Single cell method for discovering disease-resistant leader

Abstract

The present invention relates generally to the field of molecular biology and concerns a method for high throughput screening and characterization of one or more candidate plant pathogen effect genes and / or one or more candidate plant disease resistance genes in a single plant cell, the method comprises: (i) introducing into a plant protoplast one or more expression cassettes comprising at least one candidate plant pathogen effect gene and / or at least one candidate plant disease resistance gene; (ii) transiently expressing the one or more candidate plant pathogen effect genes and / or the one or more candidate plant disease resistance genes; (iii) measuring reactive oxygen species (ROS) produced by the protoplast; then (iv) separating the plant protoplast producing the ROS; and (v) identifying the introduced at least one candidate plant pathogen effect gene and / or at least one candidate plant disease resistance gene. Alternatively, the protoplast is capable of expressing the selectable marker, thereby allowing for the measurement of modulated selectable marker expression as an alternative indicator for HR activation.

Description

Technical Field

[0001] The present invention relates generally to the field of plant molecular biology and to methods for high-throughput and / or (single) cell-based screening, identification, validation and characterization of plant disease or pathogen resistance leads, including but not limited to immune receptor variants with better effector binding properties leading to more effective hypersensitive responses, and immune receptor variants that recognize a wider range of effector combinations leading to expanded activation sensitivity. Background Art

[0002] Plant diseases and plant pathogens cause significant yield losses in global crop harvests, posing a threat to food security (Savary et al., 2019). Crops cultivated worldwide are particularly vulnerable because they are exposed to a wide variety of pathogens worldwide (Nagy et al., 2021). During domestication, the diversity of disease-resistance genes in crops has decreased, placing these crops at a disadvantage against rapidly evolving pathogens (Zheng et al., 2016). Indeed, plant resistance to pathogens can be easily overcome by rapidly evolving virulent pathogen races that express a large number of avirulence genes. Therefore, there is a long-term need for plastic and adaptable plant defense systems to respond to novel pathogens, pathogen-associated molecular patterns, or pathogen effectors (Kourelis et al., 2021).

[0003] Most current strategies for conferring greater tolerance or resistance to diseases or pathogens in crops rely on time-consuming population genetics processes and QTL cloning efforts (Kawashima et al., 2016; Pedley et al., 2019). While comparative genomics (Yang et al., 2013), mutagenesis (Steuernagel et al., 2016), or reverse genetics-based gene identification (Lewis et al., 2010) represent more efficient approaches for identifying R genes, such methods also require significant time and effort. This also applies to biotechnological approaches, which rely on a deep understanding of the molecular and biochemical determinants of crop-pathogen interactions and the subsequent time-consuming in situ evaluation of candidate genes, gene variants, and gene or protein expression strategies. Given the adaptive capacity of pathogens to overcome crop resistance, such discovery strategies, which take years or at least months, are suboptimal for developing durable pathogen resistance in crops.

[0004] Plant protoplasts have proven to be versatile tools for conducting cell-based experiments. Their physiological responses and high-throughput capabilities enable cost-effective screening and hypothesis-driven experiments (Yoo et al., 2007). More specifically, protoplasts have been successfully used to study plant innate immune responses triggered by elicitors (Pachten and Barz, 1999) or pathogen-derived avirulent effectors (He et al., 2007; Su et al., 2019). Interactions between elicitors and cell surface receptors, or between avirulent effectors (AVRs) and plant nucleotide-binding leucine-rich repeat (NLR) receptors, typically lead to rapid host cell death at the site of intended infection and ultimately to plant disease resistance. This hypersensitive response can serve as a surrogate for NLR activation required for disease resistance. Given the physiological relevance of the hypersensitive response in the context of NLR / AVR interactions, assays that rely on the detection and quantification of the response are superior to more limited or indirect assays based on yeast two-hybrid screening, virus-induced gene silencing, or virus-mediated overexpression for detecting candidate AVRs or NLRs. Agrobacterium-mediated transient assays in Nicotiana spp. are a widely used method that allows for relatively rapid discovery of matching NLR / AVR pairs. However, issues related to expression regulation and AVR-independent cell death responses in this heterologous system often require extensive experimental work to detect cell death due to specific NLR / AVR interactions (Bourras et al., 2015).

[0005] WO 2022 / 218158 discloses a method for validating genes encoding putative plant pathogen effector proteins in plant protoplasts. Briefly, a putative pathogen effector gene and a LUC gene are co-expressed in plant protoplasts expressing a known resistance gene. Recognition of the effector protein by the resistance protein results in a decrease in LUC activity, presumably due to the development of a hypersensitive response in the protoplasts. Due to the low sensitivity of LUC detection, this method cannot be used for high-throughput screening of single cells, limiting it to batch measurements.

[0006] Saur et al. (2019) described a method for rapidly measuring cell death mediated by NLR / AVR pairs using barley and wheat protoplasts. The method was successfully applied to detect and quantify cell death caused by NLR / AVR interactions of two different NLR / AVR pairs in bulk protoplast samples. However, the authors observed very significant experimental variability using this protocol. Therefore, this method does not provide an effective solution for selecting, for example, immune receptor variants that exhibit higher effector binding affinity, leading to improved allergic responses, or for defining the most potent immune receptor genes present in an NLR cluster, or for quantifying the effects of multiplexed genome editing approaches on a set of sensitive genes. Methods for screening and prioritizing disease resistance genes should not only be quantitative and reproducible but also scalable, enabling the selection of optimal candidates from collections of allelic variants or from large libraries of immune receptor or effector mutants. To efficiently screen large collections of mutants, assays that provide quantitative readouts at the single-cell level or require only a limited number of cells are preferred. Furthermore, after enrichment or selection of target cells, the workflow preferably includes a single-cell genotyping step to support structure-function characterization of different lead candidates or enable rapid reconstruction of mutants in a suitable genetic background to breed crop varieties with durable disease resistance.

[0007] Therefore, there is a need for a method that allows for the rapid high-throughput identification of genes that provide plants with pathogen resistance to diseases caused by pathogens that produce pathogen effector genes. Summary of the Invention

[0008] The present invention describes a general workflow for transfection and sorting of plant protoplast cells, which can realize various screening applications, ranging from the identification and validation of candidate R gene and effector pairs, to the evaluation of R gene and / or effector function, to the screening of R gene and / or effector variant libraries, bait engineering, and the evaluation of R gene stacking or aggregation.

[0009] The workflow involves a multifaceted approach that includes: i / expressing one or more candidate effectors and / or R genes in protoplasts isolated from the crop of interest; ii / living cell sorting of the transfected protoplasts to identify cells expressing HR; iii / distributing and retrieving individual cells of interest; iv / (optional) clonal propagation of the sorted cells; v / sequencing of isolated single cells or clonal progeny to reveal one or more causal R genes and / or one or more effectors that trigger HR; and vi / (optional) regeneration of pre-selected cells / clones into whole plants.

[0010] In a first step, constructs encoding candidate effectors and / or R genes are transfected into protoplasts isolated from the crop of interest, following the procedures described herein. Depending on the specific use case and experimental objectives, different expression platforms can be used. For example, multiple pairs of candidate effectors and R genes can be transiently expressed in protoplasts derived from susceptible crop varieties / cultivars. In specific cases, candidate membrane-bound R genes can be transiently expressed in protoplasts derived from susceptible crop varieties / cultivars in combination with the expression of secreted effectors, thereby inducing a hypersensitive response. Alternatively or in addition, candidate effectors can be transformed in protoplasts from a disease-resistant strain, which is a crop variety or cultivar expressing a previously isolated (unisolated) R gene. In a third case, candidate effectors and / or R genes are heterologously expressed in protoplasts derived from different plant species.

[0011] Next, alternative reporter gene systems are used to identify protoplasts expressing HR. These systems measure the activity of fluorescent and / or luminescent (bio) reporter genes as surrogate markers of R gene activation and immune signaling. Examples of reporter gene assays for detecting single cells producing HR include, but are not limited to: i / reduced activity of co-transfected or stably expressed fluorescent or luminescent reporter genes; ii / fluorescent detection of immune-related reactive oxygen species (ROS); iii / expression of fluorescent or luminescent reporter genes under the control of HR-inducible promoters; or iv / fluorescent detection of endogenous HR marker transcripts using molecular RNA sensors.

[0012] Assays (ii) and (iii) involve a single measurement of fluorescence or light emission collected from a single cell. In contrast, when using a co-transfected or stably expressed fluorescent or luminescent reporter driven by a constitutive or inducible promoter (i.e., Assay 1), transfected cells are analyzed repeatedly to account for cell-to-cell heterogeneity in reporter gene expression. Thus, time-resolved measurements enable the sorting of cells with the greatest decrease in reporter gene activity, unaffected by variability in transfection efficiency and stochasticity in reporter gene expression.

[0013] After transfection, protoplasts are analyzed using one of several live cell sorting techniques, including fluorescence activated cell sorting (FACS), microfluidics, microfluidic containment, and microarray-based systems, or using live cell inoculation and imaging techniques, each of which has its unique characteristics and applications. Based on the above-mentioned fluorescence or luminescence readings, single positive cells are selectively sorted and assigned to 96-well plates, 384-well plates, or 1536-well plates, and subsequently analyzed via single-cell PCR and Sanger or amplicon deep sequencing to identify causal candidate R genes and / or effectors. Alternatively, the sorted cells are first grown into clonal colonies, which allows individual cells to recover and experience first mitosis from transfection and sorting. Once more robust, sustainable colonies have grown, the colonies are separated and transferred to a culture dish or container for continued colony growth, downstream genetic / molecular and / or biochemical analysis, and subsequent plant regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Measurement of GFP fluorescence (RFU) in protoplasts derived from rice ( Oryza sativa ) (A), soybean ( Glycine max ) (B), and Brassica napus ( Brassica napus ) (C). 1: empty vector control protoplasts; 2: protoplasts expressing OsRGA4.

[0015] Figure 2 : Measurement of GFP fluorescence (RFU) in protoplasts derived from wheat cv. Fielder. 1: Dummy plasmid plasmid) control protoplasts; 2: protoplasts expressing PM3A; 3: protoplasts expressing PM3A-S1335R; 4: protoplasts expressing PM3A-R1334E; 5: protoplasts expressing PM3A-D502V; 6: protoplasts expressing Avr-PM3A; 7: protoplasts expressing Avr-PM3A-Q112N; 8: protoplasts expressing PM3A and Avr-PM3A; 9: protoplasts expressing PM3A-S1335R and Avr-PM3A; 10: protoplasts expressing PM3A-R1334E and Avr-PM3A; 11: protoplasts expressing PM3A and Avr-PM3A-Q112N; 12: protoplasts expressing PM3A-S1335R and Avr-PM3A-Q112N.

[0016] Figure 3: Visualization of GFP fluorescence in rice-derived protoplasts. 1: Protoplast expressing eGFP; 2: Protoplast expressing deGFP_1; 3: Protoplast expressing deGFP_2; 4: Protoplast expressing deGFP_3; 5: Protoplast expressing eGFP and RGA4; 6: Protoplast expressing deGFP_1 and RGA4; 7: Protoplast expressing deGFP_2 and RGA4; 8: Protoplast expressing deGFP_3 and RGA4.

[0017] Figure 4 : Single-cell RGA4-specific PCR amplification of two replicate protoplasts (A and B); 1: single protoplast with high GFP intensity; 2: single protoplast with low GFP intensity.

[0018] Figure 5 : Overview of the workflow according to an embodiment of the present invention. The middle figure is modified by Union Biometica Inc.

[0019] Figure 6 : Luminescence of protoplasts (RLU×10) in undiluted samples (about 50,000 cells, A), 10-fold diluted samples (about 5,000 cells, B), 200-fold diluted samples (about 250 cells, C), and 400-fold diluted samples (about 125 cells, D). 3 1: control protoplasts expressing GFP; 2: protoplasts expressing firefly luciferase (fLUC); 3: protoplasts expressing green-enhanced nanolantern (GeNL); 4: protoplasts expressing fLUC and RGA4; 5: protoplasts expressing GeNL and RGA4.

[0020] Figure 7 : Measurement of luminescence (AU) in single protoplasts derived from rice. 1: Protoplast expressing green enhanced nanolamp (GeNL); 2: Protoplast expressing GeNL and RGA4; 3: Protoplast expressing firefly luciferase (fLUC); 4: Substrate control protoplast.

[0021] Figure 8: Measurement of luminescence (RLU) in wheat-derived protoplasts. 1: Green Enhanced Nanolight (GeNL) control protoplasts; 2: PM3A-expressing protoplasts; 3: PM3A-S1335R-expressing protoplasts; 4: PM3A-R1334E-expressing protoplasts; 5: PM3A-D502V-expressing protoplasts; 6: Avr-PM3A-expressing protoplasts; 7: Avr-PM3A-Q112N-expressing protoplasts; 8: P 9: Protoplasts expressing PM3A and Avr-PM3A; 10: Protoplasts expressing PM3A-R1334E and Avr-PM3A; 11: Protoplasts expressing PM3A and Avr-PM3A-Q112N; 12: Protoplasts expressing PM3A-S1335R and Avr-PM3A-Q112N.

[0022] Figure 9 Figure 1: Luminescence (RLU) measurements in individual protoplasts derived from wheat. 1: Protoplast expressing PM3A and Avr-PM3A; 2: Protoplast expressing PM3A-S1335R and Avr-PM3A; 3: Protoplast expressing PM3A-R1334E and Avr-PM3A; 4: Protoplast expressing PM3A and Avr-PM3A-Q112N; 5: Protoplast expressing PM3A-S1335R and Avr-PM3A-Q112N; 6: Control protoplast transfected with an empty vector.

[0023] Figure 10 In vivo measurement of reactive oxygen species (ROS) as CellRox Green fluorescence (RFU) in protoplasts derived from rapeseed during the first 24 h after transfection. 1: Control protoplasts transfected with an empty vector ("Ctrl"); 2: Protoplasts expressing RGA4 ("RGA4"); 3: Control protoplasts transfected with an empty vector treated with flg22 ("flg22"); 4: Protoplasts expressing RGA4 treated with flg22 ("RGA4+flg22"). The x-axis indicates the time in hours after transfection.

[0024] Figure 11: Time-resolved measurement of GFP fluorescence (RFU) in protoplasts derived from wheat (cultivar Fielder). A: Protoplasts expressing the GFP reporter gene alone ("GFPCtrl"); B: Protoplasts co-expressing the GFP reporter gene with PM3A and Avr-PM3A. C: Protoplasts co-expressing the GFP reporter gene with PM3A-R1334E and Avr-PM3A; D: Protoplasts co-expressing the GFP reporter gene with PM3A and Avr-PM3A-Q112N. The x-axis indicates the time in hours after transfection (hpt): 8 hpt (1); 9 hpt (2); 11 hpt (3); 13 hpt (4); 15 hpt (5); 18 hpt (6); 22 hpt (7). "n" indicates the number of individual protoplasts measured.

[0025] Figure 12 Quantification (%) of individual protoplasts derived from wheat (cultivar Fielder) showing characteristic GFP expression patterns over time: Feature A: Increased GFP signal; Feature B: Remaining GFP signal; C: Decreased GFP signal; D: Absence of GFP signal. 1: Protoplast expressing the GFP reporter gene alone ("GFPCtrl"); 2: Protoplast co-expressing the GFP reporter gene with PM3A and Avr-PM3A. 3: Protoplast co-expressing the GFP reporter gene with PM3A-R1334E and Avr-PM3A; 4: Protoplast co-expressing the GFP reporter gene with PM3A and Avr-PM3A-Q112N. "n" indicates the number of individual protoplasts measured.

[0026] Figure 13 Quantification (%) of individual protoplasts derived from wheat (cultivar Fielder) displaying specific characteristic GFP expression patterns over time. A: Characteristic C, decreased GFP signal; B: Characteristic D, absent GFP signal. 1: Protoplast expressing the GFP reporter gene alone ("GFPCtrl"); 2: Protoplast co-expressing the GFP reporter gene with PM3A and Avr-PM3A. 3: Protoplast co-expressing the GFP reporter gene with PM3A-R1334E and Avr-PM3A; 4: Protoplast co-expressing the GFP reporter gene with PM3A and Avr-PM3A-Q112N. The x-axis indicates time in hours post-transfection (hpt).

[0027] Figure 14: Measurement of GFP fluorescence (GFP / autofluorescence) in protoplasts derived from Brassica napus cv. Westar. 1: Protoplast expressing a GFP reporter gene; 2: Protoplast expressing a GFP reporter gene and a secreted effector (A: AvrLm3*, B: AvrLm1*, C: AvrLm5-9*); 3: Protoplast expressing a GFP reporter gene and a membrane-bound cell surface receptor (A: Rlm3, B: LepR3, C: Rlm9); 4: Protoplast expressing a GFP reporter gene, a secreted effector, and a matching membrane-bound cell surface receptor (A: AvrLm3* and Rlm3, B: AvrLm1* and LepR3, C: AvrLm5-9* and Rlm9). DETAILED DESCRIPTION

[0028] Recent determination of the structures of both cell surface and intracellular immune receptors in their active states in plants has provided new insights into how recognition complexes can be modified to expand recognition specificity and thereby confer resistance to other virulent pathogens. By expanding the repertoire of both cell surface and intracellular recognition systems and combining them, resistance to many diseases is expected to be enhanced and more durable (Frailie and Innes, 2021). Similarly, a better understanding of the identity of fungal effectors and how these effectors interact with intracellular immune receptors will help enhance disease resistance in crops. It is hypothesized that effective strategies to improve R gene persistence will involve alternating or clustering R genes corresponding to different structural classes of effectors (Lazar et al., 2020). Clustering strategies should not be limited to endogenous genes (Pedley et al., 2019) but could also benefit from heterologous expression of R genes from wild germplasm of crop species or from plant species related to the target crop (Kawashima et al., 2016).

[0029] Here, a rapid and efficient protoplast-based lead discovery workflow for developing disease-resistant crop species is described. Leveraging established insights into the genetic elements that define pathogen resistance, a mechanistic understanding of crop-pathogen interactions generated by conventional genetics and molecular biology methods, and crop and pathogen genome sequence data, this workflow can discover disease-resistant leads within months, weeks, or days. Key components of such a more efficient or more effective discovery and development process include single cells or protoplasts and technologies that can perform high-throughput screening of (genetically) modified cells or protoplasts.

[0030] The inventors have now found that after using the method of the present invention, the expression of screenable marker genes can be reliably and rapidly measured in single protoplasts, so the regulated expression of these marker genes can be used as a marker for the occurrence of HR in the high-throughput screening method of the present invention. In addition to cell screening methods that rely on genes encoding fluorescent or luminescent markers, label-free methods for enriching and / or selecting target cells can also be considered. For example, magnetic levitation technology (LeviCell system, Levitas) can be used to enrich or select target cells in the absence of dyes or specific markers.

[0031] The rapid generation and accumulation of ROS during the so-called oxidative burst is one of the hallmarks of effector-triggered immunity. In addition to orchestrating HR-like cell death, ROS provide multiple other immune functions. ROS production constitutes a promising surrogate for NLR activation. Therefore, ROS production, as a surrogate for the onset of HR, was measured in single protoplasts.

[0032] Individual protoplasts that exhibit regulated production of ROS, regulated expression of one or more selectable marker genes, or both, compared to control protoplasts lacking functional candidate pathogen effector genes, are subsequently isolated, and ultimately the gene or genes of interest encoding one or more pathogen effectors and / or one or more disease resistance genes in the individual protoplasts are identified.

[0033] As used herein, technical terms and expressions used within the scope of this application are generally given their commonly applicable meanings in the relevant fields of plant biology, molecular biology, bioinformatics, and plant breeding. All of the following definitions apply to the entire content of this application. In particular, the terms "essentially," "about," "approximately," "substantially," and the like, relating to an attribute or value, also precisely define the attribute or precisely define the value, respectively. In the context of the same functional activity or substantially the same function, the term "essentially" means that the functional difference is preferably within 20%, more preferably within 10%, and most preferably within 5% or less compared to the reference function. In the context of a given value or range, the term "about" specifically refers to a value or range that is within 20%, 10%, or 5% of the given value or range. As used herein, the term "comprising" also encompasses the term "consisting of." The various section headings and subheadings in this application are for convenience and reference purposes only and should not affect the meaning or interpretation of this application in any way.

[0034] By combining established insights into the genetic elements that define pathogen resistance, mechanistic understanding of crop-pathogen interactions generated by conventional genetics and molecular biology approaches, and crop and pathogen genome sequence data, the present methods enable the discovery of disease resistance leads within months, weeks, or days.

[0035] In a first embodiment, the present invention provides a method for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in a single plant cell, the method comprising the following steps:

[0036] (a) preparing at least one protoplast cell derived from a plant of interest, optionally wherein the protoplast is capable of expressing one or more selectable marker genes;

[0037] (b) introducing into the at least one protoplast one or more expression cassettes for transient expression of one or more candidate plant pathogen effector genes, and / or introducing one or more expression cassettes for transient expression of one or more candidate plant disease resistance genes;

[0038] (c) transiently expressing the one or more candidate plant pathogen effector genes and / or the one or more candidate plant disease resistance genes;

[0039] (d) measuring the production of reactive oxygen species (ROS) in the at least one protoplast, and / or optionally measuring the expression of the selectable marker gene;

[0040] (e) isolating one or more individual protoplasts that exhibit modulated production of ROS, modulated expression of the one or more selectable marker genes, or both, as compared to control plant protoplasts lacking a functional candidate pathogen effector gene;

[0041] (f) identifying the one or more candidate plant pathogen effectors and / or the one or more candidate plant disease resistance genes in the single protoplasts isolated in step (e).

[0042] The methods described herein can quickly and efficiently discover, verify and characterize pathogen effector genes and matching plant disease resistance genes, which can then be used to efficiently develop disease-resistant crop species. The method can further perform (ultra-high) high-throughput screening on large groups of genes or gene variants. The efficiency improvement of the methods described herein is mainly due to the characterization in individual plant cells, thereby obtaining data from each single plant cell. As used herein, the term "cell" refers to a plant cell. As used herein, the terms "plant cell" and "protoplast" are used interchangeably; the term "single plant cell" or single protoplast refers to a single protoplast (i.e., separated from other protoplasts) from which data is obtained (i.e., analyzed on a single basis). The plant cell that can be used in the present invention can be any type of plant protoplast. Single plant protoplasts can be produced from any type of plant used in agriculture, including, but not limited to, field crop plants such as wheat, corn, soybean, or cotton; high-value crop plants such as tobacco, tomato, lettuce, pepper, or squash plants; Brassica plants such as broccoli, mustard, Brussels sprouts, cabbage, cauliflower, kale, kohlrabi, rapeseed, rutabaga, turnip, or Arabidopsis plants; ornamental plants such as roses, petunias, poppies, lilies, lavender, miscanthus, or cactus plants; fruit trees, shrubs, or vines such as grapes, apples, oranges, strawberries, blackberries, blueberries, raspberries, plums, apricots, apricot plants, etc.; or turf or forage plants such as grasses or alfalfa plants. Methods for obtaining protoplasts are known in the art.

[0043] Although the high-throughput screening of whole plant is basically subject to the limitation of its slow growth and size, various cell sorting and distribution techniques can be used to process millions of protoplasts in a few hours. However, up to now, protoplasts are mainly extracted and analyzed in batches, which limits their use. The method of the present invention utilizes plant protoplasts derived from target plants. In an embodiment, protoplasts are derived from whole plant or seedlings, plant parts (including pollen grains), plant tissues (including leaf tissues), cell suspensions and / or callus. Removing the plant cell wall produces highly versatile protoplasts, while retaining the physiological response of intact plant cells to immune triggers, thereby forming a unique experimental system. In addition, removing the cell wall also allows the efficient introduction and efficient downstream processing of exogenous DNA in protoplasts. Therefore, in the current method, using protoplasts helps to increase the throughput of the method, and enables cost-effective screening and cell-based experiments to be carried out in plants. The method for preparing protoplasts by crops and fiber crops is known to the technician and is also provided in this article.

[0044] In a second embodiment, the present invention provides a method as described above for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in single plant cells, wherein the one or more candidate plant pathogen effectors and one or more candidate plant disease resistance genes are introduced into protoplasts derived from plants susceptible to pathogens encoding the plant pathogen effectors.

[0045] In a third embodiment, the present invention provides a method as described above for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in a single plant cell, wherein the one or more candidate plant pathogen effectors are introduced into protoplasts derived from the corresponding disease-resistant plants.

[0046] "Correspondingly disease-resistant plants" are plants that are resistant to the pathogen from which one or more candidate pathogen effector genes have been derived.

[0047] In a fourth embodiment, the present invention provides a method for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in a single plant cell as described above, wherein the one or more candidate plant pathogen effectors and the one or more candidate plant disease resistance genes are introduced into protoplasts, which are derived from a plant that is heterologous to the one or more candidate plant disease resistance genes.

[0048] The term "heterologous" polynucleotide refers to:

[0049] (a) a polynucleotide that is not native to the host cell;

[0050] (b) a polynucleotide native to the host cell in which structural modifications (e.g., deletions, substitutions, and / or insertions) have been made to alter the native polynucleotide;

[0051] (c) a polynucleotide that is native to the host cell but whose expression is quantitatively altered due to manipulation of the polynucleotide's regulatory elements (e.g., a stronger promoter) by recombinant DNA techniques; or

[0052] (d) A polynucleotide that is native to the host cell but has not been integrated into its natural genetic environment as a result of genetic manipulation using recombinant DNA techniques.

[0053] Resistance is a strategy that an organism (e.g., a plant) can use to resist infection by a pathogen. The term "resistance" refers to the ability to prevent or minimize infection or attack by a pathogen. As used herein, "disease resistance" or "resistance to disease" refers to plants that exhibit increased resistance or tolerance to disease compared to control plants (susceptible plants). Compared to susceptible plants grown under similar disease conditions, disease resistance can manifest as fewer and / or smaller lesions, improved plant health, increased yield, increased root mass, increased plant vigor, less or no discoloration, faster growth, reduced area of necrosis, or reduced wilting.

[0054] In one embodiment, one or more candidate plant pathogen effector genes and one or more candidate plant disease resistance genes are introduced into protoplasts derived from a plant susceptible to a pathogen encoding the plant pathogen effector. Preferably, the transfected one or more candidate pathogen effector genes and one or more candidate disease resistance genes are expressed simultaneously, meaning that there is an overlap in the time period for all proteins to be expressed, thereby increasing the levels of the proteins compared to a non-transfected control.

[0055] In another embodiment, one or more candidate plant pathogen effectors are introduced into protoplasts derived from a corresponding disease-resistant plant. As used herein, a corresponding disease-resistant plant refers to a crop variety or cultivar that expresses a previously isolated R gene, as well as a crop variety or cultivar that expresses an R gene that has not been previously isolated or characterized. In yet another embodiment, one or more candidate plant pathogen effectors and one or more candidate plant disease resistance genes are introduced into protoplasts derived from a plant that is heterologous to the one or more candidate plant disease resistance genes.

[0056] In the next step of the method of the present invention, one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes are transiently expressed in protoplasts. Candidate membrane-bound R genes can be transiently expressed in protoplasts derived from susceptible crop varieties or cultivars in combination with expression of secreted effectors. In another embodiment of the present invention, the one or more plant pathogen effectors are secreted effectors. In another or additional embodiment, the one or more plant disease resistance genes encode one or more membrane-bound proteins. In preferred embodiments, the R genes encode membrane-bound cell surface-localized receptor proteins, such as, but not limited to, LepR3 (SEQ ID NO:40), Rlm3 (SEQ ID NO:41), and / or Rlm9 (SEQ ID NO:42), and the transiently expressed secreted effectors are AvrLm1 (SEQ ID NO:43), AvrLm3 (SEQ ID NO:44), and / or AvrLm5-9 (SEQ ID NO:45), respectively. Recognition of effector proteins by endogenous or transiently expressed resistance genes in protoplasts most often results in activation of the HR. The method of the present invention allows for reliable and efficient detection of intracellular and extracellular interactions between effectors and disease-resistant proteins, thereby triggering the activation of HR. HR is a complex multicellular process characterized by rapid cell death at the infection site, associated with many physical, physiological and molecular changes, including rapid transcriptional reprogramming and the accumulation of ROS. Therefore, in the next step of the method of the present invention, the production of reactive oxygen species (ROS) and / or optionally the expression of one or more screenable marker genes are measured in at least one single protoplast. Without wishing to be bound by theory, it is believed that the immune response triggered by activating disease-resistant proteins by matching effector proteins requires the consumption of a large amount of cellular energy, which results in a decrease in the expression of transiently expressed screenable marker genes in those cells. It is reported that the activity of co-transformed luciferase reporter genes as markers of HR activation is reduced. However, the latter requires a large number of cell populations, and luciferase measurements are highly variable between biological replicates, thereby hindering their use for characterizing and screening single protoplasts (Baba et al., 1986; Saur et al., 2021).

[0057] Plant pathogens secrete effectors to overcome host defenses and host immune responses. "Pathogen effector gene," "effector gene," "avirulin gene," or "Avr gene" are used interchangeably herein and refer to pathogen genes that encode effector proteins that modulate plant host cell physiology, suppress basal host defense responses, and / or promote disease susceptibility. "Plant disease resistance gene," "resistance gene," or "R gene" (used interchangeably) refers to a gene that encodes a protein that recognizes one or more specific pathogen effector proteins and is capable of triggering effector-triggered immunity (ETI)-related defense mechanisms in host cells. One such defense mechanism is the hypersensitive response (HR) associated with the rapid production of reactive oxygen species (ROS) by host cells. "Reactive oxygen species" or "ROS," as used interchangeably herein, refer to extracellular and / or intracellular hydrogen peroxide (H2O2), superoxide anions (O2 - ), hydroxyl radicals (OH) and singlet oxygen, as well as ROS and nitric oxide (NO) (such as but not limited to nitrosonium ions (NO + ), nitroxyl ion (NO - ), peroxynitrite (ONOO - ) and NO x Compounds (NO2, N2O3, N2O4, NO2 - 、NO3 - ))The product produced by the reaction.

[0058] The term "gene" means a segment of DNA that contains the genetic information that is passed from parent to offspring and contributes to the phenotype of an organism. The effects of a gene on the form and function of an organism are mediated by transcription into RNA (tRNA, rRNA, mRNA, non-coding RNA) and, in the case of mRNA, by translation into peptides and proteins.

[0059] Optionally, protoplasts can express one or more screenable marker genes. Screenable markers can be used as an alternative indicator for ROS production through regulation. In one aspect of the invention, screenable marker genes are constitutively expressed in protoplasts, and in this case (by lowering expression in the case of compatible reactions), the generation of screenable markers will reduce. In another aspect of the invention, screenable marker genes can be operably connected to an induced promoter under conditions that produce ROS. Therefore, the expression of screenable marker genes will increase.

[0060] The term modulated production or modulated expression refers to increased production or expression, or decreased production or expression, compared to unmodulated (control) production or expression.

[0061] As used herein, the term screenable marker refers to a protein that gives a phenotype to the cell introduced or expressing it, so as to identify and / or select cells that show hypersensitivity reactions within the scope of the present invention. Preferably, screenable markers allow visual selection at the unicellular level. Such marker activity results in the formation of color, luminescence or fluorescence. The expression of visual marker genes results in color (such as beta-glucuronidase, GUS or beta-galactosidase and its colored substrate, such as X-Gal), luminescence (such as luciferin / luciferase system or nano lamp) or fluorescence (green fluorescent protein GFP and its derivatives, such as destabilized GFP or truncated GFP or GFP fusion protein; Cyan fluorescent protein (CYP) gene, yellow fluorescent protein (YFP) gene, DsRed gene, mCherry gene) formation. This list only represents a small amount of possible markers. The skilled person is familiar with such markers. According to organism and selection method, preferably different markers, as long as these markers can be detected in the unicellular separation.

[0062] Therefore, the present invention provides a method as described above for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in a single plant cell, wherein the one or more selectable marker genes encode a fluorescent and / or luminescent marker.

[0063] The selectable marker gene can be expressed transiently or constitutively, in which case protoplasts can be transformed with an expression cassette comprising the selectable marker gene operably linked to a suitable promoter.

[0064] Measurement of selectable marker gene expression can be performed continuously, at multiple discrete time points, or at a single time point (eg, an endpoint measurement).

[0065] Thus, the present invention provides a method as described above for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in single plant cells, wherein the regulated production of ROS and / or the regulated expression of the one or more selectable marker genes is measured continuously or at one or more time points.

[0066] In another aspect of the invention, ROS production is detected by a compound that produces fluorescence when oxidized by ROS. TM Deep Red, CellROX TM Green reagent and CellROX TM Orange is an example of such a compound.

[0067] Therefore, the present invention also provides a method as described above for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in single plant cells, wherein the production of reactive oxygen species (ROS) is measured by ROS-induced oxidation of a fluorescent dye.

[0068] The present invention also provides a method for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in single plant protoplasts, as described above, wherein a selectable marker gene is transiently or stably expressed. Measurement of selectable marker genes in single protoplasts is particularly advantageous because it allows for rapid screening of large numbers of individual cells and easy retrieval of unique HR-expressing cells for further analysis.

[0069] The present invention also provides a method as described above for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in a single plant cell, wherein the selectable marker gene is controlled by a constitutive promoter or by a hypersensitive response inducible promoter.

[0070] The term "promoter" typically refers to a nucleic acid control sequence located upstream of the start of gene transcription, and participates in identifying and binding RNA polymerase and other proteins, thereby guiding the transcription of operably connected nucleic acids. "Promoter" herein may further include any nucleic acid sequence that can drive transcription of a coding sequence. In particular, as used herein, the term "promoter" may refer to a polynucleotide sequence that is typically described as being located in the 5' regulator region of a gene near the start codon. Transcription of one or more coding sequences begins in the promoter region. The term promoter may also include a fragment of a promoter that plays a role in starting gene transcription. A "constitutive promoter" refers to a promoter that has transcriptional activity in at least one cell, tissue, or organ at most, but not necessarily all, stages of growth and development and under most environmental conditions. Examples of constitutive promoters that can be used in the methods of the present invention include the CAMV 35S promoter (Odell et al., Nature, 313:810-812, 1985; GOS2 (de Pater et al., Plant J. Nov; 2(6):837-44, 1992, WO 2004 / 065596), ubiquitin (Christensen et al., Plant Mol. Biol. 18:675-689, 1992), and the like.

[0071] "Inducible promoters" induce or increase transcription initiation in response to a chemical (for review, see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108), environmental, or physical stimulus, or can be "stress-inducible" (i.e., activated when the plant is exposed to various stress conditions), or "pathogen-inducible" (i.e., activated when the plant is exposed to various pathogens).

[0072] Examples of inducible promoters are the sugar (rhamnose, arabinose, galactose) inducible promoter or the dexamethasone inducible promoter.

[0073] Preferably, the inducible promoter operably connected to the marker gene is a hypersensitive response inducible promoter, such as the AtMYB30 promoter or the Athsr promoter. The emergence of immune-triggered hypersensitive responses (HR) in plant cells is generally associated with large-scale transcriptional reprogramming, involving the upregulation of a large set of defense-related genes. In response to HR triggering, the HR marker gene that is significantly activated is used in an embodiment of the present invention, wherein the single cell screening workflow is based on the enhanced expression of one or more screenable marker genes. Under the control of an HR inducible promoter, the expression of the screenable marker gene according to an embodiment of the present invention allows characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in a single plant cell, because the accumulation of the selection marker indicates the emergence of HR related to the resistance of the protoplast to the candidate effector. In another embodiment of the present invention, RNA sensing technology is used to measure the transcriptional induction of the HR marker gene based on the HR-dependent expression of the screenable marker gene (Kaseniit et al., 2022; Jiang et al., 2022).

[0074] The present invention also provides expression cassettes or gene constructs and vectors to promote the introduction and / or expression (de novo introduction or increase of existing expression) of any nucleic acid described herein in protoplasts. The gene construct can be inserted into a vector, which can be commercially available, suitable for transformation into plant protoplasts and suitable for expression of the gene of interest in transfected protoplasts. The nucleic acid encoding pathogen effectors, disease resistance genes or selective marker genes can be located on a separate construct, or two or more of them can be located on a single construct. When more than one expression cassette is present on a single construct, the nucleic acid can be in a tandem orientation on the construct, or in an opposite orientation. Each nucleotide sequence can be fused together or separated by coding or non-coding DNA (e.g., promoter, intron, subcellular targeting signal or filler DNA (e.g., MAR (matrix attachment region) district)).

[0075] Methods for obtaining required expression levels are well known to those skilled in the art, and include but are not limited to placing one or more proteic genes of one or more codings to be characterized in the downstream of a suitable promoter. In an embodiment, one or more proteic genes of one or more codings to be characterized are placed in the downstream of a constitutive promoter. Suitable constitutive promoters for use in the method of the present invention include but are not limited to the constitutive 35S promoter of cauliflower mosaic virus, constitutive maize ubiquitin 1 promoter and constitutive Arabidopsis ubiquitin 10 promoter.

[0076] Depending on the specific use case and experimental goals, different expression platforms can be used.

[0077] As used herein, an "expression cassette" is a DNA molecule consisting of at least one sequence of interest to be expressed, which is operably linked to one or more control sequences as described herein (at least to a promoter). Typically, an expression cassette comprises three elements: a promoter sequence, an open reading frame, and a 3' untranslated region, which, in eukaryotes, typically contains a polyadenylation site. Additional regulatory elements may include transcriptional enhancers and translational enhancers. Intron sequences may also be added to the 5' untranslated region (UTR) or coding sequence to increase the amount of mature messenger accumulated in the cytosol. It is well known to those skilled in the art that the genetic elements necessary for successful expression are present in the expression cassette. Preferably, the arrangement of the DNA or genetic elements constituting the expression cassette is at least partially artificially designed. The expression cassette may be part of a vector or may be integrated into the genome of a host cell and replicated along with the genome of the host cell. The expression cassette is capable of increasing or decreasing the expression of the target DNA and / or protein.

[0078] "Operably linked" means that the described components are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence operably linked to a coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0079] The technology for introducing such expression cassettes into plants is well known in the art. The term "transformation" mentioned herein encompasses the transfer of exogenous polynucleotides into host cells, regardless of the method used for transfer. That is, as used herein, the term "transformation" is independent of vectors, shuttle systems, or host cells, and it not only relates to polynucleotide transfer methods known in the art (see, for example, Sambrook, J. et al., (1989) Molecular Cloning: A Laboratory Manual [Molecular Cloning: Laboratory Manual], 2nd edition, Cold Spring Harbor Laboratory Press [Cold Spring Harbor Laboratory Press], Cold Spring Harbor, New York), it also encompasses any other type of polynucleotide transfer method, such as but not limited to transduction or transfection. Plant tissues that can subsequently be cloned, whether through organogenesis or embryogenesis, can be transformed with a genetic construct and regenerated into a whole plant. Polynucleotides can be introduced into host cells transiently or stably, and can remain non-integrated, for example as a plasmid. "Stable transformation" can mean that the transformed cell or organelle transfers the nucleic acid containing the foreign coding sequence to the cell or organelle of the next generation. In general, stable transformation is due to the integration of a nucleic acid comprising an external coding sequence into a chromosome or as an episome. "Transient transformation" can mean that once transformed, a cell or organelle expresses an external nucleic acid sequence within a certain period of time (mainly within a generation). In general, transient transformation is due to the non-integration of a nucleic acid comprising an external nucleic acid sequence into a chromosome or as an episome.

[0080] Methods for transformation of protoplasts are known in the art and include, for example, the calcium / polyethylene glycol method (Krens, FA et al., (1982) Nature 296, 72-74; Negrutiu I et al., (1987) Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito RD et al., (1985) Bio / Technol 3, 1099-1102). Alternatively, plants can be transformed prior to the protoplast preparation step. Suitable plant transformation methods include microinjection of plant material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); bombardment of DNA or RNA-coated particles (Klein TM et al., (1987) Nature 327: 70); (non-integrating) viral infection, etc. A well-established and preferred method is Agrobacterium-mediated transformation. An advantageous transformation method is in situ transformation of plants. To this end, Agrobacterium can, for example, be applied to plant seeds, intact plants, or at least flower primordia, or inoculated into plant meristems. Methods for Agrobacterium-mediated rice transformation include well-known methods for rice transformation, such as those described in European Patent Application EP 1198985 A1, Aldemita and Hodges (Planta [Botany] 199: 612-617, 1996); Chan et al. (Plant Mol Biol [Plant Molecular Biology] 22 (3): 491-506, 1993), Hiei et al. (Plant J [Plant Journal] 6 (2): 271-282, 1994). In the case of maize transformation, preferred methods are as described by Ishida et al. (Nat. Biotechnol 14(6):745-50, 1996) or Frame et al. (Plant Physiol 129(1):13-22, 2002).The methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. Kung and R. Wu, Academic Press, (1993) 128-143, and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol., 42 (1991) 205-225). Preferably, the nucleic acid or construct to be expressed is cloned into a vector suitable for transforming Agrobacterium tumefaciens, such as pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacterium transformed with such a vector can then be used for plant transformation in a known manner. Plant transformation by Agrobacterium tumefaciens is carried out, for example. and Willmitzer, Nucl. Acid Res. (1988) 16, 9877, or in particular from FF White, Vectors for Gene Transfer in Higher Plants; Transgenic Plants, vol. 1, Engineering and Utilization, eds. S. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[0081] Preferably, the one or more candidate plant pathogen effector genes and / or the one or more candidate plant disease resistance genes are transiently expressed in protoplasts.

[0082] The protoplasts used in the method of the present invention can be derived from any plant. Preferably, the protoplasts are derived from plants of the Viridiplantae superfamily, particularly from monocots and dicots, including crop plants, such as cereals, oilseed plants, legumes, vegetables, and fiber crops. Examples of crop plants include, but are not limited to, chicory, carrots, cassava, yams, clover, soybeans, beets, sugar beets, sunflowers, canola, alfalfa, rapeseed, linseed, cotton, cocoa, tomatoes, potatoes, coffee, and tobacco. According to another aspect of the present invention, the plant is a monocot. Examples of monocots include sugarcane, bananas, onions, asparagus, palms, sedges, and rushes. According to another aspect of the present invention, the plant is a cereal. Examples of cereals include rice, maize, wheat, barley, millet, rye, triticale, sorghum, emmer wheat, spelt, einkorn wheat, teff, milo, and oats. In particular aspects, the plant used in the methods of the present invention is selected from the group consisting of: maize, wheat, rice, soybean, cotton, rapeseed (including canola), sugarcane, sugar beet, and alfalfa. As used herein, the term "plant" encompasses whole plants, original forms and progeny of plants, and plant parts, including seeds, buds, stems, leaves, roots, flowers, and tissues and organs. The term "plant" also encompasses plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen, microspores, and propagules. Examples of plants whose cells can be used in the methods of the invention include Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp., Artocarpus spp., Asparagus officinalis, Avena spp. (e.g., Avena sativa, Avena vulgaris), and Avena spp. fatua), red oats (Avena byzantina), wild oats cultivar sativa (Avena fatua var.sativa), hybrid oats (Avena hybrida), Averrhoa carambola, Bambusa sp., winter melon (Benincasa hispida), Brazil nut (Bertholletia excelsea), sugar beet (Beta vulgaris), Brassica spp. (e.g., Brassica napus, Brassica rapa ssp. [canola, rapeseed, turnip rape]), Cadabafarinosa, tea (Camellia sinensis), canna (Cannaindica), hemp (Cannabis sativa), Capsicum spp., Carex elata, papaya (Carica papaya), Carissa macrocarpa, Carya spp., safflower (Carthamus tinctorius), Castanea spp. spp.), Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Coconut spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus spp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumber spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan longan), Dioscorea spp., Diospyros spp., Echinochloa spp.), Elaeis (e.g., Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g., Glycine max, Soja hispida, or Soja max), Gossypium hirsutum, Helianthus spp. (e.g., Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g., Hordeum vulgare), Sweet potato (Ipomoea batatas), Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Flax (Linum usitatissimum), Litchi (Litchichinensis), Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp.) (e.g., Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp.), Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g., Oryza sativa, Oryza sativa), Oryza pueraria, Oryza spp. latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Parsley (Petroselinum crispum), Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp.), Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp.), Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g., Solanum tuberosum, Solanum integrifolium, or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticose calerimpaui, Triticum spp. spp.) (e.g., wheat (Triticum aestivum), durum wheat (Triticum durum), turgidum wheat (Triticum turgidum), wheat (Triticum hybernum), mocha wheat (Triticum macha), floating wheat (Triticum sativum), einkorn wheat (Triticum monococcum), or common wheat (Triticum vulgare)), nasturtium (Tropaeolum minus), nasturtium (Tropaeolum majus), Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., etc.

[0083] Therefore, the present invention provides a method as described above for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in a single plant cell, wherein the target plant is a crop plant, preferably a cereal, oil plant, legume, vegetable or fiber crop.

[0084] The protoplasts of suitable control plants are protoplasts that do not introduce pathogen effect genes, preferably comprise the protoplasts of the carrier (that is, empty vector) lacking pathogen effector expression cassette. Alternatively, the protoplasts of control plants are protoplasts that do not introduce the pathogen effector expression cassette. Depending on the circumstances, there is or are not screenable markers in the control protoplasts. Ideally and preferably, the control protoplasts have otherwise undergone the same processing as the protoplasts to be tested. The control plant protoplasts are typically derived from the same plant species or even the same kind as the plant protoplasts to be assessed.

[0085] Advantageously, the generation of ROS can be enhanced by an allergic response elicitor (or otherwise formulated as an immune response inducing elicitor). Elicitors for causing or amplifying allergic reactions are known in the art. Preferably, the elicitor is a small molecule, such as chitin heptaose or flg22 (a 22 amino acid epitope of flagellin). Therefore, the sensitivity of the method of the present invention can be enhanced by using one or more elicitors. In addition, the method of the present invention can also be used to qualitatively or quantitatively analyze candidate elicitor molecules in an assay having a given disease resistance gene, or having a given pathogen effector gene, or having a given disease resistance gene and pathogen effector gene combination.

[0086] Therefore, the present invention provides a method as described above for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in a single plant cell, wherein the production of reactive oxygen species (ROS) is enhanced by the addition of a hypersensitive response elicitor.

[0087] The present invention also provides a method as described above for high-throughput characterization of candidate elicitor molecules of one or more given plant pathogen effector genes and / or one or more given plant disease resistance genes in a single plant cell, wherein the production of reactive oxygen species (ROS) is enhanced by adding a hypersensitive response elicitor.

[0088] There are a variety of techniques for analyzing, live cell sorting, and retrieving single protoplasts from large protoplast populations, including light scattering-based methods such as flow cytometry, which is often coupled with the detection of a fluorescent signal (fluorescence activated cell sorting or FACS) in each analyzed protoplast (e.g., Figure 5In some embodiments, the present invention provides the method for the separation of protoplasts of the present invention. For example, the fluorescent signal of auxiliary sorting can be derived from screenable markers (for example, GFP), derived from chloroplasts, derived from the use of fluorescent dyes that can be cell permeable or cell impermeable etc. Alternatively, microfluidic fluid sorters can be used to separate protoplasts, and the other advantage that these microfluidic fluid sorters have is that flux is higher, and can operate under aseptic conditions. In addition, the use of the microfluidic device combined with FACS allows separation of protoplasts under the hydrodynamic stress of reduction, further contributes to the vigor of preserving protoplasts. Other technologies that are suitable for analyzing and / or separating purpose protoplasts in the method according to the present invention adopt the temporary fixation of protoplasts (for example, on chip (for example, NanoPen chamber) or microarray) for analysis. Therefore, single protoplasts are analyzed after transfection, and selection is compared with the control plant protoplasts lacking functional candidate pathogen effector genes, showing the single protoplasts of regulated expression of screenable marker genes. Therefore, the present invention provides a method as described above for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in single plant cells, wherein the regulated expression of the fluorescent or luminescent marker in single protoplasts is measured and compared with the expression of the fluorescent or luminescent marker in control protoplasts.

[0089] In another embodiment, the present invention provides a method as described above for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in a single plant cell, wherein the isolation of one or more single protoplasts includes the steps of protoplast sorting, partitioning, and retrieving the target protoplasts.

[0090] Based on the above fluorescence or luminescence readings, single positive cells can be selectively sorted, distributed into 96-well plates, 384-well plates or 1536-well plates, and subsequently analyzed via single-cell PCR and Sanger or amplicon deep sequencing to identify causal candidate R genes and / or effectors. Therefore, the present invention provides a method for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in single plant cells as described above, wherein the one or more candidate plant pathogen effectors and / or the one or more candidate plant disease resistance genes in the single protoplast are identified by sequencing.

[0091] Alternatively, the sorted cells are first grown into clonal colonies, which allows individual cells to recover from transfection and sorting and undergo first mitosis. Once more robust, sustainable colonies have grown, the colonies are separated and transferred to culture dishes or containers for continued colony growth, downstream genetic / molecular and / or biochemical analysis, and subsequent plant regeneration. Therefore, in another embodiment, the present invention provides a method as described above for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in individual plant cells, wherein the step of isolating one or more individual protoplasts that exhibit regulated production of ROS and / or regulated expression of the one or more screenable marker genes is followed by a step of clonal propagation.

[0092] In yet another embodiment, the present invention provides a method as described above for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in a single plant cell, the method further comprising regenerating the isolated protoplasts or clones derived therefrom into plants.

[0093] In yet another embodiment, the present invention provides a method for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in single plant cells as described above, wherein the characterization further comprises genetic, molecular and / or biochemical analysis. Such regenerated plants allow for further characterization of the one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes.

[0094] In another embodiment, the present invention provides the use of the method according to any one of the preceding claims for:

[0095] (a) functional characterization of one or more candidate plant pathogen effectors or one or more candidate plant disease resistance genes, or

[0096] (b) functional characterization of multiple pairs of one or more candidate plant pathogen effectors and one or more candidate plant disease resistance genes, or

[0097] (c) screening a library of variants of one or more candidate plant pathogen effectors and / or a library of variants of one or more candidate plant disease resistance genes, or

[0098] (d) bait engineering, or

[0099] (e) Evaluation of candidate plant disease resistance gene stacks.

[0100] In yet another embodiment, the present invention provides a method for producing disease-resistant plants, comprising transforming plant cells with a construct for expressing one or more plant disease resistance genes identified using the methods described above, and then regenerating into plants.

[0101] In a final aspect, the present invention provides a method for producing disease-resistant plants, comprising genetically modifying an allele of a gene that does not confer disease resistance in a plant cell such that the modified allele expresses a plant disease resistance gene identified using the method described above, and then regenerating the plant. In preferred embodiments, the genetic modification comprises transgenic or genome editing.

[0102] "Alleles" or "allelic variants" are alternative forms of a given gene located at essentially the same chromosomal location. Allelic variants encompass single nucleotide polymorphisms (SNPs) as well as small insertion / deletion polymorphisms (INDELs). INDELs are typically less than 100 bp in size. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.

[0103] "Gene editing" or "genome editing" is a type of genetic engineering or gene modification technology in which DNA is inserted, replaced, or deleted from the genome and can be achieved by using a variety of techniques, such as "gene shuffling" or "directed evolution," which consists of iterative DNA shuffling followed by appropriate screening and / or selection to generate variants of nucleic acids or portions thereof encoding proteins with modified biological activity (Castle et al., (2004) Science 304(5674): 1151-4; U.S. Patents 5,811,238 and 6,395,547), or the use of "T-DNA activation" tags (Hayashi et al., Science (1992) 1350-1353), in which the resulting transgenic plants exhibit dominant phenotypes due to altered expression of genes near the introduced promoter, or the use of "TILLING" (Targeted Induced Local Lesions Induced Local Mutagenesis). TILLING refers to a mutagenesis technique that can be used to generate and / or identify nucleic acids encoding proteins with modified expression and / or activity. TILLING also allows for the selection of plants carrying such mutant variants. Methods for TILLING are well known in the art (reviewed by McCallum et al. (2000) Nat Biotechnol 18:455-457; Stemple (2004) Nat Rev Genet 5(2):145-50). Another technology uses artificially engineered nucleases such as zinc finger nucleases, transcription activator-like effector nucleases (TALENs), CRISPR / Cas systems, and engineered mega-nucleases, such as re-engineered homing endonucleases (Esvelt, KM.; Wang, HH. (2013), Mol Syst Biol [Molecular Systems Biology] 9(1):641; Tan, WS. et al., (2012), Adv Genet [Genetics Progress] 80:37-97; Puchta, H.; Fauser, F. (2013), Int. J. Dev. Biol [International Journal of Developmental Biology] 57:629-637). As used herein, "genome editing," "gene editing," or "genome engineering" also refers to targeted modifications to genomic DNA, where DNA can be inserted, deleted, modified, or replaced in the genome. Genome editing can use sequence-specific enzymes (e.g., endonucleases, nickases, base transfer enzymes) and / or donor nucleic acids (e.g., dsDNA, oligonucleotides) to introduce desired changes in DNA.Sequence-specific nucleases that can be programmed to recognize specific DNA sequences include meganucleases (MGNs), zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and RNA-guided or DNA-guided nucleases such as Cas9, Cpf1, CasX, CasY, C2c1, C2c3, certain Argonaut-based systems (see, e.g., Osakabe and Osakabe, Plant Cell Physiol. 2015 Mar;56(3):389-400; Ma et al., Mol Plant. 2016 Jul 6;9(7):961-74; Bortesie et al., Plant Biotech J, 2016, 14; Murovec et al., Plant Biotechnol. J. [Journal of Plant Biotechnology] 15: 917-926, 2017; Nakade et al., Bioengineered [Biological Engineering] Vol. 8, No. 3: 265-273, 2017; Burstein et al., Nature [Natural] 542, 37-241; Komor et al., Nature [Natural] 533, 420-424, 2016; all documents are incorporated herein by reference). Donor nucleic acid can be used as a template for repairing DNA breaks induced by sequence-specific nucleases. Donor nucleic acid can also be used for genome editing (without DNA breakage induction) to introduce desired changes into genomic DNA. Genome editing can also refer to the introduction of specific mutations at a specific location in the cell genome. Gene editing can be introduced by applying more advanced technologies (e.g., using CRISPR Cas systems and donor DNA, or CRISPR Cas systems associated with mutagenic activity (such as deaminases) (WO 15133554, WO 17070632)).

[0104] In general, the present invention and the experimental methods described herein provide a set of methods and assays at the single cell level (which can be used alone or in combination), including but not limited to:

[0105] - Methods for screening, sorting and selection of target protoplasts (including, for example, flow cytometry, microfluidics, cell seeding);

[0106] - Methods for phenotyping of (single) protoplasts;

[0107] - Protoplast-based hypersensitivity assay (based on ROS production, intracellular Ca 2+ levels, regulated ATP levels, regulated gene expression, regulated protein production, detection of reporter molecules, aptamer characterization);

[0108] - Methods for high-throughput sorting of protoplasts and for selecting protoplasts showing a hypersensitive reaction;

[0109] -Used for

[0110] from natural sources (via bioinformatics (including de novo structure prediction methods) and synthetic biology) or

[0111] Methods for identifying new fungal resistance genes (e.g., in soybean, rapeseed, rice, or wheat) by modifying identified receptors; receptor engineering (e.g., but not limited to, direct detection engineering; bait engineering; nucleotide-binding leucine-rich repeat (NLR) proteins with engineered integration domains; expanded effector recognition; R genes encoding immune receptors that interact with structurally distinct classes of effectors; immune receptor repositioning);

[0112] - Methods for protoplast-based identification or validation of the following disease resistance genes or pathogen resistance genes:

[0113] R genes (cell surface (receptor-like kinases or RLKs, receptor-like proteins or RLPs) and intracellular (NLR sensors, NLR auxiliaries)),

[0114] S gene (multiple genome editing);

[0115] - Methods for identifying avirulent effectors of diseases and / or pathogens;

[0116] - Methods for identifying compatible plant immune receptors and avirulent effectors of diseases or pathogens (including identification of host factors required for immune receptor function);

[0117] - Methods for identifying and selecting mutually reinforcing cell surface pattern recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat proteins (NLRs) to develop durable and enhanced disease and / or pathogen resistance in plants;

[0118] - Methods for the selection of plant immune receptor gene combinations to develop stacking concepts for generating plants with enhanced disease and / or pathogen resistance (stacking of R genes encoding immune receptors that interact with effectors of different structural classes);

[0119] - Methods for efficient functional characterization of NLRs in NLR clusters in plant genomes;

[0120] -Methods for efficient discovery and functional characterization of pathogen-inducible promoters;

[0121] - Methods for generating optimal pathogen-inducible promoters;

[0122] - Methods for optimizing promoter-NLR expression cassettes;

[0123] - Methods for selecting products obtained from the strategy of "directed evolution of disease resistance in plants";

[0124] - A method for enriching for targeted transfection events via enrichment of cells co-transfected with a reporter gene.

[0125] Those skilled in the art will appreciate that the applicability of the above methods and assays, as exemplified below, is not limited to the identification and characterization of pathogen effectors and / or plant disease resistance genes, but can also be used to answer a variety of other scientific questions for which assays at the single-cell level are beneficial. For example, the methods of the present invention can be applied to studies related to cell death.

[0126] Additional reporter gene assays may involve the use of direct or indirect biosensors to detect immune-related biochemical or physical events via (imaging-based) live cell sorting. Physiological and metabolic responses that provide potential surrogates for HR activation include, but are not limited to:

[0127] -Intracellular Ca 2+ changes in levels or alterations in the cellular redox state (for review, see Kostyuk et al., 2020 );

[0128] -Cellular proton efflux leading to matrix alkalinization;

[0129] -MAP kinase activation;

[0130] - Changes in the perception, biosynthesis, degradation, or signaling output of immune-related plant hormones (e.g., salicylic acid, jasmonic acid, and ethylene), or altered concentrations of downstream signaling molecules (e.g., azelaic acid, glycerol-3-phosphate, pipecolic acid, and N-hydroxypiperidinic acid). Other hormone-acting factors include abscisic acid, auxins, gibberellins, cytokinins, brassinosteroids, and strigolactones (for a recent review, see Bürger and Chory, 2019);

[0131] - Fluctuations in the growth-defense trade-off (e.g., changes in cell division rates, for review, see Levak et al., 2021)).

[0132] References

[0133] Shan Q,Wang Y,Li J,Gao C.Genome editing in rice and wheat using theCRISPR / Cas system.Nat Protoc.2014;9(10):2395-2410.doi:10.1038 / nprot.2014.157

[0134] Césari S,Kanzaki H,Fujiwara T,et al.The NB-LRR proteins RGA4 and RGA5interact functionally and physically to confer disease resistance.EMBOJ.2014;33(17):1941-1959.doi:10.15252 / embj.201487923

[0135] Saur IML,Bauer S,Lu X,Schulze-Lefert P.A cell death assay in barleyand wheat protoplasts for identification and validation of matching pathogenAVR effector and plant NLR immune receptors.Plant Methods.2019;15:118.doi:10.1186 / s13007-019-0502-0

[0136] McNally KE,Menardo F,Lüthi L,et al.Distinct domains of the AVRPM3A2 / F2 avirulence protein from wheat powdery mildew are involved in immunereceptor recognition and putative effector function.New Phytol.2018;218(2):681-695.doi:10.1111 / nph.15026

[0137] Lindner S,Keller B,Singh SP,et al.Single residues in the LRR domainof the wheat PM3A immune receptor can control the strength and the spectrumof the immune response.Plant J.2020;104(1):200-214.doi:10.1111 / tpj.14917

[0138] Dong W,Nowara D,Schweizer P.Protein polyubiquitination plays a rolein basal host resistance of barley.Plant Cell.2006;18(11):3321-3331.doi:10.1105 / tpc.106.046326

[0139] Binder A,Lambert J,Morbitzer R,et al.A modular plasmid assembly kitfor multigene expression,gene silencing and silencing rescue in plants.PLoSOne.2014;9(2):e88218.Published 2014Feb 13.doi:10.1371 / journal.pone.0088218

[0140] Garamella J,Garenne D,Noireaux V.TXTL-based approach to syntheticcells.Methods Enzymol.2019;617:217-239.doi:10.1016 / bs.mie.2018.12.015

[0141] N,Marr C,Swain PS.A geometric analysis of fast-slow models forstochastic gene expression.J Math Biol.2016;72(1-2):87-122.doi:10.1007 / s00285-015-0876-1

[0142] Ow DW,DE Wet JR,Helinski DR,Howell SH,Wood KV,Deluca M.Transient andstable expression of the firefly luciferase gene in plant cells andtransgenic plants.Science.1986;234(4778):856-859.doi:10.1126 / science.234.4778.856

[0143] Suzuki K,Kimura T,Shinoda H,et al.Five colour variants of brightluminescent protein for real-time multicolour bioimaging.Nat Commun.2016;7:13718.Published 2016Dec 14.doi:10.1038 / ncomms13718

[0144] Furuhata Y,Sakai A,Murakami T,Nagasaki A,Kato Y.Bioluminescentimaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescencereporter system.PLoS One.2020;15(1):e0227477.Published 2020Jan 3.doi:10.1371 / journal.pone.0227477

[0145] Apel K,Hirt H.Reactive oxygen species:metabolism,oxidative stress,andsignal transduction.Annu Rev Plant Biol.2004;55:373-399.doi:10.1146 / annurev.arplant.55.031903.141701

[0146] Zipfel C,Robatzek S,Navarro L,et al.Bacterial disease resistance inArabidopsis through flagellin perception.Nature.2004;428(6984):764-767.doi:10.1038 / nature02485

[0147] Chang M,Chen H,Liu F,Fu ZQ.PTI and ETI:convergent pathways withdiverse elicitors.Trends Plant Sci.2022;27(2):113-115.doi:10.1016 / j.tplants.2021.11.013

[0148] Shen R,Liu P,Zhang Y,et al.Sensitive Detection of Single-CellSecreted H2O2 by Integrating a MicrofluidicDroplet Sensor and AuNanoclusters.Anal Chem.2018;90(7):4478-4484.doi:10.1021 / acs.analchem.7b04798

[0149] Zhou T,Xu W,Hirani AH,Liu Z,Tuan PA,Ayele BT,Daayf F,McVetty PBE,Duncan RW,Li G.TranscriptionalInsight Into Brassica napus Resistance GenesLepR3 and Rlm2-Mediated Defense Response Against theLeptosphaeria maculansInfection.Front Plant Sci.2019 Jul 2;10:823.doi:10.3389 / fpls.2019.00823.PMID:31333690;PMCID:PMC6615431.

[0150] Becker MG,Zhang X,Walker PL,Wan JC,Millar JL,Khan D,Granger MJ,CaversJD,Chan AC,Fernando DWG,Belmonte MF.Transcriptome analysis of the Brassicanapus-Leptosphaeria maculans pathosystem identifies receptor,signaling andstructural genes underlying plant resistance.Plant J.2017 May;90(3):573-586.doi:10.1111 / tpj.13514.PMID:28222234.

[0151] Bargmann BO,Birnbaum KD.Fluorescence activated cell sorting of plantprotoplasts.J Vis Exp.2010;(36):1673.Published 2010 Feb 18.doi:10.3791 / 1673

[0152] Galbraith DW,Sun G.Flow Cytometry and Sorting in Arabidopsis.MethodsMol Biol.2021;2200:255-294.doi:10.1007 / 978-1-0716-0880-7_12

[0153] Yu Z,Boehm CR,Hibberd JM,et al.Droplet-based microfluidic analysisand screening of single plant cells.PLoSOne.2018;13(5):e0196810.Published2018 May 3.doi:10.1371 / journal.pone.0196810

[0154] Qian Y,Li J,Zhao S,et al.Programmable RNA sensing for cell monitoringand manipulation.Nature.2022;610(7933):713-721.doi:10.1038 / s41586-022-05280-1

[0155] Kaseniit KE,Katz N,Kolber NS,et al.Modular,programmable RNA sensingusing ADAR editing in living cells.NatBiotechnol.2022;10.1038 / s41587-022-01493-x.doi:10.1038 / s41587-022-01493-x

[0156] Bürger M,Chory J.Stressed Out About Hormones:How Plants OrchestrateImmunity.Cell Host Microbe.2019;26(2):163-172.doi:10.1016 / j.chom.2019.07.006

[0157] Kostyuk AI,Panova AS,Kokova AD,Kotova DA,Maltsev DI,Podgorny OV,Belousov VV,Bilan DS.In VivoImaging with Genetically Encoded RedoxBiosensors.Int J Mol Sci.2020 Oct 31;21(21):8164.doi:10.3390 / ijms21218164.PMID:33142884;PMCID:PMC7662651.

[0158] Levak V,Lukan T,Gruden K,Coll A.Biosensors:A Sneak Peek into PlantCell's Immunity.Life(Basel).2021 Mar7;11(3):209.doi:10.3390 / life11030209.PMID:33800034;PMCID:PMC7999283.

[0159] Fitt BDL, Brun H, Barbetti MJ, Rimmer SR. 2006. World-wide importance of phoma stem canker (Leptosphaeria maculans and L. biglobosa) on oilseed rape (Brassica napus). Eur. J. Plant Pathol. 114:3–15

[0160] Borhan, M Hossein et al. "Molecular Interactions Between Leptosphaeriamaculans and Brassica Species." Annual review of phytopathology vol.60(2022):237-257.doi:10.1146 / annurev-phyto-021621-120602

[0161] Ma, Lisong et al. "Leptosphaeria maculans Effector Protein AvrLm1Modulates Plant Immunity by EnhancingMAP Kinase 9 Phosphorylation." iSciencevol.3(2018):177-191.doi:10.1016 / j.isci.2018.04.015

[0162] van Esse, H Peter et al. "The Cladosporium fulvum virulence proteinAvr2 inhibits host proteases required for basal defense." The Plant cellvol.20,7(2008):1948-63.doi:10.1105 / tpc.108.059394

[0163] Sequence Listing

[0164] The sequence listing contained in the file named "221151WO01 SEQUENCE LISTING.xml" is 101 kilobytes in size (as in Microsoft The invention relates to a novel nucleic acid sequence of the present invention, comprising 49 sequences (SEQ ID NO: 1 to SEQ ID NO: 49), which is submitted electronically with the application and is incorporated herein by reference.

[0165] Throughout the specification and examples, reference is made to the following sequence:

[0166] SEQ ID NO: 1: eGFP coding sequence

[0167] SEQ ID NO: 2: Constitutive cauliflower mosaic virus 35S promoter

[0168] SEQ ID NO: 3: RGA4 coding sequence

[0169] SEQ ID NO: 4: Constitutive maize ubiquitin 1 promoter

[0170] SEQ ID NO: 5: RGA5 coding sequence

[0171] SEQ ID NO: 6: Constitutive Arabidopsis ubiquitin 10 promoter

[0172] SEQ ID NO: 7: Null plasmid

[0173] SEQ ID NO: 8: PM3A coding sequence

[0174] SEQ ID NO: 9: Avr-PM3A coding sequence

[0175] SEQ ID NO: 10: PM3A-R1334E coding sequence

[0176] SEQ ID NO: 11: PM3A-S1335R coding sequence

[0177] SEQ ID NO: 12: Avr-PM3A-Q112N coding sequence

[0178] SEQ ID NO: 13: PM3A-D502V coding sequence

[0179] SEQ ID NO: 14: deGFP_1 coding sequence

[0180] SEQ ID NO: 15: deGFP_2 coding sequence

[0181] SEQ ID NO: 16: deGFP_3 coding sequence

[0182] SEQ ID NO: 17: RGA4_FW1 primer sequence

[0183] SEQ ID NO: 18: RGA4_RV1 primer sequence

[0184] SEQ ID NO: 19: RGA4_FW2 primer sequence

[0185] SEQ ID NO: 20: RGA4_RV2 primer sequence

[0186] SEQ ID NO: 21: PM3A_FW1 primer sequence

[0187] SEQ ID NO: 22: PM3A_RV1 primer sequence

[0188] SEQ ID NO: 23: Green enhanced nanolight (GeNL) coding sequence

[0189] SEQ ID NO: 24: Firefly luciferase (fLUC) coding sequence

[0190] SEQ ID NO: 25: Flg22 amino acid sequence

[0191] SEQ ID NO: 26: eGFP protein sequence

[0192] SEQ ID NO: 27: RGA4 protein sequence

[0193] SEQ ID NO: 28: RGA5 protein sequence

[0194] SEQ ID NO: 29: PM3A protein sequence

[0195] SEQ ID NO: 30: Avr-PM3A protein sequence

[0196] SEQ ID NO: 31: PM3A-R1334E protein sequence

[0197] SEQ ID NO: 32: PM3A-S1335R protein sequence

[0198] SEQ ID NO: 33: Avr-PM3A-Q112N protein sequence

[0199] SEQ ID NO: 34: PM3A-D502V protein sequence

[0200] SEQ ID NO:35: deGFP_1 protein sequence

[0201] SEQ ID NO:36: deGFP_2 protein sequence

[0202] SEQ ID NO:37: deGFP_3 protein sequence

[0203] SEQ ID NO: 38: Green Enhanced Nano-Lamp (GeNL) protein sequence

[0204] SEQ ID NO: 39: Firefly luciferase (fLUC) protein sequence

[0205] SEQ ID NO:40: LepR3 coding sequence

[0206] SEQ ID NO:41: Rlm3 coding sequence

[0207] SEQ ID NO:42: Rlm9 coding sequence

[0208] SEQ ID NO:43: AvrLm1 (WT) coding sequence

[0209] SEQ ID NO:44: AvrLm3 (WT) coding sequence

[0210] SEQ ID NO:45: AvrLm5-9 (WT) coding sequence

[0211] SEQ ID NO:46: PR1a signal peptide coding sequence

[0212] SEQ ID NO:47: AvrLm1* (AvrLm1 with PR1a signal peptide) coding sequence

[0213] SEQ ID NO:48: AvrLm3* (AvrLm3 with PR1a signal peptide) coding sequence

[0214] SEQ ID NO:49: AvrLm5-9* (AvrLm5-9 with PR1a signal peptide) coding sequence

[0215] Examples

[0216] The present invention will now be described with reference to the following examples, which are given by way of illustration only. The following examples are not intended to fully define or otherwise limit the scope of the present invention.

[0217] DNA Manipulations: Unless otherwise indicated, recombinant DNA techniques were performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) or in Ausubel et al., (1994), Current Protocols in Molecular Biology, Current Protocols, Volumes 1 and 2. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993), by RDD Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).

[0218] Example 1: General method

[0219] Cloning methods and plasmid construction

[0220] Unless otherwise indicated, the cloning procedures carried out for the purposes of the present invention, including restriction digestion, agarose gel electrophoresis, purification and connection of nucleic acids, transformation, selection and cultivation of bacterial cells, were performed as described in (Sambrook J, Fritsch EF and Maniatis T (1989)). Sequence analysis of recombinant DNA was performed using Sanger technology (Sanger et al., 1977) by LGC Genomics (Berlin, Germany). Restriction endonucleases and Gibson assembly reagents for constructing plasmids were from New England Biolabs (Ipswich, Massachusetts, USA). Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, Iowa, USA). Codon-optimized genes were from Genewiz, South Plainfield, New Jersey, USA.

[0221] All plasmids were transformed into E. coli for propagation and isolated using the ZymoPure II Plasmid Gigaprep Kit for DNA purification (Zymo Research, Irvine, CA, USA).

[0222] Wheat protoplast preparation and transfection

[0223] Transformation of wheat protoplasts was performed as described by Shan et al. (2014) with minor modifications. Protoplasts were isolated from the youngest, fully expanded leaves of 10-day-old aseptically grown wheat seedlings. Healthy leaves were bundled into five piles and cut into thin strips with a sharp razor blade. The strips were infiltrated with a cell wall lytic enzyme solution (1.5% cellulase R10 and 0.75% maize enzyme R10 in 10 mM KCl and 0.6 M mannitol, pH 7.5) and incubated overnight at 24 ° C in the dark with gentle shaking (40 rpm). After enzymatic digestion, the released protoplasts were collected by filtering the mixture through a 40 μm nylon mesh and resuspended in W5 solution. The resuspended protoplasts were kept on ice and allowed to settle by gravity, and the cell pellet was then resuspended in MMG. For transformation, 200 μl of cells (2.5×10 5 ) were mixed with 20 μg of plasmid DNA and 220 μl of freshly prepared polyethylene glycol (PEG) solution. The mixture was incubated in the dark for 15-20 min. After removing the PEG solution, the protoplasts were resuspended in 2 ml of W5 solution, transferred to a six-well plate, and incubated at 24°C.

[0224] Rice protoplast preparation and transfection

[0225] Transformation of rice protoplasts was performed as described by Wang et al. (2014) with minor modifications. Protoplasts were prepared from the sheaths of 3-week-old sterile-grown rice seedlings. Healthy stems and sheaths were bundled into 20 piles and cut into thin strips with a sharp razor blade. The strips were then infiltrated with a cell wall lytic enzyme solution (1.5% cellulase R10 and 0.75% maize maize R10 in 10 mM KCl and 0.6 M mannitol, pH 7.5) and incubated overnight at 24°C in the dark with gentle shaking (40 rpm). After enzymatic digestion, the released protoplasts were collected by filtering the mixture through a 40 μm nylon mesh and resuspended in W5 solution. The resuspended protoplasts were washed with W5 solution, and the cell pellet was then suspended in MMG solution at a density of 2.5 million cells / ml. For transformation, 200 μl of cells (5×10 5) was mixed with 20 μg of plasmid DNA and 220 μl of freshly prepared polyethylene glycol (PEG) solution. The mixture was incubated in the dark for 15-20 min. After removing the PEG solution, the protoplasts were resuspended in 2 ml of WI solution, transferred to a six-well plate, and incubated at 24°C for at least 48 h. Finally, the protoplasts were collected by centrifugation at 12,000 rpm for 1 min at room temperature, and the precipitated fractions were stored at -80°C until further analysis.

[0226] Rapeseed protoplast preparation and transfection

[0227] Rapeseed protoplasts were isolated from leaves of 4-7 week old sterile grown plants and transfected essentially as described for wheat cells.After removal of the PEG solution, the transfected cells were resuspended in 2 ml of W5 solution and incubated at 24°C.

[0228] Soybean protoplast isolation and transfection

[0229] Protoplasts were isolated from the single leaves of 6-day-old seedlings. Healthy leaves were cut into thin strips with a sharp razor blade and transferred to a culture dish. The thin strips were vacuum infiltrated with a cell wall lytic enzyme solution (containing 0.25% cellulase R10 and 0.25% macerate R10) and incubated overnight at 24°C in the dark with gentle shaking (40 rpm). After enzymatic digestion, the released protoplasts were collected by filtering the mixture through a 40 μm nylon mesh and resuspended in a W5 solution. The resuspended protoplasts were washed with a W5 solution and the cell pellet was then resuspended in an MMG solution. For transformation, 200 μl cells (4 × 10 5 ) were mixed with 20 μg plasmid DNA and 220 μl freshly prepared PEG solution. The mixture was incubated in the dark for 13-15 min. After removing the PEG solution, the protoplasts were resuspended in a 2 ml WI solution, transferred to a six-well plate, and incubated at 24°C.

[0230] Example 2: Development of a single-cell screening workflow using fluorescent reporter genes

[0231] The corresponding plant resistance genes perceive the effector proteins secreted by pathogens, most often triggering the activation of the so-called hypersensitive response (HR). HR is characterized by rapid cell death at the infection site. HR is a complex multicellular process associated with many physical, physiological and molecular changes, including the deposition of lignin and callose in plant cell walls, and the production of antimicrobial compounds (such as phytoalexins, hydrolases and pathogenesis-related proteins). Since the generation of these immune responses requires a lot of cellular energy, the attenuation of the activity of the co-transformed luciferase reporter gene can be used as a surrogate for HR activation (Cesari et al., 2014; Saur et al., 2021). However, this method relies on the consumption of a large number of cell populations and is characterized by high variability in luciferase measurements between biological replicates (Saur et al., 2021), which makes this method unusable for characterization and screening of single protoplasts.

[0232] To determine whether cells expressing HR could also be identified based on expression of a fluorescent reporter protein, a plasmid containing enhanced GFP (eGFP, SEQ ID NO: 1) driven by the strong constitutive 35S promoter of cauliflower mosaic virus (p35S, SEQ ID NO: 2) was generated. A second construct carrying the rice self-activating immune sensor RGA4 (OsRGA4, SEQ ID NO: 3) under the control of the constitutive maize ubiquitin 1 promoter (pZmUbil, SEQ ID NO: 4) was used to activate HR. The disease resistance gene RGA4 encodes a nucleotide-binding leucine-rich repeat (NBS-LRR) domain protein that mediates resistance to the fungal pathogen Magnaporthe oryzae, the causal agent of rice blast. Previous protoplast studies have shown that constitutive RGA4 expression triggers effector-independent HR, while the presence of a second NBS-LRR, RGA5, results in repression (Cesari et al., 2014; OsRGA5, SEQ ID NO: 5). eGFP and RGA4 constructs were mixed in equal ratios and transformed into protoplasts isolated from a blast-susceptible rice variety lacking RGA5. Co-transfection of eGFP and a plasmid lacking RGA4 served as an empty vector control.

[0233] HR induction was monitored by measuring GFP fluorescence intensity 48 h after transformation ( Figure 1 GFP fluorescence was excited at 405 ± 20 nm and collected at 485 ± 9 nm. Compared with the empty vector control, the expression of OsRGA4 resulted in a more than 10-fold decrease in GFP fluorescence intensity, indicating that HR was activated ( Figure 1Similarly, in soybean and rapeseed protoplasts, RGA4 under the control of the constitutive Arabidopsis ubiquitin 10 promoter (pAtUbil0, SEQ ID NO: 6) also significantly reduced GFP fluorescence relative to the empty vector control, demonstrating the effectiveness of this reporter gene assay in homologous as well as heterologous expression systems (see Figure 1 B and Figure 1 C).

[0234] Next, the screening workflow according to an embodiment of the present invention using a GFP reporter gene assay was further validated using the wheat PM3a immune receptor (SEQ ID NO: 8) and its cognate effector Avr-PM3A. The disease resistance gene PM3a encodes a well-studied NBS-LRR that confers resistance to the wheat powdery mildew pathogen Blumeria graminis. Over the past few years, single residue changes in Avr-PM3A have been identified that are sufficient to eliminate, enhance, or alter the intensity of HR in whole plant assays (McNally et al., 2018). Similarly, it is known that polymorphic residues in PM3A determine the intensity and spectrum of HR when transiently expressed in Nicotiana benthamiana using Agrobacterium infiltration (Lindner et al., 2020).

[0235] In order to test whether GFP reporter gene can be used to measure the whole plant experiment in the cell-based method according to an embodiment of the present invention, the protoplasts lacking PM3A isolated from wheat cultivar Fielder were co-transfected with cDNAs having different Avr-PM3A and PM3A alleles. A reference sample for providing GFP expression readings in the absence of effectors and R genes was also included. This reference sample consisted of a GFP reporter gene and a "null" plasmid (SEQ ID NO:7). In the test sample, the null plasmid construct was replaced by a plasmid encoding the PM3A and Avr-PM3A cDNA of interest. Co-transfection of PM3A or Avr-PM3A with GFP and a null plasmid was used as a negative control. The concentration of the null plasmid was adjusted to ensure that the amount of the total plasmid DNA in all transfections was equal.

[0236] like Figure 2As shown, compared with the reference sample ("GFPCtrl"), co-transfection of wild-type PM3A (SEQ ID NO: 8) and Avr-PM3A (SEQ ID NO: 9) triggered a significant decrease in GFP signal, which proves that PM3A effectively recognizes Avr-PM3A. Interestingly, the combined expression of Avr-PM3a and the inactive R1334E allele of PM3A (SEQ ID NO: 10) did not trigger HR, while the expression of Avr-PM3A and PM3A gain-of-function S1335R (SEQ ID NO: 11) alleles showed that GFP decreased more significantly compared to wild-type PM3A. Replacing Avr-PM3A (McNally et al., 2018) with Q112N (SEQ ID NO: 12), a variant that significantly enhances HR triggered by PM3a recognition in intact leaves, further reduced GFP reporter gene activity. However, the most significant reduction in GFP reporter gene activity was observed after co-expression of the PM3A-S1335R gain-of-function variant and Avr-PM3A-Q112N, confirming earlier findings in Nicotiana benthamiana (Lindner et al., 2020; McNally et al., 2018). Importantly, with the exception of the self-activating PM3A-D502V mutant (SEQ ID NO: 13), which carries a mutation in the C-terminal region of the ARC2 domain (Lindner et al., 2020), neither Avr-PM3A nor the PM3A allele significantly reduced GFP activity compared to the empty vector control when expressed alone. Combined with the above analysis of RGA4 in rice, soybean, and canola protoplasts, these results clearly demonstrate that quantification of regulated expression of co-transfected GFP reporters can be used to identify the occurrence of HR in miniaturized cell-based assays while predicting immune activation in whole plants.

[0237] Example 3: Effect of GFP protein stability on assay sensitivity

[0238] With a half-life of more than 24 hours, eGFP is a highly stable protein, which allows it to accumulate and be easily detected in cells. However, this stability may also limit its application in single-cell studies, because after extended periods of time, even rapid and complete transcriptional blockade can only be detected at the level of reporter gene activity. In order to evaluate the impact of GFP stability on the performance of fluorescent reporter gene assays according to embodiments of the present invention, different eGFP variants with enhanced turnover rates were tested (see Figure 3). These variants include an eGFP protein fusion carrying the 164AA destabilization domain of the putative barley 1-aminocyclopropane-1-carboxylic acid synthase (Dong et al., 2006; "deGFP_2", SEQ ID No: 15, SEQ ID NO: 36), an unstable GFP variant reportedly carrying L64F and L231H mutations (Binder et al., 2014; "deGFP_3", SEQ ID NO: 16, SEQ ID NO: 37), and a truncated eGFP variant with an estimated half-life of less than 1 h in bacteria (Garamella et al., 2019; "deGFP_1", SEQ ID NO: 14, SEQ ID NO: 35). The destabilized eGFP variants were codon-optimized for expression in plants and transcribed from a constitutive 35S promoter. Each of the GFP variants was transfected into rice protoplasts along with a construct encoding RGA4 or an empty vector control, and GFP reporter gene activity was measured 48 h after transformation. Although expression of RGA4 resulted in a 12-fold decrease in eGFP activity compared to the empty vector control, the expression of the truncated eGFP variant ( Figure 3 The decrease in activity observed with deGFP_1 in the GFP reporter assay was even more pronounced (up to 17-fold). In contrast, expression of the other GFP variants resulted in a weaker reporter response relative to that observed with native eGFP (3-fold decrease in GFP activity for deGFP_2 and 7-fold decrease in GFP activity for deGFP_3). These findings demonstrate that destabilized protein variants can be used to optimize the sensitivity of methods according to embodiments of the present invention when using a GFP reporter assay.

[0239] Example 4: Validation of a single-cell screening workflow using fluorescent reporters

[0240] In methods for characterizing and screening single plant protoplasts according to embodiments of the present invention, the prospect of utilizing fluorescent reporter gene assays is highly attractive. While high-throughput screening of whole plants is fundamentally limited by their slow growth and size, various cell sorting and partitioning techniques can process millions of protoplasts in a matter of hours. However, to date, protoplasts have primarily been extracted and analyzed in batches, which has limited their use.

[0241] To evaluate the use of GFP as a selectable marker at the single-cell level according to examples of the present invention, soybean protoplasts were transfected with 35S-driven eGFP in combination with the autoactivating RGA4 at a ratio of 10:1. Under our experimental conditions, transfection efficiency in soybean protoplasts averaged 30%-40%. To remove untransfected cells from downstream analysis, a two-step sorting method was used. First, samples were sorted one day after transfection to enrich for transfected cells. The collected cells were then incubated for an additional 48 hours and then sorted based on GFP fluorescence levels.

[0242] The protoplasts were transferred to a microfluidic cartridge and loaded into the Hana single-cell dispenser device (Namocell). This device combines microfluidics, flow cytometry, and liquid dispensing to sort and dispense single cells directly into 96-well or 384-well plates. Much like fluorescence-activated cell sorting (FACS) devices, the Hana dispenser is capable of binning by forward and side scatter, but with significantly lower sorting pressure (<2 psi) to help maintain cell viability.

[0243] Data acquisition gates were set for live protoplasts based on forward and side scatter characteristics, and appropriate laser excitation and collection channels were selected. To detect and isolate cells expressing GFP, the sample was excited with a 488 nm laser and emission was quantified at 533 nm (FITC / GFP detection channel). To quantify autofluorescence, a PE / PI detection channel was used (excitation at 488 nm, collection at 585 nm). A scatter plot of FITC versus PE was generated with a forward scatter threshold of 100. Protoplasts transfected with an empty vector plasmid were first analyzed using WI buffer as the sheath fluid, which enabled accurate setting of the collection gate for GFP-positive protoplasts. Next, a sample containing GFP-transfected cells was run. A new protoplast population was visible in the scatter plot as a group of cells with a higher FITC / PE ratio compared to the negative control. A collection gate was drawn around this population, and approximately 2,000 GFP-positive cells were distributed in a 6-well plate and incubated at 24°C. Two days later, the collected cells were reloaded onto the Hana sorting device.

[0244] Two fluorescently enriched populations corresponding to 5.7% and 19.2% of the total population, i.e., protoplasts with low GFP intensity (20 < FITC < 500) and with high intensity (FITC > 3,000), were selected for sorting. After sorting, individual protoplasts were dispensed into 96-well plates containing 10 μl of 2x Phire Tissue Direct PCR Extraction Buffer (ThermoFisher Scientific). The presence / absence of the RGA4 plasmid in each collected cell was then verified by nested PCR using primers RGA4_FW1 (SEQ ID NO:17) and RGA4_RV1 (SEQ ID NO:18). Each reaction was run at a final volume of 20 μl and contained the following reagents: 10 μl of 2x Phire Plant Reaction Buffer, 1 μl of 10 μM forward primer, 1 μl of 10 μM reverse primer, 0.4 μl of Phire polymerase, 1 μl of single cell lysate (template), and 6.6 μl of nuclease-free water. The PCR program was: 1 cycle at 98 °C for 5 min; 40 cycles of 5 s at 98 °C, 5 s at 60 °C, and 25 s at 72 °C; and 1 cycle at 72 °C for 1 min. Then, one microliter of the PCR reaction was used as a template in a secondary PCR containing the following reagents: 12.5 μl of Q5 High-Fidelity Master Mix (NEB), 1.25 μl of 10 μM forward primer RGA4_FW2 (SEQ ID NO:19), 1.25 μl of 10 μM reverse primer RGA4_RV2 (SEQ ID NO:20), and 9 μl of nuclease-free water. The PCR program was: 1 cycle at 98 °C for 2 min; 30 cycles of 10 s at 98 °C, 30 s at 65 °C, and 32 s at 72 °C; and 1 cycle at 72 °C for 2 min. The resulting PCR products were analyzed on a 1% agarose gel stained with SybrSafe and visualized by UV transillumination, or loaded onto a Fragment Analyzer capillary electrophoresis system.

[0245] As Figure 4 shown in Figure 4 A, gating on GFP intensity successfully separated protoplasts expressing RGA4 from non-expressing cells: none of the 60 cells with high GFP intensity showed RGA4 amplification compared to 12 out of 25 cells with low GFP intensity showing RGA4 amplification. Repeating the experiment gave similar results, with 44 out of 48 cells with low GFP intensity showing RGA4 positive, and none of the 6 cells with high fluorescence showing RGA4 positive (see B).

[0246] In order to further evaluate the performance of eGFP as a selectable marker according to an embodiment of the present invention at the single cell level, the combination of Avr-PM3A and GFP reporter protein was combined with wild-type PM3A ( Figure 5 , left column, lower panel) or its “loss-of-function” PM3A-R1334E variant ( Figure 5 , left column, upper panel) were co-expressed in wheat protoplasts. Two days after transfection (2 dpt), protoplasts from the two populations were pooled at equal ratios and injected into a Biosorter device (Union Biometrica, Figure 5 The Biosorter is a continuous flow system that operates at low pressure and uses a gentle airflow splitter for sorting, and is capable of analyzing, sorting, and partitioning fragile cells (such as single plant protoplasts) ranging in size from 10 μm to 1,500 μm ( Figure 5 , middle column). In the Biosorter, pooled protoplasts are introduced into a flow cell (1) where they are surrounded by a sheath solution that is hydrodynamically focused (sheath flow 2) to the center of the flow for interrogation by multiple lasers (3). W5 buffer is used as the sheath fluid. The Biosorter further simultaneously records the extinction intensity of each protoplast, the forward scatter intensity (4) that allows for optical density and size detection, and the fluorescence intensity (5) along the length. The device also allows for brightfield image capture (camera (6), brightfield illumination (7)) of each individual protoplast sample before it leaves the flow channel (1).

[0247] Cells with high, medium, and low GFP intensities were gated separately, and single positive cells were selectively sorted and distributed into 96-well plates filled with 5 μl of 2xPhire Tissue Direct PCR Extraction Buffer (Thermo Fisher Scientific). The Biosorter uses a gentle airflow to assist sorting (8), which allows sorted (9) single protoplasts with desired characteristics to be collected in multiwell plates or large-capacity containers (10). Unsorted (11) cells are collected by the system in a waste / sample recovery container (12).

[0248] To test whether the fluorescence intensity of sorted cells could be used as a surrogate for HR induced after PM3A recognition, single-cell PCR was performed on sorted cells ( Figure 5, right column). Each reaction was run in a total volume of 21 μl and contained the following reagents: 10 μl of 2x Q5 High Fidelity Master Mix Buffer, 0.1 μl of 100 μM forward primer PM3A_FW1 (SEQ ID NO: 21), 0.1 μl of 100 μM reverse primer PM3A_RV1 (SEQ ID NO: 22), 2 μl of 10-fold diluted single cell lysate (template), and 8.8 μl of nuclease-free water. The PCR program was: 1 cycle at 98°C for 2 min; 40 cycles of 98°C for 10 s, 65°C for 20 s, and 72°C for 20 s; and 1 cycle at 72°C for 2 min. Next, the PCR products were purified and sequenced using Sanger technology to reveal the transfected PM3A allele (i.e., wild type or "loss of function"). The results are shown in Table 1. An overview of the experimental setup is shown in Figure 5 middle.

[0249] Table 1: Correlation between PM3A alleles and GFP fluorescence levels in selected protoplasts

[0250]

[0251] Although mixed sequencing peaks indicative of cell doublets were observed in some reactions, the results showed a close correlation between GFP intensity in individual cells and the strength of the PM3A-induced immune response. Indeed, while the majority of cells with high GFP intensity contained the loss-of-function PM3A-R1334E allele, and up to 90% of weakly fluorescent cells carried wild-type PM3A, the population gated for medium GFP contained a mixture of PM3A- and PM3A-R1334E-transfected cells. Combined with results obtained in rice protoplasts, these findings demonstrate the applicability of GFP as a selectable marker for automated characterization and sorting of individual protoplasts, as described in the present invention, thereby facilitating high-throughput screening and retrieval of HR-expressing cells based on expression of a fluorescent reporter gene. Further development work is underway to develop an integrated workflow that will allow rapid screening of large genetic libraries in protoplasts isolated from different crops of interest.

[0252] Example 5: Optimization of single-cell screening assays via time-resolved single-cell measurements

[0253] Gene expression is an intrinsically stochastic process that manifests as fluctuations in the abundance of expressed molecules at the single-cell level, as well as variability and heterogeneity within genetically identical cell populations. Fluctuations in the biochemistry of gene expression (intrinsic noise) and fluctuations in other cellular components (extrinsic noise) essentially contribute to the overall cell-to-cell variability (Popovic et al., 2016).

[0254] To account for this variation and normalize the differences in transfection efficiency of individual cells, time-resolved GFP reporter gene measurements were attempted using the CellCelector single-cell isolation platform (Automated Lab Solutions), which monitors individual cells over time by physically trapping them in a nanopore array. To this end, a combination of Avr-PM3A and GFP reporter protein was co-expressed in wheat protoplasts (cultivar Fielder) along with wild-type PM3A or its “loss-of-function” R1334E variant. Immediately after transfection, protoplasts from the two populations were merged in equal ratios and seeded onto six-well plates with approximately 60,000 nanopores per well. One milliliter of cell suspension containing 60,000 cells was slowly dispensed into one well of the six-well plate to achieve an average occupancy of one cell per nanopore. The cells were then allowed to settle for 5 min at room temperature and centrifuged at 300 × g for 5 min. Single nanopores were scanned in bright field and fluorescence (GFP and mCherry channels) every two hours. At least 50 cells with unchanged or greatly reduced GFP expression 12 to 48 hours after transfection were identified and selected for picking using a liquid-buffered glass capillary. The picked colonies were then transferred to a 96-well plate and subjected to single-cell PCR and Sanger sequencing as described above.

[0255] Example 6: Development of a single-cell screening workflow using luminescent screenable markers

[0256] According to the donor material used, the isolated protoplasts sometimes show strong chlorophyll-derived autofluorescence, which may hinder accurate fluorescence detection. In view of the fact that luciferase can emit light without the need for external light-induced excitation, the luminescent reporter gene system is used to provide an attractive alternative for the determination based on fluorescence. However, conventional luciferase genes (including firefly, sea renilla and sea firefly (Vargula) luciferase) produce relatively weak light emission, and therefore, these luciferase genes are doubtful in the application of single cell luminescence imaging. In fact, Ow et al., (1986) reported that tobacco cells transfected with firefly luciferase need to be exposed for more than 24h to detect luminescence. Therefore, the implementation of the single cell reporter gene assay based on luminescence will need to use a brighter and more transmittance improved luciferase reporter gene.

[0257] A recently engineered class of luciferases is Nanolantern, which is a chimera of Nanolantern (one of the brightest luciferases) and fluorescent proteins of different colors. Using bioluminescence resonance energy transfer (BRET) to shift the emission wavelength of the luminescent reporter, Nanolantern is bright enough to be detected as a single molecule and tracked as a fluorescent protein (Suzuki et al., 2016; Furuhata et al., 2020).

[0258] To assess the feasibility of luminescence imaging with single-cell resolution, rice protoplasts were transiently transfected with a construct encoding green enhanced nano-lantern (GeNL, SEQ ID NO: 23), a fusion of nano-luciferase and mNeonGreen protein as a BRET receptor (Suzuki et al., 2016). GeNL driven by the maize ubiquitin 1 promoter (SEQ ID NO: 4) was co-transfected with RGA4 (SEQ ID NO: 3) or an empty vector control, and its luminescence was compared with that of a firefly luciferase reporter gene (fLUC, SEQ ID NO: 24) expressed from the 35S promoter (SEQ ID NO: 2). To detect GeNL activity, 100 μl of protoplast culture was transferred to a white 96-well plate, to which 10 μl of Nano-Glo luciferase assay reagent (Promega; containing furazolidinone substrate) was added, and fLuc activity was detected by adding 10 μl of Dual-Glo luciferase reagent (Promega; containing D-luciferin). Luminescence was measured 3 min and 10 min after adding Nano-Glo and Dual-Glo substrates, respectively, using a Tecan microplate reader equipped with a luminescence detection unit. In order to evaluate the wavelength-independent luciferase-generated reporter gene signal, luminescence was measured without applying an optical filter.

[0259] like Figure 6As shown, in an undiluted protoplast sample containing approximately 50,000 cells, GeNL luminescence was significantly higher than fLuc luminescence (GeNL / fLUC=2.29). Interestingly, the difference in luminescence intensity between GeNL and fLUC became more pronounced when the sample was diluted (GeNL / fLUC=59.2 for a 10-fold dilution, GeNL / fLUC=54.3 for a 200-fold dilution, and GeNL / fLUC=49.36 for a 400-fold dilution). Even at the highest dilution (corresponding to approximately 125 cells), GeNL-transfected cells emitted a bright luminescence signal that was almost 50 times that of fLUC, while co-expression of RGA4 significantly inhibited GeNL luminescence. To further test the performance of GeNL-based reporter gene assays at the single-cell level, rice cells transiently expressing fLUC or GeNL and RGA4 were subsequently injected into the Hana Single Cell Dispenser device, gated based on their autofluorescence levels, and dispensed into white 96-well plates filled with 49 μl of WI buffer and 5 μl of Nano-Glo or 50 μl of Dual-Glo substrate. The results are shown in Figure 7 Unlike GeNL, which exhibited strong reporter activity in almost all sorted cells, fLUC-derived luminescence appeared too low to be reliably detected at the single-cell level, with only 3 of 42 cells transfected with the fLUC construct showing luminescence values above those in the substrate control (RLU>20). Furthermore, consistent with data from bulk samples, expression of RGA4 severely attenuated GeNL luminescence in single cells, further suggesting that GeNL reporter activity can be used as a surrogate for HR.

[0260] Next, the performance of GeNL as a selectable marker in a method according to an embodiment of the present invention was evaluated in wheat protoplasts using the PM3a / Avr-PM3A test system described in Example 2 above. The results are shown in Figure 8 In bulk samples, coexpression of wild-type PM3A and Avr-PM3A resulted in a moderate but significant decrease in GeNL luminescence relative to the positive control, whereas no decrease in luminescence was observed with the inactive R1334E allele of PM3A. In contrast, combining Avr-PM3A with the PM3A gain-of-function S1335R allele or replacing Avr-PM3A with its HR-enhancing Q112N variant resulted in a more pronounced decrease in reporter activity compared to that observed with wild-type PM3A. Importantly, when expressed individually, neither Avr-PM3A nor any of the PM3A alleles, with the exception of the autoactivating PM3A D502V mutant, substantially altered GeNL activity.

[0261] The same experimental method was then used to quantify GeNL luminescence in individual wheat protoplasts. Here, one day after transfection, samples transformed with GeNL together with PM3A and Avr-PM3A were loaded onto the Pala sorting device and gated into GeNL expressing cells (FITC>1,000 and PE>10) and non-expressing cells (FITC<1,000 and PE<10). Single positive cells were distributed into white microtiter plates containing 49 μl of W5 buffer and 5 μl of Nano-Glo substrate. As shown in Table 2 and Figure 9 As shown, the results obtained at the single-cell level largely mirrored those observed in bulk samples, where the lowest average GeNL reporter activity was observed in single cells transfected with combinations of PM3A-S1335R (“gain of function”) + Avr-PM3A-Q112N, followed by PM3A + Avr-PM3A-Q112N and PM3A or PM3A-S1335R + Avr-PM3A. Thus, single cells transfected with the PM3A-R1334E “loss of function” variant and Avr-PM3A exhibited almost 2-fold higher luminescence intensity compared to PM3A or PM3A-S1335R. Combined with the findings with RGA4 in rice, these results suggest that quantification of co-transfected GeNL reporter proteins provides a reliable alternative to fluorescence-based screenable markers for single-cell assays according to embodiments of the present invention.

[0262] Table 2: Measurement of luminescence (RLU) in single protoplasts derived from wheat

[0263] R-gene / effector combinations Luminescence (average of 96 single cells, RLU) PM3A and Avr-PM3A 7756 PM3A-S1335R and Avr-PM3A 8361 PM3A-R1334E and Avr-PM3A 15155 PM3A and Avr-PM3A-Q112N 6621 PM3A-S1335R and Avr-PM3A-Q112N 3823 Empty vector control 62

[0264] Much like fluorescence-based reporter gene systems, GeNL assays can be further optimized through real-time kinetic studies to account for variations in single-cell transfection efficiency and stochasticity in reporter gene expression. Live-cell detection of GeNL luminescence over time in single protoplasts is achieved by physically capturing isolated single cells, for example by seeding protoplasts on nano- or microwell arrays or (digital) microfluidic chips, in combination with the use of alternative furazolidone-based substrates such as Endurazine and Vivazine (Promega), both of which enable non-lytic assays over time periods of hours or days. Using this experimental approach, cells of interest can be identified based on reporter gene kinetics and selected for downstream isolation and characterization via single-cell retrieval systems such as the CellCelector platform (Automated Laboratory Solutions).

[0265] Example 7: Development of a single-cell screening workflow based on HR-induced reactive oxygen species (ROS) production

[0266] During the so-called oxidative burst, the rapid generation and accumulation of reactive oxygen species (ROS) (such as hydrogen peroxide and superoxide anion) is one of the hallmarks of effector-triggered immunity. In addition to coordinating HR-like cell death, ROS also has a variety of other immune functions, including local enhancement of cell walls by oxidative cross-linking of structural proteins, and activation of phytoalexin biosynthesis. In addition, ROS can induce an array of cytoprotective agents and defense genes and can act as secondary messengers in inducing systemic acquired resistance (Apel and Hirt, 2004). Considering the large number of defense-related responses regulated by ROS, ROS production constitutes a promising alternative for NLR activation.

[0267] To monitor the accumulation of reactive oxygen species in transfected cells, rapeseed protoplasts expressing the rice autoactivator NLR RGA4 under the control of the Arabidopsis ubiquitin 10 promoter were stained with CellRox Green reagent. This cell-permeable dye is weakly fluorescent in the reduced state but exhibits bright green, photostable fluorescence after oxidation by ROS and subsequent binding to DNA. CellRox Green fluorescence was excited at 405 ± 20 nm and collected at 485 ± 9 nm. Figure 10 As shown, in vivo ROS imaging during the first 24 hours after transfection demonstrated a moderate but sustained 2-fold increase in CellRoxGreen fluorescence in RGA4-expressing cells ("RGA4") compared to an empty vector control ("Ctrl"). While these results demonstrate the potential of CellRox Green staining for detecting immune-related oxidative stress, the overall low signal intensity raises doubts about the applicability of this assay at the single-cell level. Therefore, to attempt to enhance RGA4-induced ROS production, control and RGA4-transfected cells were treated with 1 μM flg22 ("flg22," SEQ ID NO: 25), a 22-amino acid epitope of the prototypical bacterial MAMP elicitor flagellin (Zipfel et al., 2004). Consistent with recent findings showing that immune pathways activated by cell surface and intracellular receptors potentiate each other (reviewed in Chang et al., 2022), flg22 treatment resulted in a significant increase in ROS production in RGA4-expressing cells, greatly improving the sensitivity of the ROS reporter assay.

[0268] Experiments are currently underway to establish ROS-based screening assays with spatiotemporal resolution. Here, single rice protoplasts transiently expressing RGA4 and a nuclear-localized mCherry transfected reporter were stained with CellRox Green, encapsulated in microdroplets or picoliter droplets of water-in-oil emulsions, and sorted on custom microfluidic chips, such as the Cytomine device (Sphere Fluidics). In an alternative approach, immune-related ROS were measured by integrating a droplet microfluidics system with fluorescent HRP-gold nanoclusters, synthesized by assembling gold particles with HRP molecules, as described by Shen et al. (2018).

[0269] Example 8: Optimization of single-cell screening assays via time-resolved single-cell measurements of ROS production

[0270] Single rice protoplasts transiently expressing RGA4 and a nuclear-localized mCherry reporter gene were stained with CellRox Green, encapsulated in droplets of a water-in-oil emulsion, and sorted on a microfluidic chip. In an alternative approach, immune-related ROS were measured by integrating a droplet microfluidics system with fluorescent HRP-gold nanoclusters, which were synthesized by assembling gold particles with HRP molecules, as described by Shen et al. (2018).

[0271] Example 9: Development of single-cell screening assays using genetic markers

[0272] HR induction in plant cells is generally associated with large-scale transcriptional reprogramming, involving the upregulation of a large set of defense-related genes. Identifying HR marker genes (i.e., genes that are significantly activated in response to HR initiation) is crucial for designing single-cell screening workflows based on reporter gene fluorescence enhancement. These reporter gene systems use HR-responsive promoters to drive the expression of fluorescent reporter gene proteins (e.g., GFP). The activity of the GFP reporter gene is then compared with the activity of a constitutively expressed second reporter gene (e.g., mCherry, eBFP2, dsRed, etc.), which is contained in the same box as the HR-responsive promoter. This allows normalization of transfection efficiency differences between cells and can generate ratio images comparing HR activation (GFP) with constitutively expressed reporter gene proteins. An example constitutive promoter that can be used to drive the second reporter gene is the 35S promoter of cauliflower mosaic virus or the ubiquitin (Ubi) promoter of maize or Arabidopsis, depending on the target species of interest. Candidate HR-responsive promoters can be identified by comparing differential gene expression in compatible and incompatible plant-pathogen interactions. Zhou et al. (2019) and Becker et al. (2017) provide examples of comparative defense transcriptome profiling by RNA sequencing.

[0273] Using the dual-reporter gene system described above, high-throughput sorting methods, such as flow-based microfluidics or fluorescence-activated cell sorting (FACS), can be used to gate HR-expressing cells from large single-cell populations. Various groups (Bargmann and Birnbaum, 2010; Galbraith and Sun, 2021) have applied FACS to sorting plant cells, while Yu et al. (2018) reported high-throughput microfluidic analysis and screening of protoplasts at processing rates of >100,000 cells / hour.

[0274] Alternatively, the transcriptional induction of HR marker genes in transfected single cells can also be quantitatively analyzed using recently described RNA sensing technology (Kaseniit et al., 2022; Qian et al., 2022). These molecular RNA sensors utilize RNA editing of adenosine deaminases (ADARs) acting on RNA to gate the translation of protein payloads in the presence of endogenous RNA transcripts. One such sensor, called RADAR (Kaseniit et al., 2022), uses a “sensor mRNA” containing two coding sequences (CDS), a marker (mCherry) and an output (eGFP), separated by a “sensor sequence” containing an in-frame UAG stop codon. The sensor sequence is designed to be reverse complementary to the target RNA of interest, while the stop codon prevents the downstream output eGFP coding sequence from being translated, thereby achieving the expression of only mCherry. However, in the presence of target RNA, a double-stranded RNA (dsRNA) segment is formed around the stop codon, which leads to the recruitment of the ADAR enzyme. The ADAR enzyme converts the stop codon to tryptophan, allowing translation of the downstream CDS, thereby allowing both mCherry and EGFP to be expressed. A "self-cleaving" 2A sequence is introduced to isolate the sensor sequence from the flanking CDS, thereby preventing the peptide encoded in the sensor sequence from inducing degradation, aggregation, or other undesirable side effects.

[0275] Example 10: Optimization of single-cell screening assays via time-resolved single-cell measurements

[0276] Gene expression is an intrinsically stochastic process that manifests as fluctuations in the abundance of expressed molecules at the single-cell level, as well as variability and heterogeneity within genetically identical cell populations. Fluctuations in the biochemistry of gene expression (intrinsic noise) and fluctuations in other cellular components (extrinsic noise) essentially contribute to the overall cell-to-cell variability (Popovic et al., 2016).

[0277] To account for this variation and normalize for differences in transfection efficiency of individual cells, time-resolved GFP reporter gene measurements were performed using the CellCelector single-cell isolation platform (Automated Laboratory Solutions), which enables monitoring of individual cells over time by physically trapping them in a nanopore array. To this end, a GFP reporter gene and a combination of Avr-PM3A or its HR-enhanced Q112N mutant, as well as wild-type PM3A or the "loss-of-function" R1334E variant, were co-expressed in wheat protoplasts (cultivar Fielder). Eight hours after transfection, transfected cells were diluted 20-fold in W5 buffer and seeded onto 24-well plates with approximately 3,000 nanopores per well. One milliliter of cell suspension containing approximately 5,000 cells was slowly dispensed into one well of the 24-well plate. The cells were then allowed to settle for 5 minutes at room temperature and centrifuged at 800×g for 3 minutes. Between 8 and 24 hpt, individual nanopores were scanned periodically in bright field and fluorescence channels (GFP / FITC and Cy5 channels). Single cells were selected based on multiple criteria, including the number of particles per well (1), particle area (500-4000 μm 2 ), sphericity (0.3-1), elongation and average gray value. Figure 11 An overview of the temporal changes in GFP fluorescence across all cells measured is shown. As expected, in cells expressing the GFP reporter alone ("GFP Ctrl," Figure 11 A) or a combination of Avr-PM3A and PM3A-R1334E loss-of-function alleles ( Figure 11 C) The average GFP intensity of single cells in transfected samples showed an increasing trend. In contrast, in cells expressing WT PM3A and Avr-PM3A ( Figure 11 B) or its HR-enhanced variant Avr-PM3A-Q112N ( Figure 11 D) The average GFP fluorescence value remained almost constant during the experiment.

[0278] Interestingly, different GFP expression patterns can be observed at the single-cell level, where the GFP signal increases (feature "A"), decreases (feature "C"), or remains constant (feature "B") throughout the experiment. Furthermore, cells lysed before the end of the measurement result in a complete loss of GFP signal, a response (which we designated as feature "D") that indicates cell death. Figure 12 As shown, both control samples and cells transfected with a PM3A loss-of-function allele predominantly displayed GFP signatures "A" and "B" (accounting for up to 90% of all cells), whereas protoplasts expressing wild-type PM3A were characterized by a high rate of HR-related "C" and "D" type reactions, accounting for more than 50% of all cells.

[0279] like Figure 13 As shown, comparative kinetic analysis revealed clear differences in the frequency of "C"- and "D"-type signatures between cells transfected with the loss-of-function variant PM3A-R1334E and those expressing WT PM3A at all measured time points. Furthermore, the greatest differences in the frequency of "C"- and "D"-type reactions between cells transfected with the loss-of-function variant PM3A-R1334E and those expressing WT PM3A were observed between 8 and 11 hpt. As shown in Table 3, PM3A-transfected samples showed approximately 12-fold and 8-fold increases in the percentage of cells exhibiting loss or reduction in GFP fluorescence, respectively. This detailed understanding of the real-time kinetics of reporter activity is expected to further improve the sensitivity and accuracy of GFP reporter assays by defining the optimal time window for analysis and reducing the number of false positives.

[0280] Table 3: Comparative kinetic analysis of GFP fluorescence in individual wheat protoplasts displaying GFP signature C or GFP signature D

[0281]

[0282]

[0283] Example 11: Detection of extracellular R-Avr interactions using a GFP reporter-based single-cell screening workflow

[0284] Leptosphaeria maculans is the causative agent of blackleg disease (stem canker), causing significant yield losses to Brassica napus crops worldwide (Fitt et al., 2006). During infection, Leptosphaeria maculans remains extracellular and exhibits a range of lifestyles from biotrophic to necrotrophic. To date, more than 20 species-specific blackleg R genes have been reported, all of which encode membrane-bound cell surface-localized receptor proteins for detecting apoplast effectors secreted by the pathogen. To date, cloned R genes encode receptor-like proteins (RLPs) (e.g., LepR3 and Rlm2) or wall-associated kinase-like (WAKL) proteins (including Rlm9, Rlm4, and Rlm7) (Borhan et al., 2022).

[0285] To evaluate the versatility of the methods according to embodiments of the present invention and to test whether the system can also be used to detect extracellular R-Avr interactions, protoplasts were isolated from Westar (a rapeseed variety susceptible to blackleg) and co-transfected with plasmids containing GFP (SEQ ID NO: 1) driven by the strong constitutive 35S promoter of cauliflower mosaic virus (p35S, SEQ ID NO: 2), and different membrane-bound cell surface-localized R genes and their corresponding Avr genes. The R genes RLP LepR3 (SEQ ID NO: 40) or WAKLRlm3 (WO 2023 / 004429 A1; SEQ ID NO: 41) and Rlm9 (SEQ ID NO: 42) were expressed under the control of the Arabidopsis ubiquitin 10 promoter (pAtUbil0, SEQ ID NO: 6). To promote extracellular secretion of the corresponding Avr genes AvrLm1 (SEQ ID NO: 43), AvrLm3 (SEQ ID NO: 44), and AvrLm5-9 (SEQ ID NO: 45), the natural signal peptide of the effector was replaced by the natural signal peptide of the tobacco PR1a protein (SEQ ID NO: 46), which is a motif widely used to secrete pathogen effectors in various plants (van Esse et al., 2006). The resulting Avr genes AvrLm1* (SEQ ID NO: 47), AvrLm3* (SEQ ID NO: 48), and AvrLm5-9* (SEQ ID NO: 49) were expressed behind the strong constitutive 35S promoter of cauliflower mosaic virus (p35S, SEQ ID NO: 2).

[0286] A reference sample consisting of a GFP reporter gene and a "null" plasmid (SEQ ID NO: 7) was included to provide a readout of GFP expression in the absence of the R and Avr genes. Co-transfection of the R or Avr gene with GFP and a null plasmid served as a negative control. The concentration of the null plasmid was adjusted to ensure equal amounts of total plasmid DNA in all transfections.

[0287] GFP reporter gene activity was quantified at 24 hpt and normalized to cell autofluorescence to account for differences in transfection efficiency between samples. Figure 11 As shown, all three R-Avr interactions tested resulted in a significant decrease in GFP fluorescence relative to controls containing only GFP or R genes, indicating HR activation. Furthermore, consistent with previous reports, translocated AvrLm1 was shown to induce host cell death by enhancing the accumulation and phosphorylation of BnMPK9 (Ma et al., 2018), but transient expression of AvrLm1* and AvrLm3* resulted in reduced reporter gene activity compared to controls containing only GFP, effector, or R genes, even in the absence of the corresponding R genes.

[0288] Alternatively, single protoplasts with reduced GFP expression are sorted and analyzed using methods as described herein (e.g., fluorescence activated cell sorting or flow-based microfluidics).

[0289] Together, these findings demonstrate the practicality and effectiveness of the single-cell screening workflow using GFP as a selectable marker in the present examples for both detecting effector-induced cell death and quantifying HR triggered by sensing secreted effectors via membrane-bound cell surface receptors in the Brassica napus-Pseudomonas nigricans lesion system.

Claims

1. A method for high-throughput characterization of one or more candidate plant pathogen effector genes and / or one or more candidate plant disease resistance genes in a single plant cell, the method comprising the following steps: (a) preparing at least one protoplast derived from a plant of interest, optionally wherein the protoplast is capable of expressing one or more selectable marker genes; (b) introducing into the at least one protoplast one or more expression cassettes for transient expression of one or more candidate plant pathogen effector genes, and / or introducing one or more expression cassettes for transient expression of one or more candidate plant disease resistance genes; (c) transiently expressing the one or more candidate plant pathogen effector genes and / or the one or more candidate plant disease resistance genes; (d) measuring the production of reactive oxygen species (ROS) in the at least one protoplast, and / or optionally measuring the expression of the selectable marker gene; (e) isolating one or more individual protoplasts that exhibit modulated production of ROS, modulated expression of the one or more selectable marker genes, or both, as compared to control plant protoplasts lacking a functional candidate pathogen effector gene; (f) identifying the one or more candidate plant pathogen effectors and / or the one or more candidate plant disease resistance genes in the single protoplast isolated in step (e).

2. The method of claim 1, wherein one or more candidate plant pathogen effectors and one or more candidate plant disease resistance genes are introduced into protoplasts derived from a plant susceptible to infection by a pathogen encoding the plant pathogen effector.

3. The method of claim 1, wherein one or more candidate plant pathogen effectors are introduced into protoplasts derived from the corresponding disease-resistant plant.

4. The method of claim 1 , wherein the one or more candidate plant pathogen effectors and the one or more candidate plant disease resistance genes are introduced into protoplasts derived from a plant heterologous to the one or more candidate plant disease resistance genes.

5. The method of claim 1, wherein the selectable marker gene is expressed transiently or stably.

6. The method of claim 5, wherein the selectable marker gene is controlled by a constitutive promoter or a hypersensitive response inducible promoter.

7. A method according to any preceding claim, wherein the selectable marker gene encodes a fluorescent and / or luminescent marker.

8. The method of claim 7, wherein the regulated expression of the fluorescent or luminescent marker is measured in single protoplasts.

9. The method of any preceding claim, wherein at one or more time points, the modulated production of ROS and / or the modulated expression of the one or more selectable marker genes is measured.

10. The method according to any preceding claim, wherein the plant of interest is a crop plant, preferably a cereal, oilseed plant, leguminous plant, vegetable or fiber crop.

11. A method according to any preceding claim, wherein the production of reactive oxygen species (ROS) is enhanced by a hypersensitive response elicitor.

12. A method according to any preceding claim, wherein the generation of reactive oxygen species (ROS) is measured by ROS-induced oxidation of a fluorescent dye.

13. A method according to any preceding claim, wherein the isolation of one or more individual protoplasts comprises the steps of protoplast sorting, partitioning and retrieving the protoplasts of interest.

14. The method of any preceding claim, wherein the one or more candidate plant pathogen effectors and / or the one or more candidate plant disease resistance genes in the single protoplast are identified by sequencing.

15. A method according to any preceding claim, wherein the step of isolating one or more individual protoplasts showing regulated production of ROS and / or regulated expression of said one or more selectable marker genes is followed by a step of clonal propagation.

16. A method according to any preceding claim, further comprising regenerating the isolated protoplasts or clones derived therefrom into plants.

17. The method of claim 14, wherein the identification further comprises genetic, molecular and / or biochemical analysis.

18. Use of the method according to any one of the preceding claims for: (a) functional characterization of one or more candidate plant pathogen effectors or one or more candidate plant disease resistance genes, or (b) functional characterization of multiple pairs of one or more candidate plant pathogen effectors and one or more candidate plant disease resistance genes, or (c) screening a library of variants of one or more candidate plant pathogen effectors and / or a library of variants of one or more candidate plant disease resistance genes, or (d) bait engineering, or (e) Evaluation of candidate plant disease resistance gene stacks.

19. A method of producing disease resistant plants, the method comprising transforming plant cells with a construct for expressing one or more plant disease resistance genes identified using the method of any one of claims 1 to 17, and regenerating into plants.

20. A method for producing a disease-resistant plant, the method comprising genetically modifying an allele of a gene that does not confer disease resistance in a plant cell so that the modified allele is capable of expressing a plant disease resistance gene identified using the method of any one of claims 1 to 17, and then regenerating into a plant.

21. The method of claim 20, wherein the genetic modification comprises transgenic or genome editing.

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