| Grapevine powdery mildew (Uncinula necator [Schw.] Burr.) is one of the mostimportant fungal diseases that damages grapevine production. Vitis vinifera is currently onemajor species, which possesses good quality and high yield. However, most of them aresensitive to diseases. China has abundant wild grape species that contain some valuableresistant-gene resources. In this study, one powdery mildew-resistant accession Baihe35-1ofChinese wild Vitis pseudoreticulata was used as the material to clone Chinese wild Vitispseudoreticulata UIRP1gene and its promoter. The responsive pattern of UIRP1gene and itspromoter to pathogen have been analyzed, respectively. The ubiquitin ligase activity and thesubcellular localization of the UIRP1gene have also been investigated. A transcription factorgene VpWRKY11that interacts with UIRP1has been screened, and their interactive modelwere further analyzed, as well as their roles in plant disease-resistant reaction were alsoexplored. The main results are described as followings.1. UIRP1gene was cloned from Chinese Wild V. pseudoreticulata accession Baihe35-1.The gene is1068bp in length with723bp opening read frame (ORF), encoding240aminoacids, and its GenBank accession number is JF502034. Bioinformatics analysis showedUIRP1gene possesses a C3HC4-type RING finger conserved motif at N-terminus and atransmembrane domain at C-terminus. The promoter of UIRP1gene was cloned usinghomology cloning method, in which is1182bp in length. Prediction analysis demonstratedthat UIRP1gene promoter contains the basic promoter elements and a plurality of cis-elements related to disease resistance.2. Inoculation with powdery mildew in Chinese Wild V. pseudoreticulata accessionBaihe35-1displayed the expression of UIRP1present two peaks in12hpi and48hpi,respectively, suggesting the expression of UIRP1is induced by powdery mildew. Thepromoter of UIRP1was fused with GUS, and transiently transform into grapevine leaves, theresults showed the activity of UIRP1promoter is induced by powdery mildew.3. The Chinese Wild V. pseudoreticulata UIRP1gene was expressed in Escherichia coli, and further purified to obtain the fusion protein. In vitro ubiquitination assay were conductedto analyze whether or not it possess ubiquitin ligase activity. The results showed UIRP1hasthe ubiquitin ligase activity, and mutation in conserved amino acid of the RING finger motifcould not occur ubiquitination. In contrast, mutation in non-conserved amino acid of theRING finger motif could not interfer the ubiquitination.4. Chinese Wild V. pseudoreticulata ubiquitin ligase gene UIRP1was used as the bait toscreen the library, and obtain its interactive protein VpWRKY11. Subcellular localizationdisplayed that UIRP1gene localized in nucleus, cytoplasm and cell membrane. Bimolecularfluorescence complementation (BiFC) assays further showed UIRP1interaction withVpWRKY11in the nucleus.5. The activation activity of VpWRKY11, which interacts with Chinese Wild V.pseudoreticulata ubiquitin ligase gene UIRP1, has no transcription in yeast. But VpWRKY11can combine the W-box cis-element, and has the transcription activation activity in planta.Result from subcellular localization assay showed VpWRKY11gene localized in nucleus.Inoculation with powdery mildew in Chinese Wild V. pseudoreticulata accession Baihe35-1showed the expression of VpWRKY11was induced by powdery mildew.6. The Chinese Wild V. pseudoreticulata ubiquitin ligase gene UIRP1acts in theupstream of the VpWRKY11, and UIRP1degrades VpWRKY11protein, and this degraderely on26S protease. The Chinese Wild V. pseudoreticulata ubiquitin ligase gene UIRP1negatively degrades and regulated VpWRKY11, and resulted in high expression of NPR1、AOS and LOX2disease-resistant related genes, therefore improving plant resistance.In conclusion, the ubiquitin ligase gene of Chinese Wild Vitis pseudoreticulata possessthe grape powdery mildew resistant function. |
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