A Study On Mechanism Of Priming Resistance Induced By β-Aminobutyric Acid In Postharvest Peach Fruit

Peach fruit is favored by consumers for its juiceful texture,appealing flavor and rich in nutrition.However,peaches mature in mid-summer season and are extremely susceptible to mechanical injury and pathogenic infection,with rapid postharvest physiological deterioration(PPD)under ambient temperature mainly due to its high respiration rate,thin epicarp as well as delicate in texture.Soft rot caused by Rhizopus stolonifer is the major postharvest fungal disease of peach fruit and can be effectively controlled by chemical fungicides.Nevertheless,public concern regarding chemical residues and pathogen resistance to fungicides has caused a wide range of fungicide applications to be restricted.Alternative strategies such as biological elicitor treatment seems to be sustainable and promising approachs to manage postharvest diseases of peach fruit owing to the induction effect of disease resistance in fruit by elicitors.β-aminobutyric acid(BABA),known as a non-protein amino acid,has been described to be a promising inducer that triggers defence responses in several agricultural plants.Our previous research has shown that the priming resistance elicited by BABA is beneficial to the balance of plant disease response and fitness cost,whlie the molecular mechanism underling BABA primed disease resistance is still not fully elucidated.In this study,we further investigate the mechanism of cofactor NPR1,bZIP transcription factor TGA1,MADS-box gene MADS2 and MAPK cascade in the regulation of BABA-primed defense response in peach fruit,which will contribute to elucidating the regulatory network of priming defense in peach fruit.The main results are described as follows:1.The highly conserved amino acid sequences of BRLZ(PF07716)and DOG1(PF14144)domains were used to identify the TGA family members,and 15 members of the TGA family from the peach genome have be identified through bioinformatics analysis.The TGAs were unevenly distributed on the 1st,2rd,6th and 7th Prunus persica chromosomes.The protein parameters of the determined TGA TFs listed as follows:the sizes ranging from 333 to 546 aa,molecular weights ranging from 37.07 to 61.47 kD,the isoelectric points ranging from 6.01 to 8.59,respectively.And all members of the TGA family were located in the nucleus,without signal peptide as well as transmembrane domain.According to the phylogenetic relationship,the TGA family of Arabidopsis thaliana,Glycine max,Solanum lycopersicum,Oryza sativa and Prunus persica was divided into five subfamilies,among which the peach TGA family members were mainly distributed in Ⅰ,Ⅱ and Ⅳ clades.And TGA family members in the same clade shared similar gene structure and conservative motif.After searching the promoter sequences of peach TGA genes by PlantCARE database,we found that the promoter regions of the TGA members contained at least one plant hormone or stress response-related elements.In addition,RNA-seq data analyses showed that the expression patterns of TGA gene family under BABA treatment and/or R.stolonifer infection could be divided into three types.The expressions of the first and second types of TGA family members were potentiated by the elicitor BABA and fungal pathogen and their expression peaks were presented within 12 h and 36 h after R.stolonifer inoculation,respectively.The results indicated the involvement of these TGA members in BABA-induced fungal resistance of peach friuit.2.Transcriptome sequencing showed that there were 37 DEGs up-regulated genes and 139 down-regulated genes in R.stolonifer-inoculated peaches no matter whether these fruit was pre-treated with BABA or not.And KEGG pathway enrichment analysis showed that the 176 DEGs shared between BABA+inoculation-vs-control and inoculation-vs-control pairwise comparisons were significantly enriched in ’plant-pathogen interaction’ and ’plant hormone signal transduction’.Furthermore,the TGA1 transcription factor with the highest degree in the protein-protein interaction(PPI)network of the proteins encoded by these 176 common DEGs was identified as the key target protein through degree method and centrality measurement.In addition,BABA treatment could induce the generation of redox signaling molecules(nitric oxide(NO)and SA)to promote the reduction potential of peach fruit upon infected by R.stolonifer and consequently induce the translocation of the product of the highly expressed PpNPRl gene to the nucleus,where it interacted with PpTGAl and strengthened the gene expressions of defence-related PpPRl and PpPRl-like,and thus enhancing the resistance level of peach fruit to fungal pathogen.3.The first pre-infection priming phase of the peaches was triggered after BABA elicitation alone,which enhanced the transcript abundance of SA synthesis-related and redox-regulated genes for elevating the reducing potential of peach fruit.The second post-infection priming phase was activated by attacking R.stolonifer and caused an H2O2 burst by upregulation of PpRBOH genes,resulting in a robust defence response.In addition,PpTGA1 was used as a bait to screen the Y2H library,and PpMAPKK5 was identified as a shortcut interacting protein of PpTGA1.Yeast two-hybrid(Y2H),His pull-down and coimmunoprecipitation(Co-IP)assays further confirmed the interaction of PpMAPKK5 and PpTGA1.The results of subcellular localization,yeast one-hybrid(Y1H)and dual-luciferase reporter(DLR)assays suggested that the nucleus-localized PpMAPKK5 could increase the DNA binding activity of PpTGA1 for the upregulation of SA-responsive PR gene expression.Overexpressing PpMAPKK5 in Arabidopsis caused the constitutive transcripts of SA-dependent PR genes and lowered the sensitivity to R.stolonifer.In contrast,the atmapkk5cos9 loss-of-function mutation caused an attenuated resistance to R.stolonifer.Hence,we suggest that BABA-induced priming defence in peaches linked with an augmented capacity for NPR1-related SAR defence is activated by two-phase redox homeostasis with initial elicitor-induced reductive signaling and a second pathogen-stimulated H2O2 burst accompanied by the possible phosphorylation of PpTGA1 by PpMAPKK5 for signal amplification.4.The treatment of peach fruit with BABA resulted in a significant delay in the symptomatic appearance of soft rot disease and this protection was greatly potent in R.stolonifer-inoculated peaches and equipped the intertwined defence response containing H2O2 burst,ABA accumulation and callose deposition.Moreover,nucleus-localized PpMADS2 was identified as a PpNPR1 interacting partner via cDNA library screening.Y2H,firefly luciferase complementation imaging(LCI)and Co-IP assays further verified the interaction between PpNPRl and PpMADS2.Y1H and DLR assays indicated that the DNA binding activity of PpNPR1 conferred by the PpNPR1-PpMADS2 complex was required for the transcription of SA-dependent PRs,ABA-inducible ABI1 and RAB18 and PpCalS genes to gain the augmented BABA-IR,in which the PpMAPK1-induced posttranslational modification of PpMADS2 simultaneously was involved.Accordingly,PpMADS2 overexpression potentiated the transcription of a group of PR genes and fungal resistance in transgenic Arabidopsis.Conversely,mads2 knockout lines showed high sensitivity to fungal pathogen.Therefore,the results indicate that PpMADS2 positively participates in the BABA-elicited defence in peaches through the collaboration of SA-dependent NPR1 activation and ABA-signaling callose accumulation,which is also related to the posttranslational modification of PpMADS2 by PpMAPK1 for signal amplification.5.RNA-seq analysis and qRT-PCR detection proved that mitogen-activated protein kinases PpMAPKKK3,PpMAPKK5 and PpMAPK1 are highly responsive to BABA-mediated fungal defense responses in peach fruit and their expression level was significantly induced by BABA treatment and R.stolonifer inoculation.Y2H,LCI,GST pull-down and in vitro phosphorylation assay confirmed that PpMAPKKK3 could interact with PpMAPKK5 in yeast cells,tobacco leaf epidermal cells and in vitro,and phosphorylated PpMAPKK5.At the same time,phosphorylation-activated PpMAPKK5 can directly bind to and phosphorylate PpMAPK1 located at the terminal of the MAPK cascade in vitro.Therefore,there is a complete MAPK cascade pathway,PpMAPKKK3→PpMAPKK5→PpMAPK1,in BABA-mediated disease resistance in peach fruit,in which PpMAPKKK3 located at the upstream of this MAPK cascade could transmit the received fungal stress signals to the downstream PpMAPKK5 and PpMAPKl through its phosphorylation of PpMAPKK5.Moreover,resistance phenotype analysis showed that both atmapkkk3cos9 atmapkk5cos9 and atmapkk5cos9 atmapklcos9 double mutants obtained based on CRISPR-Cas9 gene editing technology and artificial hybridization presented similar fungal susceptibility to R.stolonifer.These findings revealed that MAPKKK3 MAPKK5→MAPK1 cascade positively regulates BABA-mediated fungal resistance in peach fruit via multilevel phosphorylation.In conclusion,BABA treatment could trigger the defense response of peach fruit against fungal pathogen through a two-phase priming mechanism,and this process mainly depends on the SA and ABA signaling pathways.Meanwhile,the nuclear localization of PpNPR1 protein and the formation of the PpNPR1-PpTGAl complex contribute to the induction of BABA-induce resistance(BABA-IR)in peach fruit.Furthermore,PpMADS2 transcription factor can directly activate the ABA signaling pathway to potentiate the synthesis of callose at the infection sites.In addition,the activation of mitogen-activated protein kinase cascade,PpMAPKKK3→PpMAPKK5→PpMAPK1,leads to the amplification of the BABA-mediated fungal resistance response in peach fruit through a concatenation of three successive phosphorylation events.In this MAPK cascade,the direct signaling of PpMAPKK5 to PpTGAl can bypass the normal MAPK cascade and PpMAPK1 can enhance the transcriptional regulatory activity of PpMADS2 transcription factor.