Functional Analyses Of E3 Ubiquitin Ligase Gene PUB24 From Chinese Wild Grape Vitis Pseudoreticulata

Grape is one of the most important fruit crops and widely cultivated in the world,especially European grape(Vitis vinifera L.),which is the dominant species in the grape production and used for fresh,juice,dried and wine.European grape cultivars have excellent edible quality and great commercial interests but many of them exhibit low resistance to biotic stresses,especially to the powdery mildew.China,one centre of origin of grapes,has abundant wild grape germplasm resources.The Chinese wild species,V.pseudoreticulata accession Baihe-35-1,shows the high resistance to powdery mildew.In this study,we isolated a U-box type E3 ubiquitin ligase gene from Baihe-35-1,which was induced by powdery mildew.Then the gene structure,expression patterns and resistance mechanism were analyzed.The main results were obtained as follows:1.In this study,RT-PCR analysis was used to test the expression pattern of 56 putative U-box type E3 ubiquitin ligase genes in Baihe-35-1 in response to powdery mildew inoculation and we found that VpPUB24,a member of the U-box family,was significantly induced by powdery mildew inoculation.2.The full-length sequence of VpPUB24(GenBank accession number: KU296025)was amplified by RACE(Rapid amplification of cDNA ends)method.According to the sequencing analysis,the full length of VpPUB24 mRNA was 1457 bp and the open reading frame of VpPUB24 was 1236 bp encoding 411 amino acid sequences with a N-terminal U-box domain and 4 reduplicative ARM domains.Furthermore,the amino acids of PUB24 from V.pseudoreticulata accession Baihe-35-1,V.vinifera cv.Red Globe,V.vinifera cv.Cabernet Sauvignon and V.vinifera cv.Thompson Seedless were 100% identity.3.The promoter of VpPUB24 was cloned and inserted into the reporter vector pC0380-GUS.The recombinant vector containing the GUS reporter gene was transformed into the Agrobacterium tumefaciens GV3101 strain.The quantification of GUS activity was measured through the transient expression of PVp PUB24-GUS into grape leaves followed by biotic and abiotic treatments.The result showed that VpPUB24 promoter was induced by PM,SA,MeJA,and 4°C treatment.Furthermore,the cis-elements analysis of promoters from Baihe-35-1,Red Globe,Cabernet Sauvignon and Thompson Seedless by PlantCARE revealed that the P-box in V.vinifera was replaced by the TCA-element in V.pseudoreticulata.The quantification of GUS activity suggested that the transcriptions of PUB24 were induced by PM inoculation and SA treatment and the response pattern was different between V.pseudoreticulata and V.vinifera.4.The yeast cDNA library was constructed using V.pseudoreticulata accession Baihe-35-1 after PM infection.The interaction partner VpICE1 and VpEXO70B1 of VpPUB24 were identified by the yeast two-hybird assay.VpICE1(GenBank accession number: KU296026)was an HLH(helix-loop-helix)type transcription factor and the ORF of VpICE1 was 1551 bp encoding 516 amino acids.VpEXO70B1(GenBank accession number: KU507498)was a member of EXO70 gene family with a conserved EXO70 domain and a subunit of exocytosis complex involved in vesicle transport.The ORF of VpEXO70B1 was 1884 bp encoding 627 amino acids.5.The interaction of VpPUB24 with VpEXO70B1 was confirmed by BiFC(Bimolecular Fluorescence Complementation,BiFC)and Co-IP(Co-Immunoprecipitation)assay.However,VpPUB23,the homologous protein of VpPUB24,did not interact with VpEXO70B1,indicating that the interaction of VpPUB24 with VpEXO70B1 was specific.The degradation of VpEXO70B1 was promoted by Vp PUB24 and mediated by the 26 S proteasome.Overexpression of VpPUB24 in Arabidopsis thaliana led to the decrease of genes involved in the SA signaling pathway,which indicated that VpPUB24 negatively regulate the PM resistance.Moreover,the subcellular location of VpPUB24 and VpEXO70B1 was changed by SA treatment.The quantification of GUS activity showed that EXO70B1 promoters from Baihe-35-1,Red Globe,Cabernet Sauvignon and Thompson Seedless were induced by PM infection and SA treatment.All together,VpPUB24 interacted with VpEXO70B1 to negatively regulate the PM resistance.6.The yeast two-hybird assay revealed that the ARM domain of VpPUB24 interacted with the ACT domain of VpICE1.And yeast two-hybird assay also confirmed the interaction of VpPUB24 with VpICE2 and VpICE3.Although the degradation of VpICE1 was mediated by 26 S proteasome,overexpression of VpPUB24 increased the accumulation of VpICE1.Further studies indicated that VpHOS1 also interacted with VpICE1 and promoted the degradation of VpICE1,while VpPUB24 interacted with VpHOS1 and inhibited the accumulation of VpHOS1 to stable the level of VpICE1.Briefly,VpPUB24 interacts with VpICE1 and activates the CBF/DREB1 cascade signaling pathway to regulate the cold response.Collectively,the Chinese wild grape E3 ubiquitin ligase gene VpPUB24 not only enhances the cold tolerance but also plays the role in regulation of the resistance to powdery mildew.