Functional Analysis Of Ubiquitin Ligase Gene VpUIFP1 From Wild Chinese Vitis Pseudoreticulata In Regulation Of Disease Resistance

As one of the most widely distributed and most used economic fruit crops in the world,grape is widely cultivated and utilized.In particular,Vitis vinifera L.is the main cultivar of grape production due to its high quality,rich processing products and high nutritional value.However,the most prominent problem is that European grape is not resistant to fungal diseases,especially grape powdery mildew.China,as one of the important origin of grapevine,has extremely abundant wild grape species and varieties.In recent decades,the studies show that the wild Chinese Vitis pseudoreticulata Baihe-35-1 clone has the characteristics of high resistance to powdery mildew.In this study,V.pseudoreticulata Baihe-35-1 was used as the material.VpUIFP1,an F-box ubiquitin ligase,was cloned from the cDNA library of V.pseudoreticulata Baihe-35-1 inoculated with Uncinula necator.The full length of VpUIFP1and its promoter sequence were cloned.The expression pattern of VpUIFP1 response to U.necator was studied.The role of VpUIFP1 in regulating disease resistace was analysed by overepressing VpUFIP1 in the V.vinifera L.Red Globe.VpSKP1.1 and VpTrxz were screened out as the interacting proteins of VpUIFP1 by yeast two-hybrid（Y2H）.The results were obtained as follows:（1）The expression patterns of F-box genes from the cDNA library of V.pseudoreticulata Baihe-35-1 inoculated with U.necator and their homologous genes in V.vinifera L.Red Globe were investigated by qRT-PCR after inoculation of U.necator.The results show that the expression level of VpUIFP1 in V.pseudoreticulata Baihe-35-1 is up-regulated and higher than that of V.vinifera L.Red Globe.（2）The full length of VpUIFP1was cloned by RACE（Rapid amplification of cDNA ends）with 1068 bp ORF（Open readign frame）,encoding 355 amino acids.Its GenBank accession number is KU160540.Bioinformatcs prediction analysis indicates that an F-box conserved domain was located at N-terminus and two Kelch motifs were located at C-terminus.Subcellular localisation analysis shows that VpUIFP1 was located in the cell nucleus and cytoplasma.（3）Overexpression of VpUIFP1 in Arabidopsis and V.vinifera L.Red Globe enhances the resistance of plants to powdery midlew.Overexpression of VpUIFP1 activated ICS2,a key gene in the salicylic acid（SA）biosynthesis,NPR1 and PR1,important genes involved in hypersensitive response（HR）,as well as promoted the accumulation of H₂O₂,resulting in inhibiting the growth of powdery mildew.（4）The promoter of Vp UIFP1 in the V.pseudoreticulata Baihe-35-1 and its homologous sequence in the V.vinifera L.Red Globe were cloned.The sequence alignment analysis shows that the promoter in the V.pseudoreticulata Baihe-35-1 contains a W-box cis-element,not existed in the same position of the homologous sequence in the V.vinifera L.Red Globe.The promoters fused with GUS reporter were transiently expressed in the grape leaves.The results of GUS histochemical staining and quantitative analysis of GUS protein activity indicats that the activity of VpUIFP1 promoter in the V.pseudoreticulata Baihe-35-1 is higher than that of the V.vinifera L.Red Globe.The activity of Vp UIFP1 promoter fragment without the W-box declines after inoculation of U.necator,which indicated the W-box is an improtant cis-element for promoting the expression of VpUIFP1 in response to U.necator.（5）VpSKP1.1 and VpTrxz were screened out as the interacting proteins of VpUIFP1 by yeast two-hybrid.The interaction was verified by Y2H and BiFC（Bimolecular fluorescence complementation）.VpSKP1.1（GenBank accession no.KU160541）is an improtant component of SCF complex.The interaction of VpUIFP1 and VpSKP1.1 indicates that VpUIFP1 is an E3 ubiquitin ligase as a component of SCF complex.VpTrxz（GenBank accession no.KU160543）is z-type thioredoxin.The protein level of VpTrxz is decreased in the V.vinifera L.Red Globe leaves overexpressing VpUIFP1,which is restored by protease inhibitor MG132.The results shows that VpUIFP1,as a component of E3 SCF complex,mediates the degradation of VpTrxz via ubiquitin/26S proteosome pathway.（6）The expression of VpTrxz is decreased after inoculation of U.necator.RNAi of VpTrxz contributes to the accumulation of H₂O₂ upon inoculation of U.necator,which enhances the resistance of grapevine to U.necator.In conslusion,the ubiquitin ligase VpUIFP1 positively mediates the grapevine resistance to powdery mildew by inducing the degradation of VpTrxz via the ubiquitin/26S proteasome system.