CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U. S. Provisional Patent Application

No. 62/505,395, filed May 12, 2017, which is hereby incorporated herein in its entirety by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE

The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 070294-0139SEQLST.TXT, created on April 12, 2018, and having a size of 15.4 KB, and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant improvement, particularly to methods for the identification of plant genes that encode proteins that are involved in plant responses to elicitors such as, for example, elicitors derived from the interactions of microorganisms and herbivores with plants.

BACKGROUND OF THE INVENTION

Plants are under constant attack from a range of pathogens, yet disease symptoms are comparatively rare. Cell-autonomous innate immunity ensures each plant cell has the ability to respond to pathogen attack. In contrast, animals use a circulatory system to ensure full spatial coverage of innate immunity, while jawed vertebrates have supplemented this defense through the evolution of an adaptive immune system. Despite the presence of similar innate immunity strategies in plants and animals, the precise epitopes perceived differ. Thus, instead of divergent evolution from a common ancestor, any similarities are likely to be a result of convergent evolution (Zipfel and Felix, G. (2005) Curr. Opin. Plant Biol. 8:353-360;

Ausubel (2005) Nature Immunology 6:973-979). In plants, innate immunity consists of three main defense systems: physical, local and systemic. Physical defense include the waxy cuticle and rigid cell wall, as well as secondary metabolites and enzymes possessing antimicrobial properties. This defense is partially breached by stomata or through wounding. Recognition at the local level then relies on the specific perception of microbial compounds.

Plant cells continuously monitor their apoplastic environment notably by employing receptor-like kinases (RLKs) and receptor-like proteins (RLPs). Among them, the pattern recognition receptors (PRRs) are able to detect apoplastic elicitors ('non-self molecules, also referred as Pathogen-/Microbe-/Herbivore- Associated Molecular Patterns

(PAMPs/MAMPs/HAMPs), and 'damaged-self molecules, also referred to as damage- associated molecular patterns" (DAMPs)) and to trigger a cellular response. This response is characterized by a series of physiological events, including very early ones such as ion fluxes across the membrane causing a rapid increase in cytoplasmic Ca ²⁺ concentrations, production of reactive oxygen species (ROS) and MAPK phosphorylation. These early responses collectively contribute to the establishment of plant PRR-triggered immunity (PTI) and play an important role in plant basal defence against a broad spectrum of microbial infections (Boiler and Felix {2009) Annu. Rev. Plant Biol. 60:379-40).

Because many PAMPs are essential microbial molecules which are not easily mutated without conferring a selective disadvantage, the use of PRRs to enhance the resistance of plants to multiple pathogens holds great promise for creating durable resistance. In a groundbreaking study, Lacombe et al. first demonstrated that the interfamily transfer of a

Brassicaceae PRR to non-Brassicaceae plants confers broad-spectrum bacterial resistance in stably transformed, non-Brassicaceae plants (Lacombe et al. (2010) Nat. Biotechnol. 28:365- 369). The PRR that was transferred to non-Brassicaceae plants by Lacombe et al. is EF-Tu receptor (EFR) fromArabidopsis thaliana ecotype Col-0. Elongation Factor-Tu (EF-Tu) is a bacterial PAMP that can cause PTI in plants of the Brassicaceae family but is not known to cause PTI in plants outside of the Brassicaceae family (Zipfel and Felix, G. (2005) Curr. Opin. Plant Biol. 8:353-360). EFR recognizes a region of 18 amino-acids at the N-acetylated terminus of eubacterial EF-Tu that is highly conserved (Kunze et al. (2004) The Plant Cell 16:3496-3507; Zipfel et al. (2006) Cell 125 :749-760). Lacombe et al. demonstrated that a Brassicaceae PRR, when expressed in stably transformed, non-Brassicaceae plants, can trigger the activation of basal immune responses stably transformed, non-Brassicaceae plants leading to resistance to multiple bacterial pathogens. See also WO 2010/062751 and U. S. Pat. No. 9,222, 103. More recently, other researchers have reported that monocotyledonous plants (wheat, rice) and apple trees stably transformed with EFR have enhanced resistance to bacterial pathogens (Schoonbeek et al. (2015) New Phytol. 206:606-613; Schwessinger et al., PLoS Pathogens, 2015, 11 (3): el004809; 2Blades Foundation, Printed April 8, 2016, 2Blades Foudnation, "Pattern recognition receptors (PRRs)," available on the World Wide Web at: 2blades.org/pattem-recognition-receptors-prrs/).

The identification of new PRRs that recognize specific apoplastic elicitors has the potential to provide new sources of durable resistance for use in the genetic improvement of crop plants against multiple plant pathogens. While bioinformatics approaches can be used to rapidly select candidate polypeptides which are likely to be function as PRRs in plants, the identification of a PRR that recognizes a particular elicitor requires a functional assay to demonstrate that the exposure of a candidate polypeptide to a particular elicitor triggers one or more physiological changes associated with PTI. Heterologous, non-plant systems such as yeast or bacteria have been used to express large collections of candidate genes (Boiler and Felix (2009) Annu. Rev. Plant Biol. 60:379-40). However, beyond ligand and protein binding, activated PRRs are not able to trigger plant-specific PTI physiological events when expressed in heterologous, non-plant systems.

A range of methods is available to monitor the different early physiological PTI events in plants (Boiler and Felix (2009) Annu. Rev. Plant Biol. 60:379-40). To detect PRR activation, monitoring Ca ²⁺ cytoplasmic influx is a reliable proxy as transient changes in permeability of the plasma membrane to Ca ²⁺ are a common early event in plant defence signaling (Lecourieux et al. (2006) New Phytol. 171 :249-269, doi: 10.1 11 1/j .1469- 8137.2006.01777.x). The PRR-dependent increase of cytosolic Ca ²⁺ concentration is typically transient in response to elicitor perception (Ranf et al. (201 1) Plant J. 68: 100-113). The cytosolic Ca ²⁺ concentration arises a few seconds after elicitor treatment and returns to the resting state within ten minutes.

Recently, Keinath et al. ((2015) Mol. Plant 8: 1188-1200) reported that transgenic Arabidopsis thaliana lines stably expressing a genetically encoded calcium indicator (GECI) can be used to monitor Ca ²⁺ -dependent signal changes in roots and detached leaves following treatment with the bacterial-derived peptide elicitor fig22 and the fungal elicitor chitin. Flg22 and chitin are conserved elicitors that are recognized cell autonomously by pattern recognition receptors (Boiler and Felix (2009) Annu. Rev. Plant Biol. 60:379-40). Keinath et al. produced separate transgenic Arabidopsis lines expressing either two fluorescence-based GECIs, the Forster resonance energy transfer (FRET)-based reporter yellow camel eon NES- YC3.6 and the intensity -based sensor R-GECOl and demonstrated that in detached leaves R- GECOl shows significantly increased Ca ²⁺ -dependent signal changes compared with NES- YC3.6. Keinath et al. reported that the superior sensitivity of R-GECOl enabled the monitoring of flg22- and chitin-induced Ca ²⁺ signals on a cellular level.

While the results of Keneith et al. demonstrate that a GECI, particularly R-GECOl, can be used to monitor changes in cellular Ca ²⁺ levels associated with PTI in plants in leaves and roots of Arabidopsis seedlings, the method used by Keneith et al. requires the development of stable transgenic plants and thus, is not particularly well suited for the functional screening candidate polypeptides for a desired PRR function in a medium- to high- throughput format. A method for rapidly assaying candidate PRR polypeptides for the initiation of PTI-associated physiological changes following exposure to an elicitor of interest would aid scientists in the identification of new potential sources of durable resistance to plant diseases for use in the genetic improvement of crop plants.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for screening a candidate protein for a desired pattern recognition receptor (PRR) function. The methods involve exposing at least one plant protoplast to an effective concentration of an elicitor of interest, wherein the plant protoplast comprises the candidate protein and an effective concentration of a calcium ion (Ca ²⁺ ) sensor. Preferably, the plant protoplast comprises the candidate protein and an effective concentration of the Ca ²⁺ sensor prior to the being exposed to the effective concentration of the elicitor. The candidate protein can be directly introduced into the plant protoplast or can be expressed from a polynucleotide construct encoding the candidate protein that is present in the plant protoplast. The Ca ²⁺ sensor can be, for example, a chemical indicator or a genetically encoded calcium indicator (GECI) that can be expressed in the plant protoplast from a polynucleotide construct encoding the GECI that was introduced into the protoplast. In some embodiments, the candidate protein, the GECI, or both are expressed in the plant protoplast from one or more polynucleotide constructs introduced into the plant protoplast.

The methods for screening a candidate protein for a desired pattern recognition receptor (PRR) function further comprise measuring a signal emitted from or in the plant protoplast, wherein a change in the magnitude of the signal is indicative of an increase in the concentration of Ca ²⁺ in the plant protoplast. The methods can further comprise selecting the candidate protein as having the desired PRR function when a change in the magnitude of the signal is measured following exposure of the protoplast to the elicitor.

The present invention further provides plant protoplasts that comprise a candidate protein for a desired pattern recognition receptor (PRR) function and a Ca ²⁺ sensor and/or that are a capable of expressing one or both of the candidate protein and the Ca ²⁺ sensor from one or more polynucleotides introduced into the plant protoplasts.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graphical representation of the elfl 8-induced Ca ²⁺ burst in 96-well format using corn protoplasts transiently transfected with 10 μg ZmUbi: :R-GEC01.2: :rbcS and 10 μg of either 2x35S+Q: :AtEFR-FLAG: :rbcS or pUC19 with the protoplasts in 96-well format. Elfl 8 final concentration is 100 nM. Fluorescence values are normalized as (F-Fo)/Fo using 10 μg pUC19 as Fo. Values are averages ± SD (n=5).

FIG. 2 is a graphical representation of the elfl 8-induced Ca ²⁺ burst in 384-well format using corn protoplasts transiently transfected with 15 μg ZmUbi: :R-GEC01.2: :rbcS and 10 μg of either 2x35S+Q: :AtEFR-FLAG: :rbcS or pUC19. Elfl 8 final concentration is 100 nM. Fluorescence values are normalized as (F-Fo)/Fo using respective buffer treatments as Fo. Values are averages, «=8.

FIG. 3 is a graphical representation of the response of corn protoplasts to exogenous

Ca ²⁺ with or without pre-elicitation. The application of exogenous calcium (2 M CaCh, 20% ethanol) induces a transient increase of fluorescence in protoplasts expressing ZmUbi: :R- GEC01.2: :rbcS, but not in protoplasts that did not express this construct. Thirty minutes before exogenous calcium treatment, protoplasts have been pre-treated with either buffer or efll 8, inducing an elfl 8- dependent increase of fluorescence only in the protoplasts expressing ZmUbi: :R-GEC01.2: :rbcS + 2x35S+Q: :AtEFR-FLAG: :rbcS. In response to exogenous calcium treatment, all the protoplasts expressing ZmUbi: :R-GEC01.2: :rbcS display a comparable increase of fluorescence and the presence of PRR or pre-treatment with the elicitor was found not to affect the emission of fluorescence. Values are averages, «=8.

SEQUENCE LISTING

The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5' end of the sequence and proceeding forward (i.e. , from left to right in each line) to the 3' end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus.

SEQ ID NO: 1 sets forth the nucleotide sequence of the PRR polynucleotide construct 2x35S+Q: : AtEFR-FLAG: :rbcS.

SEQ ID NO: 2 sets forth the nucleotide sequence of the control (PRR) polynucleotide construct pUC19.

SEQ ID NO: 3 sets forth the nucleotide sequence of the GECI polynucleotide construct ZmUbi: :R-GEC01.2: :rbcS.

SEQ ID NO: 4 sets forth the amino acid sequence for the elf 18 peptide.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Overview

The present invention relates to methods that can be used to screen a group of candidate plant proteins to identify from among the members of the group of candidate proteins at least one pattern recognition receptor (PRR) that binds to or recognizes a specific elicitor, whereby cellular responses are initiated which can lead to PRR-triggered immunity (PTI). The present invention can also be used to screen a group of candidate plant proteins to identify among the members of the group of candidate proteins at least one receptor that binds to or recognizes a specific ligand, whereby cellular responses are initiated and include a transient change in cytoplasmic calcium concentration. PRRs are plant proteins that are involved in the innate immunity response of plants that can occur following the initiation of an interaction between, for example, a host plant and a plant pathogen. A PRR binds or recognizes a particular elicitor that results from the interaction between a host plant and a plant pathogen and then initiates PRR-triggered immunity (PTI) to limit plant disease in the host plant.

Thus, the methods of the present invention find use in identifying PRRs that have the potential to be useful in strategies for producing crop plants with enhanced resistance to plant diseases caused by plant pathogens. Such strategies can involve, for example, introducing into a plant, which lacks a PRR for an elicitor associated with a particular plant pathogen, a PRR that binds to or recognizes the elicitor, whereby the plant comprising the new PRR is capable of initiating PTI when exposed to the elicitor or preferably when exposed to the plant pathogen that is known to produce the elicitor.

Such a strategy has been shown to be successful with the EF-Tu receptor (EFR) from

Arabidopsis thaliana. Elongation Factor-Tu (EF-Tu) is a bacterial elicitor that can cause PTI in plants of the Brassicaceae family but is not known to cause PTI in plants outside of the Brassicaceae family (Zipfel and Felix, G. (2005) Curr. Opin. Plant Biol. 8:353-360). It has been demonstrated that stably transforming non-Brassicaceae plants— including both dicots and monocots— with EFR can enhance the resistance of the transgenic plants to bacterial pathogens (Lacombe et al. (2010) Nat. Biotechnol. 28:365-369; WO 2010/062751 ; U. S. Pat. No. 9,222, 103; Schoonbeek et al. (2015) New Phytol. 206:606-613; Schwessinger et al, PLoS Pathogens, 2015, 1 1 (3): el004809; 2Blades Foundation, Printed April 8, 2016, 2Blades Foundation, "Pattern recognition receptors (PRRs)," available on the World Wide Web at: 2blades.org/pattern-recognition-receptors-prrs/).

The present invention provides methods for screening a candidate protein for a desired PRR function that are described in further detail below. The methods of the present invention can be performed in medium-throughput and high-throughput formats for the rapid screening of candidate plant proteins for a desired PRR function, particularly the ability of cause an increase in the cytoplasmic concentration of Ca ²⁺ in the cytoplasm of at least one plant protoplast following the exposure of the plant protoplast to an effective concentration of an elicitor of interest. While methods disclosed herein may be described for use with at least one protoplast, the methods are not limited to use with a single plant protoplast but can be used with populations of plant protoplasts of varying numbers.

Non-limiting embodiments of the invention include, for example, the following embodiments.

1. A method for screening a candidate protein for a desired pattern recognition receptor (PRR) function, the method comprising:

(a) exposing at least one plant protoplast to an effective concentration of an elicitor of interest, wherein the plant protoplast comprises the candidate protein and an effective concentration of a calcium ion (Ca ²⁺ ) sensor; and

(b) measuring a signal emitted from the Ca ²⁺ sensor, wherein a change in the magnitude of the signal is indicative of an increase in the concentration of Ca ²⁺ in the plant protoplast.

2. The method of embodiment 1, wherein the candidate protein is expressed in the plant protoplast after transfecting the plant protoplast with a first polynucleotide construct comprising a nucleotide sequence encoding the candidate protein.

3. The method of embodiment 1 or 2, wherein the Ca ²⁺ sensor comprises a polypeptide.

4. The method of embodiment 3, wherein the Ca ²⁺ sensor is expressed in the plant protoplast after transfecting the plant protoplast with a second polynucleotide construct comprising a nucleotide sequence encoding the Ca ²⁺ sensor.

5. The method of embodiment 4, wherein the plant protoplast is transfected with the first polynucleotide construct and the second polynucleotide construct at the same time.

6. The method embodiment 4, wherein the plant protoplast is not transfected with the first polynucleotide construct and the second polynucleotide construct at the same time. 7. The method of any one of embodiments 2-6, wherein the transfecting comprises polyethylene glycol (PEG) mediated transfection.

8. The method of any one of embodiments 2-6, wherein the transfecting comprises electroporation.

9. The method of any one of embodiments 1-8, wherein the Ca ²⁺ sensor is a chemical indicator.

10. The method of embodiment 9, wherein the chemical indicator is selected from the group consisting of Stil-1, Stil-2, Indo-1, Fura-1, Fura-2, Fura-3, Quin-2, and Calcium Green- 1.

11. The method of any one of embodiments 1-8, wherein the Ca ²⁺ sensor is a genetically encoded calcium indicator (GECI).

12. The method of embodiment 11, wherein the GECI is selected from the group consisting of R-GECOl.2, O-GECOl, CAR-GECOl, G-GECO0.5, G-GECOl, G-GECOl. l, G-GECOl.2, B-GECO0.1, B-GECOl, R-GECOl, GEM-GECOl, GEX-GECOl, GCaMPl, GCaMPl.6, GCAMP2, GCaMP3, SyGCaMP2, GCaMP5B, GCaMP5D, GCaMP5G,

GCaMP5K, GCaMP5L, GECO, GCaMP6, RCaMP1.07, R-CaMP2, YC2, YC3, YC4, YC3.6, Camgaroo, Camgaroo-2, Flash Pericam, Inverse Pericam, Ratiometric Pericam, TN-XXL, Twitch, and Aequorin.

13. The method of embodiment 11, wherein the GECI is R-GECOl.2.

14. The method of any one of embodiments 1-13, further comprising selecting the candidate protein as having the desired PRR function when a change is the magnitude of the signal is measured.

15. The method of any of embodiments 1-14, wherein the change in the magnitude of the signal is an increase in the magnitude of the signal.

16. The method of any of one of embodiments 1-15, wherein the signal is fluorescence.

17. The method of embodiment 16, wherein the fluorescence is measured using a fluorimeter.

18. The method of any one of embodiments 1-17, wherein the candidate protein is homologous to known PRR proteins.

19. The method of any one of embodiments 1-18, wherein step (a) comprises exposing a population of plant protoplasts to the effective concentration of the elicitor of interest and step (b) comprises measuring a signal emitted from the population of plant protoplasts and wherein a change in the magnitude of the signal is indicative of an increase in the concentration of cytosolic Ca ²⁺ in the plant protoplasts.

20. The method of embodiment 19, wherein the number of plant protoplasts in the population is between about 4000 and about 400,000.

21. The method of embodiment 19 or 20, wherein the population of plant protoplasts is solution having a volume between about 1 and about 100 μί.

22. The method of any one of embodiments 19-21, wherein the population of protoplasts in a solution comprising between about 2* 10 ⁵ protoplasts per mL to about 2* 10 ⁷ protoplasts per mL.

23. The method of anyone of embodiments 1 -22, wherein step (a) and/or step (b) comprise(s) the use of a microplate.

24. The method of any one of embodiments 1-23, wherein the plant protoplast is derived from at least one plant part selected from the group consisting of a leaf, a root, a stem, a fruit, a flower, a petal, a cotyledon, a hypocotyl, an epicotyl, an embryo or a seed.

25. The method of any one of embodiments 1-23, wherein the plant protoplast is prepared from an in-vitro-cultured cell.

26. The method of any one of embodiments 1-25, wherein the plant protoplast is from a monocotyledonous plant or a dicotyledonous plant.

27. The method of embodiment 1-26, wherein the candidate protein was obtained from a monocotyledonous plant or a dicotyledonous plant.

28. The method of embodiment 26 or 27, wherein the monocotyledonous plant is selected from the group consisting of maize, wheat, rice, barley, rye, oats, sorghum, switchgrass, sugarcane, teff, banana, date, coconut, oil palm, banana, and yam.

29. The method of embodiment 26 or 27, wherein the dicotyledonous plant is selected from the group consisting of soybean, canola, cotton, alfalfa, sugar beet, potato, tomato, pepper, tobacco, eggplant, chickpea, cassava, coffee, cacao, cannabis, lettuce, poplar, eucalyptus, sweet potato, peanut, citrus trees, and cashew.

30. The method of any one of embodiments 1-29, wherein the elicitor of interest is derived from a plant pathogen.

31. The method of embodiment 30, wherein the plant pathogen is selected from the group consisting of bacteria, fungi, oomycetes, and viruses.

32. The method of any one of embodiments 1-29, wherein the elicitor of interest is derived from an herbivore or microorganism. 33. The method of embodiment 32, wherein the herbivore is selected from the group consisting of insects and nematodes.

34. The method of embodiment 32, wherein the microorganism is not known to be a plant pathogen of a plant of interest.

35. The method of any one of embodiments 1-29, wherein the elicitor of interest is derived from a plant comprising an infection with a pathogen or a plant comprising damage from an herbivore.

36. The method of embodiment 35, wherein the plant pathogen is selected from the group consisting of bacteria, fungi, oomycetes, and viruses.

37. The method of embodiment 35, wherein the herbivore is selected from the group consisting of insects and nematodes.

38. A plant protoplast comprising a first polynucleotide construct comprising a nucleotide sequence encoding a candidate PRR protein and a second polynucleotide construct comprising a nucleotide sequence encoding a Ca ²⁺ sensor.

39. A plant protoplast comprising a heterologous, candidate PRR protein and a

Ca ²⁺ sensor.

Definitions

In the context of this disclosure, a number of terms are used. The following definitions are provided immediately below. Other definitions can be found throughout the disclosure. It is understood that the definitions provided herein are the preferred definitions for the purposes of describing the present invention, unless it is stated otherwise herein or apparent form the context of usage.

A "calcium ion sensor" or "Ca ²⁺ sensor" is a molecule or a group of two or more different molecules that when present in plant protoplast is capable of emitting a signal that changes in magnitude following a change in the Ca ²⁺ level in the plant protoplast, particularly the in the cytoplasm of the plant protoplast. Preferred Ca ²⁺ sensors are those sensors that preferential bind Ca ²⁺ over other cations that are known to occur in plant cells, particularly divalent cations (e.g. Mg ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , Fe ²⁺ ) that are known to occur in cells, particularly plant cells.

A "candidate protein" is a protein that is suitable for use in the methods disclosed elsewhere herein for screening for a desired PRR function. In some embodiments of the invention, the candidate protein is selected based on homology to known PRRs and/or selected as being encoded by a nucleotide sequence with homology to nucleotide sequences encoding one or more known PRRs. In other embodiments, the candidate protein is a chimeric PRR wherein, for example, at least one domain of the candidate protein is homologous to corresponding domain of a known PRR and at least one other domain of the candidate protein is not homologous to a corresponding domain of a known PRR protein. In yet other embodiments, the candidate protein is not homologous to a known PRR nor does the candidate protein comprise a domain that is homologous to a corresponding domain of a known PRR.

A "chemical indicator" is a non-proteinaceous molecule that can bind calcium ions in a plant protoplast and is a dye, particularly a fluorescent dye. For example, binding of a Ca ²⁺ to a chemical indicator that is a fluorescent dye leads to either an increase in quantum yield of fluorescence or emission/excitation wavelength shift following excitation by exposure to radiation at an excitation wavelength.

A "desired pattern recognition receptor function" or "desired PRR function" is the ability, biological activity, or biological function of protein to cause an increase, particularly a transient increase, in the Ca ²⁺ concentration in the cytoplasm of one or more plant protoplast following exposure to an elicitor of interest. Typically, the transient increase in the cytosolic Ca ²⁺ concentration can be measured or detected as a change in the magnitude of signal emitted by the Ca ²⁺ sensor.

An "effective amount of Ca ²⁺ sensor" is an amount that is suitable for detecting or measuring changes in the Ca ²⁺ levels in a plant protoplast in the methods of the present invention. It is recognized that such an effective amount is dependent on a number of factors including, for example, the plant species from which the protoplast is derived, the tissue or cell type from which the plant protoplast was derived, the developmental stage or maturity of the tissue or cell type from which the plant protoplast was derived, the environmental conditions under which the plant or in vitro-cultured cell was grown, the protoplast isolation procedure, the solution comprising the plant protoplast when it exposed to the elicitor, and/or the particular Ca ²⁺ sensor utilized. It is further recognized that persons of ordinary skill in the art know and understand how to determine empirically an effective amount of a Ca ²⁺ sensor.

An "effective amount of an elicitor" is an amount that is suitable for initiating in a plant protoplast one or more of the cellular responses that are associated with PRR-triggered immunity (PTI), particularly an increase in the cytosolic Ca ²⁺ level in the plant protoplast in the methods of the present invention. It is recognized that such an effective amount is dependent on a number of factors including, for example, the plant species from which the protoplast is derived, the tissue or cell type from which the plant protoplast was derived, the developmental stage of the tissue or cell type from which the plant protoplast was derived, the environmental conditions under which the plant or in vitro-cultured cell was grown, the protoplast isolation procedure, the solution comprising the plant protoplast when it exposed to the elicitor, the protoplast density, and/or the particular elicitor utilized. It is further recognized that persons of ordinary skill in the art know and understand how to determine empirically an effective amount of an elicitor.

Dose-response assays have revealed that apoplastic elicitors are usually active at nanomolar ranges (Boiler et al. (2009) doi: 10.1146/annurev.arplant.57.032905.105346).

Dose-responses are presented for chitin in Felix et al., 1998 (doi: 10.1104/pp.117.2.643), for flg22 in Felix et al, 1999 (doi: 10.1046/j. l365-313X.1999.00265.x), for elf26 in Kunze et al, 2004 (doi: 10.1105/tpc.104.026765), for AtPepl in Krol et al, 2010 (doi:

10.1074/jbc.M109.097394), and for xup25 in Mott et al, 2016 (doi: 10.1186/sl3059-016- 0955-7). To improve the success of signal detection, biological assays are usually performed with saturating concentrations of elicitor (e.g. in the micromolar range).

An "elicitor" is a molecule is which is capable of inducing in a plant one or more of cellular responses associated with PRR-triggered immunity (PTI). Elicitors include both naturally occurring and synthetic or artificial (i.e. non-naturally occurring) molecules such as, for example, peptides, oligopeptides, polypeptides, and non-peptide molecules. Such naturally occurring elicitors include, but are not limited to, molecules that produced by a plant pathogen or are derived from a molecule produced by plant pathogen that is modified upon initiation of an interaction of a host plant and a plant pathogen and damaged host plant molecules which are derived from molecules produced by the host plant and are modified (e.g. degraded) either upon initiation of an interaction of a host plant and a plant pathogen or during or following the herbivore attack, so as to produce a molecule that did not occur in the plant prior to the initiation of the interaction of the host plant and the plant pathogen or prior to the herbivore attack. Such synthetic or artificial elicitors include, but are not limited to, molecules that are structurally similar to a naturally occurring elicitor or at least a part thereof and are capable of binding to or being recognized by the same PRR as a naturally occurring elicitor. An example of such a synthetic or artificial elicitor is the elf 18 peptide which is derived elongation factor Tu (EF-Tu) (Kunze et al. (2004) Plant Cell 16: 3496-3507).

Preferred elicitors of the present invention are apoplastic elicitors including, but not limited to, 'non-self (i.e. non-host plant) molecules such as, for example, pathogen-associated molecular patterns (PAMPs), microbe- associated molecular patterns (MAMPs), and herbivore- associated molecular patterns (HAMPs).

A "genetically encoded calcium indicator" or "GECI" is intended to mean a protein that is capable of binding Ca ²⁺ and emitting light or fluorescence as a result of Ca ²⁺ binding to the protein. Typically, a GECI is expressed in a plant protoplast or cell from a nucleic acid molecule encoding the GECI that was introduced into the protoplast or cell or into a progenitor protoplast or cell. However, a GECI can be directly introduced into plant protoplast or cell by, for example, injection or any method known in the art for introducing a protein into a plant protoplast.

A "plant protoplast" is a plant cell which has had its cell wall removed or partially removed by mechanical and/or enzymatic methods but retains an intact plasma membrane. Preferably, a plant protoplast of the present invention is a capable of one or more of the normal cellular activities characteristic of the living plant cell from which it was derived including, for example, respiration, transcription, translation, glycolysis, and photosynthesis. More preferably, a plant protoplast of the present invention is capable of initiating at least the early stages of a PTI response following the binding of an elicitor to a PRR. Most preferably, a plant protoplast of the present invention is capable of increasing its cytoplasmic Ca ²⁺ concentration, at least transiently, for following the binding of an elicitor to a PRR.

A "population of plant protoplasts" is intended to mean two or more plant protoplasts.

While the methods of the present invention are not dependent of a population of a certain number of plant protoplasts, it is recognized that for the present invention, a population of plant protoplasts typically comprises at least 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 1,000,000 or more plant protoplasts. Typically, a plant protoplast of the present invention and populations thereof are in an aqueous solution that contains a buffer and an appropriate concentration such that the aqueous solution is isotonic to the cytoplasm of the plant protoplasts or the cytoplasms of the plant protoplasts in the population to plant protoplasts.

"Transfection" is the process of deliberately introducing naked or purified nucleic acids (e.g. DNA or RNA) by into eukaryotic cells including, for example, animal cells, plant cells, and plant protoplasts. Typically, "transfection" does not involve the use of a bacterium or virus to facilitate the introduction of the nucleic acids into the host cells. A related term is "transformation" or "genetic transformation" which is the genetic alteration of a cell or protoplast resulting from the introduction and incorporation of exogenous nucleic acids (e.g. DNA or RNA) from its surroundings through the cell membrane(s). As used related to plant cells, "transformation" typically can often involve the use of a bacterium (e.g. Agrobacterium tumefaciens) or a virus to mediate the introduction of DNA into the plant cell. However, for the present invention, unless expressly stated otherwise or apparent from the context of usage, "transfection" and "transformation" are equivalent terms that are intended to mean the process of introducing nucleic acids into plant cells and plant protoplasts.

Description

The methods of the present invention for screening a candidate protein for a desired pattern recognition receptor (PRR) function involve the use of plant protoplasts that are derived from one or more plants, one of more plant parts, or in vitro-cultured plant cells. The methods of the present invention comprise the following steps:

(a) exposing at least one plant protoplast to an effective concentration of an

elicitor derived from a plant pathogen of interest, wherein the plant protoplast comprises the candidate protein and an effective concentration of a calcium ion (Ca ²⁺ ) sensor;

(b) measuring a signal emitted from the calcium ion sensor, wherein a change in the magnitude of the signal is indicative of an increase in the concentration of Ca ²⁺ in the plant protoplast.

Typically, a plant protoplast is in an aqueous solution when it is exposed to the elicitor. The aqueous solution will generally comprise a sufficient concentration of an osmoticum or osmotic agent or to achieve an osmotic potential of the aqueous solution that is essentially isotonic with the interior of the plant protoplast so as to prevent the plant protoplast from bursting or shrinking. Osmoticum include, for example, mannitol, sorbitol, glucose, fructose, galactose, and sucrose. Preferred osmoticum are soluble carbohydrates that are not metabolized by a protoplast derived from a particular plant species or at least only slowly metabolized by the plant protoplast including, but not limited to, mannitol and sorbitol. Typically, such preferred osmoticum are present in the aqueous solution at a concentration of between about 0.3 M and about 0.7 M. It is recognized that the

concentration of the osmoticum used will depend on a number of factors including, but not limited to, the osmoticum used, the plants species from which the protoplast is derived, the concentration(s) of any other osmotically active components of the aqueous solution, the environment conditions (e.g. assay temperature), the protoplast density in the aqueous solution, and the like.

The plant protoplast or a population of plant protoplasts can be exposed to the elicitor by adding a certain volume of a solution comprising the elicitor (i.e. "an elicitor solution") to the aqueous solution comprising one or more plant protoplasts to an effective concentration of the elicitor in the solution comprising the one or more plant protoplasts. Preferably, the elicitor solution has osmotic potential approximately the same as the aqueous solution comprising the plant protoplast or the population of plant protoplasts so as not alter substantially the osmotic potential of the resulting combined solution.

The methods of the present invention do not depend on particular volume of solution comprising the population of plant protoplasts. The volume of aqueous solution can depend on, for example, the size of vessel or container used in the assay. For example, in certain embodiments of the invention comprising the use of a 96-well microplate, the population of plant protoplasts can be in an aqueous solution of about 100 μί. In certain embodiments of the invention comprising the use of a 384-well microplate, the population of plant protoplasts can be, for example, in an aqueous solution of about 25 μί. In various embodiments of the invention, the population of plant protoplast can in an aqueous solution having a volume, before or after the addition of the elicitor solution, of about 1 μί, 2 μί, 3 μί, 4 μί, 5 μί, 10 μΐ,, 15 μΐ,, 20 μί, 25 μί, 30 μί, 40 μί, 50 μί, 75 μί, 100 μί, 125 μί, 150, 175 μί, 200 μί, 250 μί, 300 μί, 400 μί, 500 μί, 750 μί, 1000 μί, or more.

The methods of the present invention do not depend on particular protoplast density (i.e. number of protoplasts per unit volume) in the aqueous solution. Generally, the protoplast density is in the range of about 10 ³ to about 10 ⁹ protoplasts per mL of aqueous solution before or after addition of the elicitor solution. In some embodiments of the invention, the protoplast density is in the range of about 2* 10 ⁵ to about 2* 10 ⁷ protoplasts per mL of aqueous solution before or after addition of the elicitor solution. In other embodiments of the invention, the protoplast density is about 2x l0 ⁶ protoplasts per mL of aqueous solution before or after addition of the elicitor solution.

The plant protoplasts of the present invention comprise the candidate protein and an effective concentration of a calcium ion (Ca ²⁺ ) sensor. If desired, the methods of the present invention can further comprise introducing the candidate protein, the Ca ²⁺ sensor, or both into a plant protoplast. While in some embodiments of the invention the candidate protein is directly introduced into a plant protoplast by, for example, injecting a solution comprising the candidate protein into the plant protoplast, the candidate protein is preferably expressed in the plant protoplast from polynucleotide construct comprising a nucleotide sequence encoding the candidate protein. Such a polynucleotide construct comprising a nucleotide sequence encoding the candidate protein can be introduced into the plant protoplast by, for example, polyethylene glycol (PEG)-mediated transfection, electroporation, particle bombardment, or Agrobacterium-mediated transfection. Such a polynucleotide construct can further comprise a promoter operably linked to the nucleotide sequence encoding the candidate protein whereby the promoter is capable of driving expression of the operably linked nucleotide sequence in the plant protoplast. In preferred embodiments of the invention, the promoter is a constitutive promoter.

In certain embodiments of the invention, the Ca ²⁺ sensor is chemical indicator, and in certain other embodiments the Ca ²⁺ sensor comprises a polypeptide. However, in preferred embodiments of the invention, the Ca ²⁺ sensor is a genetically encoded calcium indicator (GECI) which is capable of being expressed in the plant protoplast from a polynucleotide construct comprising a nucleotide sequence encoding the Ca ²⁺ sensor that is a GECI. A

GECI is a protein that is capable of binding Ca ²⁺ and emitting a signal such as, for example, light or fluorescence that changes in the intensity or magnitude as a result of Ca ²⁺ binding to the protein. A polynucleotide construct comprising a nucleotide sequence encoding a GECI can further comprise a promoter operably linked to the nucleotide sequence encoding the GECI, whereby the promoter is capable of driving expression of the operably linked nucleotide sequence in the plant protoplast. In preferred embodiments of the invention, the promoter is a constitutive promoter. Such a GECI can be expressed in a plant protoplast from a polynucleotide construct encoding the GECI that was directly introduced into the plant protoplast by, for example, PEG-mediated transfection, electroporation, particle

bombardment, or Agrobacterium-mediated transfection. Alternatively, the GECI can be expressed in a plant protoplast that is produced from a plant cell that comprises the polynucleotide construct encoding the GECI stably integrated into its genome. Methods for producing a plant cell comprising a stably integrated polynucleotide construct are described below or at otherwise known in the art.

GECIs that can be used in the methods of the present invention include, but are not limited to, of R-GECOl.2, O-GECOl, CAR-GECOl, G-GECO0.5, G-GECOl, G-GECOl. l, G-GECOl.2, B-GECO0.1, B-GECOl, R-GECOl, GEM-GECOl, GEX-GECOl, GCaMPl, GCaMPl.6, GCAMP2, GCaMP3, SyGCaMP2, GCaMP5B, GCaMP5D, GCaMP5G, GCaMP5K, GCaMP5L, GECO, GCaMP6, YC2, YC3, YC4, YC3.6, Camgaroo, Camgaroo-2, Flash Pericam, Inverse Pericam, Ratiometric Pericam, TN-XXL, Twitch, and Aequorin. See Okorocha (2015) IOSR-JNHS 4: 13-19 for additional GECIs and the references cited therein; herein incorporated by reference. For a discussion of the use of R-GECOl and R-GEC01.2 as Ca ²⁺ sensors, see Zhao et al. (2011) Science 333(6051): 1888-1891 md Wu et al. (2013) ACS Chem. Neurosci. 4:963-972, respectively; herein incorporated by reference. Further improved variants of R-GECOl, named RCaMP1.07 and R-CaMP2, have recently been reported and can be used in the methods of the present invention (Shen et al. (2015)

Neurophotonics. 2(3):031203, doi: 10.1117/l.NPh.2.3.031203; herein incorporated by reference).

In some embodiments of the invention, the candidate protein is expressed in the plant protoplast from a first polynucleotide construct comprising a nucleotide sequence encoding the candidate protein and the Ca ²⁺ sensor is expressed in the plant protoplast from a second polynucleotide construct comprising a nucleotide sequence encoding the Ca ²⁺ sensor. In certain embodiments, the plant protoplast is co-transfected with the first polynucleotide construct and the second first polynucleotide construct. In certain other embodiments, the plant protoplast is sequentially transfected with the two polynucleotide constructs; that is, the plant protoplast is initially transfected with first polynucleotide construct followed by being transfected with the second polynucleotide construct, or the plant protoplast is initially transfected with second polynucleotide construct followed by being transfected with the first polynucleotide construct. In yet other embodiments, a plant protoplast comprising one of the first or second polynucleotide construct stably incorporated it its genome is transfected with the other (i.e. the one not stably incorporated in its genome) polynucleotide construct. For example, a plant protoplast comprising a stably incorporated polynucleotide construct comprising a nucleotide sequence encoding a GECI is transfected with a polynucleotide construct comprising a nucleotide sequence encoding a candidate protein.

In embodiments of the invention in which each of the candidate protein and the Ca ²⁺ sensor are expressed from polynucleotide constructs, each of the polynucleotide constructs will typically further comprise an operably linked promoter that is capable of driving expression in the plant protoplast. It is recognized that the depending on the desired expression level of the candidate protein and the Ca ²⁺ sensor, the same promoter or different promoters can be used to drive expression of the two polynucleotide constructs in the plant protoplast. As indicate above, the Ca ²⁺ sensor can also be chemical indicator, which is a non- proteinaceous molecule that can bind calcium ions in a plant protoplast and is a dye, particularly a fluorescent dye. Preferably, the chemical indicators are sufficiently lipophilic to be able or cross a plasma membrane or are injected into a protoplast. In certain embodiments, chemical indicators that are dyes comprise chelator carboxyl groups masked as acetoxymethyl esters, in order to render the molecule lipophilic and to allow easy entrance into the cell. Once this form of the indicator is in the plant protoplast, endogenous esterases therein can free the carboxyl groups and thus, converting the chemical indicator to form that is able to bind Ca ²⁺ . Preferred chemical indicators are capable of crossing the plasma membrane of the plant protoplast to gain access to cytoplasm of the plant protoplast.

Chemical indicators that can be used in the method of the present invention include, but are not limited to, Stil-1, Stil-2, Indo-1, Fura-1, Fura-2, Fura-3, Quin-2, and Calcium Green-1. See Okorocha (2015) IOSR-JNHS 4: 13-19 for additional chemical indicators.

The methods for screening a candidate protein for a desired PRR comprise measuring a signal emitted from the Ca ²⁺ sensor, wherein a change in the magnitude of the signal is indicative of an increase in the concentration of Ca ²⁺ in the plant protoplast. Typically, the signal is emitted from the Ca ²⁺ is readily detectable such as, for example, light or fluorescence. When the signal is light, a change in the magnitude or intensity of light emitted can be measured with, for example a photometer. When the signal is fluorescence, a change in the magnitude or intensity of fluorescence emitted at a specific wavelength can be measured with, for example a fluorimeter (also known as a "fluorometer") which is device that is capable of measuring fluorescence emitted at a specific wavelength from a sample following excitation of the sample by radiation at an excitation wavelength.

The methods of the present invention involve measuring a signal emitted from the Ca ²⁺ sensor, wherein a change in the magnitude of the signal is indicative of an increase in the concentration of Ca ²⁺ in the plant protoplast. The present invention does not depend on particularly change in magnitude of the signal. Even relatively minor changes in the magnitude of signals such as, for example, fluorescence can be detected using state-of-the-art instruments such as the fluorimeters that are available today. Preferably, for the methods of the present invention, a change in the magnitude of the signal emitted from the Ca ²⁺ sensor following exposure of the one or more plant protoplasts to the elicitor is a change of at least about 0.5%, 1%, 2%, 3% 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 750%, 1000% or more, relative to the magnitude of the signal immediately prior to the addition of the elicitor. For the methods of the present invention in which the signal is light, a change in the magnitude of the light emitted from the Ca ²⁺ sensor following exposure of the one or more plant protoplasts to the elicitor is preferably an increase from about 0 to about 50 photons/second/image field.

For the methods of the present invention in which the signal is fluorescence, a change in the magnitude of the fluorescence emitted from the Ca ²⁺ sensor following exposure of the one or more plant protoplasts to the elicitor is preferably an increase at least about 0.5%, 1 %, 2%, 3% 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, 200% or more, relative to the magnitude of the signal immediately prior to the addition of the elicitor. As described elsewhere, the signal can be measured at one, two, three, four, five, or more times following the addition of the elicitor so as to monitor transient changes in the magnitude of the signal. Typically, in such cases, the change in magnitude is determined as the peak change; that is the largest change in magnitude detected. See FIGS. 1-3.

The methods can further comprise selecting the candidate protein as having the desired PRR function when a pre-specified change in the magnitude of the signal is measured following exposure of the one or more plant protoplasts to the elicitor. The magnitude of such a pre-specified change will depend on a number of factors including the elicitor, the plant species, the assay conditions, and the like. Typically, such a pre-specified change in the magnitude of the signal is a change of at least about 1 %, 5%, 10%, 25%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 800%, 900%, 1000% or more, relative to the magnitude of the signal immediately prior to the addition of the elicitor.

The candidate proteins of the present invention are any proteins that might comprise the desired PRR function, particularly naturally occurring plant proteins. However, it is recognized that the methods of the present invention are also suitable for screening candidate proteins which are synthetic or artificial variants of naturally occurring plant proteins and other non-naturally occurring proteins. Such synthetic or artificial variants of naturally occurring plant proteins include, for example, engineered bifunctional molecules comprising the ectodomain from one receptor-like kinase (RLK) and the kinase domain from another RLK (de Lorenzo et al. (201 1) FEBS Lett. 585(1 1): 1521-1528,

doi: 10.1016/j .febslet.201 1.04.043; Kishimoto et al. (201 1) Plant Signal Behav. 6(3): 449- 451 , doi: 10.4161/psb.6.3.14655; Kaku and Shibuy (2016) Physiol. Mol. Plant Pathol. 95: 60- 65. doi : 10.1016/j .pmpp.2016.02.003). Such synthetic or artificial variants of naturally occurring plant proteins also include, for example, synthetic or artificial variants that are produced in a plant by mutation breeding or genome editing techniques and synthetic or artificial variants that are produced in vitro by, for example, site-directed mutagenesis. For a comprehensive overview of mutation breeding, see "Principals of Cultivar Development" Fehr, 1993 Macmillan Publishing Company; herein incorporated by reference.

Genome editing techniques involve inducing double breaks in DNA using TAL

(transcription activator-like) effector nucleases (TALEN), Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease (CRISPR/Cas nuclease), zinc-finger nucleases (ZFN), or homing endonucleases that have been engineered endonucleases to make double-strand breaks at specific recognition sequences in the genome of a plant, other organism, or host cell. See, for example, WO 2010/079430; Morbitzer et al. (2010) PNAS 10.1073/pnas. l013133107; Scholze and Boch (2010) Virulence 1 :428-432; Christian et al. Genetics (2010) 186:757-761 ; Li et al. (2010) Nuc. Acids Res. (2010)

doi: 10.1093/nar/gkq704; and Miller et al. (2011) Nature Biotechnology 29: 143-148; Cho et al. (2013) Nat. Biotechnol. 31 :230-232; Cong et al. (2013) Science 339:819-823; Mali et al. (2013) Science 339:823-826; Feng et al. (2013) Cell Research 23: 1229-1232; Urnov et al. (2010) Nat Rev Genet. 11 :636-46; Carroll (2011) Genetics 188:773-782; Durai et al , (2005) Nucleic Acids Res 33:5978-90; Mani et al. (2005) Biochem Biophys Res Comm 335:447-57; U.S. Pat. Nos. 7,163,824, 7,001,768, and 6,453,242; all of which are herein incorporated in their entirety by reference.

Naturally occurring candidate proteins can be identified based on homology to known

PRRs. The amino acid sequences of such known PRRs can be found, for example, in a protein database. Membrane-bound PRRs include, for example, receptor-like kinases (RLKs) and receptor-like proteins (RLPs). RLKs comprise an ectodomain, a transmembrane domain, and a kinase domain. RLPs comprise either an ectodomain and a transmembrane domain or an ectodomain and a signal for plasma membrane anchoring by post-translational modification. Methods for bioinformatic identification of RLKs have recently been published in Brustolini et al. (2017) Methods Mol. Biol. 1578: 123-132 (doi: 10.1007/978-1- 4939-6859-6_9); herein incorporated by reference.

The plant protoplasts that are used in the methods of the present invention can be from any plant species of interest. Preferably, the plant protoplasts are derived from a plant species for which an elicitor of interest does not cause, or is not known to cause, PTI and/or any one or more the PTI-associated physiological changes that are known to occur in another plant species following exposure to the elicitor of interest. More preferably, the plant protoplasts are derived from a plant species for which an elicitor of interest does not cause, or is not known to cause, in plant cells from that species a transient increase in the cytosolic Ca ²⁺ level which is known to be one of earliest PTI-associated physiological changes.

The methods of the present invention for screening a candidate protein for a desired PRR are suitable for performing in low-throughput, medium-throughput and high-throughput formats. It is recognized that low-throughput, medium-throughput and high-throughput formats are relative terms that are understood by those of skill in the art. Generally, it is recognized that a low-throughput format comprises the simultaneously processing of fewer individual samples than can be processed in a medium-throughput format and that a medium- throughput format comprises the simultaneously processing of fewer individual samples than can be proceed in a high-throughput format. In general, the level or degree of automation also increases from a low-throughput format to a medium-throughput format to a high- throughput format, with the high-throughput format typically being fully automated. For example, a low-throughput format comprises performing the methods of the present invention in a 12-well or smaller microplate or other laboratory container. Typically, with such a low- throughput format, a laboratory worker would only be able to screen about 5-8 candidate proteins per plate. A medium-throughput format comprises, for example, performing the methods of the present invention in a 96-well microplate with some of the steps being automated. Typically, with such a medium-throughput format, a laboratory worker would be able to screen of about 40-50 candidate proteins per plate. A high-throughput format comprises, for example, performing the methods of the present invention in a 96-well, 384- well, or even a 1536 well microplate format with all (i.e. fully automated) or nearly all of the steps being automated. Typically, with such a high-throughput format, a laboratory worker would be able to assess the biological activity of more than 100 candidate proteins per plate.

In certain other embodiments of the invention, the methods of the present invention are performed in a medium-throughput or a high-throughput format using a population of plant protoplasts in each well of a microplate (e.g. a 96-well microplate), wherein one or more of the steps of the methods disclosed herein are performed in the wells of the microplate. In such methods, a plate reader that is capable of measuring changes in the magnitude of the signal emitted from the Ca ²⁺ sensor is used to measure relative changes in Ca ²⁺ levels in the plant protoplasts in each well. In embodiments of the invention involving a Ca ²⁺ sensor that emits a signal comprising fluorescence, a plate reader comprises a fluorimeter that is capable of measuring relative changes in fluorescence in the wells. Preferably, the plate reader that comprises a fluorimeter is capable of measuring relative changes in fluorescence separately emitted from each of the wells on the plate

simultaneously. By using such a plate reader, relatively large numbers of candidate proteins can be rapidly screened for a PRR function of interest.

Unless expressly stated or apparent from the context of usage, the methods and compositions of the present invention can be used with any plant species including, for example, monocotyledonous plants, dicotyledonous plants, and conifers. Examples of plant species of interest include, but are not limited to, corn {Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa {Medicago sativa), rice {Oryza sativa), rye {Secale cereale), triticale (x Triticosecale or Triticum ^χ Secale) sorghum {Sorghum bicolor, Sorghum vulgar e), teff {Eragrostis tef), millet (e.g., pearl millet {Pennisetum glaucum), proso millet {Panicum miliaceum), foxtail millet {Setaria italica), finger millet {Eleusine coracana)), switchgrass {Panicum virgatum), sunflower {Helianthus annuus), safflower {Carthamus tinctorius), wheat {Triticum aestivum), soybean {Glycine max), tobacco {Nicotiana tabacum), potato {Solanum tuberosum), peanuts {Arachis hypogaed), cotton {Gossypium barbadense, Gossypium hirsutum), strawberry (e.g. Fragaria x ananassa, Fragaria vesca, Fragaria moschata, Fragaria virginiana, Fragaria chiloensis), sweet potato {Ipomoea batatus), yam {Dioscorea spp., D. rotundata, D. cayenensis, D. alata, D. polystachya, D. bulbifera, D. esculenta, D. dumetorum, D. triflda), cassava {Manihot esculenta), coffee {Coffea spp.), coconut {Cocos nucifera), oil palm (e.g. Elaeis guineensis, Elaeis oleifera), pineapple {Ananas comosus), citrus trees {Citrus spp.), cocoa {Theobroma cacao), tea {Camellia sinensis), banana {Musa spp.), avocado {Per sea americana), fig {Ficus casica), guava {Psidium guajava), mango {Mangifera indica), olive {Olea europaea), papaya {Carica papaya), cashew {Anacardium occidentale), macadamia {Macadamia integrifolia), almond {Prunus amygdalus), date {Phoenix dactylifera), cultivated forms of Beta vulgaris (sugar beets, garden beets, chard or spinach beet, mangelwurzel or fodder beet), sugarcane {Saccharum spp.), oat {Avena sativa), barley {Hordeum vulgare), cannabis {Cannabis sativa, C. indica, C. ruderalis), poplar {Populus spp.), eucalyptus {Eucalyptus spp.), Arabidopsis thaliana, Arabidopsis rhizogenes , Nicotiana benthamiana, Brachypodium distachyon vegetables, ornamentals, and conifers and other trees. In specific embodiments, plants of the present invention are crop plants (e.g. maize, sorghum, wheat, millet, rice, barley, oats, sugarcane, alfalfa, soybean, peanut, sunflower, cotton, safflower, Brassica spp., lettuce, strawberry, apple, citrus, etc.). Vegetables include tomatoes (Lycopersicon esculentum), eggplant (also known as "aubergine" or "brinj al") {Solarium melongena), pepper (Capsicum annuum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), chickpeas (Cicer arietinum), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).

Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. Fruit trees and related plants include, for example, apples, pears, peaches, plums, oranges, grapefruits, limes, pomelos, palms, and bananas. Nut trees and related plants include, for example, almonds, cashews, walnuts, pistachios, macadamia nuts, filberts, hazelnuts, and pecans.

In specific embodiments, plants of the present invention are crop plants such as, for example, maize (corn), soybean, wheat, rice, cotton, alfalfa, sunflower, canola (Brassica spp., particularly Brassica napus, Brassica rapa, Brassica juncea), rapeseed (Brassica napus), sorghum, millet, barley, triticale, safflower, peanut, sugarcane, tobacco, potato, tomato, and pepper.

The term "plant" is intended to encompass plants at any stage of maturity or development, as well as any cells, tissues or organs (plant parts) taken or derived from any such plant unless otherwise clearly indicated by context. Plant parts include, but are not limited to, fruits, stems, tubers, roots, flowers, ovules, stamens, petals, leaves, hypocotyls, epicotyls, cotyledons, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, seeds, and the like. It is recognized that the plant protoplasts of the present invention can be prepared from any one or more of the aforementioned plant parts and at any stage of development and/or maturity.

Likewise, the term "plant cell" is intended to encompass plant cells obtained from or in plants at any stage of maturity or development unless otherwise clearly indicated by context. Plant cells can be from or in plant parts including, but are not limited to, fruits, stems, tubers, roots, flowers, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, in vitro-cultured tissues, organs or cells and the like. It is recognized that the plant protoplasts of the present invention can be prepared from any one or more of the aforementioned plant cells and at any stage of development and/or maturity. As used herein, unless expressly stated otherwise or apparent from the context of usage, the term "plant cell" is intended to encompass a plant protoplast.

The elicitors can be from any organism that produces or is known to produce and an elicitor, particularly any plant pathogen that produces or is known to produce and an elicitor that is known to trigger PTI in at least one plant. Plant pathogens include, for example, bacteria, fungi, oomycetes, viruses, nematodes, and the like. Specific pathogens for the major crops include: Soybeans: Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var. caulivora,

Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p. v. glycinea, Xanthomonas campestris p. v. phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted wilt virus, Heterodera glycines Fusarium solani; Canola: Albugo Candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassicicola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganese subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis,

Leptotrochila medicaginis, Fusarium oxysporum, Verticillium albo-atrum, Xanthomonas campestris p.v. alfalfae , Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum, Leptotrichila medicaginis; Wheat: Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis fsp. tritici, Puccinia graminis fsp. tritici, Puccinia recondita fsp. tritici, Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium arrhenomanes , Pythium ultimum, Bipolaris sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat Spindle Streak Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythium

arrhenomannes , Pythium gramicola, Pythium aphanidermatum, High Plains Virus, European wheat striate virus; Sunflower: Plasmopora halstedii, Sclerotinia sclerotiorum, Aster Yellows, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis; Corn: Colletotrichum graminicola, Fusarium moniliforme var. subglutinans, Erwinia stewartii, F. verticillioides, Gibber ella zeae (Fusarium

graminearum) , Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T (Cochliobolus heterostrophus), Helminthosporium carbonum I, II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora,

Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp. nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi pv. zea, Erwinia carotovora, Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi,

Peronosclerospora philippinensis, Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium, Maize Chlorotic Mottle Virus, High Plains Virus, Maize Mosaic Virus, Maize Ray ado Fino Virus, Maize Streak Virus, Maize Stripe Virus, Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum, C. sublineolum, Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria alternata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta,

Sporisorium sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora,

Peronosclerospora sorghi, Peronosclerospora philippinensis , Sclerospora graminicola, Fusarium graminearum, Fusarium verticillioides, Fusarium oxysporum, Pythium

arrhenomanes , Pythium graminicola, etc.; Tomato: Corynebacterium michiganense pv. michiganense, Pseudomonas syringae pv. tomato, Ralstonia solanacearum, Xanthomonas vesicatoria, Xanthomonas perforans, Alternaria solani, Alternaria porri, Collectotrichum spp., Fulvia fulva Syn. Cladosporium fulvum, Fusarium oxysporum f. lycopersici, Leveillula taurica/Oidiopsis taurica, Phytophthora infestans, other Phytophthora spp.,

Pseudocercospora fuligena Syn. Cercospora fuligena, Sclerotium rolfsii, Septoria lycopersici, Meloidogyne spp.; Potato: Ralstonia solanacearum, Pseudomonas solanacearum, Erwinia carotovora subsp. Atroseptica Erwinia carotovora subsp. Carotovora,

Pectobacterium carotovorum subsp. Atrosepticum, Pseudomonas fluorescens, Clavibacter michiganensis subsp. Sepedonicus, Corynebacterium sepedonicum, Streptomyces scabiei, Colletotrichum coccodes, Alternaria alternate, Mycovellosiella concors, Cercospora solani, Macrophomina phaseolina, Sclerotium bataticola, Choanephora cucurbitarum, Puccinia pittieriana, Aecidium cantensis, Alternaria solani, Fusarium spp., Phoma solanicola f.

foveata, Botrytis cinerea, Botryotinia fuckeliana, Phytophthora infestans, Pythium spp., Phoma andigena var. andina, Pleospora herbarum, Stemphylium herbarum, Erysiphe dehor acearum, Spongospora subterranean Rhizoctonia solani, Thanatephorus cucumeris, Rosellinia sp. Dematophora sp., Septoria lycopersici, Helminthosporium solani,

Polyscytalum pustulans, Sclerotium rolfsii, Athelia rolfsii, Angiosorus solani, Ulocladium atrum, Verticillium albo-atrum, V. dahlia, Synchytrium endobioticum, Sclerotinia

sclerotiorum, Candidatus Liberibacter solanacearum; Banana: Fusarium oxysporum f. sp. cubense, Colletotrichum musae, Armillaria mellea, Armillaria tabescens, Pseudomonas solanacearum, Phyllachora musicola, Mycosphaerella fljiensis, Rosellinia bunodes,

Pseudomas spp., Pestalotiopsis leprogena, Cercospora hayi, Pseudomonas solanacearum, Ceratocystis paradoxa, Verticillium theobromae, Trachysphaera fructigena, Cladosporium musae, Junghuhnia vincta, Cordana johnstonii, Cordana musae, Fusarium pallidoroseum, Colletotrichum musae, Verticillium theobromae, Fusarium spp.. Acremonium spp., Cylindrocladium spp., Deightoniella torulosa, Nattrassia mangiferae, Dreschslera gigantean, Guignardia musae, Botryosphaeria ribis, Fusarium solani, Nectria haematococca, Fusarium oxysporum, Rhizoctonia spp., Colletotrichum musae, Uredo musae, Uromyces musae, Acrodontium simplex, Curvularia eragrostidis, Drechslera musae-sapientum, Leptosphaeria musarum, Pestalotiopsis disseminate, Ceratocystis paradoxa, Haplobasidion musae,

Marasmiellus inoderma, Pseudomonas solanacearum, Radopholus similis, Lasiodiplodia theobromae, Fusarium pallidoroseum, Verticillium theobromae, Pestalotiopsis palmarum, Phaeoseptoria musae, Pyricularia grisea, Fusarium moniliforme, Gibber ella fujikuroi, Erwinia carotovora, Erwinia chrysanthemi, Cylindrocarpon musae, Meloidogyne arenaria, Meloidogyne incognita, Meloidogyne javanica, Pratylenchus coffeae, Pratylenchus goodeyi, Pratylenchus brachyurus, Pratylenchus reniformia, Sclerotinia sclerotiorum, Nectria foliicola, Mycosphaerella musicola, Pseudocercospora musae, Limacinula tenuis,

Mycosphaerella musae, Helicotylenchus multicinctus, Helicotylenchus dihystera, Nigrospora sphaerica, Trachysphaera frutigena, Ramichloridium musae, Verticillium theobromae, Phytophthora infestans, Phytophthora parasitica, Phytophthora ramorum, Phytophthora ipomoeae, Phytophthora mirabilis, Phytophthora capsici, Phytophthora porri, Phytophthora sojae, Phytophthora palmivora, and Phytophthora phaseoli.

Bacteria, fungi, and oomycetes are known to produce elicitors recognized by PRRs to induce PTI. A few viral elicitors are also known. Animals, particularly herbivores, are also known to produce elicitors including, for example, insects, and nematodes. All presently known elicitors and the known elicitor/PRR pairs are described in Boutrot and Zipfel (2017) Annu. Rev. Phytopathol. {in press; to be available on the worldwide web at:

annualreviews. org/ doi/10.1146/ annurev-phy to-080614- 120106).

Herbivores of interest include, for example, nematodes and insects, particularly nematodes and insects that are plant pests. Nematodes of interest include, but are not limited to, parasitic nematodes such as root-knot, cyst, and lesion nematodes, including, for example, Globodera spp., Meloidogyne spp., and Heterodera spp. Globodera spp. include, but are not limited to, G. rostochiensis and G. pallida (potato cyst nematodes). Meloidogyne spp.

include, but are not limited to, M. javanica, M. arenaria, M. graminicola, M. incognita, M. hapla, and chitwood. Heterodera spp. include, but are not limited to, H. glycines

(soybean cyst nematode), H. schachtii (beet cyst nematode), Heterodera goettingiana (pea cyst nematode), Heterodera zeae (com cyst nematode), and H. avenae (cereal cyst nematode). Lesion nematodes include, for example, Pratylenchus spp. Other nematodes of interest include, for example, Radopholus similis (banana-root nematode) and Belonolaimus longicaudatus (sting nematode).

Insect pests include, but are not limited to, insects selected from the orders

Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Thysanoptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera.

Insects of the order Lepidoptera include, but are not limited to, army worms, cutworms, loopers, and heliothines in the family Noctuidae Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. segetum Denis & Schiffermiiller (turnip moth); A. subterranea Fabricius (granulate cutworm); Alabama argillacea Hiibner (cotton leaf worm); Anticarsia gemmatalis Hiibner (velvetbean caterpillar); Athetis mindara Barnes and McDunnough (rough skinned cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Egira (Xylomyges) curialis Grote (citrus cutworm); Euxoa messoria Harris (darksided cutworm); Helicoverpa armigera Hiibner (American bollworm); H. zea Boddie (corn earworm or cotton bollworm); Heliothis virescens Fabricius (tobacco budworm); Hypena scabra Fabricius (green cloverworm); Hyponeuma taltula Schaus; {Mamestra conflgurata Walker (bertha army worm); M. brassicae Linnaeus (cabbage moth); Melanchra picta Harris (zebra caterpillar); Mods latipes Guenee (small mocis moth); Pseudaletia unipuncta Haworth (army worm); Pseudoplusia includens Walker (soybean looper); Richia albicosta Smith (Western bean cutworm) ,Spodoptera frugiperda JE Smith (fall army worm); S. exigua Hiibner (beet army worm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Trichoplusia ni Hiibner (cabbage looper); borers, casebearers, webworms, coneworms, and skeletonizers from the families Pyralidae and Crambidae such as Achroia grisella Fabricius (lesser wax moth); Amyelois transitella Walker (naval

orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella

Walker (almond moth); Chilo partellus Swinhoe (spotted stalk borer); C. suppressalis Walker (striped stem/rice borer); C. terrenellus Pagenstecher (sugarcane stemp borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice leaf roller); Desmia funeralis Hiibner (grape leaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea flavipennella Box; D. grandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius (surgarcane borer); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Papaipema nebris (stalk borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hiibner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth); Hedylepta accepta Butler (sugarcane leafroller); Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma electellum Hulst (sunflower moth); Loxostege sticticalis Linnaeus (beet webworm); Maruca testulalis Geyer (bean pod borer); Orthaga thyrisalis Walker (tea tree web moth); Ostrinia nubilalis Hiibner (European corn borer); Ostrinia furnacalis (Asian corn borer); Plodia interpunctella Hiibner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenee (celery leaftier); and leafrollers, budworms, seed worms, and fruit worms in the family Tortri cidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm); Hellula phidilealis ('cabbage budworm mothj; Adoxophyes orana Fischer von Rosslerstamm (summer fruit tortrix moth); Archips spp. including^, argyrospila Walker (fruit tree leaf roller) and A. rosana Linnaeus (European leaf roller); Argyrotaenia spp.; Bonagota salubricola Meyrick (Brazilian apple leafroller); Choristoneura spp. ; Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (codling moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hiibner (vine moth); Grapholita molesta Busck (oriental fruit moth); Lobesia botrana Denis & Schiffermiiller (European grape vine moth); Platynota flavedana Clemens (variegated leafroller); P. stultana

Walsingham (omnivorous leafroller); Spilonota ocellana Denis & Schiffermiiller (eyespotted bud moth); and Suleima helianthana Riley (sunflower bud moth).

Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria J.E. Smith (orange striped oakworm); Antheraea pernyi Guerin- Meneville (Chinese Oak Silkmoth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus

Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hiibner (elm spanworm); Erannis tiliaria Harris (linden looper); Erechthias flavistriata Walsingham (sugarcane bud moth); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guerin-Meneville (grapeleaf skeletonizer); Heliothis subflexa Guenee; Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferia ly coper sicella Walsingham (tomato pinworm); Lambdina flscellaria flscellaria Hulst (Eastern hemlock looper); L.

flscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); Malacosoma spp. ; Manduca quinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth); Orgyia spp. ; Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail, orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval & Leconte (Southern cabbageworm); Sabulodes aegrotata Guenee (omnivorous looper);

Schizura concinna J.E. Smith (red humped caterpillar); Sitotroga cerealella Olivier

(Angoumois grain moth); Telchin licus Drury (giant sugarcane borer); Thaumetopoea pityocampa Schiffermiiller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick (tomato leafminer) and Yponomeuta padella Linnaeus (ermine moth).

Of interest are larvae and adults of the order Coleoptera including weevils from the families Anthribidae, Chrysomelidae, and Curculionidae including, but not limited to:

Bruchus pisorum (pea weevil), Callosobruchus maculatus (cowpea weevil), Anthonomus grandis Boheman (boll weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Diaprepes abbreviatus Linnaeus (Diaprepes root weevil); Hypera punctata Fabricius (clover leaf weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil); Metamasius hemipterus hemipterus Linnaeus (West Indian cane weevil); M. hemipterus sericeus Olivier (silky cane weevil); Sitophilus zeamais (maize weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Smicronyx fulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug); S. livis Vaurie (sugarcane weevil); Rhabdoscelus obscurus Boisduval (New Guinea sugarcane weevil); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles, and leafminers in the family Chrysomelidae including, but not limited to: Cerotoma trifurcata (bean leaf beetle), Chaetocnema ectypa Horn (desert corn flea beetle); C. pulicaria Melsheimer (com flea beetle); Colaspis brunnea Fabricius (grape colaspis); Diabrotica barberi Smith & Lawrence (northern corn rootwormj; D.

undecimpunctata howardi Barber (southern com rootworm); D. virgifera virgifera LeConte (western corn rootworm); Leptinotarsa decemlineata Say (Colorado potato beetle); Oulema melanopus Linnaeus (cereal leaf beetle); Phyllotreta cruciferae Goeze (corn flea beetle); Zygogramma exclamationis Fabricius (sunflower beetle); beetles from the family

Coccinellidae including, but not limited to: Epilachna varivestis Mulsant (Mexican bean beetle); chafers and other beetles from the family Scarabaeidae including, but not limited to: Antitrogus parvulus Britton (Childers cane grub); Cyclocephala borealis Arrow (northern masked chafer, white grub ; C. immaculata Olivier (southern masked chafer, white gruty; Dermolepida albohirtum Waterhouse (Greyback cane beetle); Euetheola humilis rugiceps LeConte (sugarcane beetle); Lepidiota frenchi Blackburn (French's cane grub); Tomarus gibbosus De Geer (carrot beetle); T. subtropicus Blatchley (sugarcane grub); Phyllophaga crinita Burmeister (white grub); P. latifrons LeConte (June beetle); Popillia japonica Newman (Japanese beetle); Rhizotrogus majalis Razoumowsky (European chafer); carpet beetles from the family Dermestidae; wireworms from the family Elateridae, Eleodes spp., Melanotus spp. including M. communis Gyllenhal (wireworm); Conoderus spp. ; Limonius spp.; Agriotes spp. ; Ctenicera spp. ; Aeolus spp. ; bark beetles from the family Scolytidae; beetles from the family Tenebrionidae; beetles from the family Cerambycidae such as, but not limited to, Migdolus fryanus Westwood (longhorn beetle); and beetles from the

Buprestidae family including, but not limited to, Aphanisticus cochinchinae seminulum Obenberger (leaf-mining buprestid beetle).

Adults and immatures of the order Diptera are of interest, including leafminers

Agromyza parvicornis Loew (corn blotch leafminer); midges including, but not limited to: Contarinia sorghicola Coquillett (sorghum midge); Mayetiola destructor Say (Hessian fly); Neolasioptera murtfeldtiana Felt, (sunflower seed midge); Sitodiplosis mosellana Gehin (wheat midge); fruit flies (Tephritidae), Bactrocera oleae (olive fruit fly), Ceratitis capitata (Mediterranean fruit fly), Oscinella frit Linnaeus (frit flies); maggots including, but not limited to: Delia spp. including Delia platura Meigen (seedcom maggot); D. coarctata Fallen (wheat bulb fly); Fannia canicularis Linnaeus, F. femoralis Stein (lesser house flies); Meromyza americana Fitch (wheat stem maggot); Musca domestica Linnaeus (house flies); Stomoxys calcitrans Linnaeus (stable flies)); face flies, horn flies, blow flies, Chrysomya spp.; Phormia spp. ; and other muscoid fly pests, horse flies Tabanus spp. ; bot flies

Gastrophilus spp. ; Oestrus spp. ; cattle grubs Hypoderma spp. ; deer flies Chrysops spp. ; Melophagus ovinus Linnaeus (keds); and other Brachycera, mosquitoes Aedes spp. ; Anopheles spp.; Culex spp.; black flies Prosimulium spp.; Simulium spp.; biting midges, sand flies, sciarids, and other Nematocera.

Included as insects of interest are those of the order Hemiptera such as, but not limited to, the following families: Adelgidae, Aleyrodidae, Aphididae, Asterolecaniidae,

Cercopidae, Cicadellidae, Cicadidae, Cixiidae, Coccidae, Coreidae, Dactylopiidae,

Delphacidae, Diaspididae, Eriococcidae, Flatidae, Fulgoridae, Issidae, Lygaeidae,

Margarodidae, Membracidae, Miridae, Ortheziidae, Pentatomidae, Phoenicococcidae, Phylloxeridae, Pseudococcidae, Psyllidae, Pyrrhocoridae and Tingidae.

Agronomically important members from the order Hemiptera include, but are not limited to: Acrosternum hilare Say (green stink bug); Acyrthisiphon pisum Harris (pea aphid); Adelges spp. (adelgids); Adelphocoris rapidus Say (rapid plant bug); Anasa tristis De Geer (squash bug); Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black bean aphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicis Forbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecola Patch (spirea aphid); Aulacaspis tegalensis Zehntner (sugarcane scale); Aulacorthum solani Kaltenbach (foxglove aphid); Bemisia argentifolii (silverleaf whitefly); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleaf whitefly); Blissus leucopterus leucopterus Say (chinch bug); Blostomatidae spp.; Brevicoryne brassicae Linnaeus (cabbage aphid); Cacopsylla pyricola Foerster (pear psylla); Calocoris norvegicus Gmelin (potato capsid bug); Chaetosiphon fragaefolii Cockerell (strawberry aphid);

Cimicidae spp.; Coreidae spp.; Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis modesta Distant (tomato bug); C. notatus Distant (suckfly); Deois flavopicta Stal (spittlebug); Dialeurodes citri Ashmead (citrus whitefly); Diaphnocoris chlorionis Say (honeylocust plant bug); Diuraphis noxia Kurdjumov/Mordvilko (Russian wheat aphid); Duplachionaspis divergens Green (armored scale); Dysaphis plantaginea Paaserini (rosy apple aphid);

Dysdercus suturellus Herrich-Schaffer (cotton stainer); Dysmicoccus boninsis Kuwana (gray sugarcane mealybug); Empoasca fabae Harris (potato leafhopper); Eriosoma lanigerum Hausmann (woolly apple aphid); Erythroneoura spp. (grape leafhoppers); Eumetopina flavipes Muir (Island sugarcane planthopper); Eurygaster spp.; Euschistus servus Say (brown stink bug); E. variolarius Palisot de Beauvois (one-spotted stink bug); Graptostethus spp. (complex of seed bugs); and Hyalopterus pruni Geoffroy (mealy plum aphid); Icerya purchasi Maskell (cottony cushion scale); Labopidicola allii Knight (onion plant bug);

Laodelphax striatellus Fallen (smaller brown planthopper); Leptoglossus corculus Say (leaf- footed pine seed bug); Leptodictya tabida Herrich-Schaeffer (sugarcane lace bug); Lipaphis erysimi Kaltenbach (turnip aphid); Lygocoris pabulinus Linnaeus (common green capsid); Lygus lineolaris Palisot de Beauvois (tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug); L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius (European tarnished plant bug); Macrosiphum euphorbiae Thomas (potato aphid);

Macrosteles quadrilineatus Forbes (aster leafhopper); Magicicada septendecim Linnaeus (periodical cicada); Mahanarva flmbriolata Stal (sugarcane spittlebug); M. posticata Stal (little cicada of sugarcane); Melanaphis sacchari Zehntner (sugarcane aphid); Melanaspis glomerata Green (black scale); Metopolophium dirhodum Walker (rose grain aphid); Myzus persicae Sulzer (peach-potato aphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid); Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stal (rice leafhopper); Nezara viridula Linnaeus (southern green stink bug); Nilaparvata lugens Stal (brown planthopper); Nysius ericae Schilling (false chinch bug); Nysius raphanus Howard (false chinch bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas (large milkweed bug); Orthops campestris Linnaeus; Pemphigus spp. (root aphids and gall aphids); Peregrinus maidis Ashmead (com planthopper); Perkinsiella saccharicida Kirkaldy (sugarcane delphacid); Phylloxera devastatrix Pergande (pecan phylloxera); Planococcus citri Risso (citrus mealybug); Plesiocoris rugicollis Fallen (apple capsid); Poecilocapsus lineatus Fabricius (four-lined plant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Pseudococcus spp. (other mealybug complex); Pulvinaria elongata Newstead (cottony grass scale); Pyrilla perpusilla Walker (sugarcane leafhopper); Pyrrhocoridae spp.; Quadraspidiotus perniciosus Comstock (San Jose scale); Reduviidae spp.; Rhopalosiphum maidis Fitch (com leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Saccharicoccus sacchari Cockerell (pink sugarcane mealybug); Scaptacoris castanea Perty (brown root stink bug); Schizaphis graminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcane aphid); Sitobion avenae Fabricius (English grain aphid); Sogatella furcifera Horvath (white- backed planthopper); Sogatodes oryzicola Muir (rice delphacid); Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Therioaphis maculata Buckton (spotted alfalfa aphid); Tinidae spp.; Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid); and T. citricida Kirkaldy (brown citrus aphid); Trialeurodes vaporariorum (greenhouse whitefly);

Trialeurodes abutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood

(greenhouse whitefly); Trioza diospyri Ashmead (persimmon psylla); Typhlocyba pomaria McAtee (white apple \eafhopper),Homalodisca vitripennis (glassy winged sharpshooter); Cicadulina mbila (maize leafhopper); Circulifer tenellus (beet leafhopper); Daktulosphaira vitifoliae (grape phylloxera); Coccus pseudomagnoliarum (citricola scale); Coccus hesperidum (soft brown scale); Pulvinaria regalis (horse chestnut scale); Pulvinaria psidii (green shield scale); Aonidiella aurantii (California citrus scale); Aonidiella taxus (Asiatic red scale); Aspidiotus excisus (Cyanotis scale); Aspidiotus nerii (oleander scale); Aulacaspis rosarum (Asiatic rose scale); Aulacaspis tubercularis (white mango scale); Chionaspis lepineyi (oak scurfy scale); Hemiberlesia lataniae (latania scale); Kuwanaspis

pseudoleucaspis (bamboo diaspidid scale; Lepidosaphes pini (pine oystershell scale);

Lopholeucaspis japonica (Japanese maple scale); Oceanaspidiotus spinosus (spined scale insect); Parlatoria ziziphi (black parlatoria scale); Pseudaonidia duplex (camphor scale); Unaspis yanonensis (arrowhead scale); Phenacoccus solani (Solanum mealybug);

Planococcus citri (citrus mealybug); Planococcus (ficus vine mealybug); Pseudococcus longispinus (long-tailed mealybug); Pseudococcus afflnis (glasshouse mealybug);

Diaphorina citri (Asian citrus psyllid); and Bactericera cockerelli (potato psyllid).

Insects of the order Thysanoptera include, but are not limited to, Thrips tabaci (potato thrips) and Frankliniella occidentalis (western flower thrips).

Other insects of interest include, but are not limited to, grasshopper species (e.g. Schistocerca americana and crickets (e,g, Teleogryllus taiwanemma, Teleogryllus emmd).

Acarids are arachnids (Class Arachnida) that are members of the subclass Arci which comprise mites and ticks. While acarids are not true insects, acarids are often grouped together with insect pests of plants because both acarids and insects are members of the phylum Arthropoda. As used herein, the term "insects" encompasses both true insects and acarids unless stated otherwise or apparent from the context of usage. Acarids of interest include, but are not limited to: Aceria tosichella Keifer (wheat curl mite); Panonychus ulmi Koch (European red mite); Petrobia latens Miiller (brown wheat mite); Steneotarsonemus bancrofti Michael (sugarcane stalk mite) spider mites and red mites in the family

Tetranychidae, Oligonychus grypus Baker & Pritchard, O. indicus Hirst (sugarcane leaf mite), O. pratensis Banks (Banks grass mite), O. stickneyi McGregor (sugarcane spider mite); Tetranychus urticae Koch (two spotted spider mite); T. mcdanieli McGregor (McDaniel mite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestani Ugarov & Nikolski

(strawberry spider mite), flat mites in the family Tenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust and bud mites in the family Eriophyidae. In the general, the polynucleotide constructs of the invention comprise coding sequences for a protein or polypeptide, particularly coding sequences for candidate proteins, PRRs and GECIs. The polynucleotide constructs comprising such coding regions can be provided in expression cassettes for expression in a plant protoplast of interest. The cassette will include 5' and 3' regulatory sequences operably linked to the coding sequence.

"Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a coding sequence or gene of interest and a regulatory sequence (i.e., a promoter) is functional link that allows for expression of the coding sequence of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional polynucleotide construct or gene to be cotransformed into the plant protoplast. For example, the expression cassette can contain a first polynucleotide construct comprising a nucleotide sequence encoding a candidate protein and a second polynucleotide construct comprising a nucleotide sequence encoding a Ca ²⁺ sensor, particularly a GECI. Alternatively, the additional polynucleotide construct(s) or gene(s) can be provided on multiple expression cassettes. Preferably, such an expression cassette comprises a plurality of restriction sites and/or recombination sites for insertion of the coding sequence to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain one or more selectable marker genes.

The expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a coding sequence of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plant cells, particularly plant protoplasts. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the coding sequence of the invention may be native/analogous to the host plant protoplast or to each other. Alternatively, the regulatory regions and/or the coding sequence invention may be heterologous to the host plant protoplast or to each other.

As used herein, "heterologous" in reference to a nucleic acid molecule or nucleotide sequence is a nucleic acid molecule or nucleotide sequence that originates from a foreign species, or, if from the same species, is modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide comprising a coding sequence is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

While it may be optimal to express a candidate protein or PRR using heterologous promoters, the native promoter of the corresponding candidate protein or PRR may be used.

The termination region may be native with the transcriptional initiation region, may be native with the operably linked coding sequence, may be native with the plant cell from which the protoplast was prepared, or may be derived from another source (i. e. , foreign or heterologous to the promoter, the protein of interest, and/or the plant cell), or any

combination thereof. Convenient termination regions are available from the Ti-plasmid of A. tumefacien , such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (\ 99\) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671 - 674; Sanfacon ei a/. (1991) Genes Dev. 5 : 141 -149; Mogen et al. (1990) Plant Cell 2: 1261- 1272; Munroe et al. (1990) Gene 91 : 151-158; Ballas et al. (1989) Nucleic Acids Res.

17:7891-7903; and Joshi a/. (19^,1) Nucleic Acids Res. 15:9627-9639.

Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant protoplast. That is, the polynucleotides can be synthesized using plant- preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1 -11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U. S. Patent Nos.

5,380,831, and 5,436,391 , and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); poty virus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) {Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology

81 :382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, chemical-regulated (also known as chemical-inducible), or other promoters for expression in plants. Such constitutive promoters include, for example, the core CaMV 35 S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen ei a/. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (\99\) Theor. Appl. Genet. 81 :581-588); MAS (Velten ef a/. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;

5,268,463; 5,608,142; and 6,177,611.

Where low level constitutive expression is desired, a weak, constitutive promoter can be used. Generally, by "weak promoter" is intended a promoter that drives expression of a coding sequence at a low level. By low level is intended at levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts. Where a promoter is expressed at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels. Such weak constitutive promoters include, for example, the core promoter of the Rsyn 7 promoter (WO 99/43838 and U. S. Patent No. 6,072,050), the core 35S CaMV promoter, and the like.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical -inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR- l a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the

glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA

88: 10421 -10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline- inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U. S. Patent Nos. 5,814,618 and 5,789, 156), herein

incorporated by reference.

The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 55: 610-9 and Fetter et al. (2004) Plant Cell 7(5:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. Cell Science 777:943-54 and Kato et al. (2002) Plant Physiol 729:913-42), and yellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al. (2004) J. Cell Science 777:943-54). For additional selectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511 ; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71 :63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. Act. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90: 1917-1921 ; Labow ei a/. (1990) o/. Cell. Biol. 10:3343-3356;

Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Bairn et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;

Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094- 1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551 ; Oliva ef al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka ei a/. (1985) Handbook of ^' Experimental Pharmacology, Vol. 78 ( Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not intended to be limiting. Any selectable marker gene can be used in the present invention.

Numerous plant transformation vectors and methods for transforming plants are available. See, for example, An, G. et al. (1986) Plant Pysiol. , 81 :301 -305; Fry, J., et al. (1987) Plant Cell Rep. 6:321 -325; Block, M. (1988) Theor. Appl Genet .1 '6: '61 '-77 '4; Hinchee, et al. (1990) Stadler. Genet. Symp.203212.203-212; Cousins, et al. (1991) Aust. J. Plant Physiol. 18:481-494; Chee, P. P. and Slightom, J. L. (1992) Gene.118:255-260; Christou, et al. (1992) Trends. Biotechnol. 10:239-246; D'Halluin, et al. (1992) Bio/Technol. 10:309-314; Dhir, et al. (1992) Plant Physiol. 99: 81 -88; Casas et al. (1993) Proc. Nat. Acad Sci. USA 90: 1 1212-1 1216; Christou, P. (1993) In Vitro Cell. Dev. Biol.-Plant; 29P: 1 19-124; Davies, et al. (1993) Plant Cell Rep. 12: 180-183; Dong, J. A. and Mchughen, A. (1993) Plant Sci. 91 : 139-148; Franklin, C. I. and Trieu, T. N. (1993) Plant. Physiol. 102: 167; Golovkin, et al.

(1993) Plant Sci. 90:41 -52; Guo Chin Sci. Bull. 38:2072-2078; Asano, et al. (1994) Plant Cell Rep. 13; Ayeres N. M. and Park, W. D. (1994) Crit. Rev. Plant. Sci. 13:219-239;

Barcelo, et al. (1994) Plant. J. 5 :583-592; Becker, et al. (1994) Plant. J. 5:299-307;

Borkowska et al. (\ 994) Acta. Physiol Plant. 16:225-230; Christou, P. (1994) ^gro. Food. Ind. Hi Tech. 5: 17-27; Eapen et al. (1994) Plant Cell Rep. 13 :582-586; Hartman, et al.

(1994) Bio-Technology 12: 919923; Ritala, et al. (1994) Plant. Mol. Biol. 24:317-325; and Wan, Y. C. and Lemaux, P. G. (1994) Plant Physiol. 104:3748. The methods of the present invention involve a number of techniques and reagents that are frequently used in the field of molecular biology are generally known in the art or described elsewhere herein. Many of such techniques and reagents are described in Ausubel et al, eds. (2002) Short Protocols in Molecular Biology (5th ed., John Wiley & Sons, New York) and/or in Green & Sambrook (2012) Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Plainview, New York); herein incorporate by reference.

The methods of the invention involve introducing one or more polynucleotide constructs into a plant. By "introducing" is intended presenting to the plant the

polynucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a polynucleotide construct to a plant, only that the polynucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

By "stable transformation" (or "stable transfection") is intended that the

polynucleotide construct introduced into a plant cell integrates into the genome of the plant cell and is capable of being inherited by progeny of a plant regenerated from such a stably transformed plant cell. By "transient transformation" (or "transient transfection") is intended that a polynucleotide construct introduced into a plant cell or plant protoplast does not integrate into the genome of the plant cell or plant protoplast.

The methods of the present invention involve the use of plant protoplasts that comprise one or more polynucleotide constructs. The methods of the present invention do not depend on any particular method of introducing one or more polynucleotide constructs into a plant protoplast. In some embodiments of the invention, one or more polynucleotide constructs are introduced into a plant cell and stably incorporated in its genome, and a plant protoplast comprising the one or more polynucleotide constructs is prepared or isolated from such a plant cell or a progenitor thereof comprising one or more of the polynucleotide constructs. In other embodiments, one or more polynucleotide constructs are introduced directly into the plant protoplast using, for example, PEG-mediated transfection.

In yet other embodiments, a first polynucleotide construct is introduced into a plant cell and stably incorporated in its genome, and a plant protoplast comprising the first polynucleotide constructs is prepared or isolated from such a plant cell or a progenitor thereof comprising the first polynucleotide construct. A second polynucleotide is introduced directly into the plant protoplast using, for example, PEG-mediated transfection. For example, a plant cell is stably transformed with a polynucleotide construct comprising a nucleotide sequence encoding a GECI and then regenerated into stably transformed plant comprising in its genome the polynucleotide construct comprising a nucleotide sequence encoding a GECI. Plant protoplasts comprising such a polynucleotide construct are then prepared from plant cells of the stably transformed plant and an additional polynucleotide comprising a nucleotide sequence encoding a candidate protein of interest is introduced directly into the plant protoplast using, for example, PEG-mediated transfection whereby a plant protoplast comprising a polynucleotide construct comprising a nucleotide sequence encoding a GECI and a polynucleotide construct comprising a nucleotide sequence encoding a candidate protein of interest is produced.

Methods for preparing and transfecting plant protoplasts have been described for numerous plant species and crop plants. Such methods include, but are not limited to: Sheen et al. (2001) Plant Physiol. 127: 1466- 1475 (Arabidopsis thaliana and maize); Fischer & Hain (1995) Methods Cell Biol. 50:401 -410 (tobacco); Zhang et al. (2011) Plant Methods 7(1):30 (rice); Burris et al. (2016) Plant Cell Rep. 35 :693-704 (switchgrass, Panicum virgatum L.); Nanjareddy et al. (2016) BMC Biotechnol. 16(1):53 (Phaseolus vulgaris); Omar et al. (2016) Methods Mol. Biol. 1359:289-327 (citrus); Masani et al. (2014) PLoS One. 9(5):e96831 (oil palm); Lelivelt et al. (2014) Methods Mol Biol. 1 132:317-30 (lettuce); Sun et al. (2013)

Chinese J. Biotechnol. 29:224-34 (wheat); and Liu & Vidali (2011) J. Vis. Exp. (50), e2569, doi: 10.3791/2560 (Physcomitrella patens). Additional methods for preparing and/or transfecting plant protoplasts are disclosed elsewhere herein or are otherwise known in the art.

If desired for the transformation of plants and plant cells, the nucleotide sequences of the invention can be inserted using standard techniques into any vector known in the art that is suitable for expression of the nucleotide sequences in a plant or plant cell. The selection of the vector depends on the preferred transformation technique and the target plant species to be transformed.

Methodologies for constructing plant expression cassettes and introducing foreign nucleic acids into plants are generally known in the art and have been previously described. For example, foreign DNA can be introduced into plants, using tumor-inducing (Ti) plasmid vectors. Other methods utilized for foreign DNA delivery involve the use of PEG mediated protoplast transformation, electroporation, microinjection whiskers, and biolistics or microprojectile bombardment for direct DNA uptake. Such methods are known in the art. (U.S. Pat. No. 5,405,765 to Vasil et al.; Bilang et al. (1991) Gene 100: 247-250; Scheid et al, (1991) o/. Gen. Genet. , 228: 104-112; Guerche et al, (1987) Plant Science 52: 111-116; Neuhause et al, (1987) Theor. Appl Genet. 75: 30-36; Klein et al, (1987) Nature 327: 70-73; Howell et al, (1980) Science 208: 1265; Horsch et al, (1985) Science 227: 1229-1231 ;

DeBlock et al, (1989) Plant Physiology 91 : 694-701 ; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988) and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989). The method of transformation depends upon the plant cell to be transformed, stability of vectors used, expression level of gene products and other parameters.

Other suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection as Crossway et al. (1986) Biotechniques 4:320-334, electroporation as described by Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-m diated transformation as described by Townsend et al, U.S. Patent No. 5,563,055, Zhao et al, U.S. Patent No. 5,981,840, direct gene transfer as described by Paszkowski et al. (1984) EMBO J. 3:2717-2722, and ballistic particle acceleration as described in, for example, Sanford et al., U.S. Patent No. 4,945,050; Tomes et al, U.S. Patent No. 5,879,918; Tomes et al, U.S. Patent No. 5,886,244; Bidney et al, U.S. Patent No. 5,932,782; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture:

Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lecl transformation (WO 00/28058). Also see, Weissinger et al. (\988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Patent No. 5,240,855; Buising et al, U.S. Patent Nos.

5,322,783 and 5,324,646; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 91 :440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (\9% ) Nature (London) 311 :763-764; Bowen et al , U.S. Patent No.

5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349

(Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker- mediated transformation); D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

The polynucleotides of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a polynucleotide construct of the invention within a viral DNA or RNA molecule. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931; herein incorporated by reference.

If desired, the modified viruses or modified viral nucleic acids can be prepared in formulations. Such formulations are prepared in a known manner (see e.g. for review US 3,060,084, EP-A 707 445 (for liquid concentrates), Browning, "Agglomeration", Chemical Engineering, Dec. 4, 1967, 147-48, Perry's Chemical Engineer's Handbook, 4th Ed., McGraw-Hill, New York, 1963, pages 8-57 and et seq. WO 91/13546, US 4,172,714, US 4,144,050, US 3,920,442, US 5,180,587, US 5,232,701, US 5,208,030, GB 2,095,558, US 3,299,566, Klingman, Weed Control as a Science, John Wiley and Sons, Inc., New York, 1961, Hance et al. Weed Control Handbook, 8th Ed., Blackwell Scientific Publications, Oxford, 1989 and Mollet, H., Grubemann, A., Formulation technology, Wiley VCH Verlag GmbH, Weinheim (Germany), 2001, 2. D. A. Knowles, Chemistry and Technology of Agrochemical Formulations, Kluwer Academic Publishers, Dordrecht, 1998 (ISBN 0-7514- 0443-8), for example by extending the active compound with auxiliaries suitable for the formulation of agrochemicals, such as solvents and/or carriers, if desired emulsifiers, surfactants and dispersants, preservatives, antifoaming agents, anti-freezing agents, for seed treatment formulation also optionally colorants and/or binders and/or gelling agents. In specific embodiments, the polynucleotide constructs and expression cassettes of the invention can be provided to a plant using a variety of transient transformation methods known in the art. Such methods include, for example, microinjection or particle

bombardment. See, for example, Crossway et al. (1986) Mo/ Gen. Genet. 202: 179-185; Nomura ei a/. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) PNAS Sci. 91 : 2176-2180 and Hush et al. (1994) J. Cell Science 107: 775-784, all of which are herein incorporated by reference. Alternatively, the polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and

Agrobacterium tumefaciens-m diated transient expression as described elsewhere herein.

A "control" or "control plant protoplast" provides a reference point for measuring changes in the magnitude of the signal emitted from the Ca ²⁺ sensor. Preferably, such a "control plant protoplast" is treated identically to plant protoplast comprising a candidate protein of interest as utilized in the methods of the present invention but the control plant protoplast lacks the candidate protein of interest. In certain embodiments of the invention in which the candidate protein is expressed from a polynucleotide construct contained in the plant protoplast, a control plant may comprise a similar, but non-identical polynucleotide construct that is engineered not to express the candidate protein by, for example, not operably linking a promoter, or is engineered to express a variant of the candidate protein that is expected to be non-functional with respect to the PRR function of interest. In certain embodiments of the invention in which the candidate protein is expressed from a

polynucleotide construct contained in the plant protoplast, a control plant protoplast is treated with a control solution that is identical or essentially identical to the solution used to dissolve the elicitor but lacking the elictor.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

EXAMPLE 1: A Screening Assay for Functional PRRs Using R-GECOl.2

Following ligand binding, PRRs are activated and induce a transient increase of cytosolic Ca ²⁺ concentration as part of a PTI response. The increase of cytosolic Ca ²⁺ concentration can be monitored in a population of plant protoplasts expressing the R- GEC01.2 fluorescent protein. In a high-throughput method, the kinetics of PRR-dependent transient variation of cytosolic Ca ²⁺ concentration is measured with a fluorimeter and in microplate formats of 96 or 384 wells. Using negative controls or candidate PRRs unable to perceive a given elicitor, the fluorescence remains unchanged following treatment with this elicitor.

In the assay, a PRR can be activated with a purified elicitor but also with a solution containing a complex mixture of compounds containing at least one eliciting component.

In absence of candidate PRRs, the protoplasts express endogenous PRRs that are able to perceive elicitors like the flg22 epitope from the flagellin of the bacteria Pseudomonas aeruginosa and chitin (polymer of N-acetylglucosamine) (Liang et al. (2013) Science

341(6152): 1384-1387, doi: 10.1126/science.1242736; Zhang et al. (2017) Theor. Appl. Genet. doi: 10.1007/s00122-017-2876-6. doi: 10.1007/s00122-017-2876-6). Also, for optimal detection of fluorescence variations, the complex mixture should not contain elicitors that are recognized by the protoplast endogenous PRRs. To ascertain the absence of a general inhibitor in the complex mixture, flg22 can be added to an aliquot of this mixture to determine if the flg22-dependent variation of fluorescence remains visible.

Using the biochemical properties of R-GECOl.2 it is possible (i) to ascertain that the protoplasts have been successfully transfected, and (ii) to normalize the specific reporter readout value in response to an elicitor.

For data analyses, fractional fluorescence changes (AF/F) for R-GECOl .2 are calculated from background corrected intensity values of R-GECOl .2 as (F-Fo)/Fo, where F represents the average fluorescence intensity of the a batch of protoplasts treated with elicitor, and Fo represents the average fluorescence intensity of the same batch of protoplasts treated with a control solution that lacks the elicitor but is otherwise identical or essentially identical in composition to the solution comprising the elicitor.

The fluorescence emitted by the R-GECOl .2 reporter depends on the availability of free calcium present in cells. Hence, adding exogenous calcium (in presence of ethanol to permeate the exogenous Ca ²⁺ ) and monitoring an increase of fluorescence allows verification that the protoplasts have been successfully transfected and expressing the R-GECOl .2 reporter.

A final exogenous calcium treatment can be performed after the elicitation assay. To normalize the readout obtained in presence of an elicitor, the fluorescence values can be divided by the final amplitude of the integrated signal obtained after application of exogenous calcium (2 M CaCh, 20% ethanol).

EXAMPLE 2: Detection of the Response to elf 18 Mediated

by EFR in a 96-Well Microplate Format

Corn protoplasts were isolated from 20-hr illuminated leaves of 10-day old maize seedlings that had been kept in the dark at 25 ^° C essentially as described in Sheen et al. (1990) Plant Cell 2: 1027-1038.

32 x 10 ⁴ corn (Zea mays) protoplasts were co-transfected with either 10 μg of PRR construct (2x35S+Q: :AtEFR-FLAG: :rbcS, SEQ ID NO: 1) or 10 μg of control DNA (pUC19, SEQ ID NO: 2), and 10 μg of reporter construct (ZmUbi: :R-GEC01.2: :rbcS, SEQ ID NO: 3) by PEG-mediated transformation using an adaptation of the method of Yoo et al. (2007) Nat Protocols 2: 1565-1572. 100 μΐ. of transfected protoplasts (32 x 10 ⁴ protoplasts) were transferred to each well of a white 96-well microplate (Greiner Bio-One, Lumitrac, model 655075), and then 50 μΐ, of 300 nM elfl 8 peptide (ac-SKEKFERTKPHVNVGTIG, SEQ ID NO: 4; prepared by diluting a 100 μΜ stock solution with incubation buffer) was added to each well. Fluorescence was monitored using a fiuorimeter equipped with a 100 Hz xenon flash lamp with excitation at 556 nm and emission measured at 585 nm for 100 ms, every 23.5 seconds for 30 minutes.

As shown in FIG. 1, transient expression of AtEFR in corn protoplasts led to a significant Ca ²⁺ increase in response to el l 8 treatment. These results demonstrate that AtEFR expression in corn protoplast leads to elfl 8 perception and that the screening assay can be successfully conducted in a 96-well format.

EXAMPLE 3: Detection of the Response to elf 18 Mediated by EFR in a 384-Well Microplate Format

32 x 10 ⁴ corn protoplasts that were prepared as described in Example 2 were co- transfected with either 10 μg of PRR construct (2x35S+Q: : AtEFR-FLAG: :rbcS, SEQ ID NO: 1) or 10 μg of control DNA (pUC19, SEQ ID NO: 2), and 10 μg of reporter construct (ZmUbi: :R-GEC01.2: :rbcS, SEQ ID NO: 3). 25 μΐ, of protoplasts (8 x 10 ⁴ protoplasts) were transferred to 192 wells of a white 384-well plate (Corning, low volume non-binding surface, model 3824), and then 12.5 μΐ. of 300 nM elfl 8 peptide (ac-SKEKFERTKPHVNVGTIG, SEQ ID NO: 4; prepared by diluting a 100 μΜ stock solution with incubation buffer) was added to each of the 192 wells. Fluorescence was monitored using a fluorimeter equipped with a 100 Hz xenon flash lamp with excitation at 556 nm and emission measured at 585 nm for 30 ms, every 27 seconds for 30 minutes.

As shown in FIG. 2, transient expression of AtEFR in corn protoplasts led to a significant Ca ²⁺ increase in response to el l 8 treatment. These results provides additional evidence that AtEFR expression in com protoplast leads to elfl 8 perception and that the screening assay can be successfully conducted in a 384-well format.

EXAMPLE 4: Response of Corn Protoplasts to Exogenous Ca ²⁺ with/without

Pre-Treatment with an Exogenous Elicitor 32 x 10 ⁴ corn protoplasts that were prepared as described in Example 2 were co- transfected with no DNA, or with 10 μg of pUC19 (SEQ ID NO: 2) and 10 μg of reporter construct (ZmUbi: :R-GEC01.2: :rbcS, SEQ ID NO: 3), or with 10 μg of 2x35S+Q::AtEFR- FLAG: :rbcS (SEQ ID NO: 1) and 10 μg of reporter construct (ZmUbi: :R-GEC01.2: :rbcS, SEQ ID NO: 3). 25 μΐ. of protoplasts (8 x 10 ⁴ ) protoplasts were transferred to 192 wells of a white 384-well plate (Corning, low volume non-binding surface, mode 3824), and then 12.5 μΐ. of buffer or 300 nM elfl 8 were added. After 30 minutes, \0 μΐ. of exogenous calcium (2 M CaCh, 20% ethanol) were added in each well, and the fluorescence was immediately monitored using a fluorimeter equipped with a 100 Hz xenon flash lamp with excitation at 556 nm and emission measured at 585 nm. Fluorescnce was measured for 30 ms, every 1.87 seconds, and for a total 3 minutes. The application of exogenous calcium (2 M CaCh, 20% ethanol) resulted in a transient increase of fluorescence in only the protoplasts expressing R- GECOl .2 (FIG. 3). The presence of PRR or pre-treatment with the elicitor was found not to affect the emission of fluorescence (FIG. 3). The article "a" and "an" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one or more element. Throughout the specification the word "comprising," or variations such as

"comprises" or "comprising," will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.