Study On The Pathogenic Mechanism Of Plasmopara Viticola RxLR Effectors

Grape is one of the most important fruit crops in the world.Grape fruits create large economic benefits and widely used for wine,raisins,juice and fresh consumption.European grape（Vitis vinifera L.）cultivars are cultivated widely around the world,but V.vinifera cultivars are generally susceptible to fungus diseases,especially downy mildew（Plasmopara viticola）.Chinese wild Vitis species showing resistance to P.viticola offer a promising pathway to develop new grapevine cultivars resistant to P.viticola.P.viticola secrets effectors to modulate plant immune responses and finally to infect host plants.Studying the molecular mechanism of P.viticola effectors interact with grapevine may be of great significance.In this study,Chinese wild Vitis genotypes V.piasezkii‘Liuba-8’was classified as highly resistant to P.viticola,then we completed whole genome sequence of P.viticola and two Rx LR effectors were identified and analyzed.The results of this study are as follows:1.Cytological and histochemical studies were made of pathogen development and host responses to Plasmopara viticola in different grapes.Transmission and scanning microscopy was used to compare the resistance responses to downy mildew of three resistant genotypes of V.davidii var.cyanocarpa,V.piasesezkii and V.pseudoreticulata and the suceptible V.vinifera cultivar‘Pinot Noir’.Following inoculation with sporangia of P.viticola on V.vinifera cv.‘Pinot Noir’,the infection was characterized by a rapid spread of intercellular hyphae,a high frequency of haustorium formation within the host’s mesophyll cells,the production of sporangia and by the absence of host-cell necrosis.In contrast zoospores were collapsed in the resistant V.pseudoreticulata‘Baihe-35-1’,secretions appeared arround stomata,or the percentage of infected stomata decreased at the beginning of the infection period in V.davidii var.cyanocarpa‘Langao-5’and V.piasezkii‘Liuba-8’.The main characteristics of the resistance responses were the rapid depositions of callose and the appearance of empty hyphae and the plasmolysis of penetrated tissue.Moreover,collapsed haustoria were observed in V.davidii var.cyanocarpa‘Langao-5’and in V.piasezkii‘Liuba-8’.Lastly,necrosis extended beyond the zone of restricted colonization in all three resistant genotypes.Sporangia were absent in V.piasezkii‘Liuba-8’and greatly decreased in V.davidii var.cyanocarpa‘Langao-5’and in V.pseudoreticulata‘Baihe-35-1’compared with in V.vinifera cv.‘Pinot Noir’.These results indicated that V.vinifera cv.‘Pinot Noir’was susceptible to P.viticola while V.davidii var.cyanocarpa‘Langao-5’and V.pseudoreticulata‘Baihe-35-1’were partially resistant to P.viticola and V.piasezkii‘Liuba-8’was highly resistant to P.viticola.2.We report the genome of the virulent P.viticola‘Pv-YL’strain,and we reconstructed a genome of 127.88 Mb with an N50 of 235.51 Kb,encoding for 41,381 putative protein-coding genes.Analysis of potential gene function revealed that genes with annotated function in RNA processing and modification comprised>5,000 genes and was the major gene functional class.We identified 151 Rx LR genes and 35 CRN genes.Comparative analysis of genomes from P.viticola,P.halstedii and Phytophtora nicotianae identified a core gene set with functions in conserved biological processes;however,all identified Rx LR or CRN genes were unique to P.viticola.3.In present study,an Rx LR effector Pv R10 was induced at early stage during the P.viticola infection.Transient expression of Pv R10 in Nicotiana benthamiana leaves suppressed the elicitors（INF1 and Bax）triggered cell death and promoted the colonization of Phytophthora capsici.Transient expression of Pv R10 in grape leaves also promoted the colonization of P.viticola.Then,Vp BPA1（Binding partner of ACD11-1）was screened from the yeast library of P.viticola infected garpe（V.piasezkii‘Liuba-8’）leaves using Pv R10 as a bait.The interaction between Pv R10 and Vp BPA1 was then confirmed by bimolecular fluorescence complementation and co-immunoprecipitation.Further analysis revealed Pv R10 also interacted with Vp BPA1 homologues in N.benthamiana（Nb BPA1）.Overexpressing Vp BPA1 enhanced susceptibility to P.capsici and P.viticola in tobacco and grape leaves,respectively.Nb BPA1 silenced N.benthamiana leaves enhanced the resistance to P.capsici and H₂O₂accumulated.Transient expression of Vp BPA1 in Nb BPA1 silenced N.benthamiana leaves could reduce the accumulation of H₂O₂.Experiments in vivo demonstrated that Pv R10 inhibits degradation of Vp BPA1.These findings showed that Pv R10 may attenuate plant immunity through reducing H2O2 accumulation by inhibit degradation of BPA1.4.A candidate P.viticola Rx LR effector,Pv Avh74 induces cell death and immunity responses in Nicotiana benthamiana.Using agroinfiltration,we found that nuclear localization,two putative N-glycosylation sites,and 427 amino acids of the Pv Avh74carboxyl terminus were necessary for cell-deathinducing activity.Using virus-induced gene silencing（VIGS）,we found that Pv Avh74-induced cell death in N.benthamiana requires EDS1,NDR1,SGT1,RAR1,and HSP90,but not BAK1.The MAPK cascade components MEK2,WIPK,and SIPK were also involved in Pv Avh74-induced cell death in N.benthamiana.Transient expression of Pv Avh74 could suppress Phytophthora capsici colonization of N.benthamiana,which suggests that Pv Avh74 elicits plant immune responses.Suppression of P.capsici colonization also was dependent on nuclear localization of Pv Avh74.Additionally,Pv Avh74-triggered cell death could be suppressed by another effector,Pv Avh8,from the same isolate.