Single-cell methods for the discovery of disease resistance leads

Abstract

The present invention relates generally to the field of molecular biology and concerns a method for high-throughput screening and characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, encompassing (i) the introduction into a plant protoplast of one or more expression cassette(s) comprising at least one candidate plant pathogen effector gene(s) and / or at least one candidate plant disease resistance gene(s), (ii) transient expression of the candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s), (iii) measurent of reactive oxygen species (ROS) produced by the protoplast, followed by (iv) isolation of the plant protoplast producing ROS and (v) identification of the introduced at least one candidate plant pathogen effector gene(s) and / or at least one candidate plant disease resistance gene(s). Alternatively, the protoplast is capable of expressing a screenable marker, allowing measurement of modulated screenable marker expression as a proxy for HR activation.

Description

[0001] Single-cell methods for the discovery of disease resistance leads

[0002] FIELD OF THE INVENTION

[0003] The present invention relates generally to the field of plant molecular biology and concerns methods for the 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 a more effective hypersensitive response, and immune receptor variants recognizing a broader panel of effectors and thus leading to expansion of activation sensitivity).

[0004] BACKGROUND OF THE INVENTION

[0005] Plant diseases and plant pathogens cause extensive yield losses in crop harvests worldwide and therefore represent a threat to food security (Savary et al., 2019). Crops with large global footprints are especially vulnerable as they are exposed to a broad diversity of pathogens around the world (Nagy et al., 2021). The diversity of disease resistance genes has been reduced in crops during domestication putting them in disadvantage against rapidly mutating pathogens (Zheng et al., 2016). Indeed, plant resistance to pathogens can be easily overcome by rapidly evolving pathogen virulent races, expressing a plethora of avirulence genes. As a consequence, there is a permanent need for plant defense systems that have plasticity and adaptability to new pathogens, pathogen-associated molecular patterns or pathogen effectors (Kourelis et al., 2021).

[0006] Most strategies currently applied to make crops more tolerant or resistant to diseases or pathogens rely on lengthy population genetics processes and QTL cloning efforts (Kawashima et al., 2016; Pedley et al., 2019). While comparative genomics (Yang et al., 2013) and mutagenesis (Steuernagel et al, 2016) or reverse genetics-based gene identification (Lewis et al., 2010) represent more efficient approaches to R-gene identification, such approaches also require a significant and time-consuming effort. This also applies to biotech approaches that rely on developing an insight in the molecular and biochemical determinants of the crop-pathogen interaction, and the subsequent time consuming in planta evaluation of gene candidates, gene variants, and gene or protein expression strategies. Considering the pathogens’ adaptation capability to overcome crop resistance, such discovery tactics taking years or at least many months are suboptimal towards developing durable pathogen resistance for crops.

[0007] Plant protoplasts have proven to be a versatile tool for conducting cell-based experiments. Physiological responses and high-throughput capability enable cost- effective screenings and hypothesis-driven tests (Yoo et al., 2007). More specifically, protoplasts have been used successfully to study plant innate immune responses triggered by elicitors (Pachten & Barz, 1999) or pathogen-derived avirulence effectors (He et al., 2007; Su et al., 2019). The interaction of elicitors with cell surface receptors, or of avirulence effectors (AVRs) with plant nucleotide-binding and leucine-rich repeat (NLR) receptors typically leads to rapid host cell death at sites of attempted infection, and ultimately to plant disease resistance. This hypersensitive response can be used as a surrogate for the NLR activation required for disease resistance. Given the physiological relevance of the hypersensitive response in the context of the NLR / AVR interaction, assays relying on the detection and quantification of the response are preferred over more limited or indirect assays based on yeast-2-hybrid screening, virus- induced gene silencing or virus-mediated overexpression for testing AVR or NLR candidates. Transient Agrobacterium-mediated testing in Nicotiana spp. is a widely applied method that enables the relatively rapid of discovery of matching NLR / AVR pairs. However, issues related to expression tuning and AVR-independent cell death responses in this heterologous system often require significant experimental efforts to detect cell death resulting from a specific NLR / AVR interaction (Bourras et al 2015).

[0008] WO2022 / 218158 discloses a method to validate genes encoding putative plant pathogen effector proteins in plant protoplasts. Briefly, a putative pathogen effector gene and LUC gene are co-expressed in plant protoplasts expressing a known resistance gene. Upon recognition of the effector protein by the resistance protein leads to a reduction in LUC activity which is presumably caused by the onset of hypersensitive response in the protoplast. Due to the low sensitivity of LUC detection, this method does not allow high- throughput screening of individual cells but is rather limited to measurement in bulk.

[0009] Saur et al. (2019) described a method using barley and wheat protoplasts for rapidly assaying cell death me-diated by NLR / AVR pairs. The method was successfully applied for detecting and quantifying the cell death resulting from the NLR / AVR interaction for 2 different NLR / AVR pairs in bulk protoplast samples. However, applying this protocol the authors observed a very significant experimental variation. As a consequence, the method does not provide an effective solution for the selection of f.ex. immune receptor variants displaying a higher effector binding affinity leading to an improved hypersensitive response, or for defining the most effective immune receptor genes present in an NLR cluster, or for quantifying the effect of a multiplex genome editing approach on a set of sensitivity genes. In addition to being quantitative and reproducible, methods for the screening and prioritization of disease resistance genes should also be scalable, enabling the selection of the best candidates from collections of allelic variants or from vast mutant libraries of im-mune receptors or effectors. For the efficient screening of large mutant collections, it is preferred to have access to assays providing a quantitative readout at the single cell level, or only requiring a limited number of cells. Furthermore, following the enrichment or selection of target cells, the workflow preferably includes a single-cell genotyping step to support structural-functional characterization of the different lead candidates, or to enable rapid recreation of the mutant in the proper genetic background, towards the development of a crop variety with durable disease resistance.

[0010] Thus, there is a need for a method that allows rapid high-throughput identification of genes providing pathogen resistance in plants to diseases caused by pathogens producing pathogen effector genes.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention describes a general workflow for transfection and sorting of plant protoplast cells enabling various screening applications ranging from identification and validation of candidate R gene and effector pairs to assessment of R gene and / or effector function to screening of R gene and / or effector variant libraries, decoy engineering and evaluation of R gene stacks or pyramids.

[0013] The workflow entails a multifaceted approach comprising i / expression of one or more effector and / or R gene candidate(s) in protoplasts isolated from a crop of interest, ii / live sorting of transfected protoplasts to identify HR-expressing cells, iii / dispensing and retrieval of individual cells of interest, iv / (optional) clonal propagation of sorted cells, v / sequencing of isolated single cells or clonal progeny to reveal the causal R gene(s) and / or HR-eliciting effector(s), and vi / (optional) regeneration of preselected cells / clones into whole plants. In the first step, constructs encoding effector and / or R gene candidates are transfected into protoplasts isolated from a crop of interest following the procedures described herein. Different expression platforms can be used depending on the specific use case and experimental objectives. For instance, pairs of effector and R gene candidates can be transiently expressed in protoplasts derived from a disease-susceptible crop variety / cultivar. In a particular case, membrane-bound R gene candidates can be transiently expressed in protoplasts derived from a disease-susceptible crop variety / cultivar, in combination with expression of effectors that are secreted, thereby inducing a hypersensitive response. Alternatively, or in addition, effector candidates can be transformed in protoplasts from a disease-resistant line, that is a crop variety or cultivar expressing a previously (un)isolated R gene. In a third scenario, candidate effectors and / or R genes are heterologously expressed in protoplasts derived from a different plant species.

[0014] Next, HR-expressing protoplasts are identified using surrogate reporter systems. These systems measure the activity of fluorescent and / or luminescent (bio)reporters as a proxy for R gene activation and immune signaling. Examples of reporter assays for detection of single cells mounting an HR include but are not limited to: i / diminished activity of a co-transfected or stably expressed fluorescent or luminescent reporter, ii / fluorescent detection of immune-associated production of reactive oxygen species (ROS), iii / expression of a fluorescent or luminescent reporter under control of an HR- inducible promoter or iv / fluorescent detection of endogenous HR marker transcripts using molecular RNA sensors.

[0015] Assays (ii) and (iii) involve single measurements of fluorescence or light emission collected from individual cells. In contrast, when using co-transfected or stably expressed fluorescent or luminescent reporters 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. Time-resolved measurements thus enable to sort for cells with the strongest reduction in reporter activity independent of transfection efficiency variability and stochasticity in reporter gene expression.

[0016] Following transfection, protoplasts are analyzed using one of several live-cell sorting technologies including fluorescence-activated cell sorting (FACS), microfluidic flow, microfluidic containment and microarray-based systems, or using live-cell seeding and imaging technologies, each having unique characteristics and applications. Based on above-mentioned fluorescent or luminescent readouts, single, positive cells are selectively sorted, dispensed into 96-well, 384-well or 1536-well plates, and subsequently analyzed via single-cell PCR and Sanger or Amplicon deep sequencing to identify the causal R gene and / or effector candidate. Alternatively, sorted cells are first grown into clonal colonies, which allows individual cells to recover from transfection and sorting and undergo the first mitotic divisions. Once a more robust, sustainable colony has grown, it is isolated and transferred to a culture plate or vessel for continued colony growth, downstream genetic / molecular and / or biochemical analysis, and subsequent plant regeneration.

[0017] BRIEF DESCRIPTION OF THE FIGURES

[0018] Figure 1: Measurement of GFP fluorescence (RFU) in protoplasts derived from Oryza sativa (A) Glycine max(^) and Brassica napus (C). 1: Empty vector control protoplasts; 2: Protoplasts expressing 0sRGA4.

[0019] Figure 2: Measurement of GFP fluorescence (RFU) in protoplasts derived from wheat cultivar Fielder. 1: dummy plasmid control protoplasts; 2: Protoplasts expressing A ? / !; 3: Protoplasts expressing PM3A-S1335R, 4: Protoplasts expressing PM3A-R1334E, 5: Protoplasts expressing PM3A-D502V, 6: Protoplasts expressing A vr- PM3A\ 7: Protoplasts expressing Avr-PM3A-Q112N\ 8: Protoplasts expressing PM3A and Avr- PM3A\ 9: Protoplatst expressing PM3A-S1335R and A vr- PM3A 10: Protoplatst expressing PM3A-R1334E and A vr-PM3A; 11: Protoplasts expressing PM3A and Avr- PM3A-Q112N', 12: Protoplasts expressing PM3A-S1335R and Avr-PM3A-Q112N.

[0020] Figure 3: Visualisation of GFP fluorescence in protoplasts derived from rice. 1: Protoplasts expressing eGFR 2: Protoplasts expressing deGFP_E 3: Protoplasts expressing deGFP_2, 4: Protoplasts expressing deGFP_3, 5: Protoplasts expressing eGFP and RGA4 6: Protoplasts expressing deGFP_l and RGA4 7: Protoplasts expressing Ve<SAA_ ’and RGA4 8: Protoplasts expressing deGFP_3 and RGA4.

[0021] Figure 4: Single cell RGA4-specific PCR amplification of protoplasts in two repeats (A and B); 1: Individual protoplasts with high GFP intensity; 2: Individual protoplasts with low GFP intensity.

[0022] Figure 5: Overview of workflow according to an embodiment of the invention. Middle panel adapted from Union Biometica Inc.

[0023] Figure 6: Measurement of luminescence (RLU xlO3) in protoplasts in an undiluted sample (approximately 50000 cells, A), in a lOx diluted sample (approximately 5000 cells, B), in a 200x diluted sample (approximately 250 cells, C) and in a 400x diluted sample (approximately 125 cells, D). 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.

[0024] Figure 7: Measurement of luminescence (AU) in individual protoplasts derived from rice. 1: protoplasts expressing green enhanced Nanolantern (GeNL); 2: protoplasts expressing GeNL and RGA4; 3: protoplasts expressing firefly luciferase (fLUC); 4: substrate control protoplasts.

[0025] Figure 8: Measurement of luminescence (RLU) in protoplasts derived from wheat. 1: Green enhanced Nanolantern (GeNL) 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.

[0026] Figure 9: Measurement of luminescence (RLU) in individual protoplasts derived from wheat. 1: Protoplasts expressing PM3A and Avr-PM3A; 2: Protoplasts expressing PM3A- S1335R and Avr-PM3A; 3: Protoplasts expressing PM3A-R1334E and Avr-PM3A; 4: Protoplasts expressing PM3A and Avr-PM3A-Q112N; 5: Protoplasts expressing PM3A- S1335R and Avr-PM3A-Q112N; 6: Control protoplasts transfected with empty vector.

[0027] Figure 10: In vivo measurement of reactive oxygen species (ROS) as CellRox Green fluorescence (RFU) in protoplasts derived from oilseed rape during the first 24 h after transfection. 1: Control protoplasts transfected with empty vector (‘Ctrl’); 2: Protoplasts expressing RGA4 (‘RGA4’); 3: Control protoplasts transfected with empty vector treated with flg22 (‘flg22’); 4: Protoplasts expressing RGA4 treated with flg22 (‘RGA4 + flg22’). The x-axis indicates time in hours post transfection.

[0028] Figure 11: Time-resolved measurement of GFP fluorescence (RFU) in protoplasts derived from wheat (cultivar Fielder). A: protoplasts expressing GFP reporter alone (“GFP Ctrl”); B: protoplasts co-expressing GFP reporter with PM3A and Avr-PM3A. C: protoplasts co-expressing GFP reporter with PM3A-R1334E and Avr-PM3A; D: protoplasts co-expressing GFP reporter with PM3A and Avr-PM3A-Q112N. The x-axis indicates time in hours post 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.

[0029] Figure 12: Quantification (%) of individual protoplasts derived from wheat (cultivar Fielder) showing signature GFP expression patterns over time: Signature A: GFP signal increases; Signature B: GFP signal remains constant; C: GFP signal decreases; D: loss of GFP signal. 1: protoplasts expressing GFP reporter alone (“GFP Ctrl”); 2: protoplasts co-expressing GFP reporter with PM3A and Avr-PM3A. 3: protoplasts co-expressing GFP reporter with PM3A-R1334E and Avr-PM3A; 4: protoplasts co-expressing GFP reporter with PM3A and Avr-PM3A-Q112N. “n” indicates the number of individual protoplasts measured.

[0030] Figure 13: Quantification (%) of individual protoplasts derived from wheat (cultivar Fielder) showing a specific signature GFP expression pattern over time: A: Signature C, GFP signal decreases; B: Signature D, loss of GFP signal. 1: protoplasts expressing GFP reporter alone (“GFP Ctrl”); 2: protoplasts co-expressing GFP reporter with PM3A and Avr-PM3A. 3: protoplasts co-expressing GFP reporter with PM3A-R1334E and Avr- PM3A; 4: protoplasts co-expressing GFP reporter with PM3A and Avr-PM3A-Q112N. The x-axis indicates time in hours post transfection (hpt).

[0031] Figure 14: Measurement of GFP fluorescence (GFP / autofluorescence) in protoplasts derived from Brassica napus cultivar Westar. 1: Protoplasts expressing GFP reporter; 2: Protoplasts expressing GFP reporter and secreted effector (A: AvrLm3*, B: AvrLml*, C: AvrLm5-9*); 3: Protoplasts expressing GFP reporter and membrane-bound cell surface receptor (A: Rlm3, B: LepR3, C: Rlm9); 4: Protoplasts expressing GFP reporter, secreted effector and matching membrane-bound cell surface receptor (A: AvrLm3* and Rlm3, B: AvrLml* and LepR3, C: AvrLm5-9* and Rlm9).

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] The recent determination of structures of both cell surface and intracellular immune receptors in plants in their activated states is providing new insights into how recognition complexes can be modified to expand recognition specificities to confer resistance to otherwise virulent pathogens. By expanding the repertoire of both cell surface and intracellular recognition systems, and combining them, it is expected that resistance to numerous diseases will be enhanced and will be more durable (Frailie & Innes, 2021). Similarly, a better understanding of the identity of fungal effectors and the way these effectors interact with intracellular immune receptors will contribute to enhanced disease resistance in crops. It is hypothesized that an efficient strategy to improve durability of R genes would consist in alternating or pyramiding R genes corresponding to different structural classes of effectors (Lazar et al., 2020). Pyramiding strategies should not be limited to endogenous genes (Pedley et al., 2019), but can also benefit from the heterologous expression expression of R genes from wild accessions of crop species, or from plant species related to the target crop (Kawashima et al., 2016).

[0034] Here, a fast and efficient protoplast-based lead discovery workflow for developing disease resistant crop species is described. Taking advantage of the established insights in genetic elements defining pathogen resistance, of the mechanistic understanding of crop-pathogen interactions generated through conventional genetics and molecular biology approaches, and of crop and pathogen genome sequence data, the workflow enables discovery of disease resistance leads within months, weeks or days. The key components of such a more efficient or effective discovery and development process include single cells or protoplasts and technologies enabling the high-througput screening of (genetically) modified cells or protoplasts.

[0035] The inventors have now found that upon using the methods of the invention, the expression of screenable marker genes can be reliably and rapidly measured in individual protoplasts and their modulated expression can therefore be used as markers for the onset of HR in the high throughput screening method of the invention. Besides cell screening methods relying on f.ex. genes encoding fluorescent or luminescent markers, one may also consider a marker-free approach for the enrichment and / or selection of target cells. For instance, magnetic levitation technology (LeviCell System, Levitas Inc.) can be used for enriching or selecting target cells in the absence of dyes or specific markers.

[0036] The rapid production 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 serve multiple other immune functions. ROS production constitutes a promising surrogate for NLR activation. Accordingly, the production of ROS is measured in the individual protoplasts as a surrogate for the onset of HR.

[0037] Individual protoplasts showing modulated production of ROS, modulated expression of one or more screenable marker gene, or both, as compared to control protoplasts lacking a functional candidate pathogen effector gene are subsequently isolated and the gene(s) of interest encoding pathogen effector(s) and / or disease resistance gene(s) in the individual protoplast are eventually identified.

[0038] As used herein, technical terms and expressions used within the scope of this application are generally to be given the meaning commonly applied to them in the pertinent art of plant biology, molecular biology, bioinformatics and plant breeding. All of the following definitions apply to the complete content of this application. The terms “essentially”, “about”, “approximately”, “substantially” and the like in connection with an attribute or a value, particularly also define exactly the attribute or exactly the value, respectively. The term “substantially” in the context of the same functional activity or substantially the same function means a difference in function preferably within a range of 20%, more preferably within a range of 10%, most preferably within a range of 5% or less compared to the reference function. The term “about” in the context of a given numeric value or range relates in particular to a value or range that is within 20%, within 10%, or within 5% of the value or range given. As used herein, the term “comprising” also encompasses the term “consisting of”. The section captions and headings in this application are for convenience and reference purpose only and should not affect in any way the meaning or interpretation of this application.

[0039] By combining established insights in genetic elements defining pathogen resistance, the mechanistic understanding of crop-pathogen interactions generated through conventional genetics and molecular biology approaches, and crop and pathogen genome sequence data, the method of the invention enables discovery of disease resistance leads within months, weeks or days.

[0040] In a first embodiment, the invention provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, which method comprises the steps of:

[0041] (a) Preparing at least one protoplast cell derived from a plant of interest, which protoplast is optionally capable of expressing one or more screenable marker gene(s);

[0042] (b) Introducing one or more expression cassette(s) for transiently expressing one or more candidate plant pathogen effector gene(s) and / or introducing expression cassette(s) for transiently expressing one or more candidate plant disease resistance gene(s) in said at least one protoplast; (c) Transiently expressing said one or more candidate plant pathogen effector gene(s) and / or said one or more candidate plant disease resistance gene(s);

[0043] (d) measuring in said at least one protoplast the production of reactive oxygen species (ROS) and / or optionally the expression of said screenable marker gene;

[0044] (e) isolating one or more individual protoplasts that show modulated production of ROS, modulated expression of said one or more screenable marker genes, or both, as compared to control plant protoplasts lacking a functional candidate pathogen effector gene;

[0045] (f) Identifying said one or more candidate plant pathogen effector(s) and / or said one or more candidate plant disease resistance gene(s) in the individual protoplast isolated in step (e).

[0046] The method described herein enables fast and efficient discovery, validation and characterization of pathogen effector genes and matching plant disease resistance genes which, in turn, can be used to efficiently develop disease resistant crop species. The method further enables the (ultra) high-throughput screening of large sets of genes or gene variants. The enhanced efficiency of the method described herein is due mainly to the characterization in individual plant cells, thus obtaining data from each single plant cell. The term “cell” as used herein refers to a plant cell. As used herein, the terms “plant cell” and “protoplast” are used interchangeably; the term “individual plant cell” or individual protoplast refers to a single protoplast (i. e. separated from other protoplasts) from which data is obtained (that is, analysed on individual basis). A plant cell capable of use in the invention may be any type of plant protoplast. Individual plant protoplasts can be generated from, for example, any type of plant used for agriculture including but not limited to broad acre crop plants such as wheat, corn, soy or cotton; high value crop plants such as tobacco, tomato, lettuce, pepper or squash plant; brassica plants such as broccoli, brown mustard, brussels sprouts, cabbage, cauliflower, kale, kohlrabi, rape, rutabaga, turnip, or Arabidopsis plant; ornamental plants, such as rose, petunia, poppy, lilly, lavender, silver grass, or cactus plant; a fruit tree, shrub, or vines, such as grape, apple, orange, strawberry, blackberry, blueberry, raspberry, plum, pluot, apricot plant, or the like; or turf or forage plants, such as grass or alfalfa plants. Methods of obtaining protoplasts are known in the art.

[0047] Whereas high-throughput screening of whole plants is substantially limited by their slow growth and size, millions of protoplasts may be processed in a matter of hours using various cell sorting and dispensing technologies. However, to date protoplasts have been predominantly extracted and analyzed in bulk, limiting their use. The method of the invention makes use of plant protoplasts derived from the plant of interest. In an embodiment, the protoplasts are derived from whole plants or seedlings, plant parts including pollen grains, plant tissues including leaf tissues, cell suspensions and / or callus. Removal of the plant cell wall yields highly versatile protoplasts while retaining physiological responses of intact plant cells to immune triggers, thus forming a unique experimental system. In addition, removal of the cell wall also allows for efficient introduction of exogenous DNA in the protoplast as well as efficient downstream processing. The use of protoplasts in the current method therefore contributes to increasing the method’s throughput and enables performing cost-effective screenings and cell-based experiments in plants. Methods for preparing protoplasts from crops and fiber crops are known by the skilled person and are also provided herein.

[0048] In a second embodiment, the invention provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells as described above, wherein one or more candidate plant pathogen effector(s) and one or more candidate plant disease resistance gene(s) are introduced in protoplasts derived from a plant that is susceptible to a pathogen encoding said plant pathogen effector.

[0049] In a third embodiment, the invention provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells as described above, wherein one or more candidate plant pathogen effector(s) are introduced in protoplasts derived from a corresponding disease resistant plant.

[0050] A “corresponding disease resistant plant” is a plant that is resistant to the pathogen from which the candidate pathogen effector gene(s) is / are derived.

[0051] In a fourth embodiment, the invention provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells as described above, wherein the one or more candidate plant pathogen effector(s) and the one or more candidate plant disease resistance gene(s) are introduced in protoplasts that are derived from a plant that is heterologous to said candidate plant disease resistance gene(s).

[0052] The term “heterologous” polynucleotide refers:

[0053] (a) to a polynucleotide that is not native to the host cell; (b) a polynucleotide native to the host cell and in which structural modifications, e.g., deletions, substitutions, and / or insertions, have been made to alter the native polynucleotide;

[0054] (c) a polynucleotide native to the host cell but whose expression is quantitatively altered as a result of manipulation of the regulatory elements of the polynucleotide by recombinant DNA techniques, e.g., a stronger promoter; or

[0055] (d) a polynucleotide native to the host cell but integrated not within its natural genetic environment as a result of genetic manipulation by recombinant DNA techniques.

[0056] Resistance is a strategy an organism, such as a plant, can employ to withstand infection with a pathogen. The term “resistance” refers to the capability to prevent or minimize infection or attack by a pathogen. As used to herein, “disease resistant” or “have resistance to a disease” refers to a plant showing increased resistance, or tolerance, to a disease compared to a control plant, which is a susceptible plant. Disease resistance may manifest in fewer and / or smaller lesions, increased plant health, increased yield, increased root mass, increased plant vigor, less or no discoloration, increased growth, reduced necrotic area, or reduced wilting compared to a susceptible plant grown under similar disease conditions.

[0057] In one embodiment, one or more candidate plant pathogen effector gene (s) and one or more candidate plant disease resistance gene(s) are introduced in protoplasts derived from a plant that is susceptible to a pathogen encoding said plant pathogen effector. Preferably, the transfected candidate pathogen effector gene(s) and the candidate disease resistance gene(s) are expressed simultaneously meaning that there is an overlap in timeframe for all the proteins to be expressed, whereby the level of said proteins is increased when compared to a non-transfected control.

[0058] In another embodiment, one or more candidate plant pathogen effector(s) are introduced in protoplasts derived from a corresponding disease resistant plant. A corresponding disease resistant plant as used herein refers to a crop variety or cultivar expressing a previously isolated R gene, as well as a crop variety or cultivar expressing an R gene which has not yet been previously isolated or characterised. In yet another embodiment, the one or more candidate plant pathogen effector(s) and the one or more candidate plant disease resistance gene(s) are introduced in protoplasts that are derived from a plant that is heterologous to said candidate plant disease resistance gene(s). In a next step of the method of the invention, the one or more candidate plant pathogen effector gene(s) and / or one or more candidate plant disease resistance gene(s) are transiently expressed in protoplasts. Membrane-bound R gene candidates can be transiently expressed in protoplasts derived from a disease-susceptible crop variety or cultivar, in combination with expression of effectors that are secreted. In a further embodiment of the invention, the one or more plant pathogen effector(s) are secreted effectors. In another or further embodiment, the one or more plant disease resistance gene(s) encode one or more membrane-bound protein. In a preferred embodiment, the R gene encodes a membrane-bound cell surface-localized receptor protein such as, but not limited to, LepR3 (SEQ ID NO: 40), Rlm3 (SEQ ID NO: 41) and / or Rlm9 (SEQ ID NO:

[0059] 42) and the transiently expressed secreted effector is, respectively, AvrLml (SEQ ID NO:

[0060] 43), AvrLm3 (SEQ ID NO: 44) and / or AvrLm5-9 (SEQ ID NO: 45). Recognition of an effector protein by an endogenous or transiently expressed resistance gene in the protoplast most often results in activation of HR. The method of the invention allows reliable and efficient detection of intracellular as well as extracellular interactions between effectors and disease resistance proteins leading to the activation of HR. HR is a complex multicellular process characterized by rapid cell death at the site of infection, correlated with numerous physical, physiological and molecular alterations including rapid transcriptional reprogramming and accumulation of ROS. Accordingly, in a next step of the method of the invention, the production of reactive oxygen species (ROS) and / or optionally the expression of one or more screenable marker gene is measured in at least one individual protoplast. Without wishing to be bound by theory, it is thought that the high amount of cellular energy needed to mount the immune responses triggered by activation of a disease resistance protein by a matching effector protein leads to a reduced expression of transiently expressed screenable marker genes in those cells. The reduced activity of a co-transformed luciferase reporter as a marker for HR activation has been reported. However, the latter required large populations of cells and luciferase measurements are highly variable across biological replicates, preventing its use for characterization and screening of individual protoplasts (Baba et al., 1986; Saur et al., 2021).

[0061] Plant pathogens secrete effectors to overcome host defenses and host immune responses. “Pathogen effector gene”, “effector gene”, “avirulence gene” or “Avr gene” are used herein interchangeably and refer to a pathogen gene encoding an effector protein that modulates plant host cell physiology, suppresses basal host defense responses and / or promotes disease susceptibility. “Plant disease resistance gene”, “resistance gene” or “R gene” (used interchangeably) refers to a gene that encodes a protein capable of recognizing one or more specific pathogen effector proteins and of triggering an effector-triggered immunity (ETI) related defense mechanisms in the host cell. One such defense mechanism is the hypersensitive response (HR) which is correlated with the rapid production of reactive oxygen species (ROS) by the host cell. “Reactive oxygen species” or “ROS” as used intercheangably herein refers to extracellular and / or intracellular hydrogen peroxide (H2O2), superoxide anion (O2), hydroxyl radicals (OH) and singlet oxygen as well as products resulting from ROS reaction with nitric oxide (NO) such as but not limited to nitrosonium (NO+), nitroxyl ion (NO-), peroxynitrite (ONOO-) and NOXcompounds (NO2, N2O3, N2O4, NO2‘, NO3‘).

[0062] The term “gene” means a segment of DNA containing hereditary information that is passed on from parent to offspring and that contributes to the phenotype of an organism. The influence of a gene on the form and function of an organism is mediated through the transcription into RNA (tRNA, rRNA, mRNA, non-coding RNA) and in the case of mRNA through translation into peptides and proteins.

[0063] Optionally, the protoplast is capable of expressing one or more screenable marker gene(s). Modulated production of screenable markers can be used as a proxy for ROS production. In one aspect of the invention, the screenable marker gene is constitutively expressed in the protoplast, in which case (by downregulation of expression in a situation of compatible reaction), production of screenable markers will diminish. In another aspect of the invention the screenable marker gene is operably linked to a promoter that is induced under conditions where ROS are produced. As a result, expression of the screenable marker gene will increase.

[0064] The term modulated production or modulated expression refers to either increased production or expression, or to decreased production or expression, compared to unmodulated (control) production or expression.

[0065] As used herein, the term screenable marker refers to a protein that confers a phenotype on a cell in which it is introduced or expressed to facilitate the identification and / or selection of cells that are, in the scope of this invention, showing a hypersensitive response. Preferably, the screenable marker allows visual selection on the level of single cells. Such marker activity results in the formation of colour, luminescence or fluorescence. Expression of visual marker genes results in the formation of colour (for example / ? -glucuronidase, GUS or / ? -galactosidase with its coloured substrates, for example X-Gal), luminescence (such as the luciferin / luceferase system or nanolanterns) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof such as a destabilized GFP or a truncated GFP or a GFP fusion protein; the cyan fluorescent protein (CYP) gene, the yellow fluorescent protein (YFP) gene, a DsRed gene, an mCherry gene). This list represents only a small number of possible markers. The skilled worker is familiar with such markers. Different markers are preferred, depending on the organism and the selection method, as long as they are detectable in isolated single cells.

[0066] Thus, the invention provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, as described above, wherein the one or more screenable marker gene encodes a fluorescent and / or luminescent marker.

[0067] The screenable marker gene can be transiently expressed or constitutively expressed, in which case the protoplast may be transformed with an expression cassette comprising a screenable marker gene operably linked to a suitable promoter.

[0068] Measurement of screenable marker gene expression can be continuously, or at multiple discrete timepoints, or at a single timepoint (e.g. endpoint measurement).

[0069] Thus, the invention provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, as described above, wherein modulated production of ROS and / or modulated expression of said one or more screenable marker genes is measured continuously or on one or more timepoints.

[0070] In another aspect of the invention, ROS production is detected by compounds that, when oxidized by ROS, become fluorescent. CellROX™ Deep Red CellROX™ Green Reagent, and CellROX™ Orange are examples of such compounds.

[0071] Thus, the invention also provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, as described above, wherein the production of reactive oxygen species (ROS) is measured through ROS induced oxidation of a fluorescent dye.

[0072] The invention also provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant protoplasts, as described above, wherein the screenable marker gene is transiently or stably expressed. Measurement of the screenable marker gene in individual protoplasts is especially advantageous as it allows fo rapid screening of large quantities of individual cells and easy retrieval of unique HR-expressing cells for further analysis.

[0073] The invention also provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, as described above, wherein the screenable marker gene is under control of a constitutive promoter or under control of a hypersensitive response inducible promoter.

[0074] The term “promoter” typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and is involved in recognising and binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. “Promoter” herein may further include any nucleic acid sequence capable of driving transcription of a coding sequence. In particular, the term “promoter” as used herein may refer to a polynucleotide sequence generally described as the 5' regulator region of a gene, located proximal to the start codon. The transcription of one or more coding sequence is initiated at the promoter region. The term promoter may also include fragments of a promoter that are functional in initiating transcription of the gene. A “constitutive promoter” refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ. Examples of constitutive promoters useful 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), amongst others.

[0075] An “inducible promoter” has induced or increased transcription initiation in response to a chemical (for a review see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89- 108), to environmental or physical stimulus, or may be “stress-inducible”, i.e. activated when a plant is exposed to various stress conditions, or a “pathogen-inducible" i.e. activated when a plant is exposed to exposure to various pathogens.

[0076] Examples of inducible promoters are sugar inducible promoters (rhamnose, arabinose, galactose), or dexamethasone inducible promoters.

[0077] Preferably the inducible promoter operably linked to the marker gene is a hypersensitive response inducible promoter, such as the AtMYB30 promoter or the Athsr promoters. The onset of immune-triggered hypersensitive response (HR) in plant cells is typically associated with massive transcriptional reprogramming, involving upregulation of large suites of defense-related genes. HR-marker genes which are strongly activated in response to HR elicitation are useful in embodiments of the invention wherein the single-cell screening workflow is based on enhanced expression of one or more screenable marker genes. Expression of a screenable marker gene under the control of a HR-inducible promoter according to an embodiment of the invention allows to characterize candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, as accumulation of the selectable marker indicates the onset of HR associated with resistance of the protoplast against the candidate effector. In another embodiment of the invention, the transcriptional induction of HR-marker genes is measured based on the HR-dependent expression of screenable marker genes using RNA-sensing technology (Kaseniit et al., 2022; Jiang et al., 2022).

[0078] The invention also provides expression cassettes or genetic constructs and vectors to facilitate introduction and / or expression (de novo introduced or increasing the already existing expression) in protoplasts of any of the nucleic acids described herein. The genetic constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plant protoplasts and for expression of the gene(s) of interest in the transfected protoplasts. The nucleic acids encoding a pathogen effector, a disease resistance gene or a selectable marker gene can be located on separate constructs 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 acids can be in tandem orientation on the construct, or in opposite orientation. The respective nucleic acid sequences may be fused together or separated by coding or non-coding DNA, such as promoters, introns, a subcellular targeting signal, or stuffed DNA such as the MARs (Matrix attachment Regions) regions.

[0079] Methods for obtaining desirable levels of expression are known to the person skilled in the art, and comprise, but are not limited to placing the gene(s) encoding the protein(s) to be characterized downstream of a suitable promoter. In an embodiment, the gene(s) encoding the protein(s) to be characterized are placed downstream of a constitutive promoter. Suitable constitutive promoters for use in the methods of the invention include, but are not limited to, the constitutive 35S promoter of Cauliflower mosaic virus, the constitutive maize Ubitquitinl promoter and the constitutive Arabidopsis UbiquitinlO promoter.

[0080] Different expression platforms can be used depending on the specific use case and experimental objectives. An “expression cassette” as used herein, is a DNA molecule composed of at least one sequence of interest to be expressed, operably linked to one or more control sequences (at least to a promoter) as described herein. Typically, the expression cassette comprises three elements: a promoter sequence, an open reading frame, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site. Additional regulatory elements may include transcriptional as well as translational enhancers. An intron sequence may also be added to the 5' untranslated region (UTR) or in the coding sequence to increase the amount of the mature message that accumulates in the cytosol. The skilled artisan is well aware of the genetic elements that must be present in the expression cassette in order to be successfully expressed. Preferably, at least part of the DNA or the arrangement of the genetic elements forming the expression cassette is artificial. The expression cassette may be part of a vector or may be integrated into the genome of a host cell and replicated together with the genome of its host cell. The expression cassette is capable of increasing or decreasing the expression of DNA and / or protein of interest.

[0081] “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 ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0082] Techniques for introducing such expression cassette into plants are well known in the art. “Transformation” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. That is, the term “transformation” as used herein is independent from vector, shuttle system, or host cell, and it not only relates to the polynucleotide transfer method of transformation as known in the art (cf., for example, Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), but it encompasses any further kind polynucleotide transfer methods such as, but not limited to, transduction or transfection. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct and a whole plant regenerated therefrom. The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. “Stable transformation” may mean that the transformed cell or cell organelle passes the nucleic acid comprising the foreign coding sequence on to the next generations of the cell or cell organelles. Usually, stable transformation is due to integration of nucleic acid comprising a foreign coding sequence into the chromosomes or as an episome. “Transient transformation” may mean that the cell or cell organelle once transformed expresses the foreign nucleic acid sequence for a certain time - mostly within one generation. Usually, transient transformation is due to nucleic acid comprising a foreign nucleic acid sequence is not integrated into the chromosomes or maintained as an episome.

[0083] Transformation methods for protoplasts are known in the art and include for example the calcium / polyethylene glycol method (Krens, F.A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio / Technol 3, 1099-1102). Alternatively, plants can be transformed before the step of protoplast preparation. Suitable methods for plant transformation include microinjection into plant material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle bombardment (Klein TM et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. A well established and preferred method is Agrobacterium-mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds, on the intact plant or at least on the flower primordia, or to inoculate the plant meristem with agrobacteria. Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in: European patent application EP 1198985 Al, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994). In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002). Said 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.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hbfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38. Preferably, candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) are transiently expressed in the protoplast.

[0084] The protoplasts used in the methods of the current invention can be derived from any plant. Preferably the protoplast is derived from a plant of the superfamily Viridiplantae, in particular from monocotyledonous and dicotyledonous plants, including crop plants, such as cereals, oilseed plants, leguminous plants, vegetables and fiber crops. Examples of crop plants include but are not limited to chicory, carrot, cassava, yams, trefoil, soybean, beet, sugar beet, sunflower, canola, alfalfa, rapeseed, linseed, cotton, cocoa, tomato, potato, coffee and tobacco. According to another aspect of the present invention, the plant is a monocotyledonous plant. Examples of monocotyledonous plants include sugarcane, banana, onion, 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, spelt, einkorn, teff, mi Io and oats. In a particular aspect the plants used in the methods of the invention, are selected from the group consisting of maize, wheat, rice, soybean, cotton, oilseed rape including canola, sugarcane, sugar beet and alfalfa. The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots, flowers, and tissues and organs. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, microspores and propagules. Examples of plants of which the cells can be used in the methods of the present 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 fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus 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), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, 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 latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., 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, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amongst others.

[0085] Thus, the invention provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, as described above, wherein said plant of interest is a crop plant, preferably a cereal, an oilseed plant, a leguminous plant, a vegetable or a fiber crop.

[0086] Suitable control plant protoplasts are protoplasts without having introduced the pathogen effector gene, preferably protoplasts comprising a vector lacking the pathogen effector expression cassette (i.e., an empty vector). Alternatively, the control protoplast is a protoplast without the introduced vector comprising the pathogen effector expression cassette. Depending on the case, the screenable marker is absent or present in the control protoplast. Ideally and preferably, the control protoplast has otherwise undergone the same treatment as the protoplast to be tested. The control plant protoplast is typically derived from the same plant species or even the same variety as the plant protoplasts to be assessed.

[0087] Advantageously the production of ROS can be enhanced by a hypersensitive response elicitor (or otherwise formulated: an immune response inducing elicitor). Elicitors for invoking or amplifying a hypersensitive response are know in the art. Preferably the elicitors are small molecules, such as chitin heptaose or flg22, the 22 amino acid epitope of flagellin. Therefore, sensitivity of the methods of the current invention can be enhanced by using one or more elicitor(s). In addition, the methods of the current invention can equally be used for the qualitative or quantitative analysis of candidate elicitor molecules in an assay with a given disease resistance gene, or with a given pathogen effector gene or with a given combination of a disease resistance gene and a pathogen effector gene.

[0088] Thus, the invention provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, as described above, wherein the production of reactive oxygen species (ROS) is enhanced by by addition of a hypersensitive response elicitor.

[0089] The invention also provides a method for high-throughput characterization of candidate elicitor molecules for a given plant pathogen effector gene(s) and / or a given plant disease resistance gene(s) in individual plant cells, as described above, wherein the production of reactive oxygen species (ROS) is enhanced by addition of a hypersensitive response elicitor.

[0090] A variety of techniques for analyzing, live-sorting and retrieving individual protoplasts from large protoplast populations exist, including methods based on light scattering such as for example flow cytometry which is often combined with the detection of a fluorescent signal (fluorescence activated cell sorting or FACS) in each analyzed protoplast (as illustrated in Figure 5). Fluorescent signals that assist sorting can for example originate from a screenable marker (e.g., GFP), from chloroplasts, from the use of fluorescent dyes which can be cell-permeable or impermeable, etc. Alternatively, protoplasts can be isolated using microfluidic flow sorters which have the additional advantage of allowing higher throughput as well as operating in sterile conditions. Furthermore, the use of microfluidic devices combined with FACS allows isolation of protoplasts under reduced hydrodynamic stress, further contributing to preserving protoplast viability. Yet other techniques suitable for analyzing and / or isolating protoplasts of interest in a method according to the invention deploy temporary immobilization of the protoplasts for analysis, for example on a chip (e.g., NanoPen chambers) or microarray. Accordingly, individual protoplasts are analyzed after transfection and individual protoplasts showing modulated expression of a screenable marker gene, as compared to control plant protoplasts lacking a functional candidate pathogen effector gene, are selected. Thus, the invention provides a method for high- throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, as described above, wherein modulated expression of said fluorescent or luminescent marker is measured in individual protoplasts and compared to fluorescent or luminescent marker expression in control protoplasts.

[0091] In another further embodiment, the invention provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, as described above, wherein the isolation of one or more individual protoplasts comprises a step of protoplast sorting, dispensing and retrieving protoplasts of interest.

[0092] Based on above-mentioned fluorescent or luminescent readouts, single, positive cells can be selectively sorted, dispensed into 96-well, 384-well or 1536-well plates, and subsequently analyzed via single-cell PCR and Sanger or Amplicon deep sequencing to identify the causal R gene and / or effector candidate. Thus, the invention provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, as described above, wherein said one or more candidate plant pathogen effector(s) and / or said one or more candidate plant disease resistance gene(s) in the individual protoplast are identified by sequencing.

[0093] Alternatively, sorted cells are first grown into clonal colonies, which allows individual cells to recover from transfection and sorting and undergo the first mitotic divisions. Once a more robust, sustainable colony has grown, it is isolated and transferred to a culture plate or vessel for continued colony growth, downstream genetic / molecular and / or biochemical analysis, and subsequent plant regeneration. Accordingly, in another further embodiment, the invention provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, as described above, wherein the step of isolating one or more individual protoplasts that show modulated production of ROS and / or modulated expression of said one or more screenable marker genes, is followed by a step of clonal propagation. In another further embodiment, the invention provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, as described above, further comprising regeneration of isolated protoplast or clones derived therefrom into plants.

[0094] In yet another further embodiment the invention provides a method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, as described above, wherein the identification furthermore comprises a genetic, molecular and / or biochemical analysis. Such regenerated plants allow the further characterisation of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s).

[0095] In a further embodiment, the invention provides the use of the method according to any of the preceding claims for

[0096] (a) Functional characterisation of candidate plant pathogen effector(s) or candidate plant disease resistance gene(s), or

[0097] (b) Functional characterisation of pairs of candidate plant pathogen effector(s) and candidate plant disease resistance gene(s), or

[0098] (c) Screening of variant libraries of candidate plant pathogen effector(s) and / or of candidate plant disease resistance gene(s), or

[0099] (d) Decoy engineering, or

[0100] (e) Evaluating candidate plant disease resistance gene stacks.

[0101] In yet another embodiment, the invention provides a method of producing a disease resistant plant comprising transforming a plant cell with a construct for expressing one or more plant disease resistance gene(s) identified with a method as described above, followed by regeneration into a plant.

[0102] In a last aspect, the invention provides a method of producing a disease resistant plant comprising genetically modifying in a plant cell an allele of a gene that does not confer disease resistance such that the modified allele is capable of expressing a plant disease resistance gene identified with a method as described above, followed by regeneration into a plant. In a preferred embodiment the genetic modification comprises transgenesis or genome editing.

[0103] “Alleles” or “allelic variants” are alternative forms of a given gene, located at substantially the same chromosomal position. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small Insertion / Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.

[0104] “Gene editing” or “genome editing” is a type of genetic engineering or genetic modification technique in which DNA is inserted, replaced, or removed from a genome and which can be obtained by using a variety of techniques such as “gene shuffling” or “directed evolution” consisting of iterations of DNA shuffling followed by appropriate screening and / or selection to generate variants of nucleic acids or portions thereof encoding proteins having a modified biological activity (Castle et al., (2004) Science 304(5674): 1151-4; US patents 5,811,238 and 6,395,547), or with “T-DNA activation” tagging (Hayashi et al. Science (1992) 1350-1353), where the resulting transgenic plants show dominant phenotypes due to modified expression of genes close to the introduced promoter, or with “TILLING” (Targeted Induced Local Lesions In Genomes) and refers to a mutagenesis technology useful to generate and / or identify nucleic acids encoding proteins with modified expression and / or activity. TILLING also allows selection of plants carrying such mutant variants. Methods for TILLING are well known in the art (McCallum et al., (2000) Nat Biotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet 5(2): 145-50). Another technique uses artificially engineered nucleases like Zinc finger nucleases, Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR / Cas system, and engineered meganuclease such as re-engineered homing endonucleases (Esvelt, KM.; Wang, HH. (2013), Mol Syst Biol 9 (1): 641; Tan, WS.et al. (2012), Adv Genet 80: 37-97; Puchta, H.; Fauser, F. (2013), Int. J. Dev. Biol 57: 629-637). “Genome editing”, “gene editing” or “genome engineering” as used herein also refers to targeted modification of genomic DNA in which the DNA may be inserted, deleted, modified or replaced in the genome. Genome editing may use sequence-specific enzymes (such as endonuclease, nickases, base conversion enzymes) and / or donor nucleic acids (e.g. dsDNA, oligo’s) to introduce desired changes in the DNA. Sequencespecific nucleases that can be programmed to recognize specific DNA sequences include meganucleases (MGNs), zinefinger nucleases (ZFNs), TAL-effector nucleases (TALENs) and RNA-guided or DNA-guided nucleases such as Cas9, Cpfl, CasX, CasY, C2cl, 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. 15:917-926, 2017; Nakade et al., Bioengineered Vol 8, No.3:265-273, 2017; Burstein et al., Nature 542, 37- 241; Komor et al., Nature 533, 420-424, 2016; all incorporated herein by reference). Donor nucleic acids can be used as a template for repair of the DNA break induced by a sequence specific nuclease. Donor nucleic acids can also be used as such for genome editing without DNA break induction to introduce a desired change into the genomic DNA. Genome editing can also refer to the introduction of a specific mutation at a specific position of the genome of a cell. The gene edit may be introduced by precise editing applying more advanced technologies e.g. using a CRISPR Cas system and a donor DNA, or a CRISPR Cas system linked to mutagenic activity such as a deaminase (WO15133554, WC17070632).

[0105] Overall, the present invention and experimental approaches described herein provide a suite of methods and assays on single cell level (which are useful either separate or in combination), including but not limited to:

[0106] - Methods for target protoplast screening, sorting and selection (comprising for example flow cytometry, microfluidics, cell seeding);

[0107] - Methods for (single) protoplast phenotyping;

[0108] - Protoplast-based hypersensitive response assays (based on ROS production, elevation of intracellular Ca2+levels, modified levels of ATP, modulated gene expression, modulated protein production, detection of reporter molecules, aptamer characterisation);

[0109] - Methods for the high-throughput sorting of protoplasts and for the selection of prototoplasts displaying a hypersensitive response;

[0110] - Methods for the identification of new fungal disease resistance genes (for example in soy, oilseed rape, rice, or wheat),

[0111] • from natural sources (via bioinformatics (including ab initio structure prediction methods) & synthetic biology), or

[0112] • through modification of an identified receptor; receptor engineering (such as, but not limited to direct detection engineering; decoy engineering; nucleotide-binding leucine-rich repeat proteins (NLR) with engineered integrated domains; extended effector recognition; R genes encoding immune receptors interacting with structurally different classes of effectors; immune receptor relocalization);

[0113] - Methods for the protoplast-based identification or validation of disease or pathogen resistance genes:

[0114] • R genes (cell surface (receptor-like kinases or RLKs, receptor like proteins or RLPs) and intracellular (NLR sensors, NLR helpers)),

[0115] • S genes (multiplex genome editing);

[0116] - Methods for the identification of disease and / or pathogen avirulence effectors; - Methods for the identification of matching plant immune receptors and disease or pathogen avirulence effectors (including identification of host factors required for immune receptor function);

[0117] - Methods for the identification and selection of mutually potentiating cell-surface pattern recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat proteins (NLRs) for the development of durable and stronger disease and / or pathogen resistance in plants;

[0118] - Method for the selection of plant immune receptor gene combinations for the development of stacking concepts for the production of plants with enhanced disease and / or pathogen resistance (stacking of R genes encoding immune receptors interacting with different structural classes of effectors);

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

[0120] - Methods for the efficient discovery and functional characterization of pathogeninducible promoters;

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

[0122] - Methods for the optimization of promoter-NLR expression cassettes;

[0123] - Methods for the selection of products obtained from “in planta directed evolution of disease resistance” strategies;

[0124] - Methods for the enrichment of targeted transfection events via enrichment of cells cotransfected with a reporter gene.

[0125] A person skilled in the art will appreciate that the applicability of the abovementioned methods and assays, and as exemplified hereunder, is not limited to identification and characterisation of pathogen effectors and / or plant disease resistance genes but can also be used to answer various other scientific questions for which assays on single cell level are beneficial. By way of example, the methods of the present invention can be applied in studies relating to cell death.

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

[0127] - changes in intracellular Ca2+levels or alterations in cellular redox status (for review see Kostyuk et al., 2020);

[0128] - cellular proton efflux leading to medium a I kal i nization ; - MAP kinase activation;

[0129] - changes in the perception, biosynthesis, degradation or signaling output of immune- associated plant hormones such as salicylic acid, jasmonic acid and ethylene, or alterations in the concentration of downstream signaling molecules such as azelaic acid, glycerol-3-phosphate, pipecolic acid, and N-hydroxypipecolic acid. Other hormonal players include abscisic acid, auxins, gibberellins, cytokinins, brassinosteroids and strigolactones (for recent review see Burger and Chory, 2019);

[0130] - fluctuations in growth vs defense trade-offs (e.g. alterations in cell division rates, for review see Levak et al., 2021)).

[0131] Reference list

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[0136] Lindner S, Keller B, Singh SP, et al. Single residues in the LRR domain of the wheat PM3A immune receptor can control the strength and the spectrum of the immune response. Plant J. 2020;104(l):200-214. doi: 10.1111 / tpj.14917

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[0142] Furuhata Y, Sakai A, Murakami T, Nagasaki A, Kato Y. Bioluminescent imaging of Arabidopsis thaliana using an enhanced Nano-lantern luminescence reporter system. PLoS One. 2020;15(l):e0227477. Published 2020 Jan 3. doi: 10.1371 / journal.pone.0227477

[0143] Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol. 2004;55:373-399. doi:10.1146 / annurev.arplant.55.031903.141701

[0144] Zipfel C, Robatzek S, Navarro L, et al. Bacterial disease resistance in Arabidopsis through flagellin perception. Nature. 2004;428(6984):764-767. doi:10.1038 / nature02485

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[0146] Shen R, Liu P, Zhang Y, et al. Sensitive Detection of Single-Cell Secreted H2O2 by Integrating a Microfluidic Droplet Sensor and Au Nanoclusters. Anal Chem. 2018;90(7):4478-4484. doi:10.1021 / acs.analchem.7b04798

[0147] Zhou T, Xu W, Hirani AH, Liu Z, Tuan PA, Ayele BT, Daayf F, McVetty PBE, Duncan RW, Li G. Transcriptional Insight Into Brassica napus Resistance Genes LepR3 and Rlm2- Mediated Defense Response Against the Leptosphaeria maculans Infection. Front Plant Sci. 2019 Jul 2;10:823. doi: 10.3389 / f pls.2019.00823. PMID: 31333690; PMCID: PMC6615431.

[0148] Becker MG, Zhang X, Walker PL, Wan JC, Millar JL, Khan D, Granger MJ, Cavers JD, Chan AC, Fernando DWG, Belmonte MF. Transcriptome analysis of the Brassica napus- Leptosphaeria maculans pathosystem identifies receptor, signaling and structural genes underlying plant resistance. Plant J. 2017 May;90(3):573-586. doi: 10.1111 / tpj.13514. PMID: 28222234. Bargmann BO, Birnbaum KD. Fluorescence activated cell sorting of plant protoplasts. J Vis Exp. 2010;(36):1673. Published 2010 Feb 18. doi:10.3791 / 1673

[0149] Galbraith DW, Sun G. Flow Cytometry and Sorting in Arabidopsis. Methods Mol Biol. 2021;2200:255-294. doi:10.1007 / 978-l-0716-0880-7_12

[0150] Yu Z, Boehm CR, Hibberd JM, et al. Droplet-based microfluidic analysis and screening of single plant cells. PLoS One. 2018;13(5):e0196810. Published 2018 May 3. doi: 10.1371 / journal.pone.0196810

[0151] Qian Y, Li J, Zhao S, et al. Programmable RNA sensing for cell monitoring and manipulation. Nature. 2022;610(7933):713-721. doi:10.1038 / s41586-022-05280-l

[0152] Kaseniit KE, Katz N, Kolber NS, et al. Modular, programmable RNA sensing using ADAR editing in living cells. Nat Biotechnol. 2022;10.1038 / s41587-022-01493-x. doi:10.1038 / s41587-022-01493-x

[0153] Burger M, Chory J. Stressed Out About Hormones: How Plants Orchestrate Immunity. Cell Host Microbe. 2019;26(2):163-172. doi:10.1016 / j.chom.2019.07.006

[0154] Kostyuk Al, Panova AS, Kokova AD, Kotova DA, Maltsev DI, Podgorny OV, Belousov VV, Bilan DS. In Vivo Imaging with Genetically Encoded Redox Biosensors. I nt J Mol Sci. 2020 Oct 31;21(21):8164. doi: 10.3390 / ijms21218164. PMID: 33142884; PMCID: PMC7662651.

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[0156] 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

[0157] Borhan, M Hossein et al. “Molecular Interactions Between Leptosphaeria maculans and Brassica Species.” Annual review of phytopathology vol. 60 (2022): 237-257. doi:10.1146 / annurev-phyto-021621-120602

[0158] Ma, Lisong et al. “Leptosphaeria maculans Effector Protein AvrLml Modulates Plant Immunity by Enhancing MAP Kinase 9 Phosphorylation.” iScience vol. 3 (2018): 177-191. doi:10.1016 / j.isci.2018.04.015 van Esse, H Peter et al. “The Cladosporium fulvum virulence protein Avr2 inhibits host proteases required for basal defense.” The Plant cell vol. 20,7 (2008): 1948-63. doi:10.1105 / tpc.108.059394 SEQUENCE LISTING

[0159] The sequence listing contained in the file named “221151WO01 SEQUENCE LISTING. xml”, which is 101 kilobytes (size as measured in Microsoft Windows®), contains 49 sequences SEQ ID NO: 1 through SEQ ID NO: 49 is filed herewith by electronic submission and is incorporated by reference herein. In the description and examples, reference is made to the following sequences:

[0160] SEQ ID NO: 1: eGFP coding sequence

[0161] SEQ ID NO: 2: Constitutive Cauliflower mosaic virus 35S promoter

[0162] SEQ ID NO: 3: RGA4 coding sequence

[0163] SEQ ID NO: 4: constitutive maize ubiquitinl promoter

[0164] SEQ ID NO: 5: RGA5 coding sequence

[0165] SEQ ID NO: 6: constitutive Arabidopsis UbiquitinlO promoter

[0166] SEQ ID NO: 7: Dummy plasmid

[0167] SEQ ID NO: 8: PM3A coding sequence

[0168] SEQ ID NO: 9: Avr-PM3A coding sequence

[0169] SEQ ID NO: 10: PM3A-R1334E coding sequence

[0170] SEQ ID NO: 11: PM3A-S1335R coding sequence

[0171] SEQ ID NO: 12: Avr-PM3A-Q112N coding sequence

[0172] SEQ ID NO: 13: PM3A-D502V coding sequence

[0173] SEQ ID NO: 14: deGFP_l coding sequence

[0174] SEQ ID NO: 15: deGFP_2 coding sequence

[0175] SEQ ID NO: 16: deGFP_3 coding sequence

[0176] SEQ ID NO: 17: RGA4_FW1 primer sequence

[0177] SEQ ID NO: 18: RGA4_RV1 primer sequence

[0178] SEQ ID NO: 19: RGA4_FW2 primer sequence

[0179] SEQ ID NO: 20: RGA4_RV2 primer sequence SEQ ID NO: 21 PM3A_FW1 primer sequence

[0180] SEQ ID NO: 22 PM3A_RV1 primer sequence

[0181] SEQ ID NO: 23 Green enhanced Nanolantern (GeNL) coding sequence

[0182] SEQ ID NO: 24 Firefly luciferase (fLUC) coding sequence

[0183] SEQ ID NO: 25 Flg22 amino acid sequence

[0184] SEQ ID NO: 26 eGFP protein sequence

[0185] SEQ ID NO: 27 RGA4 protein sequence

[0186] SEQ ID NO: 28 RGA5 protein sequence

[0187] SEQ ID NO: 29 PM3A protein sequence

[0188] SEQ ID NO: 30 Avr-PM3A protein sequence

[0189] SEQ ID NO: 31 PM3A-R1334E protein sequence

[0190] SEQ ID NO: 32 PM3A-S1335R protein sequence

[0191] SEQ ID NO: 33 Avr-PM3A-Q112N protein sequence

[0192] SEQ ID NO: 34 PM3A-D502V protein sequence

[0193] SEQ ID NO: 35 deGFP_l protein sequence

[0194] SEQ ID NO: 36 deGFP_2 protein sequence

[0195] SEQ ID NO: 37 deGFP_3 protein sequence

[0196] SEQ ID NO: 38 Green enhanced Nanolantern (GeNL) protein sequence

[0197] SEQ ID NO: 39 Firefly luciferase (fLUC) protein sequence

[0198] SEQ ID NO: 40 LepR3 coding sequence

[0199] SEQ ID NO: 41 Rlm3 coding sequence

[0200] SEQ ID NO: 42 Rlm9 coding sequence

[0201] SEQ ID NO: 43 AvrLml (WT) coding sequence

[0202] SEQ ID NO: 44 AvrLm3 (WT) coding sequence

[0203] SEQ ID NO: 45 AvrLm5-9 (WT) coding sequence

[0204] SEQ ID NO: 46 PRla signal peptide coding sequence

[0205] SEQ ID NO: 47 AvrLml* (AvrLml with PRla signal peptide) coding sequence

[0206] SEQ ID NO: 48: AvrLm3* (AvrLm3 with PRla signal peptide) coding sequence SEQ ID NO: 49: Avrl_m5-9* (Avrl_m5-9 with PRla signal peptide) coding sequence

[0207] EXAMPLES

[0208] The present invention will now be described with reference to the following examples, which are given by way of illustration alone. The following examples are not intended to completely define or otherwise limit the scope of the invention.

[0209] DNA manipulation: unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).

[0210] Example 1: General methods

[0211] Cloning methods and plasmid construction

[0212] Unless indicated otherwise, cloning procedures carried out for the purpose of the current invention including restriction digest, agarose gel electrophoresis, purification and ligation of nucleic acids, transformation, selection and cultivation of bacterial cells are performed as described (Sambrook J, Fritsch EF and Maniatis T (1989)). Sequence analysis of recombinant DNA was performed by LGC Genomics (Berlin, Germany) using the Sanger technology (Sanger et al., 1977). Restriction endonucleases and Gibson Assembly reagents used to construct plasmids are from New England Biolabs (Ipswich, MA, USA). Oligonucleotides are synthesized by Integrated DNA Technologies (Coralville, IA, USA). Codon-optimized genes are from Genewiz (South Plainfield, NJ, USA).

[0213] All plasmids were transformed in E. coli for propagation and isolated using a ZymoPure II Plasmid Gigaprep kit for DNA purification (Zymo Research, Irvine, CA, USA).

[0214] Wheat protoplast preparation and transfection

[0215] Transformation of wheat protoplast cells was performed as described by Shan et al. (2014) with minor modifications. Protoplasts are isolated from the youngest fully developed leaf of 10-day-old aseptically grown wheat seedlings. Healthy leaves are bundled in stacks of five and cut into fine strips with a sharp razor blade. The strips are infiltrated with cell wall-dissolving enzyme solution (1.5% cellulase RIO and 0.75% macerozyme RIO in 10 mM KCI and 0.6 M mannitol, pH 7.5) and incubated overnight in the dark with gentle shaking (40 rpm) at 24° C. After enzymatic digestion, the released protoplasts are collected by filtering the mixture through 40-pm nylon meshes and resuspended in W5 solution. The resuspended protoplasts are kept on ice and allowed to settle by gravity, after which the cell pellet is resuspended in MMG. For transformation, 200 pl of cells (2.5 x 105) are mixed with 20 pg plasmid DNA and 220 pl of freshly prepared polyethylene glycol (PEG) solution. The mixture is incubated for 15- 20 min in the dark. After removing the PEG solution, the protoplasts are resuspended in 2 ml of W5 solution, transferred into six-well plates, and incubated at 24° C.

[0216] Rice protoplast preparation and transfection

[0217] T ransformation of rice protoplast cells was performed as described by Wang et al. (2014) with minor modifications. Protoplasts were prepared from the sheaths of 3-week-old aseptically grown rice seedlings. Healthy stems and sheaths were bundled in stacks of 20 and cut into fine strips with a sharp razor blade. The strips were then infiltrated with cell wall-dissolving enzyme solution (1.5% cellulase R10 and 0.75% macerozyme R10 in 10 mM KCI and 0.6 M mannitol, pH 7.5) and incubated overnight in the dark with gentle shaking (40 rpm) at 24° C. After enzymatic digestion, the released protoplasts were collected by filtering the mixture through 40-pm nylon meshes and resuspended in W5 solution. The resuspended protoplasts were washed with W5 solution, after which the cell pellet was suspended in MMG solution at a density of 2.5 million cells / ml. For transformation, 200 pl of cells (5 xlO5) were mixed with 20 pg plasmid DNA and 220 pl of freshly prepared polyethylene glycol (PEG) solution. The mixture was incubated for 15-20 min in the dark. After removing the PEG solution, the protoplasts were resuspended in 2 ml of Wl solution, transferred into six-well plates, and incubated at 24° C for at least 48h. Finally, protoplasts were collected by centrifuging at 12,000 rpm for 1 min at room temperature and the pelleted fraction was stored at minus 80° C until further analysis.

[0218] Oilseed rape protoplast preparation and transfection

[0219] Oilseed rape protoplasts were isolated from the leaves of 4- to 7-week-old aseptically grown plants and transfected essentially as described for wheat cells. After removing the PEG solution, transfected cells are resuspended in 2 ml W5 solution, and incubated at 24° C. Soybean protoplast isolation and transfection

[0220] Protoplasts were isolated from the unifoliate leaves of 6-day-old seedlings. Healthy leaves are cut into fine strips with a sharp razor blade and transferred to a Petri dish. The strips are vacuum infiltrated with cell wall-dissolving enzyme solution containing 0.25% cellulase RIO and 0.25% macerozyme RIO and incubated overnight in the dark with gentle shaking (40 rpm) at 24° C. After enzymatic digestion, the released protoplasts are collected by filtering the mixture through 40-pm nylon meshes and resuspended in W5 solution. The resuspended protoplasts are washed with W5 solution, after which the cell pellet is resuspended in MMG solution. For transformation, 200 pl of cells (4 x 105) are mixed with 20 pg plasmid DNA and 220 pl of freshly prepared PEG solution. The mixture is incubated for 13-15 min in the dark. After removing the PEG solution, the protoplasts are resuspended in 2 ml of Wl solution, transferred into six-well plates, and incubated at 24 ° C.

[0221] Example 2: Development of a single-cell screening workflow using a fluorescent reporter

[0222] Perception of pathogen-secreted effector proteins by corresponding plant resistance genes most often results in activation of the so-called hypersensitive response (HR). Characterized by rapid cell death at the site of infection, HR is a complex multicellular process correlated with numerous physical, physiological and molecular alterations, including deposition of lignin and callose into the plant cell wall as well as the production of anti-microbial compounds such as phytoalexins, hydrolytic enzymes and pathogenesis-related proteins. Owing to the high amount of cellular energy needed to mount these immune responses, diminished activity of a co-transformed luciferase reporter can be used as a proxy for HR activation (Cesari et al., 2014; Saur et al., 2021). Yet, this approach relies on the consumption of large populations of cells and is characterized by high variability in luciferase measurements across biological replicates (Saur et al., 2021), preventing its use for characterization and screening of individual protoplasts.

[0223] To determine if HR-expressing cells can also be identified based on expression of a fluorescent reporter protein, a plasmid comprising enhanced GFP (eGFP, SEQ ID NO: 1) driven by the strong constitutive 35S promoter of Cauliflower mosaic virus was created (p35S, SEQ ID NO: 2). A second construct harboring the rice auto-active immune sensor RGA4 (OsRGA4, SEQ ID NO: 3) under control of the constitutive maize ubiquitinl promoter (pZmUbil, SEQ ID NO: 4) was used to activate HR. The disease resistance gene RGA4 encodes a nucleotide binding and leucine-rich repeat (NBS-LRR) domain protein that mediates resistance to the fungal pathogen Magnaporthe oryzae, causal agent of rice blast disease. Previous studies in protoplasts have shown that constitutive RGA4 expression triggers an effector-independent HR that is repressed by the presence of a second NBS-LRR, RGA5 (Cesari et al., 2014; OsRGA5, SEQ ID NO: 5). The eGFP and RGA4 constructs were mixed in equal ratio and transformed into protoplasts isolated from a blast-susceptible rice variety lacking RGA5. Co-transfection of eGFP and a plasmid lacking RGA4 served as the empty vector control.

[0224] HR induction was monitored by measuring GFP fluorescence intensity at 48 h after transformation (Figure 1). GFP fluorescence was excited at 405 ± 20 nm and collected at 485 ± 9 nm. Compared to the empty vector control, expression of OsRGA4 resulted in a more than 10-fold reduction in GFP fluorescence intensity, indicative of HR activation (Figure 1A). In a similar vein, RGA4 under control of the constitutive Arabidopsis UbiquitinlO promoter (pAtUbilO, SEQ ID NO: 6) also strongly decreased GFP fluorescence relative to the empty vector control in soybean and oilseed rape protoplasts, demonstrating the efficacy of the reporter assay in homologous as well as heterologous expression systems (see Figures IB and 1C).

[0225] Next, the screening workflow according to an embodiment of the invention using a GFP reporter 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 against 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 abolish, enhance or alter the strength of the HR in intact plant assays (McNally et al, 2018). Similarly, polymorphic residues in PM3A are known to determine the strength and the spectrum of HR when transiently expressed in Nicotiana benthamiana using Agrobacterium infiltration (Lindner et al., 2020).

[0226] To test whether whole plant experiments can be replicated in the cell-based method according to an embodiment of the invention using the GFP reporter assay, protoplasts isolated from the wheat cultivar Fielder, which lacks PM3A, were co-transfected with cDNAs of different Avr-PM3A and PM3A alleles. A reference sample providing a readout on GFP expression in the absence of an effector and R gene was included as well. This reference sample consisted of the GFP reporter and a ‘dummy’ plasmid (SEQ ID NO: 7). In the test samples, the dummy plasmid construct was substituted by plasmids encoding the PM3A and Avr-PM3A cDNAs of interest. Co-transfection of either PM3A or Avr-PM3A together with GFP and dummy plasmid served as negative control. The concentration of dummy plasmid was adjusted to ensure equal amounts of total plasmid DNA in all transfections.

[0227] As shown in Figure 2, co-transfection of wild-type PM3A (SEQ ID NO: 8) and Avr-PM3A (SEQ ID NO: 9) triggered a significant reduction in GFP signal as compared to the reference sample (‘GFP Ctrl’), demonstrating effective recognition of Avr-PM3A by PM3A. Interestingly, combining expression of Avr-PM3a with the inactive R1334E allele of PM3A (SEQ ID NO: 10) did not result in HR, while expression of Avr-PM3A and the PM3A gain-of-function S1335R (SEQ ID NO: 11) allele showed a stronger decrease in GFP relative to that observed with wild-type PM3A. Substituting Avr-PM3A with Q112N (SEQ ID NO: 12), a variant which strongly enhances the HR elicited by PM3a recognition in intact leaves (McNally et al., 2018), led to a further reduction in GFP reporter activity. The strongest decrease in GFP reporter activity was however observed upon coexpression of the PM3A-S1335R gain-of-function variant and Avr-PM3A-Q112N, corroborating earlier findings in N. benthamiana (Lindner et al., 2020; McNally et al., 2018). Importantly, except for the auto-active PM3A-D502V mutant (SEQ ID NO: 13), which carries a mutation in the C-terminal region of the ARC2 domain (Lindner et al., 2020), none of the Avr-PM3A or PM3A alleles significantly reduced GFP activity compared to the empty vector control when expressed alone. Together with abovementioned analysis of RGA4 in rice, soybean and canola protoplasts, these results clearly show that quantification of modulated expression of a co-transfected GFP reporter can be used to identify the onset of HR in miniaturized cell-based assays whilst being predictive of immune activation in whole plants.

[0228] Example 3: Effect of GFP protein stability on assay sensitivity

[0229] With a half-life of more than 24 h, eGFP is a highly stable protein which allows its accumulation and easy detection in cells. Yet, this stability may also limit its application in single-cell studies since even a rapid and complete block in transcription can only be detected at the level of reporter activity after a protracted period of time. To assess the impact of GFP stability on the performance of the fluorescent reporter assay according to an embodiment of the invention, different eGFP variants with enhanced turnover rates were tested (see Figure 3). These variants included an eGFP protein fusion harboring a 164 AA destabilization domain of a putative barley 1-aminocyclopropane-l-carboxylate synthase (Dong et al., 2006; ‘deGFP_2’, SEQ ID No: 15, SEQ ID NO: 36), a reportedly unstable GFP variant 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 halflife in bacteria of less than Ih (Garamella et al, 2019; ‘deGFP_l’, 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 in rice protoplasts along with either the RGA4-encoding construct or an empty vector control and GFP reporter activity was measured at 48 h post transformation. While expression of RGA4 resulted in a 12-fold reduction in eGFP activity compared to the empty vector control, an even stronger decrease (up to 17-fold) was observed for the truncated eGFP variant (deGFP_l in Figure 3). In contrast, expression of the other GFP variants yielded a weaker reporter response relative to that observed with native eGFP (3-fold reduction in GFP activity for deGFP_2 and 7-fold reduction in GFP activity for deGFP_3). These findings demonstrate that destabilized protein variants can be used to optimize the sensitivity of the method according to an embodiment of the invention when using a GFP reporter assay.

[0230] Example 4: Validation of a single cell screening workflow using a fluorescent reporter

[0231] The prospect of utilizing a fluorescent reporter assay in a method for characterization and screening of individual plant protoplasts according to an embodiment of the invention is highly attractive. Whereas high-throughput screening of whole plants is substantially limited by their slow growth and size, millions of protoplasts may be processed in a matter of hours using various cell sorting and dispensing technologies. However, to date protoplasts have been predominantly extracted and analyzed in bulk, limiting their use.

[0232] To evaluate the use of GFP as a screenable marker at the single-cell level according to an embodiment of the invention, soybean protoplasts were transfected with 35S-driven eGFP combined with auto-active RGA4 at a ratio of 10:1. Under our experimental conditions, transfection efficiency in soy protoplasts averages 30-40%. To remove nontransfected cells from downstream analyses, a two-step sorting approach was used where samples were first sorted at one day post transfection to enrich for transfected cells. The collected cells were then incubated for another 48h before being resorted based on GFP fluorescence level. Protoplasts were transferred to a microfluidics cartridge which was loaded into a 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 enables binning by forward and side scatter, but at significantly lower sorting pressures (< 2 psi) to help preserve cell viability.

[0233] Live protoplasts were gated for data acquisition based on their forward and side scatter properties and appropriate laser excitation and collection channels were chosen. For detection and isolation of GFP-expressing cells, samples were excited with a 488 nm laser and emission was quantified at 533 nm (FITC / GFP detection channel). To quantify autofluorescence, the PE / PI detection channel was used (excitation at 488 nm, collection at 585 nm). A scatter plot with FITC versus PE was generated with a forward scatter threshold of 100. Using Wl buffer as sheath fluid, protoplasts transfected with an empty vector plasmid were analyzed first, which enabled setting the collection gate for GFP positive protoplasts accurately. Next, the sample containing GFP-transfected cells was run. Compared to the negative control, a new protoplast population became apparent as a group of cells with a higher FITC / PE ratio in the scatter plot. A collection gate was drawn around this population and approximately 2,000 GFP-positive cells were dispensed in a 6-well plate and incubated at 24 ° C. Two days later, the collected cells were re-loaded onto the Hana sorting device.

[0234] Two fluorescent enriched populations, protoplasts with low GFP intensity (20 < FITC < 500) and with high intensity (FITC > 3,000), were selected for sorting corresponding to 5.7% and 19.2% of the total population, respectively. Following sorting, individual protoplasts were dispensed into 96-well plates containing 10 pl 2x Phire Tissue Direct PCR extraction buffer (Thermo Fisher Scientific). The presence / absence of the RGA4 plasmid in each of the collected cells 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 in a final volume of 20 pl containing the following reagents: 10 pl of 2x Phire plant reaction buffer, 1 pl of 10 pM forward primer, 1 pl of 10 pM reverse primer, 0.4 pl of Phire Polymerase, 1 pl of single-cell lysate as template and 6.6 pl of nuclease-free water. The PCR profile was 1 cycle at 98 ° C for 5 min; 40 cycles at 98 ° C for 5 s, 60 ° C for 5 s and 72 ° C for 25 s; and 1 cycle at 72 ° C for 1 min. One microliter of the PCR reaction was then used as template in a second PCR containing the following reagents: 12.5 pl of Q5 high-fidelity master mix (NEB), 1.25 pl of 10 pM forward primer RGA4_FW2 (SEQ ID NO: 19), 1.25 pl of 10 pM reverse primer RGA4_RV2 (SEQ ID NO: 20) and 9 pl of nuclease-free water. The PCR profile was 1 cycle at 98 ° C for 2 min; 30 cycles at 98 C for 10 s, 65 ° C for 30 s and 72 ° C for 32 s; and 1 cycle at 72 ° C for 2 min. The resulting PCR products were either analyzed on a 1% agarose gel stained with SybrSafe and visualized by UV-trans-illumination or loaded onto a Fragment Analyzer capillary electrophoresis system.

[0235] As shown in Figure 4A, gating on GFP intensity successfully separated RGA4-expressing protoplasts from non-expressing cells, with 0 out of 60 cells with high GFP intensity showing amplification of RGA4 compared to 12 out of 25 cells with low GFP intensity. Repeating the experiment led to similar results with 44 out of 48 cells with low GFP intensity but none of the highly fluorescent cells (0 out 6) scoring positive for RGA4 (see Figure 4B).

[0236] To further evaluate the performance of eGFP as a screenable marker at the single-cell level according to an embodiment of the invention, combinations of Avr-PM3A and the GFP reporter protein were co-expressed in wheat protoplasts together with either wildtype PM3A (Figure 5, left column, lower panel) or its ‘loss-of-function’ PM3A-R1334E variant (Figure 5, left column, upper panel). Two days after transfection (2dpt), protoplasts from both populations were pooled together in equal ratio and injected into a Biosorter device (Union Biometrica, Figure 5 middle column) which allows for sorting of large fragile cells such as individual protoplasts. The Biosorter is a continuous flowbased system which operates at low pressure and uses gentle air stream diverter for sorting and which is capable of analyzing, sorting and dispensing fragile cells, such as individual plant protoplasts, ranging in size from 10 to 1,500 pm (Figure 5, middle column). In the Biosorter, pooled protoplasts were introduced into a flow cell (1) where they were surrounded by a sheath solution which hydrodynamically (sheath flow 2) focused them into the center of the stream for interrogation by multiple lasers (3). W5 buffer was used as sheath fluid. The Biosorter further simultaneously records the intensities of extinction, forward scatter (4) allowing optical density and size detection, and fluorescence (5) along the length of each protoplast. The device also allows brightfield image capture (camera (6), bright-field illumination (7)) of each individual protoplast sample before exiting the flow channel (1).

[0237] Cells with high, moderate and low GFP intensity were gated separately, and single, positive cells were selectively sorted and dispensed in a 96-well plate filled with 5 pl 2x Phire Tissue Direct PCR extraction buffer (Thermo Fisher Scientific). The Biosorter uses a gentle air stream assisted sorting (8) which allows collection of sorted (9) individual protoplasts with desired characteristics in multiwell plates or into bulk containers (10). Unsorted (11) cells are collected in a waste / sample recovery container (12) by the system.

[0238] To test whether the fluorescence intensity of sorted cells can be used as surrogate for HR induced upon PM3A recognition, dispensed cells were subjected to single-cell PCR (Figure 5, right column). Each reaction was run in a total volume of 21 pl containing the following reagents: 10 pl of 2x Q5 High Fidelity Master Mix buffer, 0.1 pl of 100 pM forward primer PM3A_FW1 (SEQ ID NO: 21), 0.1 pl of 100 pM reverse primer PM3A_RV1 (SEQ ID NO: 22), 2 pl of lOx-diluted single-cell lysate as template and 8.8 pl of nuclease- free water. The PCR profile was 1 cycle at 98 ° C for 2 min; 40 cycles at 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.

[0239] Table 1: Correlation of PM3A-allele with the GFP fluorescence level in selected protoplasts

[0240] Although mixed sequencing peaks indicative of cell doublets were observed in some reactions, the results showed a tight correlation between the GFP intensity of individual cells and the strength of the PM3A-elicited immune response. Indeed, while most cells with high GFP intensity contained the loss-of-function PM3A-R1334E allele and up to 90% of the weakly fluorescent cells harbored wild-type PM3A, the population gated as moderate GFP comprised a mixture of PM3A and PM3A-R1334E transfected cells. Together with the obtained results in rice protoplasts, these findings illustrate the applicability of GFP as a screenable marker in an embodiment of the invention for automated characterization and sorting of single protoplasts, thus facilitating high- throughput screening and retrieval of HR-expressing cells based on expression of a fluorescent reporter. Further development work is ongoing, aiming to develop an integrated workflow that will allow rapid screening of extensive genetic libraries in protoplasts isolated from different crops of interest.

[0241] Example 5: Optimalization of single-cell screening assay via time-resolved single cell measurements

[0242] Gene expression is an intrinsically stochastic process manifested as fluctuations in the abundance of expressed molecules at the single-cell level, and variability and heterogeneity within populations of genetically identical cells. Fluctuations in the biochemical process of gene expression (intrinsic noise) and fluctuations in other cellular components (extrinsic noise) both contribute substantially to overall cel l-to-cel I variation (Popovic et al, 2016).

[0243] To account for this variation and to normalize for differences in transfection efficiency of individual cells, time-resolved GFP reporter measurements were attempted using the CellCelector single-cell isolation platform which enables individual cells to be monitored over time by physically entrapping them within nanowell arrays (Automated Lab Solutions). To this end, combinations of Avr-PM3A and the GFP reporter protein are coexpressed in wheat protoplasts (cultivar Fielder) along with either wild-type PM3A or its ‘loss-of-function’ R1334E variant. Immediately after transfection, protoplasts from both populations are pooled together in equal ratio and seeded onto a six-well plate with approximately 60,000 nanowells per well. One milliliter of the cell suspension containing 60,000 cells is slowly dispensed across one well of the six-well plate, to achieve an average occupancy of one cell per nanowell. Cells are then left to settle for 5 min at room temperature and centrifuged for 5 min at 300 x g. Individual nanowells are scanned every two hours in brightfield and fluorescence (GFP and mCherry channels). At least 50 cells showing either constant or strongly reduced GFP expression between 12 and 48 hours post transfection are identified and selected for picking using liquid buffered glass capillaries. The picked colonies are then transferred to a 96-well plate and subjected to single-cell PCR and Sanger sequencing as described above. Example 6: Development of a single-cell screening workflow using a luminescent screenable marker

[0244] Depending on the donor material used, isolated protoplasts sometimes show strong chlorophyll-derived autofluorescence, which can hamper accurate fluorescence detection. Given that luciferases can emit light without excitation induced by an external light, the use of a luminescent reporter system offers an attractive alternative to fluorescence-based assays. However, commonly used luciferase genes, including firefly, Ren i I la and Vargula luciferases, produce relatively weak light emission, questioning their application for single-cell luminescence imaging. Indeed, Ow et al. (1986) reported that tobacco cells transfected with firefly luciferase needed to be exposed for more than 24 h in order to detect the luminescence. Hence, implementation of a luminescence-based single-cell reporter assay will require the use of improved luciferase reporters that are brighter and more transmissive.

[0245] One class of recently engineered luciferases are the Nano-lanterns, which are chimeras of NanoLuciferase, one of the brightest luciferases, and fluorescent proteins of different color. Harnessing bioluminescence resonance energy transfer (BRET) to shift the emission wavelength of the luminescent reporter, Nano-lanterns are sufficiently bright to be detectable as single molecules and can also be tracked as fluorescent proteins (Suzuki et al., 2016; Furuhata et al., 2020).

[0246] To evaluate the feasibility of luminescent 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 NanoLuciferase and mNeonGreen protein as BRET acceptor (Suzuki et al., 2016). GeNL driven by the maize Ubiquitinl promoter (SEQ ID NO: 4) was co-transfected with RGA4 (SEQ ID NO: 3) or an empty vector control and its luminescence was compared to that of a firefly luciferase reporter (fLUC, SEQ ID NO: 24) expressed from a 35S promoter (SEQ ID NO: 2). To detect GeNL activity, 100 pl of protoplast culture was transferred to a white 96-well plate to which 10 pl of Nano-Gio luciferase assay reagent (Promega; containing furimazine substrate) was added, while fLuc activity was detected by adding 10 pl of Dual-Glo luciferase reagent (Promega; containing D-luciferin). Luminescence was measured at 3 and 10 min after adding Nano-Gio and Dual-Glo substrate, respectively, using a Tecan microplate reader equipped with a luminescence detection unit. To assess luciferase-generated reporter signal independent of wavelength, luminescence was measured without applying optical filters. As shown in Figure 6, GeNL showed significantly higher luminescence than fLuc in the undiluted protoplast sample (GeNL / fLUC = 2.29) which comprised approximately 50,000 cells. Interestingly, the difference in luminescence intensity between GeNL and fLUC became more pronounced when diluting the sample (GeNL / fLUC = 59.2 for lOx dilution, GeNL / fLUC = 54.3 for 200x dilution and GeNL / fLUC = 49.36 for 400x dilution). Even at the highest dilution, corresponding to approximately 125 cells, GeNL- transfected cells emitted a bright luminescent signal that was almost 50-fold higher compared to that of fLUC, while co-expressing RGA4 strongly inhibited GeNL luminescence. To further test the performance of the GeNL-based reporter assay at the single-cell level, rice cells transiently expressing fLUC or GeNL and RGA4 were then injected into a Hana single cell dispenser device, gated based on their level of autofluorescence, and dispensed into a white 96-well plate filled with 49 pl Wl buffer and 5 pl Nano-Gio or 50 pl 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 reliably detect at the single-cell level, with only 3 out of 42 cells transfected with the fLUC construct showing luminescence values above those in the substrate control (RLU>20). Moreover, consistent with data from bulk samples, expression of RGA4 severely attenuated GeNL luminescence in individual cells, further suggesting that activity of a GeNL reporter might be used as a surrogate for HR.

[0247] Next, the performance of GeNL as a screenable marker in a method according to an embodiment of the invention was evaluated in wheat protoplasts using the PM3a / Avr- PM3A test system described above in example 2. The results are shown in Figure 8. In bulk samples, co-expression of wild-type PM3A and Avr-PM3A resulted in a moderate yet significant reduction in GeNL luminescence relative to the positive control, whereas no decrease in luminescence could be observed for the inactive R1334E allele of PM3A. Conversely, combining Avr-PM3A and the PM3A gain-of-function S1335R allele or substituting Avr-PM3A with its HR-enhancing Q112N variant led to a stronger decrease in reporter activity compared to that observed with wild-type PM3A. Importantly, none of the Avr-PM3A or PM3A alleles substantially changed GeNL activity when expressed alone, save for the auto-active PM3A D502V mutant.

[0248] The same experimental approach was then also used for quantification of GeNL luminescence in individual wheat protoplasts. Here, samples transformed with GeNL along with PM3A and Avr-PM3A were loaded onto the Paia sorting device at 1 day post transfection and gated into GeNL-expressing (FITC > 1,000 and PE > 10) and nonexpressing cells (FITC < 1,000 and PE < 10). Individual, positive cells were dispensed in a white microtiter plate containing 49 pl W5 buffer and 5 pl Nano-Gio substrate. As shown in Table 2 and Figure 9, results obtained at the single cell level largely echoed the observations in bulk samples with the lowest average GeNL reporter activity observed in single cells transfected with the combination of PM3A-S1335R (‘gain-of- function’) + Avr-PM3A-Q112N, followed by PM3A + Avr-PM3A-Q112N and either PM3A or PM3A-S1335R + Avr-PM3A. Accordingly, single cells transfected with the PM3A- R1334E ‘loss of function’ variant and Avr-PM3A showed an almost 2-fold higher luminescence intensity compared to PM3A or PM3A-S1335R. Together with the RGA4 findings in rice, these results show that quantification of a co-transfected GeNL reporter protein provides a reliable alternative to fluorescence-based screenable markers for use in single cell assays according to an embodiment of the invention.

[0249] Table 2: Measurement of luminescence (RLU) in individual protoplasts derived from wheat

[0250] Much like fluorescence-based reporter systems, GeNL assays can be further optimized via real-time kinetic studies to account for variation in single-cell transfection efficiency and stochasticity in reporter gene expression. Live-cell detection of GeNL luminescence in individual protoplasts over time is realized by physically entrapping isolated single cells, for instance by seeding protoplasts on a nano- or microwell array or (digital) microfluidics chip, coupled to the use of alternative furimazine-based substrates such as Endurazine and Vivazine (Promega), both of which enable nonlytic assays for periods lasting several hours or days. Using this experimental approach, cells of interest can be identified based on reporter kinetics and selected for downstream isolation and characterization via a single cell retrieval system such as the CellCelector platform (Automated Lab Solutions). Example 7: Development of a single-cell screening workflow based on HR-induced production of Reactive Oxygen Species (ROS)

[0251] The rapid production and accumulation of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, during the so-called oxidative burst is one of the hallmarks of effector-triggered immunity. In addition to orchestrating HR-like cell death, ROS serve multiple other immune functions, including local strengthening of cell walls through oxidative cross-linking of structural proteins and activation of phytoalexin biosynthesis. Moreover, ROS can induce arrays of cellular protectant and defense genes and may function as secondary messengers in the induction of systemic acquired resistance (Apel and Hirt, 2004). Considering the myriad defense-related responses modulated by ROS, ROS production constitutes a promising surrogate for NLR activation.

[0252] To monitor the accumulation of reactive oxygen species in transfected cells, oilseed rape protoplasts expressing the rice auto-active NLR RGA4 under control of the Arabidopsis UbiquitinlO promoter were stained with CellRox Green reagent. This cell-permeant dye is weakly fluorescent while in a reduced state but exhibits bright green photostable fluorescence upon oxidation by ROS and subsequent binding to DNA. CellRox Green fluorescence was excited at 405 ± 20 nm and collected at 485 ± 9 nm. As shown in Figure 10, in vivo ROS imaging during the first 24 h after transfection demonstrated a moderate yet sustained 2-fold increase in CellRox Green fluorescence in RGA4- expressing cells (‘RGA4’) compared to the empty vector control (‘Ctrl’). While these results demonstrated the potential of CellRox Green staining for probing immune- associated oxidative stress, overall signal intensities were low, raising doubts about the applicability of the assay at the single-cell level. Therefore, to try and elevate RGA4- induced ROS production, control and RGA4-transfected cells were treated with 1 pM flg22 (‘flg22’, SEQ ID NO: 25), a 22-amino-acid epitope of the archetypal bacterial MAMP elicitor flagellin (Zipfel et al., 2004). In line with recent findings showing that immune pathways activated by cell-surface and intracellular receptors potentiate each other (for review see Chang et al., 2022), flg22 treatment resulted in strongly increased ROS generation in RGA4-expressing cells, thereby greatly improving the sensitivity of the ROS reporter assay.

[0253] Experiments aimed at establishing a ROS-based screening assay with spatio-temporal resolution are currently ongoing. Here, individual rice protoplasts transiently expressing RGA4 as well as a nuclear-localized mCherry transfection reporter are stained with CellRox Green, encapsulated in micro- or picodroplets of water-in-oil emulsion and sorted on a custom-made microfluidics chip such as the Cytomine device (Sphere Fluidics). In an alternative approach, immune-associated ROS are measured by integrating a microfluidic droplet system and fluorescent HRP-gold nanoclusters, the latter synthesized by assembling gold particles with HRP molecules as described by Shen et al. (2018).

[0254] Example 8: Optimalization of single-cell screening assay via time-resolved single cell measurement of ROS production

[0255] Individual rice protoplasts transiently expressing RGA4 as well as a nuclear-localized mCherry transfection reporter are stained with CellRox Green, encapsulated in microdroplets of water-in-oil emulsion and sorted on a microfluidics chip. In an alternative approach, immune-associated ROS are measured by integrating a microfluidic droplet system and fluorescent HRP-gold nanoclusters, the latter synthesized by assembling gold particles with HRP molecules as described by Shen et al. (2018).

[0256] Example 9: Development of a single-cell screening assay using genetic markers

[0257] HR induction in plant cells is typically associated with massive transcriptional reprogramming, involving upregulation of large suites of defense-related genes. Identifying HR marker genes (i.e. genes that are strongly activated in response to HR elicitation) is highly instrumental towards the design of single-cell screening workflows based on gain-of-reporter fluorescence. These reporter systems use HR-responsive promoters to drive expression of a fluorescent reporter protein such as GFP. The activity of the GFP reporter is then compared to a constitutively expressed second reporter (e.g. mCherry, eBFP2, dsRed, etc) that is contained on the same cassette with the HR- responsive promoter. This allows to normalize for differences in transfection efficiency across cells and enables the generation of ratiometric images comparing HR activation (GFP) to the constitutively expressed reporter protein. Example constitutive promoters that can be used to drive the second reporter are the 35S promoter of Cauliflower mosaic virus or the maize or Arabidopsis polyubiquitin (Ubi) promoters, depending on the target species of interest. Candidate HR-responsive promoters can be identified by comparing differential gene expression in compatible versus incompatible plant-pathogen interactions. Examples of comparative defense transcriptome profiling via RNA sequencing are provided by Zhou et al. (2019) and Becker et al. (2017). Using above-mentioned dual-reporter system, HR-expressing cells can be gated from large populations of single cells using high-throughput sorting methods such as flowbased microfluidics or fluorescence-activated cell sorting (FACS). FACS has been applied to sorting of plant cells by different groups (Bargmann and Birnbaum, 2010; Galbraith and Sun, 2021), while Yu et al. (2018) reported on high-throughput microfluidic analysis and screening of protoplasts at processing rates of >100,000 cells per hour.

[0258] Alternatively, 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 leverage RNA editing by adenosine deaminases (ADAR) acting on RNA to gate translation of a protein payload on the presence of endogenous RNA transcripts. One such sensor, termed RADAR (Kaseniit et al., 2022), deploys a "sensor mRNA," which contains 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 so that only mCherry is expressed. However, in the presence of the target RNA, a double-stranded RNA (dsRNA) stretch is formed around the stop codon, which results in recruitment of ADAR enzymes. ADAR enzymes will convert the stop codon into a tryptophan, enabling the translation of the downstream CDS so that both mCherry and EGFP are expressed. “Self-cleaving” 2A sequences are introduced to insulate the sensor sequence from the flanking CDSs to avoid the induction of degradation, aggregates, or other undesired side effects by the peptide encoded in the sensor sequence.

[0259] Example 10: Optimalization of single-cell screening assay via time-resolved single cell measurements

[0260] Gene expression is an intrinsically stochastic process manifested as fluctuations in the abundance of expressed molecules at the single-cell level, and variability and heterogeneity within populations of genetically identical cells. Fluctuations in the biochemical process of gene expression (intrinsic noise) and fluctuations in other cellular components (extrinsic noise) both contribute substantially to overall cel l-to-cel I variation (Popovic et al, 2016).

[0261] To account for this variation and to normalize for differences in transfection efficiency of individual cells, time-resolved GFP reporter measurements were performed using the Cel ICelector single-cell isolation platform which enables individual cells to be monitored over time by physically entrapping them within nanowell arrays (Automated Lab Solutions). To this end, combinations of GFP reporter and Avr-PM3A or its HR- enhancing Q112N mutant were co-expressed in wheat protoplasts (cultivar Fielder) along with either wild-type PM3A or the ‘loss-of-function’ R1334E variant. Eight hours post transfection, transfected cells were diluted 20-fold in W5 buffer and seeded onto a 24-well plate with approximately 3,000 nanowells per well. One milliliter of the cell suspension containing approximately 5,000 cells was slowly dispensed across one well of the 24-well plate. Cells were then left to settle for 5 min at room temperature and centrifuged for 3 min at 800 x g. Individual nanowells were scanned in brightfield and fluorescence (GFP / FITC and Cy5 channels) at regular times between 8 hpt and 24 hpt. Single cells were selected based on multiple criteria, including the number of particles per well (1), particle area (500-4000 pm2), sphericity (0.3-1), elongation and mean gray value. An overview of the temporal changes in GFP fluorescence across all cells measured is shown in Figure 11. As expected, average single-cell GFP intensities showed an increasing trend in samples transfected with either GFP reporter alone (“GFP Ctrl”, Figure HA) or the combination of Avr-PM3A and the PM3A-R1334E loss-of- function allele (Figure 11C). Conversely, in cells expressing WT PM3A together with Avr- PM3A (Figure 11B) or its HR-enhancing variant Avr-PM3A-Q112N (Figure 11D) average GFP fluorescence values remained almost constant during the experiment.

[0262] Interestingly, distinct patterns of GFP expression could be observed at the single-cell level, with the GFP signal either increasing (signature ‘A’), decreasing (signature ‘C’) or remaining unchanged (signature ‘B’) throughout the course of the experiment. In addition, cells that collapsed before the end of the measurements resulted in a complete loss of GFP signal, a reaction which we designated as signature ‘D’, and which is indicative of cell death. As shown in Figure 12, both control samples and cells transfected with the PM3A loss-of-function allele predominantly displayed GFP signatures ‘A’ and ‘B’ (up to 90% of all cells), whereas protoplasts expressing wild-type PM3A were characterized by a high ratio of HR-associated type ‘C’ and type ‘D’ reactions, accounting for more than 50% of all cells.

[0263] Comparative kinetic analysis, as shown in Figure 13, revealed that there was a clear difference in the frequency of type ‘C’ and type ‘D’ signatures between cells transfected with the loss-of-function variant PM3A-R1334E and those expressing WT PM3A at al timepoints measured. In addition, the strongest differences in the frequency of type ‘C’ and type ‘D’ reactions between cells transfected with the loss-of-function variant PM3A- R1334E and those expressing WT PM3A were seen between 8 hpt and 11 hpt. As shown in Table 3, PM3A-transfected samples showed an approximate 12-fold and 8-fold increase in the percentage of cells exhibiting a loss of or reduction in GFP fluorescence, respectively. By defining the optimal time window for analysis and reducing the number of false positives, such detailed insights into the real-time kinetics of reporter activity are expected to further improve the sensitivity and accuracy of the GFP reporter assay.

[0264] Table 3: Comparative kinetic analysis of GFP fuorescence in individual wheat protoplasts showing GFP signature C or GFP signature D

[0265] Example 11: Detection of extracellular R x Ayr interactions using a GFP reporter based single cell screening workflow

[0266] Leptosphaeria macu / ans, the causal agent of blackleg disease (phoma stem canker), causes significant yield losses on Brassica napus crops worldwide (Fitt et al., 2006). During infection, L macu / ans remains extracellular and exhibits a range of lifestyles from biotrophy to necrotrophy. To date, over 20 race-specific blackleg R genes have been reported, all of which code for membrane-bound cell surface-localized receptor proteins used to detect pathogen-secreted apoplastic effectors. R genes cloned thus far encode either receptor-like proteins (RLPs), such as LepR3 and Rlm2, or wall- associated kinase-like (WAKL) proteins, including Rlm9, Rlm4 and Rlm7 (Borhan et al., 2022). To assess the versatility of the method according to an embodiment of the invention and test whether the system can also be used to detect extracellular R x Avr interactions, protoplasts were isolated from Westar, a blackleg-susceptible oilseed rape variety, and co-transfected with plasmids comprising 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 along with their corresponding Avr genes. The R-genes RLP LepR3 (SEQ ID NO: 40) or the WAKLs Rlm3 (WQ2023 / 004429A1; SEQ ID NO: 41) and Rlm9 (SEQ ID NO: 42) were expressed under control of the Arabidopsis UbiquitinlO promoter (pAtUbilO, SEQ ID NO: 6). To facilitate extracellular secretion of the corresponding Avr genes AvrLml (SEQ ID NO: 43), AvrLm3 (SEQ ID NO: 44) and AvrLm5-9 (SEQ ID NO: 45), the effectors’ native signal peptide was substituted with that of the tobacco PRla protein (SEQ ID NO: 46), a motif which has been widely used for secretion of pathogen effectors in various plants (van Esse et al., 2006). The resulting Avr genes AvrLml* (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).

[0267] A reference sample consisting of the GFP reporter and a ‘dummy’ plasmid (SEQ ID NO: 7) was included to provide a read-out on GFP expression in the absence of R and Avr genes. Co-transfections of R or Avr genes together with GFP and dummy plasmid served as negative controls. The concentration of dummy plasmid was adjusted to ensure equal amounts of total plasmid DNA in all transfections.

[0268] GFP reporter activity was quantified at 24 hpt and normalized against cellular autofluorescence to account for differences in transfection efficiency across samples. As shown in Figure 11, all three R x Avr interactions tested resulted in a strong reduction in GFP fluorescence relative to the GFP or R gene only controls, which is indicative of HR activation. Moreover, consistent with previous reports showing induction of host cell death due to enhanced accumulation and phosphorylation of BnMPK9 by translocated AvrLml (Ma et al., 2018), transient expression of AvrLml* and AvrLm3* led to a decrease in reporter activity as compared to the GFP, effector or R gene only controls, even in the absence of the corresponding R gene.

[0269] Alternatively, individual protoplasts with reduced GFP expression are sorted and analyzed using methods as described herein (such as fluorescence activated cell sorting or flow-based microfluidics). Together these findings demonstrate the utility and efficacy of the single cell screening workflow using GFP as a selectable marker in an embodiment of the invention for both detection of effector-induced cell death and quantification of HR following perception of secreted effectors by membrane-bound cell surface receptors in the Brassica napus - Leptosphaeria macula ns pathosystem.

Claims

CLAIMS1. A method for high-throughput characterization of candidate plant pathogen effector gene(s) and / or candidate plant disease resistance gene(s) in individual plant cells, which method comprises the steps of:(a) Preparing at least one protoplast derived from a plant of interest, which protoplast is optionally capable of expressing one or more screenable marker gene(s);(b) Introducing one or more expression cassette(s) for transiently expressing one or more candidate plant pathogen effector gene(s) and / or introducing expression cassette(s) for transiently expressing one or more candidate plant disease resistance gene(s) in said at least one protoplast;(c) Transiently expressing said one or more candidate plant pathogen effector gene(s) and / or said one or more candidate plant disease resistance gene(s);(d) measuring in said at least one protoplast the production of reactive oxygen species (ROS) and / or optionally the expression of said screenable marker gene;(e) isolating one or more individual protoplasts that show modulated production of ROS, modulated expression of said one or more screenable marker genes, or both, as compared to control plant protoplasts lacking a functional candidate pathogen effector gene;(f) Identifying said one or more candidate plant pathogen effector(s) and / or said one or more candidate plant disease resistance gene(s) in the individual protoplast isolated in step (e).

2. The method of claim 1, wherein one or more candidate plant pathogen effector(s) and one or more candidate plant disease resistance gene(s) are introduced in protoplasts derived from a plant that is susceptible to a pathogen encoding said plant pathogen effector.

3. The method of claim 1, wherein one or more candidate plant pathogen effector(s) are introduced in protoplasts derived from a corresponding disease resistant plant.

4. The method of claim 1, wherein the one or more candidate plant pathogen effector(s) and the one or more candidate plant disease resistance gene(s) are introduced in protoplasts that are derived from a plant that is heterologous to said candidate plant disease resistance gene(s).

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

6. The method of claim 5, wherein the screenable marker gene is under control of a constitutive promoter or under control of a hypersensitive response inducible promoter.

7. Method according to any preceding claim, wherein the screenable marker gene encodes a fluorescent and / or luminescent marker.

8. The method of claim 7, wherein modulated expression of said fluorescent or luminescent marker is measured in individual protoplasts.

9. Method according to any preceding claim, wherein modulated production of ROS and / or modulated expression of said one or more screenable marker genes is measured on one or more timepoints.

10. Method according to any preceding claim, wherein said plant of interest is a crop plant, preferably a cereal, an oilseed plant, a leguminous plant, a vegetable or a fiber crop.

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

12. Method according to any preceding claim wherein the production of reactive oxygen species (ROS) is measured through ROS induced oxidation of a fluorescent dye.

13. Method according to any preceding claim, wherein the isolation of one or more individual protoplasts comprises a step of protoplast sorting, dispensing and retrieving protoplasts of interest.

14. Method according to any preceding claim, wherein said one or more candidate plant pathogen effector(s) and / or said one or more candidate plant disease resistance gene(s) in the individual protoplast are identified by sequencing.

15. Method according to any preceding claim, wherein the step of isolating one or more individual protoplasts that show modulated production of ROS and / or modulated expression of said one or more screenable marker genes, is followed by a step of clonal propagation.

16. Method according to any preceding claim, further comprising regeneration of isolated protoplast or clones derived therefrom into plants.

17. Method of claim 14, wherein the identification furthermore comprises a genetic, molecular and / or biochemical analysis.

18. Use of the method according to any of the preceding claims for(a) Functional characterisation of candidate plant pathogen effector(s) or candidate plant disease resistance gene(s), or(b) Functional characterisation of pairs of candidate plant pathogen effector(s) and candidate plant disease resistance gene(s), or(c) Screening of variant libraries of candidate plant pathogen effector(s) and / or of candidate plant disease resistance gene(s), or(d) Decoy engineering, or(e) Evaluating candidate plant disease resistance gene stacks.

19. Method of producing a disease resistant plant comprising transforming a plant cell with a construct for expressing one or more plant disease resistance gene(s) identified with a method according to any of claims 1 to 17, followed by regeneration into a plant.

20. Method of producing a disease resistant plant comprising genetically modifying in a plant cell an allele of a gene that does not confer disease resistance such that the modified allele is capable of expressing a plant disease resistance gene identified with a method according to any of claims 1 to 17, followed by regeneration into a plant.

21. Method of claim 20 wherein the genetic modification comprises transgenesis or genome editing.

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