Engineered plant cell-surface immune receptors and uses thereof

Abstract

The present invention relates generally to a plant cell-surface immune receptor engineered to modulate an immune response in a plant, cell or seed, as well as nucleic acid molecules encoding the engineered receptor, and methods of use. In particular, the present invention relates to a plant cell-surface immune receptor into which a coiled-coil (CC) motif is integrated to enhance or modify the plant's immune response, with utility in reducing pathogen infection and improving pathogen resistance.

Description

[0001] ENGINEERED PLANT CELL-SURFACE IMMUNE RECEPTORS AND USES THEREOF

[0002]

[0001] This application makes reference to and claims the benefit of priority of the Singapore Patent Application No. 10202500103U filed on 13 January 2025, the content of which is incorporated herein by reference for all purposes.

[0003] FIELD OF THE INVENTION

[0004]

[0002] The present invention relates generally to a plant cell-surface immune receptor engineered to modulate an immune response in a plant, cell or seed, as well as nucleic acid molecules encoding the engineered receptor, and methods of use. In particular, the present invention relates to a plant cellsurface immune receptor into which a coiled-coil (CC) motif is integrated to enhance or modify the plant’s immune response, with utility in reducing pathogen infection and improving pathogen resistance.

[0005] BACKGROUND OF THE INVENTION

[0006]

[0003] Plants rely on a multifaceted innate immune system to detect and respond to diverse pathogens. A key component of this defence strategy is the recognition of pathogen-associated molecular patterns (PAMPs) by cell-surface pattern recognition receptors (PRRs) (Jones and Dangl, 2006). In Arabidopsis thaliana, one of the most extensively characterised PRRs is FLAGELLIN SENSING2 (FLS2), which perceives the conserved bacterial flagellin epitope flg22 (Gomez-Gomez and Boiler, 2000). Upon binding flg22, FLS2 rapidly associates with its co-receptor BRI1 -ASSOCIATED RECEPTOR KINASE1 (BAK1), forming an active receptor complex that triggers downstream immune responses (Chinchilla et al., 2007; Heese et al., 2007).

[0007]

[0004] Activation of the FLS2-BAK1 complex leads to a cascade of defence-related events, including reactive oxygen species (ROS) production, activation of mitogen-activated protein kinases (MAPKs), and transcriptional reprogramming (Boiler and Felix, 2009). These responses help fortify plant cells against invading pathogens, such as Pseudomonas syringae pv. tomato DC3000 (Felix et al., 1999). This FLS2-mediated surveillance mechanism exemplifies how PRRs underpin plant immunity, highlighting opportunities to engineer or enhance such receptor-based defence pathways. By exploiting the principles of PRR activation and oligomerisation from dimerization to higher order assembly, it will become feasible to bolster disease resistance not only in Arabidopsis but also across a broader range of plant species facing bacterial threats, including but not limited to Pseudomonas and Xanthomonas genera.

[0008]

[0005] Advances in receptor engineering have significantly expanded current strategies for enhancing plant immunity. One established approach involves the overexpression of native pattern recognition receptors (PRRs), which increases plant sensitivity to pathogen-associated molecular patterns (PAMPs) and strengthens basal immune responses. For example, overexpression of the Xa21 receptor kinase in rice (Oryza sativa) enhances resistance to Xanthomonas oryzae pv. oryzae (Song et al., 1995), while elevated expression of FLS2 (FLAGELLIN SENSING2) in Arabidopsis thaliana improvesresponsiveness to flagellin-derived PAMPs (Takai et al., 2008). These studies collectively demonstrate that increasing the abundance of endogenous PRRs can reinforce basal plant defense mechanisms.

[0009]

[0006] Heterologous expression of PRRs has also emerged as a powerful strategy for broadening immune recognition capacity. Introduction of the Arabidopsis thaliana EF-Tu receptor (EFR), which perceives the bacterial elongation factor Tu, into solanaceous crops confers enhanced resistance to diverse bacterial pathogens (Lacombe et al., 2010; Zipfel et al., 2006). Such cross-species deployment of immune receptors enables crops to detect PAMPs that their native immune systems would not ordinarily recognise, thereby expanding their defensive repertoire. Further advancements include the construction of chimeric PRRs, generated by fusing or exchanging functional domains between receptors. Chimeric receptors can exhibit broadened ligand specificity or improved signaling potency. Domain-swapping within leucine-rich repeat (LRR) receptors has been shown to produce novel or enhanced PAMP-binding functions (Boutrot and Zipfel, 2017; Brutus et al., 2010), and various mosaic receptor designs have been tested in model plant species to evaluate broadened pathogen detection potential (Schwessinger and Ronald, 2012).

[0010]

[0007] In addition to PRRs, intracellular NLR (nucleotide-binding leucine-rich repeat) receptors have been engineered to alter or expand effector recognition profiles. Although promising, such modifications risk triggering inappropriate immune activation. The rice NLR pair Pik exemplifies how incompatible domain combinations can lead to unintended autoimmunity and reduced plant fitness (De la Concepcion et al., 2019). By identifying structural determinants of such incompatibilities, researchers have successfully re-engineered NLR receptors to provide expanded resistance while avoiding detrimental constitutive activation (Kroj et al., 2016).

[0011]

[0008] Beyond receptor modification itself, engineering downstream signaling components offers another avenue to amplify immune responses. For instance, constitutive activation or targeted overexpression of mitogen-activated protein kinases (MAPKs) in Arabidopsis enhances resistance to multiple pathogens, though often accompanied by growth penalties (Zhang et aL, 2007). Efforts to modulate transcription factors that orchestrate defense gene expression similarly aim to increase immunity while mitigating associated metabolic costs (Hwang et al., 2011).

[0012]

[0009] Effector-triggered immunity (ETI) has likewise been enhanced by stacking multiple NLR receptors to achieve broader effector recognition (Dangl et al., 2013) and by engineering new effector specificities through domain exchange or decoy integration (Kroj et al., 2016). The advent of targeted genome editing tools such as CRISPR / Cas9 has further enabled the modification of susceptibility (S) genes, limiting pathogen effector exploitation and thereby improving resistance (Langner et al., 2018; Zaidi et al., 2017).

[0013]

[0010] Despite these advancements, existing receptor engineering strategies exhibit several limitations. A recurring challenge is the growth-defense trade-off, where heightened immune activationdiverts metabolic resources from growth and reproduction. Overexpression of certain PRRs or NLRs has been associated with reduced seed size, biomass, and agronomic performance (Huot et al., 2014; Pajerowska-Mukhtar et al., 2012). Specificity and compatibility issues also arise when combining domains from evolutionarily distant species, as heterologous or chimeric receptors may not integrate efficiently into the host’s signaling architecture, resulting in reduced stability or impaired function (Boutrot and Zipfel, 2017).

[0014]

[0011] Another limitation concerns the risk of uncontrolled or constitutive immune activation. Overexpressed signaling components or misregulated NLRs can trigger chronic defense responses, causing energy depletion, early senescence, or heightened sensitivity to abiotic stress (Zhang et al., 2007; Chakraborty et al., 2018). Moreover, many downstream signaling proteins participate in both biotic and abiotic stress pathways, creating complex crosstalk that complicates precise engineering (Denance et al., 2013; Tsuda and Somssich, 2015). Environmental variation, including temperature, drought, or simultaneous pathogen pressure, can disrupt or override engineered immune circuits, leading to inconsistent performance under field conditions (Cheng et al., 2019).

[0015]

[0012] Therefore, there exists a need for alternative techniques and strategies for modulating a plant's immune response to a range of pathogens, particularly by targeting and engineering plant cell-surface receptors, thereby improving the innate immune system of the plant to detect and respond to diverse pathogens.

[0016] SUMMARY OF THE INVENTION

[0017]

[0013] The present invention satisfies the aforementioned need in the art by providing the polypeptides, nucleic acids, plants / plant cells / plant seeds and methods of use described herein.

[0018]

[0014] In one aspect there is provided a polypeptide for use in modulating an immune response in a plant, comprising a plant cell-surface immune receptor, and a coiled-coil (CC) motif operably fused to the plant cell-surface immune receptor, wherein the CC motif is selected from a dimeric, trimeric, tetrameric, or pentameric CC motif.

[0019]

[0015] In various embodiments, the plant cell-surface immune receptor is a pattern-recognition receptor (PRR), a PRR co-receptor, or a leucine-rich repeat (LRR)-single transmembrane domain receptor, preferably a FLS2 or PEPR1 , or BAK1.

[0020]

[0016] In various embodiments, the CC motif is a dimeric CC-motif or a tetrameric CC-motif.

[0021]

[0017] In various embodiments, the dimeric CC-motif comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6, or a functional variant thereof, and the tetrameric CC-motif comprises or consists of an amino acid sequence set forth in SEQ ID NO: 8, or a functional variant thereof.

[0018] In various embodiments, the polypeptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NO: 15, 16, 18, 19, 21 or 22, or a functional variant thereof.

[0022]

[0019] In another aspect, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide disclosed herein, preferably the nucleic acid molecule is comprised in a vector.

[0023]

[0020] In various embodiments, the nucleic acid molecule further comprises a promoter capable of driving expression in a plant operably linked to the nucleotide sequence encoding the polypeptide.

[0024]

[0021] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in any one of SEQ ID NO: 24, 25, 27, 28, 30 or 31 , or a functional or degenerate variant thereof.

[0025]

[0022] In various embodiments, the polypeptide, or the nucleic acid molecule disclosed herein, for use in reducing, treating or preventing a pathogen infection in a plant, and / or improving resistance to a pathogen infection in a plant.

[0026]

[0023] In another aspect, there is provided a plant, plant cell or plant seed comprising the polypeptide, or the nucleic acid molecule disclosed herein.

[0027]

[0024] In various embodiments, the plant is Arabidopsis thaliana or Nicotiana benthamiana.

[0028]

[0025] In various embodiments, the plant comprises or expresses a monocot or dicot LRR receptor on the cell surface.

[0029]

[0026] In another aspect, there is provided a method of producing the plant, plant cell or plant seed disclosed herein, comprising introducing the nucleic acid molecule disclosed herein into a plant, plant cell or plant seed, preferably transforming the plant, plant cell or plant seed with the nucleic acid molecule.

[0030]

[0027] In another aspect, there is provided a method for modulating an immune response of a plant, comprising introducing the nucleic acid molecule disclosed herein, into the plant and expressing the nucleic acid molecule such that the encoded polypeptide is localised to the plasma membrane of plant cells in the plant.

[0031]

[0028] In various embodiments, the plant exhibits receptor-mediated immunity following perception of a pathogen-associated molecular pattern (PAMP) or damage-associated molecular pattern (DAMP), and / or the plant does not display stunted growth, developmental defects, or phenotypes associated with chronic immune activation.

[0029] In various embodiments, the cell-surface immune receptor remains in a primed but inactive state until perception of PAMPs or DAMPs in the presence of a pathogen.

[0032]

[0030] In another aspect, there is provided a method for reducing pathogen infection in a plant, comprising introducing the nucleic acid molecule disclosed herein, into the plant, and expressing the nucleic acid molecule such that the encoded polypeptide is localised to the plasma membrane of plant cells in the plant.

[0033]

[0031] In various embodiments, the pathogen infection is a bacterial, fungal, or oomycete pathogen.

[0034]

[0032] In another aspect, there is provided a method of producing a plant, plant cell or plant seed comprising an engineered plant cell-surface immune receptor, the method comprising modifying a plant cell-surface immune receptor in the plant, plant cell or plant seed to operably associate, fuse or couple with a CC motif, wherein the CC motif is selected from a dimeric, trimeric, tetrameric, or pentameric CC motif.

[0035]

[0033] In various embodiments, the modifying step comprises genetically modifying a gene encoding the plant cell-surface immune receptor to operably link a nucleotide sequence encoding the CC motif, such that the expressed plant cell-surface immune receptor is operably associated, fused or coupled with the CC motif.

[0036]

[0034] In various embodiments, the modifying step comprises post-translationally modifying the plant cell-surface immune receptor to operably associate, fuse or couple with the CC motif, and the method further comprises introducing a nucleic acid molecule encoding the CC motif into the plant, plant cell or plant seed under suitable conditions to express the nucleic acid molecule such that the encoded CC motif associates, fuses or couples to the plant cell-surface immune receptor.

[0037]

[0035] In various embodiments, the method modulates an immune response and / or reduces pathogen infection and / or improves resistance to a pathogen infection, in the plant, plant cell or plant seed.

[0038]

[0036] In various embodiments, the CC motif is a dimeric CC-motif or a tetrameric CC-motif.

[0039]

[0037] In various embodiments, the dimeric CC-motif comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6, or a functional variant thereof, and the tetrameric CC-motif comprises or consists of an amino acid sequence set forth in SEQ ID NO: 8, or a functional variant thereof.

[0040]

[0038] In various embodiments, the plant cell-surface immune receptor is a pattern-recognition receptor (PRR), a PRR co-receptor, or a leucine-rich repeat (LRR)-single transmembrane domain receptor, preferably a FLS2 or PEPR1 , or BAK1.

[0039] It is understood that all embodiments disclosed herein in relation to one aspect of the invention are similarly applicable to all other aspects of the invention.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042]

[0040] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[0043]

[0041] FIG. 1 shows a schematic illustration for genetic engineering of FLS2 or PEPR1 for defined oligomerization.

[0044]

[0042] FIG. 2 A diagram illustrating the early plant immune signalling initiated by the plasma membrane (PM) localized PRRs (here as FLS2 / PEPER1) recognizing PAMPs or DAMPs. Those immune activation will eventually trigger multiple layers of defence responses coupled by plant growth inhibition.

[0045]

[0043] FIG.3 Secant view and single molecule images of FLS2-M / D / T-mG in the cotyledon epidermal cell of Arabidopsis. Colour bar indicates the corresponding intensity. Scale bar, 10 mm and 2 mm from upper to lower panel.

[0046]

[0044] FIG. 4 ROS productions, as monitored by relative luminescence unit (RLU), in leaf discs of indicated Arabidopsis lines elicited by 20 nM flg22. Solid lines and shaded area represent mean ± SD from multiple leaf discs.

[0047]

[0045] FIG. 5 Quantification of total photon reading in 5-30 min in (FIG. 4) representing the total ROS generation in indicated Arabidopsis genotypes, n > 13 leaf discs.

[0048]

[0046] FIG. 6 MAPK activation in fls2, WT and FLS2-CC-mG Arabidopsis seedlings elicited by 20 nM flg22, as determined over time by immunoblot analysis through anti-p-44 / 42 MAPK antibody.

[0049]

[0047] FIG. 7 Quantification of bacteria colonialization at 3-days post infection. 1 x 106CFU-mL-1of Pst DC 3000 were injected into the rosette leaves of 4-weeks-old WT, fls2 and FLS2-M / D / T-mG Arabidopsis seedling, n = 12 replicates for each genotype. Error bars = SD.

[0050]

[0048] FIG. 8 Growth assay of WT, fls2 and FLS2-M / D / T-mG Arabidopsis in half strength of MS medium supplemented with or without 1 pM flg22 for 5 days, before root length measurement. Scale bar, 0.5 cm.

[0051]

[0049] FIG. 9 Quantification of the root length in (H) at resting states without flg22 treatment. n>20 roots.

[0050] FIG. 10 Quantification of the root growth inhibition by flg22 treatment in (H). n>20 roots.

[0052]

[0051] FIG. 11 Secant view and single particle images of PEPR1-M / D / T-mGs expressing in the leaf cells of N. benthamiana. Colour bar indicates the corresponding intensity. Scale bar, 10 pm and 2 pm from upper to lower panel.

[0053]

[0052] FIG. 12 ROS productions, as monitored by relative luminescence unit (RLU), in N. benthamiana leaf discs as in (FIG. 11) elicited by 20 nM pep1. Solid lines and shaded area represent mean ± SD from multiple leaf discs.

[0054]

[0053] FIG. 13 Quantification of total photon reading in 5-30 min in (FIG. 12) representing the total ROS generation, n > 14 of leaf discs.

[0055]

[0054] FIG. 14 Schematic of BAK1 engineering. The CC domains (D and T) were integrated in between the transmembrane domain and the kinase domain (KD) of BAK1. BAK1 without CC domain was designated as M. C-terminal mG fusion was used for imaging but omitted in ROS assays.

[0056]

[0055] FIG. 15 Single-particle images of BAK1-CC-mGs in N. benthamiana bak1 mutant.

[0057]

[0056] FIG. 16 Measurement of ROS in leaf discs of N. benthamiana bak1 mutant expressing BAK1 -M / D / T (similar protein levels) or empty vector (Ctrl) after 20 nM flg22 elicitation. Solid lines and shaded area represent mean ± SD from multiple leaf discs.

[0058]

[0057] FIG. 17 Quantification of total photon reading in 5-30 min in (FIG. 16) representing the total ROS generation, n = 12 leaf discs. Box plots in (FIG. 5, 13, 17) indicate mean and median by cross symbols and centre bars, upper and lower limits represent quartiles, whiskers indicate SD. Significant differences were determined via one-way ANOVA with multiple comparisons (****p < 0.0001 , “**p 2 0.001 , **p < 0.01 , *p < 0.05, ns = not significant).

[0059] DETAILED DESCRIPTION OF THE INVENTION

[0060]

[0058] The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural, and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0061]

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" isintended to include "and" unless the context clearly indicates otherwise. The term "comprises" means "includes." In case of conflict, the present specification, including explanations of terms, will control.

[0062]

[0060] The present invention provides an innovative approach to receptor engineering based on precise, dimerisation-controlled modulation of plant receptors. In particular, the invention departs from traditional approaches that rely on receptor overexpression or domain exchange between evolutionarily distant species. Instead, the present invention focuses on control over oligomerisation state to achieve predictable and conditional activation of pattern recognition receptors (PRRs). By integrating coiled-coil (CC) motifs, the invention enables enforced receptor dimerisation with high structural precision.

[0063]

[0061] In contrast to approaches that elevate receptor expression or blend unrelated receptor domains, the present invention initiates receptor activation upon genuine pathogen challenge. This “on-demand” strategy avoids costly energy expenditures or growth penalties associated with chronic immune activation, thereby maintaining normal plant development in pathogen-free conditions. The resulting system provides a robust yet tunable defence platform, plants are primed to mount a rapid and potent immune response if a pathogen is detected but remain developmentally unaltered otherwise. This invention offers a sustainable, next-generation strategy for crop protection that could be extended beyond Arabidopsis to a variety of agriculturally important species facing bacterial, fungal, or oomycete threats.

[0064]

[0062] As a non-limiting representative example of the concept of the present invention engineered oligomeric motifs were fused to the Arabidopsis thaliana flagellin receptor FLS2, a receptor known to heterodimerize with its co-receptor BAK1 upon detection of bacterial flagellin (Chinchilla et al., 2007; Heese et al., 2007). Earlier studies demonstrated that the formation of FLS2-FLS2 receptor clustering for effective immune signalling in Arabidopsis thaliana (Sun et al., 2012; Tran et al., 2020). Building on these insights, the invention described here focuses on engineering a synthetic dimeric form of the FLS2 receptor in stably transformed Arabidopsis plants. By driving FLS2 into a dimerised “primed” configuration, the present invention results in a more robust and rapid activation of immune responses upon pathogen detection. This strategy is designed to strengthen the plant’s innate immune system specifically against bacterial pathogens, while minimising energy expenditure and maintaining normal growth in the absence of infection.

[0065]

[0063] The dimer-forming CC motif provides high specificity, reducing the likelihood of spurious interactions or unintended receptor activation, and ensuring that receptor signalling remains tightly coupled to genuine pathogen cues. This system is also inherently modular, as CC motifs can be readily customised and adapted to other receptors and plant species, offering a flexible platform for engineering improved immunity across diverse crops and pathogen types.

[0066]

[0064] This disclosure details a technology enabling precise control of PRR, or co-receptor, dimerization through coiled-coil oligomerisation domains, as demonstrated with the FLS2 receptor. Byenhancing the kinase activities of initial signalling complexes upon PAMP perception, the immune response is more robust and rapid, yet remains off until actual pathogen challenge. This contrasts with alternative methods that risk unintended autoactivation and associated growth penalties. The precise dimerization-controlled protein engineering represents a significant advancement over existing receptor modification strategies for enhancing plant immunity. By using coiled-coil-based oligomeric motifs to control the dimerization of PRRs like FLS2, or their co-receptors, a balance is achieved between strong immune responses and normal plant growth. This invention thus presents a robust, scalable, and widely adaptable method for bolstering plant immunity through controlled PRR dimerization, with strong potential for patent protection and commercial deployment across diverse agricultural sectors.

[0067]

[0065] Accordingly, in one aspect, there is provided a polypeptide for use in modulating an immune response in a plant, comprising a cell-surface immune receptor, and acoiled-coil (CO) oligomerisation motif operably fused to the cell-surface immune receptor, wherein the CC motif is selected from a dimeric, trimeric, tetrameric, or pentameric CC oligomerisation motif. It will be appreciated that the polypeptide may be termed as a modified or engineered cell-surface immune receptor, with the modification being the fusion of the CC-motif.

[0068]

[0066] As used herein, the term ‘‘cell-surface immune receptor” refers to a class of plant plasmamembrane receptors involved in the perception or transduction of extracellular pathogenic signals. These receptors mediate the early steps of innate immune activation by recognising pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), followed by forming essential signalling complexes required for defence initiation. The term is intended to encompass both ligand-binding receptors and their associated co-receptors that participate in immune signal propagation at the cell surface. In various embodiments, the cell-surface immune receptor may be a pattern-recognition receptor (PRR), a PRR co-receptor, or a leucine-rich repeat (LRR)-single transmembrane domain receptor. The coiled-coil oligomerisation strategy can readily be extended to PRRs recognising diverse PAMPs, enhancing immune response and enabling resistance against multiple pathogens in various crops.

[0069]

[0067] In various embodiments, the cell-surface immune receptor may comprise pattern-recognition receptors (PRRs), which directly recognise PAMPs or DAMPs and typically comprise an extracellular ligand-binding domain, a single transmembrane helix, and an intracellular kinase domain (in receptorlike kinases) or a short cytosolic tail (in receptor-like proteins). Examples of PRRs include FLS2, a leucine-rich repeat receptor-like kinase (LRR-RLK) that recognises bacterial flagellin; EFR, which detects EF-Tu-derived peptides; PEPR1 and PEPR2, which perceive plant-derived Pep peptides; CERK1 , a lysin-motif receptor kinase involved in chitin recognition; LYK5, a high-affinity chitin receptor; RLP23, an LRR receptor-like protein that detects necrosis-inducing peptides; and tomato receptors such as Cf-4 and Cf-9 that recognise fungal effectors. PRRs may be structurally classified as LRR-RLKs, LRR-RLPs, LysM-RLKs, LysM-RLPs, or other single-pass membrane proteins capable of ligand-induced immune activation.

[0068] In various embodiments, the cell-surface immune receptor may comprise PRR co-receptors, which do not directly bind elicitors but are essential for PRR activation upon ligand perception. Coreceptors form ligand-induced heterodimers or higher-order complexes with PRRs to initiate downstream signalling cascades. Examples include BAK1 (SERK3), a well-characterised LRR-RLKthat partners with multiple PRRs including FLS2, EFR, PEPR1, and PEPR2; BKK1 (SERK4), which functions redundantly with BAK1 ; SERK1 and SERK2, which participate in several PRR pathways; and SOBIR1 , an LRR-RLK that stabilises and activates many LRR-RLP-type PRRs such as RLP23 and Cf-4.

[0070]

[0069] In various embodiments, the cell-surface immune receptor may comprise leucine-rich repeat (LRR)-single transmembrane domain receptors, which are a plant cell-surface receptor comprising an extracellular LRR domain, a single-pass transmembrane helix, and either (I) an intracellular kinase domain, as in LRR receptor-like kinases (LRR-RLKs), or (ii) a short intracellular tail lacking a kinase domain, as in LRR receptor-like proteins (LRR-RLPs). These receptors are expressed at the plasma membrane and participate in extracellular elicitor perception, including pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) recognition. LRR single-transmembrane receptors are widely conserved across plant species and include functional orthologues, paralogues, and homologues found in both monocot and dicot plants. Accordingly, the term encompasses any monocot or dicot LRR-type receptor localised at the plant cell surface, including but not limited to LRR-RLKs such as FLS2, EFR, PEPR1 , PEPR2, and BAK1 / SERK-family co-receptors, as well as LRR-RLPs such as RLP23 and Cf-family receptors.

[0071]

[0070] In various embodiments, the cell-surface immune receptor may comprise an LRR receptor-like kinase (LRR-RLK), LRR receptor-like protein (LRR-RLP), LysM-RLK, LysM-RLP, or any single-transmembrane immune receptor involved in extracellular elicitor perception.

[0072]

[0071] In various embodiments, the cell-surface immune receptor may be selected from Table 1 below derived from the Arabidopsis thaliana plant.

[0073]

[0072] Table 1 : Sequence information of cell-surface immune receptors

[0074]

[0075]

[0076]

[0073] The gene / locus identifiers and accession numbers of non-limiting examples of the cell-surface immune receptors is provided in Table 1. It will be appreciated that orthologues, paralogues, and other homologues of the cell-surface immune receptors listed in Table 1 are also included, which belong to the same structural and functional classes present in a broad range of other plant species, including but not limited to rice, Nicotiana species, tomato, soybean, wheat, and maize. Accordingly, any orthologue, paralogue, homologue, allelic variant, isoform, splice form, or functional equivalent of these receptors from any plant species is encompassed within the present invention.

[0077]

[0074] In various embodiments, the cell-surface immune receptor comprises any orthologue, paralogue, allelic variant, isoform, or functional equivalent of a pattern-recognition receptor (PRR), a PRR co-receptor, or a leucine-rich repeat (LRR)-single transmembrane domain receptor from any plant species.

[0078]

[0075] In various embodiments, the cell-surface immune receptor comprises a FLS2 (UniProt: Q9FL28) or PEPR1 (UniProt: Q9SSL9), or BAK1 (UniProt: Q94F62) derived from Arabidopsis thaliana, or any orthologue, paralogue, allelic variant, isoform, or functional variant thereof.

[0079]

[0076] In various embodiments, the cell-surface immune receptor comprises a BAK1 , and the BAK1 comprises a BAK1 extracellular domain (BAK1 ECD) and a BAK1 kinase domain (BAK1KD), wherein the BAK1 ECD refers to the amino acid residues M1 - K253 of BAK1 and BAK1 KD refers to the amino acid residues P254 ~ R615 of BAK1 , wherein position numbering is relative to the amino acid sequence defined in UniProt: Q94F62.

[0080]

[0077] In various embodiments, the cell-surface immune receptor is FLS2 comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 1, or functional variant thereof. In variousembodiments, the cell-surface immune receptor is PEPR1 comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 2, or functional variant thereof. In various embodiments, the cellsurface immune receptor is BAK1 comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 3, or functional variant thereof. In various embodiments, the cell-surface immune receptor comprises BAK1 ECD comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 4, or functional variant thereof, and BAK1 KD comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 5, or functional variant thereof. It will be appreciated that also contemplated are any orthologue, paralogue, homologue, allelic variant, isoform, splice form, or functional equivalent of the receptors having the amino acid sequence set forth in any one of SEQ ID NO:1-5.

[0081]

[0078] As used herein, the term “orthologue" refers to a gene or protein in a different species that derives from a common ancestral gene and retains equivalent structure or biological function, such as LRR-RLK, LRR-RLP, or SERK-family receptors performing analogous immune roles across monocot and dicot plants. The term “paralogue" refers to a related gene or protein within the same species that has arisen through gene duplication and shares sequence similarity and conserved domains with the reference receptor, including members of multi-gene families such as the SERK, LRR-RLK, or PEPR receptor families. The term “allelic variant" refers to any naturally occurring or artificially generated version of a gene containing nucleotide substitutions, insertions, deletions, or polymorphisms that may give rise to minor structural variation while retaining the ability of the encoded receptor to localise to the cell surface and function in immune signalling. The term “isoform" refers to alternative protein forms derived from the same gene through mechanisms such as alternative splicing, alternative promoter usage, alternative polyadenylation, or post-translational modification, resulting in proteins that remain functionally competent as cell-surface immune receptors within the context of the present invention.

[0082]

[0079] The term “functional variant”, as used herein relates to amino acid sequences that comprise or consist of an amino acid sequence that is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.25%, or 99.5% identical or homologous to the amino acid sequence of the reference cell-surface immune receptor, such as FLS2 or PEPR1 , or BAK1 , over their entire length, but retain the functionality of the reference amino acid sequence

[0083]

[0080] The identity of amino acid sequences (or nucleotide sequences) is generally determined by means of a sequence comparison. This sequence comparison is based on the BLAST algorithm that is established in the existing art and commonly used (cf. e.g. Altschul et al. (1990) “Basic local alignment search tool”, J. Mol. Biol. 215:403-410, and Altschul et al. (1997): “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”; Nucleic Acids Res., 25, p. 3389-3402) and is effected in principle by mutually associating similar successions of nucleotides or amino acids in the nucleic acid sequences and amino acid sequences, respectively. A tabular association of the relevant positions is referred to as an "alignment." Sequence comparisons (alignments), in particular multiplesequence comparisons, are commonly prepared using computer programs which are available and known to those skilled in the art. A comparison of this kind also allows a statement as to the similarity to one another of the sequences that are being compared. This is usually indicated as a percentage identity, i.e. the proportion of identical nucleotides or amino acid residues at the same positions or at positions corresponding to one another in an alignment. The more broadly construed term "homology", in the context of amino acid sequences, also incorporates consideration of the conserved amino acid exchanges, i.e. amino acids having a similar chemical activity, since these usually perform similar chemical activities within the protein. The similarity of the compared sequences can therefore also be indicated as a "percentage homology" or "percentage similarity." Indications of identity and / or homology can be encountered over entire polypeptides or genes, or only over individual regions. Homologous and identical regions of various nucleic acid sequences or amino acid sequences are therefore defined by way of matches in the sequences. Such regions often exhibit identical functions. They can be small, and can encompass only a few nucleotides or amino acids. Small regions of this kind often perform functions that are essential to the overall activity of the protein. It may therefore be useful to refer sequence matches only to individual, and optionally small, regions. Unless otherwise indicated, however, indications of identity and homology herein refer to the full length of the respectively indicated amino acid sequence (or nucleic acid sequence).

[0084]

[0081] The “coiled-coil (CO) motif" as used herein refers to an oligomerisation domain that is a structural motif capable of mediating non-covalent association of two or more polypeptide chains to form higher-order complexes such as dimers, trimers, tetramers, or pentamers. The CC-motif may refers to a heptad-repeat-based a-helical structural motif that mediates the non-covalent association of two or more polypeptide chains through hydrophobic core packing and electrostatic interactions. CC-motifs typically comprise repeating (a-g) heptad units in which positions "a" and “d” are occupied by hydrophobic residues, thereby promoting the formation of stable, rope-like superhelical assemblies. Depending on the specific sequence pattern, register, and charge distribution, CC-motifs can drive the formation of defined oligomeric states, including parallel or antiparallel dimers, trimers, tetramers, pentamers, or higher-order bundles. In various embodiments, the CC-motif may be naturally occurring, derived from known coiled-coil proteins (e.g., GCN4, leucine zippers, Cartwright helices), or may be synthetically engineered or computationally designed to provide precise oligomerisation valencies and rigid, predictable multimeric architectures. The CC-motif thus provides a modular oligomerisation domain that can be fused to the cell-surface immune receptor to impose a predefined oligomeric state. The CC-motifs are especially attractive for bioengineering because they exhibit predictable interaction specificity and a well-characterized sequence-to-structure relationship, enabling rational design of selfassembling protein complexes.

[0085]

[0082] In various embodiments, the CC-motifs are selected from a dimeric CC-motif, a trimeric CC-motif, a tetrameric CC-motif, and a pentameric CC-motif, wherein the predefined miltimeric state is a dimeric, a trimeric, a tetrameric, and a pentameric state.

[0083] In various embodiments, the CC-motif may be selected or designed to exhibit high dimerisation specificity, such that the engineered receptor is driven into a defined dimeric state without forming higher-order or non-specific oligomers. The CC motif provides structural precision that reduces the likelihood of spurious protein-protein interactions or inappropriate clustering of the receptor at the plasma membrane. As a result, the engineered receptor remains quiescent under resting conditions and is not subject to unintended autoactivation. Upon perception of a bona fide pathogen-associated ligand, however, the CC-mediated dimeric architecture enables a more efficient or rapid assembly of the receptor-co-receptor complex, ensuring that immune signalling remains tightly coupled to genuine pathogen cues. This functional design allows for enhanced immune activation only when appropriate stimuli are detected, avoiding the deleterious growth penalties associated with chronic or mis-regulated immune activation.

[0086]

[0084] In various embodiments, the dimeric CC-motif comprises or consists of the amino acid sequence set forth in SEQ ID NO:6, or functional variants thereof. In various embodiments, the trimeric CC-motif comprises or consists of the amino acid sequence set forth in SEQ ID NO:7, or functional variants thereof. In various embodiments, the tetrameric CC-motif comprises or consists of the amino acid sequence set forth in SEQ ID NO:8, or functional variants thereof. In various embodiments, the pentameric CC-motif comprises or consists of the amino acid sequence set forth in SEQ ID NO:9, or functional variants thereof.

[0087]

[0085] As used herein, the term “operably fused” refers to any arrangement in which the coiled-coil (CC) motif and the plant cell-surface immune receptor are joined within the same polypeptide in a manner that preserves receptor expression, localisation, and signalling capability. The term includes, but is not limited to, fusions at the N-terminus or C-terminus of the receptor, fusions mediated by peptide linkers, and embodiments in which the CC motif is incorporated into the receptor at an internal position, including insertion between functional domains such as between the transmembrane domain and intracellular kinase domain of BAK1. In all cases, the CC motif remains functionally associated with the receptor so as to modulate its oligomerisation state.

[0088]

[0086] In this regard, the polypeptide disclosed herein may also be termed as a fusion protein comprising two or more polypeptides (receptor and CC-motif) linked to form a single polypeptide chain. As used herein, the term "fusion protein" refers to a chimeric protein in which two or more distinct polypeptide sequences, originating from different proteins or functional domains, are joined together within a single polypeptide chain, such that each constituent domain or moiety retains at least part of its native biological activity. As used herein, the term "single polypeptide chain" refers to a contiguous sequence of amino acids translated from a single open reading frame (ORF), such that both the cellsurface immune receptor and the CC-motif are encoded by the same mRNA transcript and are synthesized as a unified molecule by a ribosome. In various embodiments, the fusion protein may refer to a single polypeptide chain comprising a first polypeptide comprising or consisting of the cell-surfaceimmune receptor disclosed herein operably linked to a second polypeptide comprising or consisting of a CC-motif.

[0089]

[0087] In various embodiments, the cell-surface immune receptor may be indirectly linked to the CC-motif via a linker. The linker may be a linker peptide that facilitates proper folding and function of the fusion protein. As used herein, a linker refers to a peptide sequence that connects two functional domains within a fusion protein. The linker may be rigid or flexible, and may serve to: (i) facilitate correct spatial orientation and folding of each domain; (ii) minimize steric hindrance that may impede activity; and / or (iii) preserve the functional independence of each domain. In various embodiments, the linker may comprise one or more Glycine-Serine (Gly-Ser) repeats, such as (G4S)n, where n is an integer from 1 to 5. In other embodiments, the linker may comprise a rigid helical sequence, such as one or more repeats of EAAAK (SEQ ID NO: 32), to maintain fixed spatial separation between domains and prevent domain interference. In further embodiments, the linker may comprise or consist of a synthetic unstructured polypeptide composed of small polar, uncharged amino acids, such as Glycine (Gly), Serine (Ser), Alanine (Ala), Threonine (Thr), Glutamine (Gin), Proline (Pro), Glutamic acid (Glu), and Asparagine (Asn). Such linkers, including XTEN-like linkers, are designed to be flexible, hydrophilic, and resistant to folding into stable secondary structures, thereby enhancing solubility and reducing immunogenicity. In yet other embodiments, the linker may be cleavable under specific conditions (e.g., by a protease), thereby allowing controlled release or separation of the individual domains.

[0090]

[0088] In various embodiments, the linker peptide (LP) may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 10, or functional variants thereof.

[0091]

[0089] In various embodiments, the polypeptide may further comprise one or more optional protein elements selected from epitope tags, fluorescent reporters, affinity tags, or purification tags. In various embodiments, an epitope tag such as a 1xMyc, 3xMyc, FLAG, HA, V5, or similar peptide motif may be operably linked to the receptor or CC-motif to facilitate immunodetection using commercially available antibodies. For example, a 3xMyc tag may be incorporated to enhance detection sensitivity and allow reliable verification of transgene expression by western blotting or immunopurification. In various embodiments, a fluorescent reporter protein may be fused to the CC-motif or receptor to enable visualisation, localisation, or quantification of receptor expression or receptor-complex assembly in planta. These optional elements may be positioned at the N-terminus, C-terminus, or an internal site, provided that receptor localisation and signalling capability are retained. The inclusion of such epitope tags or fluorescent reporters does not form part of the functional oligomerisation mechanism but provides useful experimental, analytical, or diagnostic features for monitoring receptor expression, stability, or oligomeric state.

[0092]

[0090] In various embodiments, the polypeptide comprises a Myc comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 11, or a functional variant thereof. In variousembodiments, the polypeptide comprises a 3xMyc comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 12, or a functional variant thereof.

[0093]

[0091] In various embodiments, the polypeptide may further comprise an epitope tag such as a 1 xMyc or 3xMyc tag, and a fluorescent reporter protein. Exemplary fluorescent reporter proteins include, without limitation, green fluorescent protein (GFP) and monomeric or stabilised derivatives such as superfolder GFP (sfGFP), monomeric GFP (mGFP), or monomeric superfolder GFP (mG) and enhanced GFP variants engineered to reduce self-association; and alternative monomeric fluorescent proteins such as monomeric Cherry fluorescent protein (mCherry), monomeric Scarlet fluorescent protein (mScarlet), monomeric far-red fluorescent protein “Kate2” (mKate2), monomeric Orange fluorescent protein 2 (mOrange2), monomeric NeonGreen fluorescent protein (mNeonGreen), and monomeric Yellow Fluorescent Protein (YFP) or Cyan Fluorescent Protein (CFP) variants. In various embodiments, the fluorescent reporter protein may be monomeric GFP.

[0094]

[0092] In various embodiments, the fluorescent reporter protein may be a monomeric superfolder GFP (mG) comprising or consisting of the amino acid sequence set forth in SEQ ID NO:13, or a functional variant thereof.

[0095]

[0093] In various embodiments, the polypeptide may comprise the cell-surface immune receptor and the CC-motif operably fused to the C-terminus of the cell-surface immune receptor.

[0096]

[0094] In various embodiments, the polypeptide may comprise, from N-terminal to C-terminal, the cellsurface immune receptor and the CC-motif operably fused to the C-terminal of the cell-surface immune receptor.

[0097]

[0095] In various embodiments, the polypeptide may comprise a pattern-recognition receptor (PRR), preferably FLS2 or PEPR1, operably fused to a dimeric or tetrameric CC-motif at its C-terminal. In various embodiments, the polypeptide may further comprise an epitope tag, preferably a 3xMyc tag, operably fused to the C-terminal of the PRR and the N-terminal of the CC-motif, or operably fused to the C-terminal of the CC-motif. In various embodiments, the polypeptide may further comprise a fluorescent reporter protein, preferably a mG, operably fused to the C-terminal of the CC-motif, or to the C-terminal of the PRR and the N-terminal of the CC-motif.

[0098]

[0096] In various embodiments, the PRR may be FLS2, and the polypeptide comprises the FLS2 operably fused at its C-terminal to a CC-motif, and a fluorescent reporter protein operably fused to the C-terminal of the CC-motif, wherein optionally an epitope tag may be included in between the receptor and CC-motif. In various embodiments, the polypeptide may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 14, or a functional variant thereof (FLS2-D-mG). In various embodiments, the PRR may be FLS2, and the polypeptide may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 15, or a functional variant thereof (FLS2-T-mG) .

[0097] In various embodiments, the PRR may be PEPR1 , and the polypeptide comprises the PEPR1 operably fused at its C-terminal to a fluorescent reporter protein, and a CC-motif operably fused to the C-terminal of the fluorescent reporter protein, wherein optionally an epitope tag may be included and operably fused to the C-terminal of the CC-motif. In various embodiments, the polypeptide may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 16, or a functional variant thereof (PEPR1-mG-D). In various embodiments, the PRR may be PEPR1 , and the polypeptide may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 17, or a functional variant thereof (PEPR1 -mG-T) .

[0099]

[0098] In various embodiments, the cell-surface immune receptor may be provided as a multi-domain polypeptide comprising at least an extracellular domain (ECD), a transmembrane domain (TMD), and an intracellular signalling domain (ICD), such as a kinase domain or other effector domain. In various embodiments, the cell-surface immune receptor may be provided as a multi-domain polypeptide comprising an extracellular domain (ECD), and an intracellular signalling domain (ICD). In such embodiments, the CC-motif may be operably fused between the extracellular domain and the intracellular signalling domain of the receptor. Thus, in various embodiments, the polypeptide may comprise, from N-terminal to C-terminal, an extracellular domain (ECD) of the receptor, the CC-motif, and an intracellular signalling domain (ICD) of the receptor, wherein the CC-motif is operably fused to the C-terminal of the ECD and the N-terminal of the ICD.

[0100]

[0099] In various embodiments, the polypeptide may comprise a co-receptor of a pattern-recognition receptor (PRR), preferably BAK1, operably fused to a dimeric or tetrameric CC-motif. In various embodiments, the CC-motif may be operably fused to the C-terminal of an extracellular domain (ECD) of the co-receptor and the N-terminal of the intracellular signalling domain (ICD) of the receptor. In various embodiments, the polypeptide may further comprise an epitope tag, preferably a 1xMyc tag, operably fused to the C-terminal of the ECD and the N-terminal of the CC-motif. In various embodiments, the polypeptide may further comprise a fluorescent reporter protein, preferably a mG, operably fused to the C-terminal of the CC-motif.

[0101]

[0100] In various embodiments, the cell-surface immune receptor may be BAK1 , and the polypeptide comprises a CC-motif operably fused to the C-terminal of a BAK1 ECD and the N-terminal of a BAK1 KD, and a fluorescent reporter protein operably fused to the C-terminal of the BAK1 KD, wherein optionally an epitope tag may be included and operably fused to the C-terminal of the BAK1KD. In various embodiments, the polypeptide may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 18, or a functional variant thereof (BAK1 ECD-D-BAK1KD-mG) . In various embodiments, the polypeptide may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 19, or a functional variant thereof (BAK1 ECD-T-BAK1 KD-mG).

[0101] In various embodiments, the polypeptide may comprise or consist of an amino acid sequence set forth in any one of SEQ ID NO: 15, 16, 18, 19, 21 or 22, or a functional variant thereof.

[0102]

[0102] Nucleic acid molecules encoding the polypeptide disclosed herein are also provided. All embodiments disclosed above in relation to the polypeptide similarly apply to the nucleic acid molecules and vice versa.

[0103]

[0103] The nucleic acid molecules can be DNA molecules or RNA molecules. They can exist as an individual strand, as an individual strand complementary to said individual strand, or as a double strand. With DNA molecules in particular, the sequences of both complementary strands in all three possible reading frames are to be considered in each case. Also to be considered is the fact that different codons, i.e. base triplets, can code for the same amino acids, so that a specific amino acid sequence can be coded by multiple different nucleic acids. As a result of this degeneracy of the genetic code, all nucleic acid sequences that can encode the polypeptide are included. The skilled artisan is capable of unequivocally determining these nucleic acid sequences, since despite the degeneracy of the genetic code, defined amino acids are to be associated with individual codons. The skilled artisan can therefore, proceeding from an amino acid sequence, readily ascertain nucleic acids coding for that amino acid sequence. In addition, in the context of nucleic acids, according to the present invention one or more codons can be replaced by synonymous codons. This aspect refers in particular to heterologous expression of the enzymes contemplated herein. For example, every organism, e.g. a host cell of a production strain, possesses a specific codon usage. "Codon usage" is understood as the translation of the genetic code into amino acids by the respective organism. Bottlenecks in protein biosynthesis can occur if the codons located on the nucleic acid are confronted, in the organism, with a comparatively small number of loaded tRNA molecules. Also it codes for the same amino acid, the result is that a codon becomes translated in the organism less efficiently than a synonymous codon that codes for the same amino acid. Because of the presence of a larger number of tRNA molecules for the synonymous codon, the latter can be translated more efficiently in the organism. By way of methods commonly known today such as, for example, chemical synthesis or the polymerase chain reaction (PCR) in combination with standard methods of molecular biology or protein chemistry, a skilled artisan has the ability to manufacture, on the basis of known DNA sequences and / or amino acid sequences, the corresponding nucleic acids all the way to complete genes. Such methods are known, for example, from Sambrook, J., Fritsch, E. F., and Maniatis, T, 2001, Molecular cloning: a laboratory manual, 3rd edition, Cold Spring Laboratory Press.

[0104]

[0104] The term "degenerate variant" refers to a nucleotide sequence encoding a protein (for example, the polypeptide disclosed herein) that includes a nucleotide sequence that is degenerate as a result of the genetic code. There are twenty natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the resulting polypeptide or fusion protein disclosed herein encoded by the nucleotide sequence has the desired activity.

[0105] In various embodiments, the nucleic acid molecules comprises a nucleotide sequence set forth in any one of SEQ ID NO:24, 25, 27, 28, 30 or 31 , or a functional or degenerate variant thereof.

[0105]

[0106] The nucleic acid molecules encoding the polypeptides described herein, as well as a circular DNA molecule containing such a nucleic acid, in particular a plasmid, vector, cosmid, bacterial artificial chromosome (BAC), bacteriophage, viral vector or hybrids thereof, also form part of the present invention. In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide disclosed herein.

[0106]

[0107] In various embodiments, the nucleic acid molecule may be a vector or comprised in a vector. "Vectors" are understood for purposes herein as elements - made up of nucleic acids - that contain a nucleic acid molecule contemplated herein as a characterising nucleic acid region. They enable said nucleic acid to be established as a stable genetic element in a species or a cell line over multiple generations or cell divisions. In the context herein, a nucleic acid molecule as contemplated herein may be cloned into a vector suitable for introduction into a plant or a plant cell. Suitable vectors include, for example, plasmid vectors, viral or plant-viral vectors, binary or co-integrating Agrobacterium vectors, bacteriophage-derived vectors used during intermediate cloning steps, and predominantly synthetic vectors or plasmids incorporating functional elements of diverse origins. Such vectors typically comprise regulatory elements compatible with plant expression, such as plant-active promoters, terminators, and, where appropriate, transit peptides or introns that enhance expression in plant cells. Using the additional genetic elements present in each case, the vectors are capable of establishing themselves as stable genetic units in the relevant plant host cells, either episomally or following genomic integration, and may be maintained over multiple cell divisions or, where applicable, through plant generations.

[0107]

[0108] In various embodiments, the vector may be an expression vector. Expression vectors encompass nucleic acid molecules which are capable of replicating in the cells that contain them, and expressing therein a contained nucleic acid. In various embodiments, the vectors described herein thus also contain regulatory elements that control expression of the nucleic acids encoding a polypeptide or fusion protein of the invention. Expression is influenced in particular by the promoter or promoters that regulate transcription. Expression can occur in principle by means of the natural promoter originally located in front of the nucleic acid to be expressed, but also by means of a host-cell promoter furnished on the expression vector or also by means of a modified, or entirely different, promoter of another organism or of another host cell. In the present case at least one promoter for expression of a nucleic acid as contemplated herein is made available and used for expression thereof.

[0108]

[0109] In various embodiments, the nucleic acid molecule further comprises regulatory elements for controlling expression of said nucleic acid molecule. "Regulatory nucleotide sequences” or "regulatory elements” as used herein refer to nucleotide sequences that influence the timing and level / amount of transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatorysequences may include promoters; translation leader sequences; introns; enhancers; stem-loop structures; repressor binding sequences; termination sequences; and polyadenylation recognition sequences. Particular regulatory sequences may be located upstream and / or downstream of a coding sequence operably linked thereto. In various embodiments, the regulatory elements for gene expression in plant systems, may include plant-active promoters, untranslated regions, introns that enhance expression, and transcriptional terminators. Expression may be driven by the native promoter associated with the nucleic acid or by a heterologous promoter incorporated into the vector, including constitutive, tissue-specific, developmentally regulated, or inducible plant promoters.

[0109]

[0110] In various embodiments, the expression vector may be configured for transformation of a microbial or plant cell, such as a binary vector (e.g., pHGW) for Agrobacterium-mediated plant transformation or a bacterial expression vector (e.g., pET28a) for recombinant protein production in E. coli. In various embodiments, the nucleic acid molecules are comprised in binary plasmid vectors compatible with Agrobacfer / um-mediated delivery, each vector comprising a nucleic acid molecule disclosed herein.

[0110]

[0111] In various embodiments, the nucleic acid molecule disclosed herein may comprise an “expression construct” which refers to a functional unit built in the vector for the purpose of recombinantly expressing the polypeptide disclosed herein, when introduced into an appropriate host plant cell. The term ’'recombinant'', as used herein (e.g. a recombinant polypeptide, a recombinant nucleic acid, or the like), refers to any molecule which is prepared, expressed, created or isolated by recombinant means, and which is not naturally occurring. "Recombinant” can be used synonymously with "engineered" or "non-natural" and can refer to to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, fusion proteins, or other nucleic acid molecule additions, deletions, substitutions or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene or operon.

[0111]

[0112] In various embodiments, the nucleic acid molecule may be provided as an expression cassette, or may be comprised within an expression cassette, configured for transcription and translation in a plant or plant cell. The expression cassette may include, in operable linkage, a plant-active promoter, one or more introns that enhance expression, and a transcriptional terminator or polyadenylation signal suitable for plant systems. In various embodiments, the expression cassette may further comprise additional regulatory elements such as enhancer sequences, 5’ or 3’ untranslated regions. The expression cassette may be present as a discrete nucleic acid construct, or may be incorporated into a plasmid, viral vector, binary Agrobacterium vector, or other vector system for delivery into plant cells.

[0113] In various embodiments, the nucleic acid molecule further comprises a promoter capable of driving expression in a plant cell operably linked to the nucleotide sequence encoding the polypeptide.

[0112]

[0114] As used herein, the term “operably linked” refers to the functional connection between two or more nucleotide sequences such that they are transcribed and / or translated as intended. When the connection results in a single continuous open reading frame encoding a multi-domain polypeptide, the encoded domains are said to be “fused” at the protein level, thus the polypeptides disclosed herein may also be termed as fusion proteins.

[0113]

[0115] The term "downstream" as used herein refers to a nucleotide sequence that is located 3’ to a reference nucleotide sequence. In particular, downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene may be located downstream of the start site of transcription. The term “immediately downstream” may be used to specify that the nucleotide sequence immediately follows and is directly next to the reference nucleotide sequence in the 3’ direction, with no other intervening nucleotide sequence or genetic element therein between. The term "upstream" as used herein refers to a nucleotide sequence that is located 5’ to a reference nucleotide sequence. In particular, upstream nucleotide sequences generally relate to sequences that are located on the 5' side of a coding sequence or starting point of transcription. For example, most promoters are located upstream of the start site of transcription. The term “immediately upstream” may be used to specify that the nucleotide sequence immediately precedes and is directly next to the reference nucleotide sequence in the 5’ direction, with no other intervening nucleotide sequence or genetic element therein between.

[0114]

[0116] In various embodiments, the promoter may be any promoter capable of directing transcription in a plant cell. Suitable promoters include constitutive, inducible, tissue-specific, pathogen-responsive, or native (endogenous) promoters, and may be selected based on the desired level, spatial distribution, or timing of receptor expression. In various embodiments, the promoter may be a constitutive promoter, such as the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, Enhanced 35S promoter, Ubiquitin promoters (e.g., Arabidopsis UBQ10, maize Ubi-1 ), Actin promoters (e.g., Arabidopsis ACT2 / ACT8), or other strong viral or plant housekeeping promoters known in the art. Such promoters may be advantageous for achieving robust and uniform expression of receptors such as PEPR1 or BAK1 in transient or stable plant expression systems, including Nicotiana benthamiana. In various embodiments, the promoter may be a native or endogenous promoter, such as the FLS2 promoter for expression of the native FLS2 receptor in Arabidopsis thaliana or other plant species. Use of an endogenous promoter may preserve natural expression patterns, ligand responsiveness, or developmental regulation of the receptor. In various embodiments, the receptor may be driven by a pathogen-inducible or stress-responsive promoter, including promoters of PR genes (e.g., PR1, PR2, PR5), WRKY transcription factor promoters, FRK1 promoter, or promoters activated by microbe-associated molecular patterns (MAMPs) or damage-associated signals. These promoters enable temporal control of receptor accumulation, reducing metabolic cost and limiting unwanted growth-defense trade-offs. Promoters may also be tissue- or cell-type-specific, such as epidermis-specific promoters (e.g., CER5, LTP1), mesophyll-preferred promoters (e.g., RBCS), vascular promoters (e.g., SUC2, SWEET), or root-specific promoters (e.g., W0X5, EXPANSIN). Such promoters may be used to tailor localisation or signalling output of receptors depending on the target tissue or intended function. In various embodiments, synthetic or chimeric promoters, enhancer-modified promoters, or inducible systems such as dexamethasone-inducible, estradiol-inducible, copper-inducible, ethanol-inducible, or heat-shock-inducible promoters may be employed to regulate expression of the receptors. Accordingly, the invention encompasses the use of any promoter capable of driving transcription in a plant cell, whether constitutive or regulated, endogenous or heterologous, viral or plant -derived, for the expression of cell-surface immune receptors, including but not limited to FLS2, PEPR1 , BAK1, and their orthologues, variants, or engineered derivatives.

[0115]

[0117] In another aspect, there is provided a composition comprising the polypeptide or nucleic acid molecule disclosed herein.

[0116]

[0118] In various embodiments, the composition encompasses any formulation, mixture, or assembly of components suitable for introducing the disclosed polypeptide or nucleic acid molecule into a plant, plant cell, or plant tissue, and / or for enabling their expression therein. The composition may further comprise one or more of: a delivery vehicle such as an recombinant Agrobacterium strain, or nanoparticle-based system; a carrier, excipient, or formulation buffer suitable for plant infiltration or transformation; selectable markers or reporter constructs; additional polypeptides or nucleic acid molecules that facilitate folding, stability, membrane localisation, oligomerisation, or functional activity of the encoded polypeptides; plant-compatible transformation components such as Agrobacterium suspension media, acetosyringone, infiltration media, or biolistic particle-coating reagents; and stabilising or protective agents for maintaining the integrity of the polypeptides or nucleic acids prior to delivery. In various embodiments, the composition may be formulated for stable transformation or transient expression.

[0117]

[0119] Another aspect of the invention relates to a plant, plant cell or plant seed comprising the polypeptide, or nucleic acid molecule disclosed herein. All embodiments disclosed above in relation to the polypeptide, or nucleic acid molecule disclosed herein, similarly apply to the plant, plant cell or plant seed, and vice versa.

[0118]

[0120] As used herein, the term “plant” includes whole plants at any developmental stage, including seedlings, juvenile plants, and mature plants. It is not meant to limit a plant to any particular structure. It also refers to a unicellular plant and a plurality of plant cells that are largely differentiated into a colony or a plant structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a seed, a tiller, a sprig, a stolen, a plug, a rhizome, a shoot, a stem, a leaf, a flower petal, a fruit. The “plant" may refer to a whole photosynthetic organism comprising multiple plant cells,tissues, or organs, including angiosperms, gymnosperms, monocots, dicots, mosses, algae, or any plant capable of harbouring the polypeptides or expressing the nucleic acid molecules disclosed herein.

[0119]

[0121] In various embodiments, the plant may be termed as a transgenic plant or transformed plant. The term “transgenic plant" refers to any plant, plant tissue, plant organ, plant cell, or progeny thereof that comprises one or more heterologous nucleic acid molecules introduced by recombinant or transformation techniques. The term includes a living plant, a regenerated plant derived from transformed cells, and any vegetative or sexual progeny thereof that inherit the introduced nucleic acid molecules.

[0120]

[0122] As used herein, the term “plant cell” may refer to an individual plant cell, whereas the “plant” refers to the multicellular organism comprising such cells. A plant may include plant cells transiently or stably expressing the nucleic acid molecules disclosed herein. The term “plant tissue” includes differentiated and undifferentiated tissues of plants, including those present in roots, shoots, leaves, pollen, seeds, and flowers, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture. As used herein, the term “plant part” as used herein refers to a plant structure or a plant tissue. In some instances, the plant part can include vegetative tissues of the plant. The term “seed” refers to a ripened ovule, consisting of the embryo and a casing. The term “propagation” refers to the process of producing new plants, either by vegetative means involving the rooting or grafting of pieces of a plant, or by sowing seeds. The terms “vegetative propagation” and “asexual reproduction” refer to the ability of plants to reproduce without sexual reproduction, by producing new plants from existing vegetative structures that are clones, i.e., plants that are identical in all attributes to the mother plant and to one another. For example, the division of a clump, rooting of proliferations, or cutting of mature crowns can produce a new plant.

[0121]

[0123] In various embodiments, there is provided a plant or a plant grown from the plant seed under conditions effective to express the nucleic acid molecule in said plant or said plant grown from the plant seed.

[0122]

[0124] The plant may be any plant species amenable to recombinant molecular manipulation, transient expression, or stable transgenic production. In various embodiments, the plant may include, without limitation, monocotyledonous and dicotyledonous species, annual or perennial crops, horticultural plants, fruiting plants, ornamental plants, fibre crops, and forestry species. Non-limiting examples of compatible plants include Arabidopsis species such as Arabidopsis thaliana, Oryza sativa (rice), Zea mays (Maize), Triticum aestivum (Wheat), Nicotiana species such as N. benthamiana and N. tabacum, Solatium lycopersicum (tomato) and other Solanaceae members, Glycine max (soybean), Triticum aestivum (wheat), Zea mays (maize), Hordeum vulgare (barley), Brassica species (e.g., 8. napus, B. rapaj', Medicago truncatula, Pisum sativum (pea), Vitis vinifera (grapevine), Populus species (poplar), and Citrus species, among others. Orthologues and paralogues of LRR-RLK, LRR-RLP, LysM-RLK, LysM-RLP, and SERK-family immune receptors are broadly conserved across these plants, and theengineered receptor constructs described herein may be introduced into any such species using standard transformation approaches to generate transgenic plants, plant cells, tissues, or progeny.

[0123]

[0125] The modular nature of the CC-motifs allow customisation and modification of a variety of different plant cell-surface immune receptors (e.g. PRR-co-receptor pairs), making this modification and concept transferable to economically important crop plants (such as Soybean by engineering the GmFLS2), trees, and vegetable species (such as multiple brassica species by engineering respect FLS2).

[0124]

[0126] In various embodiments, the plant comprises or expresses a monocot or dicot leucine-rich-repeat (LRR) receptor on the cell surface, the LRR receptor being a single-pass transmembrane immune receptor selected from an LRR receptor-like kinase (LRR-RLK) or an LRR receptor-like protein (LRR-RLP). Such LRR receptors are widely conserved across monocotyledonous and dicotyledonous plant species and typically include receptors involved in extracellular elicitor perception, pathogen-associated molecular pattern (PAMP) recognition, or damage-associated molecular pattern (DAMP) signalling. In various embodiments, the plant may be selected from monocot or dicot species that encode or express endogenous LRR receptors such as FLS2, EFR, PEPR1 , PEPR2, RLP23, LYK5, RLP23 or SERK-family co-receptors, including but not limited to Arabidopsis thaliana, Nicotiana species, Oryza sativa, Zea mays, Triticum aestivum, Solanum lycopersicum, Glycine max, and Brassica species.

[0125]

[0127] The plant, plant cell, or plant seed comprising the polypeptide or the nucleic acid molecule disclosed herein is intended to encompass any plant material in which the polypeptide are present or in which the nucleic acid molecule are carried, maintained, or expressed. This includes embodiments in which the nucleic acid molecules are stably integrated into the plant nuclear genome, or, where applicable, into the plastid genome; as well as embodiments in which the nucleic acid molecules are transiently present and expressed from an episomal, extrachromosomal, or non-integrating expression vector. Accordingly, the term embraces both transient and stable transformation events, including systems in which the nucleic acid molecules or encoded polypeptides are present or expressed at any given time. In embodiments employing polypeptide delivery, the plant, plant cell, or plant seed may comprise the polypeptides directly, such as through protein uptake, transfection, or delivery by agroinfiltration, without requiring genomic integration of the corresponding nucleic acids.

[0126]

[0128] In various embodiments, when the polypeptide is expressed in the plant, plant cell, or plant seed from the nucleic acid molecule, the receptor is targeted to, and correctly integrated into, the plant plasma membrane. This is because the native signal peptide, extracellular domain, and transmembrane domain of the receptor retain their canonical trafficking and membrane-insertion functions, irrespective of the presence of the CC-motif fused thereto. Accordingly, incorporation of a CC motif does not abrogate or substantially impair membrane targeting, and the resulting chimeric receptor is expressedas a membrane-anchored, cell-surface-localised protein capable of participating in receptor complex formation, ligand perception, and downstream signalling in planta.

[0127]

[0129] In various embodiments, in the plant, plant cell, or plant seed the polypeptide disclosed herein may be expressed and maintained in its multimeric state and is in an inactive state in the absence of a PAMP or DAMP, and in an active state upon ligand binding through co-receptor recruitment to initiate downstream signalling and an immune response.

[0128]

[0130] In various embodiments, the plant cells may include isolated cells, protoplasts, cultured cells, or cells within a differentiated or undifferentiated plant tissue. Plant cells may be present within any tissue or anatomical part, including leaves, stems, roots, cotyledons, hypocotyls, flowers, reproductive organs, embryos, fruits, seed coats, vascular tissue, meristems, callus tissue, or any explant capable of regeneration into a whole plant.

[0129]

[0131] In various embodiments, the plant parts or plant materials may be derived from the transgenic plant, whether obtained directly from the plant or generated through vegetative propagation, cuttings, grafts, tissue culture, micropropagation, or embryogenic culture. Such plant parts may include, but are not limited to, harvested biomass, foliage, stems, tubers, bulbs, corms, rhizomes, roots, fibres, cuttings, rachis tissue, sap, or extracts prepared therefrom.

[0130]

[0132] In various embodiments, progeny of the transgenic plant are also contemplated here, including F1 , F2, F3, and subsequent generations, as well as hybrids and backcrossed lines, provided that the progeny retains the nucleic acid molecules or expresses the polypeptides disclosed herein. Progeny may be obtained by sexual reproduction, self-pollination, cross-pollination, or vegetative propagation. This includes plants “obtainable from” or “derived from” the initial transgenic line.

[0131]

[0133] In various embodiments, the nucleic acid molecule disclosed herein may be introduced into a recipient plant cell to create a transformed plant cell by available methods. The frequency of occurrence of cells taking up exogenous (foreign) DNA can be low, and it is likely that not all recipient cells receiving DNA segments or sequences will result in a transformed cell wherein the DNA is stably integrated into the plant genome and / or expressed. Some may show only initial and transient gene expression. However, cells from virtually any dicot or monocot species can be stably transformed, and those cells can be regenerated into transgenic plants, for example, through the application of the techniques disclosed herein.

[0132]

[0134] In various embodiments, the plant may be Nicotiana benthamiana or Arabidopsis thaliana. The plant may act as a transient expression host plant, infiltrated with A. tumefaciens strains harbouring the nucleic acid molecules disclosed herein. In various embodiments, the plant may act as a stable transformation host plant in which the nucleic acid molecules disclosed herein are integrated into the plant genome and heritably maintained.

[0135] In various embodiments, the plant exhibits normal physiology, such that the presence of the polypeptide or expression of the nucleic acid molecule does not significantly perturb growth or development. Thus, the presence of the polypeptide or expression of the nucleic acid molecule in the plant does not affect plant development and plant physiology.

[0133]

[0136] Accordingly, the scope of the invention extends to the transgenic plant, its cells, tissues, organs, seeds, reproductive materials, progeny, and any biological or industrial product produced from or incorporating such materials.

[0134]

[0137] In various embodiments, the plant, plant cell, or plant seed may comprise two or more polypeptides disclosed herein or two or more nucleic acid molecules disclosed herein. Each polypeptide or nucleic acid molecule relates to a different plant cell-surface immune receptor. In these embodiments, the multiple polypeptides or nucleic acid molecules are in the same cell or in overlapping cell populations so as to generate a stacked set of engineered receptors conferring broad-spectrum or multilayered disease resistance. Such stacking may include, for example, co-expression of engineered variants of FLS2, PEPR1, and BAK1 . Plants, plant cells, or seeds comprising such stacked receptor constructs may exhibit enhanced resilience against diverse microbial pathogens, and the stacked traits are heritable through seed propagation.

[0135]

[0138] In another aspect, there is provided a method of producing or generating a transgenic plant, plant cell, or plant seed, comprising introducing the nucleic acid molecule disclosed herein into a plant, plant cell, or plant seed.

[0136]

[0139] The step of “introducing” may refer to any process, technique, or operation by which the nucleic acid molecules disclosed herein are delivered into the plant, plant tissue, plant cell or plant seed under conditions that permit uptake, maintenance, and / or expression of the nucleic acid molecules. The introducing step may result in transient expression, episomal maintenance, or stable genomic integration of the nucleic acid molecules. The term encompasses physical, chemical, and biological delivery methods and is not limited to any particular transformation mechanism, vector backbone, or plant species. The introducing step is carried out under suitable conditions that permit expression of the nucleic acid molecules in the plant to produce the polypeptides disclosed herein.

[0137]

[0140] In various embodiments, the introducing step may comprise transforming the plant, plant cell, or plant seed with the nucleic acid molecule disclosed herein.

[0138]

[0141] Transformation of the plant, plant cell, or plant seeds may be performed using any suitable method known to those of skill in the art. Representative approaches include, without limitation, direct DNA delivery methods such as electroporation, microprojectile bombardment, or biological delivery using Agrobacterium species. Electroporation or particle bombardment may be carried out using“naked” DNA in which the nucleic acid molecule disclosed herein is carried on a standard plasmid cloning vector. In embodiments using viral vectors, it is desirable that the vector retains replication capability but lacks functions associated with pathogenicity or disease induction. For dicotyledonous species, a common transformation method involves inoculation of plant tissues with Agrobacterium tumefaciens, including but not limited to leaf-disk transformation, floral dip transformation, cotyledon or hypocotyl transformation, or co-cultivation of explants. Monocotyledonous species such as Zea mays may be transformed using microprojectile bombardment of embryogenic callus tissue or immature embryos, or by electroporation following partial enzymatic digestion of the cell wall using pectinasecontaining preparations. For example, embryogenic tissues derived from immature maize embryos may be transformed by accelerated particle delivery. Alternatively, excised immature embryos can be directly transformed prior to induction of tissue culture, selection, and regeneration. Methods for transforming monocotyledonous plants using Agrobacterium tumefaciens have also been extensively described and may be employed in certain embodiments.

[0139]

[0142] In various embodiments, the plant, plant cell, or plant seed may be transformed under conditions that permit stable or transient expression of the encoded polypeptide. Such conditions are not particularly limited, provided that they enable production of the engineered receptor at a detectable or functional level in the plant, plant cell, or progeny thereof.

[0140]

[0143] In various embodiments, the nucleic acid molecules may be introduced into any plant structure, organ, tissue, or cell type capable of receiving the nucleic acid molecules and supporting expression of the introduced nucleic acid molecules. Suitable target locations include, without limitation, leaf tissues (including epidermal, mesophyll, guard, or pavement cells), stems, hypocotyls, cotyledons, roots, meristematic regions, floral tissues (including inflorescences or developing floral buds), and isolated cells or protoplasts obtained from leaf, root, callus, or suspension cultures. In embodiments involving transient expression, the nucleic acid molecules may be introduced into the abaxial surface of the leaves by infiltration. In embodiments involving stable transformation, the nucleic acid molecules may be introduced into floral tissues using the floral dip method or into regenerable explants such as leaf disks, hypocotyl segments, or callus cultures. Any plant tissue capable of transformation and regeneration may serve as a target for introducing the nucleic acid molecules disclosed herein.

[0141]

[0144] In various embodiments, the plant is a stably transformed plant comprising the nucleic acid molecule integrated into the plant's nuclear genome. In such embodiments, the nucleic acid molecule disclosed herein is incorporated into the chromosomal DNA of the plant cell, thereby enabling heritable expression of the engineered receptor in regenerated plants and their progeny. Stable transformation may be achieved using Agrobacterium-mediated gene transfer, or any other method suitable for genomic integration in monocot or dicot species. Plants regenerated from stably transformed cells exhibit stable transmission of the integrated nucleic acid across mitotic and meiotic divisions, allowing recovery of homozygous or heterozygous transgenic lines expressing the engineered receptor in roots, leaves, flowers, seeds, or other tissues as appropriate.

[0145] In various embodiments, the introducing step comprises transiently delivering the nucleic acid molecules into leaves of the plant using an Agrabacter / um-mediated infiltration route. In this embodiment, binary plasmid vectors comprising the nucleic acid molecules are first transformed into an Agrobacterium species suitable for plant transformation, such as Agrobacterium tumefaciens strain GV3101. The resulting Agrobacterium cultures are resuspended in an infiltration buffer, to induce virulence gene expression and enhance DNA transfer efficiency. The bacterial suspension is then introduced into the abaxial surface of the plant leaf using a needleless syringe or by gentle pressure infiltration, thereby introducing the nucleic acid molecules into the epidermal and mesophyll cells of the leaf tissue.

[0142]

[0146] After introduction and delivery of the nucleic acid molecule disclosed herein, transformed plants, cells or seeds may be identified using a selectable or screenable marker gene carried on the same nucleic acid molecules. Suitable markers include antibiotic- or herbicide-resistance genes, fluorescent reporter proteins, or enzymatic reporters. Transgenic events may also be confirmed through detection of the introduced nucleic acid by PCR, hybridisation, sequencing, or by detecting the encoded protein using epitope tags (e.g., Myc or FLAG) and / or fluorescent reporters (e.g., mGFP). Thus,

[0143]

[0147] Regeneration of whole plants from transformed plant cells and tissues may be achieved by standard plant tissue culture and regeneration procedures appropriate for the species, including induction of callus, shoot regeneration, root formation, and acclimation to soil. In embodiments involving species capable of sexual reproduction, regenerated plants may be grown to maturity to produce seeds, and transgenic progeny containing the polypeptide disclosed herein may be recovered through standard breeding methods. To confirm the presence of the nucleic acid molecule in the regenerating plants, or seeds or progeny derived from the regenerated plant, a variety of assays may be performed. Such assays include, for example, molecular biological assays available to those of skill in the art, such as Southern and Northern blotting and PCR; biochemical assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf, seed or root assays; and also, by analysing the phenotype of the whole regenerated plant.

[0144] ❖ Methods of Use

[0145]

[0148] All embodiments disclosed herein in relation to the polypeptides, nucleic acids, plants, plant cells, plant seeds are similarly applicable to the uses and methods described herein and vice versa.

[0146]

[0149] It is contemplated that the polypeptides disclosed herein present, or expressed, in a plant, or plant cell may be used to modulate an immune response therein and / or to reduce, treat or prevent a pathogen infection, and / or to improve resistance to a pathogen infection.

[0150] Accordingly, in another aspect, there is provided a method of modulating an immune response in a plant, comprising introducing the nucleic acid molecule disclosed herein, into the plant, wherein the nucleic acid molecule is expressed in the plant to produce the polypeptide disclosed herein.

[0147]

[0151] The introduction step may be the same as those outlined above in the context of the generation and production of the plant, more particularly the transgenic plant, plant cell or plant seed. In particular, methods for introducing nucleic acid molecules into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. By "stable transformation" it is intended that the nucleic acid molecule introduced into the plant integrates into the genome of the plant and is capable of being inherited by progeny thereof. By "transient transformation" it is intended that the nucleic acid molecule introduced into a plant does not integrate into the genome of the plant.

[0148]

[0152] In various embodiments, the step of introducing the nucleic acid molecule disclosed herein into the plant may be carried out under conditions that permit expression of the encoded polypeptide such that the engineered cell-surface immune receptor is localised to the plant cell surface (i.e. plasma membrane of the plant cells).

[0149]

[0153] In various embodiments, the introducing step may be carried out under conditions that permit transcription and translation of the nucleic acid molecule in the plant, proper folding and post-translational modification of the encoded receptor, and trafficking of the receptor through the secretory pathway for insertion into the plasma membrane or other cell surface. Such conditions include the use of growth or culture conditions that support expression, membrane localisation, and functional presentation of the extracellular ligand-binding domain, thereby enabling detection of PAMPs and / or DAMPs and perception of pathogen infection.

[0150]

[0154] As used herein, the term “modulating" an immune response in a plant refers to altering, adjusting, enhancing, attenuating, or otherwise changing the magnitude, duration, sensitivity, or outcome of a plant innate immune response, and encompasses both increases and decreases in immune activity. Modulation may involve enhancing immune responsiveness, such as strengthening or accelerating pattern-recognition receptor (PRR) activation, promoting receptor-co-receptor complex formation, increasing or sensitising downstream signalling events, including reactive oxygen species (ROS) production, calcium influx, MARK activation, transcriptional reprogramming, or defence gene expression, or improving resistance to pathogen infection. Modulation may also involve decreasing immune responsiveness, such as dampening PRR activation, reducing signalling amplitude, suppressing defence outputs, or mitigating constitutive or inappropriate immune activation that may otherwise impair growth or development. Accordingly, modulating an immune response encompasses any intervention that increases, decreases, primes, sensitises, desensitises, or otherwise alters immune signalling in a plant cell, tissue, or whole plant, whether directly or indirectly.

[0155] In various embodiments, the method may be for enhancing or increasing the immune response in a plant. The enhancement or increase of the immune response may refer to any measurable improvement in the activation, magnitude, sensitivity, or durability of innate immune signalling in a plant expressing the nucleic acid molecule or polypeptide disclosed herein, relative to an appropriate control plant. In various embodiments, the enhancement is observed when comparing plants that express the polypeptide disclosed herein, with plants that lack the polypeptide or that express only the corresponding native, unmodified, or monomeric version of the receptor. Enhancement may be reflected in increased ligand responsiveness, elevated or faster induction of defence-related transcriptional programs, augmented MAP kinase activation, enhanced reactive oxygen species production, strengthened pathogen-associated molecular pattern (RAMP) perception, reduced pathogen burden, or increased disease resistance. Accordingly, the term "enhancing” includes improvements achieved through receptor re-engineering that modify receptor clustering, stability, multimerisation, or signalling potency relative to plants in which the receptor is not present or not engineered to adopt the desired oligomeric state.

[0151]

[0156] In various embodiments, the immune response may comprises one or more of the following (i) rapid production of reactive oxygen species (ROS), (ii) accelerated MAPK activation, (iii) enhanced transcription of defence-related genes, and / or (iv) reduced pathogen load.

[0152]

[0157] In various embodiments, the method of modulating an immune response may be characterised in that the plant exhibits receptor-mediated immunity following perception of a pathogen-associated molecular pattern (PAMP) or a damage-associated molecular pattern (DAMP). In these embodiments, expression of the polypeptide (i.e. engineered receptor) enhances the sensitivity or breadth of ligand recognition, such that immune responses are initiated when the plant encounters microbial elicitors or endogenous damage signals. The expressed polypeptides may recognise PAMPs or DAMPs directly or may act as a co-receptor that stabilises or amplifies signalling from a primary PRR.

[0153]

[0158] Pattern-recognition receptors (PRRs), such as FLS2, PEPR1, and the co-receptor BAK1, are capable of recognising a broad range of PAMPs and DAMPs. Exemplary PAMPs include flagellin-derived peptides such as flg22 and flg15, EF-Tu-derived peptides such as elf 18 and elf26, chitin oligomers, -glucans, lipopolysaccharides, peptidoglycan fragments including muramyl dipeptide, bacterial cold-shock protein-derived peptides such as csp22, oomycete elicitors such as Pep-13, and fungal proteins such as EIX. Exemplary DAMPs include endogenous plant danger peptides such as Pep1-Pep8 (canonical ligands of PEPR1 and PEPR2), oligogalacturonides generated from pectin degradation, extracellular ATP or NAD7NADP+, cell-wall-derived oligosaccharides, ROS-modified macromolecules, systemin or systemin-like peptides, heat-shock protein-derived peptides, and cutin monomers. As BAK1 functions as a co-receptor that heterodimerises with numerous ligand-binding PRRs, the range of PAMPs and DAMPs encompassed by BAK1 includes any ligand recognised by PRRs that require BAK1 for full activation or signal via SERK-family co-receptors, including all of the PAMPs and DAMPs described above.

[0159] In various embodiments, ligand binding to the expressed polypeptide in vivo triggers the intracellular signalling cascade that underlies natural PAMP-triggered immunity (PTI) , thereby activating downstream defence responses.

[0154]

[0160] In various embodiments, recognition of a pathogen-delivered effector by the expressed polypeptide, or by an immune receptor complex modulated by the expressed polypeptide, triggers effector-triggered immunity (ETI), thereby activating a heightened or sustained defence response characterised by amplified signalling outputs, transcriptional reprogramming, and enhanced restriction of pathogen growth.

[0155]

[0161] In this regard, the expressed polypeptide (i.e. engineered receptor) in the plant may be characterised in that it is in a primed but inactive state in the absence of a pathogen, wherein once a pathogen is detected the engineered receptor undergoes activation into an active state and initiates or amplifies immune responses relative to the native receptor. In these embodiments, the engineered receptor is not constitutively active and does not trigger downstream immune signalling in the absence of pathogen. Thus, the engineered receptor is inactive in the absence of a pathogen infection and active in the presence of a pathogen and upon pathogen perception, enabling controlled activation of a plant’s defense response; unlike constitutive defence systems. This regulated behaviour ensures that immune outputs are triggered exclusively under biotic stress, reducing unnecessary energy expenditure while providing rapid and robust defence upon pathogen challenge.

[0156]

[0162] For example, by maintaining FLS2 in a primed but inactive state until pathogen perception, plants conserve metabolic resources and avoid the growth penalties typically associated with continuously active immune pathways. Upon detection of pathogen signals, the dimerised resting state of FLS2 enables stronger and faster activation of downstream signalling cascades, allowing plants to mount an effective defence before infection can escalate. This strategy also avoids basal autoimmunity, as thedimerisation-based design enhances immune capability without imposing a continuous metabolic burden, thereby preventing the growth and yield penalties commonly associated with chronic immune stimulation. In comparison with existing receptor modification strategies, the present approach offers several clear advantages. Unlike methods based on PRR overexpression, which can heighten disease resistance but do not inherently improve receptor function or reduce the likelihood of defence misregulation (Yeh et al., 2015), the present method enhances the efficiency of FLS2-BAK1 complex formation without relying on excessively elevated receptor abundance. In contrast to constitutive activation strategies, which often trigger persistent immune signalling and compromise plant vitality, the engineered receptor described herein remains largely quiescent until a pathogen is encountered, thereby preventing unnecessary energy expenditure and supporting normal growth in pathogen-free conditions. Moreover, approaches that construct chimeric receptors by merging domains from unrelated receptors may suffer from folding defects, instability, or unintended signalling crosstalk. By preservingthe native FLS2 architecture, the present design avoids these compatibility issues and maintains essential regulatory controls, reducing the likelihood of unintended physiological effects.

[0157]

[0163] In various embodiments, the engineered receptor expressed in the plant does not impose growth penalties or defence-related fitness costs on the plant. Thus, the plant does not display stunted growth, developmental defects, sterility, chlorosis, or other phenotypes associated with chronic or constitutive immune activation. The presence of the engineered receptor in the plant preserves normal plant development while enabling enhanced immune activation only under pathogen challenge.

[0158]

[0164] It is also contemplated that the enhancement of a plants immune response may be used for treating, reducing or preventing a pathogen infection in a plant, and improve pathogen resistance in a plant.

[0159]

[0165] Accordingly, in another aspect, there is provided a method for reducing pathogen infection in a plant, comprising introducing the nucleic acid molecule disclosed herein into the plant, wherein the plant expresses the polypeptide disclosed herein. This method may result in increasing resistance to a pathogen upon perception by the engineered receptor of a corresponding PAMP or DAMP.

[0160]

[0166] As used herein, the term "resistance" to a pathogen is intended to refer to the plants avoiding the disease symptoms that are the outcome of plant-pathogen interactions. That is, pathogens are prevented from causing plant diseases and the associated disease symptoms, or alternatively, the disease symptoms caused by the pathogen is minimized or lessened. The plant or a plant regenerated from a plant cell of the plant may comprise enhanced resistance to at least one plant disease caused by a plant pathogen, relative to the resistance of a control plant lacking the nucleic acid molecule and encoded polypeptide (e.g. the wild-type plant).

[0161]

[0167] In various embodiments, the method may reduce infection and enhance resistance against a wide range of economically important plant pathogens, including bacterial, fungal, oomycete, viral, and nematode pathogens that affect major crops such as soybean, canola, alfalfa, wheat, sunflower, corn, sorghum, tomato, potato, banana, and related species. Exemplary pathogens include species of Pseudomonas, Xanthomonas, Ralstonia, Erwinia, Ciavibacter, Phytophthora, Pythium, Rhizoctonia, Sclerotinia, Fusarium, Alternaria, Colletotrichum, Cercospora, Macrophomina, Verticillium, and Botrytis; oomycetes such as Peronospora, Plasmopara, and Albugo; rust and smut fungi including Puccinia, Uromyces, Tilletia, and Sporisorium; nematodes such as Heterodera, Meloidogyne, and Pratylenchus. It will be appreciated that the present invention is not limited to these examples, and may be applied to modulate immunity against any pathogen for which receptor-mediated defence can be enhanced through the engineered receptors described herein. In various embodiments, the pathogen may be Pseudomonas syringae pv. tomato (Pst).

[0168] The methods of the present invention find use in agriculture, particularly in the development of crop plants with enhanced resistance to plant diseases. Such crop plants include resistant and susceptible plant varieties.

[0162]

[0169] In view of the modular nature of the CC motifs, it is appreciated that native plant cell-surface immune receptors may be engineered by operably incorporating the CC motifs at their endogenous genomic loci, rather than by transforming the plant or plant cell with the exogenous nucleic acid molecules disclosed herein. For example, such receptors may be modified using traditional transgenic approaches that introduce the CC motif as part of an expressed transgene, or by employing targeted knock-in or genome-editing techniques (e.g., CRISPR / Cas-mediated homology-directed repair) to integrate the CC motif sequence in situ at the endogenous receptor gene. Accordingly, the disclosed CC motifs may be operably fused to the optimized site in the intracellular domains of a native patternrecognition receptor (PRR) to modulate its oligomerisation state and enhance immune-signalling output.

[0163]

[0170] Accordingly, there is also provided a method of producing a plant, plant cell or plant seed comprising an engineered cell-surface immune receptor, the method comprising modifying a cellsurface immune receptor in the plant, plant cell or plant seed to operably associate, fuse or couple with a CC motif.

[0164]

[0171] In various embodiments, the CC may be operably fused to an intracellular domain rather than an extracellular domain, of the cell-surface immune receptor. The site of fusion and insertion of the CC motif within the intracellular region of the cell-surface immune receptor is not particularly limited and may be selected or optimized to preserve receptor integrity and functionality while enhancing or modulating immune signalling. By way of example, the CC motif may be fused at or near a cytoplasmic terminus of the receptor, inserted within an intracellular loop or juxtamembrane region, or positioned at a location distal from the transmembrane domain, provided that the CC motif remains accessible in the cytosolic environment. Optimization of the insertion site may involve routine design considerations, including maintenance of correct receptor folding, membrane localization, and signal transduction competence. Such optimization may be achieved empirically or through rational design, and does not require any particular insertion position or sequence context, so long as the resulting fusion polypeptide is capable of modulating an immune response in the plant.

[0165]

[0172] In various embodiments, the cell-surface immune receptor is a native or endogenous receptor of the plant, wherein the nucleotide sequence encoding the CC motif is introduced directly into the endogenous receptor gene while retaining its native promoter and regulatory context.

[0166]

[0173] In various embodiments, the cell-surface immune receptor is endogenous in the plant, such that the modification of the receptor is carried out in situ at the endogenous gene locus, or the expressed endogenous receptor is post-translationally modified, associated with, or otherwise functionally coupled to an introduced CC motif in vivo.

[0174] As used herein, the phrase “operably associate, fuse or couple with’’ refers to the functional linkage of the CC motif to the cell-surface immune receptor, whether achieved by (i) in situ modification of the endogenous receptor gene to introduce the CC-motif-encoding sequence directly into the receptor coding region, thereby producing a receptor-CC motif fusion upon expression, or (ii) post-translational association in vivo whereby the expressed endogenous receptor becomes physically or functionally associated with an introduced CC motif, wherein the CC motif becomes physically or functionally associated with the expressed receptor in a receptor-specific manner. In embodiments involving post-translational association, the specificity of the CC motif for the target cell-surface immune receptor may be ensured by design features that promote selective interaction or recruitment to the receptor, thereby avoiding non-specific clustering or signalling. Such specificity may be achieved, for example, through covalent attachment mediated by receptor-specific tags or enzymatic ligation systems, through domain-domain interactions that recognize defined intracellular regions of the receptor, or through other targeting mechanisms that preferentially associate the CC motif with the intended receptor. Accordingly, post-translational association as described herein is not a random or untargeted interaction, but rather a designed and selective association that results in targeted modulation of the clustering, oligomerisation, or signalling behaviour of the specified cell-surface immune receptor.

[0167]

[0175] In various embodiments, the method comprises genetically modifying the cell-surface immune receptor to introduce a nucleotide sequence encoding a CC motif, and expressing the genetically modified cell-surface immune receptor in the plant, plant cell or plant seed. In this regard, the method will result in the plant, plant cell or plant seed expressing the cell-surface immune receptor operably associate, fuse or couple with the CC-motif.

[0168]

[0176] As used herein, “genetic modification” or “genetically modifying” a plant, plant cell, or plant genome encompasses any molecular technique by which a nucleotide sequence encoding a CC motif is introduced into, associated with, or integrated within a gene encoding a plant cell-surface immune receptor. Such genetic modification includes, without limitation, targeted knock-in strategies in which the CC motif is inserted at a defined position within the endogenous receptor gene, or genome-editing techniques, such as CRISPR / Cas, TALENs, zinc-finger nucleases, or related systems, that mediate site-specific cleavage followed by homology-directed repair or other repair pathways to integrate the CC motif in situ. In all cases, the genetic modification is intended to operably incorporate the CC motif into the receptor coding sequence to modulate receptor oligomerisation, signalling properties, or immune function. In various embodiments, the nucleotide sequence may encode a CC-motif having an amino acid sequence selected from any one of SEQ ID NO: 6-9 (i.e. dimeric, trimeric, tetrameric, or pentameric CC motif).

[0169]

[0177] In various embodiments, the method may comprise directing a genome-editing nuclease to a selected target site within an endogenous gene encoding the cell-surface immune receptor; andproviding a donor nucleic acid molecule comprising a nucleotide sequence encoding the CC motif, wherein the donor nucleic acid molecule is introduced into the plant, plant cell, or plant seed under conditions that permit insertion of the CC motif into the endogenous gene.

[0170]

[0178] In various embodiments, the method may comprise inserting a nucleotide sequence encoding a CC motif into the endogenous gene encoding the cell-surface immune receptor using a genomeediting system selected from CRISPR / Cas9, Cas12a, TALENs, or zinc-finger nucleases, thereby producing the engineered cell-surface immune receptor with CC-motif. A guide RNA is designed to direct the nuclease to a predetermined cleavage site within the endogenous receptor gene (e.g., FLS2, PEPR1 , BAK1 , or functional homologues). A donor template comprising the nucleotide sequence encoding the CC motif and flanking homology arms is delivered into the plant cell to enable homology-directed repair (HDR) or related repair pathways, resulting in precise genomic insertion of the CC motif. The CRISPR / Cas nuclease and donor template may be delivered using Agrobacterium-mediated transformation, biolistic particle delivery, protoplast transfection with ribonucleoprotein complexes (RNPs), viral replicon-based vectors, or polyethylene-glycol-mediated DNA delivery. In some embodiments, transient expression of the editing machinery is preferred to minimise foreign DNA integration and to produce edited, transgene-free plants. This targeted knock-in approach enables precise, in situ incorporation of the CC motif into the native receptor coding sequence while retaining the receptor’s endogenous promoter, regulatory elements, and genomic context, thereby generating plants that express a modified receptor with enhanced or altered oligomerisation and immune-signalling properties.

[0171]

[0179] In various embodiments, the genome-editing step is carried out using a CRISPR / Cas system, such as Cas9, Cas12a, or high-fidelity variants thereof.

[0172]

[0180] In various embodiments, the nucleotide sequence encoding the CC-motif may be introduced at a position corresponding to the C-terminal region, transmembrane-intracellular domain junction.

[0173]

[0181] In various embodiments, the method comprises post-translationally modifying the cell-surface immune receptor in the plant, plant cell or plant seed to operably associate, fuse or couple with a CC motif. This may comprise introducing into the plant, plant cell, or plant seed a nucleic acid molecule encoding a CC motif, wherein the expressed CC motif associates with, fused to, or becomes post-translationally coupled to the endogenously expressed cell-surface immune receptor in vivo. The CC motif may be operably associated, fused or coupled with the receptor at a position that preserves the catalytic integrity and signalling competence of the receptor kinase domain. Accordingly, the CC motif is not positioned within the kinase catalytic core itself, but rather is located at a site adjacent to, or spatially separated from, the kinase domain. In various embodiments, the expressed CC motif associates with, fused to, or becomes post-translationally coupled to the receptor at either N- or C-terminal end of the receptor kinase domain or in between the receptor transmembrane domain and kinase domain. In various embodiments, kinase activity and immune signalling functionality of themodified receptor may be evaluated by transient expression in the plant. Such transient expression assays may be used to confirm that the post-translational association of the CC motif does not abolish kinase activity and that the modified receptor retains the ability to initiate or modulate an immune response.

[0174]

[0182] In various embodiments, the introduction of the CC motif may be achieved using a transgenebased approach using a recombinant nucleic acid construct comprising a nucleotide sequence encoding the CC motif. In such embodiments, the CC motif is expressed as a standalone polypeptide, peptide domain, or fusion module that associates with, recruits, or becomes covalently linked to the endogenous cell-surface immune receptor in vivo, thereby modulating its oligomerisation state or signalling properties. The CC-motif-encoding transgene may be placed under the control of a constitutive, inducible, or tissue-specific promoter and introduced into the plant genome using Agrobacterium-mediated transformation, biolistic delivery, viral vectors, or transient expression systems. Plants or plant cells generated through this transgene approach express an engineered version of the native cell-surface immune receptor that exhibits enhanced or altered oligomerisation behaviour and modified immune-signalling properties while maintaining functional compatibility with other components of the plant’s immune network, while leaving the endogenous receptor locus genetically unaltered.

[0175]

[0183] In various embodiments, the CC motif may be specifically targeted to the cell-surface immune receptor by site-specific enzymatic ligation. For example, the immune receptor may comprise a ligase-recognition sequence positioned within an intracellular or juxtamembrane region of the receptor, and the CC motif is expressed as a separate polypeptide comprising a complementary ligation substrate sequence. Upon expression, an enzymatic ligation system catalyses covalent attachment of the CC motif to the immune receptor in a post-translational manner, thereby achieving receptor-specific and site-defined coupling of the CC motif without random association with other cellular proteins.

[0176]

[0184] In various embodiments, the CC motif may be specifically targeted to the immune receptor through receptor-selective domain-domain interactions. For example, the CC motif may be operably linked to a receptor-binding domain that selectively recognises an intracellular region of the cell-surface immune receptor, such as a receptor-like kinase. Expression of the CC motif fusion results in preferential association of the CC motif with the intended immune receptor, positioning the CC motif at the receptor to modulate receptor clustering, oligomerisation, or downstream signalling.

[0177]

[0185] It will be appreciated that plants, pant cells or plant seeds generated by these methods express an engineered cell-surface immune receptor under control of the native regulatory elements. Such receptors may display enhanced oligomerisation, receptor clustering, or activation upon ligand binding, resulting in strengthened PAMP-triggered immunity (PTI), effector-triggered immunity (ETI), increased resistance to pathogens, and improved defensive output such as ROS burst, MAPK activation, callose deposition, and defence gene induction.

[0186] Accordingly, there is also provided a method of (i) modulating an immune response of a plant, or (ii) reducing pathogen infection in a plant, comprising genetically modifying a nucleotide sequence encoding a cell-surface immune receptor to introduce a nucleotide sequence encoding a CC motif, and expressing the genetically modified cell-surface immune receptor in the plant.

[0178]

[0187] The present invention is further illustrated by the following examples. However, it should be understood, that the invention is not limited to the exemplified embodiments.

[0179] EXAMPLES

[0180] Materials and Methods

[0181]

[0188] Engineering Methodology: A set of naturally derived or computationally designed Coiled-coil (CC) motifs (see below for the detailed amino acid sequences) (Fletcher et al., 2012; Kiihnel et al., 2004) were employed to drive the FLS2 receptors assembling to defined oligomerization following the valencies of CC motifs. Specifically, the dimeric (D) and tetrameric (T) CC motifs were fused at the C-terminus of FLS2 (encoded by At5g46330) and connected by 3xMyc peptides (FIG. 1). To quantitatively characterize the assembly of engineered FLS2, a monomeric super folder green fluorescence protein (mG) was further attached following CC motifs, which enable the fluorescent image (FIG. 1). Protein with no CC motif insertion was designated as monomeric (M) FLS2. To demonstrate the broad applicability of this receptor engineering method to multiple plant cell surface receptors, another typical pattern recognition receptor, PEPR1 (encoded by At1g73080), which recognizes the damage-associated molecular pattern pep1 in Arabidopsis-was also engineered using the similar methodology applied to FLS2 (FIG. 1). In addition, engineering of BAK1 (encoded by At4g33430) was carried out by integrating the above CC motifs in between BAK1 transmembrane domain and kinase domain (BAK1-M / D / T). When we determined the stoichiometries of BAK1 clusters, a mG was further added at the C-terminus (BAK1-M / D / T-mG) (FIG. 17), which allows to do a single-particle image to confirm the assembly states of engineered BAK1 (FIG. 14).

[0182]

[0189] Genetic Constructs: The DNA sequence encoding the above-designed FLS2-M / D / T-mG (i.e. SEQ ID NO: 23-25) proteins were cloned into pHGW binary vector containing the native promotor of FLS2 to drive FLS2 expression. For PEPR1 -mG-M / D / T (i.e. SEQ ID NO: 26-28), 35S promotor was applied to drive a robust expression of PEPR1 or BAK1 in Nicotiana benthamiana transient expression. Sequencing verified constructs were transformed into A. tumefaciens strain GV3101 subjected to screening by 50mg / mL spectinomycin. The nucleotide sequences of FLS2-M / D / T-mG, PEPR1 -mG-M / D / T and BAK1-M / D / T-mG, along with the translated amino acid sequences are listed in Table 2. Table 2: Nucleotide and Amino Acid sequences

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0190] Transformation Protocols: Arabidopsis transformation was conducted in Col-0 fls2 (SALK_026801 C) background through standard A. tumefaciens-wed\ateti gene transfer procedure via floral infiltration (Zhang et al 2006) . Transient expression of PEPR1-M / D / T-mG in N. benthamiana leaves were conducted by injecting A. tumefaciens suspensions in the infiltration buffer (10 mM MES, pH 5.7, 10 mM MgCl2, and 100 uM acetosy ringone) into the abaxial surface of N. benthamian leaves. Image or sample collection was done at 16-20 h post injection.

[0199]

[0191] Selection and Verification: Individual transgenic lines were selected based on hygromycin resistance. Expression of FLS2-M / D / T-mG proteins were confirmed by western blot analysis using anti-myc antibody and fluorescent image. The single molecules of FLS2 / PEPR1 / BAK1-M / D / T-mGs were recorded under variable angle-total internal reflection fluorescence microscope (VA-TIRFM) to check the FLS2 / PEPR1 / BAK1 protein assembly according to the single particle signal intensity of FLS2-M / D / T-mGs.

[0200]

[0192] Measure of Plant Defence Response: Multiple plant early immune responses were monitored to evaluate the effects of the engineered receptors (FIG. 2). The transgenic FLS2-M / D / T-mG Arabidopsis with comparable expression of FLS2 were grown under short-day cycles (10 h light / 14 h dark) at 22 °C. To check the MAPK cascade activation, two-week-old Arabidopsis seedlings grown in 1 / 2 MS medium were spray with 20 nM flg22 supplied with 0.01% (v / v) silvet-77. The samples were then collected at 0 min, 5 min, 10 min, 15 min and 30 min post flg22 spray. Total proteins were further isolated and separated by SDS-PAGE. Phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) monoclonal antibody was applied to detect the phosphorylated MAP kinases. To measure the receptor activation mediated ROS generation. Leaf discsx (Diameter = 0.4 cm) were punched from the rosette leaves of four-week-old Arabidopsis or N. benthamian leaves with transient expression of engineered PEPR1 or BAK1 receptors. Leaf discs were further floated on sterile water in a 96-well plate for overnight under continuous light. To elicit ROS production, the water was replaced by ROS elicitation solution (20 nM flg22 / pep1 , 20 mM luminal L-021 and 20 mg / mL horseradish peroxidase). The luminescence was immediately monitored by a BioTek cytation 5 multimode reader with a time interval as 80 s. To evaluate the plant defence to bacterial infection. 1 x 106CFU / mL of Pseudomonas syringae pv. tomato (Pst), DC3000, suspended in 10 mM MgCl2, were injected into the abaxial surface of rosette leaves. The seedlings were then grown for another three days. To quantify the internal bacteria colonisation, the leaf tissues with bacteria inoculation were collected and grinded in a bead beater. The tissue lysis was resuspended in 10 mM MgCl2. With a series dilution, the tissue suspension was further dropped on NYG (3 g / L yeast extract, 5 g / L peptone, 20 g / L glycerol) agar (1.5%, W / V) plates, which were further incubated in 28 °C chamber for another two days before the bacteria clone numbers were then counted.

[0201]

[0193] Growth Analysis: The surface-sterilised seeds of transgenic FLS2-M / D / T-mG Arabidopsis with comparable expression of FLS2 were plotted in the half strength of MS medium supplied with or without 100 nM flg22. They were further grown vertically under long-day cycles (16 h light / 8 h dark) at 22 °C for five days before root length measurement.Results and Discussion

[0202]

[0194] Engineering of Receptor to Defined Oligomerisation: Single-molecule image of the engineered FLS2 receptor revealed that FLS2-M / D-mGs homogenously assembled to monomeric or dimeric clusters following the valencies of CC motifs (Khairil Anuar et al., 2019), each showing homogenous single-molecule intensity (FIG. 3). However, the FLS2-T-mG was heterogeneously assembled by displaying single spots with varied intensity. Nevertheless, the overall intensities of FLS2-T-mG was higher than FLS2-M / D-mG single molecules (FIG. 3), suggesting an overall higher oligomerisation of FLS2-T-mG than FLS2-M / D-mGs.

[0203]

[0195] Enhanced Defense Responses: With those engineered FLS2 receptors with defined oligomerization stoichiometry, a series of plant immune responses following FLS2 activation were investigated to evaluate how each engineered FLS2 influences immune activation (FIG. 2). First, FLS2-D-mG and FLS2-T-mG showed significant elevation in both activating MAPK cascade and producing ROS upon flg22 elicitation, in which FLS2-T-mG showed relatively lower activities compared to FLS2-D-mG (FIG. 4-6). Consistent with the rapid PTI activation results, both dimeric FLS2 and tetrameric FLS2 demonstrated overall greater resistance to Pst DC3000 infection compared to monomeric-FLS2, with FLS2-D-mG showing the strongest effect (FIG. 7).

[0204]

[0196] Growth Analysis: At resting states, engineering of FLS2 receptor, either to a dimeric- or tetrameric- form, did not affect the plant growth as shown by the comparable root length across FLS2-M / D / T-mG compared with WT and fls2 (FIG. 8 and 9). In the presence of immune activation by adding flg22, the growth trade-offs caused by long-lasting PTI responses indicate stronger PTI activation in FLS2-D-mG and FLS2-T-mG compared to monomeric FLS2, as evidenced by a more pronounced reduction in root growth, particularly in FLS2-D-mG (FIG. 8 and 10).

[0205]

[0197] Broad Applicability of Receptor Engineering: Transient expression of engineered PEPR1 , via the similar methods applied on FLS2, showed progressively increasing single particle intensities following the CC valencies on the plasma membrane (FIG. 11), similar to FLS2-M / D / T-mGs (FIG. 3).

[0206] Those engineered PEPR1 receptors phenocopied FLS2 engineering in mediating ROS production. PEPR1-mG-D and PEPR1-mG-T both showed apparently increased ROS production upon ligand pep1 elicitation compared with PEPR1-mG-M, in which PEPR2-mG-D showed the strongest effect (FIG. 12 and 13). Similarly, transient expression of engineered BAK1 also displayed gradually increased single particle intensities (FIG. 14 and 15) following the CC valencies. ROS measurement revealed the BAK1 oligomerisation-dependent increase in flg22-triggered immune responses (FIG. 16 and 17). FLS2, PEPR1 and BAK1 are both highly conserved in land plants. In addition, they represent typical leucine rich repeat-receptor like kinases (LRR-RLKs) in plants (Chakraborty et al., 2019). The exemplified results thus can be expanded to a broader range of LRR-RLKs and plant species.

[0198] Together, all those results demonstrate receptor dimerization enhances defence responses against bacterial infections but does not affect plant growth at its resting-state. This offers an effective strategy for improving plant defence capabilities.

[0207]

[0199] The mechanism and supporting evidence for this approach demonstrate its effectiveness and robustness. Transgenic Arabidopsis plants expressing the dimerised FLS2 exhibited markedly increased resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 infection compared to wildtype plants, confirming that enforced oligomerisation enhances receptor-mediated immunity. Importantly, despite the heightened immune response that effectively restricts pathogen proliferation, the engineered plants did not display stunted growth, developmental defects, or other phenotypes typically associated with chronic immune activation, indicating that the dimeric FLS2 design avoids growth penalties. The synthetic dimeric FLS2 also enabled more efficient PAMP perception, resulting in rapid production of reactive oxygen species (ROS), accelerated MAPK activation, and elevated expression of defence-related genes. This accelerated downstream signalling cascade played a central role in restricting pathogen proliferation. Furthermore, the approach demonstrated broad applicability across multiple surface receptors and co-receptors. Engineered PEPR1 , which recognises damage-associated molecular patterns, as well as BAK1, a co-receptor for multiple surface receptors including FLS2 and PEPR1 , also exhibited enhanced immune signalling and strengthened defence responses when oligomerized.

[0208]

[0200] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0209]

[0201] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The polypeptides, nucleic acids, plants, cells, seeds, methods, and uses described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0202] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising”, "including,” containing", etc. shall be read expansively and without limitation. The word "comprise" or variations such as "comprises" or "comprising" will accordingly be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0210]

[0203] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

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Claims

CLAIMSWhat is claimed is:

1. A polypeptide for use in modulating an immune response in a plant, comprisinga plant cell-surface immune receptor, anda coiled-coil (CC) motif operably fused to the plant cell-surface immune receptor, wherein the CC motif is selected from a dimeric, trimeric, tetrameric, or pentameric CC motif.

2. The polypeptide of claim 1 , wherein the plant cell-surface immune receptor is a patternrecognition receptor (PRR), a PRR co-receptor, or a leucine-rich repeat (LRR)-single transmembrane domain receptor, preferably a FLS2 or PEPR1 , or BAK1.

3. The polypeptide of claim 1, wherein the CC motif is a dimeric CC-motif or a tetrameric CC-motif.

4. The polypeptide of claim 3, wherein the dimeric CC-motif comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6, or a functional variant thereof, and the tetrameric CC-motif comprises or consists of an amino acid sequence set forth in SEQ ID NO: 8, or a functional variant thereof.

5. The polypeptide of claim 1 , wherein the polypeptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NO: 15, 16, 18, 19, 21 or 22, or a functional variant thereof.

6. A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of any one of claims 1 -5, preferably the nucleic acid molecule is comprised in a vector.

7. The nucleic acid molecule of claim 6, further comprising a promoter capable of driving expression in a plant operably linked to the nucleotide sequence encoding the polypeptide.

8. The nucleic acid molecule of claim 6, comprising a nucleotide sequence set forth in any one of SEQ ID NO: 24, 25, 27, 28, 30 or 31 , or a functional or degenerate variant thereof.

9. The polypeptide or claim 1 , or the nucleic acid molecule of claim 6 for use in reducing, treating or preventing a pathogen infection in a plant, and / or improving resistance to a pathogen infection in a plant.

10. A plant, plant cell or plant seed comprising the polypeptide or claim 1 , or the nucleic acid molecule of claim 6.

11. The plant, plant cell or plant seed of claim 10, wherein the plant is Arabidopsis thaliana or Nicotiana benthamiana.

12. The plant, plant cell or plant seed of claim 10, wherein the plant comprises or expresses a monocot or dicot LRR receptor on the cell surface.

13. A method of producing the plant, plant cell or plant seed of claim 10, comprising introducing the nucleic acid molecule of claim 6 into a plant, plant cell or plant seed, preferably transforming the plant, plant cell or plant seed with the nucleic acid molecule.

14. A method for modulating an immune response of a plant, comprising introducing the nucleic acid molecule of claim 6, into the plant and expressing the nucleic acid molecule such that the encoded polypeptide is localised to the plasma membrane of plant cells in the plant.

15. The method of claim 14, wherein the plant exhibits receptor-mediated immunity following perception of a pathogen-associated molecular pattern (RAMP) or damage-associated molecular pattern (DAMP), and / or the plant does not display stunted growth, developmental defects, or phenotypes associated with chronic immune activation.

16. The method of claim 14, wherein the cell-surface immune receptor remains in a primed but inactive state until perception of PAMPs or DAMPs in the presence of a pathogen.

17. A method for reducing pathogen infection in a plant, comprising introducing the nucleic acid molecule of claim 6, into the plant, and expressing the nucleic acid molecule such that the encoded polypeptide is localised to the plasma membrane of plant cells in the plant.

18. The method of claim 17, wherein the pathogen infection is a bacterial, fungal, or oomycete pathogen.

19. A method of producing a plant, plant cell or plant seed comprising an engineered plant cellsurface immune receptor, the method comprising modifying a plant cell-surface immune receptor in the plant, plant cell or plant seed to operably associate, fuse or couple with a CO motif, wherein the CC motif is selected from a dimeric, trimeric, tetrameric, or pentameric CC motif.

20. The method of claim 19, wherein the modifying step comprises genetically modifying a gene encoding the plant cell-surface immune receptor to operably link a nucleotide sequence encoding the CC motif, such that the expressed plant cell-surface immune receptor is operably associated, fused or coupled with the CC motif.

21. The method of claim 20, wherein the modifying step comprises post-translationally modifying the plant cell-surface immune receptor to operably associate, fuse or couple with the CC motif, and the method further comprises introducing a nucleic acid molecule encoding the CC motif into the plant, plant cell or plant seed under suitable conditions to express the nucleic acid molecule such that the encoded CC motif associates, fuses or couples to the plant cell-surface immune receptor.

22. The method of claim 20, wherein the method modulates an immune response and / or reduces pathogen infection and / or improves resistance to a pathogen infection, in the plant, plant cell or plant seed.

23. The method of claim 20, wherein the CC motif is a dimeric CC-motif or a tetrameric CC-motif.

24. The method of claim 23, wherein the dimeric CC-motif comprises or consists of an amino acid sequence set forth in SEQ ID NO: 6, or a functional variant thereof, and the tetrameric CC-motif comprises or consists of an amino acid sequence set forth in SEQ ID NO: 8, or a functional variant thereof.

25. The method of claim 20, wherein the plant cell-surface immune receptor is a pattern-recognition receptor (PRR), a PRR co-receptor, or a leucine-rich repeat (LRR)-single transmembrane domain receptor, preferably a FLS2 or PEPR1 , or BAK1.

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