Functional Characterization And Regulating Mechanism Study Of CaAP2/ERF Genes Related To Phytophthora Capsici Resistance In Pepper

Pepper is an important vegetable crop around the world.Pepper blight is one of severe diseases devastating the production of pepper.Identification of resistance genes to P.capsici in pepper is of great importance for blight disease resistance breeding.ERF genes play a significant role in plant responses to the infection of different pathogens.In this research,pepper AP2/ERF gene family was identified by bioinformatics strategies.The expression patterns of CaAP2/ERF genes in different tissue and under biotic stresses were analyzed.What's more,cis-elements in the promoter of each CaAP2/ERF gene were predicted and analyzed as well.Subsequently,VIGS approach was used to screen CaAP2/ERF genes associated with blight disease resistance.At last,by transient expression assay in N.benthamiana,yeast one hybrid and ectopic transformation in tobacco,disease resistance functions and regulation machineries of CaAP2/ERF064,CaAP2/ERF109,and CaAP2/ERF049 gene were studied.The main results are below:1.The family of AP2/ERF gene in pepper has 175 members.Subcellular localization experiment revealed that CaAP2/ERF127 protein was located in the nucleus,CaAP2/ERF129 and CaAP2/ERF171 protein were both located in the nucleus and cytoplasm.CaAP2/ERF genes have a higher expression in root and fruit of pepper.Different CaAP2/ERF gene has its own specific expression patterns under different biotic stresses and phytohormones treatments.The analysis of cis-elements indicates that most of CaAP2/ERF genes contained elements responding to phytohormones,abiotic and biotic stresses in their promoter regions.Exogenous ETH treatment will promote transcript levels of pepper defense response genes CaBPR1,CaPR10,CaSAR82,and CaPO2.Screening of differentially expressed genes CaAP2/ERF064,CaAP2/ERF078,CaAP2/ERF086,CaAP2/ERF113,CaAP2/ERF127 and CaAP2/ERF129 with VIGS approach indicated that CaAP2/ERF064 gene was related to plant disease response to the infection of P.capsici.2.CaAP2/ERF064 protein is located in cytoplasm and nucleus.Expression analysis showed that phytohormones MeJA and ETH could regulate the transcription of CaAP2/ERF064 gene synergistically.Transactivition assay in yeast illustrated that CaAP2/ERF064 gene is a transcription activator and the activation domain of CaAP2/ERF064 protein was in the C-terminal.Transient co-overexpression of CaAP2/ERF064 gene and P19 could trigger cell death response in tobacco.CaAP2/ERF064-N2,a deletion mutant of CaAP2/ERF064 gene,will induce the collapse of epidermis but could not trigger cell death.Above all implies the N-terminal,AP2 domain and C-terminal of CaAP2/ERF064 protein are indispensable for triggering cell death.Experiment of yeast one hybrid indicated that CaAP2/ERF064 and CaAP2/ERF109 proteins could bind to the promoter of CaBPR1 gene.The expression patterns of CaBPR1 gene in pepper post treatment of different phytohormones are almost identical but delayed compared to those of CaAP2/ERF064.The transient expression of CaBPR1 in tobacco could not trigger cell death but could strengthen resistance to the infection of P.capsici.Both silencing of CaAP2/ERF064 in pepper and its homology gene NbERF1B-l in tobacco could significantly decrease the plant disease response to P.capsici.While the over-expression of CaAP2/ERF064 in tobacco could enhanced the plant disease resistance to P.capsici by incresing the expression level of PR genes(NbPR1b,NbGLA,and NbCHN).Besides,double transgenic tobacco plants pCaMV35S:CaAP2/ERF064/ProCaBPR1:CaBPR1 was obtained by the cross of pCaMV35S:CaAP2/ERF064 and Pro CaBPR1:CaBPR1 transgenic tobacco plants,and CaAP2/ERF064 was demonstrated to regulate the expression of CaBPR1 in plant.3.The combined treatment of MeJA,SA,and ETH could enhance the transcription of CaAP2/ERF109 gene when compared with that in treatment of ETH.CaAP2/ERF109 protein was located in nucleus and transient expression of CaAP2/ERF109 in tobacco will enhance disease resistance to P.capsici.Silencing of CaAP2/ERF109 in pepper could not affect disease response to P.capsici.However,over-expression of CaAP2/ERF109 in tobacco will result in increased number of branches,decreased plant height,round and chlorotic leaf,boosted tolerance to drought stress,enhanced transcript expression of NbPR1 b,NbNPR1,NbPR1 a,NbOSM,NbGLA,and NbCHN50 as well as enhanced disease resistance to the infection of P.capsici.Examination results of CaBPR1 gene in pCaMV35S:CaAP2/ERF109/ProCaBPR1:CaBPR1 double transgenic tobacco plants suggests that transcriptional expression of CaBPR1 was regulated by CaAP2/ERF109 and the regulatory activity of CaAP2/ERF109 for transcription of CaBPR1 is higher than that of CaAP2/ERF064.4.The combined treatment of SA and ETH could enhance the expression level of CaAP2/ERF049(CaPTI1)gene when compared with that in treatment of ETH.Over-expression of CaAP2/ERF049 in tobacco could enhance tobacco resistance to the infection of P.capsici.While examination results of CaBPR1 gene in pCaMV35S:CaAP2/ERF049/ProCaBPR1:CaBPR1 double transgenic tobacco plants suggests that CaAP2/ERF049 could not activate the transcription of CaBPR1 in plant.What's more,function analysis of promoter deletion fragment of CaAP2/ERF049 illustrated that-720 ~-1151 bp in promoter region could have important enhancing effect for transcription of CaAP2/ERF049 post infection of P.capsici.