ERF Transcription Factors Regulate The Expression Of The Disease Resistance Genes In Chinese Wild Vitis Quinquangularis

Grapevine is one of the most important fruit trees and widely cultivated in the world,especially Vitis vinifera,which has excellent edible quality and high economic value.However,V.vinifera has poor disease resistance.China is one of the worlds’major biodiversity centres for Vitis and has a large number of Chinese wild grapevines,which are very important germplasm resources.The Chinese wild species Vitis quinquangularis,particularly accession‘Danfeng-2’,has attracted considerable interest because of its high resistance to pathogen invasion and the high level of resveratrol found in its ripe berries.Based on the transcriptome analysis performed on‘Danfeng-2’and V.vinifera cv.Cabernet Sauvignon during the various stages of berry development,three ERF transcription factors（VqERF112,VqERF114 and VqERF072）were found to be strongly co-expressed with stilbene synthases and in response to Uncinula necator.Studies on three ERF transcription factors involved in regulating the expressions of disease resistance genes were explored.The main results are as follows:1.Three ERF transcription factors,identified as VqERF112,VqERF114 and VqERF072,were cloned from Chinese wild V.quinquangularis accession‘Danfeng-2’and functional analyses were performed.The cDNA clones of VqERF112,VqERF114 and VqERF072consist of 717 bp,1266 bp and 645 bp ORFs predicted to encode proteins of 238,421 and214 amino acids,respectively.These three ERF transcription factors contain single conserved AP2/ERF domain and belong to ERF subfamily.VqERF112,VqERF114 and VqERF072 shared 99.58%,99.05%and 98.13%amino acid identity with the predicted proteins encoded by V.vinifera VvERF112,VvERF114 and VvERF072,which are located on chromosome 1,18 and 15 of the Pinot Noir genotype,respectively.VqERF112,VqERF114 and VqERF072 can respond to powdery mildew inoculation and hormone treatments,such as ethylene,salicylic acid,methyl jasmonate and abscisic acid.VqERF112and VqERF072 can also respond to gibberellin treatment.However,VqERF114 can not respond to gibberellin treatment.Tissue specific expression analysis demonstrated that VqERF112 and VqERF114 had the highest expression levels in mature berries and VqERF072 had the highest expression level in tendril.The result of subcellular localization showed that the GFP signal of VqERF112 was found in nucleus,cytoplasm and cell membrane.VqERF114 and VqERF072 localized in the nuclei of the tobacco epidermal cells.VqERF112,VqERF114 and VqERF072 possessed trans-activation abilities in yeast cells.2.The promoters of VqERF112,VqERF114 and VqERF072 from‘Danfeng-2’and the promoters of their homologous genes VvERF112,VvERF114 and VvERF072 from Thompson seedless were cloned and conserved cis-element motifs in each promoter were predicted.The differences of cis-element motifs between‘Danfeng-2’and Thompson seedless were TC-rich repeats（defense and stress responsiveness）,ERE element（ethylene-responsive element）,TCA-element（salicylic acid responsiveness）and TGACG-motif（involved in the MeJA-responsiveness）.The promoters were inserted into the pC0380-GUS vector to fuse the GUS reporter.Each construct was transiently expressed in leaves of Thompson seedless and tested for GUS activity after powdery mildew inoculation and hormone treatments.The results indicated that the promoters of VqERF112,VqERF114and VqERF072 can be induced by powdery mildew inoculation and hormone treatments,such as ethylene,salicylic acid and methyl jasmonate.3.Functional analyses of three ERF transcription factors involved in disease resistance were performed.VqERF112,VqERF114 and VqERF072 can respond to Pseudomonas syringae pv.tomato（Pst）DC3000 and Botrytis cinerea inoculation.The CaMV35S promoter-driven overexpression（OE）transgenic Arabidopsis lines of VqERF112,VqERF114and VqERF072 were generated and inoculated with Pst DC3000 and B.cinerea.After Pst DC3000 inoculation,no obvious disease symptoms of chlorosis were observed in leaves of VqERF112-OE,VqERF114-OE and VqERF072-OE plants and the bacterial quantities in transgenic plants（approximately 10⁴ cfu/cm²）were significantly lower than in Col-0 plants（approximately 10⁶ cfu/cm²）.Overexpressions of VqERF112,VqERF114 and VqERF072 in transgenic Arabidopsis promoted cell death and H₂O₂ accumulation and improved the resistance to Pst DC3000.The SA signaling-related genes,AtNPR1 and AtPR1,were up-regulated in OE transgenic plants compared with Col-0 plants.However,the expressions of SA signaling-related genes,AtICS1 and AtPR5,were down-regulated.The expressions of JA/ET signaling-related genes,AtPDF1.2,AtLOX3,AtPR3 and AtPR4,increased in OE transgenic plants compared with Col-0 plants.After B.cinerea inoculation,smaller necrotic and chlorotic lesions appeared on leaves of VqERF112-OE,VqERF114-OE and VqERF072-OE plants compared with Col-0 plants.Overexpressions of VqERF112,VqERF114 and VqERF072 in transgenic Arabidopsis decreased cell death and H₂O₂accumulation.The resistance of OE transgenic plants to B.cinerea was improved.The expressions of SA signaling-related genes increased in OE transgenic plants compared with Col-0 plants,such as AtNPR1,AtPR1 and AtICS1.However,the expression of SA signaling-related gene,AtPR5,was down-regulated.The expressions of JA/ET signaling-related genes,AtPDF1.2,AtLOX3,AtPR3 and AtPR4,were up-regulated in OE transgenic plants compared with Col-0 plants after B.cinerea inoculation.4.The expression of stilbene synthase（STS）regulated by VqERF114 was explored.Transient overexpression of VqERF114 in‘Danfeng-2’leaves increased the expression of STS by 10.9-fold.The contents of trans-resveratrol and trans-piceid also increased by1.6-fold and 1.7-fold after VqERF114 was overexpressed.VqERF114 specifically binds to the GCC-box element.However,analysis of cis-element motifs indicates that promoters of13 VqSTSs had no GCC-box,but contained the MRE or MBS element,which can be recognised by MYB transcription factors.VqMYB35,identified as a VqERF114 interacting protein,was selected by the yeast two-hybrid assay.Bimolecular fluorescence complementation assay was carried out to further confirm the interaction between VqERF114 and VqMYB35.Furthermore,VqMYB35 showed activation effects on the expressions of VqSTS15,VqSTS28,VqSTS42 and VqSTS46 by directly binding to the MBS elements in their promoters.Co-overexpression of VqERF114 and VqMYB35 resulted in higher VqSTSs expression and more stilbene synthesis.The gene cloning and functional study of VqMYB35 from‘Danfeng-2’were carried out.The cDNA clone of VqMYB35consists of a 1113 bp ORF predicted to encode a protein of 370 amino acids and VqMYB35was predicted to be located on chromosome 14.VqMYB35 contains two conserved SANT domains and belongs to R2R3-MYB transcription factor.VqMYB35 can respond to powdery mildew inoculation and hormone treatments,such as ethylene,salicylic acid,methyl jasmonate and abscisic acid.Tissue specific expression analysis demonstrated that VqMYB35had the highest expression level in mature leaves.VqMYB35 localized in the nuclei of the tobacco epidermal cells and possessed trans-activation ability in yeast cells.In conclusion,VqERF112,VqERF114 and VqERF072 from Chinese wild V.quinquangularis positively regulate the resistances to Pst DC3000 and B.cinerea via SA and JA/ET signaling pathways.VqERF114 indirectly regulates STS expression and stilbene synthesis by interacting with VqMYB35.