Transcription Factors WRKY And BZIP Regulate The Expression Of Disease-Resistance Genes In Chinese Wild Vitis Quinquangularis

Grapevine as important fruit crop in the world creates large economic benefits.Resveratrol as important phytoalexin in grape,has been proved not only protect plants from biotic and abiotic stress,also act as cardioprotective,antiumor and neuroprotective agent.European grape?Vitis vinifera L.?as main cultivated species show high quality and yield,but poor resistance.China as one of the grape origin centers has abundant wild grape germpalsm resources.In previous studies,our research group found that the Chinese wild Vitis quinquangularis accession Danfeng-2 contains higher level of resveratrol than other grapevines.In this study,co-expression analysis was performed using transcriptomic data to identify transcription factors participating in regulating STS genes expression.Function of these genes was analyzed.The main results were listed below:1.The qRT-PCR method was used to analyze the expression of 59 WRKY transcription factor family members in Chinese wild Vitis quinquangularis accession Danfeng-2 in response to the induction of powdery mildew.Among them,the expression level of 19WRKY members including,VqWRKY2,VqWRKY3,VqWRKY6,VqWRKY7,VqWRKY10,VqWRKY18,VqWRKY21,VqWRKY23,VqWRKY26,VqWRKY27,VqWRKY28,VqWRKY29,VqWRKY30,VqWRKY31,VqWRKY32,VqWRKY48,VqWRKY49,VqWRKY52,VqWRKY53were up-regulated under the induction of powdery mildew.Homologous cloning method was used to isolate VqWRKY2,VqWRKY3,VqWRKY9,VqWRKY24 and VqWRKY53 from Chinese wild Vitis quinquangularis Danfeng-2.Coding sequences?CDSs?of the five WRKY genes were respectively 1779 bp,570 bp,780 bp,582 bp and 456 bp.The NCBI accession numbers are:VqWRKY2?MN240478?,VqWRKY3?MN240479?,VqWRKY9?MN240480?,VqWRKY24?MN240481?,VqWRKY53?MN240482?.VqWRKY2 belongs to the group IIb subfamily of the WRKY transcription factor.VqWRKY3,VqWRKY24 and VqWRKY53belong to group IIc subfamily and VqWRKY9 belongs to the group IIa.At the same time,the promoters of VqWRKY2,VqWRKY3,VqWRKY9,VqWRKY24 and VqWRKY53 were isolated and conducted sequence analysis.Subcellular localization analysis of Arabidopsis protoplasts by PEG-mediated transformation indicated that VqWRKY2,VqWRKY3,VqWRKY9,VqWRKY24 and VqWRKY53 were all located in the nucleus.2.The expression profile of the five VqWRKY genes under the infection of powdery mildew,flg22,H₂O₂ and CaCl₂ treatment were analyzed using qRT-PCR.The transcript level of VqWRKY2,VqWRKY3 and VqWRKY53 were increased after inoculation with powdery mildew.VqWRKY9 and VqWRKY24 were down-regulated under the inoculation of powdery mildew.We found that VqWRKY53 and STS genes had a synergistic expression relationship under powdery mildew infection.VqWRKY3 and VqWRKY53 may also be involved in the regulation of STS genes under pathogen infection.After flg22 treatment,VqWRKY3,VqWRKY9,VqWRKY53 and STS genes more significantly responded to flg22 induction.After H₂O₂ treatment,the transcript level of VqWRKY2,VqWRKY3,VqWRKY9,VqWRKY24,VqWRKY53 and STS genes were strongly up-regulated.After CaCl₂ treatment,only VqWRKY53 and STS genes were up-regulated.At the same time,we found that the response of VqWRKY3,VqWRKY9 and VqWRKY53 to flg22 need to be via the reactive oxygen burst and MAPK cascade pathway.3.Leaves of Chinese wild Vitis quinquangularis Danfeng-2 were treated with hormones including SA,ABA,Eth and MeJA.The response patterns of WRKY transcription factors and STS genes to hormone signals were analyzed by qRT-PCR.We found that VqWRKY2,VqWRKY3,VqWRKY53 and STS were up-regulated under the induction of SA,ABA,Eth and MeJA.The expression level of VqWRKY9 was increased under the induction of SA,ABA and MeJA.VqWRKY24 is up-regulated under the induction of SA.4.Yeast two hybrid assay and bimolecular fluorescence complementation?BiFC?assay showed that VqWRKY3 could interact with VqMYB14 and VqWRKY53 could interact with VqMYB14,VqMYB15.The promoters of VqSTS9,VqSTS16,VqSTS32,VqSTS41 and VqSTS48 were cloned from the gDNA of Danfeng-2.Sequence analysis indicated that the five STS promoter sequences contained W-box and MBS cis-elements.Yeast one-hybrid experiments demonstrated that VqWRKY2 can bind to the promoters of VqSTS9,VqSTS32,VqSTS41 and VqSTS48.VqWRKY3 can bind to the promoters of VqSTS16 and VqSTS48.VqWRKY9 and VqWRKY24 can bind to the promoters of VqSTS9,VqSTS16,VqSTS41 and VqSTS48.VqWRKY53 can bind to the promoters of VqSTS9,VqSTS16 and VqSTS41.Five WRKY transcription factors can bind to the TTGACC element,while only VqWRKY9 and VqWRKY53 can bind to the TTGACT element.5.The effect of WRKY transcription factors on promoters of STS genes was further verified by GUS activity quantitative assay.The results indicated that VqWRKY2 and VqWRKY9 can promote the promoter activity of VqSTS9,VqSTS16,VqSTS32,VqSTS41 and VqSTS48.VqWRKY3 can promote the promoter activity of VqSTS32 and VqSTS48.VqWRKY24 can promote the promoter activity of VqSTS16,VqSTS32,VqSTS41 and VqSTS48.VqWRKY53 can promote the promoter activity of VqSTS9,VqSTS16,VqSTS32,and VqSTS41.Co-expression of VqWRKY3 and VqMYB14 up-regulated the the promoter activity of VqSTS9,VqSTS16,VqSTS32 and VqSTS41.However,co-expression of VqWRKY3and VqMYB14 reduced promoter activity of VqSTS48 relative to VqWRKY3 alone.Co-expression of VqWRKY53 with VqMYB14 or VqMYB15 can promote STS promoter activity to varying degrees.The WRKY transcription factors were transiently overexpressed in the leaves of Danfeng-2,and the expression level of STS gene was increased.The content of trans-resveratrol and trans-piceid was increased at the same time.Heterologous expression of VqWRKY2 can enhance Arabidopsis resistance to PstDC3000 via the JA pathway.Heterologous overexpression of VqWRKY53 can up-regulate the expression of AtSAG12,promote the accumulation of ROS and SA and accelerate leaf senescence.Overexpression of VqWRKY5 also enhanced Arabidopsis resistance to PstDC3000 via the SA pathway.6.Using the method of homologous cloning,VqbZIP1 was isolated from Danfeng-2.The coding sequence of VqbZIP1 is 900 bp encoding 299 amino acids.The sequence of VqbZIP1 was submitted to GenBank and the accession number is AXN75965.1.Spatiotemporal expression analysis of VqbZIP1 gene showed that VqbZIP1 expressed in all tissues and organs of grape and higher in mature berry.VqbZIP1 responsed to varying degrees in all four hormone treatments.Among them,the response to ABA,Eth and MeJA is more significant.VqbZIP1 also responds to powdery mildew infection,CaCl₂ and H₂O₂small molecule signals.VqbZIP1 was localized in the nucleus and has transcriptional activation activity in yeast.Yeast two-hybrid assay and BiFC assay were used to demonstrate the interaction between VqbZIP1 and VqSnRK2.4,VqSnRK2.6.Through bioinformatic analysis,VqSnRK2.4 and VqSnRK2.6 genes were predicted located on chromosomes 7 and 3and showed highest homology to AtSnRK2.4 and AtSnRK2.6 in Arabidopsis.Subcellular localization indicated that VqSnRK2.4 and VqSnRK2.6 existed in the cytoplasm,nucleus and cell membrane.Under the induction of ABA hormone,VqSTS6,VqSTS16,VqSTS20,VqSnRK2.4 and VqSnRK2.6 were all up-regulated to different degrees.Promoters of STS6,STS16 and STS20 were cloned from the gDNA of Chinese wild Vitis quinquangularis Danfeng-2.The three promoters contain ABRE cis-elements in response to ABA signals.Three STS promoters were inserted into pC0380-GUS vector,and GUS activity assay was carried out.The result showed that VqbZIP1 could activate the promoters of the VqSTS6,STS16,and STS20.Co-expression of VqbZIP1 with VqSnRK2.4 and VqSnRK2.6 showed higherr activation of STS promoters compared with single expression of VqbZIP1.Agrobacterium-mediated transient overexpression aasay was conducted in leaves of Chinese wild Vitis quinquangularis Danfeng-2.The results showed that overexpression of VqbZIP1 could up-regulate the total transcript level of STS genes and individual STS6,STS16 and STS20.The accumulation of resveratrol and piceid was inceased at the same time.Heterologous expression of VqbZIP1 in Arabidopsis thaliana increased the plant sensitivity to ABA and up-regulated the key genes in ABA signaling pathway.In summary,Chinese wild Vitis quinquangularis transcription factors VqWRKY2,VqWRKY3,VqWRKY9,VqWRKY24,VqWRKY53 and VqbZIP1 participate in regulation of the expression of STS genes and promote plant disease resistance.