Patent Grant
10030251
This application is a U.S. National Phase Application under 35 U.S.C. § 371 of International Patent Application PCT/DE2013/000446 filed Aug. 6, 2013 which claims priority to German Patent Application 10 2012 016 009.7 filed Aug. 8, 2012. The International Application was published on Feb. 13, 2014, as International Publication No. WO 2014/023285 under PCT Article 21(2). The entire contents of these applications are hereby incorporated by reference.
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 15, 2015, is named 245761.000009_SL.txt and is 100,249 bytes in size.
The present invention relates to a transgenic plant of the species Solanum tuberosum with a resistance to an oomycete of the genus Phytophthora, to transgenic parts a plant of this type, to a method for its manufacture and to a means for external application to plants.
Even now, potato late blight caused by Phytophthora infestans is still the most prevalent and most economically important potato disease.
Throughout the globe, the pathogen results in loss of earnings, with harvest losses of more than 20 percent. This means that expensive chemical plant protection means have to be used, because the natural defence mechanisms of the potato with the help of which P. infestans is combatted or with which propagation can be slowed down and restricted is not sufficient or not permanent.
Natural plant defence mechanisms, such as the hypersensitive reaction at the infection site, lignification of the cell wall, the production of PR (pathogenesis-related) proteins and the synthesis of phytoalexins are indeed known to contribute to augmenting resistance, but they are always accompanied by an energy loss and thus a loss of earnings for affected plants.
Natural defence mechanisms in plants also include the expression of so-called resistance genes (R genes), the gene products of which interact with microbial avirulence genes (Avr genes) (gene for gene hypothesis) and thus induce a specific defence reaction. This resistance can, however, be interrupted if a pathogen such as P. infestans can dispense with the synthesis of the Avr gene and recognition of the pathogen and thus the subsequent specific defence reaction in the plant host does not occur.
Fire et al. (1998) have already demonstrated that double stranded RNA (dsRNA) can result in the sequence-specific degradation of homologous RNA. Starting from these results, transgenic plants have been developed in the meantime which, with the aid of RNA interference (RNAi) by means of host plant-induced silencing of conserved and essential genes, for example from nematodes or Lepidoptera- and Coleroptera species, can exhibit resistance to these pests in vitro as well as in vivo.
In addition, the host plant-phytopathogenic fungus interaction can constitute an application of the concept of host-induced gene silencing (HIGS) to induce resistance (EP 1 716 238).
Van West et al. (1999) initially used the gene silencing method in Phytophthora, in order to carry out functional analyses of these oomycete-specific genes.
In WO 2006/070227, the use of RNA interference to control fungal pathogens based on contact of dsRNA with fungal cells outside the fungal cell was described for the first time. It proposes a method for the manufacture of a pathogen-resistant plant. In this manner, the RNA interference can be directed against one or more genes of a pathogen as well as several pathogens. Phytophthora infestans is mentioned as a possible fungal pathogen and potato as a possible host plant.
Previous studies have given rise to the hypothesis that host plant-induced gene silencing does not work for every gene and choice of the target gene is essential for functional silencing. Thus, for example, the plasma membrane H+-ATPase PnMA1 in Phytophthora parasitica could not be reduced sufficiently by host plant-induced gene silencing to deliver efficient protection against a pathogen (Zhang et al. 2011). According to this, selection of the target genes is also decisive for effective pathogen defence (Yin et al. 2011).
Recently, a screening system was proposed which was supposed to facilitate the selection of suitable parasitic genes for silencing constructs for the production of pathogen-resistant plants (US 2010/0257634). The identification of appropriate test constructs to induce phytoresistance in potato was also proposed by the authors. In this regard, target genes were defined based on bioinformatic analyses of genome sequences or based on sequence homologies to essential genes or virulence factors from known model organisms. That document does not contain any indications of the genes disclosed in the present invention for the generation of a resistance against an oomycete of the genus Phytophthora.
A method for producing a broad spectrum resistance in transgenic plants against multiple fungi is described in WO 2009/112270. In one implementation of the method of that invention, the broad spectrum resistance is directed against Uncinula necator, Plasmopora viticola, Uromyces spec., Phakopsora pachyrhizi, Erysiphe sp. and also P. infestans.
Furthermore, the development of Phytophthora infestans-resistant potato plants through RNAi-induced silencing is disclosed in WO 2006/047495. On the one hand, plants were generated which carry gene sequences of the rRNA gene from Phytophthora infestans for RNA interference. The silencing construct described in WO 2006/047495 directed against the rRNA gene of Phytophthora infestans comprises base pairs 1-600 of Accession number AJ854293 and with it 32 bp of the coding region of the 18S rRNA as well as the complete coding region of the 5.8 S rRNA gene of the blight pathogen. When selecting the target genes for HIGS strategies, with a view to applicability, it is vital that it has as short as possible or preferably no homologies extending over more than 17 sequential base pairs to the gene sequences of non-target organisms, as if there were, gene expression of the non-target organisms in the case of consumption of the transgenic plant or its harvest product could be destroyed (“off-target” effect). However, the sequence described in in WO 2006/047495 comprises 32 bp of the P. infestans 18S rRNA, which has 100% identity with the homologous sequence of the 18S rRNA gene from man (Homo sapiens), pigs (Sus scrofa) and cattle (Bos taurus). Human potato consumption in Asia in 2005 was 26 kg, in North America it was 58 kg and in Europe it was 96 kg per person (FAOSTAT). In the light of the high human and animal consumption of potatoes, the rRNA sequences from Phytophthora infestans described in WO 2006/047495 as HIGS target genes are unsuitable for consumers on safety grounds.
On the other hand, in WO 2006/047495, plants were produced that carry gene sequences for the cathepsin B gene from Myzus persicae and the elicitin gene INF1 from P. infestans for RNA interference and thus exhibit resistance to two plant pathogens. The target gene INF1 used therein codes for an elicitor. A resistance based on an elicitor as a pathogenicity factor is a disadvantage because the elicitin gene INF1 is not always necessary for an infection of potatoes by Phytophthora infestans (Kamoun et al. 1998).
The aim of the present invention is thus to provide a transgenic plant of the species Solanum tuberosum which is pathogen-resistant to an oomycete of the genus Phytophthora and in particular is suitable for consumption.
In accordance with the invention, this aim is accomplished by the fact that a double-stranded first and second DNA are stably integrated into the transgenic plant, wherein the first DNA comprises (a) a nucleotide sequence in accordance with SEQ ID NOS: 1-43, or (b) a fragment of at least 15 successive nucleotides of a nucleotide sequence in accordance with SEQ ID NOS: 1-43, or (c) a nucleotide sequence which is complementary to one of the nucleotide sequences of (a) or (b), or (d) a nucleotide sequence which hybridizes with one of the nucleotide sequences of (a), (b) or (c) under stringent conditions.
Surprisingly, it has been found that a first DNA of this type is particularly suitable for conferring a pathogen resistance in potato plants via a host-induced gene silencing strategy.
The first and the second DNA are integrated in a stable manner into the genome of a transgenic plant of the species Solanum tuberosum. Preferably, the DNAs are integrated in a stable manner into a chromosome of the plant. However, they can also be integrated into an extra-chromosomal element. The advantage of stable integration is that the DNA can be passed on to subsequent generations of the transgenic plant.
The double-stranded DNA is composed of a coding and a non-coding strand.
Furthermore, the nucleotide sequence of the coding strand of the second DNA is the reverse complement of the nucleotide sequence of the coding strand of the first DNA. The term “reverse complement” with respect to a nucleotide sequence in the 5′-3′ direction should be understood to mean a nucleotide sequence in the 3′-5′ direction wherein, in accordance with the base pairing rules, the bases correspond to the bases of the first DNA and are in a reverse/mirrored sequence. If the nucleotide sequence of the coding strand of the first DNA is atggttc, for example, then the reverse complementary nucleotide sequence of the coding strand of the second DNA is gaaccat. This is also known as sense and corresponding antisense (reverse complementary) orientation of the nucleotide sequences.
In particular, the nucleotide sequence of the coding strand of the second DNA can be the reverse complement of the nucleotide sequence of the coding strand of the first DNA over the whole length of the sequence. However, it can also be only partially reverse complementary, i.e. reverse complementary over a limited length. The nucleotide sequence of the coding strand of the second DNA can also be reverse complementary in more than one region, for example in two or three regions of its nucleotide sequence to the nucleotide sequence of the coding strand of the first DNA.
Starting from the completely or partially reverse complementary nucleotide sequences for the coding strand of the first and second DNA, a double-stranded RNA is produced. The double-stranded structure of the RNA arises by the formation of bridging hydrogen bonds between complementary nucleotides. Double-stranded RNA regions may be formed over a single nucleic acid strand which is partially complementary to itself, or over two different, discontinuous complementary nucleic acid strands. The bridging hydrogen bond formation may thus be intramolecular as well as intermolecular.
In accordance with the invention, the first DNA comprises a nucleotide sequence in accordance with SEQ ID NOS: 1-43, wherein these sequences are nucleotide sequences from selected target genes from Phytophthora infestans. The group formed by these target genes comprises essential genes for primary metabolism as well as for amino acid synthesis, in particular the biosynthesis of aliphatic amino acids (valine, leucine, isoleucine) as well as for glutamate biosynthesis, genes for cell regulation and signal transduction as well as redox regulation, calcium signalling, G-protein signalling, MAP-kinase signalling and transcription factors, as well as genes for translation components, gene with RNA processing functions, genes which code for developmental and differentiation proteins such as, for example, with cell wall formation functions, as well as genes which code for transporters, channelling and membrane proteins. A summary of these target genes from Phytophthora which are used for designing the host-induced gene silencing is set out in Table 1.
The term “gene silencing” or silencing describes processes for switching genes off. Silencing can, for example, be transcriptional or post-transcriptional. Gene silencing also includes antisense technology, RNAi, or dsRNA.
The expression of a nucleotide sequence of a target gene in Phytophthora infestans is selectively inhibited by gene silencing. A target nucleotide sequence can in this case also be a non-processed RNA molecule, an mRNA or a ribosomal RNA sequence.
The target genes were identified by (i) publically available expression studies such as microarray data regarding oomycete differentiation or infection processes, for example, and publically available data on the investigation of metabolic processes during oomycete differentiation or infection (Grenville-Briggs et al. 2005, Judelson et al. 2009a, Judelson et al. 2009b) (A), (ii) comparative bioinformatic studies coupled with pedantic analysis (BioMax Bioinformatic Framework) (B), (iii) analyses of metabolic pathways coupled with pedantic analysis (C) as well as (iv) evaluations of publically available data regarding the characterization of homologous genes in eukaryotic organisms (Roemer et al. 1994, Inoue et al. 1995, Mazur et al. 1995, Lesage et al. 2004, Avrova et al. 2008, Wang et al. 2009, Li et al. 2010, Wang et al. 2010) (D).
When selecting the target genes, care was taken that the nucleotide sequence of these genes was specific for P. infestans in order to exclude unwanted silencing of plant and human genes. To this end, the selected target genes were compared as regards their proteins (BlastX) with the proteome of Solanum tuberosum and Solanum lycopersicum. At the same time, the target gene sequences were compared as regards their nucleotides (BlastN) with the genome of Solanum tuberosum, Solanum lycopersicum and a general BlastN (criteria: BlastN; database: human genomic+transcript; optimize for: somewhat similar sequences (blastn)). Target genes were considered to be highly suitable when they exhibited no nucleotide homologies with Solanum tuberosum and Solanum lycopersicum and no or only partial homologies in general BlastN in only short sequence regions (<17 nts), so that an interaction with endogenous plant nucleotide sequences was inhibited or did not occur.
In accordance with the invention, the nucleotide sequences used may have different lengths. Thus, the nucleotide sequences of one of SEQ ID NOS: 1-43 may, for example, have a length of between 501 and 735 nucleotides.
The nucleotide sequences used may also be one or more fragments of one or more nucleotide sequences of SEQ ID NOS: 1-43. In this regard, the fragments comprise at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200 or 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400 or 2500 successive nucleotides of one or more of the nucleotide sequences of SEQ ID NOS: 1-43. A particularly suitable fragment is a fragment of the nucleotide sequence of SEQ ID NO: 1 with 290 nucleotides.
In a preferred embodiment of the invention, combinations of two, three, four, five, six, seven, eight, nine, ten or more fragments of the same nucleotide sequence as that of SEQ ID NO: 1 or different nucleotide sequences such as those of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43 are used. A preferred combination comprises fragments of the nucleotide sequences of SEQ ID NOS: 4, 23, 27 and 28, the genes of which are involved with signal transduction. A further preferred combination comprises fragments of nucleotide sequences of SEQ ID NOS: 3, 16 and 17; these are genes for glutamate biosynthesis from P. infestans. Further advantageous combinations comprise nucleotide sequences or fragments of nucleotide sequences from genes for cell wall formation (SEQ ID NOS: 25, 36, 37), calcium signalling (SEQ ID NOS: 20, 21, 22), primary metabolism genes (SEQ ID NOS: 5, 6, 7), redox regulation genes (SEQ ID NOS: 24, 25, 26), or comprise several nucleotide sequences or fragments of nucleotide sequences from transporter genes in accordance with SEQ ID NOS: 39, 40, 41, 42. Further preferred combinations comprise nucleotide sequences or fragments of nucleotide sequences of different target gene groups such as, for example, genes for G-protein signalling, MAP kinase signalling, primary metabolism and redox regulation (SEQ ID NOS: 27, 28, 4, 23). Combining several target genes means that the possibility that the resistance of the transgenic plant could be disrupted by a natural mutation in the oomycete is avoided.
The double-stranded first DNA introduced into the potato plant of the invention may comprise a nucleotide sequence which hybridizes under stringent conditions with one of the following nucleotide sequences: (a) a nucleotide sequence in accordance with SEQ ID NOS: 1-43, or (b) a fragment of at least 15 successive nucleotides of a nucleotide sequence in accordance with SEQ ID NOS: 1-43, or (c) a nucleotide sequence which is complementary to one of the nucleotide sequences of (a) or (b), or (c). Examples of stringent conditions are: hybridizing in 4×SSC at 65° C. and then washing several times in 0.1×SSC at 65° C. for approximately 1 hour in total. The term “stringent hybridization conditions” as used here can also mean: hybridization at 68° C. in 0.25 M sodium phosphate, pH 7.2 7% SDS, 1 mM EDTA and 1% BSA for 16 hours and subsequently washing twice with 2×SSC and 0.1% SDS at 68° C.
The present invention also in particular encompasses such fragments of nucleotide sequences which have a few, for example 1 or 2 nucleotides, which are not complementary to the target gene sequence from Phytophthora infestans. Sequence variations which, for example, occur in oomycetes, which are based on a genetic mutation, for example by addition, deletion or substitution or a polymorphism in a Phytophthora infestans strain and which result in wrong pairing over a region of 1, 2 or more nucleotides, can be tolerated as long as the RNA formed by the transgenic potato plant can still interfere with the target gene RNA formed by the oomycete.
In accordance with the invention, the transgenic plant of the species Solanum tuberosum confers a pathogen resistance to an oomycete of the genus Phytophthora. To determine the resistance, the transgenic potato plant is compared with a control plant which ideally has the identical genotype to the transgenic plant and has been grown under identical conditions, but which does not contain the DNA which has been introduced into the transgenic plant. The resistance can be determined using an optical score, wherein scores of 0 (not susceptible) to 100 (very susceptible) are awarded. Preferably, the transgenic plants of the invention confer a resistance which, compared with a control plant, results in a reduced propagation of infection over the plant surface of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 percent (see the conditions under “measuring the resistance in transgenic potato plants under outdoor conditions).
The genus Phytophthora comprises various species, for example the species alni, cactorum, capsici, cinnamomi, citrophthora, clandestina, fragariae, hedraiandra, idaei, infestans, ipomoeae, iranica, kernoviae, mirabilis, megakarya, nicotianae, palmivora, parasitica, phaseoli, ramorum, pseuodotsugae, quercina, sojae or tentaculata.
In a preferred embodiment of the invention, the transgenic potato plant exhibits a resistance to Phytophthora infestans.
Already the inhibition of the biosynthesis of the aliphatic amino acids valine, leucine and isoleucine by a construct directed against acetolactate synthase from P. infestans results in a substantial reduction in blight infestation and a drastic increase in leaf resistance under laboratory and field conditions. By combining several target genes such as, for example, genes from G protein signalling, MAP kinase signalling, primary metabolism, amino acid biosynthesis and redox regulation in one construct, the resistance effect can be increased still further, since the pathogen is inhibited by the multiple action of the combination construct on the use of alternative signalling and metabolic pathways.
Surprisingly, the defensive power of the potato plants of the invention against isolates of P. infestans of varying aggressivity is changed in a manner such that a resistance is obtained which protects the plants efficiently and permanently against this most important of pathogens.
It was also surprisingly discovered that the resistance has no great energetic disadvantages or negative changes in the agronomic properties of the potato plant. The cultivation trials under near-field conditions did not have any deleterious effects on the quality of the plants. Careful selection of the target genes of P. infestans which excludes any homology extending over 17 successive base pairs with genes from non-target organisms (potato, human, pig, cattle) means that there are no restrictions on using the plants as a feed or foodstuff and no restrictions as regards sowing, cultivation, harvesting or processing the crop. The plants can freely be used as agricultural, food or feed plants.
In accordance with a preferred embodiment, the double-stranded RNA is miRNA or siRNA. MiRNA describes small interfering RNA and includes naturally produced miRNAs and synthetic miRNAs which, for example, can be produced by recombinant or chemical synthesis or by processing primary miRNA.
SiRNA describes small interfering RNA and includes naturally produced siRNAs and synthetic siRNAs which, for example, can be produced by recombinant or chemical synthesis or by processing dsRNAs.
The transgenic plant produces dsRNA from the introduced double-stranded DNA which is processed by endogenous RNAi or silencing mechanisms to form siRNAs and miRNAs.
In order to obtain dsRNA, a double-stranded first DNA with a nucleotide sequence in accordance with one of SEQ ID NOS: 1-43 or a fragment thereof in the sense orientation and a double-stranded second DNA in the antisense orientation can be used which are separated by an intron which has no similarity with the target genes in question. As an example, the DNA may be orientated with a nucleotide sequence of SEQ ID NO: 1 against the acetolactate synthase gene from Phytophthora infestans. Upon expression in a plant cell, an RNA transcript is formed which, because of the homology between the sense and antisense sequence regions, can coalesce to form a dsRNA. Because the missing base pairs in the region of the intron, the dsRNA forms a hairpin structure. A dsRNA with a hairpin structure can also be prepared by means of one double-stranded DNA with a nucleotide sequence in accordance with one of SEQ ID NOS: 1-43 in the sense orientation and a second in the antisense orientation with a different length. In this respect, the nucleotide sequence in the sense orientation may be about 190 nucleotides longer than the nucleotide sequence in the antisense orientation, or vice versa.
Defined sequence regions for the selected nucleotide sequences of the target genes are amplified by PCR and cloned both in the sense and in the antisense direction into a vector which is suitable for the synthesis of hairpin structures. In this regard, several fragments with sequence regions of different target genes can be cloned into a vector in order to construct a combination hairpin construct. The vectors can be introduced into a plant cell using transformation methods which are known in plant biotechnology. The skilled person will be aware that, for example, a selected nucleotide sequence of a target gene can also be cloned into one vector in the sense orientation and the nucleotide sequence of the target gene can be cloned into a second vector in the antisense orientation and then introduced into a plant cell by co-transformation, for example.
The silencing mechanism arises from dsRNA such as, for example, hairpin RNA structures or gene duplexes. The dsRNA will produce small dsRNAs by means of a dsRNA-specific endonuclease (dicer), which are processed by means of longer nucleotide sequences into small dsRNAs preferably of 21-25 base pairs, a process which is similar for both “stem-loop” (primary miRNA) and also for long complementary dsRNA precursors. Argonaut proteins, as central components of the RNA-induced silencing complexes (RISC), bind and unwind siRNA and miRNA so that the lead strand of the duplex binds specifically by base pairing to the mRNA and leads to its degradation. By means of miRNA, RNAi behaves in a comparatively similar process, with the difference that the miRNA produced also comprises partial regions which are not identical to the target genes.
After infestation of a host plant with Phytophthora infestans, an exchange of RNA formed in the plant which is directed against one or more Phytophthora-specific target sequences can occur between the host plant and the oomycetes. In the oomycetes, these RNAs can lead to sequence-specific gene silencing of one or more target genes. Proteins and protein complexes such as dicers, RISC (RNA-induced silencing complex) as well as RNA-dependent RNA polymerase (RdRP), can participate in this process.
The siRNA effect is known to be continued in plants when the RdRP synthesises new siRNAs from the degraded mRNA fragments. This secondary or transitive RNAi can reinforce silencing and also result in silencing of different transcripts when they share these highly conserved sequences.
In a preferred embodiment, the first DNA and the second DNA are operatively linked with at least one promoter.
A “promoter” is a non-translated DNA sequence, typically upstream of a coding region which contains the binding site for the RNA polymerase and initiates transcription of the DNA. A promoter contains special elements which function as regulators for gene expression (for example cis-regulatory elements). The term “operatively linked” means that the DNA which comprises the integrated nucleotide sequence is linked to a promoter in a manner such that it allows expression of this nucleotide sequence. The integrated nucleotide sequence may be linked with a terminator signal downstream as a further component.
The promoter can be of plant, animal or microbial origin, or it may be of synthetic origin and can, for example, be selected from one of the following groups of promoters: constitutive, inducible, development-specific, cell type-specific, tissue-specific or organospecific. While constitutive promoters are active under most conditions, inducible promoters exhibit expression as a result of an inducing signal which, for example, may be issued by biotic stressors such as pathogens or abiotic stressors such as cold or dryness or chemicals.
Examples of promoters are the constitutive CaMV 35S promoter (Benfey et al., 1990) as well as the C1 promoter which is active in green tissue (Stahl et al., 2004).
The first and second DNA may also, however, be operatively linked to a double promoter such as, for example, the bidirectionally active TR1′ and TR2′ promoter (Saito et al., 1991).
Furthermore, the first and the second DNA may each be operatively linked to a promoter.
The use of two promoters, which each flank the 3′ end and the 5′ end of the nucleic acid molecule, enables expression of the respective individual DNA strand, wherein two complementary RNAs are formed which hybridize and form a dsRNA. In addition, the two promoters can be deployed such that one promoter is directed towards the transcription of a selected nucleotide sequence and the second promoter is directed towards the transcription of a nucleotide sequence which is complementary to the first nucleotide sequence. As long as both nucleotide sequences are transcribed, a dsRNA is formed.
Further, a bidirectional promoter can be deployed which allows the expression of two nucleotide sequences in two directions, wherein one nucleotide sequence is read off in the 3′ direction and a second nucleotide sequence is read off in the 5′ direction. As long as both nucleotide sequences are complementary to each other, a dsRNA can be formed.
The present invention also concerns parts of a transgenic plant of the species Solanum tuberosum.
In the context of this application, the term “parts” of the transgenic plant in particular means seeds, roots, leaves, flowers as well as cells of the plant of the invention. In this regard, the term “cells” should be understood to mean, for example, isolated cells with a cell wall or aggregates thereof, or protoplasts. “Transgenic parts” of the transgenic plant also means those which can be harvested, such as potato tubers, for example.
Furthermore, the present invention concerns a method for the manufacture of a transgenic plant of the species Solanum tuberosum which exhibits a resistance against an oomycete of the genus Phytophthora.
Suitable methods for the transformation of plant cells are known in plant biotechnology. Each of these methods can be used to insert a selected nucleic acid, preferably in a vector, into a plant cell in order to obtain a transgenic plant in accordance with the present invention. Transformation methods can include direct or indirect methods for transformation and can be used for dicotyledenous plants and primarily also for monocotyledenous plants. Suitable direct transformation methods include PEG-induced DNA uptake, liposome-induced transformation, biolistic methods by means of particle bombardment, electroporation or microinjection. Examples of indirect methods are agrobacterium-induced transformation techniques or viral infection by means of viral vectors.
A preferred method which is employed is agrobacterium-induced DNA transfer using binary vectors. After transformation of the plant cells, the cells are selected on one or more markers which were transformed in the plant with the DNA of the invention and comprise genes which preferably induce antibiotic resistance such as, for example, the neomycin phosphotransferase II gene NPTII, which induces kanamycin resistance, or the hygromycin phosphotransferase II gene HPTII, which induces hygromycin resistance.
Next, the transformed cells are regenerated into complete plants. After DNA transfer and regeneration, the plants obtained may, for example, be examined by quantitative PCR for the presence of the DNA of the invention. Resistance tests on these plants against Phytophthora infestans in vitro and in the greenhouse are next. Routine further phenotypic investigations can be carried out by appropriately trained personnel in the greenhouse or outdoors. These transformed plants under investigation can be cultivated directly.
The method of the invention for the manufacture of a transgenic plant of the species Solanum tuberosum which exhibit a resistance against an oomycete of the genus Phytophthora comprises the following steps:
In accordance with the invention, it is a nucleotide sequence or a fragment of a nucleotide sequence in accordance with SEQ ID NOS: 1-43 from Phytophthora infestans.
In a preferred embodiment of the invention, the double-stranded RNA can be miRNA or siRNA.
The invention also concerns a composition for external application to plants.
This composition is prepared for external application to plants. It contains double-stranded RNA, wherein one strand of this RNA corresponds to the transcript of a double-stranded DNA comprising (a) a nucleotide sequence in accordance with SEQ ID NOS: 1-43, or (b) a fragment of at least 15 successive nucleotides of a nucleotide sequence in accordance with SEQ ID NOS: 1-43, or (c) a nucleotide sequence which is complementary to one of the nucleotide sequences of (a) or (b), or (d) a nucleotide sequence which hybridizes with one of the nucleotide sequences of (a), (b) or (c) under stringent conditions.
Double-stranded RNA for the manufacture of the composition in accordance with the invention can be produced in vitro using methods known to the skilled person. As an example, the double-stranded RNA can be synthesized by forming the RNA directly in vitro. The double-stranded RNA can also be synthesized from a double-stranded DNA by formation of an mRNA transcript which then forms a hairpin structure, for example.
The composition in accordance with the invention can be used as a fungicide for a plant or its seed. In this regard, the composition is used to control the growth of the pathogen, for containing the propagation of the pathogen or for the treatment of infected plants. As an example, the composition can be used as a fungicide for spraying in the form of a spray, or other routine ways which are familiar to the skilled person for external application to the plant tissue or by spraying or mixing with the cultivation substrate before or after the plants have sprouted.
In a further application, the composition in accordance with the invention is used as a pre-treatment for seed. In this regard, the composition is initially mixed with a carrier substrate and applied to the seeds in a combination which comprises the double-stranded RNA and the carrier substrate, whereby the carrier substrate has an RNA-stabilizing effect, for example. Thus, the RNA stability and thus its action on the selected target genes of Phytophthora infestans can be increased, for example by chemical modifications such as the exchange of ribose for a hexose. Liposomes which encapsulate the RNA molecules can also be used as RNA stabilizers.
Ideally, the plants treated with the composisiton are those of the species Solanum tuberosum.
The discussion above regarding the plant of the invention and the method of the invention also apply to this composition.
The present invention will now be described with reference to the figures and sequences:
Detection of sense fragment (370 bp) (primer S334 5′-ATCCCACTATCCTTCGCAAG-3′ (SEQ ID NO: 44)×S1259 5′-TTGATATCGCGGAAGGCGAGAGACATCG-3′ (SEQ ID NO: 45)) and antisense fragment (450 bp) (S 329 5′-CTAAGGGTTTCTTATATGCTCAAC-3′ (SEQ ID NO: 46)×S1259 5′-TTGATATCGCGGAAGGCGAGAGACATCG-3′ (SEQ ID NO: 45)). Mix: PCR-MasterMix, PCR monitoring. Marker: Tracklt™ 1 Kb DNA Ladder.
Preparation of Constructs
Defined sequence regions of the selected target genes were amplified using PCR and cloned in both the sense and the antisense direction into a pRNAi vector which is suitable for the system of hairpin structures (
Starting from genomic DNA from Phytophthora infestans, a sequence region of 290 bp from the coding region of the gene PITG_03410 was amplified using PCR, cleaved via the restriction enzyme cleaving sites XhoI and SmaI inserted via the primer sequences and cloned into the pRNAi vector (primer 1: cgctcgaggctggatctcgcgctgaggt (SEQ ID NO: 47), primer 2: ttgatatcgcggaaggcgagagacatcg (SEQ ID NO: 45)). This vector contains a CaMV 35S promoter, a multiple cloning site, an intron from the gene AtAAP6 which codes for an amino acid permease in Arabidopsis thaliana, a further multiple cloning site as well as a CaMV 35S terminator. This was cleaved with XhoI and Ec1136II and the 4.098 kb vector fraction was separated using agarose gel electrophoresis and then isolated. The ligation solution was transformed in E. coli strain XL1-blue (Stratagene, LaJolla, Calif.). The same PITG_03410 fragment was then cloned into the plasmid pRNAi_PITG_03410_sense in the antisense direction. To this end, the fragment was again amplified from genomic DNA from Phytophthora infestans using PCR, cleaved via the restriction enzyme cleaving sites XhoI and SmaI inserted via the primer sequences and then ligated into the vector pRNAi_PITG_03410_sense which had been cleaved with SmaI-SaII and then linearized (
Alternatively to a vector as described above which is suitable for the synthesis of hairpin structures, a section of the coding target gene region in the potato plant can be caused to carry out expression with the aid of two oppositely (reverse) orientated promoters (
Transformation and Regeneration
Transformation of the potatoes was carried out in accordance with the modified protocol by Pel et al (2009) using the antibiotic kanamycin. The donor material was cultivated in 80 mL MS(D) (25° C.; 16 h day/8 h night; 2000 lux) for 3-4 weeks. For transformation (C1), the internodes were cut out of the donor material in approximately 0.5 cm explants. These were cultivated in petri dishes with 10 mL MS(D) (15-20 Explants/dish) with 70 μl of an Agrobacterium tumefaciens culture which had been cultivated overnight at 28° C., which had earlier been transformed with the HIGS-RNAi construct as part of a binary vector such as, for example, p95N, incubated at 25° C. for 2 days in the dark. Next, the explants were dried on filter paper and placed in petri dishes on MSW-Medium with selection antibiotic (400 mg/L timentin+75 kanamycin mg/L) which were hermetically sealed and cultivated for 2 weeks (25° C.; 16 h day/8 h night; 2000 lux) (C2). This selection step was repeated every 2 weeks until the shoots had regenerated (from C3). Regenerated shoots (
Detection of Processed Double-Stranded RNA and siRNAs
In the transformed plants, the expressed hairpin or double-stranded RNAs were processed over the natural plant RNAi mechanisms in a manner such that these RNA molecules were degraded into small single stranded RNAs. These siRNAs are deposited on the mRNA of the corresponding target gene in oomycetes and thus effect silencing of this gene. The plants are thus placed in the position of protecting themselves against attacking pathogens. By means of this concept, transgenic potato plants can be produced which have an increased resistance to P. infestans.
The transformation of potato plants with constructs for the expression of hairpin or double-stranded RNAs should result in the fact that the resulting dsRNAs are processed to siRNAs in preference to the natural plant RNAi mechanisms. In order to measure the fragmentation of the dsRNA, whole RNA was isolated from the transgenic plants using the trizol method (Chomczynski and Sacchi, 1987). 15 μg of whole RNA/sample was supplemented with formamide, denatured and separated electrophoretically in a 1% agarose gel with 10% formaldehyde in 1×MOPS buffer (0.2 M MOPS (sodium salt), 0.05 M NaOAc, 0.01 M EDTA in DEPC dH2O. pH 7.0 with NaOH). The separated RNA was transferred from the gel onto a nylon membrane (positively charged) using the Northern Blot method into 20×SSC buffer (saline-sodium citrate buffer). This was hybridized with a radioactively labelled probe which was complementary to the sequence of the target gene fragment which was present in the sense or in the antisense direction in the construct transformed into the plants. In this manner, RNA fragments which are complementary to the sequence of the dsRNA fragment are labelled and detected by means of a phosphoimager.
The transformation of potato plants with constructs for the expression of hairpin or double-stranded RNAs should result in the fact that the resulting dsRNA are processed to siRNAs in preference to the natural plant RNAi mechanisms. In order to measure the fragmentation of the dsRNA into siRNAs, whole RNA was isolated from the transgenic plants using the trizol method (Chomczynski and Sacchi, 1987). 15 μg of whole RNA/sample was supplemented with formamide, denatured and separated electrophoretically in a polyacrylamide gel with 15% Tris/boric acid/EDTA (TBE) and uric acid in 0.5×TBE. The separated RNA was transferred onto a nylon membrane (neutral) from the gel using the Tank Blot method in 0.5×TBE. This was hybridized with a radioactively labelled probe which was complementary to the sequence of the target gene fragment which was present in the sense or in the antisense direction in the construct transformed in the plants. In this manner, siRNAs which are complementary to the sections of sequence of the dsRNA fragment are labelled and detected by means of a phosphoimager.
In various transgenic potato lines such as, for example, PR-H4 lines or PR-H2 lines, such siRNAs could be detected (
Measurement of Resistance in Transgenic Potato Plants in the Detached Leaf Assay
To test the resistance of the transgenic potato leaves, the transgenic plants were cultivated from in vitro plants in the greenhouse in 5 L pots. After 6-8 weeks, 2 pinnae per plant were cut off and placed in a sealed plastic box on a moist Grodan pad such that the leaf stem was in the moist Grodan material. This ensured that the humidity was high. The boxes were incubated at 18° C. using a day/night program (sunlight, February-September). The pinna leaflets were inoculated with drops of a zoospore suspension (10 μL; 104 zoospores/mL) of Phytophthora infestans. After 24 hours, the lid of the boxes was opened somewhat in order to allow a gentle circulation of air in the boxes. The optical scoring and quantification of the zoospores was carried out after 6 days. The optical scoring evaluated the degree of infection and the destruction of the pinna leaf by P. infestans. Counting the sporangia led to quantification of the reproductive ability of the pathogen in the plant. In this regard, the previously infected leaves of a pinna were incubated in 5 mL of water in Falcon tubes on a shaker for 2 h, so that the sporangia were loosened from the leaf. The sporangia were then counted with the aid of a Thoma counting chamber under a microscope.
In various HIGS potato lines which were generated by transformation of various potato genotypes, a reduced sporangia count could be determined after infection with P. infestans (6 dpi) (
The gene PITG_03410 was tested as a target gene for HIGS in the genetic background of the potato varieties Baltica, Hermes and Desirée as well as the variety Russet Burbank using a vector as shown in
As an alternative to vectors which are suitable for the synthesis of hairpin structures, a vector as shown in
Potato plants of the variety Russet Burbank which had been transformed with a combination vector against the genes PITG_00146, PITG_00708, PITG_10447 and PITG_08363 of
Similarly, the detached leaf assay showed that the reproductive ability of P. infestans on transgenic plants of the variety Russet Burbank is restricted when transformed with a vector in accordance with
Even on transgenic plants of the variety Desirée which were transformed with a vector as shown in
Transient Test System for RNAi Vectors in Potato Leaves
A transient test system in accordance with Birch et al. (2010) was developed to investigate the functionality of the RNAi vectors against selected target gene sequences of P. infestans. By means of co-bombardment, a RNAi vector targeting a target gene was expressed transiently in potato leaves together with a fusion construct consisting of a luciferase reporter gene and the test target fragment. If the dsRNA construct is processed in the RNAi vector, then the formation of dsRNA and the resulting formation of siRNAs should be ensured. These siRNAs should not only carry out the degradation of the target gene fragment transcript, but also give rise to the fused reporter gene transcript, so that with a functional RNAi construct, a reduction in the luciferase activity can be observed. The plasmid pABM-70Sluci comprises a double CaMV 35S promoter, a multiple cloning site, the coding sequence for the luc gene from Photinus pyralis, which codes for a luciferase, separated from a modified intron PIV2 from the potato gene St-LS1 (Eckes et al. 1986, Vancanneyt et al. 1990), a further multiple cloning site as well as a Nos terminator from the nopalin synthase gene from Agrobacterium tumefaciens. The PCR-amplified fragment of the coding sequence region, for example of the PITG_03410 gene, was cloned into this plasmid pABM-70Sluci, which was also cloned into the pRNAi vector to produce the dsRNA construct (
This transient test system can not only be used for validation of the general functionality of the RNAi construct, but also be used in order to investigate different sequence regions of a gene as regards its silencing effect and finally can select the best sequence regions of a gene for optimal silencing. In addition, the bombardment of transgenic plants stably transformed with a RNAi construct can serve to determine the silencing efficiency of the individual transgenic HIGS potato lines which, for example, can differ substantially depending on the integration site for the construct. In various transgenic HIGS potato lines which are obtained by transformations in various potato genotypes, and which show a reduced sporangia count after infection with P. infestans, a reduction in the luciferase activity could also be measured (
When the coding gene sequences which are to be silenced by a combination construct such as pRNAi_HIGS-CoA, for example (target genes: PITG_08393, PITG_00146, PITG_10447, PITG_00708), each cloned into the vector pABM_70 Sluci behind the coding sequence for the luc gene from Photinus pyralis (pABM_70 Sluci_PITG_08393, pABM_70 Sluci_PITG_00146, pABM_70 Sluci_PITG_10447, pABM_70 Sluci_PITG_00708) and together with the vector pRNAi_HIGS_CoA, are to be transiently expressed in potato leaves, the silencing efficiency of the combination construct can be analysed on the various target genes. This is also possible by the bombardment of transgenic plants stably transformed with the RNAi combination construct with the individual fusion constructs consisting of the luciferase reporter gene and the test coding sequences of the various target genes.
The luciferase activity determinations were carried out with the aid of Dual Luciferase® Reporter Assays (Promega, Mannheim) (Schmidt et al. 2004).
Measurement of Resistance in Transgenic Potato Plants Under Outdoor Conditions
For the resistance test for the transgenic potato plants under outdoor conditions, the transgenic plants were initially cultivated early in the year (March) from in vitro plants in the greenhouse for 3 weeks in multiport pads. Next, these plants were planted out into a greenhouse with a wire mesh roof in natural soil so that the plants were exposed to environmental conditions, for example temperature, sunlight, precipitation and humidity, which were comparable with field conditions. The plants were planted out in 3 plots each with 6 plants. After 8 weeks, one pinna from each of 2 plants in a plot was inoculated with P. infestans by spray inoculation (750 μL; 104 zoospores/mL). Plastic bags were placed over these pinnae to ensure that the humidity would be high and to promote infection. After two days, these plastic bags were removed. Proliferation of the blight by Phytophthora infestans in the greenhouse was scored optically and documented photographically every week. The criteria for scoring the infection was initially only on the infected pinna leaf (0: no infection, 1: slight infection (½ number of infected pinna leaves infected), 2: infection on more than ½ leaves of a pinna, 3: infection on all leaves of the pinna) and then the spread of the infection to the plant and the whole plot (4: infection also extends to some other leaves of the plant, 6: infection also extends to other plants, 8: infection also extends substantially to other plants, 10: 10% of the plants infected/destroyed, 20: 20% of plants infected/destroyed, 100: 100% of the plants infected/destroyed).
In various transgenic HIGS potato lines (PR-H-4-7, PR-H-4-11) which were obtained by transformations in the potato genotype Hermes, a greatly reduced degree of infection of these plants during the course of infection with Phytophthora infestans was determined compared with the transformation genotype Hermes which had been cultivated, planted out and infected as the control exactly as with the transgenic plants. The reduced degree of infection was initially reflected by a greatly reduced infection capability of the pathogen on the inoculated pinnae (scores 21 days post-infection: PR-H-4-7: 3.3; PR-H-4-11: 3.2; Hermes 7.6) and at later times by a substantially reduced propagation ability of the pathogen to these plants (scores 32 days post-infection: PR-H-4-7: 26; PR-H-4-11: 15; Hermes: 80) (
By means of the tests described, not only could processing of the HIGS construct in transgenic potato plants to siRNAs be demonstrated, but also the functionality of these constructs in respect of silencing of the target gene sequence in these transgenic plants, and an increased resistance of these plants to P. infestans could be quantified by a reduced sporangia production of the pathogen on these host plants. Since the identification of functional HIGS target genes is not possible without careful testing of their functionality, these analyses as described in detail here are particularly suitable for defining genes which are effective in the HIGS construct and for generating resistant HIGS plants.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2012 016 009 | Aug 2012 | DE | national |
| Filing Document | Filing Date | Country | Kind |
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| PCT/DE2013/000446 | 8/6/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/023285 | 2/13/2014 | WO | A |
| Number | Name | Date | Kind |
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| 8299318 | Brover et al. | Oct 2012 | B2 |
| 20100257634 | Bailey et al. | Oct 2010 | A1 |
| 20110167514 | Brover et al. | Jul 2011 | A1 |
| 20150082495 | Delebarre | Mar 2015 | A1 |
| Number | Date | Country |
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| 101107362 | Jan 2008 | CN |
| 102012016009 | Feb 2014 | DE |
| 1716238 | Nov 2006 | EP |
| 2006047495 | May 2006 | WO |
| 2006070227 | Jul 2006 | WO |
| 2009112270 | Sep 2009 | WO |
| 2014023285 | Feb 2014 | WO |
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