Identification And Functional Analysis Of CRN Effectors Of Plasmopara Viticola

Grapevine downy mildew is one of the most devastating diseases in grapevine production worldwide,which is usually controlled with chemical pesticides.However,it is costly and threatens the security of the environment and human health.Therefore,it is urgent to develop alternative strategies that are both economical and environmentally friendly.A thorough understanding of the pathogenesis of grapevine downy mildew and the resistance/susceptibility mechanism of grapevine is a prerequisite for the development of effective control measures.The causal agent of grapevine downy mildew is Plasmopara viticola[Berk.&M.A.Curtis]Berl.&De Toni,which is an obligate biotrophic pathogen belonging to the Oomycota.There are few reports about how P.viticola interacts with its host grapevine at the molecular level and how P.viticola could elude or inhibit host immune responses.A variety of effector proteins including RXLR and CRN(Crinkling and necrosis)are secreted by the oomycetes to interfere with host's resistance responses during the interaction between the oomycetes and their hosts.According to the sequence information of 35 functional CRN genes(PvCRN for short)in the genome of P.viticola isolate‘YL',27 PvCRN genes were cloned in this study.The sequence characteristics and virulence of these PvCRN genes were characterized and evaluated,and a highly virulent member,PvCRN17,was selected for a further study.The main findings are as follows:1.The N-terminal sequences of PvCRN proteins are relatively conservative while the C-terminal sequences of PvCRN proteins are quite different.Therefore,it is believed that the virulence of each PvCRN protein was determined by their C-terminal structures.Phylogenetic analysis showed that the 27 PvCRN proteins were divided into 7 clades.Gene duplication and gene recombination events have been occurred in the PvCRN gene family,indicating that PvCRN genes are evolving actively.Most PvCRN proteins do not possess classical signal peptide sequences.Yeast invertase secretion assay proved that the predicted eight PvCRN signal peptides did have the secretion activity.Subcellular localization analysis showed that the PvCRN protein family have multiple distributions in the plant cell.It suggests that there is more than one destination or more than one target molecule in the host cell for PvCRN effectors.2.When transiently expressed in Nicotiana benthamiana leaves,most PvCRN genes,except PvCRN11,failed to induce hypersensitive cell death on N.benthamiana leaves.While when transiently expressed in the downy mildew-resistant grapevine Vitis riparia Michx.leaves,none of the PvCRN genes cloned could induce hypersensitive cell death on the leaves,showing consistency with the result on N.benthamiana leaves.Hypersensitive cell death-suppressing test showed that most PvCRN proteins(23/27)could not inhibit hypersensitive cell death induced by INF1,15 PvCRN proteins significantly suppressed plant programmed cell death on N.benthamiana leaves induced by Bax.When transiently expressed in N.benthamiana leaves,PvCRN17,PvCRN19,PvCRN20,and PvCRN23respectively promoted the susceptibility of N.benthamiana to Phytophthora capsici Leonian,which is a hemibiotrophic oomycete.3.During the infection process of P.viticola‘YL'on V.vinifera Pinot Noir leaves,the transcription level of PvCRN14,PvCRN16,and PvCRN17 were higher at 72 h and 96 h post inoculation.Further studies showed that PvCRN17 could suppress some certain ETI responses and inhibit H₂O₂accumulation in N.benthamiana leaves inoculated with P.capsici.Transgenic Arabidopsis plants containing PvCRN17 showed delayed growth and development.The seeds of T2 generation could not mature or germinate normally.These findings indicate that PvCRN17 is highly virulent to plants.And it demonstrated that the plant cell plasma membrane localization contributed to the virulence of PvCRN17.4.Yeast-two hybrid assay,bimolecular fluorescence complementation assay,and co-immunoprecipitation assay identified VAE7L1(protein ASYMMETRIC LEAVES 1/2ENHANCER 7-Like 1)and VvNRPP?-X1(Nitrogen regulatory protein P-II homolog isoform X1)from grapevine as the target proteins of PvCRN17.The homologous proteins of VAE7L1,Nb AE7L1 from N.benthamiana and At AEL1 from Arabidopsis thaliana both interact with PvCRN17.Protein-protein interaction experiments proved that VAE7L1interacts with VvCIA1 and VvAE7(protein ASYMMETRIC LEAVES 1/2 ENHANCER7),respectively.VAE7 and VvAE7 may function in the cytosolic iron-sulfur cluster assembly(CIA)process by forming a heterodimer.VAE7L1 genes in the susceptible grapevine V.vinifera Pinot Noir and the resistant grapevine V.piasezkii Liuba-8 were both up-regulated at some time points in response to the infection of P.viticola on the leaves.Transient expression of VAE7L1 protein in N.benthamiana leaves significantly enhanced the resistance of the leaves to P.capsici.Therefore,VAE7L1 were concluded to function in the defense responses of grapevine against P.viticola.5.VvMET18,a component of the cytosolic iron-sulfur cluster assembly core complex in V.vinifera,was upregulated in response to the infection of P.viticola on the grapevine leaves.Three Fe-S proteins'coding genes including VvABCE2(VIT?02s0087g00770),VvACOL-12S(VIT?12s0059g02150),and VvACOL-19S(VIT?19s0090g00460),were upregulated in response to the infection of P.viticola on the grapevine leaves as well.When transiently expressed in N.benthamiana leaves,VvABCE2 and VvACOL-19S separately enhanced the resistance of the leaves to P.capsici.These results suggest that these Fe-S proteins from grapevine play important roles in the disease resistance responses of grapevine.6.PvCRN17 interacts with VvAE7,but does not interact with VvCIA1 directly.Competitive Co-IP assay revealed that PvCRN17 could compete with VvCIA1 to bind with VAE7L1 and VvAE7,indicating that PvCRN17 could impede the formation of the cytosolic iron-sulfur cluster assembly core complex.In addition,PvCRN17 and VAE7L1are co-localized at the plasma membrane of the plant cell.Then,it is speculated that since being delivered into the grapevine cell,PvCRN17 would compete with VCIA1 to bind with VAE7L1 and VAE7,disrupting the integrity of the cytosolic iron-sulfur cluster assembly core complex,and interrupting the maturation of the downstream Fe-S proteins,in the end,a suppression of Fe-S proteins mediated defense responses.