Enhance plant systemic acquired resistance using an atnpr1 variant

Abstract

The Arabidopsis NPR1 (AtNPR1) is a key regulator of systemic acquired resistance (SAR). Over-expression of AtNPR1 has been shown to enhance disease resistance in many crop plants. In citrus, over-expression of AtNPR1 confers robust HLB tolerance; however, the transgenic plants need to accumulate very high levels of AtNPR1 protein to have HLB tolerance. Most of the transgenic lines did not accumulate this high level of protein or show tolerance. This makes generating HLB tolerance in citrus cultivars difficult. Recently an AtNPR1 variant was found in which two amino acids were replaced. This modified AtNPR1 is more active and does not need to accumulate very high amounts to provide similar or even stronger protection. If this AtNPR1 variant is used in citrus for HLB tolerance, it will be possible to get tolerance in highly susceptible varieties such as grapefruit. Additionally, it can be used in other crops to enhance SAR.

Description

[0001] Attorney Docket no.10457-609PC0 Enhance plant systemic acquired resistance using an AtNPR1 variant STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under Grant No. 2022-70029-38470 and Grant No. 2020-70029-33195, awarded by the US Department of Agriculture, National Institute of Food & Agriculture. The government has certain rights in the invention. REFERENCE TO ELECTRONIC SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on September 23, 2025, is named “10457609PC0_seqlist.xml” and is 62,566 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. BACKGROUND Multicellular eukaryotes have evolved intricate immune systems to counter microbial infections. While animals possess both innate and adaptive immune systems, plants rely solely on germline-encoded cell-surface pattern recognition receptors (PRRs) and intracellular nucleotide- binding domain leucine-rich repeat receptors (NLRs) that mediate pattern- and effector-triggered immunity (PTI and ETI), respectively [1-3]. Activation of PTI and ETI leads to the production of mobile signals at the infection site, which are then transported to distal systemic tissues, initiating a long-lasting defense mechanism known as systemic acquired resistance (SAR). SAR confers broad-spectrum resistance throughout the plant against subsequent infections [4, 5]. SUMMARY A variety of potential SAR mobile signals have been identified, including DEFECTIVE IN INDUCED RESISTANCE1 [6], salicylic acid (SA) and its derivative methyl SA [7-9], dehydroabietinal

[0010] , azelaic acid (AzA)

[0011] , glycerol-3-phosphate (G3P)

[0012] , pipecolic acid (Pip) and its derivative N-hydroxy-Pip (NHP) [13-15], extracellular NAD(P) [eNAD(P)]

[0016] , Attorney Docket no.10457-609PC0 monoterpenes

[0017] , and phased small RNAs

[0018] . Additionally, nitric oxide and reactive oxygen species (ROS) have been shown to play significant roles in SAR [19, 20]. Recent studies suggest that SAR mobile signals initiate a signal amplification loop that enhances ROS production in systemic tissues [9, 17, 19-22], which in turn triggers the accumulation of eNAD(P) as shown in Examples

[0016] . Preventing systemic accumulation of eNAD(P) compromises SAR

[0016] , whereas NAD(P) treatment can restore systemic immunity in mutants defective in ROS, G3P, NHP biosynthesis, and AzA signaling

[0023] . Thus, eNAD(P) likely functions as a convergence point for various SAR signals in systemic tissues [16, 24]. NAD(P)-induced systemic immunity requires SA and its receptor, NONEXPRESSOR OF PATHOGENESIS-RELATED (PR) GENES1 (NPR1)

[0023] , consistent with the role of NPR1 as a transcriptional coactivator of SAR

[0025] . Nuclear import of NPR1 is crucial for transmitting SAR signals to the nucleus

[0026] . In the absence of SAR induction, NPR1 primarily resides in the cytosol as a high molecular weight oligomer associated by intermolecular disulfide bonds

[0027] . SAR induction or SA treatment triggers cellular redox changes that break these disulfide bonds, resulting in NPR1 dimers that are then transported to the nucleus to interact with TGACG-binding factors (TGAs) and activate target gene transcription [27-29]. NPR1 nuclear import is also regulated by phosphorylation at Thr373 and Ser589

[0030] . While SNF1-RELATED PROTEIN KINASE 2.8 is known to phosphorylate Ser589, the kinase responsible for Thr373 phosphorylation remains unidentified. Two legume-like (or L-type) lectin receptor kinases (LecRKs), LecRK-I.8 and LecRK- VI.2, have been identified as eNAD(P) receptors [23, 31]. LecRK-I.8 specifically binds NAD+, while LecRK-VI.2 binds both NAD+and NADP+. NAD(P)-induced immune responses are partially suppressed in T-DNA insertion loss of function mutants of LecRK-I.8 and LecRK-VI.2; and the lecrk-I.8 mutant exhibits reduced basal immunity, while lecrk-VI.2 has partially compromised SAR. Additionally, NAD(P)-induced immunity and biological induction of SAR are largely abrogated in a lecrk quintuple mutant

[0032] . These results indicate that eNAD(P) and its receptors are crucial for plant immunity, particularly SAR. However, the mechanism by which these LecRKs mediate eNAD(P) signaling remains unknown. Since both LecRK-I.8 and LecRK- VI.2 localize to the plasma membrane (PM) and their kinase domains are catalytically active [31, Attorney Docket no.10457-609PC0 33, 34], they could transmit eNAD(P) signaling across the PM by phosphorylating cytosolic signaling components. In this disclosure, it was demonstrated that LecRK-VI.2 physically associates with NPR1 at the PM in an uninduced state. Upon NADP+treatment, NPR1 is phosphorylated by LecRK- VI.2 and subsequently detaches from LecRK-VI.2, facilitating its interaction with TGAs. Furthermore, introducing non-phosphorylatable mutations at the NADP+-induced phosphorylation sites on NPR1 inhibits its interaction with TGAs and reduces NPR1’s transcriptional activity, while phospho-mimetic variants maintain their functionality. These findings establish a direct link between eNAD(P) binding to LecRK-VI.2 and NPR1 activation in SAR signaling and demonstrate that cell-surface receptors can directly transmit extracellular signals to transcriptional coactivators to drive transcriptomic changes in multicellular eukaryotes. In summary, extracellular nicotinamide adenine dinucleotide (phosphate) [eNAD(P)] is a critical signal in systemic acquired resistance (SAR), a plant defense mechanism that confers durable, broad-spectrum protection. During SAR induction, eNAD(P) accumulates systemically and binds to the cell-surface lectin receptor kinase (LecRK), LecRK-VI.2, triggering immune- related gene expression. However, the precise mechanism by which eNAD(P) signals through LecRK-VI.2 to initiate transcription has remained unclear. Here, it was demonstrated that LecRK- VI.2 transmits eNAD(P) signals by directly phosphorylating the transcriptional coactivator NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1). In the resting state, LecRK-VI.2 is physically associated with NPR1. Upon NADP+stimulation, NPR1 is phosphorylated by LecRK-VI.2 and subsequently dissociates from LecRK-VI.2, enabling its interaction with TGACG-binding factors (TGAs). Crucially, LecRK-VI.2-mediated phosphorylation is essential for NPR1-TGA interactions, thereby driving eNADP+signaling and SAR induction. These findings establish a direct link between the eNAD(P)-LecRK-VI.2 signaling module and NPR1, filling a key gap in our current understanding of the SAR signaling process. Since phosphorylation is essential for NPR1 function in SAR induction, a phospho- mimetic variant of NPR1 is introduced in this disclosure, NPR1-S356D / T359D, in which S356 and T359 are substituted with Asp. The data presented here indicate that the phospho-mimetic NPR1-S356D / T359D is more active than the wild-type NPR1 (Figure 5 and Figure 13), suggesting Attorney Docket no.10457-609PC0 that the phospho-mimetic form (S356D / T359) has significant advantages when used to increase disease resistance in crops. It will require less protein to confer similar or high levels of resistance. Producing transgenic plants expressing high levels of NPR1 is difficult in some crops such as citrus. It is expected the S356D / T359D NPR1 variant will significantly increase the chance to obtain disease-resistant or tolerant crops. Based on the results presented here, a phospho-mimetic mutant form of NPR1 protein is provided. In one embodiment, NPR from Arabidopsis NPR1 (AtNPR1) is provided, in which at least one of Ser (S) and / or Thr (T) amino acid residue(s) of the NPR1 is substituted with Asp (D), and the Ser and / or Thr amino acid residue(s) that can be substituted with Asp is selected from Ser356, Thr359, Thr373, Ser571, Thr572, Ser573, Ser 574, T575, S576, and Ser589. In a particular embodiment, to create a phospho-mimetic mutant protein of NPR1, Ser 356 (S356) and Thr 359 (T359) amino acid residues of the NPR1 are substituted with Asp (D) to make NPR1-S356D / T359D. The phospho-mimetic mutant protein, NPR1-S356D / T359D comprises an amino acid sequence of SEQ ID NO:1 or any amino acid sequence with at least 80% sequence identity thereto. In some embodiments, NPR1-S356D / T359D protein is optionally fused / tagged at its N- or C-terminus with fluorescence protein (e.g., GFP, superfolder GFP (sfGFP)), maltose- binding protein (MBP), glutathione S-transferase (GST), horseradish peroxidase (HRP), His (or hexa-histidine), FLAG, streptavidin-binding peptide (SBP), Strep II, calmodulin-binding peptide (CBP), chitin-binding domain (CBD), HA, c-Myc, or biotinylation. In a certain embodiment, a nucleic acid fragment is provided, which comprises a nucleotide sequence of SEQ ID NO:2 or any nucleotide sequence with at least 80% sequence identity thereto, which encodes NPR1-S356D / T359D protein or any amino sequence with at least 80% sequence identity thereto. In certain embodiments, the nucleic acid fragment encoding NPR1- S356D / T369D optionally comprises a nucleotide sequence encoding a tag at its 5’- or 3’-terminus. In some embodiments, vectors are provided, which comprise the nucleic acid fragment encoding NPR1-S356D / T359D protein or any amino sequence with at least 80% sequence identity thereto, and the vector is a plasmid for DNA replication and / or protein expression or a plant virus. In a certain embodiment, the vector is a plasmid vector for protein expression. In another embodiment, the vector is a plasmid comprising T-DNA fragment that comprises a nucleotide Attorney Docket no.10457-609PC0 sequence of SEQ ID NO:2 or any nucleotide sequence with at least 80% sequence identity thereto in its multicloning site. In a certain embodiment, Agrobacterium comprising the aforementioned plasmid vector is provided, and the Agrobacterium can be an A. tumefaciens strain. In some embodiments are provided transgenic plants with improved disease-resistance or tolerance by expressing NPR1-S356D / T359D protein comprising an amino acid sequence of SEQ ID NO:1 or any amino acid sequence with at least 80% sequence identity thereto. In a certain embodiment, the transgenic plant is citrus. In some embodiments, transgenic plants express NPR1-S356D / T359D protein fused / tagged at its N- or C-terminus with one selected from fluorescence protein (e.g., GFP, superfolder GFP (sfGFP)), maltose-binding protein (MBP), glutathione S-transferase (GST), horseradish peroxidase (HRP), His (or hexa-histidine), FLAG, streptavidin-binding peptide (SBP), Strep II, calmodulin-binding peptide (CBP), chitin-binding domain (CBD), HA, c-Myc, biotinylation. In a certain embodiment, the transgenic plant is Nicotiana benthamiana or Arabidopsis thaliana. In another embodiment, a method to improve plant immunity with NPR1-S356D / T359D protein is provided, which is delivered to the plant using liposomes. In another embodiment, a method to produce a transgenic plant expressing NPR1-S356D / T359D is provided, in which protein expression plasmid comprising a nucleotide sequence encoding NPR1-S356D / T359D is delivered to the plant using liposomes. In another embodiment, a method to produce a transgenic plant expressing NPR1-S356D / T359D by utilizing Agrobacterium and T-DNA system is provided. The aforementioned methods may comprise a step of infiltrating NAD and / or NADP+solution as well as a step of infiltrating pipecolic acid (Pip) and / or its derivative N-hydroxy-Pip (NHP) solution into a plant before, after, or simultaneously with liposome or Agrobacterium treatment to a plant. Further, a composition to improve plant immunity can be contemplated, which comprises purified protein of NPR1-S356D / T359D in liposomes and NAD / NADP+, and optionally NHP / Pip. Attorney Docket no.10457-609PC0 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. LecRK-VI.2 physically interacts with NPR1. (A) Yeast two-hybrid assay of the interaction between LecRK-VI.2KD and NPR1. The fragments encoding the kinase domains (KDs) of LecRK-VI.2 and LecRK-I.8 were cloned into the bait vector pGBKT7, and the full-length NPR1 was cloned into the prey vector pGADT7. The bait and prey vectors were co-transformed into the yeast strain AH109, and the resulting yeast cells were grown on synthetic dextrose (SD) double dropout (DDO) medium (-Leu-Trp). Interaction was determined by growth on SD quadruple dropout (QDO) medium (-Ade-His-Leu-Trp). BD, DNA binding domain; AD, activation domain. (B) Split Nano luciferase complementation assay of the interaction between LecRK-VI.2 and NPR1 in N. benthamiana. Full-length lecrk-VI.2K395Eor LecRK-I.8 fused with NlucN-HA and full-length NPR1 fused with NlucC-FLAG were transiently co-expressed in N. benthamiana. The luciferase activities were measured three days after agroinfiltration. The abundance of the fusion proteins was determined by immunoblotting with anti-HA and anti-FLAG antibodies. Ponceau S staining of Rubisco was used as the loading control. RLU, relative light unit. Bars represent means ± standard deviation (SD) (n = 6). Asterisks denote significant differences between lecrk-VI.2K395E-NlucN-HA / NPR1-NlucC-FLAG and LecRK-I.8-NlucN-HA / NPR1- NlucC-FLAG (**p < 0.01; Student’s t-test). (C) Co-immunoprecipitation assay of the interaction between LecRK-VI.2 and NPR1 in N. benthamiana. Full-length LecRK-VI.2 fused with FLAG and full-length NPR1 fused with GFP or free GFP were transiently co-expressed in N. benthamiana. Immunoprecipitation was carried out using GFP-trap magnetic agarose beads and the precipitated proteins were analyzed by immunoblotting with anti-FLAG and anti-GFP antibodies. (D) TurboID-based proximity labeling assay of the interaction between LecRK-VI.2 and NPR1 in Arabidopsis. Total protein was extracted from biotin-treated double transgenic plants expressing LecRK-VI.2-HA-TurboID or Lti6b-HA-TurboID and NPR1-GFP. Immunoprecipitation was performed using GFP-trap magnetic agarose beads and the precipitated proteins were analyzed by immunoblotting with the anti-GFP antibody. The biotinylated NPR1- GFP was detected using Streptavidin-HRP. Molecular mass markers in kilo Daltons (KD) are indicated on the right in (B)-(D). The experiments in (A)-(D) were repeated at least three times with similar results. Attorney Docket no.10457-609PC0 Figure 2. LecRK-VI.2 phosphorylates NPR1 in vitro and in vivo. (A) Phosphorylation of NPR1 by LecRK-VI.2KD in vitro. The kinase assay was performed by incubating MBP-LecRK-VI.2KD with sfGFP-NPR1-His or sfGFP-His as a substrate. Phosphorylation of NPR1 by LecRK-VI.2KD was detected using an anti-pThr antibody. The abundance of the recombinant proteins was determined by immunoblotting with anti-GFP and anti-MBP antibodies. (B) Schematic diagram of the NPR1 protein motifs with new in vivo phosphorylation sites identified by LC-MS / MS. (C) LC-MS / MS spectra of peptides harboring pSer356 identified in NADP+-treated transgenic plants overexpressing LecRK-VI.2-HA-TurboID and NPR1-GFP. (D) LC-MS / MS spectra of peptides harboring pThr359 identified in NADP+-treated transgenic plants overexpressing LecRK-VI.2-HA-TurboID and NPR1-GFP. (E) Evaluation of the specificity of the newly developed anti-pThr359 antibody. MBP- LecRK-VI.2KD was incubated with sfGFP-NPR1-His or sfGFP-npr1T359A-His in kinase reaction buffer with ATP. The abundance of the recombinant proteins was determined by immunoblotting with anti-GFP and anti-MBP antibodies. Phosphorylation at Thr359 was analyzed using the anti- pThr359 antibody. (F) Analysis of Thr359 phosphorylation in vivo using the anti-pThr359 antibody. Leaves of four-week-old transgenic plants overexpressing NPR1-GFP were infiltrated with 0.8 mM NADP+and the infiltrated leaves were collected at 0, 15, 30, and 45 min. Immunoprecipitation was carried out using GFP-trap magnetic agarose beads. The precipitated proteins were analyzed by immunoblotting with the anti-pT359 antibody, and the same membrane was stripped and probed with the anti-GFP antibody. (G) The dependence of NADP+-induced Thr359 phosphorylation on LecRK-VI.2. Leaves of four-week-old transgenic NPR1-GFP plants in the wild-type Col-0 and lecrk-VI.2-1 backgrounds were infiltrated with 0.8 mM NADP+and the infiltrated leaves were collected at 30 min. Immunoprecipitation was performed using GFP-trap magnetic agarose beads. The precipitated proteins were analyzed by immunoblotting with anti-pThr359 and anti-GFP antibodies in parallel. (H) Phos-tag gel analysis of NADP+-induced phosphorylation of Ser356 in vivo. Leaves of four-week-old transgenic plants overexpressing NPR1-GFP or npr1S356A-GFP were infiltrated Attorney Docket no.10457-609PC0 with 0.8 mM NADP+and the infiltrated leaves were collected at 30 min. Total proteins were extracted, separated in a phos-tag gel or a regular SDS-PAGE gel, and probed with the anti-GFP antibody. Molecular mass markers in kilo Daltons (KD) are indicated on the right in (A) and (E)-(H). The experiments in (A) and (E)-(H) were repeated at least twice with similar results. Figure 3. NADP+treatment triggers separation of NPR1 from LecRK-VI.2 and facilitates NPR1-TGA interactions. (A-B) Co-immunoprecipitation assay of the interaction between NPR1-GFP and LecRK- VI.2-HA-TurboID (A) or lecrk-VI.2K395E-HA-TurboID (B) in vivo. Leaves of four-week-old double transgenic plants overexpressing NPR1-GFP and LecRK-VI.2-HA-TurboID or lecrk- VI.2K395E-HA-TurboID were treated with 0.8 mM NADP+for the indicated times. Immunoprecipitation was conducted using anti-HA magnetic beads. The precipitated proteins were analyzed by immunoblotting with anti-GFP and anti-HA antibodies. Transgenic plants overexpressing NPR1-GFP without NADP+treatment were included as negative controls. (C-D) Co-immunoprecipitation assay of the interaction between NPR1-GFP and TGA1- HA-TurboID (C) or TGA3-HA-TurboID (D) in vivo. Leaves of four-week-old double transgenic plants overexpressing NPR1-GFP and TGA1-HA-TurboID or TGA3-HA-TurboID were treated with 0.8 mM NADP+for the indicated times. Immunoprecipitation was conducted using anti-HA magnetic beads. The precipitated proteins were analyzed by immunoblotting with anti-GFP and anti-HA antibodies. The molecular mass markers in kilo Daltons (KD) are indicated on the right in (A)-(D). The experiments in (A)-(D) were repeated at least three times with similar results. Figure 4. Phosphorylation of Ser356 and Thr359 is required for NPR1-TGA interactions. (A) Ser356 and Thr359 are localized at the loop between ankyrin repeat 3 (ANK3) and ANK4

[0028] . (B) Yeast two-hybrid assay of the interaction between NPR1, npr1S356A / T359A, or npr1S356D / T359Dand TGAs. The full-length NPR1, npr1S356A / T359A, and npr1S356D / T359Dwere cloned into the bait vector pGBKT7, and the full-length TGAs were in the prey vector pGADT7. The bait and prey vectors were co-transformed into the yeast strain AH109, and the resulting yeast cells were grown on synthetic dextrose (SD) double dropout (DDO) medium (-Leu-Trp). Interaction Attorney Docket no.10457-609PC0 was determined by growth on SD trip dropout (TDO) medium (-His-Leu-Trp) supplemented with 3 mM 3-amino-1,2,4-triazole (3-AT). BD, DNA binding domain; AD, activation domain. (C-D) Co-immunoprecipitation assay of the interaction between NPR1, npr1S356A / T359A, or npr1S356D / T359Dand TGA1 (C) or TGA3 (D) in N. benthamiana. NPR1-FLAG, npr1S356A / T359A- in beads. The precipitated proteins were analyzed by immunoblotting with anti-GFP and anti-FLAG antibodies. (E) Co-immunoprecipitation assay of the interaction between NPR1, npr1S356A / T359A, or npr1S356D / T359Dand TGA6 in N. benthamiana. NPR1-FLAG, npr1S356A / T359A-FLAG, or npr1S356D / T359D-FLAG were transiently co-expressed with TGA6-GFP in N. benthamiana. Immunoprecipitation was carried out using GFP-trap magnetic agarose beads. The precipitated proteins were analyzed by immunoblotting with anti-FLAG or anti-GFP antibodies. (F-H) TurboID-based proximity labeling assay of the interaction between NPR1, npr1S356A / T359A, or npr1S356D / T359Dand TGA1 (F), TGA2 (G), or TGA3 (H) in Arabidopsis. Total protein was extracted from biotin-treated double transgenic plants expressing NPR1-GFP, npr1S356A / T359A-GFP, or npr1S356D / T359D-GFP and TGA1-HA-TurboID, TGA2-HA-TurboID, or TGA3-HA-TurboID. Immunoprecipitation was performed using GFP-trap magnetic agarose beads. The precipitated proteins were analyzed by immunoblotting with the anti-GFP antibody. The biotinylated NPR1-GFP or npr1-GFP proteins were detected using Streptavidin-HRP. The molecular mass markers in kilo Daltons (KD) are indicated on the right in (C)-(H). The experiments in (B)-(H) were repeated at least three times with similar results. Figure 5. Phosphorylation of Ser356 and Thr359 is required for NPR1’s function in eNADP signaling and SAR (A) Induction of the PR1 promoter by transient expression of GFP, NPR1-GFP, npr1S356A / T359A-GFP, or npr1S356D / T359D-GFP in N. benthamiana. Agrobacteria carrying the pPR1:DUAL-LUC effector construct were co-infiltrated into N. benthamiana leaves. Samples were collected two days later for FLUC and RLUC activity assays. PR1 promoter activity was indicated by the ratio of F-LUC and R-LUC activities for each effector, normalized to free GFP, which was set to 1. Bars represent means ± SD (n = 4). Different letters denote significant differences (p < 0.01; one-way ANOVA). Attorney Docket no.10457-609PC0 (B-C) NADP+-induced PR1 protein accumulation in npr1-3 and npr1-3 complementation lines expressing NPR1-GFP, npr1S356A / T359A-GFP (B) or npr1S356D / T359D-GFP (C). Leaves of four- week-old plants were treated with 0.8 mM NADP+for 24 hr. Total proteins were extracted and analyzed by immunoblotting with an anti-PR1 antibody. Ponceau S Rubisco was used as the loading control. (D) NADP+-induced local immunity in the indicated genotypes. Two leaves on each four- week-old plant were infiltrated with 0.8 mM NADP+or water. Four hr later, the infiltrated leaves were inoculated with a Psm-lux suspension (OD600 = 0.001). Samples were collected 2.5 days later. Bars represent means ± SD (n = 8). Different letters denote significant differences (p < 0.05; two-way ANOVA). (E) NADP+-induced systemic immunity in the indicated genotypes. Three leaves on each four-week-old plant were infiltrated with 0.8 mM NADP+or water. Four hr later, two systemic leaves were inoculated with a Psm-lux suspension (OD600 = 0.001). Samples were collected 2.5 days later. Bars represent means ± SD (n = 8). Different letters denote significant differences (p < 0.05; two-way ANOVA). (F) Biological induction of SAR in the indicated genotypes. Three lower leaves on each four-week-old plant were infiltrated with Psm (OD600= 0.002) or 5 mM MgCl2. Two days later, two systemic leaves were inoculated with a Psm-lux suspension (OD600 = 0.001). Samples were collected 2.5 days later. Bars represent means ± SD (n = 8). Different letters denote significant differences (p < 0.05; two-way ANOVA). The experiments in (A)-(F) were performed three times with similar results. Figure 6. Phosphorylation of the Ser / Thr residues corresponding to the NPR1 Ser356 and Thr359 is important for the function of BrNPR1 and OsNPR1 (A) WebLogo representation of the region surrounding the conserved Ser and Thr of 18 NPR1 orthologs from 17 plant species. (B-C) Yeast two-hybrid assay of the interaction between the indicated TGA1, TGA2, or TGA3 and BrNPR1 (B), Brnpr1S340A / T342A(B), OsNPR1 (C) or Osnpr1T363A(C). The full-length BrNPR1, Brnpr1S340A / T342A, OsNPR1, and Osnpr1T363Awere cloned into the bait vector pGBKT7, and the full-length TGA1, TGA2, and TGA3 were in the prey vector pGADT7. The bait and prey vectors were co-transformed into the yeast strain AH109, and the resulting yeast cells were grown on synthetic dextrose (SD) double dropout (DDO) medium (-Leu-Trp). Interaction was Attorney Docket no.10457-609PC0 determined by growth on SD quadruple dropout (QDO) medium (-Ade-His-Leu-Trp). BD, DNA binding domain; AD, activation domain. (D-E) NADP+-induced local and systemic immunities as well as biological induction of SAR in npr1-3 and npr1-3 complementation lines expressing BrNPR1 (D), Brnpr1S340A / T342A(D), OsNPR1 (E), and Osnpr1T363A(E). For NADP+-induced local immunity, two leaves on each four- week-old plant were infiltrated with 0.8 mM NADP+or water. Four hr later, the infiltrated leaves were inoculated with a Psm-lux suspension (OD600= 0.001). Samples were collected 2.5 days later. For NADP+-induced systemic immunity, three leaves on each four-week-old plant were infiltrated with 0.8 mM NADP+or water. Four hr later, two systemic leaves were inoculated with a Psm-lux suspension (OD600= 0.001). Samples were collected 2.5 days later. For biological induction of SAR, three lower leaves on each four-week-old plant were infiltrated with Psm (OD600= 0.002) or 5 mM MgCl2. Two days later, two systemic leaves were inoculated with a Psm-lux suspension (OD600 = 0.001). Samples were collected 2.5 days later. Bars represent means ± SD (n = 8). Different letters denote significant differences (p < 0.05; two-way ANOVA). Figure 7. Proposed model for the transduction of eNAD(P) signaling by LecRK-VI.2 and NPR1. In the resting state, some NPR1 oligomers are physically associated with the eNAD(P) receptor complex LecRK-VI.2 / BAK1. Upon pathogen infection, NAD(P) is released into the extracellular space. The binding of eNAD(P) to LecRK-VI.2 triggers LecRK-VI.2-mediated phosphorylation of NPR1 at Ser356 and Thr359 and causes the dissociation of NPR1 from the plasma membrane. Additionally, eNAD(P) induces SA accumulation through unknown mechanisms (indicated by the question mark), leading to changes in the cellular redox potential [27, 35]. This change prompts NPR1 to transition from its oligomeric form to dimers. SA also binds directly to NPR1, inducing the SA-binding domain to dock onto ankyrin repeats 3 and 4

[0028] . This docking is crucial for promoting immune gene transcription. In the nucleus, phosphorylation at Ser356 and Thr359 facilitates NPR1's interaction with TGAs, initiating the transcription of immune-related genes. Figure 8. NPR1 is associated with the plasma membrane, related to Figure 1. Plasma membrane (PM) fractionation assay was conducted using double transgenic plants expressing NPR1-GFP and LecRK-VI.2-HA-TurboID. Total protein (T), cytosolic protein (S), and PM proteins were separated in 8% SDS-PADG gel and analyzed by immunoblotting with anti-GFP Attorney Docket no.10457-609PC0 and anti-HA antibodies. The PM marker H+-ATPase and the cytoplasmic marker tubulin were detected using anti-H+-ATPase and anti-tubulin antibodies, respectively. The experiment was repeated twice with similar results. Figure 9. BAK1KD interacts with NPR1, but does not phosphorylate NPR1 in vitro, related to Figure 2. (A) Yeast two-hybrid assay of the interaction between LecRK-1.8KD, LecRK-VI.2KD, or BAK1KD and NPR1. The fragments encoding the kinase domains (KDs) of LecRK-1.8, LecRK- VI.2, and BAK1 were cloned into the bait vector pGBKT7, and the full-length NPR1 was cloned into the prey vector pGADT7. The bait and prey vectors were co-transformed into the yeast strain AH109, and the resulting yeast cells were grown on synthetic dextrose (SD) double dropout (DDO) medium (-Leu-Trp). Interaction was determined by growth on SD quadruple dropout (QDO) medium (-Ade-His-Leu-Trp). BD, DNA binding domain; AD, activation domain. (B) Phosphorylation of NPR1 by LecRK-VI.2KD or BAK1KD in vitro. The kinase assay was performed by incubating MBP-LecRK-VI.2KD or GST-BAK1KD with sfGFP-NPR1-His as a substrate. Phosphorylation of NPR1 was detected using the anti-pThr antibody. The abundance of the recombinant proteins was determined by immunoblotting with anti-MBP, anti-GST, and anti-NPR1 antibodies. Figure 10. NPR1 phosphorylation sites identified by LC-MS / MS, related to Figure 2 (A) Schematic diagram of the NPR1 protein motifs with both in vitro and in vivo phosphorylation sites identified by LC-MS / MS. (B) LC-MS / MS spectra of peptides harboring pSer356, pThr359, or pThr373 derived from sfGFP-NPR1-His incubated with MBP-LecRK-VI.2KD. Figure 11. NADP+treatment induces disassociation of NPR1 and LecRK-VI.2 in N. benthamiana, related to Figure 3. NPR1-GFP and LecRK-VI.2-FLAG were transiently co- expressed in N. benthamina. Two days after agroinfiltration, the infiltrated leaves were treated with or without 0.8 mM NADP+for 30 min. Immunoprecipitation was performed using GFP-trap magnetic agarose beads. The precipitated proteins were analyzed by immunoblotting with anti- FLAG and anti-GFP antibodies. Figure 12. Phosphorylation of Ser356 and Thr359 additively contributes to the interaction of NPR1 and TGA3 in yeast, related to Figure 4. Yeast two-hybrid assay of the interaction between Attorney Docket no.10457-609PC0 NPR1 or its phospho-mutants and TGA3. The full-length NPR1 and its phospho-mutants were cloned into the bait vector pGBKT7, and the full-length TGA3 was in the prey vector pGADT7. The bait and prey vectors were co-transformed into the yeast strain AH109, and the resulting yeast cells were grown on synthetic dextrose (SD) double dropout (DDO) medium (-Leu-Trp). Interaction was determined by growth on SD quadruple dropout (QDO) medium (-Ade-His-Leu- Trp). BD, DNA binding domain; AD, activation domain. Figure 13. Phosphorylation of Ser356 and Thr359 is required for NADP+-induced PR1 gene expression, related to Figure 5. (A) NADP+-induced PR1 expression in the indicated genotypes. Leaves of four-week-old plants were infiltrated with 0.8 mM NADP+. Leaf tissues were collected at the indicated time points for gene expression assay. Expression levels of PR1 were normalized against the constitutively expressed UBQ5. Bars represent means ± SD (n = 3). Different letters denote significant differences (p < 0.05; one-way ANOVA). The comparison was made separately for each time point. (B) Expression levels of the transgenic proteins in the indicated genotypes. Total proteins were analyzed by immunoblotting with the anti-GFP antibody. Ponceau S staining of Rubisco was used as the loading control. Figure 14. Phosphorylation of Ser / Thr 571-576 is not required for NPR1’s function in eNADP signaling and SAR, related to Figure 5. (A-B) NADP+-induced local (A) and systemic (B) immunity in npr1-3 and npr1-3 complementation lines expressing NPR1-GFP or npr1-6A-GFP. 6A, S571A / T572A / S573A / S574A / T575A / S576A. Two (A) or three leaves (B) on each plant were infiltrated with 0.8 m NADP+or water. Four hr later, either the infiltrated leaves (A) or two upper systemic leaves (B) were inoculated with Psm-lux (OD600= 0.001). Samples were taken 2.5 days later. Bars represent means ± SD (n = 8). Different letters denote significant differences (p < 0.05; two-way ANOVA). (C) Biological induction of SAR in the indicated genotypes. Three lower leaves on each four-week-old plant were infiltrated with Psm (OD600= 0.002) or 5 mM MgCl2. Two days later, two systemic leaves were inoculated with a Psm-lux suspension (OD600= 0.001). Samples were collected three days later. Bars represent means ± SD (n = 8). Different letters denote significant Attorney Docket no.10457-609PC0 differences (p < 0.05; two-way ANOVA). (D) Expression levels of the transgenic proteins in the indicated genotypes. Total proteins were analyzed by immunoblotting with the anti-GFP antibody. Ponceau S staining of Rubisco was used as the loading control. Figure 15. Conservation of the Arabidopsis NPR1 Ser356 and Thr359 phosphosites across plant species, related to Figure 6. (A) Multiple alignment of 18 NPR1 orthologs from 17 plant species using the “Align” tool in UniProt with default settings. The red arrows indicate the conserved Ser and Thr. (B) The Ser / Thr residues (indicated by red arrows) corresponding to the Arabidopsis NPR1 Ser356 and Thr359 in O. sativa and B. rapa NPR1 orthologs. The Ser / Thr residues that were mutated in this study are marked with red circles. Figure 16. NHP induces de novo eNAD(P) accumulation in systemic tissues. (a-d) eNAD(P) levels in NHP-infiltrated leaves (a and b) or upper systemic leaves (c and d). For (a and b), two leaves on each wild-type plant were infiltrated with 0.5 mM NHP or water (-NHP). For (c and d), three lower leaves on each wild-type plant were infiltrated with 0.5 mM NHP or water. The infiltrated leaves (a and b) or one systemic leaf (c and d) on each plant was collected at the indicated times and then AWFs were extracted. Values are expressed relative to the eNAD(P) levels in the -NHP samples at 24 h, which were arbitrarily set to 1. Bars represent means ± SE (n = 3 independent AWF samples). NHP induced significant eNAD(P) accumulation in both the infiltrated and the upper systemic leaves (one-way ANOVA with Tukey’s test). The experiments were repeated with similar results. (e, f) NHP-induced systemic accumulation of eNAD(P) in Dex:FIN4 / fin4-3 plants with systemic leaves being pretreated with or without Dex. One upper systemic leaf on each plant was infiltrated with 50 μM Dex or 0.1% methanol (-Dex). Twenty-four hr later, three lower leaves on the plant were infiltrated with 0.5 mM NHP or water (-NHP). Twenty-four hr later, the pretreated systemic leaves were collected. Values are expressed relative to the eNAD(P) levels in the -Dex / - NHP samples, which were arbitrarily set to 1. Bars represent means ± SE (n = 3 independent AWF samples). The NHP treatment of the lower leaves induced significant eNAD(P) accumulation in the Dex-treated upper systemic leaves of the Dex:FIN4 / fin4-3 plants (one-way ANOVA with Tukey’s test). The experiment was repeated with similar results. Attorney Docket no.10457-609PC0 (g, h) SAR induction-triggered systemic accumulation of eNAD(P) in wild type (WT) and fmo1. Values are expressed relative to the eNAD(P) levels in the WT / -SAR samples, which were arbitrarily set to 1. Bars represent means ± SE (n = 3 independent AWF samples). The SAR induction-induced systemic eNAD(P) accumulation was significantly reduced in fmo1 (one-way ANOVA with Tukey’s test). The experiments were repeated with similar results. Figure 17. Total NAD(P) levels in NHP-treated leaves and upper systemic leaves. (a, b) Total NAD(P) levels in NHP-infiltrated leaves. Two leaves on each wild-type plant were infiltrated with 0.5 mM NHP or water (-NHP). The infiltrated leaves were collected at the indicated times for total NAD(P) measurement. Values are expressed relative to the NAD(P) levels in -NHP samples at 24 hr, which were arbitrarily set to 1. Bars represent means ± SE (n = 3 independent leaf samples). Same letters denote no significant differences (one-way ANOVA with Tukey’s test; p values are shown in the Source Data file). (c, d) Total NAD(P) levels in upper systemic leaves on plants with lower leaves treated with NHP. Three lower leaves on each wild-type plant were infiltrated with 0.5 mM NHP or water. One systemic leaf on each plant was collected at the indicated times for total NAD(P) measurement. Values are expressed relative to the NAD(P) levels in -NHP samples at 24 hr, which were arbitrarily set to 1. Bars represent means ± SE (n = 3 independent leaf samples). Same letters denote no significant differences (one-way ANOVA with Tukey’s test; p values are shown in the Source Data file). The experiments in (a), (b), (c), and (d) were repeated with similar results. Figure 18. The fin4-3 mutation compromises NAD(P) accumulation. (a) Morphology of three-week-old wild-type (WT), fin4-3, and two 35 S:FIN4 / fin4-3 complementation lines. (b, c) Total NAD (b) and NADP (c) levels in the indicated genotypes. Leaf samples of four- week-old plants were collected. Values are expressed relative to the NAD(P) levels in WT, which were arbitrarily set to 1, allowing comparison across experiments. Bars represent means ± SE (n = 3 independent leaf samples). The 35 S:FIN4 transgene restored total NAD(P) levels in fin4-3 (one-way ANOVA with Tukey’s test). The experiment was repeated with similar results. Figure 19. (a) GUS expression in Dex-infiltrated lower leaves and upper non-infiltrated systemic leaves of Dex:FIN4 / fin4-3 plants. Leaves were collected 24 h after the treatment. (b) Attorney Docket no.10457-609PC0 Morphology of three-week-old plants of the indicated genotypes treated with 1 μM Dex or water (-Dex) by soil drenching every other day after transplanting. Figure 20. Restoration of NAD levels in the Dex:FIN4 / fin4-3 plants by Dex application Dex application restores NAD levels in the Dex:FIN4 / fin4-3 plants. Two leaves on each four week- old wild-type (WT), fin4-3, and Dex:FIN4 / fin4-3 plants were infiltrated with 50 µM Dex (+Dex) or 0.1% methanol (-Dex). Twenty-four hr later, the infiltrated leaves were collected for total NAD measurement. Values are expressed relative to the NAD level in WT / -Dex samples. Figure 21. Pathogen-induced local accumulation of eNAD(P) in fmo1 (a, b) Three leaves on each plant were infiltrated with 1 mM MgCl2 or Psm (OD600 = 0.002). The infiltrated leaves were collected 24 hr later for AWF extraction and eNAD(P) measurement. Values are expressed relative to the eNAD(P) levels in the wild type (WT) treated with MgCl2, which were arbitrarily set to 1. Bars represent means ± SE (n = 3 independent AWF samples). Different letters denote significant differences (one-way ANOVA with Tukey’s test; p values are shown in the Source Data file). The experiment was repeated with similar results. Figure 22. NHP-induced systemic immunity depends on eNAD(P) and its receptor complex. (a) NHP-induced systemic immunity in WT and 35 S:CD38 plants. Three lower leaves were infiltrated with 0.25 mM NHP or water (-NHP). Twenty-four hr later, one upper systemic leaf on each plant was inoculated with Psm. Samples were collected at 72 hpi. Bars represent means ± SE (n = 12 independent leaf disks). The NHP-induced systemic immunity was significantly reduced in the 35 S:CD38 transgenic plants (two-way ANOVA with Sidak’s test). The experiment was performed three times with similar results. (b) NHP-induced systemic expression of PR1, ALD1, and FMO1 in the indicated genotypes. Three lower leaves on each plant were infiltrated with 0.5 mM NHP or water (-NHP). One upper systemic leaf on each plant was collected 24 h later. Bars represent means ± SE (n = 3 independent total RNA samples). Different letters denote significant differences (one-way ANOVA with Tukey’s test; p values are shown in the Source Data file). The experiment was repeated with similar results. (c, d) NHP-induced systemic immunity in the indicated genotypes. The photo in (d) was taken at 72 hpi. Bars in (c) represent means ± SE (n = 20 independent leaf disks). Different letters Attorney Docket no.10457-609PC0 denote significant differences (one-way ANOVA with Tukey’s test; p values are shown in the Source Data file). The experiment was performed three times with similar results. Figure 23. NHP treatment triggers eNAD(P) accumulation through RBOHF-generated ROS. (a). eNAD(P) levels in the leaves treated with or without MV. Values are expressed relative to the eNAD(P) levels in the -MV samples, which were arbitrarily set to 1. Bars represent means ± SE (n = 3 independent AWF samples). The MV treatment induced significant eNAD(P) accumulation (two-tailed t test). The experiment was repeated with similar results. (b, c) SAR induction-triggered systemic accumulation of eNAD(P) in wild type (WT) and rbohF. Values are expressed relative to the eNAD(P) levels in the WT / -SAR samples, which were arbitrarily set to 1. Bars represent means ± SE (n = 3 independent AWF samples). The SAR induction-induced eNAD(P) accumulation was significantly inhibited in rbohF (one-way ANOVA with Tukey’s test). The experiment was performed three times with similar results. (d, e) NAD(P)+-induced systemic immunity in the indicated genotypes. The photo in (d) and samples in (e) were taken at 72 hpi. Bars in (e) represent means ± SE (n = 8 independent leaf disks). NAD(P)+ induced similar levels of systemic immunity in the WT, rbohD, and rbohF plants (two-tailed t test). The experiment was performed three times with similar results. (f) DAB (3, 3’-diaminobenzidine) staining of NHP-induced H2O2. Arabidopsis leaves were infiltrated with 0.5 mM NHP or water (-NHP) and collected 24 h later. (g, h) NHP-induced systemic accumulation of eNAD(P) in WT and rbohF. Values are expressed relative to the eNAD(P) levels in WT / -NHP samples, which were arbitrarily set to 1. Bars represent means ± SE (n = 3 independent AWF samples). The NHP-induced eNAD(P) accumulation was significantly inhibited in rbohF (one-way ANOVA with Tukey’s test). The experiment was conducted three times with similar results. (i, j) NHP-induced systemic immunity in the indicated genotypes. The photo in (j) and samples in (i) were taken at 72 hpi. Bars in (i) represent means ± SE (n = 10 independent leaf disks). The NHP-induced systemic immunity was significantly reduced in rbohD and rbohF (one- way ANOVA with Tukey’s test; p values are shown in the Source Data file). The experiment was performed three times with similar results. (k) A proposed working model for the function of eNAD(P) in SAR. Pathogen infections activate PTI, DTI, and ETI responses in the local leaf tissues, which are accompanied by generation Attorney Docket no.10457-609PC0 of a blend of mobile signals among which are eNAD(P) and NHP. NHP likely contributes to eNAD(P) accumulation in the local leaf tissues. These mobile SAR signals are swiftly transported to systemic leaf tissues where the eNAD(P) from the local tissues binds to and activates its receptor complex LecRK-VI.2 / BAK1, but this signal appears to be too weak to trigger downstream SAR signaling. Other mobile signals including NHP initiate a signaling amplification loop, triggering accumulation of ROS that induce de novo eNAD(P) accumulation in the systemic leaf tissues. The fresh eNAD(P) further activates the receptor complex to boost the force of the SAR signaling, triggering the downstream SA / NPR1-mediated SAR responses. A modest NHP-dependent but ROS- and eNAD(P)-independent pathway to SAR seems to exist. A question marks (?) indicates that the contribution of the pathway to SAR needs further investigation. The figure was created with BioRender.com. Figure 24 shows the chemical structures of various molecules related to the embodiments described herein. DETAILED DESCRIPTION A. Overview The recently identified key SAR signal, eNAD(P), induces immune-related gene expression through the cell-surface receptor kinases LecRK-I.8 and LecRK-VI.2 [16, 23]. However, the precise mechanism by which eNAD(P) signals are transmitted from these receptors into the nucleus remains unclear. Herein, it was demonstrated that LecRK-VI.2 physically associates with and directly phosphorylates NPR1, which in turn facilitates its interaction with TGAs, leading to the activation of downstream immune gene transcription. These findings uncover a direct signaling step from LecRK-VI.2 to NPR1 and close a major gap in the current understanding of the SAR signaling process. It is unexpected that NPR1, widely known as a transcriptional coactivator, is a peripheral membrane protein associated with cell-surface receptors (Figure 1 and Figure 8). In the resting state, NPR1 exists in the cytosol as an oligomer held together by intermolecular disulfide bonds

[0027] . Upon SAR induction, cellular redox changes cause NPR1 to transition from the oligomeric form to dimers, which then migrate to the nucleus to activate target gene transcription [27, 28]. Attorney Docket no.10457-609PC0 NPR1 can thus sense cellular redox changes triggered by pathogen infection or environmental stress. NPR1 is also a receptor of SA

[0036] . SA induces docking of the SA-binding domain onto ankyrin repeats 3 and 4 in NPR1, which is crucial for promoting immune gene transcription

[0028] . The discovery herein that NPR1 associates with the PM indicates that it can transmit signals directly from the PM to the nucleus. In the case of eNAD(P) signaling, NPR1 relays signals from LecRK-VI.2 directly to the nucleus, where it interacts with TGAs and activates target gene transcription. Notably, NADP+treatment prompts the dissociation of NPR1 from LecRK-VI.2, which precedes the formation of the NPR1-TGA complex (Figure 3). This suggests that once NPR1 separates from LecRK-VI.2, it migrates to the nucleus to interact with TGAs. Interestingly, this NADP+-induced dissociation of NPR1 from LecRK-VI.2 does not require the kinase activity of LecRK-VI.2 (Figure 3B). It is plausible that other eNAD(P) receptors phosphorylate NPR1

[0032] , leading to its dissociation from LecRK-VI.2, or that NADP+-induced SA accumulation changes the cellular redox potential

[0035] , triggering this separation. On the other hand, the NADP+-induced dissociation of NPR1 from LecRK-VI.2 is transient (Figure 3A), which aligns with the short-lived nature of NAD(P)-induced immunity

[0016] . These findings suggest a mechanism for rapid attenuation of eNAD(P) signaling. Whether LecRK-VI.2 is internalized and then degraded, as seen for other PRRs like FLS2, EFR, and PEPR1 / 2 [37, 38], remains unknown. Alternatively, this attenuation may result from NAD(P) degradation in the apoplast, though specific ecto-NAD(P) hydrolases have yet to be identified. NADP+treatment induces LecRK-VI.2-mediated phosphorylation of NPR1. The kinase domain of LecRK-VI.2 phosphorylates NPR1 in vitro potentially at Ser356, Thr359, Thr373, and Ser571 (Figure 2A and Figure 10). NADP+treatment notably induces phosphorylation of NPR1 at Ser356 and Thr359 and possibly at Thr572, Ser573, and Ser 574 in vivo (Figures 2B-2D, 2F, 2H and Figure 10A). Using a novel anti-pThr359 antibody, it was confirmed that LecRK-VI.2 mediates NADP+-induced phosphorylation of NPR1 at Thr359 (Figures 2E and 2G), demonstrating that ligand binding activates LecRK-VI.2 to phosphorylate NPR1. Furthermore, in vivo phosphorylation of Thr373 was previously reported

[0039] , and genetic evidence shows that phosphorylation of Thr373 is essential for NPR1 nuclear import, although the kinase responsible was not clearly identified

[0030] . LecRK-VI.2 is a promising candidate, given that it phosphorylates Attorney Docket no.10457-609PC0 Thr373 in vitro (Figure S3). Thus, NADP+induces LecRK-VI.2-mediated phosphorylation of NPR1 at Thr359, and potentially Ser356 and Thr373 in vivo. LecRK-VI.2-mediated phosphorylation is vital for NPR1 transcriptional activity. Ser356 and Thr359 reside in the loop between ankyrin repeats 3 and 4, close to His334 (Figure 4A). Phosphorylation of Ser356 and Thr359 may facilitate formation of the His300 / His334-mediated hydrogen-bond network that stabilizes NPR1 ankyrin repeats for interaction with TGAs

[0028] . Indeed, non-phosphorylatable mutations of Ser356 and Thr359 disrupt NPR1-TGA interaction and compromise NPR1’s transcriptional activity, while phospho-mimetic mutants retain their activity (Figures 4B-4H, 5, and Figure 13). Moreover, phosphorylation of the corresponding residues in BnNPR1 and OsNPR1 is crucial for their function (Figure 6), suggesting conservation of these phosphorylation events in NPR1 orthologs. Thus, eNAD(P)-triggered LecRK-VI.2-mediated phosphorylation at Thr359, and possibly Ser356, is critical for NPR1’s interaction with TGAs and transcriptional coactivator function. Few instances of signal transduction from cell-surface receptor kinases to transcription factors (TFs) have been documented. In animals, the type I and type II receptors for transforming growth factor-β (TGF-β), TβRI and TβRII, are dual specificity kinases

[0040] . TGF-β binding triggers the formation of a heterotetrameric receptor complex in which TβRII phosphorylates and activates TβRI. The activated TβRI then phosphorylates the TFs Suppressor of Mothers Against Decapentaplegic 2 (SMAD2) and SMAD3, leading to the formation of a trimeric complex with SMAD4, which translocates to the nucleus to initiate target gene transcription. Similarly, epidermal growth factor (EGF) receptors can directly phosphorylate signal transducer and activator of transcription (STAT) TFs, facilitating EGF signaling to the nucleus

[0041] . In plants, the receptor kinase FERONIA (FER) phosphorylates and destabilizes the TF MYC2, thereby inhibiting jasmonic acid (JA) signaling

[0042] . Binding of the peptide ligand RAPID ALKALINIZATION FACTOR23 to FER stabilizes MYC2, leading to elevated JA signaling. Furthermore, the kinase domains of MERISTEMATIC RECEPTOR-LIKE KINASE and CRINKLY4 phosphorylate the TFs AGAMOUS-LIKE24 and WUSCHEL-RELATED HOMEOBOX5 in vitro, respectively; however, the biological relevance of the phosphorylation remains unclear [43, 44]. The findings disclosed here demonstrate that cell-surface receptor kinases can directly transmit signals to transcriptional coactivators. Specifically, the eNAD(P) Attorney Docket no.10457-609PC0 receptor LecRK-VI.2 directly transmits signals to NPR1, facilitating its interaction with TGAs and activation of target gene transcription (Figure 7). The SAR signaling process encompasses four distinct stages: signal generation at the initial infection site, signal translocation through vascular tissues, signal transduction in systemic tissues, and SAR execution

[0045] . One of previous research performed in this lab established that SAR mobile signals induce the accumulation of eNAD(P) in systemic tissues, where it binds to its receptor, LecRK-VI.2 [16, 23]. In this study, it was demonstrate that eNAD(P) binding activates LecRK-VI.2, which subsequently phosphorylates NPR1, a receptor / PM-bound transcriptional coactivator. Once phosphorylated, NPR1 dissociates and migrates to the nucleus, where it interacts with TGAs to activate target gene expression (Figure 7). The results reveal that the eNAD(P) / LecRK-VI.2 / NPR1 / TGAs signaling axis is central to signal transduction in systemic tissues and provide a crucial basis for further investigation into the SAR signaling process and the development of durable crop protection strategies. B. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, microbiology, genetics, protein, and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed through the present specification unless otherwise indicated. The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / −10% or less, + / −5% or less, + / −1% or less, and + / −0.1% or less of and from the specified value, insofar such variations are Attorney Docket no.10457-609PC0 appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed. Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination. The term “nucleotide” as used herein refers to a sub-unit of a nucleic acid (whether DNA or RNA or an analogue thereof) which may comprise, but is not limited to, a phosphate group, a 5-carbon sugar group and a nitrogen containing base, as well as analogs of such sub-units. Other groups (e.g., protecting groups) can be attached to the sugar group and nitrogen containing base group. It will be appreciated that, as used herein, the terms “nucleotide” and “nucleoside” will include those moieties which contain not only the naturally occurring purine and pyrimidine bases, e.g., adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U), but also modified purine and pyrimidine bases and other heterocyclic bases which have been modified (these moieties are sometimes referred to herein, collectively, as “purine and pyrimidine bases and analogs thereof”). As used herein, the term “nucleotide sequence” or “nucleic acid sequence” refers to any fragment of polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA or modified RNA or DNA, among others, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or double-stranded, or a mixture of single- and double-stranded regions. They may comprise artificial nucleic acids including peptide nucleic acids (PNA), Morpholino and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA), threose nucleic acids (TNA) Attorney Docket no.10457-609PC0 and hexitol nucleic acids (HNA). Each of these is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule. The term “vector” as used herein refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked, usually a DNA molecule that is used as a vehicle to carry a particular foreign nucleic acid sequence – usually DNA into a host / recipient cell where it can be replicated and / or expressed. The vector typically includes features to facilitate the manipulation of DNA as well as a genetic marker for their selective recognition. The most common vectors are DNA plasmids, viruses and artificial chromosomes. Certain vectors are capable of autonomous replication in a host cell into which they are introduced. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. The term “plasmid” as used herein refers to a double-stranded, covalently closed, circular DNA that can be isolated from bacterial cells, which exists in its bacterial hosts as extrachromosomal pieces of DNA that vary in size from 1 kb to >200 kb. Most of the plasmids used in molecular cloning have a multiple cloning site (MCS), also called a polylinker, which is a short segment of DNA which contains various restriction sites a standard feature of engineered plasmids for the insertion of the foreign DNA. In addition, the plasmid should have an origin of replication (ori) site – usually bacterial origin where DNA replication is initiated, marker genes – antibiotics resistance gene for selection and / or screening with antibiotics, and promoters – usually viral origin for gene expression. It should be small in size so that it can easily delivered into the host cell. Plasmids do not generally replicate in the host plant cells. By performing a process of DNA transfection or transformation, a plasmid which contains a gene of interest is efficiently delivered to the cells of interest. Numerous plasmid vectors are commercially available, and the modification thereof for specific cloning strategies is well known to the skilled person in the field. As used herein, the term “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are Attorney Docket no.10457-609PC0 substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of one and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and one. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC / GENE (Intelligenetics, Mountain View, Calif.). As used herein, the term “CRISPR-Cas endonuclease system” refers to a gene editing system utilizing a target sequence recognizing endonucleases. As for gene editing system, CRISPR-Cas (Clustered Regularly InterSpaced Palindromic Repeats-Crispr associated) system is most used to generate site-specific double-strand breaks. Cas9 is a single protein endonuclease isolated from Streptococcus pyogenes. In order to be delivered to the target site, Cas9 binds to an engineered, single-stranded guide RNA (gRNA or sgRNA) comprising crRNA and tracrRNA. In more detail, at the 5’end of gRNA, there is an approximately 20-30 nucleotides that can be customized to complement the desired target site, and at the 3’ end, there are multiple stem-loop structures, which are the structure for RNA and protein (i.e., Cas9) binding. Cas9 has molecular weight of about 158 kDa, a large protein containing two lobes, a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe binds to the hybridization duplex formed by the gRNA and the target strand of DNA, and the NUC lobe contains two nuclease domains that are responsible for two strand cuts of the target DNA, generating ds-break with a blunt end. Cas9 also contains a region that recognizes a short sequence (usually 3-5 nucleotides) known as the protospacer-adjacent motif (PAM). If the target sequence complementary to the gRNA is adjacent to a PAM, the Cas9 complex cleaves both strands. The Cas enzyme will not be able to bind or cut the target DNA if the PAM sequence isn't next to the target sequence, which guides the Cas enzyme and tells it where to bind and cut the target DNA. However, although Cas9 is the most commonly used endonuclease, other Cas endonucleases such as Cas12a, which recognizes a different PAM from that of Cas9, uses just a one-part gRNA (i.e., only crRNA), and creates Attorney Docket no.10457-609PC0 staggered cut with 5′ overhang (sticky-end cut) near the PAM sequence, or other types of sequence specific nucleases can be contemplated, including zinc-finger nuclease (ZFNs) and transcription activator-loke effector nucleases (TALENs). Generally, two types of gene editing are possible, gene knock-out and gene knock-in. These approaches utilize nucleases, which are engineered to specifically bind a target DNA sequence, e.g., in the middle of the target gene, or upstream or downstream of the target gene, and to cut ds-DNA at the target site. As a response to cut, the cell tries to repair the break via either a non-homologous end joining repair (NHEJ) or a homology-directed repair (HDR). For gene knock-out approach, non-homologous end joining (NHEJ) is preferred. If a blunt-end generating endonuclease (e.g., Cas9) is used, the break ends are directly ligated so as to introduce deletion mutations into the target gene, and if a sticky-end generating endonuclease (e.g., Cas12a) is used, a short random insertion will occur after fragment deletion so as to make the gene dysfunctional. Thus, in either case, undesirable genes can be made dysfunctional. On the contrary, for introducing a desirable trait, gene knock-in approach can be contemplated, and homology directed repair (HDR) is preferred since HDR is suitable for precise genetic modification. To guide gene repair or introduce substitution mutations as desired, a homologous template (donor sequence) is required, which comprises a gene to be inserted or replaced as well as 5’- and 3’ homology arms flanking the gene in order to induce homologous recombination between the template fragment and the recipient DNA. For gene editing, CRISPR-Cas system needs to be introduced into plant cells. There are different methods. For example, Cas endonucleases and guide RNAs can be delivered using Agrobacterium and T-DNA system or as ribonucleoprotein (RNP) complexes. For the delivery of RNP or protein as well as genes into plant cells, various biological, chemical, and physical methods can be employed, and such methods include lipofection, calcium precipitation, electroporation, heat shock and biolistic particle delivery system (gene gun) can be contemplated. In gene gun method, heavy metal particles are coated with exogenous DNA (transgenes), RNA, and / or protein of interest, and these micro-projectiles are fired into cells using mechanical force to introduce genetic information into plant cells. C. Examples of Embodiments Attorney Docket no.10457-609PC0 Here is disclosed a phospho-mimetic mutant protein of NPR1 for the establishment of a long-lasting defense mechanism called systemic acquired resistance (SAR), which provides whole-plant resistance against subsequent infections by a broad spectrum of pathogens. To this end, at least one of Ser (S) and / or Thr (T) amino acid residue(s) of the NPR1, which needs to be phosphorylated to be functional, is substituted with negative charged amino acid Asp (D). However, in some cases Glu (E) might be contemplated as another substitution amino acid, considering the negative charged functional group of both amino acids. The exemplary NPR1 protein presented here is Arabidopsis NPR1 (AtNPR1), however other proteins that have similar amino acid sequences and nucleotide sequences with at least 80% sequence identity and similar function may be candidates for such phosphor-mimetic modification. Other examples presented in this disclosure are NPR1 of Brassica rapa (BrNPR1) and NPR1 of Oryza sativa (OsNPR1). The Ser and / or Thr amino acid residue(s) that can be phosphorylated or substituted with Asp are Ser356, Thr359, Thr373, Ser571, Thr572, Ser573, Ser 574, T575, S576, and Ser589. Considering the LC-MS / MS analysis of NPR1 phosphorylation sites presented in Examples, Ser 356 (S356) and Thr 359 (T359) amino acid residues of the NPR1 are substituted with Asp (D) for phospho-mimetic modification of NPR1. However, considering that NPR1 nuclear import is also regulated by phosphorylation at Thr373 and Ser589, Thr373 and Ser589 may be substituted with Asp in addition to the substitution of Ser 356 and Thr 359. In the case of NPR1 proteins from other plants than Arabidopsis, the Ser and / or Thr amino acid residue(s) at the corresponding position(s) to the Ser 356 and / or Thr 359 residues of the Arabidopsis NPR1, when aligned together with the Arabidopsis NPR1, can be substituted with Asp to make them phospho-mimetic. For example, BrNPR1 Ser340 and OsNPR1 Thr363, correspond to Arabidopsis NPR1 Ser356 and Thr359, and can be substituted with Asp. The phospho-mimetic mutant protein of NPR1, NPR1-S356D / T359D, comprises an amino acid sequence of SEQ ID NO:1, and is optionally fused / tagged at its N- or C-terminus with a tag such as fluorescence protein (e.g., GFP, superfolder GFP (sfGFP), and other fluorescent proteins such as RFP, OFP, YFP, BFP, and the like), maltose-binding protein (MBP), glutathione S- transferase (GST), horseradish peroxidase (HRP), His (or hexa-histidine), FLAG, streptavidin- binding peptide (SBP), Strep II, calmodulin-binding peptide (CBP), chitin-binding domain (CBD), Attorney Docket no.10457-609PC0 HA, c-Myc, biotinylation, and the like for convenience in protein purification and antibody detection. When NPR1-S356D / T359D protein is expressed as a tagged protein, it can be expressed in Arabidopsis thaliana, Nicotiana benthamiana or Tobacco BY2 and purified using antibody- beads or antibody-resin columns against the tag. Since NPR1 is a cytosolic protein, NPR1-S356D / T359D protein or a gene encoding the protein, which comprises a nucleotide sequence of SEQ ID NO:2, needs to be delivered into the cytosol or nucleus of plant cells, which can be achieved using liposomes. Liposomes are artificially formed minute spherical lipid and / or phospholipid vesicles enclosing a water droplet that can contain soluble hydrophilic molecules (e.g., DNA, RNA, protein, drugs, or other substances) into the cells of an animal or plant. Liposomes have an advantage that it can protect protein and / or nucleic acids. For the delivery of desired protein or gene to plant cells, liposomes can be applied to plants in a few ways. One method is leaflet submerging, which is a method of submerging a leaf in a vial containing liposomes for 72–96 hours. The plant can remain in soil or a hydroponic solution while a leaf is submerged. Another method is foliar application, in which liposomes are directly sprayed onto the leaves and stems of plants. This method is quick and easy, and it can be used to apply small amounts of nutrients or agents. However, if need be, the liposomes are syringe-infiltrated into the crop. Liposomes applied to the leaves of plants can penetrate the leaves and release their cargo into the cytoplasm of plant cells in a 72 h period, where the cargo molecules can be translocated throughout the plant since plant cells are connected to each other through specialized channels called "plasmodesmata" which penetrate the cell walls, allowing for direct cytoplasmic communication between neighboring cells; essentially acting as tunnels between the cells to transfer nutrients and other molecules. (Karny et al., 2018: Karny, Zinger, Kajal, Shainsky- Roitman and Schroeder, Therapeutic nanoparticles penetrate leaves and deliver nutrients to agricultural crops. Scientific Reports, 8 (1) (2018), pp. 1-10) For the delivery of NPR1-S356D / T359D protein using liposomes, a method comprising the following steps can be contemplated. Attorney Docket no.10457-609PC0 i. isolate NPR1-S356D / T359D protein comprising SEQ ID NO:1 or any amino acid sequence with at least 80% sequence identity thereto, which is optionally fused with a tag at its N- or C terminus; ii. dissolve the isolated protein in a buffer, wherein buffer pH is within a range of 7.0-8.0; iii. mix a certain amount of the protein in the solution of step ii with a certain amount of liposomes; and iv. immerse leaves of a plant in a solution comprising the protein-containing liposomes of step iii, spray solution comprising the protein-containing liposomes on the leaves, or inject solution comprising the protein-containing liposomes into a plant, optionally into infection sites. To create a transgenic plant with improved immunity, NPR1-S356D / T359D gene can be delivered into the cytosol or nucleus for protein expression. For the delivery of NPR1- S356D / T359D gene, a method comprising the following steps can be contemplated. i. isolate protein expression plasmid comprising a nucleotide sequence of SEQ ID NO 2 or any nucleotide sequence with at least 80% sequence identity thereto, wherein the plasmid optionally comprises a tag-encoding sequence at the 5’- or 3’ end of the nucleotide sequence of SEQ ID NO:2; ii. dissolve the plasmid in a buffer, wherein buffer pH is within a range of 7.0-8.0; iii. mix a certain amount of the plasmid in the solution of step ii with a certain amount of liposomes; and iv. immerse a plant seed or leaves in a solution comprising the plasmid-containing liposomes of step iii, spray solution comprising plasmid-containing liposomes on the leaves, or inject solution comprising the plasmid-containing liposomes into a plant, optionally into infection sites. For gene delivery, another method, i.e., Agrobacterium-mediated plant transformation utilizing T-DNA system can be contemplated. In this method, Agrobacterium is transformed with a plasmid comprising T-DNA, which originates from tumor-inducing (Ti) plasmid of the bacterium Agrobacterium tumefaciens, which comprises right border gene (RB), a reporter gene (e.g., gfp, luc, gus), multicloning site where the gene encoding NPR1-S356D / T359D is inserted, a selection marker gene against antibiotics (e.g., kanamycin, hygromycin, phosphinothricin (or glufosinate), Attorney Docket no.10457-609PC0 glyphosate, and phosphomannose isomerase), light border gene (LB), as well as promoters and terminators for each gene. Optionally, Agrobacterium (e.g., A. tumefaciens strain EHA105) is co- transformed with helper plasmid. For T-DNA system, the vir genes of Agrobacterium, which also originate from Ti-plasmid and responsible for transferring the T-DNA into plant cells, can be included in the plasmid comprising T-DNA fragment, or the vir genes are cloned in a separate plasmid, known as the vir helper plasmid (binary vector system). Agrobacterium can be transformed with plasmids using electroporation, heat shock or other methods known to person having ordinary skill in the art. The method utilizing Agrobacterium-T-DNA system harboring the desired gene comprises the following steps. i. isolate plasmid comprising T-DNA fragment comprising a nucleotide sequence of SEQ ID NO 2 or any nucleotide sequence with at least 80% sequence identity thereto, and optionally separately isolate plasmid comprising vir genes; ii. transform Agrobacterium with the plasmid(s) of step i; iii. culture the transformed Agrobacterium in the presence of a selection antibiotic; and iv. immerse a plant seed or leaves in a solution comprising Agrobacterium of step iii, spray solution comprising Agrobacterium on the leaves, or inoculate solution comprising Agrobacterium into a plant, optionally into infection sites. In addition, gene editing in the region comprising S356 and T359 can be contemplated, in which the fragment in the plant genome comprising S356 and T359 can be replaced with a donor fragment comprising S356D and T359D using CRISPR / Cas endonuclease system, if crRNAs or gRNAs, which recognize the 5’-cutting site sequence and the 3’-cutting site sequence for donor fragment insertion, are available. In such a case, dual targeting strategy with a pair of gRNAs for Cas9 can be employed. For gene insertion by homology-directed repair (HDR), i.e., homologous recombination, the donor fragment needs to have flanking sequences complementary to the upstream region of the 5’-cutting site and to the downstream region of the 3’-cutting site. When utilizing CRISPR / Cas system, a plasmid comprising a nucleotide sequence encoding a donor fragment comprising S356D and T359D can be co-transformed with a plasmid comprising nucleotide sequences encoding crRNA / gRNA and Cas endonuclease system. Also, the crRNA / gRNA and Cas endonuclease system can be delivered as RNP complex separately from the plasmid comprising a sequence encoding the donor fragment. Attorney Docket no.10457-609PC0 Using the methods described above, plants can have improved resistance or tolerance against various diseases caused by pathogens such as viruses, bacteria, and fungi. For example, NPR1-S356D / T359D can be overexpressed in citrus to overcome HLB. In addition, improved systemic immunity in a NPR1-S356D / T359D-transgenic plant produced by the methods described above can be further enhanced by injecting NAD and / or NADP+solution into a plant. For example, 0.1-5 mM, optionally about 0.8 mM NADP+solution (pH = 5.7) is syringe-infiltrated into a plant, optionally at infection sites. Further, 1-10 mM, optionally about 4 mM nicotinic acid (NA), a cell membrane permeable intermediate that can be converted to NAD through the salvage pathway, can be syringe-infiltrated into a plant to restore intracellular NAD level. The immunity of a NPR1-S356D / T359D-transgenic plant can also be enhanced by syringe- infiltrating pipecolic acid (Pip) and / or its derivative N-hydroxypipecolic acid (NHP) solution into a plant (0.1-5 mM, optionally 0.25-0.5 mM). Considering the effects of NAD / NADP+on NPR1 through LecRK-VI.2, a composition comprising NPR1-S356D / T359D-containing liposomes, NAD / NADP+, and / or Pip / NHP can be contemplated for injection / infiltration to treat various plant diseases caused pathogen infection, and the composition can be packed in a syringe or pouch with or without a needle. Sequences NPR1-S356D / T359D protein sequence (SEQ ID NO: 1): MDTTIDGFADSYEISSTSFVATDNTDSSIVYLAAEQVLTGPDVSALQLLSNSFESVFDSPDDFYSDAK LVLSDGREVSFHRCVLSARSSFFKSALAAAKKEKDSNNTAAVKLELKEIAKDYEVGFDSVVTVLAYVY SSRVRPPPKGVSECADENCCHVACRPAVDFMLEVLYLAFIFKIPELITLYQRHLLDVVDKVVIEDTLVI LKLANICGKACMKLLDRCKEIIVKSNVDMVSLEKSLPEELVKEIIDRRKELGLEVPKVKKHVSNVHKA LDSDDIELVKLLLKEDHTNLDDACALHFAVAYCNVKTATDLLKLDLADVNHRNPRGYTVLHVAAMR KEPQLILSLLEKGASADEADLEGRTALMIAKQATMAVECNNIPEQCKHSLKGRLCVEILEQEDKREQI PRDVPPSFAVAADELKMTLLDLENRVALAQRLFPTEAQAAMEIAEMKGTCEFIVTSLEPDRLTGTKRT SPGVKIAPFRILEEHQSRLKALSKTVELGKRFFPRCSAVLDQIMNCEDLTQLACGEDDTAEKRLQKK QRYMEIQETLKKAFSEDNLELGNSSLTDSTSSTSKSTGGKRSNRKLSHRRR Attorney Docket no.10457-609PC0 NPR1-S356D / T359D DNA sequence (SEQ ID NO: 2): ATGGACACCACCATTGATGGATTCGCCGATTCTTATGAAATCAGCAGCACTAGTTTCGTCGCTAC CGATAACACCGACTCCTCTATTGTTTATCTGGCCGCCGAACAAGTACTCACCGGACCTGATGTAT CTGCTCTGCAATTGCTCTCCAACAGCTTCGAATCCGTCTTTGACTCGCCGGATGATTTCTACAGC GACGCTAAGCTTGTTCTCTCCGACGGCCGGGAAGTTTCTTTCCACCGGTGCGTTTTGTCAGCGA GAAGCTCTTTCTTCAAGAGCGCTTTAGCCGCCGCTAAGAAGGAGAAAGACTCCAACAACACCG CCGCCGTGAAGCTCGAGCTTAAGGAGATTGCCAAGGATTACGAAGTCGGTTTCGATTCGGTTGT GACTGTTTTGGCTTATGTTTACAGCAGCAGAGTGAGACCGCCGCCTAAAGGAGTTTCTGAATGCG CAGACGAGAATTGCTGCCACGTGGCTTGCCGGCCGGCGGTGGATTTCATGTTGGAGGTTCTCT ATTTGGCTTTCATCTTCAAGATCCCTGAATTAATTACTCTCTATCAGAGGCACTTATTGGACGTTGTA GACAAAGTTGTTATAGAGGACACATTGGTTATACTCAAGCTTGCTAATATATGTGGTAAAGCTTGTAT GAAGCTATTGGATAGATGTAAAGAGATTATTGTCAAGTCTAATGTAGATATGGTTAGTCTTGAAAAGT CATTGCCGGAAGAGCTTGTTAAAGAGATAATTGATAGACGTAAAGAGCTTGGTTTGGAGGTACCTA AAGTAAAGAAACATGTCTCGAATGTACATAAGGCACTTGACTCGGATGATATTGAGTTAGTCAAGTT GCTTTTGAAAGAGGATCACACCAATCTAGATGATGCGTGTGCTCTTCATTTCGCTGTTGCATATTG CAATGTGAAGACCGCAACAGATCTTTTAAAACTTGATCTTGCCGATGTCAACCATAGGAATCCGA GGGGATATACGGTGCTTCATGTTGCTGCGATGCGGAAGGAGCCACAATTGATACTATCTCTATTG GAAAAAGGTGCAAGTGCAGACGAAGCAGACTTGGAAGGTAGAACCGCACTCATGATCGCAAAA CAAGCCACTATGGCGGTTGAATGTAATAATATCCCGGAGCAATGCAAGCATTCTCTCAAAGGCC GACTATGTGTAGAAATACTAGAGCAAGAAGACAAACGAGAACAAATTCCTAGAGATGTTCCTCCC TCTTTTGCAGTGGCGGCCGATGAATTGAAGATGACGCTGCTCGATCTTGAAAATAGAGTTGCACT TGCTCAACGTCTTTTTCCAACGGAAGCACAAGCTGCAATGGAGATCGCCGAAATGAAGGGAAC ATGTGAGTTCATAGTGACTAGCCTCGAGCCTGACCGTCTCACTGGTACGAAGAGAACATCACCG GGTGTAAAGATAGCACCTTTCAGAATCCTAGAAGAGCATCAAAGTAGACTAAAAGCGCTTTCTAAA ACCGTGGAACTCGGGAAACGATTCTTCCCGCGCTGTTCGGCAGTGCTCGACCAGATTATGAAC TGTGAGGACTTGACTCAACTGGCTTGCGGAGAAGACGACACTGCTGAGAAACGACTACAAAAG AAGCAAAGGTACATGGAAATACAAGAGACACTAAAGAAGGCCTTTAGTGAGGACAATTTGGAATT AGGAAATTCGTCCCTGACAGATTCGACTTCTTCCACATCGAAATCAACCGGTGGAAAGAGGTCTA ACCGTAAACTCTCTCATCGTCGTCGGTGA EXAMPLES Example 1. METHOD DETAILS 1.1. Plasmid construction Attorney Docket no.10457-609PC0 The coding sequences of the Arabidopsis genes NPR1, BAK1, LecRK-VI.2, LecRK-I.8, Lti6b, and TGAs were amplified from the cDNA of Col-0. To generate transgenic Arabidopsis lines expressing NPR1-GFP, the coding sequence of NPR1 was amplified and inserted into SacI / SalI-linearized pCAMBIA1300S-GFP

[0023] using In- Fusion Snap Assembly (Takara). To generate transgenic Arabidopsis lines expressing BrNPR1- GFP, the coding sequence of BrNPR1 was amplified from the cDNA of B. rapa var. R500 and inserted into KpnI / BamHI-digested pCAMBIA1300S-GFP using In-Fusion Snap Assembly (Takara). To generate transgenic Arabidopsis lines expressing OsNPR1-GFP, codon-optimized OsNPR1 coding sequence was synthesized and inserted into KpnI / BamHI-digested pCAMBIA1300S-GFP using In-Fusion Snap Assembly (Takara). To express LecRK-VI.2-HA-TurboID and Lti6b-HA-TurboID, the coding sequences of LecRK-VI.2 and Lti6b were amplified and inserted into XhoI / XbaI-linearized pBASTA-HA- TurboID

[0046] using T4 ligase. To express TGAs-HA-TurboID, the coding sequences of TGA1, TGA2, and TGA3 were amplified and inserted into KpnI / XhoI-linearized pBASTA-HA-TurboID using T4 ligase. For transient expression in N. benthamiana, the coding sequences of TGAs (TGA1, TGA3, and TGA6) and NPR1 were amplified and inserted into BamHI / SalI-digested pCAMBIA1300S- GFP and SacI / SalI-digested pCAMBIA1300S-FLAG

[0047] , respectively, using T4 ligase or In- Fusion Snap Assembly (Takara). For yeast two-hybrid assays, the fragments encoding the kinase domains (KDs) of LecRK- VI.2KD, BAK1KD, and LecRK-I.8KD were amplified and inserted into pGBKT7, and the coding sequence of NPR1 was inserted into pGADT7 using In-Fusion Snap Assembly (Takara). The bait and prey vectors were linearized by NdeI and BamHI digestion before the In-Fusion reactions. For in vitro kinase assay, the coding sequence of NPR1 was amplified and inserted into pET28a-sfGFP-His using Golden Gate Assembly. pMAL-p2X-MBP-LecRK-VI.2KD and pGEX- 4T-1-GST-BAK1KD have been described previously

[0023] . For SLC assays, the coding sequence of NPR1 was inserted into SacI / BamHI digested pCAMBIA1300S-NlucC-FLAG using In-Fusion Snap Assembly. pCAMBIA1300S-LecRK- Attorney Docket no.10457-609PC0 VI.2-NlucN-HA and pCAMBIA1300S-LecRK-I.8-NlucN-HA have been described previously

[0032] . Point mutations in NPR1, OsNPR1, BrNPR1, and LecRK-VI.2 were generated using the QuikChange site-directed mutagenesis kit (Agilent) or overlap extension PCR. All the primers used for plasmid construction were listed in Table 1. To complement the fin4-3 mutant, the 35 S:FIN4 construct was generated. The full length CDS (1956 bp) of FIN4 was amplified from wild-type cDNAs using the oligos KpnI-FIN4F and XbaI-FIN4R (Table 2). The PCR products were digested with KpnI and XbaI and cloned into the kpnI / XbaI-digested pCAMBIA1300S vector to create pCAMBIA1300S-FIN4. To make Dex:FIN4 / fin4-3 and Dex:LecRK-VI.2 / lecrk-VI.2 plants, full length CDSs of FIN4 (1956 bp) and LecRK-VI.2 (2049 bp) were amplified from wild-type cDNAs using oligos attB1FIN4F / attB1FIN4R and attB1LecRK-VI.2 F / attB1LecRK-VI.2 R (Table 2), respectively. PCR products were used as templates for the second round of PCR with the oligos Adapter attB1 and Adapter attB2 to add attB1 and attB2 sites to the ends. The resulting PCR products were cloned into the pDONR221 vector by Gateway BP reactions to obtain the entry vectors pDONR221-FIN4 and pDONR221-LecRK-VI.2. Gateway LR reactions between the entry vectors and the pOpON vector were then performed to create pOpON-FIN4 and pOpON-LecRK-VI.2. All constructs were confirmed by sequencing. The A. tumefaciens strain GV3101(pMP90) was transformed with the binary constructs carrying the indicated transgenes. The floral dip method was used to transform Arabidopsis plants. T1 transgenic plants were selected on ½ MS plates containing appropriate antibiotics. Single insertion and homozygous lines were selected in the T2 and T3 generations, respectively. Table 1. Oligos used for subcloning in this study Table 1. Primers used in this study SEQ ID Attorney Docket no.10457-609PC0 TGA3-SalI-R8ACGCGTCGACAGTGTGTTCTCGTGGACGAGCT TGA6-BamHI-F9CGCGGATCCATGGCTGATACCAGTTCAAGGACT TGA6-SalI-R 10ACGCGTCGACCTCTCTTGGCCGGGCAAGOsNPR1-KpnI-F 11GCTTTCGCGAGCTCGGTACCATGGAACCACCCACATCACATGTG OsNPR1-BamHI-R CACCTCTAGAGGATCCCCTTCTTGGTCTAATGGCTCCGCTTTCGCGAGCTCGGTACCATGGATTCCTTTGCTGGATTCGGBrNPR1-KpnI-F BrNPR1-BamHI-R CACCTCTAGAGGATCCGCGACGCCGATGGTAGAG pCAMBIA1300s-FLAG NPR1-SacI-F15TCTCGAGCTTTCGCGAGCTCATGGACACCACCATTGATGGATTC NPR1-SalI-R16CTTTACCCATGTCGACCCGACGACGATGAGAGAGTTTACG pGBKT7 17GGAATTCCATATGTTCTTCGTCATGTACAAGAAGAGLecRK-VI.2KD-NdeI-F LecRK-VI.2KD-XhoI-R18CCCTCGAGCTACTGACTGATACGAGAAGTCLecRK-I.8KD19-NDeI-F GGAATTCCATATGCTATACAGAAGAAACAAGTATGCAG 20GCGTCGACTCATCGTCCAATTCCGTATTGLecRK-I.8KD-SalI-R 21GGAATTCCGAAGGAAAAAGCCGCAGGACBAK1KD-EcoRI-F 22BAK1KD-SalI-R GCGTCGACTTATCTTGGACCCGAGGGGTA23OsNPR1-NdeI-F AGGAGGACCTGCATATGATGGAACCACCCACATCACATGTG 24OsNPR1-BamHI-R GCAGGTCGACGGATCCTCACCTTCTTGGTCTAATGGCTCC 25 BrNPR1-NdeI-FAGGAGGACCTGCATATGATGGATTCCTTTGCTGGATTCGG26BrNPR1-BamHI-R GCAGGTCGACGGATCCTCAGCGACGCCGATGGTAGAGpGADT727NPR1-NdeI-F CAGATTACGCTCATATGATGGACACCACCATTGATGGATTCG 28NPR1-BamHI-R CGAGCTCGATGGATCCTCACCGACGACGATGAGAGAG pCAMBIA1300s-NlucC-FLAG 29NPR1-SacI-F CTCTCGAGCTTTCGCGAGCTCATGGACACCACCATTGATGGAT 30NPR1-BamHI-R TCCGCCGCTTCCGCCGGATCCCCGACGACGATGAGAGAGTTTA pBASTA-HA-TurboID 31CCCTCGAGATGAGTACAGCCACTTTCGTAGLti6b-XhoI-F GCTCTAGACTTGGTGATGATATAAAGAGCGLti6b-XbaI-R CCCTCGAGATGGGCACACAAAGATCCATGLecRK-VI.2-XhoI-F GCTCTAGACTGACTGATACGAGAAGTCGAAGAAAC LecRK-VI.2-XbaI-R TGA1-KpnI-F CGGGGTACCATGAATTCGACATCGACACATTTT GCGTCGACCGTTGGTTCACGATGTCGAGTTGA1-SalI-R CGGGGTACCATGGCTGATACCAGTCCGAGAATGA2-KpnI-F GCGTCGACCTCTCTGGGTCGAGCAAGCTGA2-SalI-R TGA3-KpnI-F CGGGGTACCATGGAGATGATGAGCTCTTCTTCTT GCGTCGACAGTGTGTTCTCGTGGACGAGCTTGA3-SalI-R pET-28a-sfGFP-His 41CGGGATCCGAGACCGACACCACCATTGATGGATTCNPR1-BamHI / BsaI-F NPR1-Sall / BsaI-R ACGCGTCGACGAGACCCCGACGACGATGAGAGAGTTTAC CATGCCATGGTTAGCAAAGGTGAAGAAC sfGFP-NcoI-F sfGFP-BamHH / EcoRI / SacI / SalI-R GCGTCGACGAGCTCGAATTCGGATCCGCTGCCTTTATACAGTTCATC Attorney Docket no.10457-609PC0 Site-directed mutagenesis npr1-S356A-F45GAAAAAGGTGCAAGTGCAGCAGAAGCAACTTTGGAAGnpr1-S356A-R46CTTCCAAAGTTGCTTCTGCTGCACTTGCACCTTTTTC npr1-T359A-F47GCAAGTGCATCAGAAGCAGCTTTGGAAGGTAGAACCGCnpr1-T359A-R48npr1-S356A / T49GCGGTTCTACCTTCCAAAGCTGCTTCTGATGCACTTGC359A-F GAAAAAGGTGCAAGTGCAGCAGAAGCAGCTTTGGAAGGTAGAACCGC npr1-S356A / T359A-R50GCGGTTCTACCTTCCAAAGCTGCTTCTGCTGCACTTGCACCTTTTTC npr1-S356D / T359D-F51GTGCAAGTGCAGACGAAGCAGACTTGGAAGGTAGAACCGCACTnpr1-S356D / T359D-R52AGTGCGGTTCTACCTTCCAAGTCTGCTTCGTCTGCACTTGCAC npr1- 53 S571A / T572A / S573A / S574A / T575A / S57 GGAAATTCGTCCCTGACAGATGCGGCTGCTGCCGCAGCGAAATCAACC 6A-F GGTGGAAAG npr1- 54 S571A / T572A / S573A / S574A / T575A / S57 CTTTCCACCGGTTGATTTCGCTGCGGCAGCAGCCGCATCTGTCAGGGA 6A-R CGAATTTCC LecRK-VI.2-K395E-F55ATTCGGATCCCATCGCAGTGGAGAAGATAATTCCAAGTAGCAGGCAAG LecRK-VI.2-K395E-R56CTTGCCTGCTACTTGGAATTATCTTCTCCACTGCGATGGGATCCGAAT Osnpr1-T363A-F57AGCACGACCCGCGGATGTGGCTTTTGATGGAAGAAAAGCAGTGCA Osnpr1-T363A-R58 TGCACTGCTTTTCTTCCATCAAAAGCCACATCCGCGGGTCGTGCTBrnpr1-S340A / T342A-F59GACAAAAGGGTCACGTGCAGCGGAAGCGTGTTTGGAAGGTAGAAC Brnpr1-S340A / T342A-R60GTTCTACCTTCCAAACACGCTTCCGCTGCACGTGACCCTTTTGTC Table 2. Oligos used for subcloning in this study 1.2. In vitro kinase assay The in vitro kinase assay was performed as described previously

[0048] with slight modifications. Briefly, 5 µg of indicated recombinant proteins were incubated in 30 µL kinase assay buffer (25 mM Tris-HCl, pH 7.5, 10 mM MnCl2, 10 mM MgCl2, 10 mM ATP) at 30°C for 1 hr. The reaction was stopped by adding 8 mL of Laemmli buffer, and the sample was boiled for 8 min and separated in 8% SDS-PAGE gel. Phosphorylation of NPR1 was detected by immunoblotting with an anti-pThr antibody (1:1000, Cell signaling). 1.3. LC-MS / MS detection of NPR1 phosphorylation sites Attorney Docket no.10457-609PC0 To identify in vitro LecRK-VI.2 phosphorylation sites on NPR1, purified recombinant proteins sfGFP-NPR1-His and MBP-LecRK-VI.2KD or GST-BAK1KD were incubated in kinase assay buffer (25 mM Tris-HCl, pH 7.5, 10 mM MnCl2, 10 mM MgCl2, 10mM ATP) at 30°C for 1 h with gentle shaking. The reaction was terminated by adding Laemmli buffer, and the samples were boiled for 8 min and separated in 8% SDS-PAGE gel. The SDS-PAGE gel was then stained with Coomassie Brilliant Blue (CBB) R250, and the sfGFP-NPR1-His bands were cut out for LC- MS / MS analysis. Protein in-gel trypsin-digestion, peptides extraction with acetonitrile, and phosphopeptides enrichment using a NuTip TiO2+ ZrO2(Glygen Inc. NT2TIZR) were described in detail by Perron et al [49, 50]. Ten μL of acidified phospho-enriched peptide sample was injected on Acclaim PepMap 100 C18 precolumn (20 mm by 75 μm; 3 μm) and separated on a PepMap RSLC C18 analytical column (250 mm by 75 μm; 2 μm) at a flow rate of 250 nl / min followed by LC-MS / MS on Easy-nLC and Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Mobile phase A was water with 0.1% formic acid and mobile phase B was acetonitrile with 0.1% formic acid. A 90 min gradient was used for separation in positive mode. The gradient started at 2% B, increased to 5% B till 5 min, linearly increased to 40% till 70 min, 45% till 75 min, and ended at 98% B at 90 min. A collision-induced dissociation (CID) and electron transfer dissociation (ETD) decision tree method was used for this experiment. The Orbitrap MS1 resolution was 120 k, the scan range was 350 to 1800 m / z, the nanoelectrospray voltage was 2000, the automatic gain control target was set to 200,000, microscan was 1, the RF lens was 55% and the maximum inject time was set to 50 ms. The MS / MS spectra were acquired in the linear ion trap with ETD (with charge states 6-8) and CID (with charge state 2-5), a mass window of 1.3, collision energy of 35, activation time of 10 ms, and activation q of 0.25. To identify NADP+-induced phosphorylation sites on NPR1 in vivo, leaves of four-week- old double transgenic plants overexpressing NPR1-GFP and LecRK-VI.2-HA-TurboID were infiltrated with 0.8 mM NADP+or water. Leaf tissues were collected 30 min later. Total proteins were extracted from 20 g collected leaf tissues with extraction buffer (50 mM HEPES-KOH, pH 7.5, 150 mM KCl, 1 mM EDTA, 0.5% Triton X-100, 5 mM NaF, 5 mM Na3VO4, and proteinase inhibitor cocktail). To precipitate NPR1-GFP, the total proteins were incubated with precleared protein A agarose beads (Santa Cruz) and the anti-NPR1 antibody for 3 hr at 4°C with rotation. After immunoprecipitation, the beads were washed three times with extraction buffer without proteinase inhibitor cocktail and boiled in Laemmli buffer for 8 min to elute the bound proteins. Attorney Docket no.10457-609PC0 The eluted proteins were separated in 8% SDS-PAGE gel and stained with Coomassie brilliant blue (CBB). The NPR1-GFP band was cut for trypsin digestion and LC-MS / MS analysis as described above. 1.4. Phospho-specific antibody development Rabbit polyclonal anti-pThr359 antibody was developed by Pierce / Thermo Fisher Scientific (www.thermofisher.com) using an NPR1-pThr359 peptide [C-ASASEA(pT)LEGRTA- amide]. The specificity of the newly developed anti-pThr359 antibody was confirmed using LecRK-VI.2KD-treated NPR1 and npr1T359A(Figure 2E). The anti-pThr359 antibody was diluted 1:2000 for detecting NADP+-induced phosphorylation of Thr359 in vivo and for phosphorylation of Thr359 by LecRK-VI.2KD in in vitro kinase assays. 1.5. Phos-tag gel assay Phos-tag gel assays of in vivo NPR1 phosphorylation were conducted according to the Phos-tag™ SDS-PAGE guidebook (FUJIFILM wako). Briefly, four-week-old plants were treated with 0.8 mM NADP+or water for 45 min, ground in liquid nitrogen, and homogenized in modified extraction buffer with a low concentration of salt (25 mM Tris-HCl, pH 7.5, 10 mM NaCl, 5 mM NaF, 5 mM Na3VO4, and EDTA-free protease inhibitor cocktail). The lysate was centrifuged at 16000 g for 15 min at 4°C, and the supernatant was collected. The extracted proteins were resolved in a 6% SDS-PAGE gel containing 50 μM Phos-tag acrylamide AAL-107 (NARD Institute, FUJIFILM wako) and 100 μM ZnCl2. After 6 h electrophoresis at 120V, the gel was treated with transfer buffer containing 10 mM EDTA for three times (each time for 10 min) and then soaked in transfer buffer without EDTA twice (each time for 10 min). The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using transfer buffer at 25V overnight. The membrane was blocked with 5% fat-free milk and probed with anti-GFP antibody (1:5,000, Clontech). 1.6. Yeast two-hybrid assay The yeast two-hybrid assays were performed according to the Matchmaker GAL4 Two- Hybrid System manual (Clontech). Briefly, paired bait and prey constructs were co-transformed into the yeast strain AH109, and the resulting cells were plated on SD-Trp-Leu agar plates. Yeast colonies formed on the SD-Trp-Leu medium were suspended in sterile water and then simultaneously dropped onto SD-Trp-Leu and selective agar plates (SD-Trp-Leu-His+3AT or SD- Attorney Docket no.10457-609PC0 Trp-Leu-His-Ade). The protein-protein interactions were determined by the growth rate of yeast cells on the selective medium. 1.7. Split Nano luciferase complementation (SLC) assay SLC assays were performed as previously described

[0051] . Briefly, different combinations of A. tumefaciens strain EHA105 cells harboring the indicated constructs were co-infiltrated into fully expanded leaves of five-week-old N. benthamiana plants. Three days later, 20 μM Coelenterazine h (luciferin, Nanolight 301) were applied to the infiltrated leaves by infiltration. Leaf disks (7 mm in diameter) were immediately collected with a hole punch, and each leaf disc was settled in a well of a 96-well white plate (150 μL water / well). The luciferase activities were measured using a GloMax Discover luminometer (Promega). After measurement, the leaf disks were kept for protein abundance analysis. The indicated NlucN / NlucC fusion proteins were detected by immunoblotting with anti-HA (1:3000, Roche) and anti-FLAG (1:1000, Sigma) antibodies. 1.8. Protein fractionation assay Approximately 0.3 g leave tissues of the double transgenic Arabidopsis plants expressing LecRK-VI.2-HA-TurboID and NPR1-GFP were collected and ground into powder in liquid nitrogen. The plasma membrane (PM) and cytosol fractions were isolated using the Minute Plasma Membrane Protein Isolation Kit for Plants (Invent Biotechnologies) following the manufacturer’s instructions. The presence of LecRK-VI.2-HA-TurboID and NPR1-GFP in the fractions was analyzed by immunoblotting with anti-HA and anti-GFP antibodies, respectively. The PM marker H+-ATPase and the cytoplasmic marker tubulin were detected using anti-H+-ATPase (1:5000, Agrisera) and anti-tubulin (1:1000, Agrisera) antibodies, respectively. 1.9. Co-immunoprecipitation (co-IP) assay Co-IP assays in N. benthamiana were performed as previously described

[0048] . Briefly, designated genes were transiently expressed in N. benthamiana leaves, and total proteins were extracted using an extraction buffer containing 50 mM HEPES-KOH (pH 7.5), 150 mM KCl, 1 mM EDTA, 0.5% Triton X-100, and proteinase inhibitor cocktail. For IP of GFP-fusion proteins, extracts were incubated for 2 hr at 4°C with 6 µL of GFP-trap magnetic agarose beads (Chromotek), followed by five washes with wash buffer (50 mM HEPES-KOH, pH 7.5, 150 mM Attorney Docket no.10457-609PC0 KCl, 1 mM EDTA, 0.5% Triton X-100). Precipitated proteins were eluted by adding 60 µL of Laemmli buffer and boiling for 8 min. For IP of FLAG-fusion proteins, total proteins were incubated for 2 hr at 4°C with 8 µL of anti-FLAG antibody-coupled agarose beads (Sigma), followed by six washes with wash buffer. Bound proteins were eluted using 100 µL 3 × FLAG peptides (Sigma). The precipitated proteins were separated in 8% SDS-PAGE gels and analyzed by immunoblotting with anti-GFP (1:5000, Clontech) and anti-FLAG (1:1000, Sigma) antibodies. For time-course co-IP assays, leaves of double transgenic Arabidopsis plants were infiltrated with 0.8 mM NADP+, collected at the indicated time points, and snap-frozen in liquid nitrogen. Total proteins were extracted with extraction buffer and incubated for 2 hr at 4°C with 6 µL of anti-HA antibody-coupled magnetic agarose beads (Cell signaling). Beads were washed six times with wash buffer, and precipitated proteins were eluted by boiling for 8 min in 60 µL of Laemmli buffer, separated in 8% SDS-PAGE gels, and analyzed by immunoblotting with anti-GFP (1:5000, Clontech) and anti-HA (1:3000, Roche) antibodies. 1.10. TurboID-based proximity labeling assay TurboID-based proximity labeling assays were performed as described previously

[0046] . Briefly, leaves on Arabidopsis 35S:LecRK-VI.2-HA-TurboID / 35S:NPR1-GFP and 35S:Lti6b-HA- TurboID / 35S:NPR1-GFP double transgenic plants were infiltrated with 50 µM biotin and incubated at room temperature for 3 hr to allow labeling. Total proteins were extracted using an extraction buffer containing 50 mM HEPES-KOH (pH 7.5), 150 mM KCl, 1 mM EDTA, 0.5% Triton X-100, and proteinase inhibitor cocktail. To enrich NPR1-GFP, the extracts were incubated with GFP-trap Magnetic Agarose beads (ChromoTek) for 2 hr at 4°C. The beads were then washed three times with the extraction buffer, omitting the proteinase inhibitors. The bound proteins were eluted by adding Laemmli buffer and boiling for 8 min. Biotinylation was detected using Streptavidin-HRP (1:20000, Abcam). 1.11. Promoter transactivation assay The PR1 promoter reporter (pPR1:DUAL-LUC) was co-infiltrated with 35:GFP, 35S:NPR1-GFP, 35S:npr1S356A / T359A-GFP, or 35S:npr1S356D / T359D-GFP into N. benthamiana leaves. Two days post-infiltration, 7 mm leaf disks were collected from the infiltrated leaves, ground in liquid nitrogen, and lysed with the PLB buffer from the Dual-Luciferase Reporter Assay Attorney Docket no.10457-609PC0 System (Promega E1910) for 15 min with gentle rocking at room temperature. The lysate was centrifuged at top speed for 1 min, and 20 µL of the supernatant was used to measure FLUC and RLUC activities according to the manufacturer’s instructions, using a GloMax Discover luminometer (Promega). Briefly, at 25°C, substrates for FLUC and RLUC were added using an automatic injector. Following a 2-s premeasurement delay, signals were captured for 10 s and recorded as counts per second. PR1 promoter activity was determined by calculating the ratio of F-LUC and R-LUC activities for each effector and plotting it relative to free GFP, which was arbitrarily set to 1. 1.12. RNA isolation and quantitative PCR (qPCR) Approximately 100 mg leaf tissues were snap-frozen in liquid nitrogen and ground into a fine powder in a 2 mL lysing tube with a Spex SamplePrep 2000 Geno / Grinder (OPS Diagnostics). Total mRNA was extracted using the E.Z.N.A.® Total RNA Kit (OMEGA), and reverse transcription was conducted using the SuperScript™ IV First-Strand Synthesis kit (Invitrogen), following the manufacturers’ manuals. qPCR was performed using SYBR™ Green PCR Master Mix (Applied Biosystems) on a QuantStudio 3 Real-Time PCR system (Applied Biosystems) according to the user’s manual. The 2-ΔCtmethod was used to determine the relative level of gene expression. UBQ5 was used as an internal control. The gene-specific primers used for qPCR were listed in Table S1. 1.13. NADP+-induced immunity and biological SAR assays Four-week-old Arabidopsis plants were used for NADP+-induced immunity and biological SAR assays. The previously described pathogen strain Pseudomonas syringae pv. maculicola ES4326 with an integrated luxCDABE luciferase operon (Psm_lux) was used in this study

[0016] . Psm_lux was cultured at 28°C in King’s B medium [2% (w / v) proteose peptone, 0.15% (w / v) K2HPO4, 6 mM MgSO4, and 1.5% (v / v) glycerol] containing appropriate antibiotics. Psm_lux in overnight log-phase cultures were centrifuged at 5,000 rpm for 1 min to pellet cells. The pellet was resuspended in 5 mM sterile MgCl2solution and diluted for leaf infiltration. NADP+-induced local and systemic immunity assays were performed as previously described with slight modifications

[0016] . Attorney Docket no.10457-609PC0 For NADP+-induced local immunity, 0.8 mM freshly made NADP+solution (pH = 5.7) or sterile water (negative control) was syringe-infiltrated into the 5thand 6thleaves from the bottom on each plant. After 4 hr, the NADP+-treated leaves were infiltrated with Psm_lux (OD600 = 0.001). Psm_lux growth in the leaves was determined 60 hr post inoculation (hpi). For NADP+-induced systemic immunity, 0.8 mM freshly made NADP+solution (pH = 5.7) or sterile water was infiltrated into three lower leaves on each plant (the 3rd, 4th, and 5thfrom the bottom). After 4 hr, two upper systemic leaves on the plant (the 6thand 7thfrom the bottom) were infiltrated with Psm_lux (OD600= 0.001). Psm_lux growth in the leaves was assessed 60 hpi. Biological induction of SAR was conducted as previously described with slight modifications.

[0023] Briefly, three lower leaves (the 3rd, 4th, and 5thfrom bottom) were infiltrated with 5 mM MgCl2(-SAR) or Psm (OD600= 0.002) (+SAR). After 48 hr, two upper systemic leaves (the 6thand 7thfrom the bottom) were infiltrated with Psm_lux (OD600 = 0.001). Psm_lux growth in the leaves was assessed 60 hpi. For Psm_lux growth quantification, leaf disks (7 mm in diameter) were collected using a hole punch and placed in a white, light-reflecting 96-well plate (Corning 3912). One leaf disc was taken from each leaf. Leaf disks were floated on 150 μL 1 mM MgCl2 in each well to keep them wet. The plate containing leaf disks was placed in the sample drawer of a GloMax Discover luminometer (Promega) and kept in the dark by closing the lid for 10 min to reduce background signals. The relative light unit (RLU) of each sample was then measured for 10 s. Bacterial titers were expressed as log10(RLU) per leaf disc. 1.14. Dexamethasone treatment Dexamethasone (Dex) was dissolved in methanol to make a 50 mM stock solution and preserved at -20 °C. To induce the expression of target genes, 50 µM Dex (1000-fold ddH2O dilution of the stock solution) was infiltrated into lower leaves (the 3rd, 4th, and 5th from the bottom) or upper leaves (the 6th from the bottom) using a 1 mL- needleless syringe. Methanol (0.1%) was used as the solvent control. The target gene is generally induced within several hr and the induction can last for several days. All downstream experiments were performed 24 h after Dex treatment. To rescue the mutant morphology, 1 µM Dex was applied through soil drenching Attorney Docket no.10457-609PC0 every other day for 4 weeks. Methanol (0.02%) was used as the mock control. Photos of the plants were taken at the end of the experiment. 1.15. GUS staining and DAB staining Dex-treated leaves were stained for GUS activity as previously described58. Briefly, leaves were submerged in a solution containing 1 mg / mL X-Gluc in 50 mM Na2HPO4 pH 7.0, 10 mM EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.06% Triton X-100, and vacuum infiltrated for 5 min. The staining solution was removed after overnight incubation at 37 °C, and the samples were cleared of chlorophyll by sequential changes of 75% and 95% ethanol. For DAB (3, 3’-diaminobenzidine) staining, leaves were immersed in a DAB solution (1 mg / mL, pH 3.8) overnight, and then boiled in ethanol for 10 min followed by several washes in ethanol. 1.16. NHP treatment NHP powder was dissolved in ddH2O to make a 0.5 mM solution. The NHP solution was aliquoted and frozen at −20 °C. For NHP induced local responses, 0.5 mM NHP or ddH2O (-NHP) was infiltrated using a 1 mL-needleless syringe into two leaves (the 5th and 6th from the bottom) on each four-week-old plant. After 24 h (unless otherwise indicated), the treated leaves were collected for indicated assays. For NHP induced systemic responses, 0.5 mM NHP or ddH2O was infiltrated into three lower leaves (the 3rd, 4th, and 5th from the bottom) on each four-week-old plant. After 24 h (unless otherwise indicated), one upper systemic leaf (the 6th from the bottom) was collected for indicated assays. For NHP induced systemic immunity, 0.5 mM NHP (unless otherwise indicated) or ddH2O was infiltrated into three lower leaves (the 3rd, 4th, and 5th from the bottom) on each four-week-old plant. After 24 h, one upper leaf was inoculated with Psm_lux (OD600 = 0.001) by infiltration. Psm titers were assessed 72 hpi. 1.17. Total NAD and NADP measurement To measure total NAD and NADP levels, indicated leaves were collected, weighed, and immediately boiled in 80% ethanol at 95 °C for 3 min. The supernatant was diluted 10-fold with ddH2O and used for NAD and NADP measurement with the NAD / NADH-Glo™ Assay and NADP / NADPH -Glo™ Assay kits (Promega, Cat#G9071 and G9081) according to the user’s manual. All reactions were prepared on ice. Values were normalized to the leaf weight. Attorney Docket no.10457-609PC0 1.18. eNAD and eNADP measurement To measure eNAD and eNADP levels, apoplastic wash fluids (AWFs) were extracted based on the previously described method67. Briefly, indicated leaves were cut with a sharp blade, weighed, rinsed in ddH2O, and vacuum-infiltrated with ddH2O in a 60-mL syringe. Water on the surface of infiltrated leaves was carefully removed with Kimwipes (Kimberly-Clark Professional, Cat#34120). To collect AWF, leaves were centrifuged at 500 g for 5 min at 4 °C. The volume of the AWF was brought to 500 μL with ddH2O and the solution was filtered through 3-kD MWCO filters (Sartorius, Cat#VS0192) by centrifugation at 13,000 g for 30 min at 4 °C. Filtered AWF was immediately used for NAD and NADP measurement with the NAD / NADH-Glo™ Assay and NADP / NADPH -Glo™ Assay kits according to the user’s manual. AWF was kept on ice and all reactions were prepared on ice. Values were normalized to the leaf weight. 1.19. Quantification and statistical analysis The pathogen growth results in this study are based on one experimental dataset, confirmed through at least three independent experiments conducted at different times. For quantification of Psm-lux titers in the leaves, eight Arabidopsis leaf replicates were used, with one leaf taken from each plant. In the SLC assays, six leaf disks were collected from treated N. benthamiana leaves and analyzed independently; the data were presented accordingly. This experiment was repeated three times, yielding similar results. For promoter transactivation assays, four leaf disks from four infiltrated N. benthamiana leaves were lysed together, and the lysate was analyzed four times, with data from the four technical replicates presented. The results were confirmed in three independent experiments conducted at different times. The gene expression results were derived from analyses of three independent biological samples, each taken from six Arabidopsis leaves on six plants (one leaf per plant). Statistical analyses were performed using Microsoft Excel (Student’s t-test) and Prism 10 (one-way / two-way ANOVA) on Microsoft Office 2023 for Macintosh. 1.20. Summary Arabidopsis thaliana mutants and transgenic lines used in this study were all in the Col-0 ecotype background. Transgenic Arabidopsis overexpressing GFP-fused NPR1, OsNPR1, BrNPR1, or their mutant variants are in the npr1-3 background. Transgenic Arabidopsis overexpressing NPR1-GFP in lecrk-VI.2-1 background were generated by crossing 35S:NPR1- Attorney Docket no.10457-609PC0 GFP / npr1-3 with the lecrk-VI.2-1 mutant. Double transgenic Arabidopsis lines overexpressing NPR1-GFP and LecRK-VI.2-HA-TurboID or Lti6b-HA-TurboID were generated by dipping the 35S:NPR1-GFP / npr1-3 transgenic plants with the Agrobacterium tumefaciens strain EHA105 harboring the 35S:LecRK-VI.2-HA-TurboID or 35S:Lti6b-HA-TurboID constructs, respectively. Double transgenic Arabidopsis lines overexpressing NPR1-GFP, or its phospho-mutant variants and TGAs-HA-TurboID were generated by dipping the 35S:NPR1-GFP / npr1-3, 35S:npr1S356A / T359A-GFP / npr1-3, or 35S:npr1S356D / T359D-GFP / npr1-3 transgenic plants with the A. tumefaciens strain EHA105 harboring the 35S:TGAs-HA-TurboID constructs. Arabidopsis and Nicotiana benthamiana seeds were stratified at 4°C for three days before germination, and all the plants for experiments were grown in soil in a growth room at 24°C / 22°C (day / night) and 60% relative humidity with a 14 hr / 10 hr light / dark photoperiod. For selection of transgenic Arabidopsis plants with antibiotic resistance, seeds were surface sterilized with 85% (v / v) ethanol, dried on filter paper, and germinated on sterile half-strength (½) MS medium (pH 5.7) supplemented with 1% (w / v) sucrose and 0.55% (w / v) agar with appropriate antibiotics. Example 2. LecRK-VI.2 is physically associated with NPR1 Receptor kinases generally transmit signals across the cell membrane by interacting with and phosphorylating their cytosolic substrates

[0052] . Since NPR1 is localized in the cytosol in the absence of pathogen infection and functions downstream of eNAD(P) during SAR induction [26, 35], the eNAD(P) receptor LecRK-VI.2 or LecRK-I.8 might directly interact with NPR1. To test this hypothesis, first a yeast two-hybrid (Y2H) assay was performed to determine whether the intracellular kinase domain (KD) of LecRK-VI.2 (LecRK-VI.2KD) or LecRK-I.8 (LecRK-I.8KD) interacts with NPR1. Yeast cells containing all six combinations of the bait and prey vectors grew on synthetic dextrose (SD) double dropout (DDO) medium (-Leu-Trp); however, only those harboring LecRK-VI.2KD as bait and NPR1 as prey grew on SD quadruple dropout (QDO) medium (-Ade-His-Leu-Trp) (Figure 1A). These results indicate that LecRK-VI.2KD, but not LecRK-I.8KD, physically interacts NPR1 in yeast. Then a split Nano luciferase (Nluc) complementation assay was conducted to validate the interaction between full-length LecRK-VI.2 and NPR1

[0051] . As LecRK-VI.2 triggers cell death when transiently expressed in Nicotiana benthamiana

[0032] , its kinase-dead variant, lecrk- VI.2K395E, in which the conserved Lys residue at the catalytic domain was replaced with a Glu Attorney Docket no.10457-609PC0 residue, was used. When lecrk-VI.2K395Eand NPR1 were transiently coexpressed in N. benthamiana as translational fusions with the N-terminal fragment of Nluc (NlucN) and the C- terminal fragment of Nluc (NlucC), respectively, a strong luciferase activity was detected in the N. benthamiana leaves (Figure 1B). As a negative control, replacement of LecRK-VI.2 with LecRK-I.8 resulted in dramatically reduced luciferase activity. Immunoblotting results verified that both LecRK-I.8-NlucN-HA and NPR1-NlucC-FLAG were expressed in the negative control (Figure 1B). Moreover, LecRK-VI.2-FLAG could be coimmunoprecipitated (co-IPed) with NPR1-GFP, but not GFP, when transiently coexpressed in N. benthamiana (Figure 1C), confirming the interaction between LecRK-VI.2 and NPR1. Next the recently developed TurboID-based proximity labeling method was used to further demonstrate the in vivo physical association between LecRK-VI.2 and NPR1

[0053] . To this end, stable transgenic Arabidopsis plants coexpressing NPR1-GFP and LecRK-VI.2-HA-TurboID or Lti6b-HA-TurboID were created. The PM-localized Lti6b protein was used as a negative control. Coexpression of NPR1-GFP with LecRK-VI.2-HA-TurboID, but not Lti6b-HA-TurboID, led to a significant amount of biotinylated NPR1-GFP (Figure 1D). This result substantiates that LecRK- VI.2 physically associates with NPR1 in vivo. As LecRK-VI.2 is an integral membrane protein

[0034] , the association of NPR1 with the PM was also examined through isolation of PM proteins from the Arabidopsis plants coexpressing NPR1-GFP and LecRK-VI.2-HA-TurboID. It was found that LecRK-VI.2-HA-TurboID existed exclusively in the PM fraction, whereas NPR1-GFP was detected in both soluble and PM fractions (Figure 8). This result indicates that some NPR1 proteins are constitutively associated with the PM, likely through interactions with integral membrane proteins like LecRK-VI.2. Example 3. LecRK-VI.2 phosphorylates NPR1 in vitro Next, it was tested whether LecRK-VI.2 could phosphorylate NPR1 using in vitro kinase assays with purified recombinant proteins. Maltose-binding protein (MBP)-tagged LecRK- VI.2KD (MBP-LecRK-VI.2KD) directly phosphorylated superfolder GFP (sfGFP)- and His- tagged NPR1 (sfGFP-NPR1-His), but not sfGFP-His, as revealed by an anti-phosphothreonine (pThr) antibody (Figure 2A). Interestingly, although the KD of BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1), a leucine-rich repeat receptor kinase that constitutively associates with LecRK-VI.2

[0023] , also interacts with NPR1 in yeast, Attorney Docket no.10457-609PC0 glutathione S-transferase (GST)-tagged BAK1KD (GST-BAK1KD) did not phosphorylate sfGFP- NPR1-His in the kinase assay when analyzed with the anti-pThr antibody (Figures 9A-B). The LecRK-VI.2KD- and BAK1KD-treated sfGFP-NPR1-His proteins were subsequently subjected to trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. Ser356, Thr359, Thr373, and Ser571 were identified as potential LecRK-VI.2KD phosphorylation sites on NPR1 (Figures 10A-B). Consistent with the anti-pThr antibody analysis, no BAK1KD phosphorylation site on NPR1 was identified by LC-MS / MS. Example 4. Exogenous NADP+induces LecRK-VI.2-dependent phosphorylation of NPR1 in vivo To identify in vivo phosphorylation sites on NPR1, NPR1-GFP protein was immunoprecipitated from four-week-old 35S:NPR1-GFP / npr1-3 transgenic plants treated with NADP+or water for 30 min and subjected to trypsin digestion and LC-MS / MS analysis. Ser356, Thr359, Thr572, Ser573, and Ser574 were identified as potential in vivo phosphorylation sites on NPR1 (Figures 2B-2D and Figure 10A). To determine whether eNAD(P) induces NPR1 phosphorylation at these sites and whether LecRK-VI.2 is responsible for this phosphorylation, it was attempted to develop phosphopeptide-specific antibodies recognizing these phosphorylated residues on NPR1. The antibody against Thr359 phosphopeptide recognized MBP-LecRK- VI.2KD-treated sfGFP-NPR1-His recombinant protein, but not sfGFP-T359A-His in which Thr359 was changed to an Ala residue (Figure 2E), indicating that this antibody is specific for phospho-Thr359 (pThr359). The anti-pThr359 antibody was then used to determine NADP+-induced phosphorylation and the role of LecRK-VI.2 in NPR1 phosphorylation in vivo. NPR1-GFP protein was immunoprecipitated from NADP+-treated 35S:NPR1-GFP / npr1-3 transgenic plants and analyzed by immunoblotting with the anti-pThr359 antibody. As shown in Figure 2F, NADP+-induced phosphorylation of NPR1 at Thr359 occurred as early as 15 min after the treatment. Importantly, NADP+-induced Thr359 phosphorylation was abolished in 35S:NPR1-GFP / lecrk-VI.2-1 transgenic plants (Figure 2G), indicating that LecRK-VI.2 is responsible for NADP+-induced phosphorylation of Thr359 in vivo. Moreover, as the development of antibodies for phospho- Ser356 was unsuccessful, stable 35S:S356A-GFP / npr1-3 transgenic plants were generated and analyzed NPR1 phosphorylation using Phos-tag gels to determine whether eNAD(P) induces Ser356 phosphorylation. Consistent with the previously reported basal phosphorylation of NPR1 Attorney Docket no.10457-609PC0

[0054] , an up-shifted migration band was detected in the NPR1-GFP sample (Figure 2H). NADP+treatment induced an additional up-shifted band, and the intensity of this band was dramatically weakened by the S356A mutation (Figure 2H), suggesting that NADP+also induces NPR1 phosphorylation at Ser356. Together these results demonstrate that NADP+induces LecRK-VI.2- mediated phosphorylation of NPR1 at Thr359, and likely Ser356, in vivo. Example 5. Exogenous NADP+induces dissociation of NPR1 from LecRK-VI.2 and enhances NPR1-TGA interactions To test whether NADP+-induced NPR1 phosphorylation leads to dissociation of NPR1 from LecRK-VI.2, N. benthamiana leaves transiently coexpressing LecRK-VI.2-FLAG and NPR1-GFP were treated with NADP+and their association via co-IP was assessed. Although similar amounts of NPR1-GFP were pulled down using GFP-trap magnetic agarose beads from the samples treated with or without NADP+, less LecRK-VI.2-FLAG was co-IPed from the NADP+-treated sample (Figure 11), suggesting that NADP+induces NPR1 dissociation from LecRK-VI.2. Next, a time course experiment was performed to reveal the kinetics of NADP+- induced dissociation of NPR1 from LecRK-VI.2 using the 35S:NPR1-GFP / 35S:LecRK-VI.2-HA- TurboID transgenic Arabidopsis plants. NADP+induced biphasic changes in the association between NPR1-GFP and LecRK-VI.2-HA-TurboID, which first showed a dramatic decrease 30 min after NADP+treatment, followed by a gradual increase reaching above basal levels 480 min after the treatment (Figure 3A). Since NADP+-induced LecRK-VI.2-mediated NPR1 phosphorylation occurs 15 min after NADP+treatment (Figure 2F), phosphorylation of NPR1 by LecRK-VI.2 might trigger their dissociation. To test this hypothesis, NADP+-induced dissociation between NPR1-GFP and the kinase-dead variant lecrk-VI.2K395E-HA-TurboID was examined. NADP+treatment induced similar biphasic changes in the association between NPR1-GFP and lecrk-VI.2K395E-HA-TurboID (Figure 3B), indicating that the association / dissociation between NPR1 and LecRK-VI.2 is independent of the kinase activity of LecRK-VI.2. Regardless, it was hypothesized that NADP+-induced phosphorylation of NPR1 and dissociation between NPR1 and LecRK-VI.2 might enhance the interaction between NPR1 and TGAs. To test this hypothesis, a time course experiment was conducted to assess the effects of NADP+treatment on the interaction between NPR1 and TGA1 / 3 by co-IP using 35S:NPR1- Attorney Docket no.10457-609PC0 GFP / 35S:TGA1 / 3-HA-TurboID transgenic Arabidopsis plants. A significantly enhanced interaction between NPR1-GFP and TGA1 / 3-HA-TurboID was detected 60 min after NADP+treatment (Figures 3C-3D), which followed NADP+-induced NPR1 phosphorylation and dissociation of NPR1 from LecRK-VI.2 that occurred 15 and 30 min after NADP+treatment, respectively (Figures 2F and 3A). Although the kinase activity of LecRK-VI.2 is not required for NADP+-induced NPR1 dissociation from LecRK-VI.2, the tight correlation among the three events supports a hypothesis that LecRK-VI.2-mediated phosphorylation of NPR1 might be a regulatory step required for the interaction between NPR1 and TGAs. Example 6. Ser356 / Thr359 phosphorylation facilitates NPR1-TGA interaction Ser356 and Thr359 are in the loop between ankyrin repeats 3 and 4, near His334 in the ankyrin repeat 3 of the NPR1 protein (Figure 4A). The positively charged His334 might interact with phosphorylated Ser356 and Thr359 to create a conformation favorable for interaction with other proteins including TGAs. To test this hypothesis, first the effects of non-phosphorylatable mutation(s) (substituting S or T with A) of Ser356 and Thr359 were evaluated on the interaction between NPR1 and TGA3 in yeast cells. Compared with NPR1, npr1T359Aand npr1S356A / T359Aas bait significantly reduced and completely suppressed, respectively, the growth of yeast cells carrying the bait and TGA3 as prey on QDO medium (Figures 12A-B), suggesting that phosphorylation of Ser356 and Thr359 might additively contribute to NPR1-TGA interactions. In support of this hypothesis, yeast cells harboring NPR1 or the phospho-mimetic mutant npr1S356D / T359Das bait and TGA2 / 3 / 5 / 6 as prey grew normally on SD triple dropout (TDO) medium (-Leu-Trp-His) supplemented with 3 mM 3-amino-1,2,4-triazole (3-AT), whereas those carrying npr1S356A / T359Aas bait and the TGAs as prey did not grow (Figure 4B). Interestingly, although NPR1 and TGA1 did not interact in yeast, yeast cells containing npr1S356D / T359Das bait and TGA1 as prey showed growth on the TDO medium (Figure 4B), suggesting that phosphorylation of Ser356 and Thr359 might enhance NPR1-TGA1 interaction. Next, co-IP assays were performed to validate the Y2H results. As shown in Figures 4C- 4D, significantly reduced amounts of TGA1-GFP and TGA3-GFP were co-IPed with S356A / T359A-FLAG compared with those co-IPed with NPR1-FLAG and S356D / T359D-FLAG from protein extract of N. benthamiana leaf tissues transiently coexpressing TGA1 / 3-GFP and S356A / T359A-FLAG / NPR1-GFP / S356D / T359D-FLAG. Similarly, a significantly reduced Attorney Docket no.10457-609PC0 amount of S356A / T359A-FLAG was co-IPed with TGA6-GFP compared with those of NPR1- FLAG and S356D / T359D-FLAG (Figure 4E). These results indicate that the S356A / T359A double mutations diminish NPR1-TGA interactions in N. benthamiana. TurboID-based proximity labeling method was used to further confirm the effects of the phospho-mutations on the interactions between NPR1 and TGAs with Arabidopsis 35S:NPR1- GFP / 35S:TGA1 / 2 / 3-HA-TurboID, 35S:S356A / T359A-GFP / 35S:TGA1 / 2 / 3-HA-TurboID, and 35S:S356D / T359D-GFP / 35S:TGA1 / 2 / 3-HA-TurboID transgenic plants. As shown in Figures 4F- 4H, the S356A / T359A mutations, but not the S356D / T359D mutations, significantly reduced the amount of biotinylated NPR1-GFP. These results substantiate that phosphorylation of Ser356 and Thr359 are required for NPR1-TGA interactions in vivo. Example 7. Ser356 / Thr359 phosphorylation is required for transmission of eNAD(P) signaling and establishment of SAR To determine the biological relevance of Ser356 / Thr359 phosphorylation, the transcriptional activity of the NPR1 phospho-mutants in activating the promoter of PR1, a direct target gene of NPR1, was tested using a pPR1:DUAL-LUC reporter in N. benthamiana

[0028] . As shown in Figure 5A, S356A / T359A-GFP, a loss-of-function form, is defective in activating the PR1 promoter, whereas S356D / T359D-GFP exhibited wild-type levels of transcriptional activity. Consistent with the results of the pPR1:DUAL-LUC reporter assay, NADP+-induced PR1 gene expression and PR1 protein accumulation were diminished in 35S:S356A / T359A-GFP / npr1-3 but not in 35S:S356D / T359D-GFP / npr1-3 transgenic plants (Figures 13A and B and Figures 5B-5C). Then, NADP+-induced local and systemic immunities as well as biological induction of SAR were analyzed in 35S:NPR1-GFP / npr1-3, 35S:S356A / T359A-GFP / npr1-3, and 35S:S356D / T359D / npr1-3 plants. As shown in Figures 5D-5F, while NPR1-GFP and S356D / T359D-GFP complemented the defects of npr1-3, S356A / T359A-GFP failed to do so. Furthermore, although LC-MS / MS analysis indicated potential in vivo phosphorylation on Thr572, Ser573, and Ser574, substituting Ser / Thr 571-576 with six Ala residues (6A) did not impact NPR1’s functionality in NADP+-induced immunities and SAR (Figures 14A-D). Taken together, these results demonstrate that phosphorylation of Ser356 and Thr359 is crucial for NPR1’s transcriptional activity and function in eNADP+signaling and SAR. Attorney Docket no.10457-609PC0 Interestingly in Figure 13, the protein levels in the two S356D / T359-GFP lines (#1 and #5) are significantly lower than those in the NPR1-GFP (wild-type form) line, however the induction of the defense reporter gene PR1 (pathogenesis-related protein 1) is significantly higher in the S356D / T359D-GFP plants (line #1 was used). Similarly, in Figure 5C, S356D / T359D-GFP plants accumulated significantly higher levels of the PR1 protein than in the NPR1-GFP plants. Figure 5F shows the results of biological induction of systemic acquired resistance (SAR). Although the S356D / T359D-GFP plants have lower protein levels, SAR induction is stronger than in the NPR1-GFP plants. These data indicate that the phospho-mimetic form S356D / T359D is more active than the wild-type form and has significant advantages when used to increase disease resistance in crops. It will require less protein to confer similar or high levels of resistance. Example 8. Ser356 / Thr359 phosphorylation is conserved in dicot and monocot Residues corresponding to Ser356 and Thr359 are highly conserved in NPR1 orthologs across various plant species (Figure 6A and Figure S8A)

[0055] , highlighting their potential functional importance. To investigate this, the functionality of non-phosphorylatable variants of NPR1 orthologs from Brassica rapa (BrNPR1) and Oryza sativa (OsNPR1) was examined, which have been shown to complement the Arabidopsis npr1 mutant [56, 57]. BrNPR1 Ser340 and OsNPR1 Thr363, which correspond to Arabidopsis NPR1 Ser356 and Thr359, respectively (Figure 15B), were substituted with Ala. Additionally, BrNPR1 Thr342, adjacent to the conserved Thr359 (Figure 15B), was replaced with Ala. Compared with the wild-type BrNPR1 and OsNPR1, the non-phosphorylatable mutants Brnpr1S340A / T342Aand Osnpr1T363Aas bait significantly inhibited the growth of yeast cells expressing the bait and TGA1 / 2 / 3 and TGA1 as prey, respectively, on QDO medium (Figures 6B-6C). Moreover, these mutants failed to complement the npr1-3 defects in NADP+-induced local and systemic immunities and in the biological induction of SAR, unlike the wild types (Figures 6D-6E). These findings suggest that the phosphorylation of these conserved Ser and / or Thr residues is essential for the functionality of BrNPR1 and OsNPR1. Attorney Docket no.10457-609PC0 Example 9. NHP is necessary and sufficient for triggering systemic eNAD(P) accumulation NHP has recently been shown to be a potential SAR mobile signal (Chen et al., 2018, Hartmann et al.,2018). It was shown that exogenous NAD(P)+can induce both local and systemic immunity in the NHP biosynthesis genes deficiency mutants, ald1 and fmo1 (Wang et al., 2019), suggesting that eNAD(P) might function downstream of NHP. Indeed, NHP treatment induced significant accumulation of eNAD(P) in both the treated and upper systemic leaves (Figure 16a– d), and the total NAD(P) levels did not change significantly (Figure 17a–d). Then it was tested if local application of NHP could induce de novo eNAD(P) accumulation in systemic leaves using the Dex:FIN4 / fin4-3 transgenic plants. FIN4 encodes the chloroplastic enzyme aspartate oxidase that catalyzes the first irreversible step in de novo NAD biosynthetic pathway. The fin4-3 mutant is smaller than wild type and accumulates significantly reduced NAD(P) levels (Figure 18a–c). A 35 S:FIN4 transgene complemented the fin4 morphology and restored NAD(P) levels in fin4-3 to the wild-type level (Figure 18a–c). The pOpON system was utilized to spatially control the expression of the FIN4 transgene, thus NAD biosynthesis, in the fin4-3 mutant background by Dex application. Based on the induction of the GUS reporter, two homozygous Dex:FIN4 / fin4-3 transgenic lines with strong induction and no leakage of GUS activity were selected for further investigation (Figure 19a). The transgenic plants were morphologically indistinguishable from the fin4-3 mutant and accumulated similarly reduced NAD levels as in fin4-3 (Figure 19b and Figure 20). Dex treatment restored the morphology (Figure 19b) and NAD levels of the Dex:FIN4 / fin4-3 plants but had no effects on fin4-3 or wild-type plants (Figure 20), indicating that the expression of the FIN4 transgene in the selected lines is tightly controlled by the inducible promoter. Infiltration of NHP into lower leaves induced accumulation of significantly higher eNAD(P) levels in the upper systemic leaves than the control treatment when NAD biosynthesis was restored in the systemic leaves by Dex application (Figure 16e, f). These results indicate that NHP and / or NHP-induced mobile signals not only induce eNAD(P) accumulation in treated leaves, but also trigger de novo systemic eNAD(P) accumulation. Furthermore, while eNAD(P) levels in the local Psm-infected leaves of fmo1 were not significantly different from those in the wild type (Figure 21a, b), eNAD(P) levels in the systemic leaves of fmo1 during biological induction of SAR were significantly lower than those in the wild type (Figure 16g, h). Since the Attorney Docket no.10457-609PC0 fmo1 mutant, a mutant of NHP-biosynthesis enzyme FMO1, which consequently does not synthesize NHP, is completely SAR defective

[0058] , these results indicate that NHP-mediated systemic eNAD(P) accumulation plays a crucial role in the establishment of SAR. Example 10. NHP induces systemic immunity through eNAD(P) After establishing that NHP induces eNAD(P) accumulation, it was tested whether NHP- induced systemic immunity depends on eNAD(P) and its perception at the plasma membrane. First, NHP-induced systemic immunity was tested in two previously generated 35 S:CD38 transgenic lines that express the human NAD(P)-hydrolyzing ectoenzyme CD38 (Zhang et al., 2012). As eNAD(P) levels are only slightly reduced in 35 S:CD38 transgenic plants and the 35:CD38 transgenic plants exhibit partially compromised SAR (Zhang et al., 2012), a lower concentration (0.25 mM) of NHP was used that is able to induce significant systemic immunity in wild type. NHP-induced systemic immunity was significantly inhibited in the 35 S:CD38 transgenic lines (Figure 22a). Then NHP-induced systemic immune responses including defense gene expression and disease resistance were examined in the fin4-3, lecrk-VI.2, and bak1-5 bkk1 mutants [23, 59, 60]. Except for PR1 in lecrk-VI.2, NHP-induced expression of PR1, ALD1, and FMO1 as well as NHP-induced resistance to Psm were significantly reduced in all three mutants (Figure 22b–d). Taken together, these results demonstrate that eNAD(P) functions downstream of NHP in activating systemic immunity. Example 11. NHP triggers eNAD(P) accumulation through ROS ROS has been implicated in the induction of SAR (Alvarez et al., 1998, Wang et al., 2014). Since ROS can oxidize cell membrane, leading to pore formation [61, 62], they might cause NAD(P) leakage. Surely, methyl viologen (MV) treatment, which triggers ROS production by catalyzing the transfer of electrons from photosystem I to molecular oxygen

[0063] , significantly increased eNAD(P) levels in the treated leaves (Figure 23a). To establish a cause-and-effect relationship between ROS and eNAD(P), eNAD(P) levels were measured in systemic leaves of the Arabidopsis ROS deficient mutant rbohF during biological induction of SAR

[0064] . SAR induction-triggered systemic eNAD(P) accumulation was significantly reduced in the rbohF mutant (Figure 23b, c). In line with this result, the rbohF mutant is defective in SAR (Wang et al., 2014). Then it was tested whether NAD(P)+ treatment could restore SAR in rbohF and the other ROS deficient mutant rbohD

[0064] . NAD(P)+ treatment induced similar levels of systemic Attorney Docket no.10457-609PC0 immunity in the wild type and the ROS mutants (Figure 23d, e). These results suggest that eNAD(P) functions downstream of ROS in activating systemic immunity. Next, it was tested if NHP could induce ROS accumulation. The NHP precursor, Pip, has been shown to induce ROS accumulation in Arabidopsis leaves (Wang et al., 2018). NHP also induced ROS accumulation after being infiltrated into the Arabidopsis leaves (Figure 23f). To test if NHP-induced systemic eNAD(P) accumulation depends on ROS, rbohF was treated with 0.5 mM NHP and measured systemic eNAD(P) levels 24 h later. Compared with that in the wild type, NHP-induced systemic accumulation of eNAD(P) was significantly inhibited in the rbohF mutant (Figure 23g, h). Furthermore, NHP-induced systemic immunity was significantly weakened in both rbohF and rbohD (Figure 23i, j). These results indicate that NHP induces systemic eNAD(P) accumulation and systemic immunity largely through RBOH-produced ROS. REFERENCES 1. Jones, J.D. and J.L. Dangl, The plant immune system. Nature, 2006. 444(7117): p. 323-329. 2. Jones, J.D.G., B.J. Staskawicz, and J.L. Dangl, The plant immune system: From discovery to deployment. Cell, 2024. 187(9): p. 2095-2116. 3. Zipfel, C., Plant pattern-recognition receptors. Trends Immunol, 2014. 35(7): p. 345-51. 4. Fu, Z.Q. and X. Dong, Systemic acquired resistance: turning local infection into global defense. Annu Rev Plant Biol, 2013. 64: p. 839-863. 5. Kachroo, A. and P. Kachroo, Mobile signals in systemic acquired resistance. Curr Opin Plant Biol, 2020. 58: p. 41-47. 6. Maldonado, A.M., et al., A putative lipid transfer protein involved in systemic resistance signalling in Arabidopsis. Nature, 2002. 419(6905): p. 399-403. 7. Park, S.W., et al., Methyl salicylate is a critical mobile signal for plant systemic acquired resistance. Science, 2007. 318(5847): p. 113-116. 8. Shulaev, V., P. Silverman, and I. Raskin, Airborne signalling by methyl salicylate in plant pathogen resistance. Nature, 1997. 385: p. 718-721. 9. Wang, C., et al., Pipecolic acid confers systemic immunity by regulating free radicals. Sci Adv, 2018. 4(5): p. eaar4509. 10. Chaturvedi, R., et al., Plastid omega3-fatty acid desaturase-dependent accumulation of a systemic acquired resistance inducing activity in petiole exudates of Arabidopsis thaliana is independent of jasmonic acid. Plant J, 2008. 54(1): p. 106-17. 11. Jung, H.W., et al., Priming in systemic plant immunity. Science, 2009. 324(5923): p. 89-91. 12. Chanda, B., et al., Glycerol-3-phosphate is a critical mobile inducer of systemic immunity in plants. Nat Genet, 2011. 43(5): p. 421-427. 13. Chen, Y.C., et al., N-hydroxy-pipecolic acid is a mobile metabolite that induces systemic disease resistance in Arabidopsis. Proc Natl Acad Sci USA, 2018. 115(21): p. E4920- E4929. 14. Hartmann, M., et al., Flavin Monooxygenase-Generated N-Hydroxypipecolic Acid Is a Critical Element of Plant Systemic Immunity. Cell, 2018. 173(2): p. 456-469. Attorney Docket no.10457-609PC0 15. Navarova, H., et al., Pipecolic acid, an endogenous mediator of defense amplification and priming, is a critical regulator of inducible plant immunity. Plant Cell, 2012. 24(12): p. 5123-5141. 16. Li, Q., et al., N-hydroxypipecolic acid triggers systemic acquired resistance through extracellular NAD(P). Nat Commun, 2023. 14(1): p. 6848. 17. Riedlmeier, M., et al., Monoterpenes support systemic scquired resistance within and between plants. Plant Cell, 2017. 29(6): p. 1440-1459. 18. Shine, M.B., et al., Phased small RNA-mediated systemic signaling in plants. Sci Adv, 2022. 8(25): p. eabm8791. 19. Alvarez, M.E., et al., Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell, 1998. 92: p. 773-784. 20. Wang, C., et al., Free radicals mediate systemic acquired resistance. Cell Rep, 2014. 7(2): p. 348-355. 21. Wenig, M., et al., Systemic acquired resistance networks amplify airborne defense cues. Nat Commun, 2019. 10(1): p. 3813. 22. Zottini, M., et al., Salicylic acid activates nitric oxide synthesis in Arabidopsis. J Exp Bot, 2007. 58(6): p. 1397-405. 23. Wang, C., et al., Extracellular pyridine nucleotides trigger plant systemic immunity through a lectin receptor kinase / BAK1 complex. Nat Commun, 2019. 10(1): p. 4810. 24. Liu, C., Q. Liu, and Z. Mou, Redox signaling and oxidative stress in systemic acquired resistance. J Exp Bot, 2024. 25. Rochon, A., et al., The coactivator function of Arabidopsis NPR1 requires the core of its BTB / POZ domain and the oxidation of C-terminal cysteines. Plant Cell, 2006. 18(12): p. 3670-3685. 26. Kinkema, M., W. Fan, and X. Dong, Nuclear localization of NPR1 is required for activation of PR gene expression. Plant Cell, 2000. 12(12): p. 2339-2350. 27. Mou, Z., W. Fan, and X. Dong, Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell, 2003. 113: p. 935-944. 28. Kumar, S., et al., Structural basis of NPR1 in activating plant immunity. Nature, 2022. 605(7910): p. 561-566. 29. Zhang, Y., et al., Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Proceedings of the National Academy of Science U.S.A., 1999. 96(11): p. 6523-6528. 30. Lee, H.J., et al., Systemic Immunity Requires SnRK2.8-Mediated Nuclear Import of NPR1 in Arabidopsis. Plant Cell, 2015. 27(12): p. 3425-38. 31. Wang, C., et al., A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana. Elife, 2017. 6: p. e25474. 32. Li, Q., et al., A group of L-type lectin receptor kinases function redundantly in mediating extracellular NAD(P) signaling in Arabidopsis. Plant Physiol, 2024. 33. Singh, P., et al., The Arabidopsis LECTIN RECEPTOR KINASE-VI.2 is a functional protein kinase and is dispensable for basal resistance to Botrytis cinerea. Plant Signal Behav, 2013. 8(1): p. e22611. 34. Xin, Z., et al., The Arabidopsis A4 subfamily of lectin receptor kinases negatively regulates abscisic acid response in seed germination. Plant Physiol, 2009. 149(1): p. 434-444. 35. Zhang, X.D. and Z.L. Mou, Extracellular pyridine nucleotides induce PR gene expression and disease resistance in Arabidopsis. Plant Journal, 2009. 57(2): p. 302-312. Attorney Docket no.10457-609PC0 36. Wu, Y., et al., The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid. Cell Rep, 2012. 1(6): p. 639-647. 37. Mbengue, M., et al., Clathrin-dependent endocytosis is required for immunity mediated by pattern recognition receptor kinases. Proc Natl Acad Sci U S A, 2016. 113(39): p. 11034-9. 38. Robatzek, S., D. Chinchilla, and T. Boller, Ligand-induced endocytosis of the pattern recognition receptor FLS2 in Arabidopsis. Genes Dev, 2006. 20(5): p. 537-42. 39. Yao, L., et al., High air humidity dampens salicylic acid pathway and NPR1 function to promote plant disease. EMBO J, 2023. 42(21): p. e113499. 40. Massague, J. and D. Sheppard, TGF-beta signaling in health and disease. Cell, 2023. 186(19): p. 4007-4037. 41. Leaman, D.W., et al., Roles of JAKs in activation of STATs and stimulation of c-fos gene expression by epidermal growth factor. Mol Cell Biol, 1996. 16(1): p. 369-75. 42. Guo, H., et al., FERONIA Receptor Kinase Contributes to Plant Immunity by Suppressing Jasmonic Acid Signaling in Arabidopsis thaliana. Curr Biol, 2018. 28(20): p. 3316-3324 e6. 43. Fujita, H., et al., An Arabidopsis MADS-box protein, AGL24, is specifically bound to and phosphorylated by meristematic receptor-like kinase (MRLK). Plant Cell Physiol, 2003. 44(7): p. 735-42. 44. Meyer, M.R., et al., Evidence for intermolecular interactions between the intracellular domains of the arabidopsis receptor-like kinase ACR4, its homologs and the Wox5 transcription factor. PLoS One, 2015. 10(3): p. e0118861. 45. Shah, J. and J. Zeier, Long-distance communication and signal amplification in systemic acquired resistance. Front Plant Sci, 2013. 4: p. 30. 46. Wu, Z., et al., Plant E3 ligases SNIPER1 and SNIPER2 broadly regulate the homeostasis of sensor NLR immune receptors. EMBO J, 2020. 39(15): p. e104915. 47. Wang, C., et al., The Arabidopsis Mediator complex subunit16 is a key component of basal resistance against the necrotrophic fungal pathogen Sclerotinia sclerotiorum. Plant Physiol, 2015. 169(1): p. 856-872. 48. Liu, C., et al., Two Arabidopsis Receptor-like Cytoplasmic Kinases SZE1 and SZE2 Associate with the ZAR1-ZED1 Complex and Are Required for Effector-Triggered Immunity. Mol Plant, 2019. 12(7): p. 967-983. 49. Perron, N., et al., Proteomics and phosphoproteomics of C(3) to CAM transition in the common ice plant. Methods Enzymol, 2022. 676: p. 347-368. 50. Ye, R., et al., Glucose-driven TOR-FIE-PRC2 signalling controls plant development. Nature, 2022. 609(7929): p. 986-993. 51. Wang, F.Z., et al., Split Nano luciferase complementation for probing protein-protein interactions in plant cells. J Integr Plant Biol, 2020. 62(8): p. 1065-1079. 52. Hubbard, S.R. and W.T. Miller, Receptor tyrosine kinases: mechanisms of activation and signaling. Curr Opin Cell Biol, 2007. 19(2): p. 117-23. 53. Branon, T.C., et al., Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol, 2018. 36(9): p. 880-887. 54. Spoel, S.H., et al., Proteasome-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity. Cell, 2009. 137(5): p. 860-72. 55. Jia, X., et al., The origin and evolution of salicylic acid signaling and biosynthesis in plants. Mol Plant, 2023. 16(1): p. 245-259. Attorney Docket no.10457-609PC0 56. Potlakayala, S.D., et al., Conservation of NON-EXPRESSOR OF PATHOGENESIS- RELATED GENES1 function between Arabidopsis thaliana and Brassica napus. Physiol. Mol. Plant P., 2007(71): p. 174-183. 57. Yuan, Y., et al., Functional analysis of rice NPR1-like genes reveals that OsNPR1 / NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility. Plant Biotechnol J, 2007.5(2): p. 313-24. 58. Mishina, T.E. and J. Zeier, The Arabidopsis flavin-dependent monooxygenase FMO1 is an essential component of biologically induced systemic acquired resistance. Plant Physiol, 2006. 141(4): p. 1666-75. 59. Macho, A.P., et al., Aspartate oxidase plays an important role in Arabidopsis stomatal immunity. Plant Physiol, 2012. 159(4): p. 1845-1856. 60. Schwessinger, B., et al., Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1. PLoS Genet, 2011. 7(4): p. e1002046. 61. Cwiklik, L. and P. Jungwirth, Massive oxidation of phospholipid membranes leads to pore creation and bilayer disintegration. Chem. Phys. Lett., 2010. 486: p. 99-103. 62. Tero, R., et al., Nanopore formation process in artificial cell membrane induced by plasma- generated reactive oxygen species. Arch Biochem Biophys, 2016. 605: p. 26-33. 63. Babbs, C.F., J.A. Pham, and R.C. Coolbaugh, Lethal hydroxyl radical production in paraquat-treated plants. Plant Physiol., 1989. 90: p. 1267-1270. 64. Torres, M.A., J.L. Dangl, and J.D. Jones, Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci U S A, 2002. 99(1): p. 517-22.

Claims

Attorney Docket no.10457-609PC0 CLAIMS What is claimed is:

1. A phospho-mimetic mutant protein of NPR1, wherein the NPR1 is Arabidopsis NPR1 (AtNPR1), wherein at least one of Ser (S) and / or Thr (T) residue(s) of the NPR1 is substituted with Asp (D), and wherein the Ser and / or Thr residue(s) to be substituted is selected from Ser356, Thr359, Thr373, Ser571, Thr572, Ser573, Ser 574, T575, S576, and Ser589.

2. The phospho-mimetic mutant protein of NPR1 of claim 1, wherein Ser 356 (S356) and Thr 359 (T359) residues of the NPR1 are substituted with Asp (D) to make NPR1- S356D / T359D.

3. The phospho-mimetic mutant protein of NPR1 of claim 2, wherein the amino acid sequence of NPR1-S356D / T359D comprises SEQ ID NO:1 or any amino acid sequence with at least 80% sequence identity thereto.

4. The phospho-mimetic mutant protein of NPR1 of claim 3, wherein NPR1-S356D / T359D protein is optionally fused / tagged at its N- or C-terminus with one selected from fluorescence protein (e.g., GFP, superfolder GFP (sfGFP)), maltose-binding protein (MBP), glutathione S-transferase (GST), horseradish peroxidase (HRP), His (or hexa- histidine), FLAG, streptavidin-binding peptide (SBP), Strep II, calmodulin-binding peptide (CBP), chitin-binding domain (CBD), HA, c-Myc, or biotinylation.

5. A nucleic acid fragment that comprises a nucleotide sequence of SEQ ID NO:2 or any nucleotide sequence with at least 80% sequence identity thereto, which encodes NPR1- S356D / T359D protein or any amino sequence with at least 80% sequence identity thereto.

6. The nucleic acid fragment of claim 5, wherein the nucleic acid fragment optionally comprises a nucleotide sequence encoding a tag at the 5’- or 3’-terminus of SEQ ID NO:2, wherein the tag is one selected from fluorescence protein (GFP, superfolder GFP (sfGFP), and the like), maltose-binding protein (MBP), glutathione S-transferase (GST), horseradish peroxidase (HRP), His (or hexa-histidine), FLAG, streptavidin-binding peptide (SBP), Strep II, calmodulin-binding peptide (CBP), chitin-binding domain (CBD), HA, c-Myc, or biotinylation.Attorney Docket no.10457-609PC0 7. A vector comprising the nucleic acid fragment of claim 5 or 6, wherein the vector is a plasmid for DNA replication and / or protein expression or a plant virus.

8. The vector of claim 7, wherein the vector is a plasmid for protein expression.

9. The vector of claim 8, wherein the vector is optionally a plasmid comprising T-DNA fragment that comprises a nucleotide sequence of SEQ ID NO:2 in its multicloning site.

10. Agrobacterium comprising the vector of claim 9, wherein the Agrobacterium is optionally an A. tumefaciens strain.

11. A transgenic plant with improved disease-resistance or tolerance, which expresses NPR1- S356D / T359D protein comprising an amino acid sequence of SEQ ID NO:1 or any amino acid sequence with at least 80% sequence identity thereto.

12. The transgenic plant of claim 11, wherein the plant is citrus.

13. The transgenic plant of claim 12, wherein NPR1-S356D / T359D protein is optionally fused / tagged at its N- or C-terminus with one selected from fluorescence protein (GFP, superfolder GFP (sfGFP), and the like), maltose-binding protein (MBP), glutathione S- transferase (GST), horseradish peroxidase (HRP), His (or hexa-histidine), FLAG, streptavidin-binding peptide (SBP), Strep II, calmodulin-binding peptide (CBP), chitin- binding domain (CBD), HA, c-Myc, or biotinylation.

14. The transgenic plant of claim 11 or 13, wherein the plant is Nicotiana benthamiana or Arabidopsis thaliana.

15. A method to improve plant immunity with NPR1-S356D / T359D protein, wherein the method comprises steps of: i. isolate NPR1-S356D / T359D protein comprising SEQ ID NO:1 or any amino acid sequence with at least 80% sequence identity thereto, which is optionally fused with a tag at its N- or C terminus; ii. dissolve the isolated protein in a buffer, wherein buffer pH is within a range of 7.0- 8.0; iii. mix a certain amount of the protein in the solution of step ii with a certain amount of liposomes; andAttorney Docket no.10457-609PC0 iv. immerse leaves of a plant in a solution comprising the protein-containing liposomes of step iii, spray solution comprising the protein-containing liposomes on the leaves, or inject solution comprising the protein-containing liposomes into a plant, optionally into infection sites.

16. A method to produce a transgenic plant expressing NPR1-S356D / T359D protein, wherein the method comprises steps of: i. isolate protein expression plasmid comprising a nucleotide sequence of SEQ ID NO 2 or any nucleotide sequence with at least 80% sequence identity thereto, wherein the plasmid optionally comprises a nucleotide sequence encoding a tag at the 5’- or 3’-terminus of SEQ ID NO:2; ii. dissolve the plasmid in a buffer, wherein buffer pH is optionally within a range of 7.0-8.0; iii. mix a certain amount of the plasmid in the solution of step ii with a certain amount of liposomes; and iv. immerse a plant seed or leaves in a solution comprising the plasmid-containing liposomes of step iii, spray solution comprising plasmid-containing liposomes on the leaves, or inject solution comprising the plasmid-containing liposomes into a plant, optionally into infection sites.

17. A method to produce a transgenic plant expressing NPR1-S356D / T359D protein, wherein the method comprises steps of: i. isolate plasmid comprising T-DNA fragment comprising a nucleotide sequence of SEQ ID NO 2 or any nucleotide sequence with at least 80% sequence identity thereto, and optionally separately isolate plasmid comprising vir genes; ii. transform Agrobacterium with the plasmid(s) of step i; iii. culture the transformed Agrobacterium in the presence of a selection antibiotic; and iv. immerse a plant seed or leaves in a solution comprising Agrobacterium of step iii, spray solution comprising Agrobacterium on the leaves, or inoculate solution comprising Agrobacterium into a plant, optionally into infection sites.

18. The method of any one of claims 15-17, wherein the method comprises a step of infiltrating NAD and / or NADP+solution (pH about 5.0-6.5, optionally about 5.7) into a plant before,Attorney Docket no.10457-609PC0 after or simultaneously with the step iv, wherein the concentration is 0.1-5 mM, and optionally about 0.8 mM.

19. The method of any one of claims 15-18, wherein the method further comprises a step of infiltrating pipecolic acid (Pip) and / or its derivative N-hydroxypipecolic acid (NHP) solution into a plant, wherein the concentration is 0.1-2.0 mM, and optionally 0.25-0.5 mM.

20. A composition to improve plant immunity, wherein the composition comprises purified protein of NPR1-S356D / T359D with or without a tag in liposomes, NAD or NADP+, and optionally NHP or Pip.

Images