Functional Analysis Of Grape VvNAC72 Transcription Factor On Regulation Of Glyoxalase Ⅰ-4 Expression And Resistance To Downy Mildew

Grape downy mildew is one of the most important pathogens in the production of grape.It is distributed in the main grape producing areas all over the world.In China,most grape cultivars belong to Vitis vinifera.L.However,these cultivars are not resistant to downy mildew generally.Chemical control is mainly used in the prevention of grape downy mildew in production.It is easy to cause pressure on the environment and make the pathogenic fungus resistant to fungicide.Therefore,combination of traditional and molecular breeding methods to breed disease-resistant varieties is one of the effective measures to control grape downy mildew comprehensively.Methylglyoxal（MG）is a cytotoxin that degrades with glutathione（GSH）under the action of glyoxalase I to form S-D-lactoylglutathione,which reduces the concentration of methylglyoxal in the plant.Then,S-D-lactoylglutathione is hydrolyzed to non-toxic D-lacticacid by the action of glyoxalase Ⅱ and released GSH.Glyoxalase Ⅲ can directly catalyze the conversion of methylglyoxal to D-lactic acid without any cofactor.Accumulation of methylglyoxal leads to the increasing of reactive oxygen species directly.However,the accumulation of reactive oxygen species causes hypersensitivity in plants and inhibits the growth of pathogenic fungus.Therefore,it is of great significance to explore the resistance mechanism of glyoxalase gene to grape downy mildew,to explore the regulatory factors of glyoxalase gene,and to analyze the molecular regulation network of grape downy mildew.The main results of this study are following:1.Identification and expression analysis of members of the grape glyoxalase gene families.Four glyoxalase I（GLYⅠ）,two glyoxalase Ⅱ（GLYⅡ）and three glyoxalase Ⅲ（GLYⅢ）proteins were identified from the grape genome.The phylogenetic relationships,conserved binding sites,structural properties and the relative expression levels of these genes in different tissues of Thompson seedless and 0-120 h after infection with Plasmopara viticola were further analyzed.The VvGLYⅠ-2,VvGLYⅠ-3,VvGLYⅠ-4,VvGLYⅡ-2,VvGLYⅢ-1,VvGLYⅢ-2 and VvGLYⅢ-3 genes were highly expressed at 6-12 h and 96-120 h,but VvGLYⅡ-1 gene has down-regulated expression.The VvGLYⅠ-1 gene was highly expressed48 h after inoculation.These results indicate that different glyoxalase genes play different roles in the defense of grape against Plasmopara viticola.2.Tobacco lines that overexpresses the VvGLYⅠ-4 gene is susceptible to Phytophthora capsici.The VvGLYⅠ-4 gene in Thompson seedless was cloned.VvGLYⅠ-4 was localized in the nucleus,membrane and cytoplasm by transiently transforming epidermal cells of tobacco leaves.The purified VvGLYⅠ-4 protein can degraded MG in vitro,demonstrating its function as a GLYⅠ.Phytophthora capsici can induce the accumulation of MG and increase the content of H₂O₂.The two T₃ tobacco lines overexpressing of VvGLYⅠ-4 promote the conversion of MG to D-lactic acid.After inoculation with Phytophthora capsici,the H₂O₂content was significantly lower than that of the wild type,so the lesions area of the overexpressing lines was significantly higher than that of the wild type.In addition,the activities of catalase（CAT）,superoxide dismutase（SOD）and the expression of active oxygen scavenging system genes Nb Cu/Zn-SOD,Nb CAT,Nb GR and Nb APX in overexpressing lines were significantly higher than those in the wild types.3.VvNAC72 negatively regulates VvGLYⅠ-4 gene expression.The 2000 bp promoter sequence upstream of the VvGLYⅠ-4 gene was cloned and was used as a bait for screening the Thompson seedless c DNA library by yeast one-hybrid technique and VvNAC72 was selected.GUS protein activity assay,yeast one-hybridization and dual luciferase experiments demonstrated that VvNAC72 can directly bind to the CACGTG element at the-504 bp position in the VvGLYⅠ-4 promoter and inhibits the expression of VvGLYⅠ-4 gene,but not binding with the CACGTG element in VvGLYⅠ-2,VvGLYⅠ-3,VvGLYⅡ-1 and VvGLYⅢ-1 gene promoters.The gene expression in the leaves of Thompson seedless inoculated with Plasmopara viticola was analyzed by qRT-PCR.It was found that the expression of VvNAC72 gene was negatively correlated with the expression of VvGLYⅠ-4gene.4.Grapes lines overexpressing VvNAC72 are more resistant to downy mildew.Two grape lines overexpressing of VvNAC72 were obtained.The expression of VvGLYⅠ-4 gene was inhibited in overexpressing lines,which inhibited the conversion of MG into D-lactic acid,increasing the H₂O₂ content after inoculating with Plasmopara viticola.At the same time,SA synthesis gene ICS2,defense reaction genes PR1 and NPR1 were up-regulated.In addition,CAT and SOD enzyme activity was inhibited,and the expression levels of reactive oxygen metabolism-related genes VvCu/Zn-SOD,VvCAT,VvGR,VvAPX and VvMDAR were decreased.5.Overexpression of the VvGLYⅢ-2 gene enhances grape resistance to downy mildew.Purified VvGLYⅢ protein was obtained by prokaryotic expression.MG can be degraded in vitro and producing D-lactic acid at the same time,but GSH is not involved in this process.This indicated that VvGLYⅢ-2 protein has the function of GLYⅢ enzyme and the stoichiometric ratio of the VvGLYⅢ-2 enzyme is 1:1.0-3 days after inoculated with Plasmopara viticola,the MG content in the Thompson seedless strains overexpressing of VvGLYⅢ-2 was significantly lower than that of the wild type,and the D-lactic acid content was significantly higher than that of the wild type.However,the activity of GLYⅠ and GLYⅡ was increased from the fourth day.The GLYⅠ-GLYⅡ double enzyme system plays a leading role at this moment.The content of MG and D-lactic acid is not significantly different from that of wild type.However,due to the gradual increase of MG content induced by Plasmopara viticola,the activity of CAT in overexpressing lines is significantly lower than that of the wild type,causing the H₂O₂ content to be significantly higher than that of the wild type,which prevented the further infection of Plasmopara viticola to some extent and produced a certain degree of resistance.In this study,nine glyoxalase genes were identified from the grape genome database.In grape GLYⅠ-GLYⅡ double enzyme system,VvNAC72 specifically binds to the CACGTG element in the VvGLYⅠ-4 gene promoter,inhibits the expression of VvGLYⅠ-4,inhibits the conversion of MG into D-lactic acid,increasing of H₂O₂ content induced the expression of genes related to defense signal pathway and prevented the infection of pathogenic fungus.In the GLYⅢ single enzyme system of grape,Plasmopara viticola induces the increase of MG content in grape.In the grape lines overexpressing of VvGLYⅢ-2,GLYⅢ enzyme system plays a leading role in the early stage of inoculation with Plasmopara viticola.In the late stage of inoculation when MG accumulates with a certain concentration,GLYⅠ-GLYⅡ double enzyme system plays a leading role.With the accumulation of MG,the activity of CAT enzyme is inhibited and H₂O₂accumulated,so that the plant shows disease resistance.This study preliminarily described the different molecular regulation mechanisms of GLYⅠ-GLYⅡ double enzyme system and GLYⅢ single enzyme system in the process of Plasmopara viticola infection,expanding the transcriptional regulation network of grape downy mildew.