Functional Analysis Of Effector Pst8603 And Its Homologue Pst8604 From Puccinia Striformis F.sp Tritici And Identification Of Their Interacting Proteins

Wheat stripe rust caused by Puccinia striiformis f.sp.tritici（Pst）seriously threatens the food security.Breeding resistant wheat cultivar is the most cost-effective measure to control the disease.However,the main reason why causes frequent disease epidemics is that the rapid variation of Pst leads to a loss of wheat resistance on the main wheat cultivars.Therefore,in-depth analysis of the pathogenic mechanism of Pst is of great importance for development of novel resistant wheat cultivars.Numerous studies have found that effectors are important virulence factors of pathogen.Based on the previous study on high throughput screening of candidate effectors on Pst CYR32 genome,a candidate Pst effector Pst8603 containing a nuclear localization signal（NLS）was obtained.Blast X analyses revealed a neighboring cluster gene of Pst8603 in Pst CYR32 genome,named Pst8604.Here,we intended to explore the virulence function of effector Pst8603 and its homologue Pst8604,identify their interactors and analyze the functions of interaction targets,and initially reveal the regulatory mechanism of effector Pst8603 and Pst8604.This study will further lay a foundation for creating wheat disease-resistant materials through modifying the pathogenic mechanism of the pathogenic factors.The main results are as follows:1)In Pst CYR32 genome,Pst8604,a Pst8603 homologue,was identified,which shares55.6%simiarity to Pst8603 in amino acid sequence.When transiently expressed in Nicotiana benthamiana mediated by Agrobacterium tumefaciens,the effector Pst8603 and Pst8604were unable to induce cell death,while Pst8603 could inhibit Bax-induced cell death.At the same time,it was found that only Pst8603 could inhibit callose accumulation by Flg22 and the expression of defense genes NbPR1 and NbPR2 in N.benthamiana.The result indicates that effector Pst8603 has an ability to inhibit plant PTI（PAMP-triggered immunity）.2)qRT-PCR analysis showed that Pst8603 was highly expressed at 36 h and 72 h inoculation with Pst CYR31 on the wheat cultivar Suwon 11,while Pst8604 showed only low abundance in infection stages.It indicates that Pst8603 play a crucial role in Pst-wheat interaction.Pst8603 and Pst8604 were separately or simultaneously silenced during the compatible interaction between wheat and Pst using the host-induced gene silencing system（HIGS）.Phenotypic observation showed that knockdown Pst8603 and Pst8604 or both of them did not change the pathogenicity of Pst.qRT-PCR result showed that the transcript levels of defense genes TaPR1 and TaPR2 was up-regulated in the co-silencing plant,indicating that co-silencing Pst8603 and Pst8604 increased the defense response of wheat.In addition,39 positive transgenic wheat lines of overexpressing Pst8603 have been obtained by A.tumefaciens mediated transformation method after GUS staining.This provides material for the subsequent study of the virulence of Pst8603 in wheat-Pst interaction.3)The interacting proteins of Pst8603 were screened from the cDNA library of wheat cultivar Suwon 11-Pst CYR31 interaction using yeast two-hybrid.One candidate PstUbc9from Pst was identified to interact with both Pst8603 and Pst8604.Furthermore,their interaction was also confirmed using bimolecular fluorescence complementation（BiFC）and Pull-down.Besides,mutation of SUMO-chemistry site did not affect the interaction of Pst8603^ΔSP and PstUbc9.4)Sequence analysis revealed that five consecutive repeats of HRKPTTNQ were presented at 37-76 amino acid of the Pst8603,which is rich in alkaline amino acid and encodes NLS,implying the ability of Pst8603 to bind to nucleic acids.Pst8603^ΔSP specifically localized in the nucleus in N.benthamiana.Nucleotide binding assays revealed that Pst8603^ΔSP-His could bind both double strand-DNA and single strand-DNA in vitro.It is reasonable to speculate that Pst8603 may bind nucleic acids in host nucleus to regulate host immunity.