Use of p4-atpase inhibitors for the control of fungal diseases and / or the regulation of plant growth

Abstract

This invention discloses the application of P4-ATPase inhibitors in the control of plant fungal diseases and / or the regulation of plant growth. This invention discloses a novel molecular target—P4-ATPase—for the control of plant fungal diseases and / or the regulation of plant growth. Cellular and phenotypic experiments show that inhibiting this target can lead to suppression of the growth of plant pathogenic fungi and produce a growth-regulating effect on plants. This invention demonstrates that triazole compounds can directly and specifically interact physically with P4-ATPase. Triazole compounds not only bind but also effectively block the biological function of P4-ATPase. Furthermore, based on this target, other P4-ATPase inhibitors are screened for use in the control of plant fungal diseases and / or the regulation of plant growth.

Description

Technical Field

[0001] This invention relates to the fields of agricultural biotechnology and new pesticide creation, and specifically to the application of P4-ATPase inhibitors in the prevention and control of plant fungal diseases and / or the regulation of plant growth. Background Technology

[0002] In agricultural applications, triazole compounds are primarily used as fungicides to control crop diseases. After years of development, triazole fungicides, including propiconazole (PCZ), difenoconazole (DCZ), flusilazole (FSZ), and epoxiconazole (ECZ), utilize a triazole ring structure in their molecules that can diffuse into mycelial cells and bind to the central iron in the iron porphyrin of pathogens, inactivating a key enzyme in the synthesis of ergosterol. Ergosterol is a component of the mycelial cell membrane; by preventing the pathogen's cell membrane from synthesizing normally, the fungicide is inactivated, thus hindering mycelial growth and achieving its bactericidal effect. Therefore, triazole fungicides are widely used in crop disease control. In addition to their fungicidal activity, triazole fungicides also regulate plant growth, stimulating plant resistance and enhancing their ability to withstand adverse environments. Because triazole fungicides have excellent control effects on a variety of diseases, their application range is becoming increasingly wide. However, the single site of action of these fungicides has led to the development of drug resistance in pathogens.

[0003] P4-ATPase, also known as phospholipid flipper enzyme, is a key protein for maintaining membrane function. It is a type of membrane protein that is widely found in eukaryotes and transports adenosine triphosphate (ATP) across biological membranes. By hydrolyzing ATP, it transports phospholipid molecules from the extracellular side of biological membranes (including the extracellular space and organelle cavities) to the cytoplasmic side, playing an important role in maintaining the asymmetry of membrane phospholipid distribution.

[0004] Drs2-Cdc50 is a well-studied member of the P4-ATPase family. It generates and maintains membrane lipid asymmetry by transporting phosphatidylserine (PS) from the lumen to the membrane cytoplasmic leaflets. This asymmetry is crucial for various cellular processes, including vesicle budding and intracellular vesicle transport. Previously, our research group screened a membrane transport protein gene, DRS2, associated with PCZ sensitivity using a yeast defect library. In the drs2 deletion mutant, the cells' sensitivity to high concentrations of PCZ was significantly reduced. This suggests that Drs2p may be involved in the transmembrane transport of PCZ (Chen, Zhuoyue. Mechanism of ALA3 and ENT1 gene deletion in increasing Arabidopsis thaliana sensitivity to propiconazole [D]. South China Agricultural University). The protein with the highest homology to Drs2p in Arabidopsis thaliana is ALA3, the protein with the highest homology to Drs2p in Chinese cabbage is ALA3a / b / c, and the protein with the highest homology to Drs2p in rice blast fungus is MoAPT5; all belong to the P4-ATPase family. Chinese patent application CN113584045A discloses the application of BraALA3a / b / c in regulating the absorption and accumulation of propiconazole in Chinese cabbage and in the cultivation of superior Chinese cabbage varieties.

[0005] In recent years, computational biology and structural biology methods have been widely applied in pesticide research. In particular, molecular docking and molecular dynamics simulations have provided powerful tools for exploring the mechanisms of action of pesticide molecules, optimizing lead compounds, and developing novel pesticide molecules. For example, molecular docking and crystallography have verified the binding site of abamectin to the mitochondrial respiratory complex III of ticks, elucidating its unique inhibitory mechanism. Furthermore, molecular dynamics simulations of the interaction between ryanodine receptors (nAChRs) and neonicotinoid insecticides have revealed that conformational differences caused by different substituents significantly affect the binding mode of ligands to receptor pocket residues, explaining their structure-activity relationship in terms of toxicity and selectivity. Therefore, using computational biology methods to study the interaction between phospholipid invertases and triazole compounds and their impact on PS transport activity holds promise for elucidating the molecular mechanisms by which triazole compounds regulate plant growth and development and bind to fungal phospholipid invertases, providing an important theoretical foundation and technical support for the development of novel pesticides. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of P4-ATPase inhibitors in the prevention and control of plant fungal diseases and / or the regulation of plant growth.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] This invention, through a combination of bioinformatics analysis and biological experiments, reveals and confirms for the first time that P4-ATPase (phospholipid invertase) is a novel and direct target for binding with triazole compounds, and provides its application in the agricultural field based on this discovery. Specifically, this invention constructs a complete chain of evidence by integrating multi-scale research strategies—from atomic-level computational simulations to molecular-level direct interaction verification, and then to cellular and individual-level phenotypic analysis—establishing the effectiveness of this target. Furthermore, based on this target, other P4-ATPase inhibitors are screened and obtained.

[0009] Specifically, this invention employs the AlphaFold algorithm, based on artificial intelligence, to predict the polypeptide chain complex structure of phospholipid reverserase amino acid sequences, obtaining a reliable three-dimensional structural model. The model is then evaluated and optimized using Ramachandran diagrams and the MolProbity tool, and further refined through molecular dynamics simulations to obtain a stable and reliable protein dimer complex structural model. Using molecular docking technology, with triazole compounds as ligands and the phospholipid reverserase protein dimer complex structural model as the acceptor, the binding mode and interaction between the two are analyzed. Based on the docking complex conformation, electrostatic potential energy analysis and molecular dynamics simulations are performed to further investigate the dynamic process and energetic characteristics of the interaction between the compound and the protein dimer complex structure. Surface plasmon resonance (SPR) and in vitro ATPase activity measurements are used to elucidate the affinity and inhibitory effect of the compound on the interaction with different species of phospholipid reverserases. Gene expression vectors were constructed using CRISPR gene editing technology, and *Arabidopsis thaliana* atala3 mutant plants, *Chinese cabbage* BraALA3a / b / c low / overexpression plants, and *Oryza sativa* ΔMoAPT5 mutant strain were identified. Phenotypic analysis was performed after application of triazole fungicides. Molecular docking studies revealed the key binding sites and interaction modes between triazole compounds and the phospholipid invertase complex. Molecular dynamics simulations showed that triazole compounds stably bind to the phospholipid invertase protein complex through hydrophobic and van der Waals interactions, inducing a co-adaptive change in protein conformation. Surface plasmon resonance (SPR) affinity analysis showed that the in vitro expressed phospholipid invertase protein has an affinity for triazole compounds, with varying degrees of affinity. In vitro ATPase activity assays showed that triazole compounds inhibited the phospholipid invertase protein. Experiments with gene-edited plants / fungi showed that reducing phospholipid invertase expression decreased the sensitivity of fungi or plants to triazole compounds and increased their tolerance; conversely, overexpression of phospholipid invertase increased the sensitivity of fungi or plants to triazole compounds and decreased their tolerance. This invention, for the first time, reveals and confirms through a multi-scale chain of evidence that triazole compounds can directly act on the novel target of P4-ATPase. The direct binding mode was predicted through molecular docking and kinetic simulations; surface plasmon resonance (SPR) technology confirmed the specific binding of triazole compounds to fungal and plant-derived P4-ATPase; in vitro enzyme activity experiments demonstrated that triazole compounds effectively inhibited the lipid inverting function of this enzyme; further cell and phenotypic experiments showed that inhibiting this target led to growth suppression in plant pathogenic fungi and produced growth-regulating effects on plants. This invention demonstrates that triazole compounds can directly and specifically interact physically with P4-ATPase. Triazole compounds not only bind but also effectively block the biological function of P4-ATPase.This invention also obtained 38 candidate lead compounds based on virtual screening analysis of the ALA3 protein pocket, which were classified into amide compounds, sulfonamide compounds and heterocyclic compounds, respectively. Three compounds with higher docking scores in each type, A01, A15 and A22, were selected for verification.

[0010] Therefore, the present invention provides the following uses: New uses for triazole compounds, amide compounds, sulfonamide compounds, or heterocyclic compounds as P4-ATPase inhibitors.

[0011] The use of P4-ATPase inhibitors in the prevention and control of plant fungal diseases and / or regulation of plant growth, or in the preparation of products for the prevention and control of plant fungal diseases and / or regulation of plant growth; wherein the P4-ATPase inhibitors are selected from triazole compounds, amide compounds, sulfonamide compounds, or heterocyclic compounds.

[0012] Furthermore, the triazole compound is selected from propiconazole, difenoconazole, flusilazole, flutriazole, paclobutrazol, or uniconazole.

[0013] Preferably, the amide compounds are selected from the following: A01–A14, A17, A20, A21, A23, A25, A28, A30, A32, A34, or A36; the amide compounds are selected from A15, A18, or A33; and the amide compounds are selected from A16, A19, A22, A24, A26, A27, A29, A31, A35, A37, or A38. All of these compounds are commercially available and can be obtained through PubChem CID.

[0014] .

[0015] Specifically, the product works by specifically binding to P4-ATPase and inhibiting the lipid flipping function of P4-ATPase.

[0016] Furthermore, the P4-ATPase is derived from fungi or plants.

[0017] Preferably, the P4-ATPase is yeast Drs2 protein, rice blast fungus MoAPT5 protein, Arabidopsis thaliana ALA3 protein, or Chinese cabbage ALA3a / b / c protein.

[0018] The results of this invention also show that knocking out the rice blast fungus... MoAPT5 Genes can inhibit the growth of rice blast fungus.

[0019] Therefore, this invention also provides the application of reagents for knocking out the P4-ATPase encoding gene of plant pathogenic fungi in the prevention and control of plant fungal diseases.

[0020] Furthermore, the reagent inhibits the growth of plant pathogenic fungi by targeting and knocking out the P4-ATPase encoding gene in plant pathogenic fungi, thereby inhibiting the expression and / or activity of P4-ATPase.

[0021] Furthermore, the plant pathogenic fungus is rice blast fungus, and the plant fungal disease is rice blast.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of P4-ATPase inhibitors in the control of plant fungal diseases and / or the regulation of plant growth. This invention discloses a novel molecular target—P4-ATPase—for the control of plant fungal diseases and / or the regulation of plant growth. Cellular and phenotypic experiments show that inhibiting this target can lead to suppression of the growth of plant pathogenic fungi and produce a growth-regulating effect on plants. This invention demonstrates that triazole compounds can directly and specifically interact physically with P4-ATPase. Triazole compounds not only bind but also effectively block the biological function of P4-ATPase. Furthermore, based on this target, other P4-ATPase inhibitors are screened for use in the control of plant fungal diseases and / or the regulation of plant growth. Attached Figure Description

[0023] Figure 1 Three-dimensional structural diagrams of the docking of Chinese cabbage phospholipid invertase BraALA3a / b / c, Arabidopsis thaliana phospholipid invertase AtALA3, and PCZ molecules.

[0024] Figure 2 The figure shows the 50 ns molecular dynamics simulation analysis of Chinese cabbage phospholipid invertase BraALA3a / b / c, Arabidopsis thaliana phospholipid invertase AtALA3, and different substrate molecules.

[0025] Figure 3 This represents the SPR affinity signal between propiconazole and BraALA3a / b / c and AtALA3 proteins.

[0026] Figure 4 Analysis of changes in the in vitro ATPase activity of BraALA3a / b / c and AtALA3 proteins in response to propiconazole.

[0027] Figure 5 The effect of low / overexpression of the BraALA3a / b / c gene on the sensitivity of Chinese cabbage to propiconazole.

[0028] Figure 6 To investigate the effects of propiconazole on different atala3 mutants in Arabidopsis thaliana and their slice phenotypes.

[0029] Figure 7 This is a three-dimensional structural diagram of the docking between the phospholipid invertase MoAPT5 of rice blast fungus and a triazole compound.

[0030] Figure 8 This is a 50 ns molecular dynamics simulation analysis of the phospholipid invertase MoAPT5 of rice blast fungus and triazole compounds.

[0031] Figure 9 SPR affinity signal between propiconazole and MoAPT5 protein Figure 10 Analysis of changes in MoAPT5 protein ATPase activity in response to propiconazole.

[0032] Figure 11 This represents the antibacterial phenotype of propiconazole and the MoAPT5 gene mutant.

[0033] Figure 12 Computer-aided virtual screening for potential ligands targeting the ALA3 protein.

[0034] Figure 13 The structural formulas and docking scores of 38 compounds obtained from virtual screening targeting the ALA3 active pocket are shown.

[0035] Figure 14 This represents the SPR affinity binding signal between AtALA3-AtALIS2 and lead compounds A01, A15, and A22.

[0036] Figure 15 yeast drs2 In strains ALA3 Heterogeneous verification of the sensitivity of three lead compounds.

[0037] Figure 16 In yeast strain BY4741 ALA3 Heterogeneous verification of the sensitivity of three lead compounds.

[0038] Figure 17 The inhibitory effects of three lead compounds on the growth of Chinese cabbage. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0040] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0041] Example 1: Molecular docking study of triazole compounds and protein complexes (1) Preparations before molecular docking. AutoDockTools and OpenBabel were used to preprocess the structures of the receptor protein and ligand molecules, including hydrogenation, charge calculation, and atom type definition. Based on the hydrophobic properties of the transmembrane region of the protein complex and the location of the ATP binding pocket, and with reference to the substrate binding mode of DRS2, the spatial docking site and the size of the docking box were set.

[0042] (2) Using AutoDock Vina software, with the protein complex as the receptor and propiconazole as the ligand, the ligand-receptor binding free energy was calculated based on a semi-empirical scoring function. The spatial orientation of the ligand was optimized using a genetic algorithm, and the binding conformation with the lowest energy was searched and output. The exhaustiveness value was set to 100, and num_modes output 20 binding modes.

[0043] (3) Visual analysis was performed using software such as PyMOL on the highest-scoring docking conformation in the docking pocket to examine the interaction modes between propiconazole and surrounding amino acid residues, including hydrophobic interactions, hydrogen bonds, van der Waals forces, etc.

[0044] The results showed that: First, when a docking box for propiconazole and the ALA3 protein molecule was set up (box center: X = 141.575, Y = 152.672, Z = 137.524; box size: 30 Å × 30 Å × 30 Å), propiconazole could stably bind in a hydrophobic pocket formed by the transmembrane helix of ALA3 and the β-sheet of the cytoplasmic region of ALIS1, consistent with the binding pattern of yeast DRS2-CDC50p to phospholipid substrates. The nitrogen atom on the imidazole ring of propiconazole formed a hydrogen bond with Gln88 of the Arabidopsis phospholipid invertase AtALA3-ALIS1, and formed hydrophobic interactions with Leu92, Pro331, and Val920; the triazole ring formed π-π stacking with Phe87 and Phe337, and formed hydrophobic interactions with Val1019 and Met1023. The triazole ring of propiconazole forms hydrogen bonds with amino acid residues 1045 ASN and 1129 GLN at the self-repressed C-terminus of bracken phospholipid invertase BraALA3a-BraALIS2, and with amino acid residues 1125 TYR at the self-repressed C-terminus. The dioxolane ring forms hydrophobic interactions with amino acid residues 1042 LEU, 1110 ASP, and 1114 GLN of TM10. The para-substituted benzene ring and propyl side chain form halogen bonds with 1114 GLN and 1253 PRO of TM10, and with 1255 LEU of the subunit. The nitrogen atom of the triazole ring of propiconazole forms hydrogen bonds with amino acid residues 1044 ASN, 901 LYS, and 1124 TYR at the self-repressed C-terminus of bracken phospholipid invertase BraALA3b-BraALIS2, and with amino acid residues 1113 of TM10. GLN forms hydrophobic interactions with 1127 ARG and 1253 LEU amino acid residues of the subunit; the para-substituted benzene ring and propyl side chain form hydrophobic interactions with 1041 LEU amino acid residues; the N atom of the triazole ring of propiconazole forms hydrogen bonds with 1156 GLN and 1159 GLN amino acid residues at the self-inhibitory C-terminus of BraALA3c-BraALIS2 phospholipid invertase; the dioxolane ring forms hydrophobic interactions with 989 ASP, 1010 PHE, and 1155 TYR amino acid residues; the para-substituted benzene ring and propyl side chain form π-cation interactions with 932 LYS of TM5 and hydrophobic interactions with 1144 GLN of TM10. Figure 1 ).

[0045] Second, a docking cassette was constructed between the triazole fungicide and the rice blast fungus protein MoAPT5 (cassette center: X = 130, Y = 156, Z = 126. Cassette dimensions: 30 Å × 30 Å × 30 Å). The compound was able to stably bind within the hydrophobic pocket formed by the MoAPT5 transmembrane helix and the Cdc50 cytoplasmic region. Figure 7Propiconazole forms hydrophobic interactions with amino acid residues 304 (PRO) and 563 (THR) of the MoAPT5 protein, hydrogen bonds with amino acid residues 532 (ARG) and 176 (ARG) of the subunit, π-cation interactions with amino acid residues 532 (ARG) and 559 (ARG), and halogen bonds with amino acid residues 545 (ASP), 556 (VAL), and 560 (ASP). Difenoconazole forms hydrophobic interactions with amino acid residues 305 (THR), 556 (VAL), 567 (LEU), 570 (ALA), 1087 (TYR), and 1114 (ASN) of the MoAPT5 protein, and hydrogen bonds with amino acid residues 569 (SER) and 1111 (SER). Fluorosilazole forms hydrophobic interactions with amino acid residues 290 (PHE), 310 (THR), 313 (PRO), 574 (ILE), and 1110 (LEU) of the MoAPT5 protein, and hydrogen bonds with amino acid residues 1111 (SER) and 1114 (ASN). Fluticasone forms hydrophobic interactions with amino acid residues 313PRO, 570ALA, 574ILE, and 1087TYR of the MoAPT5 protein, and hydrogen bonds with amino acid residues 1087TYR and 1114ASN. In summary, molecular docking studies have revealed the key binding sites and interaction patterns of the triazole compound-phospholipid invertase complex, providing important clues for elucidating the mechanism of action of triazole compounds and phospholipid invertase.

[0046] Example 2: Molecular dynamics simulation of the conformation of compounds and their protein complexes (1) Using the conformations of the compounds obtained by molecular docking and the protein complexes as the starting structures, a simulated system containing the complexes, POPC lipid bilayer, explicit solvent water molecules and counterions was constructed using GROMACS and CHARMM36 force fields.

[0047] (2) Minimize the energy of the complex, and then perform 100ps confined equilibrium and 100ps unconfined equilibrium in the NVT ensemble and NPT ensemble respectively to bring the system to the ideal temperature (300K) and pressure (1bar).

[0048] (3) With a time step of 2fs, perform formal sampling simulation of the balanced system for 100ns and save the trajectory at 10ps intervals.

[0049] (4) Post-processing analysis was performed on the molecular dynamics simulation trajectory to extract the changes in distance and interaction energy between the triazole compound and the binding pocket residue atoms over time, and to examine the kinetic stability of the complex. The binding free energy of the propiconazole-protein complex was calculated using the MM-PBSA method. The conformational changes of the key residue side chains of the complex in the free and bound states were compared and analyzed.

[0050] The results showed that the RMSD of BraALA3a-BraALIS2 and propiconazole / PI4P increased rapidly in the initial stage, reached equilibrium after 10 ns, and fluctuated in the range of 0.5-0.7 nm without a sustained upward trend. Figure 2 a). The RMSF of BraALA3a-BraALIS2 and propiconazole / PI4P mostly fluctuated stably in the range of 0.1-0.4 nm, except for the obvious flexible site at 6400-6800 atoms in the RMSF of the PI4P, suggesting that this site may be the region where the protein and lipid inversion enzyme regulators exert their transport activity. The fluctuation peaks of the protein and propiconazole / PI4P at other positions were almost identical, verifying the competitive binding function of propiconazole. Figure 2 b); The RMSD of BraALA3b-BraALIS2 and propiconazole / PI4P reached equilibrium after 10 ns, fluctuating in the range of 0.4-0.6 nm, without a sustained upward trend. Figure 2 c). The RMSF of BraALA3b-BraALIS2 with propiconazole / PI4P fluctuated stably in the range of 0.1-0.4 nm. Figure 2 d); The RMSD of BraALA3c-BraALIS2 and propiconazole / PI4P reached equilibrium after 10 ns, fluctuating in the range of 0.5-0.6 nm, without a sustained upward trend. Figure 2 e). The RMSF of BraALA3c-BraALIS2 and propiconazole / PI4P mostly fluctuated stably in the range of 0.1-0.4 nm. Figure 2 f), except for the obvious flexible site at atoms 7200-7300 in the RMSF of the protein with PI4P, suggesting that this site may be the region where the protein and lipid inversion enzyme regulators exert their transport activity, the fluctuation peaks of the protein and propiconazole / PI4P at other positions are almost consistent. RMSD analysis of AtALA3-ALIS1 and propiconazole / PI4P ( Figure 2 (g) It can be seen that the RMSD value of the PI4P molecule is the highest (0.2-0.8 nm), with a sharp peak (0.8 nm) around 30 ns, suggesting that the conformation of PI4P has undergone significant change or movement, which is related to its role as an allosteric activator in altering protein function. The RMSD of the PS molecule is the second highest (0.2-0.6 nm), while the RMSD of the PCZ molecule is the lowest overall (0.1-0.4 nm). This reflects the stable binding mode of propiconazole as a competitive substrate in proteins. The RMSF of the main chain atoms of the AtALA3-ALIS1 protein ( Figure 2h) The overall fluctuation range of the three systems in the distribution is 0.5-2 nm, with most residues having RMSF values ​​below 1 nm, indicating a relatively stable region. Some spikes correspond to more flexible loop regions or N / C terminal regions. Notably, the RMSF values ​​of certain residues in the PS complex (e.g., 50-100, 1300-1400) are slightly higher than those in the other two complexes, suggesting that these regions exhibit greater flexibility in binding PS substrates, possibly related to conformational changes during substrate transport. Molecular dynamics simulations of *Strombus haemolyticus* proteins extracted RMSD, RMSF, Gyrate, and SASA. It can be seen that the structures of the four triazole fungicide molecules stabilized after 50 ns of simulation. The binding of different substrate molecules significantly affected protein surface properties and potential interaction interfaces (see [link to simulation]). Figure 8 In summary, the compounds bind stably to protein complexes through hydrophobic and van der Waals interactions, inducing co-adaptive changes in protein conformation, which may be the structural basis for their biological activity.

[0051] Example 3: Surface plasmon resonance (SPR) affinity analysis of protein and propiconazole Five target membrane protein dimers expressed in vitro—MoAPT5-Cdc50 (MG), AtALA3-AtALIS1, and BraALA3a / b / c-BraALIS2—were extracted and purified. GFP was added as a control protein to prepare working solutions with concentration gradients (10 nM, 40 nM, 160 nM, 640 nM, 2560 nM). These solutions were then passed through a chip immobilized with propiconazole. The proteins bound to the chip were regenerated and dissociated using a regeneration solution. The regeneration was good, indicating that propiconazole and the tested proteins did not bind irreversibly, conforming to the Langmuir binding model. The interaction kinetics were fitted, and affinity parameters were output. Interaction analysis was performed using a Berthold bScreen LB 991. The data were fitted to a 1:1 Langmuir binding model, and the binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (kD) were calculated. The results are shown in Table 1 below. Figure 3 and Figure 9 .

[0052] Table 1. SPR affinity of propiconazole with various phospholipid invertase proteins

[0053] The results showed that: First, compared with the control DMSO, the SPR binding signals of propiconazole with its homologous proteins MoAPT5, AtALA3, and BraALA3a / b / c increased with increasing protein concentration (10 nM-2560 nM), forming concentration gradient-specific binding kinetic curves. The binding curves of each homologous membrane protein with propiconazole showed that the affinity signal increased with increasing protein concentration, with the strongest binding signal at a protein concentration of 2560 nM. Second, by plotting the specific binding kinetics of five target proteins (MoAPT5, AtALA3, BraALA3a / b / c) with propiconazole at a concentration of 2560 nM and calculating the maximum binding signal, the results showed that MoAPT5, AtALA3, BraALA3a, and BraALA3c proteins have a strong affinity for propiconazole, with binding strengths exceeding 1500 RU. BraALA3b protein has a moderate affinity for propiconazole, with an RU value between 1000 and 1500. Other negative controls showed no affinity. Figure 3 , Figure 9 Third, the values ​​of ka, kd, and kD were calculated. The results showed that the SPR equilibrium dissociation constants (Avg KD) of MoAPT5, AtALA3, BraALA3a, and BraALA3c proteins with propiconazole were 3.63 × 10⁻⁷ M, 2.04 × 10⁻⁸ M, 6.14 × 10⁻⁸ M, and 1.40 × 10⁻⁷ M, respectively, indicating strong binding affinity. The equilibrium dissociation constant of BraALA3b protein with propiconazole was 1.05 × 10⁻⁵ M, indicating moderate binding affinity. The other combinations showed no binding affinity (Table 1). In summary, this indicates that the in vitro expressed phospholipid invertase proteins have an affinity for propiconazole, and the strength of the affinity varies.

[0054] Example 4: In vitro ATPase activity assay of protein The bioactivity of the obtained protein complex was verified by measuring in vitro ATPase activity, and potential P4-ATPase activators or inhibitors were tested. Using a malachite green phosphate assay kit, the phosphate group after ATP hydrolysis was colorimetrically reacted in the presence or absence of phosphatidylserine (POPS), and the absorbance was measured at 620 nm. Each experimental condition was independently repeated three times, and the assay was calibrated using standard samples. The ATPase activity of the protein complex under different conditions could then be quantified by calculation.

[0055] The results showed that BeF3- is a known P-type ATPase inhibitor. As a negative control, the addition of 1 mM BeF3- to the reaction system reduced the ATPase activities of MoAPT5, AtALA3, and BraALA3a / b / c to extremely low levels. Compared with the positive control without inhibitors, propiconazole showed an inhibition rate of 38.87% on AtALA3 and 35.75%, 38.52%, and 32.78% on BraALA3a / b / c, respectively. In the presence of lipid phosphatidylserine (POPS), the ATPase activities of each ALA3 homolog significantly increased, indicating that POPS promoted phospholipid inversion enzyme activity. In this case, propiconazole showed an inhibition rate of 55.66% on AtALA3 and 48.45%, 52.56%, and 45.27% on BraALA3a / b / c, respectively. Figure 4 In addition, triazole compounds DCZ, ECZ, FSZ, and PCZ also exhibit the same inhibitory effect on MoAPT5. Figure 10 ).

[0056] Example 5: Statistical analysis of phenotypic indices of gene-edited plants' sensitivity to propiconazole We constructed knockdown and overexpression lines of the BraALA3a / b / c gene in Chinese cabbage. T2 generation plants of wild-type Chinese cabbage and the BraALA3a / b / c gene knockdown and overexpression lines were cultivated. At the S3 stage, the plants were treated with 50 mg / L propiconazole, and the plant phenotype was analyzed after 7 days. We also cultivated mutant lines of different T-DNA insertion types in Arabidopsis thaliana, atala3-p, atala3-l, and atala3-4, and potted them. We photographed and recorded the petiole length, leaf length, leaf width, and spread of the plants at 28 days, as well as the length of mature pods. For tissue sectioning, we embedded samples using an embedding kit and DMP-30, polymerized them at 60℃, and trimmed them to appropriate sizes after cooling to the appropriate hardness. We then sectioned the embedded plant tissues using a Leica RM2235 manual microtome, with section thicknesses of 5-10 micrometers. Finally, we observed the sections stained with toluidine blue under a microscope.

[0057] The results showed that plant growth was significantly inhibited after BraALA3 gene knockdown. Figure 5 a) Plant height was significantly reduced, main stem diameter was significantly increased, and branching angle was significantly reduced ( Figure 5 The presence of bd indicates that BraALA3KD has reduced sensitivity to propiconazole and increased tolerance. BraALA3 overexpression promotes the growth of Chinese cabbage plants (…). Figure 5 e), the plant height of BraALA3-OX was significantly increased ( Figure 5 f), the diameter of the main stem is significantly reduced ( Figure 5 g), the branch angle increases ( Figure 5 h), indicating that BraALA3a-OX plants have increased sensitivity to propiconazole and decreased tolerance. In Arabidopsis, compared to wild-type WT, mutants all exhibited a more compact plant type, with significantly reduced petiole length, leaf length, and leaf width. Compared to the wild-type treated with PCZ, atala3-p showed no significant difference in plant type, more closely resembling the growth-inhibiting effect of PCZ application, while atala3-4 showed more severe growth defects, and atala3-l showed differences in petiole length and leaf width ( Figure 6 ab). PCZ treatment and the atala3 mutant delayed bolting, resulting in a significant reduction in bolting height on day 9. Compared to wild-type treatment, the atala3 mutant treatment group showed a lower bolting height inhibition rate ( Figure 6 c) indicates that the atala3 mutant has reduced sensitivity to PCZ. Histological results showed that, compared to the wild type, both the wild-type and the atala3 mutant exhibited reduced xylem and increased phloem in the stem base cross-section after treatment. No significant changes were observed in the phloem cells of the mutant after treatment. Figure 6 d). Regardless of whether it was wild-type or mutant, longitudinal sections of thin-walled cells after drug application were more compact, and both cell length and width were inhibited, but the inhibition rate of mutants was lower than that of wild-type. Figure 6 The results (ef) indicate that atala3 has reduced sensitivity to PCZ. In conclusion, both the phenotypic and microstructural changes in the Arabidopsis mutants demonstrate that reducing ALA3 expression decreases the sensitivity of Arabidopsis to PCZ.

[0058] Example 6: Phenotypic statistics of MoAPT5 gene mutants' sensitivity to propiconazole A gene knockout and replenishment vector for *Magnaporum oryzae* was constructed. Knockout and replenishment strains were obtained through protoplast transformation, resistance screening, and PCR identification. The antifungal activity of propiconazole against *Magnaporum oryzae* was determined using a mycelial growth inhibition method. *Magnaporum oryzae* was activated on PDA plates. After 7 days, fresh mycelial discs were punched along the periphery of the colonies. One mycelial disc was selected, mycelial side up, and inoculated into the center of a PDA plate containing a series of concentration gradients of the fungicide (0.5, 1, 2, 4, 6 μg / mL). An equal volume of DMSO was added as a control. Each plate was incubated in triplicate. After 10 days at 28℃, the colony diameter was measured. The mycelial growth inhibition rate (%) of the fungicide against *Magnaporum oryzae* at each concentration gradient was calculated based on the measurement results. The EC50 of the fungicide against *Magnaporum oryzae* was calculated using Graphpad software. The analytical results are shown in Table 2 below. Figure 11 .

[0059] The results showed that, in the blank control, the hyphal diameter of the △Moapt5 mutant was smaller than that of the wild-type A60 and the complement ( Figure 10This indicates that the growth rate of the mutant is slower than that of the wild type and complement. After inoculation with different concentrations of propiconazole, the mycelial growth of both the wild type and complement was significantly inhibited. After statistical analysis of the inhibition rate, it was found that the inhibitory effect of propiconazole on the mutant was lower than that on the wild type and complement. The effective inhibition concentration EC50 of propiconazole on mycelial growth was more than twice that of the wild type and complement (Table 2), indicating that knocking out the MoAPT5 gene affects the development of resistance to propiconazole by rice blast fungus.

[0060] Table 2. Effects of propiconazole on the inhibitory effect of rice blast fungus mycelial growth.

[0061] Example 7 Screening of P4-ATPase Inhibitors 1. Method (1) Virtual screening based on ALA3 protein pocket The ChemBridge database contains 710,000 commercial compounds for screening. Large-scale virtual screening based on the binding pocket of the target protein ALA 3 and propiconazole was performed using Vina-GPU-Rigid. A second round of flexible docking was conducted using QuickVina-Flexible, and 2D similarity searches were performed based on Morgan and Atomicker fingerprint molecular characterization methods (Tanimoto, Dice, Cosine). The resulting compounds were then screened using the RDKit descriptor (Cut-off: -0.4) to eliminate compounds with complex molecular structures and low ligand efficiency. Finally, high-precision screening was performed based on structural features and pharmacophores to obtain the structures of the target lead compounds.

[0062] (2) Determination of SPR affinity between ALA3 protein and lead compound The affinity of ALA3 protein for lead compounds A01, A15, and A22 was verified using SPR technology. The lead compounds were used as the stationary phase sample, and the AtALA3-AtALIS1 protein complex was used as the mobile phase sample; the specific steps were the same as in Example 3.

[0063] (3) Plate validation of the sensitivity of ALA3 expressing yeast strain to lead compound and triazole pesticide. Replenishment type drs2-BraALA3a / b / c and overexpression BY4741 -BraALA3a / b / c The yeast strain was used to verify its sensitivity to the lead compound and triazole pesticides, following the same method as in Example 6. The concentrations of the lead compound in the culture medium were set to 0.5, 1, and 10 µM. The concentrations of the triazole pesticides in the culture medium were set to 0.05, 0.1, and 0.5 µM.

[0064] (4) Phenotypic statistics of the inhibitory effects of lead compounds and triazole pesticides on the growth of Chinese cabbage The inhibitory effects of lead compounds and triazole pesticides on the growth of Chinese cabbage were verified through a tissue culture experiment. The planting method of tissue-cultured Chinese cabbage was the same as in 4.2.8. Seeds were sown in square petri dishes (13 cm × 13 cm) containing 1 / 2 MS medium. After normal culture for 2 days, seedlings with uniform germination were transferred to 1 / 2 MS medium containing DMSO (control), different concentrations of lead compounds, and different concentrations of triazole pesticides for further culture. After 4-5 days, the phenotype of tissue-cultured Chinese cabbage was photographed and recorded, and the hypocotyl length and root length were counted.

[0065] 2. Results (1) Virtual screening analysis based on ALA3 protein pocket To achieve green growth control, besides breeding new varieties through molecular design, compounds targeting specific proteins can be designed to replace propiconazole for growth control. Based on a virtual screening strategy using the ALA3 protein activity pocket and two-dimensional molecular fingerprint similarity, the ChemBridge database containing 710,000 compounds was screened using Vina, with propiconazole as the similarity screening molecule. This resulted in 1449 candidate ligands. Then, compounds with complex structures and low efficiency were filtered out based on ligand efficiency, yielding 197 lead compounds. Finally, high-precision screening, depending on structural features and pharmacophores, yielded 38 candidate lead compounds. Figure 12 ).

[0066] The highest docking energy of these compounds with ALA3 was -10.87 kcal / mol, and the lowest was -7.96 kcal / mol. Among them, A01-A14, A17, A20, A21, A23, A25, A28, A30, A32, A34, and A36 were classified as amides, A15, A18, and A33 as sulfonamides, and the others as heterocyclic compounds. Figure 13 Further, compounds A01, A15, and A22, which had the highest docking scores among these three categories, were selected for verification.

[0067] (2) SPR affinity analysis of ALA3 protein and lead compound The affinity of lead compounds A01, A15, and A22 for ALA3 protein was determined by SPR. The results showed that the SPR binding signal of AtALA3 protein to the three lead compounds increased with increasing concentration from 10 nM to 2560 nM, forming interaction kinetic curves. Figure 14 The SPR equilibrium dissociation constants of AtALA3 with A01, A15, and A22 are 5.11 × 10⁻⁶. -9 M, 1.34×10 -8 M and 1.18×10 -7M, all showed strong binding affinity (Table 3). The results indicated that the three lead compounds A01, A15, and A22 selected in the virtual screening all exhibited strong affinity for the ALA3 membrane protein, with A01 showing the strongest affinity, followed by A15, and A22 showing slightly weaker affinity. This is consistent with the docking binding energy fraction ranking in the virtual screening. Figure 14 Furthermore, the SPR equilibrium dissociation constants of A01 and A15 with AtALA3 protein are lower than those of propiconazole, indicating that A01 and A15 have a stronger affinity for AtALA3 protein than propiconazole.

[0068] Table 3. SPR affinity of virtual screening lead compounds to AtALA3 protein

[0069] (3) ALA3 Sensitivity analysis of yeast strains expressing lead compounds Complements and overexpressing yeast strains containing the ALA3 homolog were inoculated onto SD-Gal-3aa medium containing different concentrations of propiconazole, A01, A15, and A22 to observe sensitivity. Results showed that, compared to yeast transfected with the pYES2 empty vector, ALA3 expressed [a higher level of expression]. AtALA3 and BraALA3a / b / c The yeast strains showed significantly inhibited growth on media containing propiconazole, A01, A15, and A22. Compared to the mutants, the overexpression strains were more significantly inhibited, indicating that at the same compound concentration, the growth of the yeast strains was significantly inhibited. ALA3 Higher expression levels correlated with greater sensitivity to propiconazole. Compared to propiconazole, lead compounds A01, A15, and A22 showed slightly better tolerance at the same concentration, indirectly confirming that the lead compounds may have lower toxicity. Compared to homologous genes, expression levels of… BraALA3a The yeast strains were more significantly inhibited ( Figure 15 and Figure 16 ).In summary, DRS2 The deletion caused resistance in yeast strains to propiconazole and three lead compounds, while expression of [the specific compound] resulted in resistance to propiconazole and three lead compounds. AtALA3 and BraALA3a / b / c The strain regained sensitivity to propiconazole and three lead compounds, and overexpressed... ALA3 The strains are more sensitive, and the homologous genes of Chinese cabbage are... BraALA3a The above results indicate that the sensitivity is higher. ALA3 Sensitive to three lead compounds selected by virtual screening.

[0070] (4) Analysis of the inhibitory effect of the lead compound on the growth of Chinese cabbage Young shoots of Chinese flowering cabbage were cultured on half MS medium containing different concentrations of PCZ, A01, A15, and A22. Phenotypic observation showed that, compared with DMSO, the three lead compounds had a certain inhibitory effect on shoot and root growth in the young shoots, but the inhibitory effect was relatively weaker than that of propiconazole. Among the lead compounds, A01 had the strongest inhibitory effect, followed by A15, and A22 was slightly weaker. Figure 17 This is related to SPR affinity (Table 3) and virtual screening docking score ( Figure 13 This is consistent with the conclusion.

Claims

1. The use of P4-ATPase inhibitors in the prevention and control of plant fungal diseases and / or the regulation of plant growth, or in the preparation of products for the prevention and control of plant fungal diseases and / or the regulation of plant growth, characterized in that, The P4-ATPase inhibitor is selected from triazole compounds, amide compounds, sulfonamide compounds, or heterocyclic compounds.

2. The application according to claim 1, characterized in that, The triazole compounds are selected from propiconazole, difenoconazole, flusilazole, fluconazole, paclobutrazol, or uniconazole.

3. The application according to claim 1, characterized in that, The amide compounds are selected from A01 to A14, A17, A20, A21, A23, A25, A28, A30, A32, A34, or A36. 。 4. The application according to claim 1, characterized in that, The sulfonamide compound is selected from A15, A18, or A33: 。 5. The application according to claim 1, characterized in that, The heterocyclic compound is selected from A16, A19, A22, A24, A26, A27, A29, A31, A35, A37 or A38: 。 6. The application according to any one of claims 1 to 5, characterized in that, The product works by specifically binding to P4-ATPase and inhibiting the lipid flipping function of P4-ATPase.

7. The application according to any one of claims 1 to 5, characterized in that, The P4-ATPase is derived from fungi or plants.

8. Application of reagents that knock out the P4-ATPase encoding gene of plant pathogenic fungi in the prevention and control of plant fungal diseases.

9. The application according to claim 8, characterized in that, The reagent inhibits the growth of plant pathogenic fungi by targeting and knocking out the P4-ATPase encoding gene in plant pathogenic fungi, thereby inhibiting the expression and / or activity of P4-ATPase.

10. The application according to claim 8, characterized in that, The plant pathogenic fungus is rice blast fungus, and the plant fungal disease is rice blast.

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