WO1993014213A1

WO1993014213A1 - Novel plant gene regulatory element

Info

Publication number: WO1993014213A1
Application number: PCT/GB1993/000071
Authority: WO
Inventors: Andrew Peter Goldsbrough
Original assignee: Unilever Plc; Unilever N.V.
Priority date: 1992-01-14
Filing date: 1993-01-14
Publication date: 1993-07-22
Also published as: GB9200710D0; AU3264593A

Abstract

The invention relates to a novel plant gene regulatory element having substantially the sequence 5'-TCATCTTCTT-3' which confers stress-responsiveness upon the expression of operably-linked protein-coding sequences. Substances which recognise this novel regulatory element are also disclosed. The invention provides a method of regulating the expression of protein-coding sequences and of monitoring the stress to which a plant is exposed.

Description

Title : Novel Plant Gene Regulatory Element

Field of the Invention

This invention relates to a novel nucleotide sequence, vectors containing the sequence , substances which recognise said sequence , methods of regulating gene expression and methods of detecting stress in plant cells , tissue cultures or whole plants .

Background to the Invention

Regulation of gene expression in higher eukaryotes such as plants is under bipartite control involving cis-acting regulatory DNA sequences and trans-acting transcription factors .

The most important regulatory DNA sequences are the promoter (which contains specif ic cis-acting regulatory sequence elements ) and enhancer and upstream promoter sequences (which also contain specific cis-acting regulatory sequence elements ) . The distinction between promoters and enhancers is somewhat vague since the specific sequences contained within them may be the same . However , promoters are always located immediately 5 ' to the start site of transcr iption, whereas enhancers can be located at much greater distances both 5 ¹ and 3 ' to the transcription start site.

Trans-acting transcr iption factors are generally proteins which recognise and bind to the specific cis-acting DNA elements described above . It is the specif ic combination of cis- and trans-acting factors in a given cell at a giver- time or under particular conditions- that determines v?hether or not a gene is expressed.

The expression of several plant genes is known to be induced or switched on under conditions of stress. The most well-studied of these genes are the so-called pathogenesis related (PR) proteins which are induced by pathogen invasion or stress conditions. For example, in tobacco, PR proteins are produced after infection with tobacco mosaic virus (TMV) or after treatment with salicylic acid. Beta-1 ,3-glucanases and chitinases are two examples of proteins which fall into this category. Another well-studied group of similarly induced proteins are enzymes involved in phenylpropanoid metabolism such as 4-coumarate lyase (4-CL), phenylalanine lyase (PAL), and Chalcone synthase (CHS) . Activation of phenylpropanoid metabolism is a common defence response to attempted pathogen attack, light stress and wounding. Through the activation of general phenylpropanoid pathway enzymes, and end-product specific branch pathways.^" an astounding array of secondary metabolites are elaborated from phenylalanine - including antimicrobial phytoalexins, UV-protective flavenoids, activators and inhibitors of symbiotic plant- microbe interactions and insect repellants inhibitors of symbiotic plant-microbe interactions and insect repellants and attractan s.

Summary of the Invention

In one aspect the invention provides a nucleotide sequence, comprising substantially the sequence 5'- TCATCTTCTT-3' or functional equivalents thereof.

Examples of such functional equivalents are shown in Table _ T

1

In another aspect the invention provides a recombi nant vector compr is ing the sequence def ined above .

It is clear , to those skilled in the art, with the knowledge of the disclosure herein made, that the novel sequence def ined above is naturally associated with plant genes whose expression is regulated in response to var ious stresses .

In a specific embodiment the invention provides the nucleotide sequence def ined above , in isolation from surrounding sequences with which it is naturally associated .-

In another aspect therefore, the invention provides a recombinant vector comprising the novel sequence def ined above or functional equivalents thereof , in isolation from surrounding sequences with which it is naturally associated .

It is obvious to a man skilled in the art, with knowledge of the disclosure herein made , that a protein-coding nucleotide sequence may, by means of recombinant DNA technology, be brought into position around and/or proximal and/or adjacent to the novel sequence in such a manner as to cause the expression of the protein-codi ng sequence to be regulated wholly or partly by the novel regulatory sequence.

Thus in a further aspect the invention provides a method of regulating the expression of a pr otein-codi ng seque nce comprising the step of positioning a protein-codi ng sequence around and/or proximal and/or adjacent to the novel regulatory sequence.

Clearly, the method described above is reciprocal, in that the expression of a protein-coding sequence may be regulated by positioning the novel regulatory sequence proximal and/or adjacent to and/or within the protein- coding sequence.

A barley genomic clone (g19-9) encoding a beta-1,3- glucanase has been isolated and sequenced. Within a 200bp region (shown in Figure 1 and identified as SEQ ID No. 1 in the attached sequence listing), approximately 600bp upstream of the putative TATA box of this gene, is a 1Obp motif (shown in block capitals in Figure 1 and identified as SEQ ID No. 2 in the attached sequence listing) termed the "TCA" motif, with the consensus sequence 5'- TCATCTTCTT-3¹ , repeated four times. In a survey of available literature it was found that the same or a similar motif is present in the non- ranslated regions of over 30 different plant genes which are all known to be induced by one or more forms of stress. Data obtained from this survey are presented in Table 1. The list comprises genes for the pathogenesis related (PR) family of proteins from tobacco and parsle ; enzymes of the phenylpropanoid and flavenoid biosynthetic pathways; several wound-inducible genes from potato and tomato (Win!, Wun1 and proteinase inhibitors I and II); ubiquitin, which is known to be a stress related protein throughout the biological kingdom; a heat shock protein from soybean; nitrite and nitrate reductase genes which are induced by water stress; an osmotic stress-inducible Abscissic acid-responsive gene, and an Arabidopsis sp. alcohol dehydrogenase gene which is induced under anaerobic stress.

The motif is present in genes from both dicotyledonous and monocotyledonous plants. In some genes it is present as a single copy (eg tobacco PR-1 [Payne et al. , 1989]) and in others it is repeated several times - up to seven in the case of 4-CL (Douglas et al. , 1987). With the exception of one of the parsley PR2 sequences, all those listed in Table 1 share at least 8 nucleotides in common with the consensus sequence. In some of the examples given (those marked with an asterisk in Table 1), the motif occurs on the complementary DNA strand. In one case it occurs 3' to the coding sequence and in two cases it is found within an intron. It is also frequently observed within the transcribed region upstream of the ATG initiator codon. The motif itself does not show dyad symmetry and has an unusual base composition, being 5'-TC-3' rich in its forward orientation.

In view of the possible regulatory function of the TCA motif, it was decided to perform protein-binding studies.

Tobacco (Nicotiana tabacum cv Samsun NN) was selected as a model system for these experiments because methods for nuclear protein extraction and induction of PR proteins using salicylic acid (5m ) from this species are well documented (Jensen et al., 1988). Salicylic acid has recently been confirmed as a systemic signal and inducer of PR proteins in virus-infected tobacco (Yalpani et al., 1991).

A 357 bp Aval-BamHI restriction fragment (termed "AB"), incorporating several copies of the TCA motif, was excised from the promoter region of a barley B-1 ,3-glucanase gene (g19-9) (Figure 1). This was made blunt using Klenow polymerase and dNTPs, and subcloned into the Smal site of pBI221.8 (a pUC19 derivative containing the polylinker region and CaMV-GUS-Nos cassette from the vector pBI121.8 [Goldsbrough ε- Bevan, 1991]) to produce the plasmid pAG281.8. The fragment was subsequently excised from pAG281.8 as an Asp718-BamHI fragment and purified from a gel. The cloned AB fragment contains 4 copies of the TCA motif: two are identical to the consensus sequence and two have 8 bases in common with the consensus sequence. The AB fragment could be 3' end labelled (specific activity 10'cpm/ug) using alpha^J P dCTP (purchased from Amersham International) and Klenow polymerase.

Radiolabelled AB fragment was incubated with 10ug of nuclear proteins extracted from leaves of salicylic acid- treated tobacco plants and water-treated control plants prepared using the method of Jensen et al., (1988). Protein/DNA complexes were analysed by polyacrylamide gel electrophoresis (Figure 2).

Gel retardation assays were carried out essentially as described by Jensen et al. , (1988). Labelled DNA fragments, competitor DNA (where present), 2.5ug poly(dI- dC) and nuclear extracts were mixed in 25ul of 5mM HEPES pH7.5, 2mM MgC1-,, 0.2mM DTT, 1mM CaCl₂, 2% glycerol (DNA binding buffer). The mixtures were incubated--^"'for 20 minutes at room temperature and loaded onto 4% polyacrylamide gels. Gels were fixed in 5% glycerol, 5% methanol, 5% acetic acid. They were then transferred to Whatman 3MM paper, dried under vacuum and autoradiographed.

The AB probe reacted to form complexes with nuclear proteins from both control and salicylic acid-treated plants (Figure 2, lanes 2 & 3), but the complex was more predominant when extracts from salicylic acid-treated plants were used. No complexes were observed when the nuclear extracts were boiled or treated with proteinase K prior to reacting with the labelled probe, confirming that the complex is the result of a DNA/protein interaction.

Thus in another aspect, the invention provides a substance which recognises the TCA motif or functional equivalents thereof.

Generally the substance which recognises the TCA motif is proteinaceous. Typically it is a nuclear protein.

Such ubstances might have the ability of modulating the resp .siveness to stress of certain gene expression systems.

To determine whether or not the TCA motif is the specific binding site for the nuclear protein which binds to the beta-1 ,3-glucanase promoter fragment, further gel retardation assays were carried out using a cloned synthetic 82 bp DNA fragment incorporating six copies of the TCA motif. To make this fragment, two 26bp complementary oligonucleotides incorporating two copies of the TCA sequence were synthesised, using a P armacia Gene Assembler Plus. The oligonucleotides were annealed and ligated into the BamHI site of pϋC19. One clone, pAG280.1 contained 3 copies of the annealed fragment and thus contained 6 copies of the TCA motif. This multimer was subsequently excised as a Hindlll - Asp718 fragment known as TCAxβ. TCAxβ fragment was 3' end labelled in the same manner as AB fragment. The sequence of this DNA is presented in Figure 3 (and is identified as SEQ ID No. 3 in the attached sequence listing).

Sequence-specific binding was demonstrated by competition binding experiments, the results of which are shown in Figure 4. ITuclear protein extracts from salicylic acid- treated plants were used, prepared as before. 10ug of nuclear protein extract was reacted with radiolabelled AB fragment (Figure 4, lane 2). Competition with 5Ox and lOOx molar excess of non-radiolabelled AB fragment resulted in the virtual disappearance of the protein/DNA complex (Figure 4, lanes 3 and 4). Competition with 50x and 100x molar excess of a non-radiolabelled "non¬ specific* 100bp fragment (excised from the coding region of beta-1,3-1 ,4-glucanase gene), resulted in only a small decrease in the observed binding (Figure 4, lanes 5 and 6). Competition with 50x and lOOx molar excess of non- radiolabelled TCAxβ DNA again resulted in the virtual disappearance of the protein/DNA complex (Figure 4_r lanes 7 & 8), indicating that the TCAxβ fragment specifically competes for binding of the nuclear protein which recognises the AB promoter fragment.

In a similar experiment, 10ug of nuclear protein extract from salicylic acid-treated plants was reacted with radiolabelled TCAxβ probe. The results of this experiment are shown in Figure 5. A protein/DNA complex was observed (Figure 5, lane 2). Competition with 50x and-^'TOOx molar excess of both non-labelled TCAxβ and AB fragments resulted in the virtual disappearance of the complex (Figure 5 lanes 3 & 4, 7 & 8). Competition with 50x and 10Ox molar excess of the non-specific 100bp fragment resulted only in a small decrease in the observed bound complex (Figure 5, lanes 5 & 6) . Taken together, these resuls confirm that the TCA motif is the specific recognition sequence for a tobacco nuclear protein, designated TCA-1.

South-western blotting experiments were carried out to determine the rnolecular weight of the protein which binds to the TCA motif.

The method employed was essentially that described by Miskimins et al. , (1985) . However, the following changes were introduced: SDS-PAGE was carried out at room temperature using a 10% acrylamide separating gel. The incubation buffer used was the same as DNA binding buffer used in the gel retardation assays. Filters were washed^' two times (20-30 minutes) in incubation buffer.

30ug of tobacco nuclear protein extracts from plants treated with either salicylic acid or water (control) were separated by SDS-PAGE and then transferred to a nitrocellulose membrance. The membrane was incubated in the presence of radiolabelled TCAxβ probe. The results are shown in Figure 6.

A strong signal was observed for the salicylic acid- treated plant extracts (Figure 6, lanle 1) whereas a considerably weaker signal was observed for the control plant extracts (Figure 6, lane 2). These results are consistent with the gel retardation assays (Figure 2), confirming that binding activity is increased when nuclear extracts from salicylic acid-treated plants are used. The protein has an apparent molecular weight of 40kDa.

In a specific embodiment therefore, the invention provides a plant cell nuclear protein having a molecular weight of substantially 40 kDa as judged by SDS-PAGE and having the ability to recognise specifically the TCA motif or functional equivalents thereof.

It is possible that, by placing a reporter gene under the control of the TC^. motif, one may be able to detect and/or assay the stress to which a plant or plant cell is exposed.

Thus in a further embodiment the invention provides a method for detecting and/or assaying the stress to which a plant or plant cell is exposed comprising the steps of placing a reporter gene under the regulatory control of the novel regulatory sequence or functional equivalent thereof, inserting the reporter gene and regulatory sequence (in functional relationship) into a plant or plant cell and detecting or measuring the level of expression of the reporter gene in the plant or plant cell.

Insertion of the 10 bp novel regulatory element into a promoter which already confers some form of specificity (eg tissue specificity) and/or regulation may render that promoter stress inducible under the specific circumstances as defined by that promoter.

For example, the CaMV 35S promoter A domain confers root tip specificity of expression in potatoes. Insertion of the TCA motif upstream of the A domain may result in a large induction of root tip specific expression under conditions of stress.

Similarly, insertion of the TCA motif upstream of the patatin promoter, the rubisco small sub^' unit promoter, the rape acyl carrier protein promoter or the wheat high molecular weight glutenin promoter may result in those promoters being highly inducible in tubers, leaves, seeds and endosperm respectively under conditions of stress. Alternatively, increasing the level of TCA-1 protein in plants under conditions of stress may lead to enhanced induction of defence-related proteins or an increased response to stress-

One possible method of demonstrating the utility of this technique would be to clone the gene encoding the TCA-1 protein and use it to transform potato plants where it is expressed under the control of a fully active, constitutive CaMV 35S promoter.

The potato plant would then be co-transformed with a reporter gene cassette comprising a reporter gene (eg GUS, a reporter gene well known to those skilled in the art) under the joint control of any of the promoters detailed above and the TCA motif. This should result in high levels of GUS expression in a tissue specific manner but without the need for stress induction. This is because in such a system, the TCA-1 protein is expressed continuously under the control of the CaMV 35S promoter. Conversely, if the CaMV 35S promoter in the system described above is replaced with the WIN2 (potato wound inducible promoter 2), GUS expression would be- induced to very high levels in a tissue specific manner, but only under conditions of stress. This is because the TCA-1 protein would only be synthesised when the WIN2 promoter is activated, the WIN2 promoter being itself stress- responsive.

The various aspects of the invention may be better understood by reference to the following figures, of which:

Figure 1 shows the nucleotide sequence of the AB ragment,

Figure 2 is an autoradiograph of a gel retardation assay.

Figure 3 shows the nucleotide sequence of TCAxβ,

Figures 4 and 5 show autoradiographs of gel retardation assays and

Figure 6 is a photograph of a South- estern blot.

Detailed description of the Figures

Figure 1

This shows the DNA sequence of the Asp718-BamHl AB fragment from the upstream region of the beta-1 ,3- glucanase gene g19-9. The repeated TCA motifs are shown in bold type.

Figure 2

This figure shows the results of an experiement to investigate the binding of tobacco (Samsun NN) leaf nuclear proteins to the barley glucanase (g19-9) AB fragment. Lane 1 contains 1 ng of the AB probe alone. Lane 2 contains 1ng of AB probe and 1Oug of nuclear extracts from salicylic acid-treated tobacco leaves. Lane 3 contains Ing of AB probe and 10ug of nuclear extracts from water-treated (control) tobacco leaves. The arrow indicates the position of the retarded complex.

Figure 3 This shows the DNA sequence of the cloned TCAxβ fragment.

Figure 4

This figure shows the binding of nuclear proteins with specific and non-specific competitors. Lanes 1-8 contain 1ng of AB probe. Lane 1 is AB probe alone. Lanes 2-8 contain 10ug of nuclear extracts from salicylic acid- treated tobacco leaves. Lanes 3 and 4 contain 50x and 100x molar excess of unlabelled AB fragment. Lanes 5 and 6 contain 50x and 100x molar excess of an unlabelled 100bp fragment from the coding region of a barley beta-1 ,3-1 ,4- glucanase gene. Lanes 7 and 8 contain 50x and 100x molar excess of unlabelled TCAxβ fragment. The arrow indicates the position of retarded complexes.

Figure 5

This figure shows the results of a further experiment to investigate the binding of nuclear proteins with specific and non-specific competitors. Lanes 1-8 contain 1ng of TCAxβ probe. Lane 1 is TCAxβ probe alone. Lanes 2-8 contain 1Oug of nuclear extracts from salicylic acid- treated tobacco leaves. Lanes 3 and 4 contain 50x and 100x molar excess of unlabelled TCAxβ fragment. Lanes 5 and 6 contain 50x and 100x molar excess of an unlabelled 100bp fragment from the coding region of a barley beta- 1 ,3-1 ,4-glucanase gene. Lanes 7 and 8 contain 50x and 100x molar excess of unlabelled AB fragment. The arrow indicates the position of retarded complexes.

Figure β

This figure shows the results of South- estern blot analysis. Tobacco nuclear proteins -were separated by polyacrylamide gel electropnoresis, transferred to nitrocellulose and hybridised with radiolabelled TCAxβ probe. Lane 1 contains 30ug of nuclear extracts from salicylic acid-treated tobacco leaves. Lane 2 contains 30ug of nuclear extracts from water-treated (control) leaves.

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Table 1. A comparison of TCA sequences from various stresss-induced plant genes Sequence, cf barley TCA motif position Reference

Barley β-1,3-glucanase (G19-9) T C A T C T J C T T This paper T - C

C C

N.plumbag. β-1,3-glucanase (Gnl) - - T - - -325 Castresana et al 1990

- A - - - - - - - A -346

- - - - - - - A A - +1

N.tabacum β-1,3-glucanase (Gln2) _ - - -163 Ohie-Takagi & Shinshi 1990 A A - -12

A A - - -518

N.tabacum β-1,3-glucanase (gl9) _ __ _ _ _ _ _ _ _ Q ATG-97 Liπthorst et al 1990

N.tabacum β-1,3-glucanase (gglb50) _ - - - - - _ - _ - ATG-216 Linthorst et al 1990

- _ _. _ _ _ _ _{A A} _ ATG-65

A A - - ATG-571

- - - - T - - - - - ATG-971 *

N.plumbag. β-1,3-glucanase (gn2) _ _ _ _ _ _ _ _ _ _ -178 Gheysen et al 1991

- - - - - - - A A - -18

N. abacum β-1,3-glucanase (Gla) _ _ _ _ _ _ _ _ _ _ -203 Sperisen et al 1991 - - A A - -16

A A - - -577

N.tabacum PR-1 - - - - ATG-592 Payne et al 1989

N.tabacum PR-la - - j . - - - - -^{• ■}- -532 * Payne et al 1988

_ ._ _ _ 6 - - -. .-^■ -66 * N.tabacum PR-la - - - - J. - - T -. -^' -1403 Cornelissen et al 1987

_ _^"_ _ _ .. c - - C -1070 *

- A - ^'- -■^'•-. - A •- - -360 *

Table 1 (Continued)

N.tabacum PR-S (E2) _ _ _ _ _ C - - - - -174 van Kan et al 1989

N.tabacum chitinase (CHN17) G - - - - - - - C - -44 Shinshi et al 1990

N.tabacum chitinase (CHN50) _ - - _ - - - _ c - -47 Fukuda et al 1991

- A - - T - - - - - -599

Arabidopsis PR1 _ _ _ χ _ _ _ _ _ ATG-187 Metzler et al 1991

Parsley PR1 - G - - T - - - - - +38 Somssich et al 1988

- - T - - - - T - - +52

Parsley PR2 - - - - - - - T G A -153 van de Locht et al 1990

- _ - _ _τ _ _ _ _ _ ₊ 2

Arabidopsis chalcone synthase _ _ _ _ _ _ _ _ _ _ -57 Feinbautn & Ausubel 1988

- - - - - - - T - - -1215

Mustard chalcone synthase _ _ _ _ _ _ _ G _ _ -75 Batshauer et al 1991

Bean chalcone synthase - _ - - _ - _ γ _ _A +73 Dron et al 1988 ^'

Antirrhinum chalcone synthase - - - - - - - - C A -514 Sommer & Saedler 1986

Parsley 4-coumarate ligase (Pc4CL-l) - - - - - A - - G - +22 Douglas et al 1987

- - - A A 637

Parsley phenylalanine ammonia lyase - - - - A - - - - C -123 Lois et al 1989

- - - - A - - - G - -405

N.tabacum glycine rich protein _ - _ 6 j - _ _ _ _ _n Van Kan et al 1988

- - - - - - - T -^: - -248

G A -989 *

- T T - - -1689

Table 1 (Continued)

Bean glycine rich protein _ _ _ _ _ _ _ G _ _ ATG-220 * Keller et al 1988

Bean hydroxyproline rich glycoprotein - - - - - - - - - - TAA+375 Corbin et al 1987

Potato wound inducible gene ( unl) - - - - - - - - Q - -209 Siebertz et al 1989

- - G +35

- - - - A - - A - - -433 * Potato wound inducible gene (Winl) _ _ - - - _ _A T _ _ -440 * Stanford et al 1989

- - - - - - - A A - -830 *

Soybean heat shock protein G - _ _ _ _ _ _ _ _ -256 Czarnecka et al 1988

- - - - A - - T - - -229 r A A - - - - - - - - intron

Arabidopsis alcohol dehydrogenase _ - τ - - _ - _ _ -57 Chang & Meyorowitz 1986

- G T - - -220 *

Tomato proteinase inhibitor I _ _ _ γ _ _ _ _ _ ATG-439 Lee et al 1986

Potato proteinase inhibitor II _A _ _ _ _ _ _ τ - _ -127 Keil et al 1986

A A - - - -227

- T - - - C 621

G - T - - - - - - - -166 *

Spinach nitrite reductase _ _ _ _ _ _ _ _ _A _ +37 Back et al 1991 A - +93 A - +99

Tobacco nitrate reductase _ _ _ _ _A _ _ _ _ _ ATG-25 Vaucheret et al 1989

Rice ABA responsive gene - - - - - - C - - C . -53 Mundy et al 1991

Sunflower ubiquitin _ γ _ _A - - - _ _ _ -353 Binet et al 1991

G - C -49

- - - C -24

- - _ _ > C - - j _ intron

Claims

1 . A nucleotide sequence comprising substantially the sequence 5 ' -TCATCTTCTT-3 * or functional equivalents thereof .

2. A sequence according to claim 1 , in isolation from surrounding sequences with which it is naturally associated .

3. A sequence according to claims 1 or 2 incorporated into a vector .

4. A sequence according to any one of the preceding claims, operably linked to a protein-coding nucleotide sequence.

5 . A sequence according to claim 4 , wherein the protein- coding nucleotide sequence encodes a plant cell protein whose expression is not normally regulated by stress.

6. A method of regulating the expression of a protein- coding nucleotide sequence, comprising operably linking the sequence of any one of the preceding claims to a protein-coding sequence.

7. A substance which recognises the sequence of any one of claims 1 to 5.

8. A substance according to claim 7 which is a protein.

9. A substance according to claim 8, which is a plant cell nuclear protein with an apparent molecular weight of substantially 40kDa.

10. A method of monitoring the stress to which a plant or part thereof is exposed, comprising the steps of:

operably linking a reporter gene to the sequence of any one of claims 1 to 5;

introducing said operably linked sequence and reporter gene into a plant or part thereof and monitoring a product of said reporter gene.