| European cultivated grape species have the best fruit quality but show almost no resistance to powdery mildew(PM).PM caused by Uncinula necator is a harmful disease that has a significant impact on the economic value of the grape crop;the Chinese wild grapevine V.pseudoreticulata accession Baihe-35-1 was selected as our research target because of its resistance to multiple fungi,particularly U.necator.The cDNA library of V.pseudoreticulata accession Baihe-35-1 inoculated with U.necator contained EST sequence(GR883881),named VpRH2,the full length cDNA and the promoter were obtained;VpGRP2A was selected out as the interacting protein of VpRH2,the potential links between VpRH2 and VpGRP2 A were determined by subcellular localizations and BiFC(bimolecular fluorescence complementation),over-expression of VpRH2 in transgenic Thompson Seedless was used to survey the responding to U.necator,the conclusions are as follows:1.The full length cDNA of VpRH2 was obtained by RACE(rapid-amplification of cDNA ends).The ORF(open reading frame)of VpRH2 was 1,164 bp and encoded 387 amino acids,covers a RING-H2 and transmembrane.VpRH2 belonged to the ATL subfamily and was the homologous gene of ATL20 in V.vinifera,which shared 93.02% amino acid identity;residue 154 is an aspartic acid,which is an insertion mutation compared to VvRH2;VpRH2was predicted to be located on chromosome 2 using the Grape Genome Browser.2.The expression of VpRH2 was clearly induced by U.necator inoculation compared with its homologous gene VvRH2 in a PM-susceptible grapevine V.vinifera cv.Thompson Seedless.VpRH2 was located at the cell membrane,cytoplasm,ER,TGN/EE,and endormembrane compartments.The expression of VpRH2 was inhibited by ABA treatment,but reduced by SA and MeJA treatments.3.The promoter of VpRH2 was cloned and several stress-regulated elements,such as ABRE,Box-W1,TC-rich repeats,TCA-element and TGACG-motif were found in the VpRH2promoter;compared to the VvRH2 promoter,three sections of sequences were deleted in the VpRH2 promoter,and one was inserted.The VpRH2 promoter and VvRH2 promoter were transiently transformed into grapevine V.vinifera Carignane leaves;powdery mildewinoculation,MeJA,SA,ABA,heat stress and cold stress treatments were used,as a result VpRH2 promoter activity was up-regulated by pathogen infection,SA and MeJA treatments comparing with VvRH2 promoter;the activity of VpRH2 and VvRH2 was induced by heat treatment,and the increased activity of VvRH2 promoter was higher than that of VpRH2promoter;the activity of two promoters were inhibited by ABA treatment and no obviously changed by cold treatment.4.VpGRP2 A was selected out by Y2 H as the interacting protein,and BiFC indicated that VpRH2 physically interacted with VpGRP2 A on the cell membrane and cytoplasm.The degradation of VpRH2 and VpGRP2 A was via UPS,but when VpRH2 increased,the protein level of VpGRP2 A did not obviously declined.VpGRP2 A enhanced the expression of VpRH2 but VpRH2 suppressed the expression of VpGRP2 A.5.The amino acid sequence of VpGRP2 A shared 100% similarity to VvGRP2A;the expression of VpGRP2 A and VvGRP2 A after U.necator inoculation showed a similar trend in the two grapes,clearly decreased from 6 hpi to 24 hpi;VpGRP2A was located at the nuclear,ER,TGN/EE,and endormembrane compartments.The expression of VpGRP2 A was inhibited by SA and MeJA treatments,but the expression was up-regulated by ABA treatments.6.OERH2-positive grape plants showed enhanced resistance to powdery mildew compared with the wild-type,displayed an accelerated growth phenotype,semi-thin sections of OERH2 showed that polymerized macromolecules was found in the OERH2 plants’ spongy parenchyma.In the ultra-thin section,polymerized macromolecules were found in the large central vacuole and tonoplast.OERH2 Arabidopsis plants showed enchanced tolerance to powdery mildew and ABA treatment.In summary,the ubiquitin ligase gene VpRH2 from Chinese wild grape V.pseudoreticulata has the function in resistance to powdery mildew. |
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