Study On Effect Of WRKY Transcription Factors In Regulating Methyl Jasmonate Primed Defense Against Rhizopus Stolonifer In Peach Fruit

Peach（Prunus persica（L.）Batsch）is deeply accepted by costomers due to its juicy and nutritious,peach is ripen and soften rapidly after harvest,and perished due to R.stolonifer infection.Fungicides intervention is the most widely used streagy to reduce postharvest disease of fruit and vegetables,however,due to the environmental and human health risks caused by indiscriminate use of fungicide,alternative strategies such as chemical and/or biological elicitors seems to be sustainable and promising strategies to manage postharvest diseases.As a naturally occurring pivotal biological signaling molecule,methyl jasmonate（MeJA）give advantages over conferring increased resistance to fungal infection in several fruits and vegetables.We have found that 10μM MeJA primed defense response against Rhizopus stolonifer in peach fruit previously,but the molecular mechanism underling MeJA primed disease resistance still not elucidated.This paper took peach fruit treated with 10μM MeJA as experimental materials to explore the mechanism underling MeJA induced priming of defense.Different expressed WRKY transcription factors were scaned via analyzing the RNA-seq result and then identified.The regulation of WRKY TFs to key genes in different metabolic pathway were confirmed by dual luciferase reporter assay（DLR）and electrophoretic mobility shift assay（EMSA）.Furthermore,the interaction between different WRKYs were revealed by yeast two-hybrid（Y2H）,bimolecular fluorescence complementation（Bi FC）and pull down assays,to provide new perspective on the transcriptional regulation regarding MeJA-primed defense in peach fruit,facilitated us to understand the transcriptional regulation mechanism of the induced disease resistance of harvested fruit.The results were as follows:1.A transcriptome database associated with MeJA primed disease resistance of peach fruit was established via RNA-seq.The different expressed genes（DEGs）were screened with fold change≥2 and P≤0.05 as the standard.Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway classification and functional enrichment of DEG showed that the DEGs were significantly enriched in plant hormone signal pathways,biosynthesis of secondary metabolites and metabolics.1695 TFs were identified,and 60WRKY TFs were further identified,among them,the expression pattern of PpWRKY45,PpWRKY46 and PpWRKY70 were significantly induced by exogenous MeJA treatment,while PpWRKY53 were deeply repressed after exogenous MeJA treatment.2.PpWRKY45,a nucleus-localized protein possessed trans-activation ability,was induced by MeJA treatment at an early stage and activated the transcription of PpCHI,PpGLU and PpPR-like as well as the JA biosynthesis-related genes PpLOX,PpAOS and PpOPR3 by binding to their promoters,leading to the subsequent enhancement of the CHI and GLU activities as well as the accumulation of JA content,confers stronger defense responses against pathogen attack.3.PpWRKY70,a nucleus-localized protein possessed trans-activation ability,was significantly induced by MeJA treatment at an early stage and activated the expression of phenylpropanoid metabolic related genes,including PpPAL and Pp4CL,leading to the subsequent enhancement of PAL and 4CL activities,the accumulation of total phenolics,total flavonoids and lignin content as well as the main individual phenolic compounds,increased the resistance towards R.stolonifer in peach fruit.4.PpWRKY46 is a nucleus-localized protein possessed trans-activation ability while PpWRKY53 is a trans-depressor after MeJA treatment which located in nucleus.The energy metabolic genes PpSDH and PpCOX15 were transactivated by PpWRKY46 via binding to their promoters,whereas the transcription of these two genes were repressed by PpWRKY53.Y2H,Bi FC and pull-down results showed that PpWRKY46 has direct physical interaction with PpWRKY53 in vivo and in vitro.The transient overexpression of PpWRKY46 attenuated the transcriptional repression of PpSDH and PpCOX15 caused by PpWRKY53.Which further indicated that PpWRKY46 interacted with PpWRKY53 to regulated the transcriptional of PpSDH and PpCOX15.As a result,the activities and genes expression of energy metabolic enzymes(H⁺-ATPase,Ca²⁺-ATPase,SDH and CCO)were promoted,the increase of ATP and ADP content and the decrease of AMP was alleviated by MeJA treatment and R.stolonifer inoculation during 60 h storage at 20°C.These results indicated that PpWRKY46 counteracts with PpWRKY53 to regulate energy metabolism in MeJA primed disease resistance of peaches.