GB2505718A - Chimeric plant receptors comprising LRR domains from two different receptors - Google Patents
Chimeric plant receptors comprising LRR domains from two different receptors Download PDFInfo
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- GB2505718A GB2505718A GB1216183.2A GB201216183A GB2505718A GB 2505718 A GB2505718 A GB 2505718A GB 201216183 A GB201216183 A GB 201216183A GB 2505718 A GB2505718 A GB 2505718A
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Abstract
The present invention pertains to novel artificially designed plant receptors and artificially designed ligands binding thereto. Based on the molecular architecture of plant leucine rich repeat receptor kinases (LRR-RK), new functional receptor-ligand pairs were designed by using the sequences of elongation factor Tu receptor (EFR) and its ligand epitope peptide elf18. The artificial plant receptors of the invention comprise an extracellular leucine rich repeat domain, a transmembrane domain and an intracellular kinase domain, wherein the LRR domain comprising two LRR domains, one from receptor R1 that binds ligand L1, and one from receptor R2 that cannot bind L1. The intracellular domain and transmembrane domain are preferably from the brassinosteroid-insensitive receptor. A further embodiment provides artificial proteins that can bind the receptor, such as variants and mutants of the elf18 peptide
Description
Artificial Plant Receptors and Ligands
FIELD OF THE INVENTION
The present invention pertains to novel artificially designed plant receptors and artificially designed ligands binding thereto. Based on the molecular architecture of plant leucine rich repeat receptor kinases (LRR-RK), new fttnctional receptor-ligand pairs were designed by using the sequences of elongation factor Tu receptor (EFR) and its ligand epitope peptide elfI 8. Thus, the present invention describes the new artificial receptors and ligands, as well as genetic constructs expressing same, and plants, plant tissues and plant cells transformed therewith. Furthermore provided are processes for the design and expression of the inventive artificial plant receptors and artificial protein ligands, and the use of the inventive constructs for improving resistance of a plant against phytopathogens, or for promoting growth, cell death andior development ofplants. Also provided are methods for the specific isolation and enrichment of plant cells comprising the use of the herein described novel artificial plant re-ceptor and ligand pairs.
DEs(:RIPTION Due to the constantly-increasing human population, and the declining area of land available for agriculture, it remains a major goal to improve the efficiency of agriculture and to increase the range of exploitable plants in agriculture. Conventional approaches for crop and horticul-tural improvements utilise classical plant breeding techniques in order to identify new plant varieties having desirable characteristics, like higher yield, resistance to pests and unfavour-able weather conditions, root development, nutrient uptake and stress tolerance in general.
However, such classical breeding techniques have several negative aspects, namely that these techniques are typically labour intensive and often include alteration of multiple traits.
Genetic engineering ofplants entails the isolation and manipulation of the genetic and epige-netic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Genetic engineering of plants at the industrial and laboratory scale is well established and has led to the development of plants having various improved economic, agronomic or horticultural traits. These technological advances have yielded crops that reduce food production costs through resistance to pests, herbicide, drought, and flood or generally enhance the growth and resistance of plants. Furthermore crops were engineered to produce substances such as vitamins that could improve human health. These approaches could help treat health issues in countries where people suffer from malnutrition such as vita-min A deficiency in countries where available foods do not provide the necessary nutrients necessary for people.
Traits of particular economic interest are growth characteristics such as high yield of plant material such as fruits. Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, for example, the number and size of the organs, plant architec-ture (for example, the number of branches), seed production and more. Root development, nutrient uptake and stress tolerance may also be important factors in determining yield.
Cellular signalling upon environmental or developmental signals in plants and animals alike are coordinated to a tremendous extend by membrane associated proteins -receptors -which provide an extracellular binding domain to which a signal-molecule can attach, for example pathogenic structures of pests or growth factors, and intracellular signalling domains, specifi-cally kinase domains, which can transmit the extracellular signal intracellularily by initiating a signalling cascade. In the plant kingdom many of such membrane-bound receptor kinases were identified in the past decade, e.g. in the genomes of rice or Arabidopsis are about 600 to 1000 genes that encode for these proteins.
In plants lcucin-rich-rcpcat receptor kinases (LRR-RK) are key in the recognition and trans-mittal of exogenic as well as endogenic signals. LRR-RKs are involved in developmental processes. immune reactions upon pathogenic attacks, growth, cell death, abscission and rip-ening. Each LRR-RlC is involved in one specific biological process, and binds through the extracellular portion only to one specific signal-molecule, the specific ligand. Yet, amongst the 600 to 1000 genes encoding for LRR-RlCs in Arabidopsis, specific ligand molecules or biological function could be attributed only to a minor fraction of these receptors.
LRR-RKs are composed of a multi-domain architecture in which each domain provides a spe-cific function for the receptor. The extracellular part of the receptor contains a leucine rich repeat (LRR) domain. The domain is named according to a series of repeating sequence mo- tifs comprising about 20 to 30 amino acids with a high content of leucine. Leucine-rich re-peats are frequently involved in the formation of protein-protein interactions, and not only found in plant receptors but also in a large variety of functionally unrelated proteins. Exam- ples comprise the ribonuclease inhibitor, tropomodulin and toll-like receptors. Toll-like recep-tors are involved in innate immunity and developmental (Toll) processes. The LRR domain mediates the binding of the receptor to a ligand molecule, and hence can be classified as the input domain of the LRR-RK.
On the intracellular side the LRR-RK has a protein kinase domain, that enzymatically cataly-ses the transfer of a phosphate group to a substrate protein. Usually the addition of phosphate groups to intracellular substrates results in the initiation of a signal cascade that eventually induces into a cellular response, for example the induction of immune related genes in order to fight off a pathogenic attack. Thus, the kinase domain could be classified as the output do-main of the LRR-RK. Kinase and LRR domains are interconnected by the juxtamembrane and transmembrane domains, the latter spanning the cellular membrane, which allow the receptor to be membrane bound.
Although the function of many LRR-RKs remains elusive, examples of LRR-RKs that recog-nize exogenous signals are Flagellin Sensing 2 (FLS2) and Elongation Factor Tu Receptor (EFR) of Arahidopsis. The corresponding ligands of the receptors are bacterial fiagellin (fig) and the elongation factor Tu (EF-Tu) respectively. The specific binding epitope with which EF-Tu binds to the EFR is a small 18 amino acid long peptide motif called elflS (SEQ ID No. 1). The EFR and FLS2 receptors function as immune receptors reeognising the presence of their ligands as "non self' and initiating a defense response in the plant cell. Other defense response related receptors include XA2I from rice, CERKI and AtPEPRI, the latter recogniz-ing endogenous danger signals related to wound healing processes.
A known LRR-RK that binds endogenous signals of a plant is the brassinosteroid receptor BRII (brassinosteroid insensitive 1) that is an essential component of a membrane bound multi-receptor complex recognizing the steroid brassinolide. This steroid hormone in plants is important for physiological and developmental regulation. He Z and colleagues showed in a study with chimerie receptors composed of extraeellular BRI1 domains and intracellular kinase domain of XA2I, a rice defense response receptor, that artificial produced receptor ehimeras could initiate a defense response in rice upon treatment with brassinosteroids. This study therefore shows that LRR-RKs constitute an interesting starting point for the rational design of new receptor proteins in plants.
More novel chimeric receptors were constructed in Arahidopsis thaliana consisting of the complete ectodomain of EFR and the intracellular FLS2 kinase. These chimeras were shown to be sensitive towards the elfl8 peptide, the natural ligand of EFR (Albert et al 2010). The study shows that both LRR-RKs EFR and FLS2 although they recognize different ligand molecules, still share all ifinctional aspects for initiating the intracellular signal output of LRR-RKs. A similar chimeric receptor approach was used in the elucidation of the function of wall associated kinases (WAK), which are involved in the detection of oligogalacturonides, molecular signals of ccli wall damages, growth and development. Swapping of the ecto-or endo-domains of WAK and EFR resulted in a substitution of rcccptor function. EFR ectodo-main WAK1 kinase domain receptors sense elfIS but display a signal output typical for native WAKI and vice versa (Brutus Act al., 2010).
Chimeric receptors based on the LRR-RKs domain structure, in which the ectodomain and the intracellular kinase domain are derived from different receptors can be used for the controlled activation of cellular responses like immune response reactions in transgenic. A chimeric re-ceptor that exploits the peculiarities of two receptors associated with plant defense, like FLS2, EFR or WAK1 receptors, was designed with the intend to increase plant resistance to phyto-pathogenic organisms (WO 2010/139790). However, state of the art approaches exploit only natural occurring and known ligand-receptor interactions which may induce unwanted side effects due to uncontrolled activation of the chimeric constructs by their naturally occurring
signal molecules in the field.
In view of the above, it is an object of the present invention to provide novel approaches for the rational design of artificial chimerie plant receptors and their ligands which are not re-stricted to only naturally occurring receptor-ligand interactions. It is further the intent of the present invention to provide novel receptor-ligand pairs which allow for the controlled, exclu-sive and specific activation of specific cellular responses in plants.
In a first aspect of the present invention, the above object is solved by an artificial plant recep-tor comprising an extracellular Leucine Rich Repeat (LRR)-domain, a transmembrane domain and an intracellular kinase domain, characterized in that said LRR-domain of said artificial plant receptor comprises at least one portion of an LRR-domain of a plant receptor Ri that is able to bind a protein ligand Li, and said LRR-domain of said artificial plant receptor com-prises at least one portion of an LRR-domain of a plant receptor R2 that is not able to bind said protcin ligand Li. Prcfcrably whcrcin Ri and R2 arc diffcrcnt.
The LRR domain of said artificial plant receptor of the invention in one first embodiment is an artificial LRR domain which is still functional, and which does not result in a miss-folded non-functional receptor protein. The term "artificial" in the context of the present invention shall denote such constructs or compounds which are made by human intervention and in that form do not occur in nature. For peptides/proteins or nucleic acid constructs, the term "artifi-cial" in preferred aspccts relates to thc such peptides/proteins or nucleic acid constructs that havc a mutatcd or altered scqucncc comparcd to compounds found in naturc.
The inventors surprisingly found that by rationally exchanging parts of the leucine rich repeat domains of plant LRR-RlCs novel plant receptors are created with new receptor-ligand bind-ing functions. In onc prcfcrrcd cmbodimcnt of thc abovc invcntion Ri and R2 arc prcfcrably different plant receptors selected out of the LRR-RK family of proteins. Most preferred is that Ri and R2 have a maximum of 80% sequence identity in the amino acid sequence of the cx-traccllular LRR domain; morc prefcrably RI and R2 havc a maximum of 70%, 60% and most preferably a maximum of 50% sequence identity in the amino acid sequence of the extracellu-lar LRR domain.
In another embodiment of the invention the RI and R2 share an amino acid sequence identity in thcir LRR domains of at Icast 30%, prefcrably 35%, morc prcfcrably 40%, 45 or 50%. Or, ahernativeiy or cumulatively, a minimum sequence identity of the solvent exposed amino acids of the LRR domains of at least 15%, preferably 20%, more preferably 25 or 30%.
In one other embodiment of the present invention, an artificial receptor is preferred wherein RI and R2 have an LRR-domain consisting of nearly the same number of LR repeats, or whcrcin thc diffcrcncc of thc numbcrs of LR rcpcats in thc LRR domains ofRi and R2 docs not cxcccd 4, or 3 or prcfcrably 2, or 1.
In one embodiment of the invention Ri is not FLS2 from Arabidopsis thaliana.
In another embodiment of the invention R2 is not FLS2 from Arabic/apsis thaliana.
In yet another embodiment of the present invention an artificial plant receptor is provided wherein additionally a point mutation is introduced into said LRR-domain of said artificial receptor. A point mutation might be any mutation selected from deletion, substitution, addi-tion, insertion or chemical modification of at least one amino acid residue.
In another embodiment of the present invention the artificial plant receptor comprises a transmembrane domain and/or intracellular kinase domain which is derived from the plant receptor Ri or R2, or, in one further embodiment, from a third plant receptor R3. By inter-mixing the extracellular LRR domain of two plant receptors RI and R2, the inventors created a new receptor binding function; In the here described embodiment exchanging the output domain -such as the juxta-membrane, transmembrane and/or intracellular kinase domain -with portions of a third receptor R3 allows for the integration of a new output function into the artificial plant receptor of the present invention.
Thus, preferred is that the receptor R3 is a receptor that is involved in any plant cellular proc- ess other than RI and/or R2, for example when RI and R2 are associated to plant pathogen-specific responses, it is preferred in one embodiment that R3 is involved or associated to processes like growth, cell death and/or development; and vice verca. R3 therefore has in a preferred embodiment a different -not identical -output domain, or juxta-membrane, trans-membrane and/or intracellular kinase domain compared to the respective domains of Ri and R2. In a most preferred embodiment at least one, or two or all three domains of the juxta-membrane, transmembrane and/or intracellular kinase domain share a sequence similarity between Ri and/or R2 with R3 of maximal 90%, 80%, 70%, 60%, and most preferably 50% sequence identity in their amino acid sequence.
In one specific embodiment of the present invention R3 is brassinosteroid-insensitive 1 (BRII), BRI like I (BRLI), BRI like 2 (BRL2) or BRI like 3 (BRL3).
In one further embodiment of the present invention the artificial plant receptor comprises fur-ther mutations compared to the sequences derived from Rl, R2 or R3, wherein said mutations are selected from the group consisting of deletion, addition, insertion, chemical modification or substitution ofat least one amino acid, preferably ofa maximum ofSO or 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9,8,7,6,5,4,3,2 amino acids. Most preferred is that the artificial plant receptor does not contain more than 10 modified amino acid residues compared to the corresponding sequence of either RI, R2 or R3.
Still another embodiment of the present invention pertains to the artificial plant receptor wherein Li is a protein ligand. Preferably Li comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and most preferably 100% identity to the amino acid sequence of elflS (SEQ ID No. 1). Depend-ing on the selected RI and R2, LI can also be a different ligand, for example Li in certain embodiments comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, and most preferably 100% iden-tity to the amino acid sequence of bacterial flagellin, preferably of the peptide epitope flg22 (ligand to FLS2). In other embodiments Li comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, and most preferably 100% identity to the amino acid sequence of the Avr2l peptide (ligand to XA2 1). Or any other known protein ligand of a plant LRR-RK known to the person of skill in the art.
Yet another embodiment of the artificial plant receptor according to the invention is an artifi-cial plant receptor wherein said at least one portion of an LRR-domain of a plant receptor R2 is flanked by at least two portions of said LRR-domain of said plant receptor Ri. Thus, in this embodiment plant artificial receptors are excluded wherein only the N-terminal or C-terminal part of an LRR domain of RI is fused to the N-terminal or C-terminal portion of an LRR do-main of R2. In this preferred embodiment of the present invention the artificial plant receptor comprises an LRR domain which is not only a fusion between two domain parts but contains at least one insertion of a sequence of one receptor LRR domain into another receptor LRR domain.
A further embodiment of the present invention is the artificial plant receptor, wherein said portion of said LRR-domain of said plant receptor RI and/or R2 is a consecutive stretch of at least 5,6,7, 8, 9, 10, II, 12, 13, 14, 15, 16, 17, 19 or 20 amino acids ofthe sequence of the LRR-domain ofthe respective receptor, or, alternatively, at least one single leucine rich repeat of the respective receptor, or a consecutive sequence of 2, 3, 4, 5 or more single leucine rich repeats of the respective receptor.
In another embodiment of the invention the artificial plant receptor does not bind to Li, and in certain specifically preferred embodiments the artificial plant receptor binds to a protein ligand comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of Li, preferably wherein Li is elfi8 (SEQ ID No. 1) and!or EF-Tu. Or any of the other above men-tioned known protein ligands of plant LRR-RKIs.
Preferably the artificial plant receptor according to the invention binds to an artificial protein ligand comprising a mutated sequence of Li, or a mutated sequence of a protein ligand com-prising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and most prcfcrably 100% of thc amino acid scqucncc of Li, wherein preferably Li is elfi8 (SEQ ID No. i). Further preferred is that the artificial plant receptor according to the aforementioned embodiment is obtained by substitution, addi- tion, deletion, insertion or chemical modification of at least one residue in the amino acid se-quence ofLi, preferably of elfi8 (SEQ ID No. i).
Yet one additional embodiment of the present invention relates to the artificial plant receptor, wherein said plant receptor RI that is able to bind a protein ligand LI comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, more preferably 100% identity to the sequence of EF-Tu Receptor (EFR), most preferably to a protein en-coded by the gene with the accession No. At5g20480, which is Arabiclopsis thaliana EFR Yet one additional embodiment of the present invention relates to the artificial plant receptor, wherein said plant receptor R2 that is not able to bind a protein ligand Li comprises a se- quence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, more pref-erably 100% identity to the sequence of EFR-like (L), most preferably to a protein encoded by the gene with the accession No. At3g471 10, Arabiclopsis thaliana EFR-like (L).
In certain very specific embodiments of the invention, which are most preferred, the artificial plant receptor is a receptor as dcpticted in Figure 1, according to the description of the exam-ples section.
The examples of the present invention show four artificial plant receptors that were generated based on Arabidopsis EFR and EFR-like receptors. The construct EFR-S298P (SEQ ID No. 4) has the sequence of EFR but contains a point mutation at nucleic acid residue 892 (the mu-tated sequence is depicted in SEQ ID No. 4).
Construct E6/L9/E (SEQ ID No. 5) comprises at positions 1 to 699 the sequence of EFR, then at position 700 to 918 sequences derived from the EFR-like receptor. The remaining nucleic acid residues are again from EFR.
Construct L9/E (SEQ ID No. 6)contains in positions 1 to 942 sequences derived from EFR-like, and the remaining sequence from EFR.
Construct L9/E R231Q (SEQ ID No. 7) also contains in positions Ito 942 sequences derived from the EFR-like receptor, and the remaining sequence from EFR. This construct also con-tains one point mutation at position 692.
Also preferred is a artificial plant receptor according to the invention, wherein said artificial protein ligand comprises a sequence selected from SEQ ID No. 2 (eIfAVNV) or SEQ ID No. 3 (eltKl4).
In order to allow for an easy isolation or purification of the artificial plant receptors of the present invention, said artificial plant receptor is in preferred embodiments conjugated to a tag, for example a poiy histidine tag or to a detectable tag, like for example a fluorescent or luminescent protein, preferably to GFP. Also any other frision protein derived from the artifi-cial plant receptors and/or ligands of the invention are comprised by the present invention.
Preferred is also the artificial plant receptor according to the invention that when expressed in a plant cell induces, upon binding to a ligand, intracellular signalling in said plant cell, pref- erably yielding into an immune response of said plant cell, such as ethylene synthesis, expres-sion of immune genes or production of reactive oxygen species (oxidative burst).
On the other hand another preferred embodiment of the invention is the artificial plant recep- tor that when expressed in a plant cell, induces upon binding to a ligand intracellular signal- ling, preferably yielding into plant growth, plant cell death or a promotion of plant develop-ment or any other plant cellular response besides pathogen-specific responses.
In certain embodiments of the invention the artificial plant receptor is preferred, wherein the ligand is an artificial ligand.
In a second aspect, the object of the present invention is also solved by providing an artificial protein ligand which is able to bind to the LRR-Domain of the artificial plant receptor as de-scribed herein before.
In one preferred embodiment of the second aspect of the invention the artificial protein ligand compriscs a mutatcd sequence of Li, or a protein ligand comprising an amino acid scqucncc having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and most preferably 100% identity to the amino acid sequence of Li, wherein prefera-bly LI is elfI8 (SEQ ID No. I) or alternatively any of the aforementioned protein ligands in plants. Further preferred is the artificial protein ligand according to the aforementioned em- bodiment wherein said mutated.sequence is obtained by substitution, addition, deletion, ütser-tion or chemical modification of at least one residue in the amino acid sequence of elfl8 (SEQ ID No. I).
In specifically preferred embodiments of the second aspect of the invention the artificial pro-tein ligand comprises a sequence selected flx,m SEQ ID No. 2 (e1fAVNV) or SEQ ID No. 3 (elfKI 4).
In a third aspect of the present invention the above object is solved by providing a consiruct -preferably a genetic construct -comprising a nucleotide sequence encoding for an artificial plant receptor as described herein before, or a nucleotide sequence encoding for an artificial protein ligand as described herein before.
One embodiment of the above third aspect of the invention relates to a construct which allows for the expression of said nucleotide sequences in a plant, plant ccli or plant tissue. It is spe-cifically preferred that said construct is usable for stably or transiently transforming a plant, a plant cell or plant tissue with said construct.
-11 -The expression constructs of the invention comprise preferably gene regulatory sequences that direct the expression of the genes under their controL Gene regulatory sequences can be se-lected from regulatory sequences %r constitutive expression, which maintain gene expression at a relative level of activity (basal level), or can be inducible regulatory sequences. Regula- tory sequence tbr constitutive expression can be used in any cell type, or can be tissue spe- cific, cell type or phase specific. The latter direct expression only during particular develop- mental or growth stages of a plant cell, or the like. A regulatory sequence such as a tissue spe- cific or phase specific regulatory sequences or an inducible regulatory sequence useful in con-structing a recombinant polynucleotide or in practicing a method of the invention can be a regulatory sequence that is ibund in nature in a plant genome. However; the regulatory se-quence also can be from an organism other than a plant, including, lbr example, from a plant virus, an animal virus, or a ccli from an animal or other multicellular organism.
A prefrrred regulatory sequence useful fbr expression ofpolynucleotides of the invention is a promoter element. UsefW 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 fbr a high level of gene expression, whereas a weak promoter provides lbr a very low level of gene expression. An inducible promoter is a promoter that provides fbr 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 se- qucncc that is a strong promoter tbr hctcrologous nucleic acid is advantageous bccausc it pro-vides fbr a sufficient level of gene expression to allow fbr 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 tbr ex- pression, and also depending on the target species. In some cases, expression in multiple tis- sues 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, ide- ally dicotyledonous promoters are selected fbr expression in dicotyledons, and monocotyle-donous promoters lbr expression in monocotyledons. There is, however, no restriction to the -12 -origin or source of a selected promoter. It is sufficient that the promoters are operational in driving the expression of a desired niicleotide sequence in the particular cell.
Other sequences that have been found to enhance gene expression in transgcnic plants include intron sequences (e. g., from Adh 1, bronze 1, actin 1, actin2 (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 AJVIV). For example, a number of non-translated leader se-quences 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; Skuzcski ct al., 1990). Other leaders known in the art include but are not limited topi-comavirus 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 bind-ing protein (BiP) leader, (Macejak et al., 1991); untranslated leader from the coat protein mRNA of AMy (AMV RNA 4; Jobling et al., 1987), TMV (Gallie et al.,1989), and MCMV (Lonimel et al., 1991; see also, della Cioppa et al., 1987).
A preferred expression construct 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) as describe herein before. The recombinant vector of the invention comprises a sequence encoding for any of the inventive receptors/ligand as described herein before. Also additional genes can be expressed through the recombinant vector, such as selection markers. A regulatory sequence can be isolated from a naturally occuning genomic DNA sequence or can be synthetic, for example, a synthetic promoter. Such promoters are well known in art.
For the expression of any constructs or vectors as described herein in a plant, plant tissue or plant cell, the invention preferably embodies that the described polynucleotides are operable linked to a promoter and to a polyadcnylation 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 cle- ment is sufficient that induces transcription of the downstream sequence. The minimal re- quirements of promoters are very well known in the art and many of such promoters are con-ventionally used for gene expression in plants.
-13 -In this respect, in a further aspect of the present invention a plant cell or plant is provided comprising an artificial plant receptor as described herein before, or an artificial protein ligand as dcscribcd herein bcfore, prcfcrably whercin said plant ccli is transformcd with a construct as described herein before.
Accordingly, yet another aspect of the invention is the use of an artificial plant receptor ac- cording as described herein before, andior an artificial protein hgand as described herein be-fore, in a plant or plant cell or plant tissue.
One embodiment of the above use according to the invention is preferred, wherein said artifi-cial protein ligand is able to bind to said artificial plant receptor and wherein thc binding of said ligand to said receptor illduces intracellular signalling. In certain specific embodiments it is preferred that the binding of said ligand to said receptor induces an immune response in said plant, plant cell or plant tissue. Said immune response is in a preferred embodiment char- acterizcd by thc differcntial expression of immunc gdncs, releasc of ethylene and/or the pro-duction of reactive oxygen species.
In one embodiment the use according to the invention is preferably a use for priming a plant against infections. In plant defense, priming is a process by which a plant is brought into first contact with a pathogen (priming) whereupon on subsequent contacts with the pathogen the plant is able to muster a more quickly and more aggressive response, the so called hypersensi-tive response. Because priming initiates a state of readiness that does not confer resistance per sc but rather allows for accclcratcd induced dcfensc reaction when an attack occurs, priming is beneficial in agriculture to increase resistance of a crop in advance.
Still another aspect of the present invention relates to a method for screening new receptor ligands, comprising the steps of i. Expressing in a plant cell an artificial plant receptor as described herein before, ii. Contacting said plant ccfl with a candidate ligand moiccule, and iii. Determining the binding of said candidate ligand molecule to said artificial plant receptor, wherein, when said candidate ligand molecule binds to said artificial plant receptor, said can-didate ligand molecule is a ligand of said artificial plant recoptor.
-14 -Also included in the present invention is method as above, wherein in step i. an artificial plant receptor as described herein before is provided outside the context of a plant cell, e.g. bound to a matrix surface, and wherein in step ii, said artificial plant receptor as described herein before is brought into contact with a candidate ligand molecule.
In one embodiment of the invention the above method is preferred, wherein when said candi-date ligand molecule is not a ligand of Ri and/or R2. Said candidate ligand molecule is an artificial ligand that does not occur in nature, for example a proteinaceous ligand that is ob- tained by altering via human induced mutagenesis the amino acid sequence of a naturally oc-curring ligand.
The invention also relates to the use of a LRR-RK in the screening of novel artificial ligands.
In one embodiment of the above method according to the invention, it is preferred that the binding in step iii. is determined by monitoring the immune response of said plant cell, pref- erably by monitoring the expression of immune related genes or suitable reporter genes, re-lease of ethylene and/or the production of reactive oxygen species. Reporter gene assays Also preferred in the context of the above method is to determine the binding of said artificial plant receptor to said candidate ligand molecule by binding assays, such as described in Al-bert et al. 2010, or via competition assays.
Furthermore provided is a use of an artificial plant receptor as described herein before, andlor an artificial protein ligand as described herein before, in a plant or plant cell or plant tissue to stimulate and/or promote plant growth, development or cell death.
In another aspect the invention provides a method for the isolation of a plant cell, comprising the steps of a. Transforming a plant or plant tissue or plant cell with an expression construct comprising an artificial plant receptor as described herein before, b. Bringing into contact a population of cells derived from said transformed plant, plant tissue or plant cell, with an artificial ligand as described herein before, -15 -wherein said artificial protein ligand specifically binds to said artificial plant receptor, c. Removing any non-bound cell, and d. Isolating cells which are bound to said artificial protcin ligand couplcd to a ma-trix.
Since the herein described novel receptor-ligand interactions are unique, the constructs of the invention allow for a specific isolation of plant cells, like plant protoplasts. This isolation method will be preferably used in tissue engineering.
Particularly preferred is the method as described above, wherein said expression constructs allows for a plant cdl type-specific or plant tissuc-specific exprcssion of said artificial plant receptor. In this way it is possible to specifically flag certain plant cells of choice which then, by using the above method for isolation can be isolated/enriched and/or purified. In this re-spect it is preferred that said expression construct allows for a constitutive expression or a inducible expression aficr thc transformation of the construct.
The matrix of choice in the above methods is selected from the group comprising tissue cul-ture plates and dishes, beads or membranes.
Plants for use in the above methods of the invention and in the context of the other aspects and embodiments of the present invention as described herein before are pepper, rice, citrus, cotton, tomato, soybeans, tobacco. Further plants for use in context with all aspects and em-bodiments of thc prcscnt invcntion arc corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), alfalfa (Medicago sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sor-ghum vulgare), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticurn aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Rachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsu- turn), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), co- conut (Cocos nucifcra), pineappc (Ananas comosus), citrus trecs (Citrus spp.), cocoa (Theo- broma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaultilanc), fig (Fi-cuscasica), guava (Psidium guava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew(Anacardium occidentale), macadamia (Macadamia integrifo-lia),ahnond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), -16-oats, duckweed (Lemna), barley, tomatoes (Lycopersicon esculentum), lettuce (e. g., Lactuca sativa), green beans (Phaseolus vulgaris, lima beans (Phaseoluslirnensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C sativus), cantaloupe (C. canta- lupensis), and musk melon (C. melo). Ornamentals such as azalea (Rhododendron spp.), hy-drangea 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 omarnentals within the scope of the invention include impatiens, Begonia, Pelar- gonium, Viola, Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum, Amaran- thus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea, Dahlia, Datura, Delphin-ium, Gerbera, Gladiolus, Gloxinia, 1-lippeastrum, Mesembryanthemum, Salpiglossos, and Zinnia. Conifers that may be employed in practicing the present invention include, for exam-ple, 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); Westem hemlock (Tsugaultilane); Sitka spruce (Pieea glauca); red-wood (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).
The present invention will now be frirther 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 refer-ence in their entireties. In the Figures and Sequences, Figure 1: Schematic representation of novel artificial plant receptors. These receptors possess a kinase domain of EFR which triggers the plant's pathogen-or stress related signalling cascades. The kinase part is exchangeable by any kinase of any receptor, feeding in a plant signalling pathway of interest.
Figure 2: Oxidative burst induced by natural or artificial ligands via newly designed arti-ficial receptors. Shown is the responsiveness to original EFR ligand elfl8.
Figure 3: Oxidative burst induced by natural or artificial ligands via newly designed arti-ficial receptors. Shown is the responsiveness to original EFR ligand eIfAVNV. -17-
Figure 4: Oxidative burst induced by natural or artificial ligands via newly designed arti-ficial receptors. Shown is the responsiveness to original EFR ligand eIfK 14.
Also part of the description of this application forms the enclosed sequence listing in which the following sequences are disclosed: SEQ ID NO. I: elfl8 SKEKFERTKPHVNVGTIG SEQ ID NO.2: e1fAVNV SICEKFERTKPH---GTIG SEQ ID NO. 3: eltKl4 SKEKFERTKPHVNK SEQ ID NO. 4: DNA receptor construct EFR-S298P SEQ ID NO. 5: DNA receptor construct E6/L9/E SEQ ID NO. 6: DNA receptor construct L9/E SEQ ID NO. 7: DNA receptor construct L9/E-R231Q SEQ ID NO. 8-11: primer sequences -18-
EXAMPLES
Materials and Methods Construction of novel art jficial plant LI?!? receptor kinases New receptors were constructed on basis of the DNA sequences via PCR with fusion primers as described in (Albert NI, Jehie AK, Mueller K, Eisele C, Lipschis M, Felix 0.; J Biol Chem. 2010 Jun 18;285(25):19035-42). Sequence of fusion primers for L9/E, termed as fu-sion primer 1 (fjl): aaccatcttactggaaagatacctttgagctttggaaagtta (SEQ ID No. 8), parts for sequence of EFRlike (L) in bold, for EFR (E) in italics. E6/L9/E has been constructed via f, 1 and fp2, whereas sequence of fp2 is: gaatagtttttcaggtggttttcctcctccaatttacaacctg (SEQ ID No. 9) (scqucncc parts of EFR (E) in italics and of EFRlikc (L) in bold letters). As a template for the PCRs scrved DNA-constructs containing EFR (At5g20480) or EFRlike (At3g471 10), respectively. Pointmutations in EFR_5298P or L9/E_R23 IQ were introduced via PCR using the primers caagccttgaaaggtttgatatcCcatetaattacctgtctggtagtate (SEQ ID No. 10) for S298P or gactgaaaeagatgatetttttccAaatagcattaaacaagtttaatgg (SEQ TD No. 11) for R23 1Q (in bold capi-tals: nucleotide exchange leading to the desired aa exchange). Schematic representations of the constructed receptors are shown in Figure 1.
Transient Expression in N benthamiana and oxidative burst measurement (Albert et al /BC, 2010) A. twnejbciens (strain 0V3 101) harboring the gene constructs to be expressed were grown for 48 h in LB medium, collected byccntrifugation, and transferred to induction medium 10 mM Magnesiumehloride with 150 pm acetosyringone and 10 mm MgCI2 at an 0D600 of 0.1. After further incubation at room temperature for 2-3 h, bacteria were pressure-infiltrated into leaves of 4-5-week-old Nicotiana henthainiana plants grown in the greenhouse (16-h day at 22 °C/8-h night at 18 °C). The next day (24-36 h after infiltration), leaves were cut in pieces of x 3 run and floated on water in Petri dishes overnight at room temperature. Leaf pieces (-44-48 h after infiltration) were then used to study ethylene biosynthesis and oxidative burst as follows. Ethylene biosynthesis was assayed by placing leaf samples (four pieces with -25 mg fresh weight) in 6-mi tubes with 500 p1 of water or water containing the appropriate con-centration of elflS. Tubes were sealed with rubber caps, and ethylene accumulating in the headspaee within 3 h of incubation was determined by gas chromatography. -19-
For oxidative burst, leaf pieces (one piece/well) were placed in wells of 96-well plates con-taining 100 R' of water, -10 ng/ml peroxidase (horseradish peroxidase; Applichem), 20 im luminol, and clii 8 at the concentration to be tested. Light emission was measured as relative light units in a 96-well luminometer (Mithras LB 940; Berthold Teclmologics).
Results: Oxidative burst (indicated in RLU = relative light units) in N henthamiana leaf pieces which express the constructs for the new receptors E6/L9/E, L9/E, L9/E R23 1Q, EFR S298P or EFR. Samples were treated with ligand peptides elflS (Figure 2), e1fAVNV (Figure 3) or elflCl4 (Figure 4) in a dose dependent manner (x-axis: concentration in nM).
The oxidative burst is a cellular response characteristic for very early plant stress reactions related to pathogen invasion. Under laboratory conditions it serves as one of the most sensi-tive tools to measure the functionality of MAMP receptors and thus indicates the sensitivity of a ligand-reeeptor system.
Following heterologous expression in N benthatniana, the novel designed plant LRR-RKs have been tested in the oxidative burst assay in respect to their responsiveness to elfl8, el\VNV or elfKl4 peptides. Ligands have been applied in increasing doses and the detection limits of each receptor-ligand pair (represented by the minimal active concentration) have been determined from the results represented in Figures 2 to 4.
Claims (38)
- -20 -CLAIMSAn artificial plant receptor comprising an extracellular Leucine Rich Repeat (LRR)-domain, a transmembrane domain and an intracellular kinase domain, characterized in that said LRR-domain of said artificial plant receptor comprises at least one portion of an LRR-domain of a plant receptor RI that is able to bind a protein ligand LI, and said LRR-domain of said artificial plant receptor compriscs at least one portion of an LRR-domain of a plant receptor R2 that is not able to bind said protein ligand LI, wherein RI and R2 are different.
- 2. The artificial plant receptor according to claim 1, wherein said transmembranc domain and/or said intracellular kinase domain are derived from the plant receptor RI or R2, orathird plant receptor R3.
- 3. The artificial plant receptor according to claim 2, wherein R3 is involved in any plant cellular process other than pathogen-specific responses, such as growth, development or cell death.
- 4. The artificial plant receptor according to claim 2 or 3, wherein R3 is brassinosteroid-insensitive 1 (BRI 1).
- 5. An artificial plant receptor according to any one of claims 1 to 4, wherein Ll comprises the sequence of elfl8 (SEQ ID No. I).
- 6. The artificial plant receptor according to any one ofclaims I to 5, wherein said at least one portion of an LRR-domain of a plant receptor R2 is flanked by at least two portions of said LRR-domain of said plant receptor Ri. -21 -
- 7. The artificial plant receptor according to any one of claims I to 6, wherein said portion of said LRR-domain of said plant receptor Ri and/or R2 is a consecutive stretch ofat least 5,6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 19 or 20 amino acids of the sequence of the LRR-domain of the respective receptor, or, alternatively, at least one single leucine reach repeat of the respective receptor, or a consecutive sequence of 2, 3, 4, 5 or more single leucine reach repeats of the respective receptor.
- 8. The artificial plant receptor according to any one of claims 1 to 7, that does not bind elfiS (SEQ ID No. 1) and/or EF-Tu.
- 9. Thc artificial plant receptor according to any one of claims I to 8, wherein said artificial plant receptor binds to an artificial protein ligand comprising a mutated sequence of elfIS (SEQ ID No. 1).
- 10. The artificial plant receptor according to claim 9, wherein said mutated sequence is obtained by substitution, addition, deletion, insertion or chemical modification of at least one residue in the amino acid sequence of elfl8 (SEQ ID No. 1).
- 11. The artificial plant receptor according to any one of claims 1 to 10, wherein said plant receptor Ri that is able to bind a protein ligand Li comprises a sequence having at least 70% identity to the sequence of EF-Tu Receptor (EFR), preferably to a protein encoded by the gene with the accession No. At5g20480.
- 12. The artificial plant receptor according to any one of claims 1 toil, wherein said plant receptor R2 that is not able to bind a protein ligand LI comprises a sequence having at least 70% identity to the sequence of EFR-like (L), preferably to a protein encoded by the gene with the accession No. At3g471 10.
- 13. The artificial plant receptor according to any one of claims I to 12 wherein said artificial plant receptor is a protein encoded by an nucleic acid sequence comprising a sequence according to SEQ ID No. 4 -7.-22 -
- 14. The artificial plant receptor according to any one of claims 9 to 13, wherein said artificial protein ligand comprises a sequence selected from SEQ ID No. 2 (eIfAVNV) or SEQ ID No.3 (elfKl4).
- 15. The artificial plant receptor according to any one of claims 1 to 14. that is conjugated to a detectable tag, preferably GFP.
- 16. The artificial plant receptor according to any one of claims 1 to 15, wherein the artificial plant receptor when expressed in a plant cell, induces upon binding to a ligand intracellular signalling, preferably yielding into an immune response of said cell, such as ethylene synthesis, expression of immune genes or production of reactive oxygen species (oxidative burst).
- 17. The artificial plant receptor according to any one of claims 1 to 15, wherein the artificial plant receptor when expressed in a plant cell, induces upon binding to a ligand intracellular signalling, preferably yielding into plant growth, plant cell death or a promotion of plant development or any other plant cellular response besides pathogen-specific responses.
- 18. The artificial plant receptor according to any one of claims 1 to 17, wherein the ligand is an artificial ligand.
- 19. An artificial protein ligand able to bind to the LRR-Domain ofan artificial receptor according to any one of claims ito 18.
- 20. The artificial protein ligand according to claim 19, comprising a mutated sequence ofelfl8 (SEQ ID No. 1).
- 21. The artificial protein ligand according to claim 20, wherein said mutated sequence is obtained by substitution, addition, deletion, insertion or chemical modification ofat least one residue in the amino acid sequence ofelfl8 (SEQ ID No. 1).
- 22. The artificial protein ligand according to any one of claims 19 to 21, comprising a sequence selected from SEQ ID No. 2 (elfAYNY) or SEQ ID No. 3 (eltKl4).-
- 23 - 23. A construct comprising a nucleotide sequence encoding for an artificial plant receptor according to any one of claims 1 to 18, or a nucleotide sequence encoding for an artificial protein ligand according to any one of claims 19 to 22.
- 24. The construct according to claim 23, which allows the expression of said nucleotide sequences in a plant cell or tissue.
- 25. Use of a construct according to claim 23 or 24 for stably or transiently transforming a plant, a plant cell or plant tissue.
- 26. A plant cell or plant comprising an artificial plant receptor according to any one of claims 1 to 18, or an artificial protein ligand according to any one of claims 19 to 22, preferably wherein said plant cell is transformed with a construct according to claim 23 or 24.
- 27. Usc of an artificial plant receptor according to any one of claims ito 18, and/or an artificial protein ligand according to any one of claims 19 to 22, in a plant or plant cell or plant tissue.
- 28. The use according to claim 27, wherein said artificial protein ligand is able to bind to said artificial plant receptor and wherein the binding of said ligand to said receptor induces intracellular signalling.
- 29. Thc usc according to claim 28, wherein the binding of said ligand to said receptor induces an immune response in said plant, plant cell or plant tissue.
- 30. The use according to claim 28, wherein the immune response is characterized by the differential expression of immune genes, release of ethylene and/or the production of reactive oxygen species.
- 31. The use according to any one of claims 27 to 30, for priming a plant against infections.
- 32. A method for screening new receptor ligands, comprising the steps of -24 -i. Providing an artificial plant receptor according to any one of claims 1 to 18.ii. Contacting said artificial plant receptor with a candidate ligand molecule, iii. Determining the binding of said candidate ligand molecule to said artificial plant receptor, Wherein, when said candidate ligand molecule binds to said artificial plant receptor, said candidate ligand molecule is a ligand of said artificial plant receptor.
- 33. The method according to claim 32, wherein said candidate ligand molecule is not a ligand of RI and/or R2, preferably wherein said candidate ligand molecule of an artificial ligand that docs not occur in nature, for example a protcinaccous ligand that is altered in its amino acid sequence by human induced mutagenesis.
- 34. The method according to claim 32 or 33, wherein said artificial plant receptor is in step b. provided in a plant, plant cell or plant tissue, and/or wherein the binding in step iii. is determined by monitoring the immune response of said plant cell, preferably by monitoring the expression of immune related genes or suitable reporter genes, release of ethylene and/or the production of reactive oxygen species.
- 35. Use of an artificial plant receptor according to any one of claims Ito 18, and/or an artificial protein ligand according to any one of claims 19 to 22, in a plant or plant cell or plant tissue to stimulate and/or promote plant growth, development or cell death.
- 36. Method for the isolation of a plant cell, comprising a. Transforming a plant or plant tissue or plant cell with an expression construct comprising an artificial plant receptor according to any one ofclaims I to 18, b. Bringing into contact a population of cells derived from said transformed plant, plant tissue or plant cell, with an artificial ligand according to any one of claims 19 to 22, wherein said artificial protein ligand specifically binds to said artificial plant receptor, c. Removing any non-bound cell, d. Isolating cells which are bound to said artificial protein ligand coupled to a maa
- 37. The method according to claim 35, wherein said expression constructs allows thr a plant cell type-specific or plant tissue-specific expression of said artificial plant receptor.
- 38. The method according to claim 35 of35, wherein said expression construct allows fbr a constitutive expression or an inducible expression.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB1216183.2A GB2505718A (en) | 2012-09-11 | 2012-09-11 | Chimeric plant receptors comprising LRR domains from two different receptors |
| PCT/EP2013/068831 WO2014041026A1 (en) | 2012-09-11 | 2013-09-11 | Artificial plant receptors and ligands |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB1216183.2A GB2505718A (en) | 2012-09-11 | 2012-09-11 | Chimeric plant receptors comprising LRR domains from two different receptors |
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| Publication Number | Publication Date |
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| GB201216183D0 GB201216183D0 (en) | 2012-10-24 |
| GB2505718A true GB2505718A (en) | 2014-03-12 |
| GB2505718A9 GB2505718A9 (en) | 2014-03-26 |
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| GB1216183.2A Withdrawn GB2505718A (en) | 2012-09-11 | 2012-09-11 | Chimeric plant receptors comprising LRR domains from two different receptors |
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| GB (1) | GB2505718A (en) |
| WO (1) | WO2014041026A1 (en) |
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| EP3656784A1 (en) * | 2018-11-20 | 2020-05-27 | Eberhard Karls Universität Tübingen | Chimeric pattern recognition receptor kinases |
| CN112080516B (en) * | 2020-08-05 | 2022-02-15 | 四川农业大学 | Application and method of BRI1 in plant immune signal verification |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001009283A2 (en) * | 1999-07-28 | 2001-02-08 | The Regents Of The University Of California | Chimeric receptors and their uses |
| WO2002010367A1 (en) * | 2000-07-28 | 2002-02-07 | The Salk Institute For Biological Studies | Chimeric receptors and their uses |
| WO2010139790A1 (en) * | 2009-06-04 | 2010-12-09 | Università Degli Studi Di Roma "La Sapienza" | Constructs expressing chimeric receptors and use thereof for the controlled activation of defence response to pathogens in plants |
-
2012
- 2012-09-11 GB GB1216183.2A patent/GB2505718A/en not_active Withdrawn
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2013
- 2013-09-11 WO PCT/EP2013/068831 patent/WO2014041026A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001009283A2 (en) * | 1999-07-28 | 2001-02-08 | The Regents Of The University Of California | Chimeric receptors and their uses |
| WO2002010367A1 (en) * | 2000-07-28 | 2002-02-07 | The Salk Institute For Biological Studies | Chimeric receptors and their uses |
| WO2010139790A1 (en) * | 2009-06-04 | 2010-12-09 | Università Degli Studi Di Roma "La Sapienza" | Constructs expressing chimeric receptors and use thereof for the controlled activation of defence response to pathogens in plants |
Non-Patent Citations (4)
| Title |
|---|
| Functional Plant Biol; Vol 33, pp 723-730 (2006). Dievart et al. "Brassinosteroid-independent function of BRI1/CLV chimeric receptors" * |
| J Biol Chem; Vol 285, pp 19035-19042 (2010). Albert et al. "Arabidopsis thaliana pattern recognition receptors for bacterial elongation factor....." * |
| Proc Natl Acad Sci USA; Vol 107, pp 9452-9457 (2010). Brutus et al. "A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides" * |
| Science; Vol 288, pp 2360-2363 (2000). He et al. "Perception of brassinosteroids by the extracellular domain of the receptor kinase BRI1" * |
Also Published As
| Publication number | Publication date |
|---|---|
| GB201216183D0 (en) | 2012-10-24 |
| WO2014041026A1 (en) | 2014-03-20 |
| GB2505718A9 (en) | 2014-03-26 |
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