Novel engineered immune receptors to protect plants from pathogens

Abstract

The present invention provides for an engineered, genetically modified, and / or synthetic plant receptor comprising a binding sequence that is capable of binding a fungal pathogen Avr ligand; wherein the non-engineered, genetically unmodified, and / or naturally occurring plant receptor does not bind the fungal pathogen Avr ligand, or bind with a lower binding affinity.

Description

Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory Novel engineered immune receptors to protect plants from pathogens Inventors: Pamela C. Ronald, Ellen Youngsoo Rim CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 63 / 692,024, filed September 6, 2024, which is hereby incorporated by reference. STATEMENT OF GOVERNMENTAL SUPPORT

[0002] The invention was made with government support under Contract No. DE-AC02- 05CH11231 awarded by the U.S. Department of Energy. The government has certain rights in the invention. FIELD OF THE INVENTION

[0003] The present invention is in the field of protecting plants from pathogens. REFERENCE TO SEQUENCE LISTING

[0001] Reserved. BACKGROUND OF THE INVENTION

[0002] Plant immune receptors can recognize pathogen-secreted ligands, or effectors, and protect the plant host from the invading pathogen. However, pathogens constantly evolve new effector variants that evade immune recognition (Figure 1, Panel A). Therefore, there is a need to identify immune receptors that show broad-spectrum binding of new effector variants has the potential to protect vulnerable crops from a wide range of pathogens.

[0003] Pathogens and host organisms are locked in an evolutionary competition: the host immune system strives to detect attacks while pathogens evolve to escape detection. At the molecular level, host immune recognition often hinges on direct interaction between a pathogen-derived molecule and an immune protein. Pathogens can accumulate mutations that escape such interactions, with their short lifecycles and large populations giving them an advantage. Directed evolution harnesses high throughput screening of synthetic protein sequences and effectively compresses the evolutionary timescale. Therefore, directed evolution can shift the balance in favor of the host by accelerating the development ofAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory immune proteins that recognize a wide array of pathogen-derived molecules, including those that evade detection by naturally evolved immune proteins.

[0004] Directed evolution can benefit plants in a unique manner. Unlike animals with their adaptive immune systems, plants carry hundreds to thousands of genome-encoded immune receptors. Many of these receptors are activated by direct binding to effectors, pathogen- secreted molecules that facilitate infection 1. Developing novel immune receptors that can bind previously unrecognized effectors, therefore, can protect crops against a broader spectrum of pathogens. Increasing global temperatures, which are predicted to enhance pathogen spread and reduce plant immunity, add urgency to development of such receptors 2,3. While gain-of-function random mutagenesis screens have led to improved immune receptors, their utility has been constrained by the limited throughput of in planta testing 4–7. SUMMARY OF THE INVENTION

[0005] The present invention provides for an engineered, genetically modified, and / or synthetic plant receptor comprising a binding sequence that is capable of binding a fungal pathogen Avr ligand; wherein the non-engineered, genetically unmodified, and / or naturally occurring plant receptor does not bind the fungal pathogen Avr ligand, or bind with a lower binding affinity.

[0006] The engineered, genetically modified, and / or synthetic plant receptor when expressed in a plant cell displays the binding sequence outside of the plant cell. In some embodiments, the plant receptor is a Pik receptor, such as a Pikh1 receptor.

[0007] In some embodiments, the fungal pathogen Avr ligand is Avr-PikA, Avr-PikD, Avr- PikC, Avr-PikF, and / or AvrPiz-t. In some embodiments, the fungal pathogen Avr ligand is Avr-PikC, Avr-PikF, and / or AvrPiz-t.

[0008] The present invention provides for a nucleic acid encoding the engineered, genetically modified, and / or synthetic plant receptor or binding sequence of the present invention.

[0009] A platform is developed to generate novel plant immune receptors that can protect rice crops from a devastating disease. Introduction of these immune receptor genes into rice cultivars, either through genetic modification or precise genome engineering, could protect against rice blast, which is caused by the fungal pathogen Magnaporthe oryzae and can leadAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory up to 30% loss in crop yield. In some embodiments, these immune receptors, which are called engineered Pik receptors, are about 1,142 amino acid-long proteins. The engineered Piks are able to protect rice crops from all rice-infecting strains of the fungal pathogen Magnaporthe oryzae, whereas existing natural or laboratory-generated Pik receptors can only protect rice from a subset of these disease-causing strains.

[0010] The immune receptors are unique, engineered sequences of amino acids that have neither been found in any cultivated or wild rice varieties nor previously generated through laboratory research. The immune receptor sequences are developed and tested through a protein engineering technique called yeast surface display. These immune receptors are the first application of high-throughput protein engineering to plant receptor proteins. In some embodiments, the engineered Pik proteins are about 1,142 amino acids long and different from the naturally occurring rice Pik receptor proteins by several amino acids at key positions that allow immune recognition of Magnaporthe oryzae strains.

[0011] The present invention provides for a method to engineer the rice Pik1 receptor using high-throughput protein engineering through yeast surface display. An initial experiment confirmed the possibility of engineering the rice Pik1 receptor for better immune function. The engineered Pik receptors can recognize two Magnaporthe oryzae ligands (Avr-PikC and Avr-PikF that are not recognized by the natural Pik receptor, expanding the range of the fungal pathogens that can be detected by these rice immune receptors.

[0012] The present invention provides for a nucleic acid encoding the engineered, genetically modified, and / or synthetic plant receptor of the present invention operatively linked to a promoter. The present invention also provides for a nucleic acid encoding the binding sequence of the present invention operatively linked to a promoter. In some embodiments, the promoter is a tissue-specific or inducible promoter, or is a constitutive promoter.

[0013] The present invention provides for a vector comprising the nucleic acid of the present invention. In some embodiments, the vector is capable of stably integrating into a chromosome of a host cell or stably residing in a host cell. In some embodiments, the vector is an expression vector. In some embodiments, the host cell is a plant cell. In some embodiments, the plant cell is a monocot or a dicot.

[0014] The present invention provides for a host cell, such as a plant cell, comprising the vector of the present invention, wherein the host cell is capable of expressing the engineered,Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory genetically modified, and / or synthetic plant receptor of the present invention.

[0015] The present invention provides for a method to confer on a plant or plant cell resistance to a plant pathogen, comprising: introducing into a plant or plant cell with a nucleic acid of the present invention encoding the engineered, genetically modified, and / or synthetic plant receptor of the present invention operatively linked to a promoter, whereby the nucleic acid encoding the engineered, genetically modified, and / or synthetic plant receptor operatively linked to a promoter stably integrates into the genome of the plant or plant cell.

[0016] In some embodiments, the plant pathogen is a fungal pathogen. In some embodiments, the fungal pathogen comprises or expresses the Avr ligand. In some embodiments, the fungal pathogen Magnaporthe species, such as Magnaporthe oryzae. In some embodiments, the fungal pathogen is one decribed in B. Valent, “The Impact of Blast Disease: Past, Present, and Future,” Chapter 1, in Stefan Jacob (ed.), Magnaporthe oryzae: Methods and Protocols, Methods in Molecular Biology, vol.2356 (2021), hereby incorporated by reference. In some embodiments, the host cell is any plant or plant cell of any plant category or species described in B. Valent, “The Impact of Blast Disease: Past, Present, and Future,” Chapter 1, in Stefan Jacob (ed.), Magnaporthe oryzae: Methods and Protocols, Methods in Molecular Biology, vol.2356 (2021). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.

[0018] Fig.1. (A) Crop pathogens evolve to evade immune receptor binding, rendering plants susceptible to diseases. Immune receptors can be engineered to protect the plant host from such pathogens. (B) Receptor engineering schematcs. Receptor variants were generated through random mutagenesis and expressed on yeast cells. Variants that have gained binding to a fluorescently labeled target ligand were selected through FACS sorting and sequencing. Introduction of improved immune receptors into the crop host can confer immunity against pathogens with the target ligand.

[0019] Fig.2. (A) One of the selected receptor variants shows binding to all tested effectors, including AvrPiz-t, Avr-PikC, and Avr-PikF, and retains binding to the original ligands Avr-Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory PikA and Avr-PikD, as shown in immunofluorescence images of yeast cells expressing the variant. (B) Directed evoluHon of a rice immune receptor Pikh1 expanded its ligand recogniHon range to previously unrecognized effector variants, potentially expanding its immune capability.

[0020] Fig, 3. FACS sort: 100 nM AvrPikC / F binding.

[0021] Fig.4. Wildtype Pikh immune receptor does not bind Avr-PikC, Avr-PikF, or AvrPiz- t.

[0022] Fig.5. Engineered receptor A4.1 binds to Avr-PikC and Avr-PikF.

[0023] Fig.6. Engineered receptor B4.1 binds to Avr-PikC and Avr-PikF.

[0024] Fig.7. Engineered receptor 7.1 binds to Avr-PikC, Avr-PikF, and AvrPiz-t.

[0025] Fig.8. Engineered receptor 7.2 binds to Avr-PikC, Avr-PikF, and AvrPiz-t.

[0026] Fig.9. Engineered receptor 7.2 triggers immune response to target ligands in planta.

[0027] Fig.10. Engineered receptor 7.2 binds to Avr-PikC, Avr-PikF, and AvrPiz-t.

[0028] Fig.11. Yeast surface display of the rice receptor heavy metal associated (HMA) domain recapitulates its endogenous ligand binding properties. a, A schematic representation of directed evolution of a plant immune receptor with desired binding properties through yeast surface display. b, A schematic representation of Pikh-1 yeast surface display. Pikh-1 HMA domain expression and its interaction with target Avr-Pik are assessed through two-color fluorescence detection. c, Allelic variants of the M. oryzae effector Avr-Pik. Rice receptor Pikh-1 elicits immune response in the presence of Avr-PikD and Avr-PikA, but not Avr-PikC and Avr-PikF. d, Immunofluorescence images of yeast cells expressing Pikh-1 HMA domain. Left: biotin-labeled effector binding at specified concentrations detected through Alexa Fluor 488-conjugated streptavidin. Middle: HMA domain expression detected through anti-Myc tag binding to Alexa Fluor 568. Right: merged images of effector binding (cyan) and receptor expression (magenta). Scale bar, 10μm. e, Flow cytometry quantification of the interaction between yeast surface-displayed Pikh-1 HMA domain and biotin-labeled Avr-Pik variants.

[0029] Fig.12. Rounds of FACS selection identify Pik-1 HMA domain variants withAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory affinity for novel ligands. a, Directed evolution workflow to develop binding to both Avr- PikC and Avr-PikF. b, Flow cytometry quantification of the interaction between Pikh-1 HMA domains and 1μM Avr-PikC or 1μM Avr-PikF. Compared to the wildtype Pikh-1 HMA domain (top), variants collected at the end of selection path A (middle) and path B (bottom) show increased affinity for Avr-PikC (left) and Avr-PikF (right). c, Immunofluorescence images of yeast cells expressing Pikh-1 HMA domains in the presence of 1μM Avr-PikC (left) or 1μM Avr-PikF (right). Merged images show biotin-labeled effector binding detected through Alexa Fluor 488-conjugated streptavidin (cyan) and receptor expression detected through anti-Myc tag binding to Alexa Fluor 568 (magenta) of the wildtype Pikh-1, A4.1, and B4.1 HMA domains. Scale bar, 10μm. d, Amino acid sequences of the wildtype Pikh-1 HMA domain and variants A4.1 and B4.1 enriched at the end of selection paths A and B, respectively. Sequence logos depict the 10 most frequently mutated residues after sort 4 of each selection path (red: acidic, blue: basic, black: hydrophobic, purple: neutral, green: polar). Interfaces known to interact with Avr-Pik are indicated below 10,16.

[0030] Fig.13. Additional FACS selection identifies Pik-1 HMA domain variants with affinity for an evolutionarily divergent ligand. a, Overlay of crystal structures of Avr-PikC (blue, PDB:7A8X) and AvrPiz-t (grey, PDB: 2LW6). b, Directed evolution workflow to develop binding to AvrPiz-t. Variants were selected for binding to AvrPiz-t at decreasing concentrations, with an additional round of random mutagenesis to enhance library diversity. c, Flow cytometry quantification of the interaction between Pikh-1 HMA domains and 1μM AvrPiz-t. Compared to the wildtype Pikh-1 HMA domain (top), variants collected at the end of sort 7 (middle) and selected variant 7.2 (bottom) show increased affinity for AvrPiz-t. d, Immunofluorescence images of yeast cells expressing Pikh-1 HMA domains in the presence of 1μM AvrPiz-t (left), 1μM Avr-PikC (middle), or 1μM Avr-PikF (right). Merged images show biotin-labeled effector binding detected through Alexa Fluor 488-conjugated streptavidin (cyan) and receptor expression detected through anti-Myc tag binding to Alexa Fluor 568 (magenta) of the Pikh-1 wildtype (top) and variant 7.2 (bottom) HMA domains. Scale bar, 10μm. e, Amino acid sequences of the wildtype Pikh-1 HMA domain and variant 7.2 are shown. Sequence logo depicts the 10 most frequently mutated residues after sort 7 (red: acidic, blue: basic, black: hydrophobic, purple: neutral, green: polar). Interfaces known to interact with Avr-Pik are indicated below 10,16.

[0031] Fig.14. Recognition of target ligands by engineered Pik-1 immune receptors inAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory N. benthamiana. a, A schematic representation of cell death assay in N. benthamiana to assess immune receptor function. b, Representative UV images of cell death assay. Pikh-1 harboring wildtype, A4.1, or B4.1 HMA domain was co-expressed with Avr-PikC (left leaf) or Avr-PikF (right leaf). YFP was co-expressed as a negative control. c, Quantification of cell death induced by Pikh-1 harboring wildtype, A4.1, or B4.1 HMA domain scored on a 0-6 scale. Center line denotes median value; box edges delineate 25th and 75th percentiles; whiskers extend from 10thto 90th percentiles. Each receptor-effector pair was tested in three independent experiments with six plants per experiment. d, Representative UV images of cell death assay. Pikh-1 harboring wildtype or 7.2 HMA domain was co-expressed with Avr-PikC (left leaf), Avr-PikF (middle leaf), or AvrPiz-t (right leaf). YFP was co-expressed as a negative control. e, Quantification of cell death induced by Pikh-1 harboring wildtype or 7.2 scored on a 0-6 scale. Center line denotes median value; box edges delineate 25th and 75th percentiles; whiskers extend from 10th to 90thpercentiles. Each receptor-effector pair was tested in three independent experiments with six plants per experiment.

[0032] Fig.15. Yeast surface-displayed Pik-1 alleles recapitulate their endogenous ligand binding properties. a, Immunofluorescence images of yeast cells expressing Pikp-1 or Pikm-1 HMA domain in the presence of Avr-Pik variants. Merged images show biotin- labeled effector binding detected through Alexa Fluor 488-conjugated streptavidin (cyan) and HMA domain expression detected through anti-Myc tag binding to Alexa Fluor 568 (magenta). Scale bar, 10μm. b, Flow cytometry quantification of Pikh-1, Pikp-1, and Pikm-1 HMA domain surface expression and interaction with Avr-Pik variants shown in bivariate contour plots. Clusters of cells that do not express the target protein 35 do not exhibit Avr-Pik binding. c, Flow cytometry quantification of Pikp-1 and Pikm-1 HMA domain interaction with Avr-Pik variants shown in univariate histograms. d, Exemplary flow cytometry ancestry plots showing the gating strategy applied to histograms in this study, as described in the Methods section. Plots from Pikm-1 HMA domain interaction with 1μM Avr-PikF are shown as an example.

[0033] Fig.16. Iterative selection leads to Pik-1 HMA domain variants with improved affinity for Avr-PikC and Avr-PikF. a, Frequency of variants harboring the specified number of substitutions over the 78-amino acid HMA domain in the library of 2.2×107Pikh-1 HMA domain variants. b, Bivariate dot plot of Pikh-1 HMA domain surface expression and interaction with 1μM Avr-PikF showing an exemplary FACS selection window. Cyan dotsAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory indicate cells expressing the wildtype Pikh-1 HMA domain, and red dots indicate cells expressing Pikh-1 HMA domain variants in the library. c, Flow cytometry quantification of Pikh-1, A4.1, and B4.1 HMA domain surface expression and interaction with 1μM Avr-PikC or Avr-PikF shown in bivariate contour plots. d, Immunofluorescence images of yeast cells expressing Pikh-1, A4.1, or B4.1 HMA domain in the presence of each effector at specified concentrations. Merged images show effector binding (cyan) and expression (magenta) of HMA domains. Scale bar, 10μm. e, Amino acid sequences of Avr-Pik and AvrPiz-t effectors from Magnaporthe oryzae.

[0034] Fig.17. Characterization of the second library and sequences of natural and engineered Pik-1 variants. a, Frequency of variants harboring the specified number of substitutions over the 78-amino acid HMA domain in the library of 2.1×107Pikh-1 HMA domain variants following the second round of mutagenesis. b, Bivariate dot plot of Pikh-1 HMA domain surface expression and interaction with 200nM AvrPiz-t showing an exemplary FACS selection window. Cyan dots indicate cells expressing the wildtype Pikh-1 HMA domain and red dots indicate cells expressing Pikh-1 HMA domain variants in the library following second round of mutagenesis. c, Amino acid sequences of Pik-1 HMA domains from rice alleles Pik-1, Pikg-1, Pike-1, Piks-1, Pikm-1, Pik-W25-1, Pikp-1, and Pikh-1 and engineered variants A4.1, B4.1, and 7.2. Interfaces known to interact with Avr- Pik are indicated below 10,16,36,37.

[0035] Fig.18. Additional rounds of selection lead to a Pik-1 HMA domain variant with affinity for Avr-Pik and AvrPiz-t. a, Flow cytometry quantification of Pikh-1 and 7.2 HMA domain surface expression and interaction with 1μM AvrPiz-t, Avr-PikC, or Avr-PikF shown in bivariate contour plots. b, Immunofluorescence images of yeast cells expressing Pikh-1 or 7.2 HMA domains in the presence of each effector at specified concentrations. Merged images show effector binding (cyan) and expression (magenta) of HMA domains. Scale bar, 10μm.

[0036] Fig.19. Engineered Pik-1 immune receptors induce cell death response in N. benthamiana. a, Representative images of N. benthamiana cell death scores on the 0-6 scale used in this study. b, Representative images of cell death assay. Pikh-1 harboring wildtype, A4.1, or B4.1 HMA domain was co-expressed with Avr-PikC, Avr-PikF, Avr-PikA (positive control), or YFP (negative control). c, Representative images of cell death assay. Pikh-1 harboring wildtype or 7.2 HMA domain was co-expressed with Avr-PikC, Avr-PikF, AvrPiz-Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory t, Avr-PikA (positive control), or YFP (negative control). DETAILED DESCRIPTION OF THE INVENTION

[0037] Before the invention is described in detail, it is to be understood that, unless otherwise indicated, this invention is not limited to particular sequences, expression vectors, enzymes, host microorganisms, or processes, as such may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.

[0038] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0039] The terms "optional" or "optionally" as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

[0040] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0041] The term “about” refers to a value including 10% more than the stated value and 10% less than the stated value.

[0042] As used herein, the term "promoter" refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell. Thus, promoters used in the polynucleotideAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory constructs of the invention include cis- and trans-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and / or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on / off, regulate, modulate, etc.) gene transcription. Promoters are located 5' to the transcribed gene, and as used herein, include the sequence 5' from the translation start codon.

[0043] A "constitutive promoter" is one that is capable of initiating transcription in nearly all cell types, whereas a "cell type-specific promoter" initiates transcription only in one or a few particular cell types or groups of cells forming a tissue. In some embodiments, the promoter is secondary cell wall-specific and / or fiber cell-specific. A "fiber cell-specific promoter" refers to a promoter that initiates substantially higher levels of transcription in fiber cells as compared to other non-fiber cells of the plant. A "secondary cell wall-specific promoter" refers to a promoter that initiates substantially higher levels of transcription in cell types that have secondary cell walls, e.g., lignified tissues such as vessels and fibers, which may be found in wood and bark cells of a tree, as well as other parts of plants such as the leaf stalk. In some embodiments, a promoter is fiber cell-specific or secondary cell wall-specific if the transcription levels initiated by the promoter in fiber cells or secondary cell walls, respectively, are at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 000-fold higher or more as compared to the transcription levels initiated by the promoter in other tissues, resulting in the encoded protein substantially localized in plant cells that possess fiber cells or secondary cell wall, e.g., the stem of a plant. Non- limiting examples of fiber cell and / or secondary cell wall specific promoters include the promoters directing expression of the genes IRX1, IRX3, IRX5, IRX7, IRX8, IRX9, IRX10, IRX14, NST1, NST2, NST3, MYB46, MYB58, MYB63, MYB83, MYB85, MYB103, PAL1, PAL2, C3H, CcOAMT, CCR1, F5H, LAC4, LAC17, CADc, and CADd. See, e.g., Turner et al 1997; Meyer et al 1998; Jones et al 2001; Franke et al 2002; Ha et al 2002; Rohde et al 2004; Chen et al 2005; Stobout et al 2005; Brown et al 2005; Mitsuda et al 2005; Zhong et al 2006; Mitsuda et al 2007; Zhong et al 2007a, 2007b; Zhou et al 2009; Brown et al 2009; McCarthy et al 2009; Ko et al 2009; Wu et al 2010; Berthet et al 2011. In some embodiments, a promoter is substantially identical to a promoter from the lignin biosynthesisAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory pathway. A promoter originated from one plant species may be used to direct gene expression in another plant species.

[0044] A polynucleotide or amino acid sequence is "heterologous" to an organism or a second polynucleotide or amino acid sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a polynucleotide encoding a polypeptide sequence is said to be operably linked to a heterologous promoter, it means that the polynucleotide coding sequence encoding the polypeptide is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety, or a gene that is not naturally expressed in the target tissue).

[0045] The term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a DNA or RNA sequence if it stimulates or modulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0046] The terms “host cell” of “host organism” is used herein to refer to a living biological cell that can be transformed via insertion of an expression vector.

[0047] The terms "expression vector" or "vector" refer to a compound and / or composition that transduces, transforms, or infects a host cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell, or in a manner not native to the cell. An "expression vector" contains a sequence of nucleic acids (ordinarily RNA or DNA) to be expressed by the host cell. Optionally, the expression vector also comprises materials to aid in achieving entry of the nucleic acid into the host cell, such as a virus, liposome, protein coating, or the like. The expression vectors contemplated for use in the present inventionAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory include those into which a nucleic acid sequence can be inserted, along with any preferred or required operational elements. Further, the expression vector must be one that can be transferred into a host cell and replicated therein. Particular expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of the nucleic acid sequence. Such plasmids, as well as other expression vectors, are well known to those of ordinary skill in the art.

[0048] The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones; non- ionic backbones, and non-ribose backbones. Thus, nucleic acids or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase. "Polynucleotide sequence" or "nucleic acid sequence" includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein byAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0050] In some embodiments, the amino acid sequence of binding sequence comprises one of SEQ ID NO:1-15. In some embodiments, the binding sequence comprises GLKXXIVXKV XXEGNXXRXX AMALVASTGG VXSXALXGDX XDKIXVVGXG IDPXKLXSAL RKKVGXAXLL XXSXXKKX (SEQ ID NO:17), wherein X is any amino acid.

[0051] In some embodiments, the amino acid sequence of binding sequence comprises SEQ ID NO:1 or SEQ ID NO:17, except the amino acid sequence has one or more of the following amino acid residue substitutions (wherein all of the following positions are according to SEQ ID NO:1): an amino acid with a positive charged side chain at position 4; an amino acid with a hydrophobic side chain at position 5; an amino acid with a hydrophobic side chain at position 8; an amino acid with a positive charged side chain at position 11; an amino acid with a positive charged side chain, hydrophobic side chain, or amino acid G at position 12; an amino acid with a negative charged side chain at position 16; an amino acid with a positive charged side chain at position 17; an amino acid with a bulky aromatic side chain, or the amino acid is P, at position 19; an amino acid with a negative charged side chain at position 20; an amino acid with a negative charged side chain at position 32; an amino acid with a hydrophobic side chain at position 34; an amino acid with a hydrophobic side chain at position 37;Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory an amino acid with a polar uncharged side chain or bulky aromatic side chain at position 40; an amino acid with a bulky aromatic side chain at position 41; an amino acid with a positive charged side chain at position 45; an amino acid with a positive charged side chain, or amino acid C, at position 49; an amino acid with a polar uncharged side chain at position 54; an amino acid with a hydrophobic side chain at position 57; amino acid G at position 66; an amino acid with a positive charged side chain or hydrophobic side chain at position 68; an amino acid with a polar uncharged side chain at position 70; an amino acid with a positive charged side chain at position 71; an amino acid with a hydrophobic side chain at position 72; an amino acid with a positive charged side chain or hydrophobic side chain at position 74; an amino acid with a hydrophobic side chain at position 75; an amino acid with a negative charged side chain at position 77; and / or amino acid G at position 78.

[0052] In some embodiments, the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acid residue substitutions. In some embodiments, the amino acid with a positive charged side chain is R, H, or K. In some embodiments, the amino acid with a negative charged side chain is D or E. In some embodiments, the amino acid with a polar uncharged side chain is S, T, N, or Q. In some embodiments, the amino acid with a hydrophobic side chain is A, V, I, L, or M. In some embodiments, the amino acid with a bulky aromatic side chain is F, Y, or W.

[0053] In some embodiments, the amino acid residue at position 4 is R. In some embodiments, the amino acid residue at position 5 is I. In some embodiments, the amino acidAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory residue at position 8 is V. In some embodiments, the amino acid residue at position 11 is R. In some embodiments, the amino acid residue at position 12 is K, G, or V. In some embodiments, the amino acid residue at position 16 is D. In some embodiments, the amino acid residue at position 17 is R. In some embodiments, the amino acid residue at position 19 is F or P. In some embodiments, the amino acid residue at position 20 is R. In some embodiments, the amino acid residue at position 32 is E. In some embodiments, the amino acid residue at position 34 is A. In some embodiments, the amino acid residue at position 37 is I. In some embodiments, the amino acid residue at position 40 is S or W. In some embodiments, the amino acid residue at position 41 is W. In some embodiments, the amino acid residue at position 45 is K. In some embodiments, the amino acid residue at position 49 is C or H. In some embodiments, the amino acid residue at position 54 is T. In some embodiments, the amino acid residue at position 57 is V. In some embodiments, the amino acid residue at position 66 is G. In some embodiments, the amino acid residue at position 68 is K or V. In some embodiments, the amino acid residue at position 70 is S. In some embodiments, the amino acid residue at position 71 is R. In some embodiments, the amino acid residue at position 72 is I. In some embodiments, the amino acid residue at position 74 is R or L. In some embodiments, the amino acid residue at position 75 is V. In some embodiments, the amino acid residue at position 77 is E. In some embodiments, the amino acid residue at position 78 is G.

[0054] The amino acid sequence of Clone 7.e1 is: GLKRIIVIKVARKGNNCRSKAMALVASTGGVDSVALVGDLRDKIEVVGYGIDPIKLI SALRKKVGDAKLLQVSQAKKD (SEQ ID NO:1).

[0055] The amino acid sequence of Clone 7.1 is: GLKRIIVIKVARGGNNCRSKAMALVASTGGVDSVALVGDLRDKIEVVGYGIDPIKLI SALRKKVGDAELLQVSQAKKG (SEQ ID NO:2).

[0056] The amino acid sequence of Clone 7.A is: GLKRIIVIKVARGGNNCRSKAMALVASTGGVDSVALVGDLRDKIEVVGYGIDPIKLI SALRKKVGDAELLQVSQAKKD (SEQ ID NO:3).

[0057] The amino acid sequence of Clone 7.4 is: GLKRIIVIKVAREGNNCRSKAMALVASTGGVDSVALVGDLRDKIEVVGYGIDPIKLIS ALRKKVGGAELLQVSQAKKD (SEQ ID NO:4).Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory

[0058] The amino acid sequence of Clone 7.2 is: GLKRIIVIKVAREGNNCRSKAMALVASTGGVDSVALVGDLRDKIEVVGYGIDPIKLIS ALRKKVGDAELLRVSQAKKD (SEQ ID NO:5).

[0059] The amino acid sequence of Clone 6.1 is: GLKRIIVIKVAREGNNCRSKAMALVASTGGVDSVALVGDLRDKIEVVGYGIDPIKLIS ALRKKVGDAELLQVSQAKKD (SEQ ID NO:6).

[0060] The amino acid sequence of Clone 6.3 is: GLKRKIVIKVAVEGNNCRSKAMALVASTGGVDSVALVGDLRDKIEVVGCGIDPIKLI SALRKKVGGAVLLQISQAKKD (SEQ ID NO:7).

[0061] The amino acid sequence of Clone 5.6 is: GLKQKIVVKVAMEGNNCRFKAMALVASTGGVDSVALVGDLRDKIKVVGYGIDPIK LVSALRKKVGDAELLQVSQAKKD (SEQ ID NO:8).

[0062] The amino acid sequence of Clone 6.4 is: GLKRIIVIKVAREGNNCRSKAMALVASTGGVDSVALIGDSRDKIEVVGYGIDPIKLIS ALRKKVGDAELLQVSQAKKD (SEQ ID NO:9).

[0063] The amino acid sequence of Clone FCFC1 (=B4.1) is: GLKQKIVIKVAMEGNNCRSKAMALVASTGGVDSVALVGDLRDKIEVVGYGIDPTKL ISALRKKVGGAELLQVSRVKKG (SEQ ID NO:10).

[0064] The amino acid sequence of Clone CFCF2 (=A4.1) is: GLKQKIVIKVAKEGNDCRSRAMALVASTGGVDSVALVGDLRDKIEVVGYGIDPIKLI SALRKKVGDAELLQVSRVKKD (SEQ ID NO:11).

[0065] The amino acid sequence of Clone FCFC8 is: GLKQKIVIKVAVEGNNRRSKAMALVASTGGVDSVALVGDWRDKIEVVGYGIDPIKL ISALRKKVGDAELLQVSQVKKG (SEQ ID NO:12).

[0066] The amino acid sequence of Clone FCFC4 is: GLKQKIVIKVAMEGNNCRSKAMALVASTGGVDSVALVGDLRDKIEVVGYGIDPTKL ISALRKKVGDAKLLQVSRVKEG (SEQ ID NO:13).

[0067] The amino acid sequence of Clone FCFC2 is:Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory GLKQKIVIKVAMEGNNCRPKAMALVASTGGVESAALVGDLRDKIEVVGHGIDPIKLI SALRKKVGDAELSQVSLVKKD (SEQ ID NO:14).

[0068] The amino acid sequence of Clone CFCF1 is: GLKQKIVIKVAMEGNNCRSKAMALVASTGGVDSVALVGDLWDKIEVVGYGIDLIKL ISALRKKVGDAELLQVSQVKKG (SEQ ID NO:15).

[0069] A wild-type amino acid sequence of the binding sequence is: GLKQKIVIKV AMEGNNCRSK AMALVASTGG VDSVALVGDLR DKIEVVGYGI DPIKLISALR KKVGDAELLQ VSQAKKD (SEQ ID NO:16).

[0070] The amino acid sequence of Pikh1 is (the 78-amino acid residue binding sequence is bold and underlined): MEAAAMAVTAATGALAPVLVKLAALLDDGECNLLEGSRSDAEFIRSELEAVHSLLTPNILGR MGDDDAACKDGLIAEVRELSYDLDDAVDDFLELNFEQRRSASPFGELKARVEEHVSNRFSDW KLPAASLPPSSVHRRAGLPPPDAELVGMDKRMEELTKLLEQGSNDASRWRKRKPHFPLRKTG LKQKIVIKVAMEGNNCRSKAMALVASTGGVDSVALVGDLRDKIEVVGYGIDPIKLISALRKK VGDAELLQVSQAKKDVKETTPMLAPVKSICEFHKVKTVCILGLPGGGKTTVARELYDALGTH FPCRVFVSVSPSSSPSPNLTKTLADIFAQAQLGVTDTLSTPYGGSGTGRALQQHLIDNISAF LLNKKYLIVIDDIWHWEEWEVIRKSIPKNDLGGRIIMTTRLNSIAEKCHTDDNDVFVYEVGD LDNNDALSLSWGIATKSGAGNRIGTGEDNPCYDIVNMCYGMPLALIWLSSALVGEIEELGGA EVKKCRDLRHIEDGILDIPSLQPLAESLCLGYNHLPLYLRTLLLYCSAYHWSNRIERGRLVR RWIAEGFVSEEKEAEGYFGELINRGWITQHGDNNSYNYYEIHPVMLAFLRCKSKEYNFLTCL GLGSDTSTSASSPRLIRRLSLQGGYPVDCLSSMSMDVSHTCSLVVLGDVARPKGIPFYMFKR LRVLDLEDNKDIQDSHLQGICEQLSLRVRYLGLKGTRIRKLPQEMRKLKHLEILYVGSTRIS ELPQEIGELKHLRILDVRNTDITELPLQIRELQHLHTLDVRNTPISELPPQVGKLQNLKIMC VRSTGVRELPKEIGELNHLQTLDVRNTRVRELPWQAGQISQSLRVLAGDSGDGVRLPEGVCE ALINGIPGATRAKCREVLSIAIIDRFGPPLVGIFKVPGSHMRIPKMIKDHFRVLSCLDIRLC HKLEDDDQKFLAEMPNLQTLVLRFEALPRQPITINGTGFQMLESFRVDSRVPRIAFHEDAMP NLKLLEFKFYAGPASNDAIGITNLKSLQKVVFRCSPWYKSDAPGISATIDVVKKEAEEHPNR PITLLINAGYKEISTESHGSSENIAGSSGIDTEPAQAQHDNLPAVRDDYKGKGILLDGRCPT CGRATKIEEETQDRVADIEIQTETTS (SEQ ID NO:18)

[0071] The amino acid sequence of Avr-PikA is:Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory MRVTTFNTFLLTLGTVAVVNAETGNKYIEKRAIDLSRERDPNFFDNADIPVPECFWF MFKNNVRQDAGTCYSSWKMDMKVGPNWVHIKSDDNCNLSGDFPPGWIVLGKKRP GF (SEQ ID NO:19).

[0072] The amino acid sequence of Avr-PikC is: MRVTTFNTFLLTLGTVAVVNAETGNKYIEKRAIDLSRERDPNFFDNPGIPVPECFWF MFKNNVRQDDGTCYSSWKMDMKVGPNWVHIKSDDNCNLSGDFPPGWIVLGKKRP GF (SEQ ID NO:20).

[0073] The amino acid sequence of Avr-PikD is: MRVTTFNTFLLTLGTVAVVNAETGNKYIEKRAIDLSRERDPNFFDHPGIPVPECFWF MFKNNVRQDAGTCYSSWKMDMKVGPNWVHIKSDDNCNLSGDFPPGWIVLGKKRP GF (SEQ ID NO:21).

[0074] The amino acid sequence of Avr-PikF is: MRVTTFNTFLLTLGTVAVVNAETGNKYIEKRAIDLSRERDPNFFDNADIPVPECFWF MFKNNVRQDAGTCYSSWKMDKKVGPNWVHIKSDDNCNLSGDFPPGWIVLGKKRP GF (SEQ ID NO:22).

[0075] The amino acid sequence of AvrPiz-t is: MQFSTIITVCLFTGLASASFVQCNHHLLYNGRHWGTIRKKAGWAVRFYEEKPGQPK RLVAICKNASPVHCNYLKCTNLAAGFSAGTSTDVLSSGTVGSIGNDPQAQRQ (SEQ ID NO:23).

[0076] In some embodiments, the promoter is a tissue-specific promoter. Examples of tissue- specific promoters under developmental control include promoters that initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, cell walls, including e.g., roots or leaves. A variety of promoters specifically active in vegetative tissues, such as leaves, stems, roots and tubers are known. For example, promoters controlling patatin, the major storage protein of the potato tuber, can be used (see, e.g., Kim, Plant Mol. Biol. 26:603-615, 1994; Martin, Plant J.11:53-62, 1997). The ORF13 promoter from Agrobacterium rhizogenes that exhibits high activity in roots can also be used (Hansen, Mol. Gen. Genet.254:337-343, 1997). Other useful vegetative tissue-specific promoters include: the tarn promoter of the gene encoding a globulin from a major taro (Colocasia esculenta L. Schott) corm protein family, tarin (Bezerra, Plant Mol. Biol.28:137-144, 1995); the curculin promoter active during taro corm development (de Castro, Plant Cell 4:1549-1559, 1992) andAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory the promoter for the tobacco root-specific gene TobRB7, whose expression is localized to root meristem and immature central cylinder regions (Yamamoto, Plant Cell 3:371-382, 1991).

[0077] Leaf-specific promoters, such as the ribulose biphosphate carboxylase (RBCS) promoters can be used. For example, the tomato RBCS1, RBCS2 and RBCS3A genes are expressed in leaves and light-grown seedlings, only RBCS1 and RBCS2 are expressed in developing tomato fruits (Meier, FEBS Lett.415:91-95, 1997). A ribulose bisphosphate carboxylase promoters expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels (e.g., Matsuoka, Plant J.6:311-319, 1994), can be used. Another leaf- specific promoter is the light harvesting chlorophyll a / b binding protein gene promoter (see, e.g., Shiina, Plant Physiol.115:477-483, 1997; Casal, Plant Physiol.116:1533-1538, 1998). The Arabidopsis thaliana myb-related gene promoter (Atmyb5) (Li, et al., FEBS Lett. 379:117-1211996), is leaf-specific. The Atmyb5 promoter is expressed in developing leaf trichomes, stipules, and epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds. Atmyb5 mRNA appears between fertilization and the 16 cell stage of embryo development and persists beyond the heart stage. A leaf promoter identified in maize (e.g., Busk et al., Plant J.11:1285-1295, 1997) can also be used.

[0078] Another class of useful vegetative tissue-specific promoters are meristematic (root tip and shoot apex) promoters. For example, the "SHOOTMERISTEMLESS" and "SCARECROW" promoters, which are active in the developing shoot or root apical meristems, (e.g., Di Laurenzio, et al., Cell 86:423-433, 1996; and, Long, et al., Nature 379:66-69, 1996); can be used. Another useful promoter is that which controls the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase HMG2 gene, whose expression is restricted to meristematic and floral (secretory zone of the stigma, mature pollen grains, gynoecium vascular tissue, and fertilized ovules) tissues (see, e.g., Enjuto, Plant Cell.7:517- 527, 1995). Also useful are kn1-related genes from maize and other species which show meristem-specific expression, (see, e.g., Granger, Plant Mol. Biol.31:373-378, 1996; Kerstetter, Plant Cell 6:1877-1887, 1994; Hake, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 350:45-51, 1995). For example, the Arabidopsis thaliana KNAT1 promoter (see, e.g., Lincoln, Plant Cell 6:1859-1876, 1994) can be used.

[0079] In some embodiments, the promoter is substantially identical to the native promoter of a promoter that drives expression of a gene involved in secondary wall deposition. ExamplesAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory of such promoters are promoters from IRX1, IRX3, IRX5, IRX8, IRX9, IRX14, IRX7, IRX10, GAUT13, or GAUT14 genes. Specific expression in fiber cells can be accomplished by using a promoter such as the NST1 promoter and specific expression in vessels can be accomplished by using a promoter such as VND6 or VND7. (See, e.g., PCT / US2012 / 023182 for illustrative promoter sequences). In some embodiments, the promoter is a secondary cell wall-specific promoter or a fiber cell-specific promoter. In some embodiments, the promoter is from a gene that is co-expressed in the lignin biosynthesis pathway (phenylpropanoid pathway). In some embodiments, the promoter is a C4H, C3H, HCT, CCR1, CAD4, CAD5, F5H, PAL1, PAL2, 4CL1, or CCoAMT promoter. In some embodiments, the tissue-specific secondary wall promoter is an IRXl, IRX3, IRX5, IRX8, IRX9, IRX14, IRX7, IRX10, GAUT13, GAUT14, or CESA4 promoter. Suitable tissue-specific secondary wall promoters, and other transcription factors, promoters, regulatory systems, and the like, suitable for this present invention are taught in U.S. Patent Application Pub. Nos.2014 / 0298539, 2015 / 0051376, and 2016 / 0017355.

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[0081] It is to be understood that, while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0082] All patents, patent applications, and publications mentioned herein are herebyAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory incorporated by reference in their entireties.

[0083] The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation. Example 1 Engineering plant immune receptors that bind novel pathogen-derived ligands

[0084] High-throughput single cell techniques are applied to engineer novel immune receptors that bind previously unrecognized ligands from a rice pathogen. The overarching goal of my postdoctoral research is to engineer plant immune receptors that bind novel pathogen-derived ligands and thus protect crop hosts from new pathogens. In the past year, I applied high-throughput single cell techniques to engineer novel immune receptors that bind previously unrecognized ligands from a rice pathogen. Ability of the engineered receptors to protect rice from new pathogens will be tested in the next year. Plant immune receptors can recognize pathogen-secreted ligands, or effectors, and protect the plant host from the invading pathogen. However, pathogens constantly evolve new effector variants that evade immune recognition (Figure 1, Panel A). Therefore, identifying immune receptors that show broad-spectrum binding of new effector variants has the potential to protect vulnerable crops from a wide range of pathogens. The first goal of my project was to set up a novel high- throughput method to identify such immune receptors. To this end, I expressed plant immune receptors on yeast cells, which are robust and amenable to single-cell techniques, and confirmed that yeastexpressed plant immune receptors function as expected and bind target effectors. I then applied this method to engineer a rice immune receptor that simultaneously binds effectors from multiple strains of Magnaporthe oryzae, a fungal pathogen that can lead to 10-30% crop loss (Figure 1, Panel B). Such immune receptor would protect rice from multiple strains of M. oryzae. Specifically, I searched for receptors that simultaneously bind multiple previously unrecognized effectors among a large number (2x107) of receptor variants generated through random mutagenesis in a process called directed evolution (Figure 1B). This approach allowed me to identify two rice receptor variants with increased binding to multiple new M. oryzae effectors (Avr-PikC, Avr-PikF, and AvrPiz-t) that are not recognized by any known natural immune receptors (Figure 2, Panels A and B). Notably, one of the newly targeted effectors, AvrPiz-t, is structurally dissimilar to the original set of ligands for the starting receptor. This demonstrates the flexibility of the yeast-based directedAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory evolution approach and opens the possibility to design immune receptors that bind a divergent set of pathogen-derived ligands and thus protect from a wide variety of crop pathogens. The steps and results of Paths 1 and 2 are shown in Figure 3.

[0085] Table 1. Amino acid sequence of receptor variants.

[0086] Table 2. In vitro ligand binding.Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory

[0087] References: Example 2 20210910 codon optimized OsPikp1 and OsPikm1 constructs cloning

[0088] ObjectivesAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory

[0089] Clone yeast codon optimized Pikp1 and Pikm1 sequences into yeast display vectors. OsPikp1 HMA domain codon optimized (Thermo) from Twist biosciences. OsPikm1 HMA domain codon optimized (Thermo) from Twist biosciences.

[0090] Test expression on yeast: stain N- and C-terminal ends: pCTcon2 and pCHA.

[0091] If expressed, can test binding to AVR-Pik effectors.

[0092] Procedure

[0093] 1. Amplify DNA fragment from Twist order.

[0094] Table 3. Primers.

[0095] Table 4. Buffer ingredients.

[0096] Table 5. Thermcycling program.Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory

[0097] 1. Restriction digest Twist order Q-132614 and vectors: DNA (1 μg total) = X μL; NheI-HF = 1 μL; BamHI-HF = 1 μL; CutSmart 10x buffer = 2 μL; dH2O = To 20 μL.

[0098] Vector: ER5 pCTcon2, ER19 pCHA.

[0099] Incubate at 37 °C for 2-3h.

[0100] 2. Run digested DNA on gel ^ OsPikm1 HMA digest: 243bp ^ OsPikp1 HMA digest: 240bp ^ -> clone in one reaction and sequence later.

[0101] 3. Gel purify cut bands: stain in fresh EtBr solution and place gel on saran wrap.

[0102] 4. DNA ligation ^ Calculate amount of vector and insert: see webpage at: insilico.uni-duesseldorf.de / Lig Input.html. ^ 1:8 vector:insert ratio

[0103] Table 6. DNA Ligation.

[0104] Melting ice protocol overnight ^ 4C allows sticky ends to hybridize (melting temp is 12 °C for NheI / BamHI ends).Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory ^ Raising up temperature then allows ligase to anneal the ends.

[0105] 5. Transformation: ^ Thaw vials of DH5a chemically competent E. coli (My stock in -80 °C). ^ Add 3 μL ligation mix to 50 μL competent cells. ^ Mix gently. DO NOT VORTEX OR PIPETTE. ^ Incubate on ice for 30 min. ^ Heat-shock cells for 30s at 42 °C. ^ Immediately transfer the tubes to ice and incubate for 2 min. ^ Add 200 μL of pre-warmed LB. ^ Shake the tube horizontally (200 rpm) at 37 °C for 1h . ^ Pre-warm selective agar plates at 37 °C. ^ Carb (=Amp) plates for pCHA. ^ Spread 100 μL culture on pre-warmed selective agar plates.

[0106] 1. Start cultures.4 mL LB + 4μL 1000x Carb.

[0107] 2. Miniprep Qiagen kit - elution in 30uL MilliQ water.

[0108] 3. Submit sequencing reactions. Gal1.

[0109] 4. Good.20210922: pCTcon2 OsPikm1 HMA #5 Example 3 In vitro and in planta binding of the engineered receptors

[0110] Pikh immune receptor from rice cultivar K3 does not recognize Avr-PikC, Avr-PikF, and AvrPiz-t from the fungal pathogen Magnaporthe oryzae. Engineered receptors expand immune recognition to Avr-PikC, Avr-PikF, and AvrPiz-t. These receptors couldAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory protect rice from Magnaporthe oryzae strains that express. Avr-PikC, Avr-PikF, and AvrPiz- t, from which there is no known protection. The in planta experiments are performed on tobacco plants (Nicotiana benthamiana). The results are shown in Figures 4-10. Example 4 Directed Evolution of a Plant Immune Receptor for Broad Spectrum Effector Recognition

[0111] Rapid development of immune receptors that protect crops from emerging pathogens is a critical challenge 1,2. While novel immune receptors that recognize previously undetected pathogen effectors could provide protection against a wider range of pathogens, engineering such receptors has been constrained by the low throughput and speed of in planta testing. We established yeast surface display as a high throughput platform to recapitulate plant immune receptor-ligand interactions and evolve new binding capabilities. Using this directed evolution platform, we engineered the ligand binding domain of the rice immune receptor Pik-1 to recognize diverse effectors from the fast-evolving fungal pathogen Magnaporthe oryzae. Our approach yielded Pik-1 ligand binding domains with affinity for variants of the M. oryzae effector Avr-Pik that previously escaped detection by known rice alleles of Pik-1, with in planta assays confirming functional recognition of these effectors. Additional rounds of mutagenesis and selection led to a Pik-1 domain that binds all tested Avr-Pik variants as well as the evolutionarily divergent effector AvrPiz-t. These results demonstrate the potential of directed evolution to engineer immune receptors with new-to- nature recognition of a wide range of pathogen-derived ligands and accelerate development of broad spectrum resistance in crops.

[0112] Our goal was to establish a rapid, high throughput method to explore vast synthetic sequence spaces and identify variants with broad spectrum ligand recognition. We chose Pik-1, an intracellular NOD-like receptor (NLR) in rice with a well-characterized effector binding domain 8–10, as our engineering target.

[0113] Rice blast, a fungal disease caused by Magnaporthe oryzae, leads to annual loss of rice enough to feed 60 million people 11,12. Introduction of immune receptors that protect against M. oryzae into crops presents an effective strategy to combat this global threat. Pik-1 recognizes the M. oryzae effector Avr-Pik through its heavy metal associated (HMA) domain, which has been coopted from rice endogenous targets of Avr-Pik 8,13,14.Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory Due to its modular nature, the Pik-1 HMA domain has been engineered extensively to alter ligand binding properties while retaining Pik-1 function in immunity 15–19. While Pik-1 recognition of Avr-Pik effectively triggers disease resistance, some M. oryzae strains harbor Avr-Pik variants with point mutations that escape detection by all known Pik-1 alleles; some strains lack Avr-Pik altogether. Eighty-five percent of sequenced rice-infecting M. oryzae strains that lack Avr-Pik variants harbor AvrPiz-t, a sequence divergent effector that belongs to the same family as Avr-Pik (Magnaporthe AVRs and ToxBlike, or MAX effector family) 20,21.

[0114] Coupling heterologous single cell expression with rounds of directed evolution (Fig.11, Panel a), we engineered Pik-1 HMA domains that bind all known Avr-Pik alleles. Pik-1 containing these engineered domains triggered an immune response when co-expressed with Avr-Pik effectors in plant assays. We then further optimized the HMA domain for affinity to AvrPiz-t, reasoning that an engineered immune receptor that binds both sets of effectors would lead to broad spectrum resistance. Our results reveal that directed evolution can yield plant immune receptors with the new-to-nature ability to recognize multiple sequence divergent ligands. These findings open a new avenue for enhancing crop resistance to emerging pathogens.

[0115] Plant immune receptor-ligand interactions are recapitulated in a yeast expression platform. We first set out to test whether the Pik-1 HMA domain could retain its effector interaction properties when expressed on Saccharomyces cerevisiae. In yeast surface display, recombinant proteins fused to a cell wall protein are displayed on the surface, allowing screening of up to ~109variants in one experiment. This approach has identified proteins with desired ligand interactions, including novel receptor domains that bind pathogen-derived molecules or antibodies with antitumor or pathogen neutralizing activities 22–24. We expressed three wellcharacterized rice genome-encoded alleles of the HMA domain of Pik-1 (Pikp-1, Pikm-1, and Pikh-1) tethered to the yeast Aga2p cell wall protein and flanked by epitope tags (Fig.11, Panel b).

[0116] We tested whether effector binding specificity and relative affinity were recapitulated by yeast surface displayed Pik-1 HMA domains (Fig.11, Panel c). Fluorescence imaging of Myc epitope-stained yeast cells showed expression of Pik-1 HMA domains on the cell surface and their interaction with biotin-labeled effectors (Fig.11, Panel d and Fig.15, Panel a). The Pikh-1 allele exhibits one of the widest effector recognition ranges. The HMAAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory domain of Pikh-1 showed strong affinity for Avr-PikD, moderate affinity for Avr-PikA, but no significant interaction with Avr-PikC and Avr-PikF variants of the effector, reproducing the effector binding properties of this allele in rice 25. Flow cytometry analysis of Pik-1 HMA domain-expressing yeast cells recapitulated these results (Fig.11, Panel e, and Fig.15, Panels b,c). These results validated that our yeast expression platform reflects the specificity and affinity of Pik-1 immune receptor-ligand interactions.

[0117] Directed evolution of Pik-1 HMA domain variants that bind previously unrecognized ligands. We then sought to engineer yeast displayed Pikh-1 HMA domain to bind previously unrecognized effectors through directed evolution. We first subjected the HMA domain to random mutagenesis through error prone PCR. Mutagenized amplicons were introduced into yeast cells to generate a library of Pikh-1 HMA domain variants. Colony dilution estimated the library to contain 2.2x107variants and sequencing a subset of the library showed that the average number of amino acid mutations was 2.1 over the 78aa in the effector binding domain, with the majority of variants containing one to three amino acid changes (Fig.16, Panel a).

[0118] To identify Pikh-1 HMA domain variants that bind the previously unrecognized effectors Avr-PikC and Avr-PikF, we performed four rounds of FACS selection (Fig.12, Panel a). Starting with the library, we exposed yeast cells displaying Pikh-1 HMA domain variants to either biotin-labeled Avr-PikC or Avr-PikF and selected those showing the highest affinity for the target effector and robust HMA domain expression. In subsequent selection rounds, variants were incubated with lower concentrations of the target effector alternating between Avr-PikC or Avr-PikF, with the goal of identifying variants that bind both effectors with high affinity (Fig.12, Panel a). In each selection round we sorted through enough cells to oversample the population diversity by five- to ten-fold to avoid losing unique variants, selecting cells in the top 0.5-1% for target binding and HMA domain expression to carry onto the next round (Fig.16, Panel b).

[0119] Following four rounds of selection, variants gained binding to both Avr-PikC and Avr-PikF at the population level regardless of the order in which they were exposed to the two effectors (Fig.12, Panel b). Illumina sequencing of these populations showed that several variants had increased in representation over selection rounds (Supplementary Table 2). Of these, variants we named A4.1 and B4.1 showed the greatest enrichment relative to the starting library at the end of selection path A and selection path B, respectivelyAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory (Supplementary Table 3).

[0120] These variants indeed exhibited high affinity for both Avr-PikC or Avr-PikF (Fig.12, Panel c). Flow cytometry and imaging analysis indicated that their affinities for Avr- PikC and Avr-PikF were comparable to or exceeded the binding affinity required for Pik-1- induced in planta immune response (Figs.15, Panel b and 16, Panel c). Importantly, A4.1 and B4.1 retained the ability to bind all effectors recognized by the wildtype Pikh-1 HMA domain: Avr-PikA, Avr-PikD, and Avr-PikE (Fig.16, Panel d). A4.1 and B4.1 also showed binding to a novel allele of Avr-Pik we identified in M. oryzae isolates TW-6-2-2-B-1 and JS- 10-6-1-2 and named Avr-PikJ (Fig.16, Panel d). An Avr-Pik-like effector with ~60% sequence identity to Avr-Pik, Avr-PikL2A, was previously identified in rice-infecting M. oryzae (Fig.16, Panel e) 26. Variants A4.1 and B4.1 bound Avr-PikL2A whereas the wildtype Pikh-1 HMA domain failed to do so (Fig.16, Panel d).

[0121] A4.1 and B4.1 harbor five substitutions, with two of them (Q74R and A75V of the HMA domain sequence) shared by both variants (Fig.12, Panel d). A75V is also present in interface 3 of the natural allele Pikm-1 and related variants, which is thought to contribute extensively to the interaction with Avr-Pik effectors in these alleles (Fig.17, Panel a) 27. However, the rest of the mutations that conferred broad spectrum Avr-Pik binding in A4.1 and B4.1 had not been observed in rice genome-encoded or previously engineered alleles of Pik-1. Therefore, these two Pikh-1 HMA domain variants gained the ability to bind all known alleles of Avr-Pik through unique, new-to-nature mutations.

[0122] Directed evolution of Pik-1 HMA domain variants with broad spectrum ligand binding. Directed evolution can yield a receptor that binds allelic variants of its native ligand, but can additional rounds of selection lead to affinity for a more divergent ligand? To test this, we targeted AvrPiz-t. While Avr-Pik and AvrPiz-t both belong to the MAX family of M. oryzae effectors characterized by their β-sandwich structures, they exhibit no detectable sequence similarity (Fig.13, Panel a, and Fig.16, Panel e). Avr-Pik and AvrPiz-t are recognized by two distinct rice genome-encoded immune receptors and interact with different sets of host proteins to suppress immunity, highlighting different evolutionary trajectories and functions of the two effectors 13,20,28–30. AvrPiz-t is present in 85% of rice-infecting M. oryzae that lack all alleles of Avr-Pik; 193 out of 201 rice-infecting isolates with genomes available on NCBI harbor either Avr-Pik or AvrPiz-t. Its divergence from Avr-Pik, high conservation in M. oryzae strains that lack Avr-Pik, and role in facilitating pathogen infectionAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory rendered AvrPiz-t both a good test case and a practical target for broad spectrum M. oryzae recognition by an engineered receptor.

[0123] To identify Pikh-1 HMA domain variants that bind AvrPiz-t in addition to Avr-PikC and Avr-PikF, we pooled variants collected after four rounds of selection through paths A and B. These variants were subjected to two subsequent rounds of selection to identify those with affinity for decreasing concentrations of AvrPiz-t (Fig.13, Panel b). As top HMA variants identified at the end of the selection process did not exhibit strong binding to AvrPiz-t, we further diversified the post-sort 6 pool through error-prone PCR. This new library was estimated to contain 2.1x107variants through colony dilution, with an average of 4.4 mutations over the 78aa in the effector binding domain (Fig.17, Panel b). Variants collected after one additional round of selection with 200nM AvrPiz-t showed enhanced affinity for AvrPiz-t at the population level (Fig.13, Panel c, and Fig.17, Panel c). We evaluated Pikh-1 HMA variants that were significantly enriched following sort 7 relative to their frequencies in the post-sort 6 mutagenized library (Supplementary Table 3). Among these, variant 7.2 demonstrated strong binding to AvrPiz-t as well as Avr-PikC and Avr-PikF in confocal imaging and flow cytometry analyses (Fig.13, Panels c,d, and Fig.18, Panel a). Additionally, 7.2 showed affinity for all tested alleles of Avr-Pik in confocal imaging (Fig. 18, Panel b).

[0124] The HMA domain variant with three (Q4R, K5I, and M12R) of the substitutions in 7.2 was present at low frequencies after four rounds of selection for Avr-PikC and Avr-PikF binding (Supplementary Table 2). This then became the predominant variant following selection rounds 5 and 6 for AvrPiz-t binding. However, these changes alone were insufficient to confer robust affinity for Avr-PikC, Avr-PikF, and AvrPiz-t (data not shown). The additional E13K and E68K substitutions in variant 7.2 appear to account for improved binding to all three ligands. Therefore, iterative cycles of stringent selection and diversity enrichment through random mutagenesis yielded a HMA domain variant capable of binding all Avr-Pik alleles and AvrPiz-t.

[0125] Recognition of target ligands by engineered Pik-1 immune receptors in N. benthamiana. Binding interactions between the rice Pik-1 and M. oryzae Avr-Pik can be recapitulated in the model plant Nicotiana benthamiana 10. Interaction between Agrobacterium tumefaciensdelivered Pik-1 and Avr-Pik triggers immune activity and cell death at the site of A. tumefaciens infiltration (Fig.14, Panel a) 10. We transiently expressedAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory full-length Pikh-1 harboring wildtype or engineered HMA domain, its helper receptor Pikh-2 required for immune function, and various Avr-Pik effectors in N. benthamiana. Despite plant-to-plant variability in cell death severity, especially near the ligand recognition threshold, A4.1 or B4.1 consistently triggered cell death in response to Avr-PikC and Avr- PikF (Fig.14, Panels b,c). The 7.2 HMA variant induced strong cell death with Avr-PikF and showed small but significant (p < 0.005, two-tailed Mann-Whitney test) improvement over wildtype HMA with AvrPiz-t (Fig.14, Panels d,e). However, 7.2 did not enhance Avr-PikC recognition despite showing binding in yeast-based assays (Fig.14, Panels d,e).

[0126] Changes to NLR effector binding domains can sometimes lead to autoactivity and trigger immune activation without the cognate effector 15. The engineered variants did not induce cell death in the absence of a target effector, demonstrating their specific activity (Fig.14, Panels b,d, Fig.19, Panels b,c). Therefore, Pikh-1 variants evolved to bind previously unrecognized ligands triggered immune activation to target ligands in N. benthamiana. We note that transferring robust recognition of a more divergent set of ligands proved more challenging. DISCUSSION

[0127] Our results demonstrate the first successful application of high throughput directed evolution to engineer a plant immune receptor domain that can bind previously unrecognized ligands. Lab-evolved Pikh-1 HMA domain variants displayed binding to all tested M. orzyae effectors in the Avr-Pik family as well as a sequence divergent effector, AvrPiz-t. An immune receptor that binds evolutionarily divergent effectors with distinct roles in pathogen infection is predicted to confer broad spectrum resistance in crops. If functionally validated in rice for immune function, such a receptor will likely maintain its effectiveness over time barring mutation or loss of expression of all target effectors in the pathogen.

[0128] Multiple evolutionary paths led to expanded effector perception in our experiments. A4.1 and B4.1 were obtained from different selection schemes and shared two out of their five amino acid substitutions. Yet both variants showed affinity for all known alleles of the effector Avr-Pik.Variant 7.2 additionally gained the ability to recognize AvrPiz- t through a yet different set of five synergistic changes. Substitutions in these engineered variants revealed residues relevant for effector recognition outside of the interfaces identified in receptor-effector crystal structures 10,15,25,27.Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory

[0129] When tested in N. benthamiana, engineered variants A4.1 and B4.1 activated immune responses to Avr-PikC and Avr-PikF, effectors that evade detection by known natural alleles of Pik-1. For variant 7.2, which was further engineered to bind AvrPiz-t, improvements in immune activation did not fully correlate with the binding strength observed in yeast assays for some effectors.7.2 likely binds AvrPiz-t through a different interface than Avr-Pik, as demonstrated for HMA domains that bind sequence divergent MAX effectors 31–33. The substitutions in 7.2 that enable AvrPiz-t binding may affect conformational changes required for Pikh-1 activation, particularly near ligand recognition thresholds. These results highlight both the challenge and importance of validating engineered protein functions in plant systems.

[0130] Plant immune receptor engineering has largely relied on grafting ligand binding domains or subsets thereof from genome-encoded variants and testing chimeric receptors in individual plants. While effective, this approach is labor intensive and requires extensive structural knowledge of the receptor-effector interface, which is not available yet for most plant immune receptors 34. Directed evolution in a heterologous system can expand effector recognition spectra of immune receptors at a faster pace even without prior structural knowledge. For instance, a loss-of-function random mutagenesis screen performed in yeast can quickly identify the receptor domain required for interaction with a cognate effector. A subsequent gain-of-function screen, as described here, searches for receptor variants with affinity for previously unrecognized effectors.

[0131] Enhancing effector recognition through substitutions identified through high throughput screening offers additional advantages over introducing exogenous immune receptors or replacing large receptor segments. These small changes are more amenable to current crop genome editing technologies. Introducing precise amino acid changes into a genome-encoded receptor such as Pik-1 through prime editing, for example, presents a feasible path to develop and deploy improved crops (31, 32). Our results demonstrate how high throughput directed evolution can explore large synthetic sequence spaces to develop immune recognition of divergent effectors, a function that seemed initially far-fetched. We have established directed evolution as a strategy to develop broad spectrum resistance and protect crops from fast evolving pathogens. METHODSAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory

[0132] DNA constructs. For yeast surface display: Yeast codon optimized sequences of rice Pik-1 HMA domains (aa186-263) was synthesized by Twist Biosciences and cloned into the yeast display vector pCTcon2 using NheI / BamHI restriction sites. pCTcon2 (Addgene plasmid #41843) was a gift from Dane Wittrup 38. For E. coli expression of effectors: pOPIN-GG vectors encoding Avr-PikD / A / C tagged with 6xHis and GB1 were gifts from Adam Bentham and Mark Banfield 39. Vectors encoding Avr-PikE and Avr-PikF were generated by site-directed mutagenesis of Avr-PikD and Avr-PikA, respectively. Avr-PikB and Avr-PikJ sequences were synthesized by Twist Biosciences and cloned into pOPIN-GG using NotI / BamHI restriction sites. AvrPiz-t and AvrPikL2A sequences PCR amplified from pUC57-Kan_GY11_00131271 (Addgene plasmid #121355) and pUC57-Kan_FR13_APikL2 (Addgene plasmid #123648), which were gifts from Sophien Kamoun 40, and Gibson cloned into pOPIN-GG. For cell death assay in N. benthamiana: Rice Pikh2 sequence was synthesized by Twist Biosciences and cloned into the binary vector pGFPGUSPlus (Addgene plasmid #64401), which was a gift from Claudia Vickers 41, using SacI / XbaI restriction sites. CaMV 35S promoter followed by rice Pikh1 sequence was synthesized by Twist Biosciences and introduced into pGFPGUSPlus Pikh2 using SbfI / BstEII restriction sites. This resulted in a binary vector with Pikh2 and Pikh1 with wildtype or engineered HMA domain, each flanked by CaMV 35S promoter and Nos terminator. Avr-PikA, Avr-PikC, Avr-PikF, AvrPiz-t, and P19 were introduced into pEarleyGate10442 using PspXI / PacI restriction sites. This resulted in a vector with the coding sequence flanked by CaMV 35S promoter and OCS terminator. Sequences of all primers and DNA constructs are listed in Supplementary Table 4.

[0133] Library preparation. Pikh-1 HMA domain in the yeast surface display vector pCTcon2 was subjected to error prone PCR following published library generation protocols 38,43,44. Briefly, Pikh-1 HMA domain sequence was amplified using Taq polymerase in mutagenic reactions with 1μM to 2μM of 8-oxo-dGTP (TriLink N-2034) and dPTP (TriLink N-2037) for 15 cycles. These reactions were pooled and further amplified to yield ~20μg DNA. pCTcon2 vector DNA was digested with SalI, NheI, and BamHI at 37°C overnight. 4μg of digested vector was mixed with 12μg of the mutagenized insert and precipitated using Pellet Paint following the manufacturer’s protocol (Millipore 69049). Mutagenized library of yeast surface-displayed Pikh-1 HMA domain was introduced into yeast strain EBY100 (ATCC MYA-4941) via electroporation 24,44,45.100 mL of YPD media [1% (w / v) yeast extract, 2% peptone, 2% D-glucose] was inoculated with stationary phase EBY100 to anAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory OD600 of 0.3. Cells were incubated at 30°C with shaking at 225rpm until OD600 of 1.6 was reached. Cells were pelleted at 3000 xg for 3 minutes, washed twice with 50mL cold MilliQ water, once with 50mL cold electroporation buffer [1M sorbitol, 1mM CaCl2], and incubated in 20mL of lithium buffer [0.1M LiAc, 10mM DTT] for 30 minutes at 30°C with shaking at 225rpm. Cells were resuspended in 1mL electroporation buffer.400μL of the electrocompetent EBY100 was mixed with 4μg linearized vector and 12μg mutagenized insert. Following electroporation using a Bio-Rad GenePulser Xcell at 2.5 kV and 25 μF in a 2mm cuvette, cells were suspended in 10mL of 1:1 YPD:1M sorbitol and incubated at 30°C for 1 hour. Cells were centrifuged and resuspended in 100mL SDCAA [2% (w / v) D-glucose, 0.54% disodium phosphate, 0.86% monosodium phosphate, 0.5% casamino acids, 0.67% yeast nitrogen base]. Serial dilution from 1 / 100 to 1 / 100,000 estimated library size of 2.2 × 107.

[0134] Yeast surface display. Single clones of wildtype or mutated Pikh-1 HMA domain were introduced into yeast strain EBY100 using Frozen-EZ Yeast Transformation II kit (Zymo Research) following the manufacturer’s protocol. Transformed yeast was grown on SDCAA agar plates [SDCAA with 1.5% agar and 18.2% sorbitol] at 30°C for 2-3 days. Individual colonies were picked and grown in 5mL SDCAA at 30°C with shaking at 225rpm for 16 hours. To induce expression of Pikh-1 HMA domain on yeast, SDCAA cultures were centrifuged at 1800 xg for 3 minutes and washed with SGCAA [2% (w / v) galactose, 0.54% disodium phosphate, 0.86% monosodium phosphate, 0.5% casamino acids, 0.67% yeast nitrogen base]. Cells were resuspended in SGCAA to a final concentration of 1 X 107cells / mL and grown at 25°C with shaking at 225 rpm for 24 hours.

[0135] Confocal microscopy. Approximately 106yeast cells were pelleted at 3000 xg at 4°C for 3 minutes and resuspended in 100μL PBSA [1% bovine serum albumin in PBS pH 7.4]. After adding a biotinylated effector to the specified concentration, cells were incubated rotating at 25°C for 1.5 hours. In the last 30 minutes, 1:100 Myc tag rabbit monoclonal antibody (Cell Signaling 2278) was added to detect Pikh-1 HMA domain expression. Cells were pelleted and washed with 500μL PBSA and resuspended in 100μL PBSA with 1:100 anti-rabbit IgG secondary antibody Alexa Fluor 568 conjugate (Thermo Fisher A-11010) and 1:100 streptavidin Alexa Fluor 488 conjugate (Thermo Fisher S11223). Secondary staining was performed on ice in the dark for 30 minutes. After a final wash with 500μL PBSA, cell pellet was resuspended in 30μL PBSA.6-7μL of the stained cell suspension was mounted onAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory a glass slide and covered with 22mm x 22mm #1 thickness coverslip. Samples were imaged on Leica TCS SP8 confocal microscope with 63x oil immersion lens and excitation with 488nm and 552nm lasers.

[0136] Fluorescence-activated cell sorting. Published yeast library FACS protocols were modified for selection of Pikh-1 HMA domain variants that bind target effectors 43,44,46,47. For the initial sort, 108yeast cells were pelleted at 3000 xg at 4°C for 3 minutes and resuspended in 1mL PBSA and divided into five 1.7mL tubes. After adding a biotinylated effector to the specified concentration, cells were incubated rotating at 25°C for 1.5 hours. In the last 30 minutes, 1:100 Myc-tag rabbit monoclonal antibody (Cell Signaling 2278) was added to detect Pikh-1 HMA domain expression. Cells were pelleted and washed with 1mL PBSA and resuspended in 200μL PBSA with 1:100 anti-rabbit IgG secondary antibody Alexa Fluor 488 conjugate (Thermo Fisher A-11008) and 1:100 streptavidin PE conjugate (Thermo Fisher S866). Secondary staining was performed on ice in the dark for 30 minutes. After a final wash with 1mL PBSA, cell pellet was resuspended in 4mL PBSA and passed through 35μm strainer mesh into FACS tubes (Corning 352235). Cells expressing the wildtype Pikh-1 HMA domain and exposed to 10nM Avr-PikD were used to prepare unstained, 488 or PE single stained, and double stained control samples. Top 1% cells with the high PE and 488 fluorescence values, representing target effector binding and Pikh-1 HMA domain expression, respectively, were sorted into SDCAA. Approximately 106sorted cells were grown in 50mL SDCAA at 30°C with shaking at 225 rpm until saturation. These cells were frozen down in 15% glycerol in SDCAA or passaged in SDCAA for use in subsequent rounds of FACS. Each subsequent round started with at least tenfold the number of cells collected in the previous round to ensure adequate sampling of the variant diversity. Reagent to detect target effector binding alternated between streptavidin PE conjugate and anti-biotin PE conjugate (Thermo Fisher 50-169-246) to prevent selection for secondary reagent binding. Experiments were performed on Beckman Coulter Astrios EQ cell sorter and Becton Dickinson FACS Aria II cell sorter at the UC Davis Flow Cytometry Shared Resource. FACS data were analyzed and plots were generated using FlowJo software v10.9.0 (Becton Dickinson). To generate histograms, yeast cells were gated first using forward versus side scatter, then single cells were gated using side scatter height versus width. Receptor expressing cells gated using Alexa Fluor 488 area versus PE area were then plotted in a histogram, with same gates applied to all samples (Fig.15, Panel d).Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory

[0137] Yeast DNA extraction and sequencing. For yeast DNA extraction, Zymoprep Yeast Plasmid DNA Miniprep II kit (Zymo Research) was used following the manufacturer’s protocol. Briefly, >106yeast cells were grown in 2mL SDCAA at 30°C with shaking at 225rpm for 4 hours. Cells were pelleted at 16000 xg for 3 minutes and resuspended in 200μL digestion buffer with 5μL Zymolase. Following a 2-hour incubation at 37°C, cell lysate was prepared using lysis and neutralization buffers from Zymoprep Yeast Plasmid DNA Miniprep II kit. DNA was extracted using GenElute Plasmid Miniprep Kit (Sigma) and columns and eluted in 20μL MilliQ water.50ng of the eluted DNA was used as template in a 100μL PCR reaction with 500nM pCTcon2 Illumina Fwd / Rev primers, 200μM dNTP, 3% DMSO, and 2 units of Phusion High Fidelity DNA polymerase (New England Biolabs) in 1x Phusion HF buffer. Reaction was initially heated to 98°C for 5 minutes then cycled 30 times at 98°C for 10 seconds, 60°C for 30 seconds, and 72°C for 1 minute, and finally extended at 72°C for 10 minutes, yielding 410bp amplicons containing the Pikh-1 HMA domain insert. PCR product was purified using DNA Clean & Concentrator Kit (Zymo Research) and eluted in 20μL MilliQ water. Up to 1μg of purified DNA was sent for library preparation, sequencing reaction, and initial bioinformatics analysis at AZENTA, Inc. DNA library was prepared with NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs) following the manufacturer’s protocol and sequenced using a 2x 250 paired-end (PE) configuration on an Illumina instrument. Raw sequence reads were trimmed of their adapters and nucleotides with poor quality using Trimmomatic v.0.36. This returned 338000 reads per sample on average. After further filtering reads with indels or nonsense mutations, reads that encode unique Pikh-1 HMA domain amino acid sequences were counted. Pikh-1 HMA domain variants detected and their read counts in the starting library and FACS selected samples can be found in Supplementary Table 1 and Supplementary Table 2.

[0138] Effector purification and biotinylation. Avr-Pik and AvrPiz-t effectors were expressed using pOPIN-GG vectors generated and shared by Bentham and colleagues 39. Effectors tagged with 6xHis and GB1 in pOPIN-GG were introduced into Shuffle T7 Express Competent E. coli (New England Biolabs C3029). Single colonies were grown in 5mL Terrific Broth [1.2% (w / v) tryptone, 2.4% (w / v) yeast extract, 0.94% (w / v) dipotassium phosphate, 0.22% (w / v) monopotassium phosphate, 0.4% (v / v) glycerol, 1mM magnesium sulfate] with 100μg / mL carbenicillin at 30°C with shaking at 150rpm for 20 hours. These cultures were used to inoculate 500mL fresh TB with 100μg / mL carbenicillin and grown at 30°C with shaking at 100rpm to an OD600 of 0.6-0.8. To induce protein expression, IPTGAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory was added to 1mM for 18 hours at 20°C with shaking at 100rpm. Cells were harvested by centrifugation at 4000 xg at 4°C for 20 minutes and frozen at -80°C. Thawed cell pellet was mixed with 10mL lysis buffer [50mM HEPES pH 8.0, 500mM NaCl, 50mM glycine, 5% (v / v) glycerol, 30mM imidazole, 1 tablet EDTA-free protease inhibitor cocktail (Millipore Sigma 11873580001)], incubated on ice for 30 minutes, and sonicated with approximately 600J energy. Lysate was collected following centrifugation at 10000 xg at 4°C for 20 minutes. Effectors were affinity purified using Ni-NTA Spin Columns (Thermo Fisher 88224) following the manufacturer’s protocol and eluted in 500mM imidazole in lysis buffer. Samples were desalted using Spin Desalting Columns (Thermo Fisher 89892) and buffer exchanged into A4 buffer [10mM HEPES pH 7.4, 150mM NaCl] using Amicon Ultra-0.5 Centrifugal Filter Unit 10KDa (Millipore UFC501024) following manufacturers’ protocols. 6xHis and GB1 tag was cleaved by adding 20U / mg protein 3C Protease (Thermo Fisher 88946) and rotating for 16 hours at 4°C. Cleaved effectors were collected in the flow-through after binding to Ni-NTA Spin Columns, desalted, and buffer exchanged into PBS pH 8.0. Sample purity was confirmed in a Coomassie stained gel and BCA Protein Assay (Thermo Fisher 23225) measured effector amount of 200μg-1mg per prep. Effectors were biotin labeled by adding 40-fold molar excess NHS-PEG4-biotin (Thermo Fisher 21455) and rotating for 1 hour at 24°C. To quench the reaction, Tris pH 8.0 was added to 50mM final concentration and rotated for 30 minutes at 24°C. Excess reagents were eliminated from samples using G-10 Macro SpinColumns (Harvard Apparatus 74-3904). Biotin labeled and purified effectors were stored in -80°C until use in FACS or imaging experiments.

[0139] Nicotiana benthamiana cell death assay. N. benthamiana plants were grown in a growth chamber with 16-hour light cycle with 150μmol / m2 light intensity and 24°C day and 22°C night temperatures. Plants were germinated and grown in Sunshine Mix #1 (Sun Gro Horticulture) and watered with 2:1:2 N:P:K macronutrient ratio fertilizer. Agrobacterium tumefaciens GV3101 carrying desired constructs were resuspended in infiltration media [10 mM MES pH 5.7, 10 mM MgCl2, 500 μM Acetosyringone]. Sequences of all DNA constructs are listed in Supplementary Table 4. A. tumefaciens carrying Pikh-2 and wildtype or engineered Pikh-1 (final OD6000.6) was mixed with Avr-Pik, AvrPiz-t, or YFP (final OD6000.6) and P19 (final OD6000.2). The fourth leaf from the top of each five-week-old N. benthamiana was infiltrated using a needleless syringe and the plants were kept in the dark post infiltration 48. Leaves were collected at 3 days post infiltration for white light and UV autofluorescence imaging. Cell death response of each infiltrated spot was blindly scoredAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory using a 0-6 scale (Fig.19, Panel a) and two independent scores were averaged. Each engineered Pikh-1 was tested in three experiments with six plants per experiment.

[0140] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory What is claimed is:

1. An engineered, genetically modified, and / or synthetic plant receptor comprising a binding sequence that is capable of binding a fungal pathogen Avr ligand; wherein the non-engineered, genetically unmodified, and / or naturally occurring plant receptor does not bind the fungal pathogen Avr ligand, or bind with a lower binding affinity.

2. The engineered, genetically modified, and / or synthetic plant receptor of claim 1, wherein the plant receptor is a Pik receptor.

3. The engineered, genetically modified, and / or synthetic plant receptor of claim 2, wherein the Pik receptor is a Pikh1 receptor.

4. The engineered, genetically modified, and / or synthetic plant receptor of claim 1, wherein the fungal pathogen Avr ligand is Avr-PikA, Avr-PikD, Avr-PikC, Avr-PikF, and / or AvrPiz-t.

5. The engineered, genetically modified, and / or synthetic plant receptor of claim 1, wherein the binding sequence comprises SEQ ID NO:

17.

6. The engineered, genetically modified, and / or synthetic plant receptor of claim 5, wherein the binding sequence comprises one or more of the following, wherein all of the following positions are according to SEQ ID NO:1: an amino acid with a positive charged side chain at position 4; an amino acid with a hydrophobic side chain at position 5; an amino acid with a hydrophobic side chain at position 8; an amino acid with a positive charged side chain at position 11; an amino acid with a positive charged side chain, hydrophobic side chain, or amino acid G at position 12; an amino acid with a negative charged side chain at position 16; an amino acid with a positive charged side chain at position 17; an amino acid with a bulky aromatic side chain, or the amino acid is P, at position 19; an amino acid with a negative charged side chain at position 20; an amino acid with a negative charged side chain at position 32; an amino acid with a hydrophobic side chain at position 34; an amino acid with a hydrophobic side chain at position 37; an amino acid with a polar uncharged side chain or bulky aromatic side chain at position 40; an amino acid with a bulky aromatic side chain at position 41; an amino acid with a positive charged side chain at position 45; an amino acid with a positive charged side chain, or amino acid C, at position 49; an amino acid with a polar uncharged side chain at position 54; an amino acid with a hydrophobic side chain at position 57; amino acid G at position 66; anAttorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory amino acid with a positive charged side chain or hydrophobic side chain at position 68; an amino acid with a polar uncharged side chain at position 70; an amino acid with a positive charged side chain at position 71; an amino acid with a hydrophobic side chain at position 72; an amino acid with a positive charged side chain or hydrophobic side chain at position 74; an amino acid with a hydrophobic side chain at position 75; an amino acid with a negative charged side chain at position 77; and / or amino acid G at position 78.

7. The engineered, genetically modified, and / or synthetic plant receptor of claim 6, wherein the binding sequence comprises one or more of the following: the amino acid residue at position 4 is R; the amino acid residue at position 5 is I; the amino acid residue at position 8 is V; the amino acid residue at position 11 is R; the amino acid residue at position 12 is K, G, or V; the amino acid residue at position 16 is D; the amino acid residue at position 17 is R; the amino acid residue at position 19 is F or P; the amino acid residue at position 20 is R; the amino acid residue at position 32 is E; the amino acid residue at position 34 is A; the amino acid residue at position 37 is I; the amino acid residue at position 40 is S or W; the amino acid residue at position 41 is W; the amino acid residue at position 45 is K; the amino acid residue at position 49 is C or H; the amino acid residue at position 54 is T; the amino acid residue at position 57 is V; the amino acid residue at position 66 is G; the amino acid residue at position 68 is K or V; the amino acid residue at position 70 is S; the amino acid residue at position 71 is R; the amino acid residue at position 72 is I; the amino acid residue at position 74 is R or L; the amino acid residue at position 75 is V; the amino acid residue at position 77 is E; and / or the amino acid residue at position 78 is G.

8. The engineered, genetically modified, and / or synthetic plant receptor of claim 7, wherein the binding sequence comprises one of SEQ ID NOs:1-15.

9. A nucleic acid encoding the engineered, genetically modified, and / or synthetic plant receptor or binding sequence of claim 1.

10. A nucleic acid encoding the engineered, genetically modified, and / or synthetic plant receptor of claim 1 operatively linked to a promoter.

11. A vector comprising the nucleic acid of claim 10.Attorney Docket: 2023-147-02 Lawrence Berkeley National Laboratory 12. A plant or plant cell comprising the vector of claim 11, wherein the plant or plant cell is capable of expressing the engineered, genetically modified, and / or synthetic plant receptor.

13. A method to confer on a plant or plant cell resistance to a fungal pathogen, comprising: introducing into a plant or plant cell with a nucleic acid encoding the engineered, genetically modified, and / or synthetic plant receptor of claim 1 operatively linked to a promoter, whereby the nucleic acid encoding the engineered, genetically modified, and / or synthetic plant receptor operatively linked to a promoter stably integrates into the genome of the plant or plant cell.

14. The method of claim 13, wherein the fungal pathogen is a Magnaporthe species, 15. The method of claim 14, wherein the Magnaporthe species is a Magnaporthe oryzae.

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