Gene Mining And Functional Analysis Of Resistance Gene VvPYL4 From Grapevine Resistant To Plasmopara Viticola

Grape downy mildew is one of the most damaging diseases that harms grapevine plants cultivation and productivity.Chemical control is a common measure to prevent and treat the disease.Long-term use of chemical pesticides is likely to lead to drug resistance and lead to product and environmental pollution.It has become a research hotspot to explore disease-resistant genes and select disease-resistant grapevine cultivars.The mechanism of resistance to grape downy mildew has been reported to some degree,but it has not been reported that research on disease resistance genes by transcriptome sequencing of resistant and susceptible grapevine and functional analysis of the candidate resistance gene VvPYL4.In this study,resistance of grapevine cultivars to downy mildew,transcriptome analysis of grapevine leaves infected by Plasmopara viticola,screening and functional analysis of differentially expressed genes related to disease resistance were carried out.The main research results were as follows:1.The identification of resistance of grapevine cultivars to downy mildew showed that4 species were high resistance（HR）,31 species were resistance（R）,32 species were susceptible（S）and 18 species were high sensitivity（HS）under the indoor inoculation test.While,there were 1 high resistance（HR）species,25 resistance（R）species,45 susceptible（S）species and 14 high sensitivity（HS）species under the filed survey.The immune species were as yet unfound in cultivars.The trend of resistance to downy mildew was American species,Euramerican hybrid species（lines）and Eurasian hybrid（lines）.2.Leaves of Vitis vinifera cv.gentenial seedless‘Centennial Seedless’（susceptible）and Vitis labrusca×Vitis riparia‘Beta’（resistant）were inoculated with Plasmopara viticola,and were used the Illumina HiSeqTM²⁰⁰⁰platform for deep transcriptome sequencing.We performed transcriptome analysis and identified a total of 1091 differentially expressed genes（DEGs）in the disease-resistant cultivar and 849 in the susceptible cultivar.To increase understanding of the DEGs,the datasets were analyzed using Gene Ontology（GO）and the Kyoto Encyclopedia of Genes and Genome（KEGG）database.Most of them were found to be associated with plant–pathogen interaction,flavonoid synthesis,phenylpropanoid synthesis metabolism and phytohormone signal transduction.The expression of 18 differentially expressed genes was detected by RT-q PCR.3.The results of the study on the differentially expressed disease-resistant genes in resistant and susceptible grapevine cultivars infected by Plasmopara viticola showed that there were two different of immune system in grapevine leaves which could induce defense reaction.The response reactions of grapevine to P.viticola infection could be regulated by the expression of key genes in the pathways,such as abscisic acid signal,jasmonic acid signal,salicylic acid signal,brassinol signal,phenyl-propane biosynthesis,phenylalanine metabolism,flavonoids synthesis.The number of transcription factor families responsed to downy mildew was more in Beta than in Centennial Seedless.The expression of transcription factor showed strong cultivar specificity between two cultivars tolerant to Downy mildew.HMMER 3.0 program and The Plant Resistance Gene Database were used to screen disease-related genes from the differentially expressed genes.A total of 196 genes were identified as the candidate resistant genes.4.In this study,we cloned a putative abscisic acid receptor gene,VvPYL4,from the‘Beta’grapevine cultivar.An analysis of the subcellular localization demonstrated that overexpressed VvPYL4 was localized in both the cytoplasm and the nucleus.The resistance of grapevine cultivars to downy mildew increased after the application of the exogenous hormones Me JA and ABA.The expression of VvPYL4 was significantly induced when exposed to Plasmopara viticola,Me JA,and ABA.The transcript of the VvPYL4 gene in‘Beta’grapevine leaves treated with tobacco rattle virus-induced gene silencing（VIGS）was significantly down-regulated.Silencing VvPYL4 had a slight effect on P.viticola resistance.Furthermore,silencing VvPYL4 significantly altered the transcripts of ABA and JA responsive genes,including the ABA signaling pathway-related genes ABF1 and Sn RK2.2,as well as the genes related to the JA signaling pathway,MYC2 and JAR1.This study also discusses the regulation mechanism of VvPYL4 on ABA and JA signaling transduction.