Genetic Rules Of Powdery Mildew Resistance In Wild Chinese Grapevines And Molecular Mechanisms Of VpsCDPKs In Regulating Disease Resistance

Grapevine is a global fruit tree that has significant economic value.Currently,most grapevine cultivars are derived from the European grapevines（Vitis vinifera）,which are susceptible to multiple diseases,especially to powdery mildew（PM）.Although fungicide applications could effectively limit PM in vineyards,which might be harmful to the ecosystem and human health.Compared to chemical controls,improving the PM resistance of the European grapevines via employing disease resistance（R）genes of wild relative species would be safer and more efficient.China has massive wild Vitis species,several accessions of them show excellent PM resistance.However,the cytological characteristics and genetic rules of PM resistance shown in these wild Chinese grapevines are still not clear.The recent studies revealed that Ca²⁺influx from the apoplastic spaces into the cytosol is a common physiological process after activation of diverse R proteins in plant cells.However,as a group of calcium message decoders,whether calcium-dependent protein kinases（CDPKs）of grapevines play roles in the interactions between grapevines and Erysiphe necator is waiting to be elucidated.In this study,using a native PM isolate En.NAFU1 as a pathogen,we studied the cytological characteristics shown in fourteen PM-resistant wild（Chinese）grapevines and four lines of RPW8.2（a broad-spectrum disease resistance gene cloned from Arabidopsis）transgenic Thompson Seedless,explored the genetic rules of disease resistance in V.piasezkii var.pagnucii accession Baishui-40,then studied the disease-resistant function of two VpsCDPKs from V.pseudoreticulata accession Baihe-35-1.Following are the main results:1.Hypersensitive responses-like cell death and accumulations of antifungal metabolites could be essential cytological characteristics of foliar PM resistance shown in wild Chinese Vitis species.Field observations suggested that four accessions of wild Chinese Vitis species show durable and broad-spectrum foliar PM resistance.The cytological observations suggested that the leaf structural parameters,such as the thickness of the wax layer,cuticles,and primary cell wall,are divergent in different grapevines,but these factors might not be directly related to PM resistance levels.However,in the leaves of Baishui-40 and Baihe-35-1,En.NAFU1 infections could trigger strong immune responses,including secondary thickening of the cell wall and/or tightly encasement of haustoria by antifungal metabolites.Meanwhile,En.NAFU1 infections induced strong hypersensitive responses-like cell death and H₂O₂ burst at the early infection stages in a few wild Chinese grapevines that show highly efficient resistance to PM.To further confirm that whether these above physiological processes were elicited by R genes,we analyzed the cytological characteristics of PM resistance shown in RPW8.2 transgenic Thompson Seedless grapevines.The ectopic expression of RPW8.2 enhanced the salicylic acid signaling,promoted PM-induced callose accumulation,and resulted in hypersensitive responses-like cell death and H₂O₂ burst in transgenic grapevine leaves.These similar phenotypes suggested that there are R genes functioning in wild Chinese grapevines.2.A CC-NB-LRR type R gene might underlie the genetic resistance in V.piasezkii var.pagnucii accession Baishui-40 against En.NAFU1.Using Baishui-40 as a male parent,we constructed a mapping population of 218individuals from a cross between’Pearl of Csaba’（a white table grape cultivar）and Baishui-40,and constructed a mapping population of 609 individuals from a cross between Carignan（a red wine-making cultivar）and Baishui-40.The PM resistance was segregated in these two F₁populations.Through the Chi-square test,the segregation ratio in the progeny of’Pearl of Csaba’×Baishui-40 was in accord with 1:1（HR:HS）,whereas the segregation ratio in the progeny of Carignan×Baishui-40 was only close to 1:1:1:1（HR:R:S:HS）.Meanwhile,the chromosome-level genomes of Baishui-40（NAFU＿BS40）and Baihe-35-1（NAFU＿BH351）were obtained by using Nanopore long-read sequencing and Hi-C methods.These two assembled grapevine genomes were 578 Mb and 548 Mb in size,with 41251 and40225 protein-coding genes were annotated,respectively.In these two wild grapevines,multiple types of disease-resistance associated genes,such as NLRs,have undergone tandem duplications.Through whole-genome resequencing analyses and genome-wide association studies,a major effective R gene locus that underlies the PM resistance in Baishui-40 against En.NAFU1 was mapped at 0.75 Mb to 1.2 Mb on Chromosome 1,which contains 12tandemly repeated CC-NB-LRR genes.According to the international nomenclatures,this locus was named Ren12（Resistance to Erysiphe necator 12）.3.Two Calcium-Dependent Protein Kinase genes from V.pseudoreticulata（VpsCDPKs）could be involved in the regulation of foliar PM resistance.In NAFU＿BH351,17 typical CDPKs were identified.To further study their molecular function,11 VpsCDPKs were cloned from Baihe-35-1.VpsCDPKs showed multiple subcellular localizations in protoplasts.Among them,VpsCDPK9 and VpsCDPK13 could be simultaneously located in endomembrane organelles such as endoplasmic reticulum,Golgi apparatus,oil bodies,and peroxisomes,and their N-terminal variable domains direct their subcellular localizations.After inoculations of En.NAFU1,the transcript levels of VpsCDPK9,VpsCDPK13,and their orthologous genes were differently up-regulated in the leaves of Baihe-35-1 and PM-susceptible Thompson Seedless.Overexpression of VpsCDPK9-YFP and VpsCDPK13-YFP in Thompson Seedless could significantly enhance the foliar PM resistance,and caused leaf abscission and premature aging of plants,which might be related to the hypersensitive responses-like cell death,accumulations of antifungal metabolites（callose,H₂O₂,and phytohormones）,and the dysfunction of endomembrane system in the mesophyll cells and epidermis cells.However,when VviCDPK13 alleles were mutated in Thompson Seedless via using the CRISPR/Cas9 system,the biallelic mutant line also showed elevated foliar PM resistance and leaf premature senescence post PM infections,while the PM-induced callose accumulation was reduced compared to wild type.4.The defense-associated roles of VpsCDPK9 and VpsCDPK13 rely on the salicylic acid and ethylene signaling pathways.Using mbSUS yeast-hybrid system,we confirmed the physical interactions between four VpsCDPKs with twenty-two potential substrate proteins.When VpsCDPK9-YFP was overexpressed in Arabidopsis,both transcript and protein levels of fusion genes were accumulated with leaf aging.Abnormal accumulation of VpsCDPK9-YFP could cause spontaneous cell death and H₂O₂ accumulation in leaves,leading to premature senescence of leaves and plant stunting.By contrast,these transgenic Arabidopsis lines with lower expression levels of VpsCDPK9-YFP could grow normally and showed enhanced PM resistance levels compared to the wild type,which could be attributed to PM-induced hypersensitive responses-like cell death,increased salicylic acid and ethylene production,and H₂O₂ accumulation.When VpsCDPK13-YFP was overexpressed in Arabidopsis,the protein levels of foreign genes were too low to induce obvious phenotypes.However,when VpsCDPK9-YFP and VpsCDPK13-YFP were overexpressed in Arabidopsis mutants in which the salicylic acid（pad4sid2）and ethylene（ein2-1）signaling were interrupted,the expression levels of VpsCDPK9-YFP and VpsCDPK13-YFP were too low to be detected by Western blot assays,undoubtedly,which could not cause premature aging of plants,and could not improve PM resistance.Through bimolecular fluorescence complementation（Bi FC）assays and Co-immunoprecipitation（Co-IP）assays in protoplasts of grapevine,the molecular interactions between VpsCDPK9/VpsCDPK13 with VpsACS1,VpsACS2,VpsMAPK3 and VpsMAPK6 were further confirmed in vivo.VpsCDPK9/13 could differently phosphorylate their four common substrates and thus affect their protein abundance.Meanwhile,VpsMAPK3/6 could also have effects on the protein abundance of VpsACS1/2 and VpsCDPK9/13,respectively.Moreover,ethephon and H₂O₂ treatments could elicit the promoter of VpsCDPK9/13.In field conditions,the leaves of PM-resistant wild grapevine lines showed higher ethylene production than PM-susceptible lines within the same species.These results suggested that ethylene could play essential roles in regulating PM resistance in wild Chinese grapevines.Taken together,our results preliminarily defined the cytological characteristics and the genetic rules of foliar PM resistance shown in wild Chinese Vitis species,firstly mapped a PM resistance gene（locus）in Baishui-40,and revealed the molecular mechanisms of two VpsCDPKs in regulating disease resistance.These results might provide insights into the defense mechanisms of grapevines,and provide gene resources and a theoretical basis for precise molecular breeding of PM-resistant grapevines.