Development and application of FER kinase specific inhibitor FRV

Abstract

The invention belongs to the technical field of plant biology and agricultural biology, and relates to development and application of an FER kinase specific inhibitor FRV. Specifically, the invention provides an agricultural composition containing Forovicin, and provides an application of the agricultural composition in regulating and controlling an FER mediated signal channel and improving plants by combining the agricultural composition with an ATP binding pocket of FER to inhibit kinase activity. The invention provides an important tool for analyzing an FER signal mechanism and promoting agricultural application research.

Description

Technical Field

[0001] This invention belongs to the fields of plant biology and agricultural biotechnology, specifically relating to the development and application of FER kinase-specific inhibitor FRV. Background Technology

[0002] Studying small molecules related to the extension and growth of plant root cells is a key entry point for understanding plant survival adaptation mechanisms, promoting agricultural production innovation and ecological restoration. Its core value lies in two dimensions: breakthroughs in basic science and transformation into practical applications.

[0003] From a basic research perspective, the root, as the core organ for plant water and nutrient absorption, directly determines the root system structure through cell elongation and growth. Small molecules (such as plant hormones like auxins, gibberellins, and brassinolide, as well as osmotic regulators like proline and betaine) act as "molecular switches" regulating this process. They activate intracellular signaling pathways, regulate the activity of cell wall relaxants, control cell osmotic pressure, and thus determine the rate and direction of cell elongation. Studying these substances can reveal how plants sense soil environments (such as drought, salinity, and nutrient distribution) and dynamically adjust root growth, filling the gaps in the molecular mechanisms regulating plant growth and development.

[0004] From an agricultural application perspective, once the roles of key small molecules are clearly defined, crop varieties capable of efficiently synthesizing or responding to these substances can be cultivated through gene editing. For example, wheat and corn roots can accumulate specific small molecules under drought conditions to maintain cell extension capacity, thereby enhancing drought resistance, improving nutrient absorption efficiency, and reducing fertilizer and water consumption. Simultaneously, novel plant growth regulators based on small molecules can be developed to precisely regulate crop root development and address the problem of decreased root vitality in continuous cropping obstacles. In the field of ecological restoration, for saline-alkali land and heavy metal contaminated soil, screening or modifying plants that can regulate root extension and promote the secretion of detoxification substances through small molecules can accelerate soil improvement.

[0005] In addition, this type of research provides inspiration for synthetic biology, helping to design artificial photosynthetic systems that can efficiently fix carbon and adapt to extreme environments, and has important scientific and social value.

[0006] Therefore, from revealing the mysteries of basic biology to driving agricultural technological innovation, small molecules that improve plant root systems constitute a crucial link, and their achievements have irreplaceable strategic value for realizing green and efficient agriculture. Summary of the Invention

[0007] This invention provides the use of small molecule inhibitors of FERONIA (FER) receptor kinase, such as Ferovicin (FRV) and its analogues, in regulating FER signaling, and their application in studying the growth, development, and stress processes of various plant species.

[0008] In a first aspect of the invention, an agricultural composition is provided, the agricultural composition comprising:

[0009] (i) a first active ingredient, the first active ingredient comprising ferovicin (FRV) or an agronomically acceptable salt thereof; and

[0010] (ii) Any agronomically acceptable carrier.

[0011] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, or combinations thereof.

[0012] In another preferred embodiment, the composition contains 0.0001-99 wt%, preferably 0.1-90 wt%, of Ferovicin (FRV) based on the total weight of the composition.

[0013] In another preferred embodiment, Ferovicin (FRV) is the only active ingredient in the agricultural composition.

[0014] In another preferred embodiment, the agricultural composition is a plant amendment.

[0015] In another preferred embodiment, the agricultural composition is a plant root conditioner.

[0016] In another preferred embodiment, the concentration of the FRV in the agricultural composition is 10-1000 nM, more preferably 20-800 nM, even more preferably 30-500 nM, and most preferably 40-300 nM.

[0017] In another preferred embodiment, the agricultural composition further comprises:

[0018] (iii) A second active ingredient comprising: optionally, a substance for improving plants.

[0019] In another preferred embodiment, the substance for improving plants comprises: Tesevatinib, Brigatinib, AZD-7762, or an agronomically acceptable salt thereof, or a combination thereof.

[0020] In a second aspect of the invention, an agricultural reagent kit is provided, the kit comprising the agricultural composition described in the first aspect of the invention, and AHA1 / 2.S899 Test reagents.

[0021] In a third aspect of the invention, use of the agricultural composition described in the first aspect of the invention is provided for one or more uses selected from the group consisting of:

[0022] (a) Improve plants;

[0023] (b) Inhibit the activity of FER kinase;

[0024] (c) Used as a positive reference for screening FER kinase inhibitors;

[0025] (d) Analysis of FER-dependent signaling pathways in plants.

[0026] In another preferred embodiment, the modified plant comprises: RALF1-FER mediated physiological processes in the modified plant.

[0027] In another preferred embodiment, the inhibition of FER kinase activity is to reduce the activity of FER kinase by ≥30%, more preferably ≥50%, more preferably ≥70%, even more preferably ≥90%, and most preferably 100%.

[0028] In another preferred embodiment, the RALF1-FER-mediated physiological processes include: cell expansion, apoplast alkalization, root growth, stress response, or a combination thereof.

[0029] In another preferred embodiment, it is used to prepare an agricultural composition for improving plants.

[0030] In another preferred embodiment, the improved plant comprises: an improved plant root system.

[0031] In another preferred embodiment, the modified plant comprises a modified RALF1-induced plant.

[0032] In another preferred embodiment, the modified plant comprises a plant modified under mechanical stress or a plant with impaired cell wall integrity.

[0033] In another preferred embodiment, the modified plant comprises one or more modifications selected from the group consisting of:

[0034] (Z1) inhibits FER kinase activity;

[0035] (Z2) promotes cell growth in the root meristem;

[0036] (Z3) inhibits rhizosphere alkalization;

[0037] (Z4) Increase root length

[0038] In another preferred embodiment, the root cells comprise: primary root cells and / or lateral root cells.

[0039] In another preferred embodiment, the root cells comprise: epidermal cells.

[0040] In another preferred embodiment, the root cells comprise: primary root epidermal meristem cells.

[0041] In another preferred embodiment, the inhibition of rhizosphere alkalization comprises: inhibiting RALF1-induced rhizosphere alkalization.

[0042] In another preferred embodiment, the promotion of root meristem cell growth comprises: alleviating RALF1-induced inhibition of root meristem cell growth.

[0043] In another preferred embodiment, the promotion of root pictorial cell growth means that, after applying the agricultural composition, the inhibition rate A1 of RALF1 on root pictorial cell growth is ≤80%, more preferably ≤50%, even more preferably ≤40%, and most preferably ≤30%, compared with the inhibition rate A0 of RALF1 on root pictorial cell growth before applying the agricultural composition.

[0044] In another preferred embodiment, the inhibition rate of RALF1 on the growth of root meristem cells refers to the ratio L1 / L0 of the length L1 of the root meristem cells after RALF1 treatment and the length L0 of the root meristem cells in the normal control.

[0045] In another preferred embodiment, the increase in root length means that, after applying the agricultural composition, the root length C1 is greater than or equal to the root length C0 of the normal control, with C1 / C0 ≥ 1.01, preferably ≥ 1.03, and most preferably ≥ 1.05.

[0046] In another preferred embodiment, the normal control refers to plants grown in an environment conducive to plant growth.

[0047] In another preferred embodiment, the normal control is a plant grown in a control group without RALF1.

[0048] In another preferred embodiment, the increase in root length includes mitigating RALF1-induced root shortening.

[0049] In another preferred embodiment, the increase in root length means that, after applying the agricultural composition, the root length B1 is B1 / B0 ≥ 1.1, preferably ≥ 1.2, and most preferably ≥ 1.3 compared to the root length B0 before applying the agricultural composition, wherein the root is a root induced by RALF1.

[0050] In another preferred embodiment, the inhibition of FER kinase activity comprises: specifically binding to the ATP-binding pocket of the FER kinase.

[0051] In another preferred embodiment, the ATP-binding pocket that specifically binds to the FER kinase comprises one or more residues selected from the group consisting of Lys565, Tyr610, Tyr612, Met613, and Asp679.

[0052] In another preferred embodiment, the plant comprises plants with highly conserved amino acid residues selected from the group consisting of K565, Y610, Y612, M613, and D679.

[0053] The amino acid residue numbers are based on the amino acid sequence of Arabidopsis thaliana FER kinase.

[0054] In another preferred embodiment, the Uniprot accession number for Arabidopsis thaliana FER kinase is Q9SCZ4.

[0055] In another preferred embodiment, the TAIR accession number for Arabidopsis thaliana FER kinase is AT3G51550.

[0056] In another preferred embodiment, the plant comprises: Arabidopsis thaliana, Brassica napus, Chinese cabbage, rice, corn, tomato, white pear, Selaginella tamariscina, Mossula simonii, Larix grusonii, or a combination thereof.

[0057] In a fourth aspect of the invention, a method for improving plants is provided, the method comprising the steps of:

[0058] Apply an effective amount of the agricultural composition described in the first aspect of the present invention.

[0059] In another preferred embodiment, the effective amount is 10-1000 nM, more preferably 20-800 nM, even more preferably 30-500 nM, and most preferably 40-300 nM.

[0060] In another preferred embodiment, the application includes spraying, watering, dripping, misting, coating, injection, or a combination thereof.

[0061] In another preferred embodiment, the application may be a single application, repeated application, or continuous application.

[0062] In another preferred embodiment, the application method is to apply it to the plant or to the soil surrounding the plant.

[0063] In a fifth aspect of the invention, a method for screening substances that improve plants is provided, comprising the steps of:

[0064] (S1) In the experimental group, incubation with FER kinase in the presence of the test substance; and

[0065] (S2) Determine whether a FER kinase-test substance complex is formed in the experimental group. If a FER kinase-test substance complex is formed in the experimental group, it suggests that the test substance is a potential therapeutic agent for improving plants.

[0066] In the FER kinase-test compound, the test compound binds to Lys565, Tyr610, Tyr612, Met613 and Asp679 residues of the FER kinase.

[0067] The residue numbers are based on the amino acid sequence of Arabidopsis thaliana FER kinase.

[0068] In another preferred embodiment, step (S1) further includes: incubating with FER kinase in a negative control group under the same conditions but in the absence of the test substance.

[0069] In another preferred embodiment, step (S1) further includes: incubating with FER kinase in a positive control group under the same conditions but without the test substance and in the presence of FRV.

[0070] In another preferred embodiment, the method further includes the steps of: determining the level Y2 of FER kinase-FRV in the positive control group and comparing the FER kinase-test compound Y1 and Y2.

[0071] In another preferred embodiment, the method further includes the step of comparing the binding affinity X1 of the test substance to FER kinase with the binding affinity of FRV and FER kinase.

[0072] In another preferred embodiment, the method further includes: (S3) further performing activity testing or functional testing on the potential therapeutic agent.

[0073] In another preferred embodiment, prior to step (S1), the method further includes screening test substances from a library of IRAK4 kinase inhibitors.

[0074] In another preferred embodiment, screening test substances from the IRAK4 kinase inhibitor library means screening test substances with FER kinase inhibitory activity from the IRAK4 kinase inhibitor library.

[0075] In another preferred embodiment, the IRAK4 kinase inhibitor library comprises: artificially designed IRAK4 kinase inhibitors and optionally known IRAK4 kinase inhibitors.

[0076] In another preferred embodiment, the substance used to improve the plant is a FER kinase inhibitor.

[0077] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0078] Figure 1 This demonstrates the screening of candidate FER kinase inhibitors using root hair growth and root growth experiments. Among them, Figure 1 A shows root hair imaging of wild-type (WT) and FER-5 mutant seedlings with and without FRV treatment; Figure 1 B shows the quantitative analysis of root hair length in wild-type (left) and FER-5 mutant (right) under control treatment (MOCK) or specified candidate inhibitor treatment. 100 nM treatment group, n = 399 / 14, 328 / 11, 309 / 11, 427 / 14, 305 / 12, 116 / 9 (root hair number / root number); 250 nM treatment group, n = 339 / 14, 387 / 13, 430 / 10, 313 / 12, 284 / 12, 234 / 13 (root hair number / root number). FER-5 mutants: 100 nM concentration treatment group, n = 191 / 13, 225 / 12, 194 / 12, 194 / 12, 229 / 14, 229 / 13, 8 / 13 (root hair number / root number); 250 nM concentration treatment group, n = 191 / 13, 248 / 12, 242 / 11, 165 / 11, 209 / 11, 109 / 12 (root hair number / root number); 500 nM concentration treatment group, n = 191 / 14, 247 / 10, 263 / 13, 185 / 9, 261 / 14, 86 / 10 (root hair number / root number). Figure 1 C shows that among all the small molecules and broad-spectrum kinase inhibitors tested, OTSSP167 was the most effective in inhibiting the RALF1 effect.

[0079] Figure 2 The co-crystal structure shown indicates that FRV acts on the ATP-binding pocket of FER kinase.

[0080] Figure 3 The inhibitory mechanism of FRV on FER kinase was elucidated through enzyme activity assays and binding affinity analysis. Among these, Figure 3 A shows the comparison of the inhibitory efficacy of FRV against different FER kinase mutants (K565A, Y610A, Y612A, M613A, D679A) by the ADP-Glo ​​kinase assay (50 μM ATP, incubation for 60 min). Figure 3 B- Figure 3 F respectively shows K565A ( Figure 3B), Y610A ( Figure 3 C), Y612A ( Figure 3 D), M613A Figure 3 E), D679A ( Figure 3 F) The equilibrium dissociation constant (K) of the interaction between mutation and FRV D ).

[0081] Figure 4 This study demonstrates the impact of FRV on the RALF-FER signaling pathway through phosphaomics analysis. Specifically, Figure 4 A shows the results of phosphorylated proteomics analysis of the control group (CK), RALF1, and FRV+RALF1 treatment group. Figure 4 B shows the intergroup differences between RALF1 / CK and RALF1 / FRV+RALF1; Figure 4 C shows the GO analysis of proteins that are co-upregulated or co-downregulated by RALF1 / CK and RALF1 / FRV+RALF1.

[0082] Figure 5 The RALF1-FER was shown to pass through AHA1 / 2 S899 Mediated extracellular alkalization regulates cell elongation. Among these, Figure 5 A showed that 1 μM RALF1 treatment significantly inhibited the elongation of cells in the epidermal meristem of the primary root, while the fer-4 mutant showed no significant change; among them, 200 nM FRV could weaken the inhibitory effect of RALF1; Figure 5 B showed that AHA1 / 2 plays a central role in the RALF1-FER signaling network, and that RALF1 induces a significant upregulation of phosphorylation at the S899 site of AHA1 / 2, a process that can be inhibited by FRV. Figure 5 C shows AHA2 S899D The transgenic plants, like the fer-4 mutant, were insensitive to RALF1. Figure 5 D displayed AHA2 using ultra-high resolution laser confocal imaging. S899D Transgenic seedlings showed no sensitivity to RALF1 in the epidermal meristem cells of the main root, similar to the fer-4 phenotype; application of RALF1 significantly increased the extracellular pH of the wild type, while pretreatment with 200 nM FRV effectively inhibited this effect. Figure 5 E shows the extracellular pH levels of various FER point mutant transgenic lines.

[0083] Figure 6The results show that the degree of rhizosphere alkalization in seedlings was characterized using bromocresol purple staining. Compared with the control group (Mock), treatment with 1 μM RALF1 significantly alkalized the rhizosphere of Arabidopsis seedlings; however, treatment with 50-200 nM FRV significantly inhibited the alkalization of the seedling rhizosphere.

[0084] Figure 7 The study showed that RALF1 regulates cell elongation by activating FER-S695A phosphorylation. Figure 7 A shows that FRV treatment effectively inhibits RALF1-induced FER phosphorylation; Figure 7 B shows that using the prepared FER Ser695 / Thr696 phosphorylation-specific antibody for detection, it was found that RALF1 treatment significantly promoted the increase of phosphorylation level in wild-type FER, but in FER S695A / T696A No obvious changes were observed in the mutant; Figure 7 C shows RALF1 against FER and FER respectively. S695A The effect of FER on the cell length of the epidermal meristem in the primary root. S695A The mutant does not respond to RALF1 signaling; Figure 7 D shows RALF1 against FER and FER respectively. S695A The effect of extracellular pH levels, including the effect of RALF1 application on FER. S695A The extracellular pH level of the mutant lines did not change significantly. Detailed Implementation

[0085] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time ferovicin (FRV), an ATP-competitive inhibitor of FER, which can specifically inhibit FER kinase activity. This enables the elucidation of the mechanism of FER and provides potential new tools and applications for crop growth regulation and stress resistance enhancement in agriculture. Based on this, the present invention was completed.

[0086] Specifically, cocrystal complex structure analysis and mutant enzyme activity experiments showed that FRV binds to the ATP-binding pocket of the FER kinase domain and interacts with five key amino acids in the pocket, thereby inhibiting FER kinase activity. Using FRV tools and quantitative phosphorylated proteomics, the RALF1-FER regulated signaling pathway network was systematically analyzed. It was found that RALF1 activates a novel FER phosphorylation site, Ser695, thereby activating its function. Furthermore, it inhibits proton pump activity through the Ser899 site of ATPase1 / 2, inducing extracellular alkalization and regulating cell size in the primary root epidermal meristem.

[0087] the term

[0088] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0089] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0090] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0091] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value, every tenth of every intermediate integer of the value, any other intermediate value between the upper and lower limits of the range, and any other intermediate value within the specified range are included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered within the scope of this invention, but are subject to any express exclusions within the specified range. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0092] FERONIA receptor kinase

[0093] FERONIA receptor kinases (FERs) are typical members of the RLK1 (CrRLK1L) family in Vinca minor and have become star molecules in the field of plant kinases in recent years. They play important roles in various aspects of plant growth and development, stress resistance, and disease resistance. They act like a molecular switch, regulating the activity of key effector proteins in downstream signaling pathways through their kinase activity, thereby precisely controlling plant growth and development. Currently, research on FERs faces a significant challenge: the lack of chemical tools capable of precisely regulating their activity greatly limits in-depth exploration of the FER signaling mechanism.

[0094] Numerous reports on FER have revealed its diverse functions, many of which depend on its kinase activity. The groundbreaking discovery that FER is a receptor for Rapid Alkalizing Factor 1 (RALF1) reveals that FER can control cell elongation rate, thereby preventing cell bursting due to excessive elongation. Following RALF1 treatment, FER is activated and forms a complex with its co-receptor LLG1, leading to the activation of both FER itself and the plasma membrane H... + -Phosphorylation of ATPase 2 (AHA2). This series of events induces apoplast alkalization and ultimately inhibits cell expansion.

[0095] During pollination, pollen-derived PCP-B peptides and RALF23 / 33 peptides secreted by the stigma competitively interact with FER, regulating the level of reactive oxygen species in the stigma through downstream signal transduction, thereby controlling pollen hydration, similar to a "lock" mechanism that maintains or unlocks the stigma. FER regulates root hair development through guanine exchange factors (GEFs) / plant Rho GTPases (ROPs) and receptor-like cytoplasmic kinases, RIPK, and MARIS.

[0096] Furthermore, RALF1 promotes the phosphorylation of the FER-mediated translation initiation factor eIF4E1, thereby enhancing its mRNA affinity and regulating mRNA translation. RALF1 also induces FER-dependent phosphorylation of glycine-rich RNA-binding protein 7 (GRP7), enhancing its interaction with the spliceosome component U1-70K to regulate dynamic alternative splicing. RALF1 induces, at the cellular biological level, a response most similar to plant stress, particularly to mechanical stress or impaired cell wall integrity. More specifically, this process mimics the adaptive growth response of roots when encountering physical barriers such as hard soil.

[0097] FER phosphorylation disrupts the stability of MYC2, a major transcription factor in the jasmonic acid (JA) signaling pathway that negatively regulates JA-mediated host susceptibility.

[0098] The above studies collectively highlight the central role of FER kinase activity in plant life activities.

[0099] Furthermore, some studies present differing views on whether FER signals through its kinase activity and the associated mechanisms. During pollen tube reception, studies have shown that the kinase domain (rather than kinase activity) is essential for FER function, evidenced by the successful compensation of the FER-1 mutant phenotype observed with mutations in the S695A, T696A, and S701A regions of the activation loop. Additionally, compared to wild-type FER, the inactivating mutation K565R in the FER kinase region demonstrates a reduced ability to compensate for the FER-4 knockout phenotype in roots (rather than in ovules). Transgenic lines carrying the K565R mutation indicate that RALF23 inhibition is mediated by active FER signaling, while the positive role of FER in immune signaling appears to be independent of kinase activity. These studies reveal complex regulatory mechanisms that underscore the importance of elucidating at the molecular level how the FER kinase region precisely regulates specific signaling pathways.

[0100] Exogenous small molecules can help researchers uncover the complex regulatory mechanisms of plant physiological processes. The most classic example is the discovery of the abscisic acid receptor: abscisic acid (ABA) plays a crucial role in protecting plants against adverse conditions and has significant agricultural applications. However, due to ABA's instability and the high cost of its synthesis, there is a need to develop functional analogs of ABA to realize its agricultural applications. Pyrabactin, a reported ABA analog selective for PYL protein, is a sulfonamide-based agonist. While it has no chemical structural similarity to ABA, the two are functionally very similar. Pyrabactin specifically binds to the PYL receptor, enabling protein-protein interactions between PYR1 and downstream effector molecules, thereby inhibiting seed germination and hypocotyl growth. Furthermore, multiple reports have confirmed the role of pyrabactin in studying ABA-related signaling in stomatal guard cells and other plant tissues, as well as its role in mediating stomatal movement, maintaining plant water balance, and regulating water deficit-related stress in crops. The identification of ABA agonists such as pyrabactin and its derivatives has played a crucial role in characterizing the ABA signaling pathway, ushering in a new era of using exogenous small molecules to regulate the ABA signaling pathway. Similarly, in the field of auxin research, several small molecule inhibitors have also played key roles. For example, the auxinole antagonist has been used to competitively block the interaction between auxin and the TIR1 (transport inhibitor response 1) receptor, thereby enabling more precise study of complex auxin signaling pathways.

[0101] To date, the analysis of the FER signaling pathway and the development of related agricultural applications have faced bottlenecks due to the lack of highly selective chemical probes that can precisely manipulate FER kinase activity.

[0102] OTSSP167 (i.e., Ferovicin, FRV)

[0103] OTSSP167 (CAS No. 1431697-89-0) is a highly effective and selective small molecule inhibitor of MELK (maternal embryonic leucine zipper kinase), with an IC50 value of [missing information]. 50 The value reached 0.41 nM, exhibiting extremely strong MELK kinase inhibitory activity.

[0104] This compound primarily interferes with the function of MELK kinase in key signaling pathways such as cell mitosis, DNA damage response, and tumor stem cell self-renewal by specifically targeting MELK kinase.

[0105] In preclinical studies, OTSSP167 has shown significant inhibitory effects on proliferation and apoptosis induction in various malignant tumor cells, including breast cancer, lung cancer, and leukemia. It has shown particular potential to target tumor stem cells and is expected to overcome the drug resistance problem of traditional chemotherapy.

[0106] Agricultural Composition

[0107] The active material (containing FRV) of the present invention can be prepared into agricultural compositions, such as solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, natural and synthetic materials impregnated with the active material, microcapsules in polymers, and seed coating agents, using conventional methods.

[0108] These agricultural compositions can be produced by known methods, for example, by mixing the active substance with a expander, which can be a liquid, liquefied gas, or solid diluent or carrier, and can be any type of surfactant, i.e., emulsifier and / or dispersant and / or foaming agent. For example, when water is used as the expander, organic solvents can also be used as adjuvants.

[0109] Liquid solvents are generally suitable as diluents or carriers, such as: aromatic hydrocarbons, such as xylene, toluene, or alkylnaphthalene; chlorinated aromatic or chlorinated aliphatic hydrocarbons, such as chlorobenzene, vinyl chloride, or dichloromethane; aliphatic hydrocarbons, such as cyclohexane or paraffins, such as mineral oil fractions; alcohols, such as ethanol or ethylene glycol and their ethers and esters; ketones, such as acetone, methyl ethyl ketone, methyl isobutyl ketone, or cyclohexanone; or less commonly used polar solvents, such as dimethylformamide and dimethyl sulfoxide, and water.

[0110] In the case of liquefied gas diluents or carriers, it refers to liquids that become gaseous at normal temperature and pressure, such as aerosol propellants, such as halogenated hydrocarbons, as well as butane, propane, nitrogen, and carbon dioxide.

[0111] Solid carriers can be ground natural minerals such as kaolin, clay, talc, quartz, activated clay, montmorillonite, or diatomaceous earth, and ground synthetic minerals such as highly dispersed silica, alumina, and silicates. Solid carriers for granulation are crushed and graded natural zircon, such as calcite, marble, pumice, sepiolite, and dolomite, as well as granules synthesized from inorganic and organic coarse powders, and granules of organic materials such as sawdust, coconut husks, corncobs, and tobacco stalks.

[0112] Nonionic and anionic emulsifiers can be used as emulsifiers and / or foam forming agents. Examples include polyoxyethylene-fatty acid esters, polyoxyethylene-fatty alcohol ethers, such as alkylaryl polyethylene glycol ethers, alkyl sulfonates, alkyl sulfates, aryl sulfonates, and albumin hydrolysates. Dispersants include, for example, lignin sulfite waste and methylcellulose.

[0113] In the formulation, binders such as carboxymethyl cellulose and natural and synthetic polymers in the form of powders, granules or emulsions, such as gum arabic, polyvinyl alcohol and polyvinyl acetate, can be used.

[0114] Coloring agents such as inorganic dyes, such as iron oxide, cobalt oxide and Prussian blue; organic dyes, such as azo dyes or metallic titanium cyanide dyes; and trace nutrients, such as salts of iron, manganese, boron, copper, cobalt, aluminum and zinc, can be used.

[0115] In this invention, the "agricultural composition" is typically an agricultural plant growth regulator whose FRV contains an active ingredient for improving plants, and an agriculturally acceptable carrier.

[0116] As used herein, "agriculturally acceptable carrier" refers to an agronomically acceptable solvent, suspending agent, or excipient for delivering the FRV of the present invention to plants. The carrier may be liquid or solid. Agriculturally acceptable carriers suitable for use in the present invention are selected from the group consisting of water, buffer solutions, DMSO, surfactants such as Tween-20, or combinations thereof. Any agriculturally acceptable carrier known to those skilled in the art may be used in the present invention.

[0117] The agricultural compositions of the present invention can be mixed with other plant growth regulators in their commercial formulations or in formulations prepared from these formulations, including (but not limited to): humic acid, chlormequat chloride, chlormequat chloride, naphthaleneacetic acid, or combinations thereof.

[0118] Furthermore, the agricultural formulations of the present invention can also be mixed with fertilizers in their commercial formulations or in application formulations prepared from these formulations, the fertilizers being selected from the group consisting of nitrogen fertilizers, phosphate fertilizers, potassium fertilizers, compound fertilizers, or combinations thereof.

[0119] The formulation of the agricultural composition described in this invention can be diverse, as long as it enables the active ingredient to effectively reach the plant. From the perspective of ease of preparation and application, the preferred agricultural formulation is a spray, solution, or granule.

[0120] The agricultural compositions of the present invention typically contain 0.001-99.99 wt%, preferably 0.01-99.9 wt%, and more preferably 0.05-90 wt% of the compound of the present invention by weight of the total agricultural formulation. The concentration of the compound of the present invention in commercial formulations or application formulations can vary over a wide range. The concentration of the compound of the present invention in application formulations can range from 0.0000001-100% (g / v), preferably between 0.0001 and 1% (g / v).

[0121] Uses of the present invention

[0122] This invention provides the application of Ferovicin (FRV) in plant improvement. FRV specifically targets and inhibits the kinase activity of the FERONIA (FER) receptor kinase.

[0123] The Ferovicin (FRV) of the present invention includes its stereoisomers, tautomers, stable isotope derivatives, metabolites or prodrugs, or pharmaceutically acceptable salts, cocrystals, polymorphs or solvates.

[0124] The present invention also provides agricultural compositions comprising FRV.

[0125] Specifically, FRV small molecule inhibitors were initially identified from the IRAK4 kinase inhibitor library. Using homologous sequence alignment, FER showed a high degree of kinase domain similarity (approximately 60%) with human interleukin-1 receptor-associated kinase 4 (IRAK4). Therefore, other small molecule inhibitors targeting IRAK4 or structurally or functionally related kinases may also inhibit FERONIA kinase activity.

[0126] Specifically, high-throughput virtual screening was used to identify FER kinase inhibitors. In particular, the Gnina molecular docking software was used to screen a library containing 4378 IRAK4 kinase inhibitor compounds to identify small molecules capable of binding to the FER kinase domain. Based on this high-throughput screening strategy, inhibitors targeting IRAK4 or similar kinases may also have cross-reactivity and potential applications in regulating the FER signaling pathway in plants.

[0127] Phenotypic screening of FER kinase inhibitors using a root hair growth assay revealed that four of the five selected candidate compounds (as shown in Table 1) effectively inhibited root hair growth in wild-type seedlings, exhibiting phenotypic characteristics consistent with FER-4 and FER-5 mutant plants. Specifically, the compound named OTSSP167 showed the most significant inhibitory activity among all tested molecules and was further confirmed to effectively inhibit RALF1 ligand-induced plant physiological effects. Based on its highly efficient inhibitory activity and selectivity, OTSSP167 was defined as an FER-specific inhibitor and named FRV (CAS No.: 1431697-89-0).

[0128] This invention provides the use of FRV or its derivatives in regulating FER-mediated growth processes and antagonizing RALF1-FER signaling in plants.

[0129] In a specific implementation, FRV works by selectively binding to the ATP-binding pocket within the FER kinase domain. Cocrystal structure and mutant enzyme activity experiments demonstrate the interaction mode between FRV and FER kinase and its ability to effectively inhibit FER kinase.

[0130] In a specific implementation, the present invention uses FRV to... The co-crystal structure of the resolution-resolved FER kinase domain (residues 518-820) elucidates the structural model of FRV-FER action, forming a typical kinase conformation consisting of a β-sheet-rich N-terminal region and an α-helix-dominated C-terminal region interconnected by a hinge region that forms an ATP-binding pocket.

[0131] In a specific implementation, the FRV selectively binds within the ATP-binding pocket of the FER. Specifically, it interacts with key amino acid residues Lys565, Tyr610, Tyr612, Met613, and Asp679 within the pocket through intermolecular forces such as hydrogen bonding and π-π stacking, thereby stabilizing the inhibitor-kinase complex and competitively displacing ATP.

[0132] In a specific implementation, surface plasmon resonance (SPR) confirmed the high-affinity binding between FRV and FER, and the dissociation constant (Ki) was [missing information]. D The concentration was 0.4 μM, significantly stronger than ATP (K). D =18.37 μM), demonstrating the efficacy of FRV as an ATP competitive inhibitor and its practicality in selectively blocking FER-dependent signaling.

[0133] In a specific embodiment, this invention provides a quantitative method for evaluating the inhibitory activity of FRV against FER kinase and its variants using an ADP-Glo ​​kinase activity assay, wherein FRV exhibits a strong inhibitory effect on wild-type FER kinase, IC50... 50 The value is 70 nM.

[0134] In a specific embodiment, this invention further demonstrates, through mutation of five key amino acid binding sites (K565A, Y610A, Y612A, M613A, D679A) in the FER kinase domain and enzyme activity detection, that mutations in K565A, Y610A, and D679A severely impair the binding affinity between FRV and FER, leading to IC50. 50 Values ​​exceeding 100 μM (virtually no binding) and Y612A and M613A mutations also impaired the catalytic activity of the kinase to some extent.

[0135] In a specific implementation, surface plasmon resonance (SPR) analysis confirmed that the Y612A mutation eliminated FRV binding affinity by more than 1000-fold, establishing Tyr612 as an essential residue for FRV to recognize FER molecules, while Lys565 and Tyr610 were also confirmed as key residues regulating the binding of FER and FRV small molecules.

[0136] In a specific implementation, in vitro enzyme activity assays and SPR binding affinity analyses of the FER mutants showed that key amino acids in the ATP-binding pocket can synergistically regulate FRV-FER interactions through conformational spatial combinations. This provides a guiding strategy for designing resistance inhibitors against FER kinases.

[0137] In specific implementations, binding affinity and enzyme activity assays demonstrated that FRV exhibits high selectivity for FER kinases, with binding affinity to non-target kinases, including TMK4 and BRI1, reduced by tens of times compared to FER. Furthermore, the binding affinity to CrRLK1L receptor families homologous to FER, such as ANX1, BUPS1, and ANJ, was also significantly reduced by tens to hundreds of times.

[0138] In a specific implementation, molecular docking results showed that the synergistic effect among conserved residues (K565, Y610, Y612, M613, D679) in the ATP binding pocket of FRV and FER is the key to its specific binding, revealing the molecular mechanism of its highly selective inhibition of FER.

[0139] In specific implementations, FRVs are widely applicable across evolutionarily diverse plant species, including (but not limited to): Arabidopsis thaliana, Brassica napus, Brassica genus, rice, maize, tomato, pear, Selaginella tamariscina, potato, and Strychnos nux-vomica. The high conservation of FRV binding sites among FER orthologs of different terrestrial plant species allows FRVs to regulate FER-mediated signaling pathways across species.

[0140] Structural insights gained from the selective mechanism of FRV provide a theoretical basis and framework for designing next-generation inhibitors with high specificity against other plant receptor-like kinases (RLKs).

[0141] This invention provides a phosphoproteomics analysis method for identifying FER kinase-dependent signaling pathways by using FRV as a chemical probe.

[0142] Specifically, by detecting 106 newly emerging phosphopeptides and 456 dephosphorylation events induced by RALF1, as well as 345 new phosphopeptides and 313 dephosphorylation events regulated by FRV pretreatment, the RALF1-FER signaling network can be comprehensively mapped, thereby revealing the dynamic changes in FER-dependent phosphorylation proteome in plant cells.

[0143] This invention (phosphoproteomics features obtained through FRV-mediated inhibition) provides a theoretical basis for identifying key downstream components of FER kinase and developing strategies to regulate plant growth and stress response pathways.

[0144] This invention provides a set of FER-dependent phosphorylation targets and demonstrates the specific inhibitory effect of FRV on FER kinase activity. The phosphorylation targets include Ser695 and Ser866, which are activated by RALF1 treatment and inhibited by FRV inhibition, thereby providing molecular targets for regulating FER-mediated signaling pathways.

[0145] Gene ontology analysis in this invention shows that FER-dependent phosphorylated proteins are significantly enriched in the following key biological processes, including cell localization, cell growth, cell division, plant-pathogen interactions, regulation of fungal defense responses, pollen acceptance, and abiotic stress, thereby identifying the potential agricultural application value of FRV.

[0146] FER protein interaction network analysis identified direct interacting factors of the RALF-FER complex, including ROPGEF4, AHA2, RIPK, BIG5, ATL6, and BRG5, providing key targets for manipulating FER-mediated physiological processes in plant development and stress responses.

[0147] This invention provides a method for mechanically dissecting FER-mediated signal transduction pathways using FRV as a chemogenetic tool. By combining FRV with quantitative phosphoproteomics, this invention elucidates key regulatory pathways in the RALF1-FER signaling pathway, including the phosphorylation cascade from FER-Ser695 to AHA-Ser899, leading to the regulation of apoplast alkalization and root meristem cell proliferation.

[0148] This invention elucidates the complete RALF1-FER-AHA signaling cascade pathway, in which RALF1 ligand-activated FER kinase can phosphorylate the Ser899 residue of the AHA1 / 2 proton pump, subsequently inducing apoplast alkalization and regulating the expansion of root meristem cells. As shown by phosphoproteomics data, RALF1 ligand-induced phosphorylation of the S899 site of AHA1 / 2 is significantly upregulated, and this process can be inhibited by the FRV small molecule.

[0149] In this invention, FER kinase activation is combined with AHA proton pump-mediated H... + The regulatory mechanism linking flux and apoplastic pH changes provides a targeted approach for chemically intervening in the RALF1-FER-AHA signaling axis to manipulate plant root structure and root cell extension and growth.

[0150] This invention provides a series of FER kinase domain mutants with functional characteristics (including single mutants of K565R, Y610A, Y612A, M613A, and D679A in FER-4 and their combined variants), and demonstrates that transgenic plants expressing Y610A / D679A double mutants, K565R / D679A double mutants, Y610A / K565R / D679A triple mutants, and K565R / Y610A / Y612A / M613A / D679B quintuplet mutants are completely insensitive to RALF1-induced physiological responses, thereby identifying the key residue combinations for FER signaling function.

[0151] Based on the functional importance of FER kinase residues (Y610 / K565 > Y612 / M613 / D679), FER variants that can decouple signaling pathways can be rationally designed for use in agriculture to regulate RALF1-related growth processes and achieve precise regulation of crop growth.

[0152] Due to the high conservation of FRV binding sites in FER orthologs of different terrestrial plant species, this invention also demonstrates the potential agricultural applications of FRV in the broad regulation of development and stress responses in a wide range of plants.

[0153] The main advantages of this invention include:

[0154] (a) This invention provides the application of FRV in plant improvement. Specifically, FRV promotes the extension and growth of plant root cells, which improves the efficiency of water and nutrient absorption, thereby reducing irrigation and fertilization needs and lowering production costs; enhances the drought resistance, salt and alkali resistance, and lodging resistance of crops with well-developed root systems, and improves the crop's resilience to adverse environmental conditions; robust root systems promote overall plant growth, increase crop biomass and economic yield, and improve the quality of agricultural products; and have potential application value in optimizing root structure, promoting water conservation and fertilizer reduction, and reducing soil erosion, thus contributing to sustainable agricultural development and resource-efficient agricultural practices.

[0155] (b) Cross-species applicability: By leveraging the high conservation of FRV binding residues (K565, Y610, Y612, M613, D679) in terrestrial plants (from bryophytes to angiosperms), this invention provides a universal tool for regulating FER signaling in important economic crops (including rice, maize, and tomato).

[0156] (c) The small molecule inhibitor FRV of the present invention promotes the extension and growth of plant root cells by inhibiting FER kinase. Specifically, FRV has excellent selectivity for FER kinase, with a binding affinity (K... D=0.4μM) significantly outperformed other plant receptor kinases (e.g., selectivity for ANJ and TMK4 reached 38.3-fold and 48.5-fold, respectively), minimizing off-target effects in biological applications.

[0157] (d) This invention provides the precise mechanism by which FRV improves plants through FER: the co-crystal structure of the FER-FRV complex ( The resolution helps to understand the FRV-FER interaction at the atomic level, especially through hydrogen bonding with K565 / D679 and π-π stacking with Y612, providing a rational basis for structure-guided inhibitor optimization.

[0158] (e) This invention provides the dual functionality of FRV, which can be used as a chemical genetic tool to analyze FER-mediated phosphorylation networks (identifying 709 phosphorylation events) and as an agricultural regulator to manipulate root structure and stress response through apoplast pH regulation.

[0159] (f) Identification of key residues (Y610 / K565) can guide the design of next-generation inhibitors that target conserved ATP-binding regions, while avoiding resistance development through multi-residue targeting strategies.

[0160] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0161] Example 1: Experimental Method

[0162] Plant growth conditions. Arabidopsis thaliana plants of ecotype Columbia-0 (Col-0) were grown under strictly controlled environmental conditions, with 16 hours of light and 8 hours of darkness at 22°C, and a relative humidity of 60% throughout the growth cycle. All plants were grown on 1 / 2 MS solid medium. The wild-type Col-0 line served as a universal control for all comparative experiments in this study.

[0163] Methods for constructing genetic material and mutant lines. The construction of homozygous FER-4 and FER-5 mutant lines was primarily achieved through T-DNA insertion mutation technology. After obtaining FER-4 and FER-5 mutant seeds, they were surface-sterilized and sown on MS medium. After seedlings had grown for approximately 1-2 weeks, genomic DNA was extracted. PCR amplification was performed using specific primers flanking the T-DNA insertion site to screen genotypes—homozygous mutants could only amplify the T-DNA boundary sequence band, but not the wild-type FER gene band. Finally, after thorough genomic validation through PCR amplification and sequencing, these lines were used in this study. These well-documented mutant lines provided the genetic background for subsequent complementation studies and functional analyses, ensuring the reliability and reproducibility of experimental results based on previously validated genetic material.

[0164] Methods for vector construction and site-directed mutagenesis. Site-directed mutagenesis was performed using the pCAMBIA1300 vector system to introduce specific point mutations (K565R, Y610A, Y612A, M613A, D679A) into the FER kinase domain. The successful incorporation of these mutations was confirmed by comprehensive DNA sequencing analysis. Subsequently, the mutated FER sequence was fused with the GFP reporter gene to generate a C-terminal fusion construct, enabling functional evaluation and subcellular localization studies of various FER mutant variants in plant systems.

[0165] Methods for plant transformation and selection. The serine 899 residue of AHA2 was genetically engineered to aspartic acid (S899D) via precise site-directed mutagenesis, and then cloned into the pCAMBIA1300 vector under the control of the natural AHA2 promoter 2.0 kilobases upstream of the coding sequence. This construct was named pAHA2:AHA2S899D-eGFP. Subsequently, an AHA2-4 mutant background was introduced via Agrobacterium tumefaciens-mediated flower dip transformation to generate transgenic lines expressing phosphorylated AHA2 variants for functional characterization studies.

[0166] Root cell growth assay method. Seedlings were grown on 1 / 2 MS plates for 4 and 4.5 days, then pretreated in 1 / 2 MS liquid medium for 30 minutes, followed by transfer to liquid medium or a simulant solution containing 200 nM FRV for 3 hours. Next, seedlings were transferred to liquid medium supplemented with 1 μM RALF1 and gently shaken for 16, 18, or 12 hours. Seedlings were treated with 40 μg / ml propidium iodide staining buffer for 10 minutes and washed three times with ddH2O. Roots were then placed on slides and observed under a STELLARIS 8 confocal microscope (Leica). A WLL laser and HyD2 detector were used to capture images with a 20x objective lens (Ex: 535 nm, Em: peak 617 nm). Distal meristem root epidermal cells (meristem cells about to elongate) were defined as the four cells below the first cell, the length of which was twice its width, and were selected for measurement as described in the literature. ImageJ software was used to measure the length of the selected cells in the vertical direction and record the average length of the four cells as the cell length of each root.

[0167] HPTS staining method for root cells. For RALF1 and FRV treatments, 4-day-old seedlings were placed in 1 / 2 MS liquid growth medium supplemented with 1 μM RALF1 for 6 hours, with or without 200 nM FRV pretreatment for 2 hours. In the *N. benthamina* leaf system, the FER mutant protein was introduced into *N. benthamina* leaves by *Agrobacterium* strain GV3101 for transient expression for 48 hours, followed by treatment with or without 1 μM RALF1 for 30 minutes. HPTS staining was performed by incubating seedlings in 1 / 2 MS liquid growth medium containing 1 mM HPTS for 15 minutes. The samples were then washed three times with ddH2O and mounted on microscope slides in the same growth medium supplemented with HPTS, and covered with coverslips. Seedling images were captured in line scan mode (Ex 458 nm and 405 nm, Em 505–550 nm) using a Leica STELLARIS 8 ultra-high resolution laser confocal microscope equipped with a WLL laser and HyD2 detector. Image acquisition was performed using a 40x objective lens. Notably, no fluorescence signal was detected without staining to avoid interference from other fluorescence sources. TauGating and counting techniques were employed as the capture mode to ensure accurate and specific detection. Acquired images were processed using ImageJ software with various tools such as segmentation, Gaussian blur (value 1.5), and background subtraction (value 20). The relative intensity of the fluorescence signal was quantified by dividing the pixel value of the 458nm channel by the pixel value of the 405nm channel.

[0168] Methods for expression and purification of recombinant proteins containing the FER kinase domain and its homologs. The kinase domains of FER (residues 518-820; AT3G51550), TMK4 (residues 578-928; AT3G23750), BUPS1 (residues 462-878; AT4G39110), ANX1 (residues 451-850; AT3G04690), and BRI1 (residues 883-1158; AT4G39400) were cloned as N-terminal hexahistine-tagged recombinant proteins into the pFastBac vector for baculovirus-mediated expression. Using the Bac-to-Bac baculovirus expression system, SF9 insect cells were maintained at 27°C and infected with recombinant baculovirus according to a standard protocol to express the FER kinase domain and its mutant variants. Forty-eight hours after viral infection, cells were collected by centrifugation at 3000×g for 15 minutes, and the resulting cell particles were immediately frozen at -80°C until purification. For protein extraction, frozen cell particles were thawed and resuspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole, followed by cryogenic sonication at 200 W with 5-second pulses interspersed with 5-second cooling intervals for a total duration of 20 minutes. Cell debris was removed by centrifugation at 15000 × g for 30 minutes at 4 °C. The supernatant was then loaded onto a 5 mL Ni-NTA affinity column (GE Healthcare) pre-equilibrated with lysis buffer and washed with twice the column volume of wash buffer consisting of 50 mM HEPES NaOH (pH 8.0), 300 mM NaCl, 3 mM TCEP, and 30 mM imidazole. Bound proteins were eluted using elution buffer containing 50 mM HEPES NaOH (pH 8.0), 300 mM NaCl, 3 mM TCEP, and 300 mM imidazole. The eluted protein fractions were concentrated using a 10 kDa molecular weight cutoff centrifuge (Millipore, UFC901096) and further purified by size exclusion chromatography on a Superdex 75 preparative-grade column (GE Healthcare) using a buffer system of 50 mM HEPES NaOH (pH 8.0), 300 mM NaCl, and 3 mM TCEP. For crystallization purposes, the purified FER (518-820) protein was concentrated to approximately 8 mg / mL. To form the FER kinase domain-FRV complex, 8 mg / mL of the purified FER kinase domain was incubated with 10 mM FRV compound at 277 K with gentle stirring for 1 h. The same expression and purification procedures were applied to all FER mutant proteins.

[0169] Crystallization and Data Collection Methods for the FER-FRV Complex. Initial crystallization conditions for the FER-FRV complex were determined by systematically screening a commercial crystallization kit (Hampton Research, Aliso Viejo, California) using a hanging drop vapor diffusion method. After initial screening, optimized crystals were successfully grown for 8 days at 16°C by mixing 1 μL of the protein complex solution with 1 μL of a stock solution consisting of 0.3 M ammonium sulfate, 0.1 M Bes-Tris buffer (pH 7.0), and 20% PEG 3350. The highest quality crystals were carefully selected and directly immersed in a cryoprotectant solution consisting of the original stock solution supplemented with 30% (v / v) glycerol, followed by immediate rapid cooling in liquid nitrogen at 100 K. X-ray diffraction data were collected at beamline BL41XU of the SPring-8 synchrotron facility in Japan using a 39 × 23 μm microfocused beam. Data were collected using a wavelength of [wavelength missing]. Monochromatic X-rays were used to rotate the single crystal 360°, with each image oscillating at an angle of 0.2°, thus obtaining a complete dataset. The collected diffraction data was then processed using the HKL2000 software package through indexing, integration, and scaling procedures.

[0170] The structure of the FER-FRV complex was determined and refined using molecular substitutions performed via the Phaser program in the CCP4 suite. Initial electron density maps showed clear densities of the FRV inhibitor bound within the ATP-binding pouch, allowing for manual construction of the ligand into the density using the Coot software. Iterative refining cycles were performed using phenix.refine, with parameters tailored to specific parameters. Resolution data were optimized, including individual atom displacement parameters, translation-calibration screw motion sets, and an optimized X-ray / stereochemical weight ratio. The final model exhibits excellent geometry, with 98.2% of residues in favorable regions of the Ramachandran diagram and no residues in disallowed regions, confirming the high quality of the structure determination.

[0171] The ADP-Glo ​​kinase assay method is used for in vitro testing of kinase activity. Luminescent analysis is used to monitor the phosphotransferase activity of FER-KD, TMK4-KD, and BRI1-CD kinases. Upon activation, FER phosphorylates peptide substrates (RRRVTSPARRS) by converting ATP to ADP. The kinase reaction is stopped, and unreacted ATP is removed using the ADP-Glo ​​reagent. Kinase assay reagents are added to convert ADP to ATP, which is then converted to a luminescent signal via a luciferase reaction. The assay mixture contains a final volume of 20 μL of 50 mmol / L Tris-HCl (pH 7.5), 1 mmol / L CaCl2, 5 mmol / L MgCl2, 1 mM DTT, 0.05 mmol / L ATP, 1 μM RALF1, 0.1 g / mL bovine serum albumin, and 6 ng / μL target proteins (FER-KD, BAK1-CD, TMK4-KD, and BRI1-CD). The kinase reaction is carried out at room temperature (25–27 °C) for 60 minutes. DMSO was used as a negative control for the small molecule inhibitor, and an ATP-free system was used to measure the background signal. Efficacy was measured using an EnVision microplate reader. Data were analyzed using GraphPadPrism 8 software, and inhibition curves were fitted to determine the IC50. 50 value.

[0172] Example 2: Small molecule inhibitors targeting FER kinase based on structure screening.

[0173] To identify inhibitors targeting FER, this embodiment utilized the Gnina molecular docking software to perform high-throughput virtual screening of 4378 compounds, identifying small molecules that can bind to the FER kinase domain. The ranking using a convolutional neural network (CNN) scoring function reflects the binding affinity between the small molecules and the FER kinase.

[0174] Homology sequence alignment of FER kinase revealed that FER shares a high degree of similarity (approximately 60%) with the kinase domain of human interleukin-1 receptor-associated kinase 4 (IRAK4). Therefore, further investigation was conducted on the compounds screened above that exhibit significant IRAK4 inhibitory activity.

[0175] Taking into account the core framework, membrane permeability and commercial availability of the compounds, this embodiment ultimately screened out 5 compounds that showed significant FER inhibitory activity (Table 1).

[0176] Table 1. Structures and corresponding CAS numbers of the five compounds.

[0177]

[0178] The two FER mutants (FER mutants) exhibited significant root hair growth arrest, and their root growth was unresponsive to RALF1. Subsequently, root hair growth experiments were conducted using these five screened small molecules.

[0179] It is worth noting that among these five candidate inhibitors, four effectively inhibited root hair growth in wild-type seedlings, with OTSSP167 showing the most significant inhibitory effect. Figure 1 A and Figure 1 B).

[0180] Furthermore, among all the small molecule and broad-spectrum kinase inhibitors tested, K-252a (CAS No.: 99533-80-9), OTSSP167 showed the best effect in inhibiting the RALF1 effect. Figure 1 C). Given the potency and selectivity of OTSSP167 for FER, it was named the FERONIA kinase inhibitor (FRV).

[0181] To investigate the mechanism of action of FRV in inhibiting FER, the FER kinase domain (FER-KD, amino acid residues 518-820) was expressed using an insect expression system. The co-crystal structure of the FER-KD-FRV complex was obtained, with a resolution of [resolution missing]. The structure reveals that FER possesses a typical kinase sandwich structure, comprising a smaller, β-sheet-rich N-terminal domain (residues 518-611) and a larger, predominantly α-helical C-terminal domain (residues 618-816). These two domains are connected by a six-amino acid hinge region (residues 612-617), forming an ATP-binding pocket at their junction. Notably, the activation fragment (residues 678-708) extends outward from the core structure, indicating that the FER kinase domain is in the activated conformation. Figure 2 The co-crystal structure revealed that the activation fragment covering residues 686-799 was not fully resolved, indicating an inherent disorder and high flexibility in this region. Furthermore, the co-crystal structure showed that FRV occupies the ATP-binding pocket and interacts with key residues in the FER kinase (including Lys565, Tyr610, Tyr612, Met613, and Asp679) through hydrogen bonds and π-π stacking, which play a crucial role in stabilizing the FER-FRV complex. Figure 2 Among them, Lys565, Asp679, and Tyr610 are key residues in the FER kinase catalyzing substrate phosphorylation process.

[0182] The binding affinity between the FRV inhibitor and FER was further evaluated using surface plasmon resonance (SPR) experiments. The results showed that the equilibrium dissociation constant (K0) for FER binding was [missing information].D The concentration was 0.4 μM. Furthermore, the binding affinity of FRV to FER-KD was higher than its binding affinity to ATP (ATP's K+). D The concentration was 18.37 μM, indicating that FRV can act as an ATP-competitive inhibitor, inhibiting FER kinase activity by targeting the ATP-binding pocket.

[0183] Example 3: Inhibitory effect and binding affinity of FRV on FER kinase mutants

[0184] To evaluate the inhibitory effect of FRV on FER kinase, ADP-Glo ​​was used. TM A reliable method for detecting kinase activity was established. Recombinant FER kinase was co-incubated with different concentrations of FRV, and the results showed that the luminescence intensity decreased in a dose-dependent manner, based on which dose-response curves were plotted.

[0185] The half-maximal inhibitory concentration (IC50) of FRV against FER kinase 50 The concentration was 70 nM, indicating that it has potent inhibitory activity. Figure 3 A).

[0186] To further elucidate its inhibitory mechanism, the binding affinity and inhibitory effect of FRV on FER kinase mutants were investigated. Based on the co-crystal structure of the FER kinase-FRV complex and after analyzing key amino acid residues in the binding pocket, five FER-KD point mutants (K565A, Y610A, Y612A, M613A, and D679A) were selected for study. These mutants were cloned into the pFastBac vector and expressed in SF9 insect cells using the Bac-to-Bacbaculovirus expression system.

[0187] FRV dose-response curves for these mutants showed that the inhibitory effects of the K565A, Y610A, and D679A mutants were significantly reduced, with IC50 values ​​of [missing value]. 50 Values ​​exceeding 100 μM, and Y612A and M613A mutations impair the catalytic activity of FER kinase. Figure 3 A).

[0188] To assess the binding affinity of FRV to the FER-KD mutant, this embodiment purified these five mutants and performed quantitative surface plasmon resonance (SPR) analysis. The results showed that K565A ( Figure 3 B), Y610A ( Figure 3 C), Y612A ( Figure 3 D), M613A Figure 3 E), D679A ( Figure 3The effects of F) mutations on FRV-FER interaction vary.

[0189] The study found that K565 and Y610 are key ATP-binding residues that regulate the binding of FRV to FER through allosteric effects.

[0190] Furthermore, in the SPR experiment, the Y612A mutation resulted in a more than 1000-fold reduction in the binding affinity of FRV, which confirms the crucial role of Y612A in the inhibitor recognition process.

[0191] A systematic comparison of the pharmacological parameters of different mutants revealed that spatial combinations of mutations within the pocket may synergistically disrupt the interaction between FRV and FER.

[0192] Example 4: FRV-specific inhibition of FER kinase.

[0193] To evaluate the selectivity of FRV, this embodiment employs surface plasmon resonance (SPR) experiments and ADP-Globe assays. TM The binding affinity and inhibitory effect of the kinase activity assay on key receptor-like kinases (RLKs) in plants were analyzed. These receptor-like kinases include transmembrane kinase 4 (TMK4), ANJEA (ANJ), ANXUR1 (ANX1), Buddha's PaperSeal1 (BUPS1), and brassinolide-insensitive protein 1 (BRI1).

[0194] SPR results show that the dissociation constant (K) of FRV binding with TMK4 and ANJ is... D The effective molecular weights (FMW) were 15.3 μM and 19.4 μM, respectively. Notably, compared to FER, FRV showed significantly reduced binding affinity to ANX1, BUPS1, and BRI1 (more than 100-fold decrease). These findings indicate that FRV possesses significant specificity for FER, exhibiting selectivity for FER that is at least tens of times higher than that of other receptor kinases.

[0195] Structural analysis in this embodiment explains the differences in binding affinity between FRV and FER and other kinases. Previous studies on kinase inhibitors have shown that even small differences in hydrophobic interactions or hydrogen bond networks can significantly alter inhibitor specificity. Comparative structural analysis revealed that subtle differences in key residues (such as K565, Y610, Y612, M613, and D679) may explain why FRV preferentially binds to FER. Molecular docking simulations further support this view, showing that FRV forms a more stable interaction with the ATP-binding pocket of FER compared to other members of the CrRLK1L family. These findings confirm that FRV is a potent FER-specific inhibitor and also provide structural insights for designing selective inhibitors of other receptor-like kinases (RLKs).

[0196] FERs are evolutionarily conserved across plant species ranging from algae to angiosperms, and are abundant in terrestrial plants adapted to complex growth, development, and environmental changes. Currently, extensive research has been conducted on FERs in Arabidopsis thaliana, as well as other terrestrial plants such as rice, pear, and Brassica. To further investigate the kinase domains of representative plant species, this study selected species including Arabidopsis thaliana, Brassica napus, Brassica rapa, rice (Oryza sativa), maize (Zeamays), tomato (Solanum lycopersicum), white pear (Pyrus bretschneideri), Selaginella moellendorffii, Physcomitrium patens, and Marchantia polymorpha. The study found that key amino acids interacting with FRVs are highly conserved in these species. These results suggest that FRVs may be applicable to a wide variety of terrestrial plant species.

[0197] Example 5: FRV-assisted phosphorylated proteomics analysis of FER signal.

[0198] Given that FER is involved in multiple biological processes and its absence alters the phosphorylation levels of over a thousand proteins, this embodiment aims to leverage the powerful chemical tool of FRV to comprehensively understand the FER kinase-dependent signaling pathway. Previously, a phosphorylated proteomics study of seedlings treated with 1 μM RALF1 for 5 minutes identified four highly abundant proteins, including FER, AHA2, calcium-dependent protein kinase 9 (CAP9), and the PEN3 / ABCG36 transporter, as well as one protein with reduced phosphorylation levels, namely the FER homolog ERULUS. However, subsequent studies have significantly expanded our understanding of the RALF1-FER-regulated signaling pathway, thus necessitating in-depth phosphorylated proteomics analysis of the FER kinase-dependent pathway.

[0199] Therefore, this study used 7-day-old Arabidopsis thaliana seedlings as the research subject and conducted phosphoproteomics analysis, focusing on the inhibitory effect of FRV on RALF1 signaling. After treatment with 1 μM RALF1 for 30 minutes, a total of 769 upregulated phosphopeptides and 456 downregulated phosphopeptides were identified, including 106 newly emerging phosphopeptides and 456 newly emerging dephosphopeptides. In contrast, in samples pretreated with 1 μM FRV, 460 upregulated phosphopeptides and 451 downregulated phosphopeptides were identified, including 345 newly emerging phosphopeptides and 313 newly emerging dephosphopeptides. Notably, there was an overlap of 153 upregulated phosphopeptides and 169 downregulated phosphopeptides between the FRV+RALF1 / CK group and the FER-4 / WT group reported in previous phosphoproteomics studies, indicating that FRV has an inhibitory effect on FER. Figure 4 B).

[0200] Based on the RALF1-FRV experimental results, it was confirmed that RALF1 exerts its function by activating FER kinase activity. Therefore, the research focus was placed on the phosphorylation of FER kinase.

[0201] Mass spectrometry data analysis revealed two activation sites for FER—Ser695 and Ser866. These two sites were phosphorylated after RALF1 treatment (phosphorylation levels increased by 1.79-fold and 4.71-fold, respectively), while FRV treatment inhibited phosphorylation at these two sites. Figure 4A) Ser695 is located in the activation fragment of the FER kinase domain. In FER-KD expressed in vitro, this site has been shown to be one of the major autophosphorylation sites, and mutating it to alanine (S→A) does not affect the function of FER in pollen tube acceptance. Ser866 is located close to Ser871 and Ser874, sites previously reported to be phosphorylated at the FERC end after 5 minutes of RALF1 treatment. Furthermore, 345 upregulated and 364 downregulated overlapping phosphorylation proteins were identified between the RALF1 / CK group and the RALF1 / FRV+RALF1 group. These proteins are considered to be downstream signaling components dependent on the FER kinase.

[0202] Gene ontology (GO) analysis of these differentially expressed phosphoproteins revealed their enrichment in multiple biological processes, such as intracellular localization (GO:0051649), cell growth (GO:0016049), cell division (GO:0051301), plant-pathogen interactions (GO:ath04626), regulation of fungal defense responses (GO:1900150), pollen acceptance (GO:0060321), and mechanostimulation responses (GO:0009612). Figure 4 C).

[0203] Example 6: RALF1-FER regulates the expansion of root meristem cells through AHA1 / 2Ser899-mediated alkalization.

[0204] Phenotypically, the RALF1 mutant consistently exhibited longer root epidermal cells and taproots than the wild type during the seedling stage. However, the phenotype of the FER-4 mutant was more complex: 6 days prior, the taproot of the FER-4 mutant was longer than that of the wild type, but from the 7th day onwards, its taproot length became shorter than that of the wild type. Although FER is the receptor for RALF1, it is also involved in multiple plant hormone signaling pathways, including auxin, ethylene, brassinolide, abscisic acid, and jasmonic acid signaling pathways. Regarding root meristem cells, studies have shown that auxin induces transient alkalization of the extracellular matrix, thereby inhibiting the elongation of root meristem cells.

[0205] Because of these differences, the focus of the next study will be on the expansion process of root distal meristem cells. In this study, "root meristem cells" refers to the epidermal cells (the last four cells of the meristem) in the distal meristem region.

[0206] The results showed that treatment with 1 μM RALF1 significantly inhibited the growth of root meristem cells in wild-type seedlings, but had no inhibitory effect on the FER-4 mutant; the inhibitory effect of RALF1 was alleviated after the addition of 200 nM FRV. Figure 5A) This indicates that the process requires the participation of FER kinase activity.

[0207] Protein-protein interaction (PPI) analysis revealed that AHA1 / 2 plays a central role in the RALF1-FER signaling network. Further analysis of phosphorylated proteomics data showed that RALF1 induction upregulated phosphorylation at the Ser899 site of AHA1 and AHA2, while FRV treatment inhibited phosphorylation at this site. Figure 5 B).

[0208] In addition, the length of epidermal cells in the root meristem was examined, and it was found that the insensitivity of cells to RALF1 treatment was similar in both the AHA2^S899D transgenic plant and the FER-4 mutant. Figure 5 C). Consistent with this, the cell length of the elongation zone in the AHA2^S899D transgenic line was significantly less sensitive to RALF1 treatment, while the cell length of the wild-type elongation zone was significantly inhibited by RALF1 treatment, and the elongation zone cells of the FER-4 mutant were insensitive to RALF1 treatment.

[0209] Furthermore, this embodiment also investigated the effects of RALF1 and FRV on rhizosphere pH. Using bromocresol purple staining experiments, it was found that treatment with 1 μM RALF1 led to rhizosphere alkalization in seedlings, while FRV at concentrations of 50-200 nM significantly inhibited this alkalization. Figure 6 These results indicate that RALF1 activates the kinase activity of FER, leading to phosphorylation at the Ser899 site of AHA1 / 2, which in turn affects the expansion of meristematic cells by regulating apoplast pH changes.

[0210] pH changes in the apoplast of root meristem cells were detected using 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), a membrane-impermeable ratiometric pH indicator. This indicator has been proven to be a reliable tool for studying root cell expansion associated with apoplast pH changes. High-resolution laser confocal microscopy analysis of the signal intensity ratio between the 458 nm and 405 nm channels revealed that the AHA2^S899D transgenic plant was insensitive to RALF1 in terms of apoplast alkalization. Figure 5 D). RALF1 treatment significantly increases the apoplast pH of meristematic cells, while pretreatment with 200 nM FRV effectively inhibits this pH increase. The FER-4 mutant also shows a similar pH change trend. Figure 5 D). These findings indicate that FRV small molecules can effectively inhibit RALF1-FER-AHA1 / 2-mediated cell expansion.

[0211] To further investigate the functional importance of K565R, Y610A, Y612A, M613A, and D679A residues, a series of FER mutants were constructed and introduced into the FER-4 background. In this study, RALF1-induced primary root growth experiments were conducted. The results showed that the Y612A / M613A / D679A triple mutant exhibited a weakened response to RALF1, while the Y610A / D679A and K565R / D679A double mutant, the Y610A / K565R / D679A triple mutant, and the K565R / Y610A / Y612A / M613A / D679A quintuple mutant were insensitive to RALF1 treatment.

[0212] The lengths of the meristematic and elongation zones in epidermal cells of the Y610A, K565R, Y610A / D679A, and K565R / D679A mutants were further examined. Compared to wild-type FER, which fully complements the FER-4 mutant, these transgenic plants were insensitive to RALF1 treatment. To assess the functional contributions of FERY612 and M613 residues, the FER mutant was expressed in *N. benthamiana* leaves, and its response to RALF1 was examined to determine pH changes. The results showed that the Y610A, Y610A / Y612A, Y610A / M613A, and Y610A / Y612A / M613A mutants were unresponsive to RALF1, while the Y612A and M613A mutants showed similar responses to RALF1 to the wild type. Further pH testing of various FER mutant transgenic plants showed that single mutants at the Y610 and K565 sites, as well as the corresponding double mutants and higher-order mutants, were insensitive to RALF1. Figure 5 These results suggest that, among these five key residues, Y610 and K565 may play a more critical role in RALF1 signaling than Y612, M613, and D679 residues.

[0213] Example 7: RALF1-FER regulates cell expansion via FER-S695 phosphorylation.

[0214] Phosphorylated proteomics data revealed that FER-S695 is a potential phosphorylation site induced by RALF1. To further validate this finding, pFER:FER-Flag seedlings were treated with RALF1, and their phosphorylation was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0215] To further investigate the in vivo function of S695 phosphorylation, this study constructed a pFER:FER^S695A-GFP fer-4 transgenic plant. The results showed that RALF1 treatment induced an increase in the phosphorylation abundance of FER-S695. Further analysis using parallel response monitoring (PRM) revealed that the phosphorylation level of FER-S695 in the RALF1-treated group was 2.4 times that of the control group.

[0216] Subsequently, pFER:FER-GFP transgenic seedlings were pretreated with 1 μM FRV for 3 hours, followed by treatment with 1 μM RALF1 for 30 minutes, and then FER-GFP immunoprecipitation was performed. The results showed that FRV treatment effectively inhibited RALF1-induced FER phosphorylation. Figure 7 A).

[0217] Furthermore, using self-made phosphorylation antibodies targeting the Ser695 and Thr696 sites of FER, it was found that RALF1 treatment significantly increased the phosphorylation level of FER in wild-type seedlings, but had no such effect in FER^S695A / T696A seedlings. Figure 7 B).

[0218] Furthermore, compared to pFER:FER-GFP fer-4 plants, RALF1 treatment failed to induce expansion of meristematic and elongation zone cells in pFER:FER^S695A-GFPFER-4 transgenic plants. Figure 7 C). Similarly, no significant pH changes were detected in the FER^S695A mutant line. Figure 7 D).

[0219] These findings suggest that S695 is one of the key phosphorylation sites involved in the FER-RALF1 signaling pathway (which regulates meristematic cell expansion).

[0220] discuss

[0221] The multifunctional receptor kinase FER regulates a variety of biological processes through its kinase activity. This invention identifies a small molecule tool called FRV that can effectively and specifically inhibit FER kinase activity.

[0222] High-resolution co-crystal structure analysis of the FER kinase domain and FRV successfully determined the binding mode of FRV in the ATP-binding pocket.

[0223] Furthermore, SPR experiments confirmed the role of key amino acids, including Lys565, Tyr610, Tyr612, Met613, and Asp679, in stabilizing the FER-FRV interaction.

[0224] Phenotypic analysis of transgenic mutants (through primary root growth, cell expansion, and apoplast pH detection) revealed that these residues are crucial for FER activity, with residues at positions 565 and 610 being key functional amino acid sites in FER.

[0225] This embodiment employed surface plasmon resonance (SPR) analysis to evaluate the binding affinity of FRV for various important receptor kinases, including members of the CrRLK1L family. The results showed that the binding affinity of FRV to FER was 48.5 times and 38.3 times that to ANJ kinase and TMK4 kinase, respectively. Furthermore, the binding affinity of FRV to ANX1, BUPS1, and BRI1 was significantly reduced, almost undetectable. This stronger binding affinity may stem from the unique spatial complementarity of the FER active site or additional electrostatic interactions. For example, molecular docking studies and structural comparisons revealed a key molecular difference in the FER-FRV co-crystal structure, despite FRV occupying the ATP-binding pockets of both kinases: BRI1 lacks the crucial hydrogen bond interaction between FRV and the conserved tyrosine residue (Y612 position) in FER. This absence of specific molecular interactions may explain why the binding affinity of FRV to BRI1 is significantly lower than its affinity to FER. Although FRV also binds to other receptor kinases to some extent, the differences in binding affinity may reflect subtle structural differences between these proteins.

[0226] This invention applies a broad-spectrum inhibitor along with several candidate inhibitors to seedlings and evaluates their effectiveness by measuring taproot length. Notably, compared to the broad-spectrum inhibitor K-252a and other candidate inhibitors, FRV exhibits superior efficacy in inhibiting RALF1-induced root shortening, achieving significant inhibition at a concentration of 100 nM. These results demonstrate that FRV is a potent FER kinase inhibitor in plants, effectively inhibiting FER-mediated biological processes within the nanomolar concentration range.

[0227] FER kinase activity is involved in a variety of biological processes, which has always been a major challenge in small molecule inhibitor research. One of the basic goals of developing small molecule inhibitors is to apply these inhibitors locally to specific plant tissues and cells within a specific time period, thereby precisely regulating biological processes.

[0228] In this study, a transgenic plant with a point mutation at the Ser899 site of AHA2 was constructed. Comprehensive phenotypic analysis of roots and root meristem cells treated with RALF1 and FRV was performed to verify the specific mechanism by which AHA1 / 2 responds to RALF1 signaling and FER activation. Therefore, using FRV as a chemical probe is a viable phosphoproteomics method for identifying FER kinase-dependent signaling pathways.

[0229] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An agricultural composition, characterized in that, The agricultural composition comprises: (i) a first active ingredient, the first active ingredient comprising ferovicin (FRV) or an agronomically acceptable salt thereof; and (ii) Any agronomically acceptable carrier.

2. The agricultural composition according to claim 1, characterized in that, The agricultural composition also includes: (iii) A second active ingredient comprising: optionally, a substance for improving plants.

3. The agricultural composition according to claim 2, characterized in that, The substances used to improve plants include: Tesevatinib, Brigatinib, AZD-7762, or an agronomically acceptable salt thereof, or a combination thereof.

4. The use of the agricultural composition according to claim 1, characterized in that, For one or more uses selected from the following group: (a) Improve plants; (b) Inhibit the activity of FER kinase; (c) Used as a positive reference for screening FER kinase inhibitors; (d) Analysis of FER-dependent signaling pathways in plants.

5. The use as described in claim 4, characterized in that, The improved plant includes: an improved plant root system.

6. The use as described in claim 4, characterized in that, The improved plant comprises one or more improvements selected from the group consisting of: (Z1) inhibits FER kinase activity; (Z2) promotes cell growth in the root meristem; (Z3) inhibits rhizosphere alkalization; (Z4) Increase the root length.

7. The use as described in claim 4, characterized in that, The plant is a plant containing highly conserved amino acid residues selected from the group consisting of: K565, Y610, Y612, M613, and D679. The amino acid residue numbers are based on the amino acid sequence of Arabidopsis thaliana FER kinase.

8. A method for improving plants, characterized in that, The method includes the following steps: Apply an effective amount of the agricultural composition according to claim 1.

9. A method for screening substances that improve plants, characterized in that, Including the following steps: (S1) In the experimental group, incubation with FER kinase in the presence of the test substance; and (S2) Determine whether a FER kinase-test substance complex is formed in the experimental group. If a FER kinase-test substance complex is formed in the experimental group, it suggests that the test substance is a potential therapeutic agent for improving plants. In the FER kinase-test compound, the test compound binds to Lys565, Tyr610, Tyr612, Met613 and Asp679 residues of the FER kinase. The residue numbers are based on the amino acid sequence of Arabidopsis thaliana FER kinase.

10. The method as described in claim 9, characterized in that, Step (S1) further includes: incubating with FER kinase in the positive control group under the same conditions but without the test substance and in the presence of FRV.

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