Pathogen-inducible synthetic promoter

Abstract

The invention relates to a pathogen-inducible synthetic promoter which is suitable for regulating the transcription of a nucleic acid, and includes a minimal promoter, characterized in that the minimal promoter includes a sequence motif a) dbrmwa or b) twcccmt which is disposed downstream from a TATA region and in front of a transcription starting point which is located on the minimal promoter and at which transcription of the nucleic acid to be regulated starts.

Claims

1. A pathogen inducible synthetic promoter for regulating the transcription of a nucleic acid, which includes at least one cis-regulatory element and a minimal promoter, wherein the minimal promoter is heterologous to the at least one cis-regulatory element, wherein the minimal promoter comprises a TATA-box, a transcription starting point, and a sequence motif twcccmt, and wherein the sequence motif twcccmt is disposed downstream from the TATA-box and in front of the transcription starting point at which transcription of the nucleic acid to be regulated starts and wherein the minimal promoter includes the nucleotide sequence according to SEQ ID NO: 9.

2. The pathogen inducible synthetic promoter according to claim 1, wherein the sequence motif twcccmt occurs two or more times in the minimal promoter.

3. The pathogen inducible synthetic promoter according to claim 1, wherein the at least one cis-regulatory element comprises the nucleotide sequence according to SEQ ID NO: 10.

4. A recombinant gene with a pathogen inducible synthetic promoter according to claim 1.

5. A plant cell, in which a pathogen inducible synthetic promoter according to claim 1 has been integrated into DNA of the plant cell.

6. A transgenic plant with a plant cell according to claim 5.

7. A seed of a transgenic plant comprising the pathogen inducible synthetic promoter according to claim 1.

8. A vector or an expression cassette comprising a minimal promoter wherein the minimal promoter comprises a TATA-box, a transcription starting point, and a sequence motif twcccmt, wherein the sequence motif twcccmt is disposed downstream from the TATA-box and in front of the transcription starting point at which transcription of the nucleic acid to be regulated starts, and wherein the TATA-box is the binding location for the TATA-box binding protein (TBP) associated with transcription of said nucleic acid and wherein the minimal promoter is heterologous to at least one element of the vector or the expression cassette and wherein the minimal promoter includes the nucleotide sequence according to SEQ ID NO: 9.

9. The vector or expression cassette according to claim 8, wherein the sequence motif occurs two or more times in the minimal promoter.

10. A plant cell, in which an expression cassette according to claim 8 has been integrated into DNA of the plant cell.

11. A transgenic plant with a plant cell according to claim 10.

12. A seed of a transgenic plant comprising the expression cassette according to claim 8.

13. A pathogen inducible synthetic promoter for regulating the transcription of a nucleic acid, which includes at least one cis-regulatory element and a minimal promoter, wherein the minimal promoter is heterologous to the at least one cis-regulatory element and wherein the minimal promoter comprises a sequence motif twcccmt which is disposed downstream from a TATA-box and in front of a transcription starting point which is located on the minimal promoter and at which transcription of the nucleic acid to be regulated starts, and wherein the TATA-box is the binding location for the TATA-box binding protein (TBP) associated with transcription of said nucleic acid and wherein the minimal promoter includes the nucleotide sequence according to SEQ ID NO: 9.

14. The vector or expression cassette according to claim 8, wherein the minimal promoter is operably linked to a recombinant gene.

15. The vector or expression cassette according to claim 9, wherein the minimal promoter is operably linked to a recombinant gene.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) The invention is explained in more detail below with reference to the drawing in which:

(2) FIG. 1 shows in SEQ ID NOs: 1-10 a sequence comparison between the preferred minimal promoters for dicotyledonous plants with the conserved TATA-regions and the dbrmwa-motives as well as the cleavage site Pstl and Xhol employed for cloning in the plasmid pMS23luc+;

(3) FIG. 2 shows in SEQ ID NOs: 8 and 9 a sequence comparison between the minimal promoters, preferred for monocotyledonous plants, which are employed for the transient transformation of wheat leaves, and the sequence motive twcccmt as conserved region;

(4) FIG. 3 shows an overview of the average reporter gene activity of non-infected, transgenic potato plants with a synthetic promoter comprised of the 4xGst1 element and the indicated minimal promoters, cloned ahead of the luciferase gene from Photinus pyralis as reporter gene;

(5) FIG. 4 shows a overview of the individual inductions following in vitro infection of stable, transgenic potato plants with the synthetic promoter comprised of the 4xGst1 element and the indicated minimal promoters;

(6) FIG. 5 shows that when transgenic sprouts were multiplied and inoculated under in vitro conditions with the zoospore suspension of Phytophthora infestans, also with use of the cis-regulatory element 2xS/2xD a reduced background activity could be achieved with the inventive minimal promoters in comparison to 35S-minimal promoters

(7) FIG. 6 shows that at the same time a stronger induction of the synthetic promoters following inoculation of the transgenic potatoes with P. infestans could be observed.

(8) FIG. 7 shows a comparison of the normalized activity of pathogen inducible synthetic promoters comprised of an element 2xS/2xD and the minimal promoters ubi1 (comparison promoter), TaPAL and TaACS following biologic transformation in primary leaves of the wheat type Taifun.

(9) FIG. 8 shows the binary vector p4xGst1-luc-kan used for the stable transformation of potato plants of the variety Baltica.

(10) FIG. 9 shows the plasmid pubiTATARucll, which contains the cDNA with the luciferase gene from Renilla reniformis, as it exists in the commercially available plasmid pRL-Null.

DETAILED DESCRIPTION OF THE INVENTION

(11) The symbols of the sequence motive as used herein have the following meaning:

(12) d=nucleotide a or g or t/u

(13) b=nucleotide c or g or t/u

(14) r=nucleotide g or a

(15) m=nucleotide a or c

(16) w=nucleotide a or t/u

(17) a=nucleotide a

(18) t=nucleotide t

(19) c=nucleotide c

(20) In the sense of the invention a minimal promoter is a DNA-sequence of the promoter, which is necessary for promoter function. General transcription factors such as for example TFII-D, TFII-A, TFII-B, TFII-E and TFII-F could bond at this DNA-sequence, and form the platform for the bonding of the RNA-polymerase 11/TFII-F complex. Since the transcription of the DNA into the mRNA starts in this region, the transcription start point (TS) lies within the minimal promoter and is identified as position +1. The minimal promoter encompasses the TS and can extend for example from position 50 through position +15. Frequently a so-called TATA-box is found at the position 30, which however does not occur in all promoters. The TATA-box is a region of a sequence of thymine and adenine bases. The TATA-box is the binding location for the TATA-box binding protein (TBP).

(21) Characterized as synthetic promoters are those promoters which do not occur in nature, are assembled from multiple elements and contain a minimal promoter as well as, upstream of the minimal promoter, at least one cis-regulatory element, which serves as the bonding location for special transcription factors. Synthetic promoters are designed according to the desired requirements and are induced or repressed by various factors.

(22) Derivatives of a promoter are shortened or lengthened or partially identical versions of this promoter or homologs with the same, modified or singular characteristics. The expression homology herein means a homology of at least 70% based on DNA, which can be determined by known processes, for example, a computer supported sequence comparison (Altschul, S. F. et al., 1990).

(23) The inventive pathogen inducible synthetic promoter results after transient biologic transformation in a reduced base activity in the leaf tissue of the respective plants in comparison to conventionally employed promoters with a minimal promoter such as the 35S-minimal promoter in dicotyledonous, and the corn-ubi1-minimal promoter in monocotyledonous, plants. Beyond this it was discovered that in the inventive pathogen inducible synthetic promoters the induction rate is also higher.

(24) The inventive pathogen inducible synthetic promoters can thus be employed for production of transgenic plants which have a broad resistance against numerous pathogens, such as fungi, oomycetes, bacteria, virus, insects and nematodes.

(25) The sequence motives dbrmwa and twcccmt lie in sense orientation on the codogenic strand between the TATA-box and the transcription start point and can also occur two or more times. Preferred sequences for minimal promoters are indicated in SEQ ID NOS: 1 through 9.

(26) Cis-regulatory elements for production of pathogen inducible synthetic promoters are primarily those elements which occur in natural pathogen inducible promoters and they are responsible for pathogen induction. Their identification is described in Rushton et al. (2002).

(27) Preferred cis-regulatory elements for production of synthetic promoters with use of the inventive minimal promoters are also described in WO 00/29592. From the cis-regulatory elements mentioned there, the D-box (SEQ ID NO: 10) is particularly suitable, in particular in the combination 2xS/2xD (SEQ ID NO: 11), as well as the Gst1-element, preferably in the combination 4xGst1 (SEQ ID NO: 12).

(28) Preferred cis-element combinations include in general combinations of the D-box (SEQ ID NO: 10) with the S-box or, as the case may be, the Gst1-element. Particularly preferred are, besides the above-mentioned combination 2xS/2xD (SEQ ID NO: 11), the combination 2xS/4xD (SEQ ID NO: 13); 4xS/2xD (SEQ ID NO: 14) and 2xGst1/2xD (SEQ ID NO: 15). The combination of the 2xS/4xD element (SEQ ID NO: 13) with the minimal promoter according to SEQ ID NO: 2 shows in transgenic potatoes following infection with Phytophthora infestans an average elevation of the reporter gene activity by a factor of 253,000 in comparison to a non-infected control.

(29) If the element 4xS/2xD (SEQ ID NO: 14) was cloned ahead of the minimal promoter (SEQ ID NO: 2), an average increase in the reporter gene activity by a factor of 2,892 could be detected. With element 2xGst1/2xD (SEQ ID NO: 15) an average increase by a factor of 2,967 in comparison to control was achieved.

(30) With the inventive promoters transgenic plant cells can be produced, which can be regenerated to complete plants with improved defensive characteristics against pathogens. The inventive promoters are likewise contained in the seeds of such transgenic plants. The invention is not limited to particular types of plants.

(31) The present invention is thus concerned with the process for production of a plant resistant against pathogens, in which a gene suitable for production of a pathogen resistance is introduced into a plant cell, which is under the control of a pathogen inducible synthetic promoter, and subsequently this plant cell is regenerated into a plant, characterized in that the pathogen inducible synthetic promoter is a pathogen inducible synthetic promoter as described above.

Examples

(32) FIG. 1 shows a sequence comparison between the preferred minimal promoters (SEQ ID NOS: 1 through 7) for dicotyledonous plants with the conserved TATA-regions and the dbrmwa-motives as well as the cleavage site Pstl and Xhol employed for cloning in the plasmid pMS23luc+.

(33) FIG. 2 shows a sequence comparison between the minimal promoters (SEQ ID NOS: 8 and 9), preferred for monocotyledonous plants, which are employed for the transient transformation of wheat leaves. In addition to the TATA-region the sequence motive twcccmt is shown as conserved region.

(34) It could be shown that the minimal promoters StGst (SEQ ID NO: 6), NtTGAA (SEQ ID NO: 5), StPSBR (SEQ ID NO: 7), NpCABE (SEQ ID NO: 2), NtRBS (SEQ ID NO: 3), NpATP2 (SEQ ID NO: 1) and Nt5EAS (SEQ ID NO: 4) exhibited a clearly reduced activity (<70%) in comparison to the 35S-minimal promoter.

(35) FIG. 3 shows an overview of the average reporter gene activity of non-infected, transgenic potato plants with a synthetic promoter comprised of the 4xGst1 element (SEQ ID NO: 12) and the indicated minimal promoters, cloned ahead of the luciferase gene from Photinus pyralis as reporter gene (RLU=relative light unit). Stable, transgenic lines with the minimal promoters, which carry the sequence motive dbrmwa showed under controlled conditions a clearly reduced expression of the reporter gene in comparison to the 35S-minimal promoter. The smallest average activity was achieved with use of the minimal promoter of the NpATP2 gene (SEQ ID NO: 1). In these plants only 9.7% of the average activity of the 35S-minimal promoter could be measured. With use of the minimal promoters StPSBR (SEQ ID NO: 7), NtTGAA (SEQ ID NO: 5) or StGst (SEQ ID NO: 6) 18% of the activity of the 35S-minimal promoter was measured, with NtRBS-minimal promoter (SEQ ID NO: 3) 26%, with NpCABE-minimal promoter (SEQ ID NO: 2) 39% and with Nt5EAS-minimal promoter (SEQ ID NO: 4) 41%.

(36) For the person of ordinary skill the manufacture of suitable constructs for transformation of plants with the inventive promoters is no problem. Thus for example the binary vectors p4xGst1-luc-kan (FIG. 8) could be produced, which was used for the stable transformation of potato plants of the variety Baltica. This vector is a derivative of the binary vector pGPTV (Becker et al., 1992). The binary vector p4xGst1 luc-kan carries the luciferase gene from Photinus pyralis under the control of the synthetic promoter 4xGst1:35S minimal promoter (Rushton et al., 2002). As the termination sequence the plasmid is given the terminator of the nopalinsynthase gene from Agrobacterium tumefaciens. The described expression cassette is localized on the T-DNA together with a functional expression cassette for the neomycinphosphotransferase gene (nptll) as selection marker. The neomycinphosphotransferase imparts to the transgenic plants resistance against kanamycin or paromycin. In order to exchange the 35S-minimal promoter with the above-described minimal promoters, the binary vector p4xGst1 luc-kan was digested with Xhol/Sall, whereby the 35S-minimal promoter was removed, the tetramer of the Gst1-element however remained intact. The Sall cleavage location was filled with the aid of the enzyme Klenow polymerase and dNTP's in order to achieve a blunt end. The minimal promoters, cloned in the plasmid pMS23luc+, were excised using Pdil/Xhol-digestion and ligated in the binary vector and subsequently transformed in E. coll. Binary vectors with the new sequence were transformed in the Agrobacterium type GV3101::pMP90 (Koncz and Schell, 1986) (An, 1987) and selected using the antibiotic kanamycin (50 mg/l). The transgenic Agrobacterium were employed for the transformation of potatoes of the type Baltica (Dietze et al., 1995).

(37) FIG. 4 shows a overview of the individual inductions following in vitro infection of stable, transgenic potato plants with the synthetic promoter comprised of the 4xGst1 element (SEQ ID NO: 12) and the indicated minimal promoters. The infection occurred in in vitro plants with a zoospore suspension of Phytophthora infestans.

(38) At various times following inoculation leaf samples of in vitro plants were removed, the sample weight was determined and 10 volumes IxCCLR buffer (Promega, Mannheim) was added.

(39) The material was homogenized with the aid of a RIA/90 Hnrnflga.ni.sup.>.cr OKA Labortechnik, Staufen) in buffer on ice. By centrifugation at >10,000g for 10 minutes the homogenate was clarified and 10 pl of the supernatant was suspended with 50 pl of the substrate LAR (Promega, Mannheim) in a luminometer tube and the light emission was determined as value for the activity of the luciferase in the luminometer (Sirius, Berthold Detection System GmbH, Pforzheim). For control or comparison in vitro plants were employed, which were raised under the same conditions and, in place of zoospores, were subject to a sham treatment with water. The average value of the quotients, in 5 independent lines, of the luciferase activity of the infected to the sham treated variants, indicates the induction of the synthetic promoter by the infection. As can be seen in FIG. 4, with use of the 35S-minimal promoter, a maximal induction of the luciferase activity by a factor of only 10 could be achieved 72 hours after infection. All new minimal promoters in contrast showed a clearly improved induction. The strongest induction after infection with a factor 395 was achieved 72 hours post-infection with the StPSBR minimal promoter (SEQ ID NO: 7). In general, the induction by use of the new minimal promoters could be improved at time 72 hours post-induction by a factor of 3.5 with StGst minimal promoter (SEQ ID NO: 6) to a factor of 39.5 with StPSBR minimal promoter (SEQ ID NO: 7) in comparison to 35S-minimal promoter. Interestingly, clear differences in the kinetics of the induction following pathogen induction exist between the minimal promoters. While the most discernible induction is measurable with use of the 35S-minimal promoter 72 hours post induction, this also applies with use of the StPSBR, NtTGAA, StGst, NtRBS as well as NpATP2 minimal promoter. For NpCABE and Nt5EAS promoters in comparison a strong activation is already detectable at time interval 9 and the induction remains at approximately the achieved level over the remaining test period.

(40) The preferability of the new minimal promoters was shown following fusion with the cis-element combination 2xS/2xD. For this, potato plants were stably transformed with the binary vectors p2xS/2xDluc-kan, p2xS/2xDNpCABEluc-kan and p2xS/2xDNtTGAAluc-kan. The binary vectors were produced in that the 4xGst1-element from the above-described binary vector with the new minimal promoter and the 4xGst1-element were eliminated via Bcul/Eco1471-digestion and the element 2xS/2xD (SEQ ID NO: 11) was introduced as Bcul/Eco32l-fragment. Binary vectors with the new sequence were transformed in the Agrobacterium type GV3101::pMP90 (Koncz and Schell, 1986) (An, 1987) and selected using the antibiotic kanamycin (50 mg/l). The transgenic Agrobacterium were employed for the transformation of potato of the type Baltica (Dietze et al., 1995). Transgenic sprouts were multiplied and inoculated under in vitro conditions with the zoospore suspension (50,000 spores/ml) of Phytophthora infestans. It could be shown, that also with use of the cis-regulatory element 2xS/2xD (SEQ ID NO: 11) a reduced background activity could be achieved with the inventive minimal promoters in comparison to 35S-minimal promoters (FIG. 5). At the same time a stronger induction of the synthetic promoters following inoculation of the transgenic potatoes with P. infestans could be observed (FIG. 6). The amplification of the induction was not so pronounced at the later time (3 days post infection=3 dpi) as could be observed following use of the 4xGst1-element. Two days following infection however by the use of the new minimal promoter a clearly stronger induction following pathogen attack could be observed. Herewith the use of these minimal promoters has as a consequence an improvement of the kinetics of the synthetic promoter, so that the reaction to pathogen attack occurs earlier, in comparison to the synthetic promoter using the 35S-minimal promoter.

(41) FIG. 7 shows a comparison of the normalized activity of pathogen inducible synthetic promoters comprised of an element 2xS/2xD (SEQ ID NO: 11) and the minimal promoters ubi1 (comparison promoter), TaPAL (SEQ ID NO: 9) and TaACS (SEQ ID NO: 8) following biologic transformation in primary leaves of the wheat type Taifun. As can be seen, the new minimal promoters TaPAL and TaACS in wheat have a reduced base activity in comparison to ubi1-minimal promoter. While a normalized activity of 0.17 was measured with the ubi1-minimal promoter, with use of the TaPAL-minimal promoter this could be reduced to 0.072, and with use of the TaACS-minimal promoter it could be reduced to 0.13.

(42) FIG. 9 shows the plasmid pubiTATARucll, which contains the cDNA with the luciferase gene from Renilla reniformis, as it exists in the commercially available plasmid pRL-Null. The cDNA is under the control of the ubi1-minimal promoter. The ubi1-minimal promoter includes the sequence range from 45 through +76 relative to the transcription start point. For elevating the expression strength the first intron of the ubi1-gene is maintained in its natural context in the plasmid ahead of the reporter gene. The plasmid serves for cloning the cis-regulatory element 2xS/2xD (SEQ ID NO: 11), in order thereby to produce a pathogen inducible synthetic promoter. The ubi1-minimal promoter was exchanged for the new minimal promoter for improving the characteristics of the synthetic promoter.

REFERENCES

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