Genome-wide Identification And Expression Of WRKY And MAPK Gene Family In The Diploid Woodland Strawberry Fragaria Vesca And Functional Characterization Of FvWRKY42

Strawberry is economically one of the most important fruits crops worldwide and its genome's sequecing has been completed.WRKY transcription factors are one of the most extensively transcription factor in plants that play critical roles in biotic and abiotic stress and hormone signaling transduction pathways.It has been reported that MAPKs can interact with WRKY transcription factors,play an important role in biotic and abiotic stress and various signaling pathways.However,little is known about the genome-wide WRKY and MAPK gene family in strawberry.In this study,all members of strawberry(Fragaria vesca)WRKY and MAPK gene family were identified in the strawberry genome by bioinformatics approaches.Subsequently,phylogenetic analysis,conserved motif,gene structure,protein interaction network and subcellular localization of two gene familys were systematacially investigated.Moreover,expression patterns of strawberry WRKY and MAPK gene family were examined following various biotic or abiotic stress and hormone treatments using semi-quantitative RT-PCR and RT-qPCR.Furthermore,we examined the function of FvWRKY42 gene in overexpression transgenic Arabidopsis.The main results are as follows:1.We identified 62 WRKY genes(FvWRKYs)in a wild diploid woodland strawberry(Fragaria vesca,2n = 2x = 14)accession Heilongjiang-3.According to the phylogenetic analysis and structural features,these identified strawberry FvWRKY genes were classified into three main groups.In addition,eight FvWRKY-GFP fusion proteins showed distinct nuclear localization in Arabidopsis mesophyll protoplasts.Furthermore,we examined the expression of the 62 FvWRKY genes in ‘Heilongjiang-3' under various conditions,including biotic stress(Podosphaera aphanis),abiotic stresses(drought,salt,cold,and heat),and hormone treatments(abscisic acid,ethephon,methyl jasmonate,and salicylic acid).The expressions of 33 FvWRKY genes were up-regulated,while 12 FvWRKY genes were down-regulated during powdery mildew infection.FvWRKY genes responded to drought and salt stresses much more strongly than to temperature stresses,and FvWRKY genes responded to ABA and MeJA treatments much more strongly than to ETH and SA treatments.11 FvWRKY genes were markedly induced by various stimuli on the transcriptional level,indicating versatile roles in the responses to biotic and abiotic stresses.Interaction networks revealed that the crucial pathways controlled by WRKY proteins may be involved in the differential response to biotic stress.2.We performed a genome-wide survey and identified 12 MAPK genes in the Fragaria vesca genome.Protein domain analysis indicated that all FvMAPKs had typical protein kinase domains.Sequence alignments and phylogenetic analysis classified the FvMAPK genes into four different groups.Conserved motif and exon-intron organization supported the evolutionary relationships inferred from the phylogenetic analysis.Stress-related cis-regulatory element in the promoters and subcellular localization predictions of FvMAPKs were also performed.Further,gene transcript profiles analysis showed that the majority of FvMAPK genes were ubiquitously transcribed in strawberry leaves after treatments with Podosphaera aphanis inoculation,cold,heat,drought,and salt as well as exogenous hormone abscisic acid,ethephon,methyl jasmonate,and salicylic acid.RT-qPCR showed that six selected FvMAPK genes could comprehensively respond to various stimuli.Additionally,interaction networks revealed that the crucial signaling transduction controlled by FvMAPKs may be involved in the biotic and abiotic stress responses.3.A various stress-inducible gene,FvWRKY42(GenBank accession number: KU207053),was isolated from the wild diploid woodland strawberry accession Heilongjiang-3.The full-length cDNA of FvWRKY42 is 1554 bp,which encodes a protein of 517 amino acids.Multiple sequence alignment analysis indicated that FvWRKY42 contained two WRKY domains,a D domain and an SP cluster in the N terminal.Phylogenetic analysis indicated that FvWRKY42 was belonging to group I WRKY superfamily.FvWRKY42 protein has transcriptional activation in yeast.Protein interaction network analysis showed that FvWRKY42 protein interact with various stress-related proteins.Overexpression vector 35S::FvWRKY42-YFP was constructed and transformed into Arabidopsis by floral dip methods.Overexpression of FvWRKY42 in Arabidopsis showed enhanced resistance to powdery mildew presumably via the SA-signaling pathway.Ectopic overexpression of FvWRKY42 in Arabidopsis showed enhanced salt and drought stress tolerance,which is accompanied by increase of the seedling root mass and protection of the integrity of plasma membrane,as well as more effective active oxygen removal system.Additionally,Overexpression of FvWRKY42 in Arabidopsis enhanced ABA sensitivity and promoted leaf closure after ABA and drought treatments.The above results showed that the FvWRKY42 genes may improve the osmotic stress resistance through an ABA-dependent signaling pathway.