A small molecule compound capable of inducing plant immune response and improving plant disease resistance

Abstract

The present application provides a small molecule compound capable of inducing plant immune response and improving the disease resistance of plants. Specifically, it is found for the first time that 2'cADPR or its analogues represented by formula I, or a pharmaceutically acceptable salt thereof can significantly enhance the resistance of plants to pathogenic microorganisms, pests and viruses.

Description

Technical Field

[0001] This invention relates to the field of agronomy, and more specifically to a small molecule compound that can induce plant immune responses and enhance plant disease resistance. Background Technology

[0002] Photosynthesis in green plants provides most of the energy for life on Earth, with rice and wheat being essential food crops for human survival. In agricultural production, plant diseases such as rice blast caused by *Magnaporthe grisea*, rice bacterial blight caused by *Xanthomonas oryzae*, wheat blast caused by *Magnaporthe oryzae*, and wheat scab caused by *Fusarium graminearum* seriously threaten my country's food security. Currently, plant disease control mainly relies on the external application of fungicides to inhibit the growth of pathogenic microorganisms. However, the emergence of drug-resistant pathogens has gradually weakened the effectiveness of fungicides, and the long-term use of chemical fungicides has also brought challenges to land management and food safety.

[0003] In crop production, Fusarium head blight (FHB), caused by the facultative parasitic fungus *Fusarium graminearum*, is one of the major diseases threatening wheat production in my country. On the one hand, infection with *Fusarium graminearum* during the heading and flowering stages causes disease in the wheat ears, leading to a decrease in wheat yield and grain quality. On the other hand, *Fusarium graminearum* can secrete fungal toxins such as deoxynvalenol (DON, also known as vomitoxin) and zearalenone (ZEN) into wheat grains, seriously threatening the safety and quality of wheat grains. Furthermore, *Fusarium graminearum* can also infect corn stalks, causing stalk rot after multiplication, resulting in corn stalk rot.

[0004] Over long periods of evolution, plants have developed various mechanisms to defend against external pathogens, primarily including pattern-triggered immunity (PTI) mediated by cell surface pattern recognition receptors (PRRs) and effector-triggered immunity (ETI) mediated by intracellular nucleotide-binding domain leucine-rich-repeat (NB-LRR) containing receptors (NLRs). Plant immunity research and disease-resistant breeding have shown that screening and identifying disease-resistant proteins and activating immune signaling pathways through exogenous application of immune inducers can enhance plant resistance to diseases. To reduce the risks associated with chemical fungicides, inducing plant immune responses using exogenous sugars, proteins, and small molecules to resist plant diseases has become a safe and effective method.

[0005] Plant disease prevention mainly relies on pesticides, but large-scale pesticide use poses a significant threat to the ecosystem. Furthermore, the emergence of drug-resistant pathogens makes plant disease prevention even more challenging.

[0006] In recent decades, significant progress has been made in research on wheat scab resistance genetics and breeding, host resistance, pathogen growth and reproduction mechanisms, agricultural occurrence and development patterns of scab, and control technologies. However, a series of challenges remain: breeding is constrained by the scarcity of highly resistant wheat germplasm resources and the lack of susceptible varieties. Furthermore, wheat resistance to scab controlled by quantitative trait loci limits its application in breeding. The lack of synergy between high yield and high resistance in agronomic traits hinders the promotion and agricultural application of resistant varieties. Regarding scab control, a series of highly effective and low-risk chemical insecticides—carbendazim, cyazofamid, and fluopyram—have been applied. Carbendazim (and other benzimidazole fungicides) has been a major chemical pesticide for scab control in my country since its industrialization in the last century. Its broad-spectrum, high-efficiency, and systemic action provides excellent control, and for a period, it solved the environmental pollution problem associated with protective fungicides. However, long-term use has led to drug resistance in pathogens. These resistant strains exhibit resistance to carbendazim and also possess stronger pathogenicity and toxin-producing capabilities. Studies have shown that the detection rate of these resistant strains has been steadily increasing over the past decade. This situation presents a greater challenge to the control of Fusarium head blight.

[0007] Therefore, there is an urgent need in this field to develop a small molecule compound that can induce plant immune responses and enhance plant disease resistance. Summary of the Invention

[0008] The purpose of this invention is to provide a small molecule compound that can induce plant immune responses and enhance plant disease resistance, and to apply this compound to the prevention and control of crop diseases.

[0009] The first aspect of this invention provides that 2'cADPR and its analogues can induce plant immune responses and enhance plant resistance to pathogenic microorganisms, pests, and viruses, and also provides the concentration and method of application of 2'cADPR. Simultaneously, this invention also applies this compound to the control of wheat scab and maize stalk rot.

[0010] The first aspect of this invention provides the use of 2'cADPR or its analogues, or pharmaceutically acceptable salts thereof, for the preparation of a composition or formulation for enhancing plant resistance to pathogenic microorganisms, pests, and viruses. This composition or formulation can improve wheat resistance to Fusarium head blight and maize resistance to stalk rot. The structure of the 2'cADPR or its analogues, or pharmaceutically acceptable salts thereof, is shown in Formula I:

[0011]

[0012] In the formula,

[0013] R1, R2, and R3 are each independently selected from -OH, halogen, C1-C3 alkyl, C2-C3 alkenyl, and C2-C3 alkynyl.

[0014] R4 is selected from -R a (R b N-, phosphate groups, -OH, =O, halogens, where R a and R b Each of the following is independently H, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C2-C6 alkenyl, a substituted or unsubstituted C2-C6 alkynyl, or a C1-C6 haloalkyl, wherein the substitution is having one or more substituents selected from the group consisting of -NH2, halogen, -OH, -CN, and nitro.

[0015] R5 is selected from the following group: H, halogens;

[0016] R6 is selected from the following group: H, -OH, halogens, R d (R e )N-、-XR c , where R d and R e Each can be independently H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or C1-C6 haloalkyl;

[0017] X is selected from -O-, -S-, R cSelected from hydrogen, halogen, cyano, nitro, C 1- C6 alkyl, C 3- C6 cycloalkyl, C 2- C6 alkenyl, C 2- C6 ynyl group, C 6-8 The aryl group may be further replaced by one or more substituents selected from halogens.

[0018] In another preferred embodiment, R1, R2, and R3 are each independently selected from -OH.

[0019] In another preferred embodiment, R4 is selected from -R a (R b N-, phosphate groups, -OH, =O, halogens, where R a and R b Each of the following is independently H, a substituted or unsubstituted C1-C4 alkyl, a substituted or unsubstituted C2-C4 alkenyl, a substituted or unsubstituted C2-C4 alkynyl, or a C1-C4 haloalkyl, wherein the substitution is having one or more substituents selected from the group consisting of -NH2, halogen, -OH, -CN, and nitro.

[0020] In another preferred embodiment, R6 is selected from the group consisting of: H, -OH, halogens, R d (R e )N-、-XR c , where R d and R e Each is independently H, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl or C1-C3 haloalkyl;

[0021] X is selected from -S-, R c Selected from C 1- C5 alkyl, C 4- C6 cycloalkyl, C 2- C5-olefin, C 2- C5 alkynyl, C6 aryl, the above groups may optionally be further replaced by one or more substituents selected from halogens.

[0022] In another preferred embodiment, the halogen includes F, Cl, Br, or I.

[0023] In another preferred embodiment, the halogen is Br or Cl.

[0024] In another preferred embodiment, the structure of the 2'cADPR or its analogues, or its pharmaceutically acceptable salts, is as follows:

[0025]

[0026]

[0027] In another preferred embodiment, the 2'cADPR or its analogues, or its pharmaceutically acceptable salts, are chemically synthesized, biosynthesized, or purified or derived from biofermentation or are initial products.

[0028] In another preferred embodiment, the plants include dicotyledonous plants and monocotyledonous plants.

[0029] In another preferred embodiment, the plants include Arabidopsis thaliana, tobacco, rice, wheat, corn, potato, soybean, citrus, grape, and kiwifruit, as well as different varieties of these plants.

[0030] In another preferred embodiment, the pathogenic microorganisms include *Pseudomonas syringaepv. tomato*, *Xanthomonas campestris*, *Magnaporthegrisea*, *Xanthomonas oryzae*, *Magnaporthe oryzae*, *Fusarium graminearum*, *Phytophthora festans*, *Liberbacter asianticum*, *Physodermamaydis*, *Fusarium verticillioides*, *Bipolarisnzeicol*, *Fusarium proliferatum*, *Cercospora zeae-maydis*, and *Cercospora maize*. The fungi include *Physoderma maydis*, *Exerohilum turcicum*, *Cochliobolus heterostrophus*, *Pythium aphanidermatum*, *Aspergillus flavus*, *Penicillium spp.*, *Sclerotinia sclerotiorum (Lib.) de Bary*, *Plasmodiophorabrassica*, and *Plasmopara viticola*.

[0031] In another preferred embodiment, the pests and diseases include: rice blast, rice bacterial blight, rice leaf streak / stripe rust, wheat blast, wheat scab, corn stalk rot, corn stalk base rot, corn large leaf spot, corn small leaf spot, corn leaf spot of varying sizes, corn gray spot, corn brown spot, corn bacterial wilt, corn ear rot, corn rough dwarf disease, potato late blight, soybean scab, citrus Huanglongbing, kiwi root rot, grape downy mildew, rapeseed sclerotinia rot, and rapeseed clubroot.

[0032] In another preferred embodiment, the viruses include: tobacco mosaic satellite virus, millet mosaic satellite virus, aumaivirus, citrus roughness virus, rice dwarf virus, rice black-streaked dwarf virus (RBSDV), and soybean stay-green-associated virus (SoSGV).

[0033] In another preferred embodiment, the composition or formulation is capable of inducing immune responses in different types of plants, thereby conferring resistance to pathogenic microorganisms, pests, and viruses.

[0034] In another preferred embodiment, the composition or formulation is capable of inducing an immune response in plants with the EDS-PAD4-ADR1 signaling pathway, thereby conferring resistance to pathogens, pests, and viruses.

[0035] In another preferred embodiment, the composition or formulation enhances the plant's resistance to pathogens, pests, and viruses by inducing an EDS1-PAD4-ADR1-dependent immune response in the plant.

[0036] In another preferred embodiment, the composition comprises an agricultural composition.

[0037] In another preferred embodiment, the composition contains (i) 2'cADPR or an analogue of Formula I, or a pharmaceutically acceptable salt thereof; and (ii) an agronomically acceptable carrier.

[0038] In another preferred embodiment, the component (i) accounts for 0.001-99.9 wt% of the total weight of the composition, more preferably 0.1-99 wt%, and even more preferably 1%-90 wt%.

[0039] In another preferred embodiment, the formulation includes an agricultural formulation.

[0040] In another preferred embodiment, the dosage form of the formulation is selected from the group consisting of: solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, natural and synthetic materials impregnated with active substances, microcapsules in polymers, seed coatings, or combinations thereof.

[0041] In another preferred embodiment, the composition or formulation further includes other substances for enhancing the plant’s resistance to pathogenic microorganisms, pests and viruses.

[0042] In another preferred embodiment, the other substances used to enhance the plant’s resistance to pathogens, pests and viruses include benzimidazole fungicides, carbendazim, amino oligosaccharides and plant endophytic bacteria fermentation extract ZNC.

[0043] A second aspect of the present invention provides a composition or formulation comprising:

[0044] (a) 2'cADPR or its analogues, or pharmaceutically acceptable salts thereof, wherein the structure of said 2'cADPR or its analogues, or pharmaceutically acceptable salts thereof, is shown in Formula I:

[0045]

[0046] (b) An agriculturally acceptable carrier.

[0047] In another preferred embodiment, the composition comprises an agricultural composition.

[0048] 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, encapsulating agents for other agricultural substances, or combinations thereof.

[0049] In another preferred embodiment, the composition or formulation contains 0.0001-99 wt%, preferably 0.1-90 wt%, of component (a) based on the total weight of the composition or formulation.

[0050] In another preferred embodiment, the content of component (a) in the composition or formulation is 5-200 μM, more preferably 10-100 μM, more preferably 30-80 μM, and even more preferably 40-60 μM.

[0051] In another preferred embodiment, the formulation includes an agricultural formulation.

[0052] In another preferred embodiment, the composition or formulation further includes other substances for enhancing the plant’s resistance to pathogenic microorganisms, pests and viruses.

[0053] In another preferred embodiment, the other substances used to enhance the plant’s resistance to pathogens, pests and viruses include benzimidazole fungicides, carbendazim, amino oligosaccharides and plant endophytic bacteria fermentation extract ZNC.

[0054] In another preferred embodiment, the content of component (a) in the formulation is 0.0001-99 wt%, more preferably 0.1-90 wt%, and even more preferably 1-90 wt%.

[0055] A third aspect of the present invention provides the use of the composition or preparation described in the second aspect of the present invention for enhancing the resistance of plants to pathogenic microorganisms, pests and viruses.

[0056] A fourth aspect of the present invention provides a method for enhancing the resistance of plants to pathogenic microorganisms, pests, and viruses, comprising the steps of:

[0057] The plant may be administered 2'cADPR of Formula I or an analogue thereof, or a pharmaceutically acceptable salt thereof, or the composition or formulation described in the second aspect of the invention.

[0058] In another preferred embodiment, the application includes spraying, irrigating, drip irrigation, sprinkler irrigation, spraying, smearing, soaking, or local injection.

[0059] In another preferred embodiment, the applied dose is 2-100 mM / plant, more preferably 8-80 mM / plant, and even more preferably 10-50 mM / plant.

[0060] In another preferred embodiment, the applied dose is 5-200 μM, more preferably 10-100 μM, even more preferably 30-80 μM, and still more preferably 40-60 μM.

[0061] The fifth aspect of this invention provides 2'cADPR and its formulations for the control of wheat scab and maize stalk rot, comprising the following steps:

[0062] The plant may be administered 2'cADPR of Formula I or an analogue thereof, or a pharmaceutically acceptable salt thereof, or the composition or formulation described in the second aspect of the invention.

[0063] In another preferred embodiment, the application includes dripping, spraying, irrigating, drip irrigation, sprinkler irrigation, spraying, smearing, soaking, or local injection.

[0064] In another preferred embodiment, the applied dose is 0.001-10 mM / plant, more preferably 0.01-8 mM / plant, and even more preferably 0.05-5 mM / plant.

[0065] In another preferred embodiment, the applied dose is 5-200 μM, more preferably 10-100 μM, even more preferably 30-80 μM, and still more preferably 40-60 μM.

[0066] 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

[0067] Figure 1 The structural formulas of the small molecules 3'cADPR, 2'cADPR and pRib-AMP are shown.

[0068] Figure 2 The conversion of 2'cADPR to pRib-AMP is shown.

[0069] Figure 3 It was shown that 2'cADPR induces PR1 expression and is dependent on EDS1-PAD4-ADR1.

[0070] Figure 4 The study showed that 2'cADPR induces an EDS1-PAD4-ADR1-dependent immune response.

[0071] Figure 5 GO analysis showed that 2'cADPR induced a plant immune response.

[0072] Figure 6 Transcriptome analysis showed that the 2'cADPR-induced immune response was dependent on EDS1-PAD4-ADR1.

[0073] Figure 7 The study showed that 2'cADPR induced resistance in Arabidopsis thaliana to Pseudomonas syringae DC3000 (D36E).

[0074] Figure 8 The results show that 2'cADPR induces resistance in wheat coleoptiles against Fusarium graminearum infection and lesion formation. In the image, A is a photograph taken 5 days after incubation with the 2'cADPR formulation, and B is a statistical result of lesion length in image A.

[0075] Figure 9 This study demonstrates the resistance induced by 2'cADPR in the ear of wheat (Bobwhite variety) to Fusarium head blight caused by Fusarium graminearum. In the image, A shows a photograph taken 14 days after inoculation with the 2'cADPR formulation, and B shows the statistical results of disease incidence in A.

[0076] Figure 10This study demonstrates the resistance induced in the spike of another wheat variety (Filder) by 2'cADPR to Fusarium head blight caused by Fusarium graminearum. In the figure, A is a photograph taken 14 days after inoculation with the 2'cADPR formulation, and B is a statistical chart showing the disease incidence in variety A.

[0077] Figure 11 The results show that 2'cADPR improves maize's resistance to stalk rot. In this diagram, A is a photograph taken 14 days after inoculation with the 2'cADPR formulation, and B is a statistical chart showing the disease incidence at point A. Detailed Implementation

[0078] Through extensive and in-depth research, the inventors unexpectedly discovered that 2'cADPR or its analogues, or pharmaceutically acceptable salts thereof, having the structure of Formula I, can enhance plant resistance to pathogenic microorganisms, pests, and viruses. Based on this discovery, the inventors completed this invention.

[0079] Group definition

[0080] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, or iodine, preferably chlorine and bromine.

[0081] As used herein, the term “substituted or unsubstituted” means that the group may be unsubstituted, or that the H in the group is substituted by one or more (e.g., 1-10, preferably 1-5, more preferably 1-3, most preferably 1-2) substituents.

[0082] As used herein, “substituted” or “substituted” means that the group has one or more (preferably 1-6, more preferably 1-3) substituents selected from the group consisting of -NH2, halogen, -OH, -CN, and nitro.

[0083] As used herein, the terms "C1-C6 alkyl" or "C1-C5 alkyl" refer to straight-chain or branched alkyl groups having 1-6 or 1-5 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C1-C6 alkyl or C1-C5 alkyl groups.

[0084] As used herein, the term "C1-C4 alkyl" refers to a straight-chain or branched alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C1-C4 alkyl groups, and so on.

[0085] As used herein, the term "C2-C6 alkenyl" refers to a straight-chain or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C2-C6 alkenyl chains, and so on.

[0086] As used herein, the term "C2-C6 alkenyl" refers to a straight-chain or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C2-C6 alkenyl groups, and so on.

[0087] As used herein, the term "C2-C4 alkenyl" refers to a straight-chain or branched alkenyl group having 2 to 4 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C2-C4 alkenyl groups, and so on.

[0088] As used herein, the term "C2-C6 ynyl" refers to a straight-chain or branched ynyl group having 2 to 6 carbon atoms, such as ethynyl, propynyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C2-C6 ynyl groups, and so on.

[0089] As used herein, the term "C2-C4 ynyl" refers to a straight-chain or branched ynyl group having 2 to 6 carbon atoms, such as ethynyl, propynyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C2-C4 ynyl groups, and so on.

[0090] As used herein, the term "C1-C3 alkyl" refers to a straight-chain or branched alkyl group having 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C1-C3 alkyl groups.

[0091] As used herein, the term "C2-C3 alkenyl" refers to a straight-chain or branched alkenyl group having 2-3 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C2-C3 alkenyl chains.

[0092] As used herein, the term "C2-C3 ynyl" refers to a straight-chain or branched ynyl group having 2-3 carbon atoms, such as ethynyl, propynyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C2-C3 chain ynyl groups.

[0093] As used herein, the terms “C3-C6 cycloalkyl” or “C4-C6 cycloalkyl” refer to cyclic alkyl groups having 3-6 or 4-6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C3-C6 cycloalkyl or C4-C6 cycloalkyl groups, and so on.

[0094] As used herein, the term "C1-C6 haloalkyl" refers to a straight-chain or branched alkyl group having 1-6 carbon atoms, in which one or more hydrogen atoms are substituted with a halogen, such as halomethyl, haloethyl, halopropyl, haloisopropyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C1-C6 haloalkyl groups, and so on.

[0095] As used herein, the term "C1-C4 haloalkyl" refers to a straight-chain or branched alkyl group having 1-4 carbon atoms in which one or more hydrogen atoms are substituted, such as halomethyl, haloethyl, halopropyl, haloisopropyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C1-C4 haloalkyl groups, and so on.

[0096] As used herein, the term "C1-C3 haloalkyl" refers to a straight-chain or branched alkyl group having 1-3 carbon atoms in which one or more hydrogen atoms are substituted, such as halomethyl, haloethyl, halopropyl, haloisopropyl, or similar groups. Unless otherwise specified, the term includes substituted or unsubstituted C1-C3 haloalkyl groups, and so on.

[0097] As used herein, "aryl" refers to a monocyclic aromatic hydrocarbon ring, preferably a monocyclic aromatic hydrocarbon ring containing 6-8 carbons, for example, "C 6-8 "Aryl" refers to a fully carbon-based aryl group containing 6-8 carbons, including but not limited to phenyl.

[0098] As used herein, the term "halogenated" refers to a group substituted with one or more of the same or different halogen atoms described above, which may be partially or fully halogenated, such as trifluoromethyl, pentafluoroethyl, heptafluoroisopropyl, or similar groups.

[0099] In the compounds of the present invention, since the C and N atoms of the two groups R4 and R6 can be replaced by different groups, the structure shown in Formula I is used to generate various substituted products and derivatives that have similar effects on stimulating plant immune responses.

[0100] Active ingredients

[0101] As used herein, the terms “active ingredient of the invention”, “2'cADPR of the invention or its analogues or derivatives thereof, or pharmaceutically acceptable salts thereof”, and “compound of formula I of the invention” are used interchangeably and all refer to active ingredients that can enhance the resistance of plants to pathogenic microorganisms, pests and viruses.

[0102] In this invention, the compound of formula I can be synthesized chemically or biosynthesized, for example, by cleaving NAD through plant or bacterial TIR proteins. + NAD is produced, or added by strains expressing plant or bacterial TIR proteins. + Produced through fermentation, in this invention, the active ingredient has the general formula shown in Formula I:

[0103]

[0104] In the formula,

[0105] R1, R2, and R3 are each independently selected from -OH, halogen, C1-C3 alkyl, C2-C3 alkenyl, and C2-C3 alkynyl.

[0106] R4 is selected from -R a (R b N-, phosphate groups, -OH, =O, halogens, where R a and R b Each of the following is independently H, a substituted or unsubstituted C1-C6 alkyl, a substituted or unsubstituted C2-C6 alkenyl, a substituted or unsubstituted C2-C6 alkynyl, or a C1-C6 haloalkyl, wherein the substitution is having one or more substituents selected from the group consisting of -NH2, halogen, -OH, -CN, and nitro.

[0107] R5 is selected from the following group: H, halogens;

[0108] R6 is selected from the following group: H, -OH, halogens, R d (R e )N-、-XR c , where R d and R e Each can be independently H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or C1-C6 haloalkyl;

[0109] X is selected from -O-, -S-, R c Selected from hydrogen, halogen, cyano, nitro, C 1- C6 alkyl, C 3- C6 cycloalkyl, C 2- C6 alkenyl, C 2- C6 ynyl group, C 6-8The aryl group may be further replaced by one or more substituents selected from halogens.

[0110] In a preferred embodiment, the structure of the compound is as follows:

[0111]

[0112]

[0113] Improve plant traits

[0114] In this invention, a method for improving plant traits is also provided, specifically, by applying 2'cADPR or its analogues, or pharmaceutically acceptable salts thereof, as shown in Formula I of this invention, thereby improving plant traits, including enhanced plant resistance to pathogenic microorganisms, pests and diseases and viruses.

[0115] In a preferred embodiment of the present invention, in Arabidopsis thaliana, NLRs with an N-terminal TIR (N-terminal Toll / interleukin-1 receptor) domain can oligomerize themselves to form NAD+ domains after recognizing pathogen effector factors. + The disease-resistance body, with its lysin activity, produces the pRib-AMP / ADP (phosphoribosyl adenosine monophosphate / diphosphate) or cADPR (cyclic ADPR) isomers. pRib-AMP is unstable and only activates the EDS1-PAD4-ADR1 signaling module-mediated plant immune response upon binding to EDS1-PAD4. In tobacco, the EDS1-PAD4-ADR1 signaling module also plays an important role in plant immune responses, indicating that this signaling pathway is highly conserved across different plants. A recent study infected wild-type tobacco and eds1 (EDS1 mutant) tobacco with Tobacco mosaic virus (TMV) showed that the absence of EDS1 reduced the TMV virus content in tobacco leaves (Rao et al., 2024). Plant or bacterial TIR lysis of NAD... +The bacteria produce ADPR, cADPR, and cADPR isoforms. The cADPR isoform exists in two forms: 3'cADPR and 2'cADPR. Studies have shown that 3'cADPR, produced by the TIR protein ThsB, which constitutes the Thoeris system, acts as a signaling molecule to activate the bacterial antiviral immune response. Furthermore, the effector protein HopAM1, from *Pseudomonas syringae* pv. *tomato* DC3000 and containing a TIR domain, can also produce 3'cADPR and participate in the suppression of plant immune responses. The effector factor HopBY from *Pseudomonas syringae* can produce 2'cADPR and may also participate in the suppression of plant immune responses. The regulation and mechanisms of 2'cADPR and 3'cADPR in plant immune responses require further investigation.

[0116] This invention is the first to discover that plant and bacterial TIR lysis of NAD + The generated small molecule signaling substance 2'cAPDR can be converted into pRib-AMP, which is then bound to and activated by the EDS1-PAD4 complex. 2'cAPDR treatment can induce the expression of genes related to SA synthesis, response, and systemic acquisition resistance in plants, enhancing plant resistance and enabling them to better resist the invasion of external pathogens. Furthermore, this study added 2'cAPDR to wheat coleoptiles, improving the resistance of different wheat varieties and corn stalks to the pathogen *Fusarium graminearum*. The disease resistance induced by 2'cAPDR can be applied to the management of diseases in both dicotyledonous and monocotyledonous plants.

[0117] Furthermore, this invention first demonstrates that plant or bacterial TIR lysis of NAD + The generated 2'cADPR can be converted into the unstable pRib-AMP in vivo, which is bound to and activated by the EDS1-PAD4 complex. 2'cADPR treatment can induce the expression of EDS1-PAD4-ADR1-dependent immune-related genes in plants. 2'cADPR treatment in Arabidopsis thaliana improved its resistance to *Pseudomonas syringae* DC3000 (D36E). Treatment of wheat, wheat coleoptiles, and maize with 2'cADPR formulations improved these plants' resistance to *Fusarium graminearum*.

[0118] This invention also proposes a small molecule compound that can replace pesticides. In practical applications, 2'cADPR (structure see...) Figure 1) and its derivatives or analogues, such as iodination and bromination of the hydroxyl groups on ribose, substitution reaction products of amino or phosphate groups on adenosine, and substitutions of amino and carbon side chain groups on purine groups, can be used as small molecule immune inducers to directly treat plants, such as crops like rice, wheat, and corn, cash crops like potatoes, cotton, and soybeans, and horticultural plants like citrus and kiwifruit. These induce the activation of the plant's immune system, endowing the plant with resistance to various diseases and pests, such as rice blast, wheat scab, corn stalk rot, potato late blight, soybean phytosis, and kiwifruit root rot. In agricultural production, these small molecules can be combined with specific agricultural management methods to achieve disease resistance, such as field drip irrigation or sprinkler irrigation, drone spraying, or local injection.

[0119] Furthermore, this invention is the first to discover that the plant immune response mediated by the EDS-PAD4-ADR1 signaling pathway is highly conserved across different plant species. Currently reported examples include dicotyledonous tobacco, Arabidopsis thaliana, and monocotyledonous rice. These plants, whose immune responses are regulated by this signaling pathway, can enhance their resistance to pathogenic microorganisms, plant pests, and viruses through exogenous application of 2'cAPDR. Therefore, 2'cAPDR can serve as a chemical pesticide alternative and, in conjunction with different field management practices, be applied in various ways to control various pests and diseases in agricultural production.

[0120] Agricultural compositions or formulations

[0121] The active substances of the present invention (such as 2'cADPR or its analogues, or pharmaceutically acceptable salts thereof, as shown in Formula I) can be prepared into agricultural formulations, such as solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, natural and synthetic materials impregnated with the active substances, microcapsules in polymers, and seed coating agents, using conventional methods.

[0122] These formulations can be produced using known methods, such as 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] In this invention, the “agricultural formulation” is typically an agricultural plant growth regulator containing 2'cADPR or its analogues, or a pharmaceutically acceptable salt thereof, as an active ingredient that enhances the plant’s resistance to pathogenic microorganisms, pests and viruses; and an agriculturally acceptable carrier.

[0130] As used herein, an "agriculturally acceptable carrier" is an agriculturally acceptable solvent, suspending agent, or excipient for delivering 2'cADPR or its analogues, or pharmaceutically acceptable salts thereof, of Formula I 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.

[0131] The agricultural formulations of the present invention can be mixed with other substances that resist pathogenic microorganisms, pests and viruses in their commercial formulations or in formulations prepared from these formulations. These other substances that resist pathogenic microorganisms, pests and viruses include (but are not limited to): benzimidazole fungicides, carbendazim, amino oligosaccharides and plant endophytic bacteria fermentation extract ZNC.

[0132] Furthermore, the agricultural formulations of the present invention can also be mixed with synergists and exist in their commercial formulations or in application formulations prepared from these formulations. These synergists are compounds that enhance the activity of the active substance. Since the active substance itself is active, it is not necessary to add synergists.

[0133] The formulation of the agricultural preparation described in this invention can be of various types, as long as the active ingredient can be effectively delivered to the plant. From the perspective of ease of preparation and application, the preferred agricultural preparation is a spray or solution.

[0134] The agricultural formulations described in this invention typically contain 0.0001-99 wt%, preferably 0.1-90 wt%, of the active ingredient of this invention, accounting for 0.0001-99 wt% of the total weight of the agricultural formulation. The concentration of the active ingredient of this invention in commercial formulations or application formulations can vary within a wide range. The concentration of the compound of this invention in commercial formulations or application formulations can range from 0.0000001-100% (g / v), preferably between 0.0001 and 1% (g / v).

[0135] Methods to enhance plant resistance to bacteria, fungi and viruses

[0136] The present invention provides a method for enhancing the resistance of plants to pathogenic microorganisms, pests and / or viruses, comprising the steps of: administering 2'cADPR of Formula I or an analogue thereof, or a pharmaceutically acceptable salt thereof, or applying a corresponding agricultural composition or formulation.

[0137] Application can be performed using various known methods, such as by applying, spraying, misting, dusting, sowing, drip irrigation, sprinkler irrigation, foliar spraying, soaking, or locally injecting 2'cADPR or its analogues, or pharmaceutically acceptable salts thereof, or agricultural compositions or formulations containing 2'cADPR or its analogues, or pharmaceutically acceptable salts thereof, as shown in Formula I onto plant leaves or propagation material; or by otherwise exposing plants to 2'cADPR or its analogues, or pharmaceutically acceptable salts thereof, or agricultural formulations containing 2'cADPR or its analogues, or pharmaceutically acceptable salts thereof, as shown in Formula I; and, if seeds, by coating, wrapping, or otherwise treating the seeds. Another method of directly treating plants or seeds before planting is to introduce the agricultural compositions or formulations of the present invention into soil or other culture media containing the seeds to be sown. In some embodiments, a carrier may also be used, which may be solid or liquid as described above.

[0138] In a preferred embodiment, 2'cADPR of Formula I or its analogues, or pharmaceutically acceptable salts thereof, or agricultural compositions or formulations containing 2'cADPR of Formula I or its analogues, or pharmaceutically acceptable salts thereof, can also be delivered to the plants via field drip irrigation or sprinkler irrigation, drone spraying, or local injection.

[0139] The main advantages of this invention include:

[0140] 1. Through extensive screening, it was first discovered that 2'cADPR or its analogues, or pharmaceutically acceptable salts thereof, represented by Formula I, can significantly enhance plant resistance to pathogenic microorganisms, pests, and viruses.

[0141] 2. This invention is the first to discover that plant or bacterial TIR proteins cleave NAD. + The resulting stable small molecule compound can be converted into pRib-AMP, activating the function of EDS1-PAD4. Figure 1 ,2).

[0142] 3. This invention is the first to discover that 2'cADPR treatment can induce the expression of EDS1-PAD4-ADR1-dependent immune-related genes in plants, activating SA synthesis, signal transduction, and systemic acquired resistance in plants. Figure 6 Treatment of Arabidopsis thaliana with 2'cADPR improved its resistance to Pseudomonas syringae DC3000 (D36E). Figure 7 ). 2'cADPR treatment of wheat and maize improved their resistance to Fusarium graminearum and mitigated or reduced the occurrence of Fusarium head blight and maize stalk rot in different wheat varieties.

[0143] 4. This invention is the first to discover that 2'cADPR can act as an immune inducer to activate the immune response of all plants with the EDS-PAD4-ADR1 signaling pathway. By applying it exogenously, it can enhance the plant's ability to resist pathogenic microorganisms, plant pests and viruses, reduce or even replace the use of fungicides in agricultural disease control, and reduce the safety and ecological risks caused by the latter.

[0144] 5. This invention is the first to discover that 2'cAPDR can be converted into pRib-AMP to activate an EDS1-PAD4-ADR1-dependent immune response in plants, thereby enhancing plant resistance to pathogenic microorganisms. 2'cAPDR is stable and suitable for long-term storage. 2'cAPDR can be used as a chemical pesticide alternative and applied in agricultural production using various application methods in conjunction with different field management practices.

[0145] 6. This invention is the first to discover that 2'cADPR can be produced by plant or bacterial TIR, and can be prepared by in vitro reaction, or obtained by large-scale fermentation and purification using strains expressing TIR proteins.

[0146] 7. This invention is the first to discover that 2'cADPR can be used as an immune inducer to enhance plant disease resistance with broad spectrum, applicable to typical dicotyledonous plants such as Arabidopsis thaliana, monocotyledonous plants such as wheat and maize, and can be extended to all monocotyledonous and dicotyledonous plants.

[0147] 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 as 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 specified, all materials and reagents used in the embodiments are commercially available products.

[0148] Example 1: 2'cADPR conversion to form pRib-AMP

[0149] This invention demonstrates for the first time that 2'cADPR is converted into pRib-AMP in plants. Total protein was extracted from Arabidopsis leaves (plant tissue was ground into powder in liquid nitrogen and then added to the Arabidopsis total protein extract), and 100 μL of 50 μM 2'cADPR (purchased from BIOLOG Life Science Institute, Bremen, Germany) was added. After incubation at 4°C for 24 h, in vitro purified EDS-PAD4 protein (PDB code: 7XDD) was added, followed by NiA beads. After incubation at 4°C for 1 h, the NiA beads were collected and eluted to obtain EDS1-PAD4 protein. The protein was denatured in 80% methanol, and mass spectrometry analysis of the supernatant showed that EDS-PAD4 contained pRib-AMP, indicating that 2'cADPR can be converted into pRib-AMP. Figure 2 ).

[0150] (1) Total protein extract of Arabidopsis thaliana: 25mM Tris-HCl (pH=7.5), 150mM NaCl, 5mM DTT, 1mM MPMSF, 0.5% NP40, 1x Cocktail;

[0151] (2) Purification of EDS1-PAD4 protein: Using an insect baculovirus expression system, insect sf9 cells (sf9 cells, virus expression system vector purchased from Thermo Fisher Scientific, virus construction procedure referred to product instructions) were co-infected with constructed StrepII-tagged EDS1 and His-tagged PAD4 viruses. Cells were collected after 60 h. The cells were resuspended in 50 mM Hepes (pH=7.5), 150 mM NaCl, 1 mM DTT, and 1 mM PMSF, sonicated, centrifuged, and the supernatant was collected. NiA beads were added and incubated for 1 h. The beads were then collected and eluted to obtain the EDS-PAD4 protein complex.

[0152] (3) Mass spectrometry detection of small molecule compounds: Samples were analyzed using an ultra-high performance liquid chromatography (UHPLC) Dionex Ultimate 3000 RSLC (HPG) coupled with a Q Exactive Orbitrap high-resolution mass spectrometry system (Thermo Fisher Scientific) and a HESI ionization source in positive ion mode. The chromatographic column was an ACQUITY UPLC HSS T3, and the mobile phase was 2 mM ammonium acetate and methanol.

[0153] The results showed that 2'cADPR could be converted into pRib-AMP within plant cells ( Figure 1Mass spectrometry analysis revealed that pRib-AMP could be detected in the treated group after adding 2'cADPR, but not in the blank group. Figure 2 ).

[0154] Example 2: 2'cADPR induces an EDS1-PAD4-ADR1-dependent immune response in plants.

[0155] To test whether 2'cADPR can induce an immune response in plants, this invention injected 2'cADPR into Arabidopsis Col-0 leaves. Compared with 3'cADPR (3'cADPR was purified in the laboratory; purification method is described in the literature) and Mock treatment, 2'cADPR could induce the expression of the resistance gene PR1 (pathogenesis-related protein 1). Upregulation of PR1 is an important marker of activation of plant immune signaling pathways. In the Arabidopsis resistance signaling pathway mutants eds1-12 (EDS1 gene deletion mutant), pad4-1 (PAD4 gene deletion mutant), and adr1 triple (ADR1, ADR1-L1, and ADR1-L2 gene deletion mutant), 2'cADPR could not induce the expression of the pathogenesis-related protein PR1. This induction is still present in the Arabidopsis cell death pathway SGA101 gene deletion mutant sag101 and the Arabidopsis NRG1A / B gene deletion mutant nrg1a / 1b, meaning that 2'cADPR induction of PR1 depends on EDS1-PAD4-ADR1 (PDB code: 8ZW9). Figure 3 In addition, 2'cADPR can induce FRK1 ( Figure 4 A),PBS3( Figure 4 B), NPR1 Figure 4 C),ALD1( Figure 4 F),NIMIN1( Figure 4 G),SARD4( Figure 4 E) and FMO1 Figure 4 D) and other genes related to SA signaling and systemic acquired resistance, and this induction depends on EDS1-PAD4-ADR1 ( Figure 4 At the transcriptomic level, 2,593 genes were upregulated in Arabidopsis Col-0 leaves treated with 2'cADPR. GO enrichment results indicated that these genes were related to plant responses to microorganisms. However, almost none of these genes responded in pad4, suggesting that 2'cADPR induces an EDS1-PAD4-ADR1-dependent immune response. Figure 5-6 ).

[0156] Table 1 Primer List

[0157]

[0158]

[0159] (1) Arabidopsis leaf treatment and gene expression detection: dH2O, 2'cADPR (50 μM), and 3'cADPR (50 μM) were injected into the leaves of 4-5 week old Arabidopsis Col-0 plants. Samples were taken 6 h after treatment, RNA was extracted and reversed to cDNA, and the expression level of PR1 was detected (Table 1). 2'cADPR (50 μM) was injected into the leaves of 4-5 week old Arabidopsis Col-0, eds1-12, pad4-1, sag101, nrg1a / 1b, and adr1 triple. Samples were taken before and 6 h after treatment, RNA was extracted and reversed to detect the expression of candidate genes (Table 1).

[0160] (2) Transcriptome analysis: dH2O and 2'cADPR (50μM) were injected into 4-5 week old Arabidopsis thaliana Col-0,pad4-1. Samples were taken 6 hours later, and RNA was extracted and sent to OE Biotech for library construction, sequencing and analysis.

[0161] In Example 2, direct treatment of Arabidopsis thaliana with 2'cADPR induced the expression of the disease resistance-related gene PR1, while the blank group Mock and the negative control group 3'cADPR did not produce the same effect. Figure 3 (Left), further investigation revealed that 2'cADPR-induced PR1 upregulation was absent in mutants eds1-12, pad4-1, and adr1 triple, but still present in Col-0, sag101, and nrg1a / 1b. Figure 3 (Right). Other disease resistance-related genes FRK1 after 2'cADPR treatment. Figure 4 A),PBS3( Figure 4 B), NPR1 Figure 4 C),ALD1( Figure 4 F),NIMIN1( Figure 4 G),SARD4( Figure 4 E) and FMO1 Figure 4 The expression level detection results of 2'cADPR also showed a similar trend to PR1, namely, it was missing in eds1-12, pad4-1, and adr1 triple, but still present in Col-0, sag101, and nrg1a / 1b. These results indicate that 2'cADPR can induce disease resistance responses in plants, and this induction depends on the EDS-PAD4-ADR1 signaling pathway in plants.

[0162] Transcriptome analysis of Col-0 leaves treated with 2'cADPR revealed that the upregulated genes in Col-0 treated with 2'cADPR were associated with resistance pathways such as plant responses to bacteria, fungi, and oomycetes, and plant SA responses. Figure 5 Comparison of the transcriptomes of Col-0 and pad4-1 leaves after 2'cADPR treatment revealed that PCA analysis of the transcriptomes of the two plants showed no significant changes in pad4-1, but significant changes in Col-0. Venn diagrams of differentially expressed genes also showed that among the genes that responded in Col-0, the vast majority of genes in pad4-1 did not respond. Figure 6 ).

[0163] Example 3: Inducing plant resistance to *Pseudomonas syringae* (D36E) using 2'cADPR

[0164] To test whether 2'cADPR could enhance plant resistance to pathogenic microorganisms, this invention co-injected Arabidopsis thaliana Col-0 leaves with *Pseudomonas syringae* pv. tomato DC3000 (D36E) (strain construction see reference (Yuan et al., 2021)) along with dH2O and 2'cADPR (final concentration 50 μM). Four days later, the number of D36E microorganisms in the leaves was counted. The results showed that 2'cADPR could enhance Arabidopsis thaliana resistance to D36E. Figure 7 ).

[0165] Resistance test:

[0166] (1) Inoculate D36E into 3 ml LM medium (Table 2) containing antibiotic (Rifampicin, 25 mg / L) and incubate at 30°C and 220 rpm for about 12 hours.

[0167] (2) Centrifuge at 10000g for 1 min, discard the supernatant and adjust the bacterial concentration to OD600 = 0.01 with 10mM MgCl2. Then add equal volumes of 2'cADPR (final concentration 50μM) and dH2O respectively.

[0168] (3) Leaves of Arabidopsis thaliana Col-0 seedlings with a seedling age of 4 weeks were selected for injection. Four days after injection, samples were taken for leaf colony count. The samples were aseptically washed with water and homogenized, diluted with aseptic water and dropped onto LM plates. After 36 hours of incubation, the samples were used for microbial count.

[0169] Table 2 Components of LM Culture Medium

[0170]

[0171]

[0172] Example 3 involved direct treatment of Arabidopsis thaliana Col-0 leaves with 2'cADPR. The results showed that the microbial content in the leaves of the treated group was significantly lower than that of the blank Mock group. Figure 7 This result indicates that 2'cADPR enhances plant resistance to Pseudomonas syringae (D36E) by activating plant disease resistance.

[0173] Example 4: Enhancing the Resistance of Wheat Coleoptiles to Fusarium graminearum Infection Using 2'cADPR. To test whether 2'cADPR could induce disease resistance in wheat, wheat coleoptiles were used. The inoculation method followed (Jia et al., 2017). Soaked wheat seeds were rinsed overnight with tap water and germinated in the dark for one day. Afterward, they were transferred to 24-well culture plates (25℃, 12h light / 12h dark) and grown for one day before use. Conidia of Fusarium graminearum (strain PH-1), activated on V8 medium (V8 ORIGINAL) and cultured in mung bean medium at 25℃ and 150 rpm for 3-5 days, were resuspended in sterile water and the conidia number was adjusted to 1×10⁻⁶. 6 Prepare for later use. Inoculate the resuspended conidia onto wheat coleoptiles. After 2 hours, apply 2 μL of 50 μM 2'cADPR formulation (containing 0.5% Tween-20) to the wheat coleoptiles and incubate in a light incubator (25℃, 12h light / 12h dark). After 24 hours, add the above 2'cADPR formulation again. Take photos after 5 days of incubation. Figure 8 A) and count the length of the lesions ( Figure 8 B).

[0174] Example 5: Inducing wheat resistance to Fusarium head blight caused by Fusarium graminearum using 2'cADPR

[0175] To test whether 2'cADPR can induce resistance to Fusarium head blight in wheat, tests were conducted using wheat ears at the heading and flowering stage.

[0176] 10 μL of resuspended Fusarium graminearum conidia were inoculated into the middle florets of a wheat ear (wheat variety: Bobwhite). One hour later, 10 μL of 100 μM 2'c ADPR formulation was added to the inoculation site. 72 hours later, an additional 10 μL of 200 μM 2'c ADPR formulation was added to the inoculation site. A photograph was taken 14 days later. Figure 9 A) and statistics on the incidence of the disease ( Figure 9 B).

[0177] To further test its effect on the control of Fusarium head blight in different wheat varieties, 10 μL of resuspended Fusarium graminearum conidia were inoculated into the florets in the middle of wheat ears (wheat variety: Filder). One hour later, 10 μL of 100 μM 2'cADPR formulation was added to the inoculation site. 72 hours later, an additional 10 μL of 200 μM 2'cADPR formulation was added to the inoculation site. Photos were taken 14 days later. Figure 10 A) and statistics on the incidence of the disease ( Figure 10 B).

[0178] Example 6: Using 2'cADPR to improve maize's resistance to stalk rot caused by Fusarium graminearum infection.

[0179] To test whether 2'cADPR could induce a defensive response in maize against Fusarium graminearum and enhance its resistance to stalk rot, a 60-day-old maize stalk was used for the test. 20 μL of resuspended Fusarium graminearum conidia were inoculated into the middle of the maize internodes. Then, 20 μL of 100 μM 2'cADPR formulation ba was added to the inoculation site. 72 hours later, 10 μL of 200 μM 2'cADPR formulation was added to the inoculation site. Photos were taken 14 days later. Figure 11 A) and statistics on the incidence of the disease ( Figure 11 B).

[0180] Examples 1-2 above demonstrate that 2'cADPR, as a small molecule compound, can induce plant immune responses and enhance plant disease resistance. Examples 3-6 demonstrate that 2'cADPR induces resistance in different plant species to various pathogenic microorganisms, endowing plants with broad-spectrum disease resistance. The disease resistance induced by 2'cADPR has extremely strong universality, covering various diseases of dicotyledonous and monocotyledonous plants, such as maize large leaf spot caused by *Heterospora spp.* and *Heterospora heterospira*, maize small leaf spot and the combined large and small leaf spot diseases of maize, maize brown spot caused by *Heterospora spp.*, and maize ear rot caused by *Fusarium verticillata*, *Fusarium graminearum*, *Penicillium*, and *Aspergillus flavus*. Fusarium verticillata, Fusarium lead Corn stalk rot The following diseases were identified: corn gray leaf spot caused by *Cercospora zeylans* and *Cercospora maize*; corn rough dwarf virus caused by rice black-streaked dwarf virus; corn bacterial wilt caused by *Pythium spp.*; corn circular spot caused by *Helicobacter zeylans*; potato late blight caused by *Phytophthora spp.*; soybean scab caused by soybean scab virus; rapeseed sclerotinia rot and rapeseed clubroot caused by *Sclerotinia sclerotiorum* and *Platycodon brassicae*; citrus Huanglongbing caused by *Bacillus asiatica*; and rice dwarf virus caused by rice dwarf virus. Applying 2'cADPR to these plants for disease control achieved similar effects to those shown in Examples 4-6 in improving wheat resistance to *Fusarium graminearum*-induced wheat scab and corn resistance to stem rot.

[0181] 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.

[0182] References

[0183] Rao, W., Ma, T., Cao, J., Zhang, Y., Chen, S., Lin, S., Liu, X., He, G., and Wan, L. (2024). Recognition of a salivary effector by the TNL protein RCSP promotes effector-triggered immunity and systemic resistance in Nicotianabenthamiana. Journal of Integrative Plant Biology.

[0184] Yuan,M.,Jiang,Z.,Bi,G.,Nomura,K.,Liu,M.,Wang,Y.,Cai,B.,Zhou,J.-M.,He,SY,and Xin,X.-F.(2021).Pattern-recognition receptors are required for NLR-mediated plant immunity.Nature 592,105-109.

[0185] Jia,L.-J.,Wang,W.-Q.,and Tang,W.-H.(2017).Wheat ColeoptileInoculation by Fusarium graminearum for Large-scale Phenotypic Analysis.Bio-Protocol 7.

Claims

1. The use of a 2'cADPR or a pharmaceutically acceptable salt thereof, characterized in that, For the preparation of a composition or formulation for enhancing plant resistance to pathogenic microorganisms and viruses, the structure of 2'cADPR or a pharmaceutically acceptable salt thereof is shown in the following formula: 。 2. The use as described in claim 1, characterized in that, The plants include dicotyledons and monocotyledons.

3. The use as described in claim 1, characterized in that, The plants mentioned include: Arabidopsis thaliana, tobacco, rice, wheat, corn, potato, soybean, citrus, grape and kiwi, as well as different varieties of these plants.

4. The use as described in claim 1, characterized in that, The pathogenic microorganisms include: *Pseudomonas syringae* (… Pseudomonas syringae pv. tomato Xanthomonas aeruginosa (), Xanthomonas campestris Rice blast fungus ( Magnaporthe grisea ), rice bacterial blight pathogen ( Xanthomonas oryzae Wheat blast fungus ( Magnaporthe oryzae Fusarium graminearum (or Fusarium graminearum) Fusarium graminearum ), Phytophthora infestans ( Phytophthora infestans ) and Asian phloem ( Liberbacter asianticum Corn chytrid fungus ( Physoderma maydis ), Fusarium verticillata ( Fusarium verticillioides ), Corn umbelliferous spores ( Bipolarisn zeicol Fusarium moniliforme ( ), Fusarium proliferatum Cercospora maize ( Cercospora zeae-maydis ) and Cercospora maize ( Cercosporazeina), Corn sorghum chytridum ( Physoderma maydis ), corn leaf blight pathogen ( Exerohilum turcicum ), Heterospora ( Cochliobolusheterostrophus ), Pythium spp. ( Pythium aphanidermatum Aspergillus flavus Aspergillus flavus ), Penicillium ( Penicillium spp.), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum (Lib.) de Bary), Plasmodium brassicae ( Plasmodiophorabrassica ) and grape downy mildew ( Plasmoparaviticola ).

5. The use as described in claim 1, characterized in that, The viruses include: Tobacco Mosaic Satellite Virus (Tobacco Mosaic Satellite Virus) Virus ), Millet Leaf Satellite Virus ( Papanivirus Green Ivy Satellite Virus ( Aumaivirus ), Citrus roughness virus ( Cilevirus Rice dwarf virus ( Rice dwarf virus Rice dwarf virus ( Rice dwarf virus Rice black-streaked dwarf virus ( Rice black-streaked dwarf virus RBSDV and soybean stay-green-associated virus (SoSGV).

6. The use as described in claim 1, characterized in that, The dosage form of the preparation is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, and aerosol.

7. The use of a composition or formulation, characterized in that, The composition or formulation is used to enhance the resistance of plants to pathogenic microorganisms and viruses, wherein the composition or formulation comprises: (a) 2'cADPR or a pharmaceutically acceptable salt thereof, wherein the structure of 2'cADPR is shown in the following formula: ； (b) An agriculturally acceptable carrier.

8. A method for enhancing plant resistance to pathogenic microorganisms and viruses, characterized in that, Including the following steps: The plant was treated with 2'cADPR or a pharmaceutically acceptable salt thereof, which has the structure shown in the following formula. 。 9. The method as described in claim 8, characterized in that, The application includes: irrigation, spraying, smearing, soaking, or local injection.

10. The method as described in claim 8, characterized in that, The dosage applied is 2-100 mM / plant.

11. The method as described in claim 8, characterized in that, The dosage applied is 5-200 μM.

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