DOI: 10.5061/dryad.fbg79cp37
title: Data from: β-aminobutyric acid-induced resistance in postharvest peach fruit involves interaction between the MAPK cascade and SNARE13 protein in salicylic acid-dependent pathway

description: The inducer β-aminobutyric
 acid (BABA) is capable of immune
 response in various plants. However, the specific
 mitogen-activated protein kinase (MAPK) cascade involved in
 BABA-induced resistance (BABA-IR) has not yet been elucidated.
 Here, peach fruits treated with the BABA exhibited a
 pattern-triggered immunity (PTI) defense
 against Rhizopus stolonifer, accompanied by the generation of
 reactive oxygen species (ROS) and activation of MAPK
 cascade. Transcriptome sequencing suggested a total of
 fifteen PpMAPKKK/PpMAPKK/PpMAPK genes involved in
 BABA-IR in peach fruit.
 Further qRT-PCR analysis showed that the transcript
 profiles of PpMAPKKK3,
 PpMAPKK5 and PpMAPK1 were obviously
 potentiated. Subsequently, yeast two-hybrid (Y2H), luciferase
 complementation imaging (LCI), pull-down and in vitro
 phosphorylation assays were conducted to characterize the
 complete MAPK cascade (PpMAPKKK3-PpMAPKK5-PpMAPK1) involved in peach
 fruit. Moreover, the downstream events of MAPK1 include the involvement
 of SNARE13 and the
 corresponding NPR1-responsive defense.
 Single silencing of MAPKKK3, MAPKK5 or MAPK1 and double silencing
 of MAPKKK3/MAPKK5 or MAPKK5/MAPK1 resulted in enhanced susceptibility to
 the fungus R. stolonifer in mutants and attenuated salicylic
 acid (SA)-dependent defense gene expression; in contrast, the
 homologous or heterologous overexpression of
 PpSNARE13 in peach fruit or Arabidopsis led to an
 enhanced SA pool and elevated expression of PR genes.
 Reciprocally, the ppsnare13cas9 mutants are generally
 compromised in the priming of
 SA-dependent resistance. Therefore, the
 MAPKKK3-MAPKK5-MAPK1 cascade contributes to PTI signal
 transduction in BABA-elicited peach fruit, by combination with
 downstream events such as SNARE13, NPR1, and SA-dependent signaling.
description: # Data from:β-aminobutyric acid-induced resistance in postharvest peach
 fruit involves interaction between the MAPK cascade and SNARE13 protein in
 salicylic acid-dependent pathway
 [https://doi.org/10.5061/dryad.fbg79cp37](https://doi.org/10.5061/dryad.fbg79cp37) ## Description of the data and file structure 1\. Title of Dataset: Data from: β-aminobutyric acid-induced resistance in postharvest peach fruit involves interaction between the MAPK cascade and SNARE13 protein in salicylic acid-dependent pathway. 2\. Author Information ``` Corresponding Investigator 1 Name: Prof Kaituo Wang Institution: Institute of Fruit Function and Disease Management, Department of Public Health and Management, Chongqing Three Gorges Medical College, Chongqing 404000, P.R. China; College of Biology and Food Engineering, Chongqing Three Gorges University, Chongqing 404000, P.R. China Email: kaituo_wang@sanxiau.edu.cn Co-investigator 1 Name: Dr Chunhong Li Institution: Institute of Fruit Function and Disease Management, Department of Public Health and Management, Chongqing Three Gorges Medical College, Chongqing 404000, P.R. China; College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 Jiangsu, P.R. China Co-investigator 2 Name: Dr Changyi Lei Institution: College of Biology and Food Engineering, Chongqing Three Gorges University, Chongqing 404000, P.R. China Co-investigator 3 Name: Dr Yanyu Zou Institution: College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 Jiangsu, P.R. China; College of Biology and Food Engineering, Chongqing Three Gorges University, Chongqing 404000, P.R. China; College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 Jiangsu, P.R. China Co-investigator 4 Name: Dr Sisi Yang Institution: College of Biology and Food Engineering, Chongqing Three Gorges University, Chongqing 404000, P.R. China Co-investigator 5 Name: Dr Fei Xiang Institution: College of Biology and Food Engineering, Chongqing Three Gorges University, Chongqing 404000, P.R. China Co-investigator 6 Name: Dr Meilin Li Institution: College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 Jiangsu, P.R. China; College of Food, Shenyang Agricultural University, Shenyang 110866 Liaoning, P.R. China Co-investigator 7 Name: Prof Yonghua Zheng Institution: College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 Jiangsu, P.R. China ``` 3\. Date of data collection: 2020-2022 4\. Geographic location of data collection: Chongqing, China 5\. Funding sources that supported the collection of the data: National Natural Science Foundation of China 6\. Recommended citation for this dataset: Li CH, Wang KT, Lei CY, Zou YY, Yang SS, Xiang F, Li ML, Zheng YH. 2025. Data from: β-Aminobutyric acid-induced resistance in postharvest peach fruit involves interaction between the MAPK cascade and SNARE13 protein in salicylic acid-dependent pathway. Dryad Digital Repository. [https://doi.org/10.5061/dryad.fbg79cp37](https://doi.org/10.5061/dryad.fbg79cp37) DATA & FILE OVERVIEW 1\. Description of dataset These data were generated to investigate the combination of MAPK cascade and SNARE13 protein in BABA-induced resistance in postharvest peach. the MAPKKK3-MAPKK5-MAPK1 cascade contributes to PTI signal transduction in BABA-elicited peach fruit, by combination with downstream events such as SNARE13, NPR1, and SA-dependent signalling. 2\. File List: ``` File 1 Name: File1_Raw_images_of_DAB_staining__Y2H__LCI__pulldown__phosphorylation_assay__Co-ip__BiFC__expressor_and_mutants-Final.docx File 2 Name: File2_raw_data_of_LCI.xlsx File 1 includes the data: ``` ROS accumulation was detected by DAB staining and observed in the pulp of 24-, 48- and 72-hour-old peaches treated with the BABA formulation and/or R. stolonifer conidia (data in Figure 1 in published article). The GAL4 DNA binding domain (BD) vectors fused PpMAPKKKs (PpMAPKKK1, PpMAPKKK2, PpMAPKKK3, PpMAPKKK5 and PpMAPKKK7) were transformed with the activation domain (AD) vectors fused PpMAPKKs (PpMAPKK2, PpMAPKK3, PpMAPKK5 and PpMAPKK9) into AH109 yeast cells or cotransformed into AH109 cells with BD-fused-PpMAPKK5 and AD-fused-PpMAPKs, and the transformed cells, including those on TDO (triple dropout media, SD/-Trp/-Leu/-His) and QDO (quadruple dropout media, SD/-Trp/-Leu/-His/-Ade), were further cultured on selection media supplemented with or without X-α-gal (data in Figure 4 in published article). The indicated cLUC (PpMAPKKK3-cLUC or PpMAPKK5-cLUC) and nLUC (PpMAPKK5-nLUC or PpMAPK1-nLUC) constructs were transiently coexpressed in tobacco leaves for LCI assays; In vitro GST pull-down assays confirmed the interaction of PpMAPKK5 with PpMAPKKK3/PpMAPK1 using anti-His or anti-GST antibodies; In vitro kinase assay to detect the phosphorylation of PpMAPKK5 by PpMAPKKK3 and PpMAPK1 by PpMAPKK5. GST-PpMAPKKK3 was incubated with His-PpMAPKK5 or coincubated with GST-PpMAPKK5 and His-PpMAPK1 in kinase assay buffer, either in the presence (+) or absence (–) of ATP as indicated, and further detected by PhoS-tag SDS-PAGE (data in Figure 5 in published article). Disease symptoms of representative ppmapkkk3cas9, ppmapkk5cas9 and ppmapk1cas9 mutant fruits and wild-type fruits (data in Figure 6 in published article). The atmapkkk3cas9 and atmapkk5cas9 mutants display increased susceptibility to the fungus R. stolonifer. Disease symptoms were imaged 6 days after inoculation with R. stolonifer conidia; Disease lesions of atmapkkk3cas9 atmapkk5cas9 mutants were recorded 6 days after inoculation with R. stolonifer conidia; Assessment of fungal colonization in atmapkkk3cas9 atmapkk5cas9 mutants via trypan blue staining at 6 dpi (data in Figure 7 in published article). Disease symptoms of the representative leaves of the mapkk5cas9 and mapk1cas9 mutants after punch inoculation with R. stolonifer conidia; Growth status and transcript levels of AtMAPKK5 and AtMAPK1 in four-week-old atmapkk5cas9 atmapk1cas9 double mutants; Disease symptoms, lesion diameter and relative fungal biomass of the representative leaves of the atmapkk5cas9 atmapk1cas9 mutant plants 6 days after inoculation with R. stolonifer; Level of fungal colonization in the trypan blue-stained leaves of atmapkk5cas9 atmapk1cas9 mutants and the related staining ratio 6 days after inoculation with R. stolonifer (data in Figure 8 in published article). Y2H screening of the potential targets of the PpMAPK1 protein and characterization of the interaction; PpMAPK1 phosphorylates PpSNARE13 in the presence of ATP. His-PpMAPK1 was incubated with GST-PpSNARE13 in kinase assay buffer, and total protein was subjected to SDS-PAGE with phosbind acrylamide and detected by immunoblot analysis with an anti-GST antibody (data in Figure 9 in published article). Coimmunoprecipitation assay to detect the in vivo interaction of PpSNARE13 with PpNPR1. Proteins isolated from tobacco leaves transfected with the indicated plasmid combinations were immunoprecipitated with the anti-GFP or anti-myc antibody. IP, immunoprecipitation; The BiFC assay revealed the interaction between PpSNARE13 and PpNPR1 in vivo in the nucleus (data in Figure 10 in published article). Four-week-old Arabidopsis plants overexpressing PpSNARE13 in the growth chamber; Disease symptoms of the representative leaves of PpSNARE13 overexpression lines were imaged 6 days after punch-type inoculation with R. stolonifer; Qualitative determination of the fungal colonization level through trypan blue staining of PpSNARE13 overexpression lines at 6 dpi; The trypan blue staining ratio and fungal biomass of PpSNARE13 overexpression lines relative to those of Col-0 plants at 6 days after inoculation with R. stolonifer conidia (data in Figure 11 in published article). Infection phenotypes of representative fruits of PpSNARE13-overexpressing and wild-type fruits; Lesion diameter, relative fungal biomass and trypan blue staining of representative fruits of PpSNARE13 overexpressors 2 d after inoculation (dai) (data in Figure 12 in published article). Disease symptoms of the representative fruit of ppsnare13 mutants and wide type fruit; Lesion diameter and relative fungal biomass of the representative fruit of ppsnare13 mutants and wild-type fruit at 2 days after inoculation with R. stolonifer conidia; Trypan blue staining of representative fruits of ppsnare13 mutants 2 d after inoculation (data in Figure 13 in published article). ``` File 2 includes the data: The indicated cLUC (PpMAPKKK3-cLUC or PpMAPKK5-cLUC) and nLUC (PpMAPKK5-nLUC or PpMAPK1-nLUC) constructs were transiently coexpressed in tobacco leaves for LCI assays. (data in Figure 5 in published article). ``` METHODOLOGICAL INFORMATION Peach fruit with an identical size, maturity and no visual mechanical damage was surface-sterilized for 2 min in 75 % alcohol, dried at 20 °C for approximately 2 h and divided into four subsets of 360 fruit each. Two 3-mm-deep and 3-mm-diameter holes were punched by sterilized dissecting needles at the centre of symmetry of each fruit. The optimal concentration of BABA (Sigma Co, MO, USA) at 50 mmol/L was selected. Then, different conditions were applied to each wound of each fruit from the four sets: 1) treatment with 20 μL of ddH2O (Control); 2) pre-treatment with an aqueous solution of 50 mmol/L BABA (BABA); 3) inoculation with R. stolonifer spore suspension at a 1.0 × 105 spores/mL concentration (Inoculation); and 4) pre-treatment with 50 mmol/L BABA followed by the fungal inoculation (BABA + inoculation). Following the treatments, all fruit were distributed on consoles for at least 5 h and were further seal-packaged in polyethylene (PE) film (0.06 mm in thickness) and stored at 20 °C for up to 72 hours. At each 12-hour interval from 0 to 72 hours, 30-50-gram samples of the uninfected sarcocarp (2 mm away from the infected area) from 60 fruit in each subset were collected. Fruit pulps from each individual were flash frozen with liquid nitrogen and stored at -80 °C until use. The experiment was laid out twice in a completely randomized design with three replicates per treatment. The* PpMAPKKK3*/*PpMAPKK5* and *PpMAPKK5*/*PpMAPK1* cDNAs were inserted into the pCAMBIA1300-cLUC and pCAMBIA1300-nLUC vectors, respectively, using the primers provided in Table S1. The combinations (*PpMAPKKK3*-cLUC + *PpMAPKK5*-nLUC, *PpMAPKKK3*-cLUC + nLUC, cLUC + *PpMAPKK5*-nLUC or *PpMAPKK5*-cLUC + *PpMAPK1*-nLUC, *PpMAPKK5*-cLUC + nLUC, cLUC + *PpMAPK1*-nLUC) were expressed in tobacco plants via *Agrobacterium tumefaciens*-mediated transformation (ATMT). The LUC signals and activities of the injected *N. tabacum* plants were recorded after 2-3 days of cultivation under a circadian photoperiod at 22 °C, in which the combination of *AtFLS2*-nLUC + *AtAGB1*-cLUC were treated as positive control.