/or mutageneses (Kawagoe et al., Plant J. 5(6) (1994), 885-890). The test for functionality of such a sub-domain or cis-element can take place in pianta by the detection of the reporter activity in transformed cells. In the context of the present invention, the functional part of the promoter sequence also includes sub-domains and/or cis-elements of the nucleotide sequences depicted under SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No.
4, SEQ ID No. 5 or SEQ ID No. 6 which mediate a root specificity.
Moreover, the efficiency of a sub-region or of a cis-element can be increased significantly by means of multimerisation. In the case of the CaMV 35S
promoter, the dimerisation of a fragment with a length of 250 by in tandem, for example, led to a 10-fold increase of the promoter activity (Kay et al., Science 230 (1987), 1299-1302).
In the case of the sub-domain B5 of the CaMV 35S promoter, there was a clear increase in the activity of the promoter construct when this domain was present as tetramer and not as monomer (Benfey et al., EMBO J. 9 (1990), 1685-1696).
In another embodiment, the present invention particularly relates to di- and multimers of sub-domains and/or cis-elements of the nucleotide sequences depicted under SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID
No. 6.

a In a further embodiment of the invention, the increase in promoter activity, in comparison with the wild type, is achieved by the combination of the promoter according to the invention with a so-called enhancer.
In the literature, various enhancers have been described which, as a rule, cause a tissue-specific increase of the expression, wherein the tissue specificity is usually determined by the relevant enhancer used (Benfey et al., Science 250 (1990), 966; Benfey et al., EMBO J. 8 (1989), 2195-2202; Chen et al, EMBO J. 7, (1988), 297-302; Simpson et al., Nature 323 (1986), 551-554).
Moreover, there are enhancers, such as the PetE enhancer (Sandhu et al., Plant Mol. Biol. 37 (1998), 885-896) which do not have a tissue-specific effect and can, thus, be put before the promoter according to the invention as purely quantitative enhancer elements in order to increase the expression in roots without changing the quality of the tissue specificity of the promoter according to the invention.
A root-specific enhancer which is based on the multimerisation of a specific box (Box II) was described, for example, in "DNA-Protein Interactions in the Auxin regulated Promoter of T-DNA gene 5" (Author: Sirpa Nuotio, Acta Univeritatis Ouluensis, A Scientiae Rerum Naturalium 299 (1997), Oulu University Press, pp.
38).
Furthermore, synthetic enhancers may be used, too, which are, for example, derived from naturally-occurring enhancers and/or can be obtained by combination of various enhancers.
The present invention also relates to promoters having a nucleotide sequence which hybridizes with the nucleotide sequence shown under SEQ ID No. 1, SEQ ID No.
2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 and which cause a root-specific expression of a coding nucleotide sequence controlled by said promoters in plants. Such sequences are preferred to hybridize the sequence shown under SEQ
ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No.

under stringent conditions.
The term "stringent conditions" preferably refers to hybridization conditions as described, for example, in Sambrook et al. (Molecular Cloning, A Laboratory Manual, 2"d edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). In particular, hybridization takes place under the following conditions:
hybridisation buffer: 2 x SSC; 10 x Denhardt's solution (Fikoll 400 + PEG +
BSA; ratio 1:1:1 ); 0.1 % SDS; 5 mM EDTA; 50 mM Na2HP04; 250 ~g/ml herring sperm DNA;
50,ug/ml tRNA; or 0.25 M sodium phosphate buffer, pH 7.2, 1 mM EDTA, 7% SDS
hybridization temperature T = 65 to 68°C;
washing buffer 0.2 x SSC; 0.1 % SDS;
washing temperature T = 65 to 68°C.
s a Preferably, such promoters have a sequence identity of at least 30%, preferably of at least 40%, preferably of at least 50%, particularly preferably at least 60%, more particularly preferably of at least 70% and even more preferably of at least 80%, preferably at least 90% and most preferably at least 95% to the promoter sequence shown under SEQ ID No. 1 or parts thereof. The sequence identity of such promoter sequences is preferably determined by comparison with the nucleotide sequence shown under SEQ ID No. 1. If two sequences that are to be compared have a different length, the sequence identity preferably refers to the percentage proportion of the nucleotide residues of the shorter sequence, which are identical to the nucleotide residues of the longer sequence. The sequence identity can be determined conventionally by using computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, WI 53711 ).
Bestfit uses the local homology algorithm by Smith and Waterman, Advances in Applied Mathematics 2 (1981 ), 482-489 to find the segment with the highest sequence identity between two sequences. When Bestfit or another sequence alignment program is used to determine whether a specific sequence is, for example, 95%
identical to a reference sequence of the present invention, the parameters are preferably adjusted in such a way that the percentage proportion of the identity is calculated over the complete length of the reference sequence and that gaps of homology of up to 5% of the total number of nucleotides in the reference sequence are permitted. When Bestfit is used, the so-called optional parameters preferably keep their default values. The deviations which occur during comparison of a given sequence with the above-described sequences according to the invention, can be caused, for example, by addition, deletion, substitution, insertion or recombination.
Promoter sequences hybridizing, as described above, to the sequence shown under SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID
No. 6 or exhibiting a sequence identity to SEQ ID No. 1 are preferred to originate from plant organisms, preferably from higher plants, particularly preferably from dicotyledonous plants, most preferably from plants of the genus Lycopersicon.
In a preferred embodiment of the invention, the promoter according to the invention has the complete sequence shown under SEQ ID No. 3 (about 1.4 kb fragment), SEQ ID No. 4 (about 1.1 kb fragment), SEQ ID No. 5 (0.9 kb fragment) or SEQ ID
No. 6 (0.55 kb fragment).
Furthermore, the present invention also relates to promoters exhibiting a functional part of said sequences and causing a root-specific expression of a coding nucleotide sequence controlled by said promoters in plants.

a In a particularly preferred embodiment of the invention, the promoter according to the invention has the complete or a functional part of the sequence shown under SEQ ID
No. 4 (1.1 kb fragment).
Without considering to be bound to a certain theory, it is assumed that the promoter shown under SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No.
or SEQ ID No. 6 originates from a plant gene belonging to a group of extensin-like proteins.
Thus, the present invention also relates to promoters of genes encoding a protein, preferably from the group of extensin-like proteins and exhibiting a homology, i.e.
identity, of at least 60%, preferably of at least 70%, more preferably of at least 80%, particularly preferably of at least 90% and most preferably of at least 95% to the complete amino acid sequence depicted under SEQ ID No. 8, wherein these promoters cause a root-specific expression of a coding nuleotide sequence controlled by said promoters in plants.
In a particularly preferred Pmbodiment, the present invention relates to promoters of genes encoding a protein with an amino acid sequence indicated under SEQ ID
No.
8, wherein said promoters cause a root-specific expression of a coding nuleotide sequence controlled by said promoters in plants.
The nucleotide sequence depicted under SEQ ID No. 7 encodes a polypeptide (SEQ
ID No. 8) from L. esculentum which can be assumed to belong to the group of the extensin-like proteins.
In the context of the present invention, an extensin-like protein is a plant protein whose amino acid sequence exhibits at least one of the following sequence motifs:
SPPPPP, SPPPPYY, SPPPPY, SPPPPVY, PPPPPYY, PPPPPSY, PPPPPTY, PPPPPAY, PPPPPEY, PPPPPXY, SO000, SPPPPKH, SPPPPKK, SPPPPKKPYYPP, SPPPPSP, SPPPPSPKYVYK, SPPPPSPSPPPP, SPPPPYYYH, SPPPPYYYK, SOOOOTOVYK, SPPPPTPVYK, SOOOOVYK, SPPPPVYK, SPPPPVYSPPPP, SPPPPVHSPPPPVA, SPPPPVK, SPPPPVKSPPPP, SOOOOVKP.
Within the meaning of the present invention, in particular, plant proteins, exhibiting at least one of the SPPPP- and/or (P)PPPPYY-motifs contained in the SEQ ID No. 8 are called extensin-like proteins.
DNA sequences exhibiting homology to the sequence shown under SEQ ID No. 7 can be identified, for example, by means of computer-based sequence comparisons with known sequences or also by means of screening of, for example, cDNA or io s genomic libraries with the sequence depicted under SEQ ID No. 7 or parts thereof.
Such techniques are known to the person skilled in the art (see Sambrook et al., loc.
cit.). Computer-based sequence comparisons can also be carried out on the amino acid level with the amino acid sequence indicated in SEQ ID No. 8 or parts thereof.
The cell wall influences both the form and the function of the cell. The protein fraction of the cell wall contains both enzymes and structural proteins. The structural proteins of the cell wall that are best characterized include the so-called extensins (Cassab and Varner, Annu. Rev. Plant Physiol. Plant Mol. Biol. 39 (1988), 321-353;
Showalter, Plant Cell 5 (1993), 9-23). Extensins belong to the family of the hydroxyproline-rich glycoproteins (HRGPs). Genes and cDNAs encoding extensins could so far be characterized from carrot (Chen and Varner, EMBO J. 4 (1985), 2145-2151 ), bean (Corbin et al., Mol. Cell Biol. 7 (1987), 4337-4344), rape (Evans et al., Mol. Gen. Genet. 223 (1990), 273-287), tomato (Showalter et al., Plant Mol. Biol.
16 (1991 ), 547-565) and tobacco (Memelink et al., EMBO J. 6 (1987), 3579-3583).
The gene expression of the extensins is development-dependent and is more likely to be regulated tissue-specifically than constitutively (Sommer-Knudsen et al., Phytochemistry 47 (1998), 483-497; Ye et al., Plant Cell 3 (1991 ), 23-37).
During comparison of tissue specificity of different extensin genes of different plants, it is found that the expression pattern of extensin genes can clearly vary dependent on the species of the plant analyzed, on the cell type and on the tissue type (Showalter et al., Plant Mol. Biol. 19 (1992), 205-215).
In soy bean, HRGPs are expressed most intensively in meristematic cells. The HRGPnt3-gene from tobacco is expressed in the pericycle and in the endodermis, particularly in specific cells which participate in the formation of side roots (Keller et al., Proc. Nat. Acad. Sci. 86 (1989), 1529).
It has been described that the gene expression of extensins can be regulated by environmental factors such as light, pathogen infestation, wounding, heat stress (cf.
for example Cassab and Varner, Annu. Rev. Plant Physiol. Plant Mol. Biol. 39 (1988), 321-353; Cortin et al., Mol. Cell Biol. 7 (1987), 4337-4344; Niebel et al., Plant Cell 5 (1993), 1697-1710). This, however, does not hold true for all extensins (Sommer-Knudsen et al., Phytochemistry 47 (1998), 483-497).
The mature extensin protein is normally rich in hydroxyproline (Hyp), serine and specific combinations of the amino acids valine, tyrosine, lysine and histidine.
The primary structure of extensins (for a survey see, for example, Sommer-Knudsen, Phytochemistry 47 (1998), 483-497) is usually characterized by at least one Ser-Pro3_ 6-peptide unit which can also occur repeatedly or in connection with similar sequences such as the following sequence motifs SO000, SPPPPKH, SPPPPKK.
SPPPPKKPYYPP, SPPPPSP, SPPPPSPKYVYK, SPPPPSPSPPPP, SPPPPYYYH, m s SPPPPYYYK, SOOOOTOVYK, SPPPPTPVYK, SOOOOVYK, SPPPPVYK, SPPPPVYSPPPP, SPPPPVHSPPPPVA, SPPPPVK, SPPPPVKSPPPP, SOOOOVKP (cf. also Sommer-Knudsen et al., ibid.).
Extensins are subject to an intense post-translational modification. With carrot-extensin, for example, most proline residues are hydroxylated by prolyl-hydroxylases.
The hydroxyproline residues serve as target for glycosylations (Lamport et al., Biochem. J 133 (1972), 125-132; van Holst, Plant Physiol. 74 (1984), 247-251).
The carbohydrates, especially galactose and arabinose (Smith et al., Phytochemistry 25 (1986), pp. 1021; Holst et al., Plant Physiol. 74 (1984), 247; Smith et al., Phytochemistry 23 (1984), 1233) serve the stabilization of the proteins (Showalter, Plant Cell 5 (1993), 9-23). The arabinose mainly occurs in the form of Hyp-Ara~_4, the galactose in the form of Ser-Gal.
Furthermore, a linkage of various extensins via isodityrosine bonds has been discussed which, for example, can lead to a further strengthening of the cell wall as a reaction to pathogen attack (Brisson, Plant Cell 6 (1994), 1703-1712; Epstein, Phytochemistry 23 (1984), 1241-1246). Intramolecular isodityrosine bonds could be detected in extensins (Epstein et al., Phytochemistry, 23 (1984), pp. 1241 ).
Intermolecular isodityrosine bonds, however, could only be detected in vitro (Everdeen et al., Plant Physiol. 87 (1988), pp. 616; Huystee et al., Plant Phys.
Biochem. 33 (1995), pp. 55).
By means of the proline analogue 3,4-dehydro-L-proline (Dhp), the prolyl-hydroxylase can be selectively inhibited so that the biosynthesis of the hydroxyproline can be reduced by means of Dhp. After treatment of root slices of carrot (Daucus carota) with Dhp, it could be shown that they synthesize HRGPs being structurally modified. The treatment of tobacco protoplasts with Dhp also led to the regeneration of cell walls with a modified structure (Cooper, Plant Physiol. 104 (1994), 747-752).
The present invention further relates to expression cassettes containing a promoter according to the invention. The term "expression cassette" relates to the combination of a promoter according to the invention with a nucleic acid sequence to be expressed. This nucleic acid sequence may, for example, be a sequence encoding a polypeptide, i.e. a structural gene. It can be linked to the promoter in sense-or in antisense-orientation. The nucleic acid sequence can also encode a non-translatable RNA, for example an antisense-RNA or a ribozyme. These nucleic acid sequences can be used in connection with the promoter according to the invention to produce plants with a modified, preferably improved phenotype. Moreover, the metabolism of the plant in the root can be influenced by means of the promoter according to the invention. Some examples of the heterologous (over)expression and of antisense-inhibition with the aim to manipulate metabolism flows in transgenic plants have been summarized in Herbers and Sonnewald (TIBTECH 14 (1996), 198-205). An example of ribozymes was published by Feyter (Mol. Gen. Genet. 250 (1996), 329-228).
Various possible applications of transgenic plants which can be produced by means of the promoters and vectors according to the invention are also described in TIPTEC
Plant Product & Crop Biotechnology 13 (1995), 312-397.
The expression cassettes according to the invention can further contain a transcription termination sequence downstream of the 3' end of the nucleic acid sequence linked to the promoter. In this context, "transcription termination sequence"
is a DNA sequence which is located at the 3' end of a coding gene region and which is able to cause the termination of transcription and optionally the synthesis of a polyA-tail. An example of such a termination sequence is the one of the octopine synthase gene.
According to the invention, there can be one or more restriction sites between the promoter and the nucleic acid sequence and/or the nucleic acid sequence and the terminator.
Moreover, the present invention relates to vectors containing at least one promoter according to the invention.
In a preferred embodiment, the promoter according to the invention in such a vector is linked to a polylinker which allows for the integration of any sequences downstream of the promoter. In this context, "polylinker" is a DNA sequence containing the recognition sequences of at least one restriction enzyme, preferably of two or more restriction enzymes.
In a particularly preferred embodiment, the vector according to the invention further contains a sequence for the termination of the transcription, for example the one of the octopine synthase gene, downstream of the promoter and/or the polylinker.
The present invention also relates to vectors containing the expression cassettes according to the invention. Advantageously, the vectors according to the invention contain selection markers which are suitable to identify and optionally to select cells containing the vectors according to the invention.
In a preferred embodiment, the vectors according to the invention are suitable for the transformation of plant cells and particularly preferably for the integration of foreign DNA into the plant genome. Such vectors include, for example, binary vectors which partly are available commercially.

The present invention further relates to host cells genetically engineered with a promoter according to the invention or with an expression cassette or a vector according to the invention.
In this context, "genetically engineered" means that the host cells contain a promoter according to the invention or an expression cassette or a vector according to the invention, preferably stably integrated into the genome and the promoter or the expression cassette was introduced as foreign DNA into the host cell or in a predecessor of this cell. This means that the cells according to the invention themselves can be the direct product of a transformation event or cells descending therefrom which contain a promoter according to the invention or an expression cassette according to the invention. Both prokaryotic, particularly bacterial, as well as eukaryotic cells are possible as host cells. Eukaryotic cells can be, for example, fungal cells, in particular from the genus Saccharomyces, preferably from the species Saccharomyces cerevisiae.
In another embodiment, the invention relates to the use of vectors according to the invention, expression cassettes according to the invention or host cells according to the invention for the transformation of plants, plant cells, plant tissues or parts.
In a particularly preferred embodiment, the host cells according to the invention are plant cells which are called transgenic plant cells in the following.
Moreover, the present invention also relates to plants containing plant cells according to the invention. These can belong to any plant species, genus, family, order or class.
They may be both monocotyledonous and dicotyledonous plants. The plants according to the invention are preferred to be useful plants, i.e. plants which are of interest to humans as regards agriculture, forestry and/or horticulture. In this context, agricultural useful plants such as cereals (e.g. wheat, oat, barley, rye), maize, rice, potato, turnips, tobacco, sugar cane, sugar beet, sunflower, banana, rape or forage or pasture grasses (such as alfalfa, white clover, red clover), flax, cotton, soy, millet, bean, pea etc., vegetable plants (such as tomato, cucumber, courgette, aubergine, cabbage species, artichoke, chicory etc.), fruit trees, hop, wine and so on.
Also of interest are herbs and medicinal plants such as Catharanthus roseus, Datura stramonium, Taxus SSP.I, Dioscorea deltoidea, Papaver somniferum, Atropa belladonna, Rauwolfia serpentina, Hyoscyamus niger, Digitalis lanata, Datura metel, Digitalis purpurea, Pilocarpus jaborandi, Cinchona ledgeriana, Aconitum napellus.
In another embodiment, the present invention also relates to methods for the production of transgenic plants characterized in that plant cells, tissues or parts or w protoplasts are transformed with a vector according to the invention or with an expression cassette according to the invention or with a microorganism according to the invention, the transformed cells, tissues, parts of plants or protoplasts are cultivated in a growth medium and optionally plants are regenerated from the culture.
In another embodiment, the invention relates to the use of vectors according to the invention, expression cassettes according to the invention or host cells according to the invention for the production of transgenic hairy roots by means of Agrobacterium rhizogenes.
The plants according to the invention can be produced according to methods known to the person skilled in the art, for example by transformation of plant cells or tissue and regeneration of whole plants from the transformed cells and/or the tissue.
For the introduction of DNA in a plant host cell, there is a plurality of techniques at disposal. These techniques comprise the transformation of plant cells with T-DNA
using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of DNA by means of the biolistic approach as well as other possibilities.
Methods for the transformation of plant cells and plants, preferably dicotyledonous plants, preferably using the Agrobacterium-mediated transformation, have been analysed intensively and described sufficiently in EP 0 120 516; Hoekema (In:
The Binary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam (1985), chapter V); Fraley et al. (Crit. Rev. Plant Sci. 4 (1993), 1-46) and An et al.
(EMBO J.
4 (1985), 277-287). For the transformation of potato see e.g. Rocha-Sosa et al.
(EMBO J. 8 (1989), 29-33).
The transformation of monocotyledonous plants by means of Agrobacterium-based vectors has also been described (Chan et al., Plant Mol. Biol. 22 (1993), 491-506;
Hiei et al., Plant J. 6 (1994), 271-282; Deng et al., Science in China 33 (1990), 28-34;
Wilmink et al., Plant Cell Reports 11 (1992), 76-80; May et al., Bio/Technology 13 (1995), 486-492; Conner and Domisse, Int. J. Plant Sci. 153 (1992), 550-555;
Ritchie et al., Transgenic Res. 2 (1993), 252-265}. Alternative systems for the transformation of monocotyledonous plants are the transformation by means of the biolistic approach (Wan and Lemaux, Plant Physiol. 104 (1994), 37-48; Vasil et al., Bio/Technology 11 (1993), 1553-1558; Ritala et al., Plant Mol. Biol. 24 (1994), 317-325; Spencer et al., Theor. Appl. Genet. 79 (1990), 625-631 ), the protoplast transformation, the electroporation of partially permeabilized cells or the introduction of DNA by means of glass fibres. The transformation of maize, in particular, has been described in the literature various times (e.g. WO 95/06128, EP 0 513 849, EP

875, EP 0 292 435; Fromm et al., Biotechnology 8 (1990), 833-844; Gordon-Kamm et al., Plant Cell 2 (1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200;
Moroc et al., Theor. Appl. Genet. 80 (1990), 721-726).
The successful transformation of other cereals has also been described, e.g.
for barley (Wan and Lemaux, loc. cit.; Ritala et al., loc. cit.; Krens et al., Nature 296 (1982), 72-74) and for wheat (Nehra et al., Plant J. 5 (1994), 285-297).
Various transformation methods have been described for rice, e.g. the Agrobacterium-mediated transformation (Hiei et al., Plant J. 6 (1994), 271-282; Hiei et al., Plant Mol. Biol. 35 (1997), 205-218; Park et al., J. Plant Biol. 38 (1995), 365-371 ), the protoplast transformation (Datta, In: "Gene transfer to plants", Potrykus, Spangenberg (eds.), Springer-Verlag, Berlin, Heidelberg, 1995, 66-75; Datta et al., Plant Mol. Biol. 20 (1992), 619-629; Sadasivam et al., Plant Cell Rep. 13 (1994), 394-396), the biolistic approach for the plant transformation (Li et al., Plant Cell Rep. 12 (1993), 250-255; Cao et al., Plant Cell Rep. 11 (1992), 586-591; Christou, Plant Mol.
Biol. (1997), 197-203) as well as the electroporation (Xu et al., In: "Gene transfer to plants", Potrykus, Spangenberg (eds.), Springer-Verlag, Berlin, Heidelberg, 1995, 201-208).
Moreover, the present invention also relates to propagation and harvest material of plants according to the invention containing plant cells according to the invention. In this context, the term "propagation material" comprises those parts of the plant which are suitable for the production of descendants via the vegetative or generative way.
Suitable for the vegetative propagation are, for example, cuttings, callus cultures, rhizomes, root-stock or tubers. Further propagation material comprises, for example, fruits, seeds, seedlings, protoplasts, cell cultures etc. The propagation material is preferred to be tubers and seeds.
The present invention further relates to a method for the identification and isolation of promoters causing a root-specific expression of a coding nucleic acid sequence controlled by them in plants, comprising the following steps:
a) hybridizing of a plant genomic library with a cDNA coding for an extensin-like protein;
b) isolating of positive clones;
c) testing the isolated clones for promoter activity The hybridization carried out in step a) preferably takes place under stringent conditions. For this purpose, known sequences which encode an extensin-like protein or parts thereof are used for the hybridization with a corresponding library, preferably under stringent conditions. The sequence shown under SEQ ID No. 7 or parts thereof is/are preferred to be used. The methods are known to the person skilled in the art and are described in detail, e.g. in Sambrook et al. (loc.
cit.).
As described earlier, within the meaning of the present invention, an extensin-like protein is a protein whose amino acid sequence exhibits at least one of the sequence motifs SPPPPP, SPPPPYY, SPPPPY, SPPPPVY, PPPPPYY, PPPPPSY, PPPPPTY, PPPPPAY, PPPPPEY, PPPPPXY, SO000, SPPPPKH, SPPPPKK, SPPPPKKPYYPP, SPPPPSP, SPPPPSPKYVYK, SPPPPSPSPPPP, SPPPPYYYH, SPPPPYYYK, SOOOOTOVYK, SPPPPTPVYK, SOOOOVYK, SPPPPVYK, SPPPPVYSPPPP, SPPPPVHSPPPPVA, SPPPPVK, SPPPPVKSPPPP, SOOOOVKP and which exhibits a homology of at least 60%, preferably of at least 70%, preferably of at least 80%, particularly preferably of at least 90% and most particularly of at least 95% to the coding region indicated.under SEQ ID No.
7.
The identification of positive clones and isolation of the promoter sequence stated in step b) is carried out according to methods known to the person skilled in the art and are described, for example, by Sambrook et al. (loc. cit.).
The expression properties of the isolated promoter can be analyzed by means of reporter gene experiments. For the test of the isolated promoter sequence stated in step c) for promoter activity in root cells, the promoter can, for example in an expression cassette or a vector for plant transformation, be operatively linked to a reporter gene such as the f3-glucoronidase gene from E. coli. This construct is used for the transformation of plants. Subsequently, the organ-specific expression of the C3-glucuronidase is determined, as e.g. by Martin et al. (The GUS Reporter System as a Tool to Study Plant Gene Expression, In: GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression, Academic Press (1992), 23-43). The promoters which exhibit a root specificity are selected and then isolated.
In this context, an operative link is the sequential arrangement of promoter, coding sequence, terminator and optionally further regulatory elements with each of the elements mentioned being able to fulfil its function during gene expression in accordance with the requirements.
The present invention further relates to the use of the promoters according to the invention or of the promoters identified by means of the method according to the invention for the root-specific expression of transgenes in plants.
In this context, the term "transgene" means a DNA sequence artificially introduced into a plant.
m These and other embodiments are disclosed and obvious to the person skilled in the art and embraced by the description and the Examples of the present invention.
Additional literature regarding one of the methods, means and uses stated above which can be used within the meaning of the present invention can be obtained from the state of the art, e.g. from public libraries, e.g. using electronic aids.
For this purpose, public data bases, such as the "Medfine", which are accessible via the Internet, e.g. under the address http://www.ncbi.nlm.nih.gov/PubMed/medline.html, can be used. Further data bases and addresses are known to the person skilled in the art and can be obtained from the Internet, e.g. under the address http://www.lycos.com. A survey of sources and information with regard to patents and/or patent applications in biotechnology is given in Berks, TIBTECH 12 (1994), 352-364.
The Figures show:
Figure 1: Northern blot analysis of RNA in roots ,ug total RNA of stripped-off roots and root hairs and 10 ,ug total RNA of the tomato organs stated were applied per lane for the Northern blot analysis.
Hybridization of the RNA with radioactively labelled LeExtl cDNA or 25S rDNA cDNA. The transcript sizes are given on the right-hand side. 25S rDNA served as control for an even loading of the lanes and transfer onto the nylon membrane. Stripped-off roots are roots after root hair isolation with a clearly reduced number of root hairs.
The isolation of plant RNA was carried out according to the hot phenol extraction method by Verwoerd et al. (Nucleic Acids Research 17 (1989), 2362). The RNA was separated electrophoretically, transferred onto a nylon membrane and fixed thereon by UV radiation. After hybridizing and washing, the radioactive membrane was exposed on X-ray film. This film was developed after an overnight exposition at -80°C. The intensity of the signals correlates with the concentration of the specific mRNA on the membrane.
Figure 2: Northern blot analysis of the expression of LeExt1 l0,ug total RNA (5,ug in the case of lanes 3 and 4: 18 days and soil) of 7, 14 and 18 days old seedlings (7 days, 14 days, 18 days), roots grown on soil (soil), seedling roots which were incubated in the dark (-light) or in light (+light), and controls (control), as well as wounded leaves (wounding) were used for the RNA Northern blot analysis. Radioactively labelled LeExtl cDNA was used for the hybridization.
The isolation of plant RNA was carried out according to Verwoerd et al. (1989) (see above). The RNA was separated electrophoretically, transferred onto a nylon membrane and fixed thereon by UV radiation. After hybridizing and washing, the is radioactive membrane was exposed on X-ray film. This film was developed after an overnight exposition at -80°C. The intensity of the signals correlates with the concentration of the specific mRNA on the membrane.
Figure 3: In-situ detection of the LeExt1 mRNA
Localisation of the LeExt1 transcript in tomato roots. Light-microscopic pictures of young tomato roots under sterile conditions. A, B cuts imbedded in paraffin were hybridized to non-radioactively labelled LeExt1 anti-sense RNA, according to the in situ hybridizatiori method as described in Daram et al. (Planta 206 (1998), 225-223).
A Young seedling roots with growing root hairs in the differentiation zone of the root.
B root hair zone of a young seedling root. The root tip is located on the left. The size of the bar is 200 ,um.
The following Examples illustrate the invention.
Example 1: Isolation of a root-specific gene Plant material and extraction of root hairs Various organs of tomato plants Lycopersicon esculentum Mill. cv. Moneymaker grown on earth in the green house served as plant material. For the extraction of roots and root hairs, 8000 surface-sterilized seeds plated on a metal grid on paper (No. 0858, Schleicher & Schuell, Germany) in petri dishes under sterile conditions.
Prior to that, the paper was moistened in 0.5 x Hoagland's solution.
Germination took place at 22°C at a day/night cycle of 16h/8h. On day 3 after germination, the grid was lifted approximately 4 mm above the paper by pushing sterile glass beads between the grid and the paper. This allowed for vertical growth of the seedling roots and optimum root hair formation. On day 5, the seedlings were immersed with the grid in liquid nitrogen and the roots were stripped off the grid with a spatula. The root hairs were then brushed off the roots in liquid nitrogen and purified through filters over a 250,um sieve for analysis, as described in Rohm and Werner (Physiologia Plantarum 69 (1987), 129-136).
For the Northern blot analysis of seedling roots of different age, the seedlings were incubated over 7, 14 and 18 days, as described above, and the roots were isolated accordingly. For the isolation of hairy roots of plants grown on earth, plant pots containing densely grown seedlings from the green house were removed from the earth. The roots growing on the surface of the earth on the side were harvested with tweezers and immediately frozen in liquid nitrogen. For light/dark tests, seedlings were incubated in a day/night cycle of 16h/8h exposure, as described above. 7-day-old roots were harvested after 4-hour exposure after beginning of the day cycle. In parallel, seedlings were incubated during 7 days in the permanent dark and, subsequently, their roots were also harvested. Tomato leaves were wounded as described in Peria-Cortes et al. (Planta 186 (1992), 495-502) and harvested after 24 h. Non-wounded leaves were used as control.
RNA extraction and construction of a cDNA library Total RNA from the organs mentioned was extracted according to the method by Verwoerd et al. (Nucleic Acid Research 17 (1989), 2362). 7-60 ,ug total RNA
from root hairs was further used for the isolation of the poly(A)+ RNA by using magnetic oligodT beads (Dynabeads, Dynal, Germany). 700 ng poly(A)+ RNA was used for the production of double-stranded cDNA (Pharmacia, Germany). After attaching an EcoRllNotl linker, the cDNA was cloned into the expression vector AZAPII
(Stratagene, Germany). Packaging into the A phages took place using the Gigapack II Gold Packaging Extract Kit from Stratagene (Germany).
Differential screenina of the root hair-specific cDNA library 500,000 cDNAs packaged in phages were plated on YT agar in agar dishes and screened, according to a standard protocol (Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, 2"d edition, Cold Spring Harbour Press., Cold Spring Harbour, NY), with 680 ng radioactively labelled reverse-transcribed mRNA from either root hairs or brushed-off roots. 100 putative positive plaques were subjected to a second screening to avoid artefacts. Finally, 76 positive plaques were used for in vivo excision. The bacteria cultures resulting therefrom were shaken in a 96-well microtitre plate in 2 x YT medium (Sambrook et al., loc. cit.) at 28°C
overnight.
Plasmid DNA from these cultures was used for a second screening by using a kind of reverse Southern method, as described in Bucher et al. (Plant Molecular Biology 35 (1997), 497-508). After EcoRl digest of promising plasmids, the corresponding inserts were isolated and further used for the Northern blot analysis.
Northern blot analysis and DNA seauencina 5-10 Ng total RNA from the organs mentioned were loaded onto a 1.2% agarose glyoxal gel after glyoxylation (Hull (1985), Purification, biophysical and biochemical characterisation of viruses with special reference to plant viruses. In: Mahy, (ed.), Virology. A Practical Approach. IRL Press, Oxford, UK, pp. 1-24). Northern blot analysis and hybridization conditions were taken from standard protocols (Sambrook et al., loc. cit.). The blots were hybridized at 68°C. Detection of LeExt1 mRNA was achieved by using the LeExt1 cDNA as radioactive probe. Washing the blot for the last time took place using 0.1 x SSC at 68°C. After washing, the blots were exposed on X-ray film (Kodak, Germany) at -80°C. The transcript sizes were determined by comparison with glyoxylated marker DNA (BRL, Germany). The results are shown in Figures 1 and 2.
Sequencing of the LeExtt cDNA (=SEQ ID No. 7) took place by using dideoxy sequencing with T7 DNA polymerase (Amersham, Germany). Sequence analysis was carried out by means of the University of Wisconsin GCG Packet (Devereux et al., A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12 (1984), 387-395).
Example 2: Isolation of a genomic fragment comprising the promoter region of the root-specific gene A genomic DNA library from tomato (Clontech Laboratories, USA) was screened with radioactively labelled LeExtl cDNA (cf. Example 1 ). 500,000 plaque forming units (PFU) of the DNA libraries were plated onto YT agar in agar dishes, incubated for 6 to 8 hours at 37°C and transferred onto nylon membranes (Hybond N, Amersham, Germany) (Sambrook et al., see above). Hybridization conditions were taken from standard protocols (The specific activity Sambrook et al., see above). 30 plaques from the first screening were selected for a second round. The phage suspension of two plaques selected after the second screening were plated again to produce phage lysates. From these A DNA was prepared according to Lockett (Analytical Biochemistry 185 (1990), 230-234). 600 ng A DNA each was then digested with various restriction enzymes and the fragments resulting therefrom were made visible on an agarose gel using ethidium bromide. After Southern blot hybridization (Sambrook et al., see above) with radioactively labelled LeExt1 cDNA, at last, a genomic fragment with a length of approximately 3.4 kb was isolated which hybridized to LeExt1 cDNA and which was cloned into pBluescript II SK~ plasmid also digested with Asp718. First, the fragment was partially sequenced via dideoxy sequencing with T7 DNA polymerase (Amersham, Germany). It was found that the genomic fragment overlapped with the LeExt'1 cDNA sequence over 204 base pairs and further extended 5' upstream into the non-coding region of the LeExt1 gene.
Subsequently, both strands of the genomic fragment (approximately 3.4 kb) were sequenced (SEQ ID No. 13).

Example 3: Production of GUS expression cassettes for the analysis of the functionality of the promoter In total, three different GUS expression cassettes were produced.
A. Translational fusion of the 3.4 kb genomic fragment with a GUS cassette, wherein the fragment contains approximately 200 base pairs of the coding sequence of the LeExtl cDNA. The genomic fragment was excised by a Kpnl digest from the pBluescript II SK-plasmid, blunted by a filling reaction using the T7 DNA polymerase (Ausubel et al., Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York, 1998) and cloned into the binary vector pB1101.3 (Clontech, USA) which was digested with Smal and dephosphorylated. pB1101.3 is a plasmid containing a GUS cassette without a promoter in the binary vector pBINl9. This construct was designated rohl .
B. Transcriptional fusion of a modified genomic fragment with a GUS cassette.
A
genomic fragment with a length of 3200 by was synthesized which lacks the ORF
(open reading frame) which overlaps with the LeExtl cDNA using the oligonucleotides 42tnc3 (5'-GAGAGTCGACGATATCGGGCGAATTGGGTACC-3', SEQ ID No. 9) containing the Sall restriction site, and 42trx4 (5'-GAGATCTAGAGGTACCGGACTTTATATAACATAAC-3', SEQ ID No. 10) containing the Xbal restriction site by means of PCR (30 sec at 94°C melting of the DNA, 1 min at 60°C annealing of the primers, 1 min at 72°C with 3 sec extension per cycle DNA
synthesis by means of Taq DNA polymerase [Fermentas, Lithuania]. The fragment was digested with SaA and Xbal and cloned into the accordingly digested and dephosphorylated pBluescript II SK- plasmid. After isolation of the plasmid from an E. coli DHSa culture (Sambrook et al., see above), the fragment was isolated by SalllXbal digest and blunted by a filling reaction using T7 DNA polymerase. The end product was then cloned into the binary vector pBl 101.3 which was digested with Smal and deposphorylated. This construct was designated rohtnc.
C. A PCR product of approximately 3270 by was produced by means of the following primers:
42Xba (5'-GAGATCTAGACCATGGAGAAGAATTGG-3', SEQ ID No. 11) and 42Sa1 (5'-GAGAGTCGACGGGCGAAlTGGGTACCG-3', SEQ ID No. 12). The PCR conditions were: 20 sec at 94°C melting of the DNA, 45 sec at 50°C
annealing of primers, 2 min at 72°C DNA synthesis by means of Pfu DNA polymerase (Stratagene, Germany). The PCR product was directly cloned into the plasmid pCR-Script according to the manufacturer's instructions (Stratagene, Germany). After SaA/Noi1 digest, the ends of the PCR product were blunted with the Klenow enzyme [Sambrook, 1989 #68] and cloned in pBluescript II SK- plasmid which was digested with Smal and dephosphorylated.
After plasmid preparation (Sambrook et al., see above), the plasmid was digisted with BsflCl and linearized. By means of serial shortening of the PCR product via exonuclease III and S1 nuclease from the 5' end according to the manufacturer's protocol (Fermentas, Lithuania), shortened genomic fragments of approximately the following lenghts were produced: 2.2 kb, 1.7 kb, 1.4 kb, 1.1 kb, 0.9 kb and 0.55 kb. The AKT1-GUS-3'NOS
cassette from the plasmid 5'-AKT1-320.X (Lagarde et al., Plant J. 9 (1996), 195-203) was isolated using Sad, the ends were blunted by means of mung bean nuclease treatment (New Englands BioLabs Inc., Bioconcept, Switzerland) and afterwards digested with Ncol. The GUS-3'NOS cassette obtained in that way was cloned into NcollEcoRV
digested and dephosphorylated pBluescript II SK- plasmids containing the shortened genomic fragments. The promoter-GUS-3'NOS cassettes produced in that way were isolated by digest with Sad and Sall from each plasmid and cloned into the binary vector Bini 9 (Bevan et al., Nucleic Acids Research 12 (1984), 8711 ) that was Sad/SaA digested and dephosphorylated. The constructs obtained were designated BIN-Ogenx-GUS
with x indicating the corresponding length of the fragment 2.2 kb (SEQ ID No. 1), 1.7 kb (SEQ
ID No. 2), 1.4 kb (SEQ ID No. 3), 1.1 kb (SEQ ID No. 4), 0.9 kb (SEQ ID No. 5) or 0.55 kb (SEQ ID No. 6).
Example 4: Plant transformation The constructs described which contain the promoter fragments of various sizes were introduced, by means of electroporation, into the Agrobacterium strain containing the plasmid pGV2260 (Deblaere et al., Nucleic Acids Research 13 (1985), 4777-4788). The Agrobacteria were cultured on YEB medium (Vervliet et al., Journal of General Virology 26 (1975), 33-48). The transformation of tobacco and tomato plants was carried out in accordance with the method of the Agrobacterium tumefaciens-mediated gene transfer as described by Rosahl et al. (EMBO J. 6 (1987), 1155-1159) for tobacco and by Lillatti et al. (Biotechnology 5 (1987), 730) for tomato.
Furthermore, the transformation of potato was carried out as described in Rocha-Sosa et al. (EMBO J. 8 (1989), 23-29).
Maize was transformed as described by Omirulleh et al. (in "Gene Transfer to Plants", Potrykus, Spangenberg (eds.), Springer Verlag, Berlin, Heidelberg, 1995, pp. 99).
Moreover, the constructs described above were introduced into the A.
rhizogenes strain 15834 by electroporation (Jung et al., Biotechnol. Lett. 14 (1992), 695-700) and subsequently used for transforming Catharanthus roseus by means of Agrobacterium rhizogenes following the instructions in Toivonen et al. (Plant Cell Tissue Org. Cult.
18 (1988), pp. 79).

A strong expression of GUS could be detected in the hairy roots. Rice was transformed by means of particle bombardment as described, for instance, in Christou (Plant Mol. Biol. 35 (1997), 197-203).
Example 5: Histochemical localisation of the LeExt1 promoter activity The plant material was vacuum-infiltrated with an 0.1 % X-gluc solution (pre-solving in 0.1 g X-gluc in 1 ml dimethylformamide, adding 1 ml 10% Triton and 5 ml 1 M
sodium phosphate buffer, pH 7.2 and filling up with distilled water to obtain 100 ml) and incubated overnight at 37°C. After dyeing, the plants were fixed in ethanol : acetic acid (3:1 ) and decolorized in 100% ethanol. As a result, the green parts of the plants became colourless, whereas the blue colouring remained stable; cf. Fig. 3.
Example 6: Analysis of the specificity and the activity of the promoter For analyzing the specificity of the promoter, the GUS activity in the leaves of potato and tomato plants was determined in comparison with the activity in roots according to the method by Jefferson et al. (EMBO J. 6 (1987), 3901-3907).
In tomato, the GUS activity in the roots is between 20 and 300 times higher than the activity in mature leaves. Most of the GUS-positive potato plants that were examined had a high GUS activity in the roots but no GUS activity in mature leaves.
For determining the activity of the promoter, the GUS activities were compared to the activities of the 35S promoter. The lines with the highest activity of the promoter exhibited an activity that was approximately half of the activity of the CaMV

promoter.

SEQUENCE LISTING
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<141>
<160> 13 <170> PatentIn Ver. 2.1 <210> 1 <211> 2205 <212> DNA
<213> Lycopersicon esculentum <400> 1 aaagttatta acacaacttc catcacatta aagacatcat ttgataacat tcatgtcagc 60 atacttatac taaagttgac gggcttcaat ataaaattaa taaggataaa ttttaagaat 120 cacatagttt ttaactatga aattacaatc tattcgtctt tagtttgtaa ttacattaat 180 accttaaaaa gcaataaaaa tcgattatat taatatgcag cttgaataca ttaaagagta 240 tctcagatac attacaatat aaatcgggta aataatatgt agctcggata cattatatta 300 ataagtagct tatatacaat aaagaaataa gagattttag aggtttatag aaatagaagg 360 gaatattgaa aataaggaaa agaaaggtgt gtatttaagt attttttcca attaagaata 420 catttttcac gaaagaggtg ttcaagccaa atgaaagaag attgggccac ccaaaaccca 480 ataaaagagt ctaaaaagct gaacgcttta agtcaagagt tcctatttta aaagtttcct 540 agtttaaaaa gtttgttagt ttaaaaagtt tcctagttaa agtttttcag ctttcttagt 600 ttaatgtttc ctagtttaaa agttttcata aaagtaggat ttaaatcgag gaaaggctca 660 aaataatctc tcatctttag gttaagactc aaagtgatcc ttaagttttc acatggagca 720 ctagtaatct atcatgtttg caatattggt acacttttgg tcctaaaaaa actctaacat 780 acttcaaaaa taggtaacta agaataatac tattgatata ttacgcaaaa taatcgatac 840 aatttgtcta atctgtttga atttaaaaga agaaaaataa attgattgtt atagttataa 900 acatatgatt cctgagagat aaaagaaaca aaaggaatcg tagtgaatca ttttaatcaa 960 ataaaaagaa aatcgaagat aaatgttcca caaagataaa atatcacgat ttttttcata 1020 agcccttcaa tattaatcat ttagtgattg tttaggtgta tttgatttat attgcagaag 1080 ttttaagctc taagtatgaa tttttttaat atcggaactt ctaaaatatt ttcagaaaat 1140 tttcaaaata atttgatcta ctcaattttt ttccggtggg gttcgatatt cgggaatgaa 1200 gtaatatctc atacaacact tttttctcaa gactagatca tacgggacac aaatttgaat 1260 tattataatt aataaccaaa aatatgaaaa tatgacatta aaaacaatag cgtctaatta 1320 ttcccgttgg tatcattaac taatcaaacc caaaatattg tcattcttgg tgatgaaaag 1380 tatgtatggt actcgcaagg gtacgcaaaa taatcgatac aatttgtcta atctgtttga 1440 atttaaaaga agaaaaataa attgattgct attgttataa acatatgatt cctgagagat 1500 aaaagaaata aagcgaatcg tagtgaatca ttttaatcaa ataaaaacaa actcgtagat 1560 aaatgttcca caaagataaa atattacgat ttttttcata agcccttcaa tattcagtga 1620 atcgatttct tctatcaacg ttttttcagt tgatttgtca caacggagtt gctcaaagca 1680 gcttttatat gatttgcaag taaatgcaca tgagcaattt atcggcgggc acccgaagaa 1740 tagcttaccc atttattttt ttaaaaaaag attaagtaca ataccatgat gtggattgta 1800 agttgtgctc aacaagtaca aataattaat cgacaccaaa taatggacag tatttgttaa 1860 gcctacacat atctcaactt ttaaatatta attttatcaa tttttcacaa ccaaaagaaa 1920 tgaacaaaca acattcttgc aagtcaacaa ataatcgatt caaagtttag aaataggtga 1980 gtcaagcaaa tgtgtatgaa tagtttatga cttcccatta tctcaaaacc aaccttagtg 2040 gaacagcatt aaccaaaaaa cggctgatat gtttggatta tttaatttct aatttatcaa 2100 atcacatgtt agttatgtta tataaagtcc taattcctcc attctaaaac acacaagaaa 2160 aagaaaaaca aagatatatc tagccttaga atccaattct tctcc 2205 <210> 2 <211> 1727 <212> DNA
<213> Lycopersicon esculentum <400> 2 caataaaaga gtctaaaaag ctgaacgctt taagtcaaga gttcctattt taaaagtttc 60 ctagtttaaa aagtttgtta gtttaaaaag tttcctagtt aaagtttttc agctttctta 120 gtttaatgtt tcctagttta aaagttttca taaaagtagg atttaaatcg aggaaaggct 180 caaaataatc tctcatcttt aggttaagac tcaaagtgat ccttaagttt tcacatggag 240 cactagtaat ctatcatgtt tgcaatattg gtacactttt ggtcctaaaa aaactctaac 300 atacttcaaa aataggtaac taagaataat actattgata tattacgcaa aataatcgat 360 acaatttgtc taatctgttt gaatttaaaa gaagaaaaat aaattgattg ttatagttat 420 aaacatatga ttcctgagag ataaaagaaa caaaaggaat cgtagtgaat cattttaatc 480 aaataaaaag aaaatcgaag ataaatgttc cacaaagata aaatatcacg atttttttca 540 taagcccttc aatattaatc atttagtgat tgtttaggtg tatttgattt atattgcaga 600 agttttaagc tctaagtatg aattttttta atatcggaac ttctaaaata ttttcagaaa 660 attttcaaaa taatttgatc tactcaattt ttttccggtg gggttcgata ttcgggaatg 720 aagtaatatc tcatacaaca cttttttctc aagactagat catacgggac acaaatttga 780 attattataa ttaataacca aaaatatgaa aatatgacat taaaaacaat agcgtctaat 840 tattcccgtt ggtatcatta actaatcaaa cccaaaatat tgtcattctt ggtgatgaaa 900 agtatgtatg gtactcgcaa gggtacgcaa aataatcgat acaatttgtc taatctgttt 960 gaatttaaaa gaagaaaaat aaattgattg ctattgttat aaacatatga ttcctgagag 1020 ataaaagaaa taaagcgaat cgtagtgaat cattttaatc aaataaaaac aaactcgtag 1080 ataaatgttc cacaaagata aaatattacg atttttttca taagcccttc aatattcagt 1140 gaatcgattt cttctatcaa cgttttttca gttgatttgt cacaacggag ttgctcaaag 1200 cagcttttat atgatttgca agtaaatgca catgagcaat ttatcggcgg gcacccgaag 1260 aatagcttac ccatttattt ttttaaaaaa agattaagta caataccatg atgtggattg 1320 taagttgtgc tcaacaagta caaataatta atcgacacca aataatggac agtatttgtt 1380 aagcctacac atatctcaac ttttaaatat taattttatc aatttttcac aaccaaaaga 1440 aatgaacaaa caacattctt gcaagtcaac aaataatcga ttcaaagttt agaaataggt 1500 gagtcaagca aatgtgtatg aatagtttat gacttcccat tatctcaaaa ccaaccttag 1560 tggaacagca ttaaccaaaa aacggctgat atgtttggat tatttaattt ctaatttatc 1620 aaatcacatg ttagttatgt tatataaagt cctaattcct ccattctaaa acacacaaga 1680 aaaagaaaaa caaagatata tctagcctta gaatccaatt cttctcc 1727 <210> 3 <211> 1357 <212> DNA
<213> Lycopersicon esculentum <400> 3 taatctgttt gaatttaaaa gaagaaaaat aaattgattg ttatagttat aaacatatga 60 ttcctgagag ataaaagaaa caaaaggaat cgtagtgaat cattttaatc aaataaaaag 120 aaaatcgaag ataaatgttc cacaaagata aaatatcacg atttttttca taagcccttc 180 aatattaatc atttagtgat tgtttaggtg tatttgattt atattgcaga agttttaagc 240 tctaagtatg aattttttta atatcggaac ttctaaaata ttttcagaaa attttcaaaa 300 taatttgatc tactcaattt ttttccggtg gggttcgata ttcgggaatg aagtaatatc 360 tcatacaaca cttttttctc aagactagat catacgggac acaaatttga attattataa 420 ttaataacca aaaatatgaa aatatgacat taaaaacaat agcgtctaat tattcccgtt 480 ggtatcatta actaatcaaa cccaaaatat tgtcattctt ggtgatgaaa agtatgtatg 540 gtactcgcaa gggtacgcaa aataatcgat acaatttgtc taatctgttt gaatttaaaa 600 gaagaaaaat aaattgattg ctattgttat aaacatatga ttcctgagag ataaaagaaa 660 taaagcgaat cgtagtgaat cattttaatc aaataaaaac aaactcgtag ataaatgttc 720 cacaaagata aaatattacg atttttttca taagcccttc aatattcagt gaatcgattt 780 cttctatcaa cgttttttca gttgatttgt cacaacggag ttgctcaaag cagcttttat 840 atgatttgca agtaaatgca catgagcaat ttatcggcgg gcacccgaag aatagcttac 900 ccatttattt ttttaaaaaa agattaagta caataccatg atgtggattg taagttgtgc 960 tcaacaagta caaataatta atcgacacca aataatggac agtatttgtt aagcctacac 1020 atatctcaac ttttaaatat taattttatc aatttttcac aaccaaaaga aatgaacaaa 1080 caacattctt gcaagtcaac aaataatcga ttcaaagttt agaaataggt gagtcaagca 1140 aatgtgtatg aatagtttat gacttcccat tatctcaaaa ccaaccttag tggaacagca 1200 ttaaccaaaa aacggctgat atgtttggat tatttaattt ctaatttatc aaatcacatg 1260 ttagttatgt tatataaagt cctaattcct ccattctaaa acacacaaga aaaagaaaaa 1320 caaagatata tctagcctta gaatccaatt cttctcc 1357 <210> 4 <211> 1121 <212> DNA
<213> Lycopersicon esculentum <400> 4 aagctctaag tatgaatttt tttaatatcg gaacttctaa aatattttca gaaaattttc 60 aaaataattt gatctactca atttttttcc ggtggggttc gatattcggg aatgaagtaa 120 tatctcatac aacacttttt tctcaagact agatcatacg ggacacaaat ttgaattatt 180 ataattaata accaaaaata tgaaaatatg acattaaaaa caatagcgtc taattattcc 240 cgttggtatc attaactaat caaacccaaa atattgtcat tcttggtgat gaaaagtatg 300 tatggtactc gcaagggtac gcaaaataat cgatacaatt tgtctaatct gtttgaattt 360 aaaagaagaa aaataaattg attgctattg ttataaacat atgattcctg agagataaaa 420 gaaataaagc gaatcgtagt gaatcatttt aatcaaataa aaacaaactc gtagataaat 480 gttccacaaa gataaaatat tacgattttt ttcataagcc cttcaatatt cagtgaatcg 540 atttcttcta tcaacgtttt ttcagttgat ttgtcacaac ggagttgctc aaagcagctt 600 ttatatgatt tgcaagtaaa tgcacatgag caatttatcg gcgggcaccc gaagaatagc 660 ttacccattt atttttttaa aaaaagatta agtacaatac catgatgtgg attgtaagtt 720 gtgctcaaca agtacaaata attaatcgac accaaataat ggacagtatt tgttaagcct 780 acacatatct caacttttaa atattaattt tatcaatttt tcacaaccaa aagaaatgaa 840 caaacaacat tcttgcaagt caacaaataa tcgattcaaa gtttagaaat aggtgagtca 900 agcaaatgtg tatgaatagt ttatgacttc ccattatctc aaaaccaacc ttagtggaac 960 agcattaacc aaaaaacggc tgatatgttt ggattattta atttctaatt tatcaaatca 1020 catgttagtt atgttatata aagtcctaat tcctccattc taaaacacac aagaaaaaga 1080 aaaacaaaga tatatctagc cttagaatcc aattcttctc c 1121 <210> 5 <211> 891 <212> DNA
<213> Lycopersicon esculentum <400> 5 taattattcc cgttggtatc attaactaat caaacccaaa atattgtcat tcttggtgat 60 gaaaagtatg tatggtactc gcaagggtac gcaaaataat cgatacaatt tgtctaatct 120 gtttgaattt aaaagaagaa aaataaattg attgctattg ttataaacat atgattcctg 180 agagataaaa gaaataaagc gaatcgtagt gaatcatttt aatcaaataa aaacaaactc 240 gtagataaat gttccacaaa gataaaatat tacgattttt ttcataagcc cttcaatatt 300 cagtgaatcg atttcttcta tcaacgtttt ttcagttgat ttgtcacaac ggagttgctc 360 aaagcagctt ttatatgatt tgcaagtaaa tgcacatgag caatttatcg gcgggcaccc 420 gaagaatagc ttacccattt atttttttaa aaaaagatta agtacaatac catgatgtgg 480 attgtaagtt gtgctcaaca agtacaaata attaatcgac accaaataat ggacagtatt 540 tgttaagcct acacatatct caacttttaa atattaattt tatcaatttt tcacaaccaa 600 aagaaatgaa caaacaacat tcttgcaagt caacaaataa tcgattcaaa gtttagaaat 660 aggtgagtca agcaaatgtg tatgaatagt ttatgacttc ccattatctc aaaaccaacc 720 ttagtggaac agcattaacc aaaaaacggc tgatatgttt ggattattta atttctaatt 780 tatcaaatca catgttagtt atgttatata aagtcctaat tcctccattc taaaacacac 840 aagaaaaaga aaaacaaaga tatatctagc cttagaatcc aattcttctc c 891 <210> 6 <211> 575 <212> DNA
<213> Lycopersicon esculentum <400> 6 tctatcaacg ttttttcagt tgatttgtca caacggagtt gctcaaagca gcttttatat 60 gatttgcaag taaatgcaca tgagcaattt atcggcgggc acccgaagaa tagcttaccc 120 atttattttt ttaaaaaaag attaagtaca ataccatgat gtggattgta agttgtgctc 180 aacaagtaca aataattaat cgacaccaaa taatggacag tatttgttaa gcctacacat 240 atctcaactt ttaaatatta attttatcaa tttttcacaa ccaaaagaaa tgaacaaaca 300 acattcttgc aagtcaacaa ataatcgatt caaagtttag aaataggtga gtcaagcaaa 360 tgtgtatgaa tagtttatga cttcccatta tctcaaaacc aaccttagtg gaacagcatt 420 aaccaaaaaa cggctgatat gtttggatta tttaatttct aatttatcaa atcacatgtt 480 agttatgtta tataaagtcc taattcctcc attctaaaac acacaagaaa aagaaaaaca 540 aagatatatc tagccttaga atccaattct tctcc 575 <210> 7 <211> 1420 <212> DNA
<213> Lycopersicon esculentum <220>
<221> CDS
<222> (256)~.(1197) <400> 7 taaacaaaga tatatctagc cttagaatcc aattcttctc aatgggaagc caaataaaga 60 agcaatggct tcaatttgct tgtcttttga catttttctt gattgccaca tgctctatgg 120 catattcacc ttacaattct tatgaatcat cagactcaac atataataaa gtaccaacca 180 cagtagtcaa aagtgaagac ttcaaggtac cctcagagtc ggaaaaggaa tataagtcgt 240 catttttgcc aaaaa atg att act ata aga agc cat caa ttt cag agg ata 291 Met Ile Thr Ile Arg Ser His Gln Phe Gln Arg Ile actataaga aagtatcat ttgttcccg aacatgaat cattcctgc caa 339 ThrIleArg LysTyrHis LeuPhePro AsnMetAsn HisSerCys Gln agaatgact actacaaga agccattat tttcggaag ataactaca aga 387 ArgMetThr ThrThrArg SerHisTyr PheArgLys IleThrThr Arg aggagtcat atgttcaag aggtaccct cgaaggcta aaccagaat ata 935 ArgSerHis MetPheLys ArgTyrPro ArgArgLeu AsnGlnAsn Ile aggagtcat ttttttcaa aatttgact acttcaaga agccatcat ttt 483 ArgSerHis PhePheGln AsnLeuThr ThrSerArg SerHisHis Phe tcgaagaca actacaaga acgacgtca tatgttcca aaggtaccc tcg 531 SerLysThr ThrThrArg ThrThrSer TyrValPro LysValPro Ser atg get aaa cca gaa tat aag gag tca ttt ttt cca aaa ttt gac tac 579 Met Ala Lys Pro Glu Tyr Lys Glu Ser Phe Phe Pro Lys Phe Asp Tyr tttaagaagcca tcagtt tcagaagat aactacaag aagacgtca tat 627 PheLysLysPro 5erVal SerGluAsp AsnTyrLys LysThrSer Tyr gtttcagaggtg ccctcg atggetaaa ccagaatat aaggagtca ttt 675 ValSerGluVal ProSer MetAlaLys ProGluTyr LysGluSer Phe tttccaaaattt gactac tttaagaag tcattaget cctgaagat aaa 723 PheProLysPhe AspTyr PheLysLys SerLeuAla ProGluAsp Lys tacaagaaggcg ccatat gttccagag gtatccaca gagcctaaa ccg 771 TyrLysLysAla ProTyr ValProGlu ValSerThr GluProLys Pro gaa tac aag gta cca tct ttg cca aag aat gac tac tat aag aag cca 819 Glu Tyr Lys Val Pro Ser Leu Pro Lys Asn Asp Tyr Tyr Lys Lys Pro aca att cca gaa gat aac tat aaa aag gtg tca tat gtt tca aag gtg 867 Thr Ile Pro Glu Asp Asn Tyr Lys Lys Val Ser Tyr Val Ser Lys Val ccc tca gtg cct aaa gaa gaa tac aag gca cct act ttg cca aag aat 915 Pro Ser Val Pro Lys Glu Glu Tyr Lys Ala Pro Thr Leu Pro Lys Asn gat tac tac aag aag cca tca gtt caa gaa gaa aac tac aaa aag gta 963 Asp Tyr Tyr Lys Lys Pro Ser Val Gln Glu Glu Asn Tyr Lys Lys Val cca ctt att tca aag ctg ccc tca gtg cct aaa gaa gaa tac aag gtg 1011 Pro Leu Ile Ser Lys Leu Pro Ser Val Pro Lys Glu Glu Tyr Lys Val cct tct ttg tca aaa aaa gac tac tac aag aag cca tta gtt tct gaa 1059 Pro Ser Leu Ser Lys Lys Asp Tyr Tyr Lys Lys Pro Leu Val Ser Glu gat aac tac aaa aag gtt tca tat gtt cca aag gtg ccc tcc gtg cct 1107 Asp Asn Tyr Lys Lys Val Ser Tyr Val Pro Lys Val Pro Ser Val Pro aaa gaa gaa tac aag gcc cct tct ttg tca aag aat gac tac tac aag 1155 Lys Glu Glu Tyr Lys Ala Pro Ser Leu Ser Lys Asn Asp Tyr Tyr Lys aag tca tcg cct tct cca tca cca cca cca cct cca tat tat 1197 Lys 5er Ser Pro Ser Pro Ser Pro Pro Pro Pro Pro Tyr Tyr taaatttctt ttcatgttaa gcatggctgt ttaattaaag tccttctcca gctcaacatt 1257 tataactcat ggagttctca tattatgagt tagaaggcat gatataaaaa tttgcattta 1317 tttatttatt tttccttttc ctaccttggt ttgtaattga tttgtcattt aatttctata 1377 ttttgtatga gaaatttatt tcaattgaat acaagttttt tta 1420 <210> B
<211> 314 <212> PRT
<213> Lycopersicon esculentum <400> 8 Met Ile Thr Ile Arg Ser His Gln Phe Gln Arg Ile Thr Ile Arg Lys Tyr His Leu Phe Pro Asn Met Asn His Ser Cys Gln Arg Met Thr Thr Thr Arg Ser His Tyr Phe Arg Lys Ile Thr Thr Arg Arg Ser His Met Phe Lys Arg Tyr Pro Arg Arg Leu Asn Gln Asn Ile Arg Ser His Phe Phe Gln Asn Leu Thr Thr Ser Arg Ser His His Phe Ser Lys Thr Thr Thr Arg Thr Thr Ser Tyr Val Pro Lys Val Pro Ser Met Ala Lys Pro Glu Tyr Lys Glu Ser Phe Phe Pro Lys Phe Asp Tyr Phe Lys Lys Pro Ser Val Ser Glu Asp Asn Tyr Lys Lys Thr Ser Tyr Val Ser Glu Val Pro Ser Met Ala Lys Pro Glu Tyr Lys Glu Ser Phe Phe Pro Lys Phe Asp Tyr Phe Lys Lys 5er Leu Ala Pro Glu Asp Lys Tyr Lys Lys Ala Pro Tyr Val Pro Glu Val Ser Thr Glu Pro Lys Pro Glu Tyr Lys Val Pro Ser Leu Pro Lys Asn Asp Tyr Tyr Lys Lys Pro Thr Ile Pro Glu Asp Asn Tyr Lys Lys Val Ser Tyr Val Ser Lys Val Pro Ser Val Pro Lys Glu Glu Tyr Lys Ala Pro Thr Leu Pro Lys Asn Asp Tyr Tyr Lys Lys Pro Ser Val Gln Glu Glu Asn Tyr Lys Lys Val Pro Leu Ile Ser Lys Leu Pro Ser Val Pro Lys Glu Glu Tyr Lys Val Pro Ser Leu Ser Lys Lys Asp Tyr Tyr Lys Lys Pro Leu Val Ser Glu Asp Asn Tyr Lys Lys Val Ser Tyr Val Pro Lys Val Pro Ser Val Pro Lys Glu Glu Tyr Lys Ala Pro Ser Leu Ser Lys Asn Asp Tyr Tyr Lys Lys Ser Ser Pro Ser Pro Ser Pro Pro Pro Pro Pro Tyr Tyr <210> 9 <211> 32 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: artificial <400> 9 gagagtcgac gatatcgggc gaattgggta cc 32 <210> 10 <211> 35 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: artificial <400> 10 gagatctaga ggtaccggac tttatataac ataac 35 <210> 11 <211> 27 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: artificial <400> 11 gagatctaga ccatggagaa gaattgg 27 <210> 12 <211> 27 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: artificial <400> 12 gagagtcgac gggcgaattg ggtaccg 27 <210> 13 <211> 3259 <212> DNA
<213> Lycopersicon esculentum <400> 13 ggtaccattg tttctccatc aggccccttt ttcataggaa tagtaggacc atctaagact 60 acactcaaga gatcaggatt ttctcctatc agatgatcca tcaagcgatt cttccaccat 120 ccatagtatt ttccattgaa caatgggggc cgagtttgag aagctccttc ttggggggta 180 ggtggtgctg ccatttaatt ttcaagatgt taatctttta gtgaaaagat cctgctctga 240 taccaattga agaaacctta cccctaaatc aagaaccagg ttcttgattg tttgcctaag 300 aaaagagtta attaaaacaa tatactttaa ctcaaacctt cctatctcaa cgaaggaaaa 360 actcaacttc gttttattac tttttcttac tcaataataa ttttcgatta actctaatcg 420 aattctctaa atttacaaaa agccaaaccc acttcttgca aacttactca ctaatctcct 480 ctctccacaa gaagccaaac ccacttcttg tttacaatca cacaagctta agatcttact 540 aagaacaaag ctcacaagtt aacaaaatac acagaactta gaaaaacgct ctttgctgcc 600 tctctctttg tcttacctca ccctttcttt cattgcaaag aactgctctc tttgtcttga 660 aatctctgtt tatatagact tttgaacaag agttatttcc tctctttgaa gttgataata 720 attagatttc tgtatccttc tttgatatga aaaagaattg ctttcttttt tctgtttata 780 cgcaaccttt tttgaaaagg ttttgtcttc atataaagta gacaactttt tcagaaaaga 840 ttttatctta tttatttatg gaaataatta ttatattcat ttttttcatc aatacatttg 900 tgccttaata tttgtccttc gcctcttttc aattagaacg acaactgctt taataaatga 960 gcctggttca taagtgagtc aacatcaaaa gtatttgtca ttatcaatga acctggttca 1020 tgattgatga catcaaaagt atttgtcatt ataaaaagtt attaacacaa cttccatcac 1080 attaaagaca tcatttgata acattcatgt cagcatactt atactaaagt tgacgggctt 1140 caatataaaa ttaataagga taaattttaa gaatcacata gtttttaact atgaaattac 1200 aatctattcg tctttagttt gtaattacat taatacctta aaaagcaata aaaatcgatt 1260 atattaatat gcagcttgaa tacattaaag agtatctcag atacattaca atataaatcg 1320 ggtaaataat atgtagctcg gatacattat attaataagt agcttatata caataaagaa 1380 ataagagatt ttagaggttt atagaaatag aagggaatat tgaaaataag gaaaagaaag 1490 gtgtgtattt aagtattttt tccaattaag aatacatttt tcacgaaaga ggtgttcaag 1500 ccaaatgaaa gaagattggg ccacccaaaa cccaataaaa gagtctaaaa agctgaacgc 1560 tttaagtcaa gagttcctat tttaaaagtt tcctagttta aaaagtttgt tagtttaaaa 1620 agtttcctag ttaaagtttt tcagctttct tagtttaatg tttcctagtt taaaagtttt 1680 cataaaagta ggatttaaat cgaggaaagg ctcaaaataa tctctcatct ttaggttaag 1740 actcaaagtg atccttaagt tttcacatgg agcactagta atctatcatg tttgcaatat 1800 tggtacactt ttggtcctaa aaaaactcta acatacttca aaaataggta actaagaata 1860 atactattga tatattacgc aaaataatcg atacaatttg tctaatctgt ttgaatttaa 1920 aagaagaaaa ataaattgat tgttatagtt ataaacatat gattcctgag agataaaaga 1980 aacaaaagga atcgtagtga atcattttaa tcaaataaaa agaaaatcga agataaatgt 2040 tccacaaaga taaaatatca cgattttttt cataagccct tcaatattaa tcatttagtg 2100 attgtttagg tgtatttgat ttatattgca gaagttttaa gctctaagta tgaatttttt 2160 taatatcgga acttctaaaa tattttcaga aaattttcaa aataatttga tctactcaat 2220 ttttttccgg tggggttcga tattcgggaa tgaagtaata tctcatacaa cacttttttc 2280 tcaagactag atcatacggg acacaaattt gaattattat aattaataac caaaaatatg 2340 aaaatatgac attaaaaaca atagcgtcta attattcccg ttggtatcat taactaatca 2400 aacccaaaat attgtcattc ttggtgatga aaagtatgta tggtactcgc aagggtacgc 2460 aaaataatcg atacaatttg tctaatctgt ttgaatttaa aagaagaaaa ataaattgat 2520 tgctattgtt ataaacatat gattcctgag agataaaaga aataaagcga atcgtagtga 2580 atcattttaa tcaaataaaa acaaactcgt agataaatgt tccacaaaga taaaatatta 2640 cgattttttt cataagccct tcaatattca gtgaatcgat ttcttctatc aacgtttttt 2700 cagttgattt gtcacaacgg agttgctcaa agcagctttt atatgatttg caagtaaatg 2760 cacatgagca atttatcggc gggcacccga agaatagctt acccatttat ttttttaaaa 2820 aaagattaag tacaatacca tgatgtggat tgtaagttgt gctcaacaag tacaaataat 2880 taatcgacac caaataatgg acagtatttg ttaagcctac acatatctca acttttaaat 2940 attaatttta tcaatttttc acaaccaaaa gaaatgaaca aacaacattc ttgcaagtca 3000 acaaataatc gattcaaagt ttagaaatag gtgagtcaag caaatgtgta tgaatagttt 3060 atgacttccc attatctcaa aaccaacctt agtggaacag cattaaccaa aaaacggctg 3120 atatgtttgg attatttaat ttctaattta tcaaatcaca tgttagttat gttatataaa 3180 gtcctaattc ctccattcta aaacacacaa gaaaaagaaa aacaaagata tatctagcct 3240 tagaatccaa ttcttctca 3259

Claims (14)

Claims:

1. A promoter selected from the group consisting of a) promoters comprising the nucleic acid sequence indicated under SEQ ID
No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6;
b) promoters comprising a functional part of the nucleic acid sequence indicated under SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID
No. 4, SEQ ID No. 5 or SEQ ID No. 6 and causing a root-specific expression of a coding nucleic acid sequence controlled by them in plants;
c) promoters having a sequence which hybridizes with the one shown under SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ
ID No. 5 or SEQ ID No. 6 and causing a root-specific expression of a coding nucleotide sequence controlled by them in plants; and d) promoters of genes encoding a protein whose amino acid sequence exhibits a homology of at least 60% to the amino acid sequence indicated under SEQ ID No. 8, wherein these promoters cause a root-specific expression of a coding nucleotide sequence controlled by them in plants.

2. The promoter according to claim 1, which is a plant promoter.

3. Expression cassettes containing a promoter according to claim 1 or 2.

4. A vector containing a promoter according to claim 1 or 2 or an expression cassette according to claim 3.

5. The vector according to claim 4, which is suitable for the transformation of plant cells.

6. A host cell genetically engineered with a promoter according to claim 1 or 2, with an expression cassette according to claim 3 or a vector according to claim 4 or 5.

7. The host cell according to claim 6 which is a plant cell.

8. Hairy roots containing plant cells according to claim 7.

9. A plant containing plant cells according to claim 7.

10. Propagation or harvest material of plants according to claim 9 containing plant cells according to claim 7.

11. A method for the production of transgenic plants according to claim 9 characterized in that the plant cells, tissues or parts or protoplasts are transformed with a vector according to claim 4 or 5 or with an expression cassette according to claim 3 or with a host cell according to claim 6, the transformed cells, tissue, plant parts or protoplasts are cultivated in a growth medium and optionally plants are regenerated from the culture.

12. A method for the identification and isolation of promoters causing a root-specific expression of a coding nucleotide sequence controlled by them, comprising the following steps:
(a) hybridizing of a plant genomic library with a cDNA coding for an extensin-like protein;
(b) isolating positive clones;
(c) testing isolated clones for promoter activity.

13. The method according to claim 12, wherein the cDNA used is identical to a part of the nucleotide sequence indicated under SEQ ID No. 7.

14. Use of a promoter according to claim 1 or 2 or of a promoter identified according to a method according to any one of claims 11 to 13 for the root-specific expression of transgenes in plants.