Patent Application
20170058292
The present invention relates to the finding that an until now orphan protein named receptor-like protein 1 (RLP1) in plants mediates an immune response to bacterial infections. Specifically, the invention relates to RLP1, now named receptor of enigmatic microbe-associated molecular pattern (MAMP) of Xanthomonas (REMAX), in Arabidopsis thaliana. REMAX was found to recognise the presence of Xanthomonas and to initiate an immune signalling that eventually yields into a typical plant immune responses to bacterial infections. Furthermore, the invention relates to chimeric pattern recognition receptors (PRRs) composed of the extracellular domain of REMAX, which is the recognition site for sensing infection, and C-terminal portions of PRRs of other plant species. Also provided is a method to modulate the immune response of a plant to a bacterial infection by either increasing or decreasing the expression of REMAX or REMAX-like proteins, or the inventive chimeric PRRs in plants. Finally, the invention provides methods for the identification and purification of new microbe-associated molecular patterns in plants using REMAX or REMAX-like proteins and/or the novel chimeric PRRs of the present invention.
Plants are exposed to microbial pathogens but, in general, they are resistant to most of these microbes. Obviously, plants have evolved efficient barriers and defense mechanisms to successfully ward off these microbial pathogens. In turn, pathogenic microbes have found ways for entering through wounds, pores and stomata or by active penetration through the leaf or root surface. However, once inside the plant host, the pathogen encounters further preformed barriers such as the plant cell wall or antimicrobial secondary metabolites (Nürnberger and Lipka, 2005). In addition to these permanent barriers, plants have an array of inducible defense systems that come into action when plants sense attack by microbes. For pathogen detection, plants possess pattern recognition receptors (PRRs) at the plasma membrane that recognize microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) via their extracellular domains and initiate intracellular signaling and induction of defense reactions. These processes are defined as PAMP-triggered immunity (PTI) (Chisholm et al., 2006; Jones and Dangl, 2006). Recognition of MAMPs induces multiple cellular responses such as altered ion fluxes across the plasma membrane causing extracellular alkalinization and increased concentrations of the cytoplasmatic Ca2+ (Boller and Felix, 2009) and biosynthesis of the stress hormone ethylene caused by the fast activation of the ACC-synthase (Spanu et al., 1994). A further early response is the induction of mitogen-activated protein kinase (MAPK) cascades causing transcriptional activation of transcription factors (Nühse et al., 2000; Asai et al., 2002). Thereby, the transcription of genes encoding proteins such as defensins (antimicrobial proteins), lytic enzymes or enzymes for the synthesis of phytoalexins (anti-microbial secondary metabolites) is induced (Nürnberger et al., 2004). Additional typical plant defense reactions are callose deposition at the plant cell wall and the production of reactive oxygen species (ROS) that can be toxic for pathogens and cause cross-linking of the plant cell wall (Apel and Hirt, 2004). To counteract PTI, some pathogens have evolved effectors that suppress this response. This process is called effector-triggered susceptibility (ETS). In turn, some plants hold specialized R (resistant) proteins that recognize these effectors. This recognition causes effector-triggered immunity (ETI) which in general leads to hypersensitive response (HR), a cell death of the plant tissue surrounding the infection site (Dangl and Holub, 1997), and thus limits the pathogen to the infection site (Chisholm et al., 2006; Jones and Dangl, 2006; Caplan et al., 2008). Many R proteins belong to the NB-LRR protein family that consists of a nucleotide binding site (NB) and a leucine-rich repeat (LRR) domain. These NB-LRRs can either interact directly with effectors via their LRR domains or indirectly via an additional protein (host factor) which binds the pathogen effector.
For initiating active defense reactions it is crucial that plant hosts can detect the attack by potential pathogens. As is the case for the well studied innate immune system of animals, plants can recognize chemical structures that are characteristic for microbial organism in general. In analogy also, these structures that serve as signals for the immune system of plants are termed MAMPs for microbe-associated molecular patterns. MAMPs often represent highly conserved molecular structures that carry essential functions for the microbes. Typically, these MAMPs are representative for whole classes of microbes but do not occur in the plant host. Recognition of MAMPs follows principles of recognition of ‘non-self’ (Medzhitov and Janeway, 2002; Nürnberger et al., 2004; Zipfel, 2008).
Pattern recognition receptors (PRRs) on the plant cell surface recognize MAMPs and DAMPs and transduce the signal into the cell. Until now several receptors have been identified belonging to the leucine-rich repeat receptor-like kinase (LRR-RLK) and protein (LRR-RLP) family and to the LysM-RLKs/RLPs.
The Gram-negative bacterium Xanthomonas axonopodis pv. citri (Xac) is a plant pathogen that causes citrus canker on most Citrus species. Originating in Asia, Xac currently causes Canker A also in South America, USA and Australia (Graham et al., 2004) with considerable economic losses (Gottwald et al., 2002). Xac enters the plant through stomata or lesions. About 9 days post infection blister-like lesions appear on the leaves that later turn brown with water-soaked margins, necrotic surrounded, becomes visible. The lesion center becomes raised and corky. Xac can also proliferate and cause similar symptoms on fruits, thus rendering the fruits unmarketable. Bacteria proliferate and exude from stomata during wet weather. Xac is then distributed mainly by wind-driven rain (Graham et al., 2004). Many members of the Xanthomonas genus show a high host range and race specificity with most strains restricted to a small number of hosts (Brunings and Gabriel, 2003). Xanthomonas axonopodis pv. citri is limited to Citrus and relatives of Citrus in the family Rutacea.
Several MAMPs and their corresponding pattern recognition receptors have been described so far. However, there are indications for many additional MAMPs for which the corresponding PRRs have not been identified. Some PRRs are restricted to specific plants families and a couple of MAMPs are in some microbes altered or masked, consequently are not all plants able to recognize all microbes. Therefore, transformation of PRRs to other plant species can increase the resistance to pathogens as shown for the transformation of tomato and tobacco with the PRR EFR (Lacombe et al., 2010).
In view of the above, it is an object of the present invention to provide novel means to enhance the resistance of plants to bacterial infections, specifically to infections with the pathogenic bacterium Xanthomonas.
In a first aspect of the present invention, the above object is solved by a chimeric pattern recognition receptor (PRR) for recognizing plant pathogen-associated molecular patterns, comprising at least an ectodomain, a juxtamembrane domain, a transmembrane domain and a cytoplasmatic domain, characterized in that the ectodomain and at least one of the other domains are derived from different PRRs.
In the context of the present invention the term “ectodomain” shall refer to the extracellular parts of a transmembrane receptor which harbour the receptor's function to bind to its ligands. Specifically, in certain embodiments of the present invention, the term “ectodomain” shall preferably refer to the LRR domain of a plant PRR.
In the context of the present invention and in all of its embodiments the term “pathogen-associated molecular patterns” shall refer to any molecular structure derived from a plant pathogenic organism and/or any other micro organism, such as bacteria, fungi or unicellular eukaryotic organisms and/or small multi-cellular organisms.
In the context of the present invention the term “transmembrane domain” shall refer to parts of a transmembrane receptor which form a stable fold in a cellular membrane. The term shall refer in case of single pass transmembrane receptors only to the domain which spans the cellular membrane. In case for multi pass transmembrane domains the term also denotes the region containing multiple single transmembrane domains.
In the context of the present invention the term “juxtamembrane domain” shall refer to the stretch of amino acids in a transmembrane receptor which is located on the extracellular side of the receptor, directly adjacent to the transmembrane domain. The juxtamembrane domain therefore connects the ectodomain with the transmembrane domain.
In a preferred embodiment the ectodomain of the chimeric receptor of the present invention is derived from the protein shown in SEQ ID No. 1, or a protein that is at least 50% identical to the protein shown in SEQ ID No. 1.
The ectodomain of the chimeric PRR according to the present invention is in another embodiment preferably derived from one PRR-PRR “A”—whereas the cytoplasmatic domain, the juxtamembrane domain and/or the transmembrane domain are derived from another (a different) PRR-PRR “B”. Preferably the different PRRs (“A” and “B”) are derived from different plant species.
The chimeric PRR according to the present invention is for use in the treatment and/or protection of a plant against a bacterial infection, preferably against a Xanthomonas infection.
In a further preferred embodiment the chimeric PRR of the invention comprises an ectodomain that has a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identical to the ectodomain of REMAX—SEQ ID No. 1 or At1g07390.
Furthermore preferred is a chimeric PRR according to the invention, wherein the juxtamembrane domain, transmembrane domain and/or the cytoplasmatic domain are derived from PRRs selected from leucine-rich repeat receptor-like kinases (LRR-RLK), LRR receptor-like proteins (LRR-RLP) or LysM-RLK/RLP.
Finally, in a further embodiment of the first aspect of the present invention, the chimeric PRR, comprises a juxtamembrane domain, transmembrane domain and/or a cytoplasmatic domain which each comprise a sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to LeEix2 (SEQ ID No. 2), preferably to the c-terminal part of LeEix2, more preferably to the juxtamembrane domain, transmembrane domain and/or a cytoplasmatic domain domains of LeEix2 (SEQ ID No. 2).
The object of the present invention is furthermore solved in a second aspect by a method for modulating the resistance of a plant to a pathogen infection, comprising, modulating in said plant the expression of a protein comprising an amino acid sequence of at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity to REMAX. Preferably, wherein REMAX is a protein comprising the sequence shown in SEQ ID No. 1.
In one embodiment a method is preferred, wherein modulating constitutes either an increase or a decrease of the resistance of a plant. An increase of expression of said protein results in the increase of the resistance of said plant to a pathogen infection. The decrease of expression results in a decrease of the resistance of said plant to a pathogen infection. Most preferably the expression of said protein in said plant is increased by ectopic expression of said protein.
On the other hand, and in another preferred embodiment, the expression of said protein in said plant is decreased by mutagenesis, RNA interference or RNA mediated DNA methylation. Such techniques are well known to the person of skill in the art.
RNA mediated silencing mechanisms can interfere with gene expression at different levels: some RNA-directed mechanisms act at a post-transcriptional level through degradation of targeted messenger RNAs. However, dsRNA-derived species can also direct changes in the chromatin structure of DNA regions with which they share sequence identity. For example, plants use such RNA species to lay down cytosine methylation imprints on identical DNA sequences, providing a fundamental mark for the formation of transcriptionally silent heterochromatin. This process is generally referred to as RNA-directed DNA methylation (RdDM).
RdDM is initiated by the presence of double stranded RNA (dsRNA) molecules in the cell nucleus. They potentially trigger the de novo methylation of all cytosine bases which are located in DNA regions complementary to the sequence of the RNA double strand. As a consequence, in mammals and in plants, the methylated DNA positions serve as flags for the remodelling of the surrounding chromatin in a way, that dense heterochromatin can be formed at these loci. Due to the dense chromatin environment other proteins are prohibited from contacting the DNA. In particular transcription factors or components of the transcriptional machinery cannot assemble on the methylated promoter sequences and thus no transcription can occur in these regions. In effect, genes that have methylated regulatory sequences are less transcribed and therefore less expressed.
Preferably, the modulation of the expression of REMAX, or a REMAX-like protein or a chimeric PRR of the invention is done by RNA mediated DNA methylation targeting a sequence selected from, but is not limited to, an endogenous regulatory sequence that regulates plant DNA transcription. As used herein, the term “regulatory sequence” means a nucleotide sequence that, when operatively linked to a coding region of a gene, affects transcription of the coding region such, that a ribonucleic acid (RNA) molecule is transcribed from the coding region. A regulatory element generally can increase or decrease the amount of transcription of a nucleotide sequence, for example, a coding sequence, operatively linked to the element with respect to the level at which the nucleotide sequence would be transcribed absent the regulatory element. Regulatory elements are well known in the art and preferably include promoters, enhancers, silencers, inactivated silencer intron sequences, 3′-untranslated or 5′-untranslated sequences of transcribed sequence, preferably a poly-A signal sequence, or other protein or RNA stabilizing elements, insulators which restrict the regulatory effect of these sequences to defined regions, or other gene expression control elements known to regulate gene expression or the amount of expression of a gene product. A regulatory element can be isolated from a naturally occurring genomic DNA sequence or can be synthetic, for example, a synthetic promoter.
The terms “polynucleotide”, “oligonucleotide,” and “nucleic acid sequence” are used interchangeably in the context of the present invention to refer to a polymeric (two or more monomers) form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Although nucleotides are usually joined by phosphodiester linkages, the term also includes polymers containing neutral amide backbone linkages composed of aminoethyl glycine units. The terms are used only to refer to the primary structure of the molecule. Thus, the term includes double stranded and single stranded DNA molecules as above. It will be recognized that such polynucleotides can be modified, for example, by including a label such as a radioactive, fluorescent or other tag, by methylation, by the inclusion of a cap structure, by containing a substitution of one or more of the naturally occurring nucleotides with a nucleotide analogue, by containing an internucleotide modification such as having uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, or the like), by containing a pendant moiety such as a protein (e.g., a nuclease, toxin, antibody, signal peptide, poly-L-lysine, or the like), by containing an intercalator such as acridine or psoralen, by containing a chelator, which can be a metal such as boron, an oxidative metal, or a radioactive metal, by containing an alkylator, or by having a modified linkage (e.g., an alpha anomeric nucleic acid).
Preferred polynucleotide species according to the present invention are selected from ssDNA, dsDNA tDNA, ssRNA, dsRNA, shRNA, siRNA, and mRNA. Preferably, the polynucleotide is a DNA that codes for a dsRNA molecule, preferably a dsRNA-hairpin. DsRNA hairpins are preferably generated by expression of a DNA construct that encodes contiguous sense and anti-sense sequences that are separated by a spacer. Upon transcription of such a construct, the generated ssRNA molecule forms a double strand by base pairing of the sense and anti-sense sequences.
The present invention provides in a further embodiment the above method, wherein said protein is a chimeric protein comprising an ectodomain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the ectodomain of REMAX (SEQ ID No. 1) and juxtamembrane domain and/or a transmembrane domain and/or a cytoplasmatic domain derived from at least one different receptor-like-protein (RLP) which is not REMAX.
A pathogen infection is preferably a bacterial infection, such as a Xanthomonas infection. Xanthomonas species in the context of all aspects and embodiments of the present invention are preferably selected from Xanthomonas axonopodis, Xanthomonas campestris, Xanthomonas stewartii, Xanthomonas oryzae and Xanthomonas translucens.
Plants for use in the above methods of the invention and in the context of the other aspects and embodiments of the present invention are pepper, rice, citrus, cotton, tomato, soybeans, tobacco. Preferably wherein the plant is susceptible to a Xanthomonas infection. Further plants for use in context with all aspects and embodiments of the present invention are corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), alfalfa (Medicago sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Rachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaultilane), fig (Ficuscasica), guava (Psidium guava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), ahnond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, duckweed (Lemna), barley, tomatoes (Lycopersicon esculentum), lettuce (e. g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals such as azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum are also included. Additional ornamentals within the scope of the invention include impatiens, Begonia, Pelargonium, Viola, Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum, Amaranthus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea, Dahlia, Datura, Delphinium, Gerbera, Gladiolus, Gloxinia, Hippeastrum, Mesembryanthemum, Salpiglossos, and Zinnia. Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata), Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsugaultilane); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata), and Alaska yellow-cedar (Chamaecyparis nootkatensis).
Yet another aspect of the present invention relates to a method for producing a transgenic plant having enhanced resistance to a bacterial infection, comprising the steps of (i) transforming a plant or plant cell with a nucleotide sequence encoding for a REMAX or REMAX-like protein comprising an amino acid sequence of at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity to SEQ ID No 1, or with a chimeric PRR according to invention as described herein.
It is preferred in a further embodiment that said chimeric PRR comprises a juxtamembrane domain and/or a transmembrane domain and/or a cytoplasmatic domain of an autologous PRR of said plant.
In another aspect the problem posed by the present invention is solved by providing a plant, characterized in that the plant expresses a chimeric PRR according to the invention as described herein.
In still another aspect of the present invention the objective is solved by a gene, comprising a nucleotide sequence encoding for a chimeric PRR according to the invention as described herein.
The term “gene” as used in the context of the invention describes any DNA sequence element that can be transcribed into RNA and that might encode for a heritable trait in an organism. Most genes are protein coding genes, wherein the nucleotide sequence of the gene codes for the amino acid sequence of the protein product. However, other genes might code for RNAs which are not translated into proteins—so called non-coding RNAs (ncRNAs). For example, ncRNA genes encode for transfer RNAs (tRNAs), or structural RNAs as found in large protein complexes like the ribosome (rRNAs). Further, the term “gene” includes coding regions for small none-coding RNAs species. Small non-coding RNA genes include snoRNAs, microRNAs, siRNAs and piRNAs and long ncRNAs that include examples such as Xist and HOTAIR.
Yet another aspect of the invention relates to an expression cassette characterized in that the expression cassette allows for the expression of a gene according to the invention.
A further preferred aspect of the present invention is then directed at a vector comprising a nucleic acid according to the present invention, for example a gene or an expression cassette according to the present invention. A vector within the meaning of the present invention is a protein or a nucleic acid or a mixture thereof which is capable of being introduced or of introducing the polynucleotides comprised into a cell. It is preferred that the proteins encoded by the introduced nucleic acid are expressed within the cell upon introduction of the vector.
In a preferred embodiment, the vector of the present invention comprises recombinant vectors, plasmids, phagemids, phages, cosmids, viruses, in particular but not limited to virus-derived amplicon vectors, potato virus X based vectors, tobacco rattle virus based vectors, geminivirus-based vectors such as cabbage leaf curl virus and barley stripe mosaic virus based vectors and vectors based on satellite viruses (reviewed in Curtin, S. J., Wang, M.-B., Watson, J. M., Roffey, P., Blanchard, C. L. and Waterhouse, P. M. (2007), chapter 12, p 291-332 in “Rice Functional Genomics; Challenges, Progress and Prospects”. Upadhyaya, Narayana M. (Ed.), ISBN: 978-0-387-48903-2), virosomes, and nucleic acid coated particles, in particular gold spheres.
The term “recombinant nucleic acid molecule” refers to a polynucleotide produced by human intervention. A recombinant nucleic acid molecule can contain two or more nucleotide sequences that are linked in a manner such that the product is not found in a cell in nature. In particular, the two or more nucleotide sequences can be operatively linked and, for example, can encode a fusion polypeptide, or can comprise a nucleotide sequence and a regulatory element. A recombinant nucleic acid molecule also can be based on, but different, from a naturally occurring polynucleotide, for example, a polynucleotide having one or more nucleotide changes such that a first codon, which normally is found in the polynucleotide, is replaced with a degenerate codon that encodes the same or a conservative amino acid, or such that a sequence of interest is introduced into the polynucleotide, for example, a restriction endonuclease recognition site or a splice site, a promoter, a DNA replication initiation site, or the like.
Preferred is a recombinant vector according to the present invention, which is an expression vector, optionally comprising one or more genes to be expressed. Preferably, said expression is driven by a regulatory sequence (or sequences). A regulatory sequence can be isolated from a naturally occurring genomic DNA sequence or can be synthetic, for example, a synthetic promoter.
Regulatory sequences can be constitutively expressed regulatory sequences, which maintain gene expression at a relative level of activity (basal level), or can be regulated regulatory sequences. Constitutively expressed regulatory sequence can be expressed in any cell type, or can be tissue specific, which are expressed only in particular cell types, phase specific, which are expressed only during particular developmental or growth stages of a plant cell, or the like. A regulatory sequence such as a tissue specific or phase specific regulatory sequences or an inducible regulatory sequence useful in constructing a recombinant polynucleotide or in a practicing a method of the invention can be a regulatory sequence that generally, in nature, is found in a plant genome. However, the regulatory sequence also can be from an organism other than a plant, including, for example, from a plant virus, an animal virus, or a cell from an animal or other multicellular organism.
A preferred regulatory sequence useful for expression of polynucleotides of the invention is a promoter element. Useful promoters include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, spatially-regulated, chemically regulated, stress-responsive, tissue-specific, viral and synthetic promoters. Promoter sequences are known to be strong or weak. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus. A bacterial promoter can be induced to varying levels of gene expression depending on the level of isothiopropyl galactoside added to the transformed bacterial cells. An isolated promoter sequence that is a strong promoter for heterologous nucleic acid is advantageous because it provides for a sufficient level of gene expression to allow for easy detection and selection of transformed cells and provides for a high level of gene expression when desired.
The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species. In some cases, expression in multiple tissues is desirable. While in others, tissue-specific, e.g., leaf-specific, seed-specific, petal-specific, anther-specific, or pith-specific, expression is desirable. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons. There is, however, no restriction to the origin or source of a selected promoter. It is sufficient that the promoters are operational in driving the expression of a desired nucleotide sequence in the particular cell.
Other sequences that have been found to enhance gene expression in transgenic plants include intron sequences (e. g., from Adh 1, bronze 1, actin 1, actin 2 (WO 00/760067), or the sucrose synthase intron), poly adenylation signals in the 3′ prime UTR and viral leader sequences (e.g., from TMV, MCMV and AMV). For example, a number of non-translated leader sequences derived from viruses are known to enhance expression. Specifically, leader sequences from tobacco mosaic virus (TMV), maize chlorotic mottle virus (MCMV), and alfalfa mosaic virus (AMV) have been shown to be effective in enhancing expression (e.g., Gallie et al., 1987; Skuzeski et al., 1990). Other leaders known in the art include but are not limited topicornavirus leaders, for example, EMCV leader (encephalomyocarditis virus 5′-non-coding region; Elroy-Stein et al., 1989); potyvirus leaders, for example, TEV leader (tobacco etch virus); MDMV leader (maize dwarf mosaic virus); human immunoglobulin heavy chain binding protein (BiP) leader, (Macejak et al., 1991); untranslated leader from the coat protein mRNA of AMV (AMV RNA 4; Jobling et al., 1987), TMV (Gallie et al., 1989), and MCMV (Lommel et al., 1991; see also, della Cioppa et al., 1987).
For the expression of any constructs as described herein in a plant or plant cell, the invention preferably embodies that the described polynucleotides are operable linked to a promoter and to a polyadenylation site, wherein said promoter is characterized in that it is functional in said cell of said plant. As a promoter in this context, any sequence element is sufficient that induces transcription of the downstream sequence. The minimal requirements of promoters are very well known in the art and many of such promoters are conventionally used for gene expression in plants.
In a preferred embodiment of the invention, the transformation of a plant or plant cell with any polynucleotide as described herein, is performed by a method selected from standard procedures known in the art. Transformation of plant tissue can be achieved preferably by particle bombardment (Klein et al., “High-Velocity Microprojectiles for Delivering Nucleic Acids Into Living Cells,” Nature 327:70-73 (1987)), also known as ballistic transformation of the host cell, as disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., and in Emerschad et al., “Somatic Embryogenesis and Plant Development from Immature Zygotic Embryos of Seedless Grapes (Vitis vinif era)” Plant Cell Reports 14:6-12 (1995). In particle bombardment, tungsten or gold microparticles (1 to 2 μ m in diameter) are coated with the DNA of interest and then bombarded at the tissue using high pressure gas. In this way, it is possible to deliver foreign nucleotides into the nucleus. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells. Other variations of particle bombardment, now known or hereafter developed, can also be used. Another preferred method of stably introducing the nucleic acid construct into plant cells is to infect a plant cell with Agrobacterium tumefaciens or Agrobacterium rhizogenes previously transformed with the polynucleotide construct. As described above, the Ti (or RI) plasmid of Agrobacterium enables the highly successful transfer of a foreign nucleic acid molecule into plant cells. A preferred variation of Agrobacterium transformation uses vacuum infiltration in which whole plants are used (Senior, “Uses of Plant Gene Silencing,” Biotechnology and Genetic Engineering Reviews 15:79-119 (1998)). Yet another referred method of introduction is fusion of protoplasts with other entities, either mini-cells, cells, lysosomes, or other fusible lipid-surfaced bodies (Fraley et al., Proc. Natl. Acad. Sci. USA 79:1859-63 (1982), ). Also preferred in a method, wherein the nucleic acid molecule is introduced into the plant cells by electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985)). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate. Other preferred methods of transformation include chemical-mediated plant transformation, microinjection, physical abrasives, viral transduction and laser beams (Senior, “Uses of Plant Gene Silencing,” Biotechnology and Genetic Engineering Reviews 15:79-119 (1998)). The precise method of transformation is not critical to the practice of the present invention. Any method that results in efficient transformation of the host cell of choice is appropriate for practicing the present
The problem of the present invention is solved in a further aspect by a screening method for identifying microbe associated molecular patterns (MAMPs), comprising the method steps of (i) expressing in a plant or plant cell or plant tissue a protein comprising an amino acid sequence of at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity to SEQ ID No 1 (REMAX), or a chimeric PRR as described herein, (ii) contacting said plant or plant cell with a candidate compound, (iii) measuring the response of said plant or plant cell, preferably the immune response, in comparison with a control plant or plant cell or plant tissue, wherein an elevated (immune) response of said plant or plant cell indicates that said candidate compound is a MAMP.
In a further embodiment, in step (iii) of the above method, the immune response of said plant or plant cell is measured by means of assessing the oxidative burst, ethylene production and/or the expression of immune responsive genes or reporter genes. Reporter genes usable in the present context are composed of an immune responsive promoter operably linked to a reporter gene which allows for an easy readout of the reporter gene expression, e.g. luciferase enzymes or fluorescent proteins. The person of skill in the art has access to a wide selection of enzymes that can be used as reporter genes.
A further aspect of the invention constitutes a method for purifying a MAMP, comprising the use of a REMAX protein, or extracellular parts thereof, such as the ectodomain, specifically the LRR domain, wherein the REMAX protein comprises a sequence of at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity to SEQ ID No. 1.
In a further embodiment in the method for purifying a MAMP, said protein is coupled to a solid carrier medium, preferably to a membrane or a bead. The purification of the MAMP can then be performed by incubating a plant extract on said membrane or bead to allow the binding of the MAMP to the receptor. Thereafter the complex is washed to remove non bound material. The MAMP then can be eluted under salt conditions which impairs the ligand-receptor binding.
The objective of the present invention is solved in an additional aspect by an elicitor of a defense reaction in a plant obtainable by a method comprising the steps of lysing Xanthomonas cells, binding proteins of said lysate to an anion exchange column and eluting the elicitor at low salt concentrations.
One embodiment of the invention relates to the elicitor as described above, wherein the lysing of said Xanthomonas cells is performed by sonication. However, in the art other methods for preparing a cellular extracts are known, like the use of chemicals such as detergents or mechanical force. It is preferred that the protocol used for lysing the cells is sufficient to allow for the releases of cellular proteins from the bacterial cells.
In one preferred embodiment the sonication is performed with a sonicator operating with an energy of at least 30 W, preferably 40 W, most preferably about 50 W. To achieve a complete lysis, the bacteria can be sonicated for multiple times, for example for at least two times, preferably three times. Bacteria are preferably cooled during the sonication. A preferred device for sonication is a SONOPLUS® HD UW2070, supplied from Bandelin, Berlin).
In another preferred embodiment of the invention, the method used to obtain the elicitor comprises a step of removing the bacterial debris from the lysed, preferably sonicated, samples. The step preferably includes a separation by centrifugation. (for example about 20 min, at about 4° C. with about 13000×g).
Even more preferably the supernatant of the above step of separation is subsequently dialyzed with a molecular weight cut off of about 2 to 10 kD, preferably 4 to 6 kD, against about 20 mM TRIS, pH8. The dialyzed material is then preferably used for loading on to an anion-exchange column.
A preferred anion exchange column for use in the present invention is a sepharose column, for example Q Sepharose, Fast Flow supplied by GE Healthcare. Preferably the column is pre-equilibrated before use.
Furthermore preferred is that in the above method the elicitor is eluted at a salt concentration of about 0 to 50%, preferably of about 0 to 40%, more preferably of about 20 to 35% most preferably of about 30% salt.
Yet another embodiment of the invention relates to the elicitor, wherein the method comprises the further steps of reloading the elicitor on an anion-exchange column and eluting with a salt gradient of 0 to 30% salt.
In one embodiment the elute from the first elution step is first dialysed a second time with a molecular weight cut off of about 2 to 10 kD, preferably 4 to 6 kD, against about 20 mM TRIS, pH8, and then re-loaded on the anion-exchange column.
In the context of the present invention the salt used for eluting the elicitor from the anion exchange column can be any salt. Preferred is however that the salt is sodium chloride (NaCl).
In one additional embodiment, the elicitor can be eluted from the second anion-exchange step using a salt gradient ranging from 0 to 30% salt, preferably NaCl, 20 mM TRIS, pH8).
The elicitor according to the present invention in a most preferred embodiment does not contain the known MAMPs Elongation factor Tu (EF-Tu) and/or flagellin.
Furthermore it is preferred that the elicitor as described herein induces an immune response in a plant, e.g. the release of ethylene, via the activation of REMAX. Therefore the elicitor of the present invention obtainable as described above is or contains in a preferred embodiment a ligand of REMAX.
In a final aspect of the present invention, a method is provided for sensitizing a plant against bacterial infections, the method comprises the steps of treating a plant, plant cell or plant tissue with the elicitor or extract according to the present invention and/or with a REMAX protein and/or receptor or chimera according to the present invention.
It has been established in the past years that plants can be sensitized to pathogenic infections. After a first attack of the pathogen it was observed that upon a second penetration such a plant displays a hypersensitive response compared to plants that had not experienced a pathogen attack before. This effect is called “priming”.
Therefore, the chimeric PRRs, the methods of modulation of the resistance of a plant to pathogen infections, the elicitors or MAMPs of the present invention can be used to treat plants, plant cells or plant tissues in order to prime said plants immune system.
As used herein, the term “homologous” or “homology” denotes structural similarity between two macromolecules, particularly between two polynucleotides, irrespective of whether said similarity is or is not due to shared ancestry. Conventionally, homology denotes the level of sequence identity measured in percent. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., 1989, supra.
The regeneration, development and cultivation of plants from single plant protoplast, transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, In: Methods for Plant Molecular Biology, Academic Press, San Diego, Calif., (1988)). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
In yet another aspect, plants derived from any method of the present invention can be used for the production of seeds, fruits, stems, roots and leaves, or other plant derived products.
The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures and Sequences,
SEQ ID NO. 1 shows the amino acid sequence of REMAX
SEQ ID NO. 2 shows the genomic nucleic acid sequence of REMAX
SEQ ID NO. 3 shows the amino acid sequence of LeEix2
SEQ ID NO. 4 shows the nucleic acid sequence of LeEix2
Partial purified extracts of Xanthomonas axonopodis pv. citri and other Xanthomonas species induce MAMP responses in Arabidopsis. These extracts contain a MAMP, called emax for enigmatic MAMP of Xanthomonas, which is different from the known MAMPs flagellin, EF-Tu or peptidoglycan that are recognized from A. thaliana and dominant in the partial purified extracts. emax can be solubilized from different Xanthomonas species by sonication of the bacteria. The active protein binds to anion exchanger media and can be eluted at low salt concentrations (Q SepharoseQFast Flow, pH 8.0):
Xanthomonas axonopodis pv. citri was grown on Kings B plates (1.5% Agar) for 48 hours at 30° C. The bacteria were harvested with a spatula and 2 volumes of H2O were added. For breaking the bacterial envelope, aliquots of 30 ml of the bacterial suspension was cooled on ice and sonicated three times for two minutes (50 W, SONOPULS HD UW2070, Bandelin, Berlin,). Insoluble bacterial debris was removed by centrifugation (20 min, 4° C., 13000×g). Supernatant was extensively dialyzed (Molecular weight cut off: 4-6 kDa, Roth, Karlsruhe) against 20 mM Tris, pH 8.0 and loaded on a pre-equilibrated anion exchange column (Q Sepharose, Fast Flow, GE Healthcare, München). Emax was eluted with 30% NaCl (1 M, 250 mM Tris, pH 8.0) and after dialysis (20 mM Tris, pH 8.0, Molecular weight cut off: 4-6 kDa, Roth, Karlsruhe) reloaded on Q-Sepharose column. Elution of proteins was performed with NaCl gradient (0-30% NaCl, 20 mM Tris, pH 8.0) and tested for activity on the double mutant fls2×efr (ethylene biosynthesis or extracellular alkalinization).
While for initial characterization of the activity from Xanthomonas interference by the MAMPs flagellin and EF-Tu was excluded by the use of fls2×efr double mutants, further work with wild-type plants relies on absence of such MAMPs that could interfere in the assays. Active fractions eluted from anion exchange columns were pooled and tested for contamination by the known bacterial MAMPs EF-Tu and flagellin which are potent inducers of extracellular alkalinization of cell cultures as well. Flg22-Δ2, a C-terminally truncated version of flg22, has been described as a specific antagonists for flg22 in A. thaliana (Bauer et al., 2001). In cell cultures derived from wild-type plants, a preincubation (2 min) with the antagonist flg22-Δ2 can completely block the response of the cells to subsequent treatment with flg22 (
Mapping with the emax-insensitive accession Shakhdara resulted in a single locus on chromosome 1. The receptor-like protein 1 (RLP1, At1g07390) is located in this region and two independent T-DNA insertion lines, rlp1-2 (SALK_116923) and rlp1-3 (SALK_049403C), were insensitive to emax. The responsiveness was tested with the increase of ethylene and the induction of the gene FRK1 (FLG22-INDUCED RECEPTOR-LIKE KINASE1, At2g19190).
Searching for homologues of RLP1 with program blastp (http://blast.ncbi.nlm.nih.gov/Blast.cgi) revealed clear homologues of RLP1 with >80% sequence identity only in Arabidopsis lyrata and Brassica rapa. Both species were indeed sensitive to emax treatment (
For the gene At1g07390 encoding REMAX/RLP1 three gene models with two different starting positions have been predicted (www.tair.de,
To confirm the role of REMAX, complementation experiments with REMAX-constructs were performed in rip 1-2 mutants (
Furthermore, for testing functionality of REMAX in the accession Shakhdara, which showed a deletion in the region of RLP1, protoplasts derived from Shakhdara were co-transformed with FRK1::luc and REMAX and tested for luciferase activity in response to emax. This experiment clearly showed complementation of emax response by transformation of REMAX (
The alignment of the two RLPs REMAX and LeEix2 shows a similar overall structure of both proteins. Heterologous expression of LeEix2 in a species of tobacco that has no endogenous perception provided convincing evidence for the function of LeEix2 as the receptor for xylanase (Ron and Avni, 2004). Their large LRR-domains are subdivided by island domains inserted before the last four of the LRRs. Sequence homology between REMAX and LeEix2 is highest for these four LRRs and comprising the cysteine pair at the end of the LRR domain. Their cytoplasmatic tails show little resemblance in length or sequence.
In order to figure out whether it is the C-terminal part of these RLPs that determines functionality in N. benthamiana chimeric forms with swaps of their C-terminal parts were produced as summarized in
| Number | Date | Country | Kind |
|---|---|---|---|
| 1210484.0 | Jun 2012 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2013/062254 | 6/13/2013 | WO | 00 |
