Identification Of Candidate AvrYr26 And Functional Analysis Of Pathogenicity-Related Effectors In Puccinia Striiformis F.sp.tritici

Stripe rust,caused by Puccinia striiformis f.sp.tritici?Pst?,is a destructive wheat disease worldwide.After a long competition between plants and pathogens,a series of immune responses and methods to overcome the immune response have been evolved in plants and pathogens.During this competition,the susceptible plants and the pathogens continued to co-evolve,and the plants began to recognize compounds from the pathogens,resulting in host resistance and avirulence of the pathogens.Therefore,effector proteins that can be recognized by plants immunity system are called avirulence?Avr?genes,and plant genes that recognize avirulence genes secreted by these pathogens are called disease resistance?R?genes.However,many Avr proteins also have virulence functions,which can facilitate infection and colonization of pathogens in susceptible plants,indicated that the Avr gene can also exist as a virulence gene in susceptible plants.Therefore,the effector proteins secreted by pathogens are both avirulence and virulence,which can not only help pathogens infect them,but also serve as targets for starting plant immune responses.For better understanding of Pst effectors,analysis of their mechanisms in regulating the host immune response will help expand our in-depth exploration of the pathogenic mechanism of pathogens and provide new strategies and theoretical basis for creating new durable stripe rust resistant materials.Plant pathogenic effectors have been reported that involved in many immunity ways to regulate the host defense response,and have played an extremely important role in the process of pathogens infection and colonization.With the deepening and expansion of research,more effectors and their regulatory mechanisms will be revealed in the further.Compared with other plant pathogens,the understanding of Pst is limited.Fortunately,with the development of next-generation sequencing technology,molecular biological technology and genetic transformation technology,more and more effectors can be identified,and it will further deepen our understanding of the pathogenic mechanism of rust.In this study,we performed the following studies on avirulence genes and virulence effectors:Firstly,we re-sequecing 20 isolates of Pst form china.Cluster analysis and evolution analysis of the 20 Pst isolates showed that these isolates can be clearly divided into two groups,one of which is CYR32-HB,CYR32,CYR31,G862-7,CYR20,V26-CM42-2-1,V26-CM42-2-2,V26-CM42-1-2,BHPST,and the other group is CYR25,CYR29,CYR33,CYR32-HN,CYR32-GS,CYR32-SNCN-1,CYR32-SNCN-2,V26-PL17-7,V26-Qu45-1,V26-Qu45-3,V26-GN21-3-1,V26-GN21-3-5.It is suggested that the selection by different disease resistance genes and the screening effect on Pst caused the accelerating emergence of new races.Comparative genomic analysis and association analysis were performed on 20resequencing isolates and finally identified 10 candidate AvrYr26 genes.At the same time,a time series dual RNA-seq data in compatible and incompatible wheat-Pst interactions were also performed and identified 3 candidate AvrYr26 genes.This work has accelerated the research on avirulence genes of Pst and laid the foundation for characterized the Avr gens.It is believed that with the identification of more and more avirulence genes,it will play an active role in the breeding of disease-resistant and the prediction and prevention of wheat resistance to Pst.Secondly,cutinases play important roles during plant-pathogen interactions,including attachment of the pathogen to plant surfaces and disturbing plant immunity pathways.In this study,we analyzed the cutinase gene family in Pst and presented evidence that Pst22751 plays a role as an effector for improving virulence of the wheat stripe rust pathogen.Ten cutinase genes were identified and Pst22751 is the only one that is highly expressed during early infection.The cutinase Pst22751 with its signal peptide is secreted and the localization without its signal peptide is in cytoplasm and nucleus both in wheat protoplast and N.benthamiana.Furthermore,Pst22751 suppresses PTI-associated callose deposition and Pst-triggered H₂O₂ accumulation in wheat and Pst322-induced cell death in both N.benthamiana and wheat.In addition,silencing of Pst22751 demonstrated that Pst22751 contributes to Pst infection and can interrupt expression of plant PR genes.Thirdly,we identified the glycine-serine-rich m9 region,PstGSRE1-m9,of PstGSRE1,and found that it has similar function of PstGSRE1.PstGSRE1-m9 can suppress PTI-associated ROS and callose deposition in wheat and suppresses Pst322-and Bax-triggered cell death in wheat and N.benthamiana.Meanwhile,PstGSRE1-m9 can also interact with TaLOL2 and disrupt the subcellular localization of TaLOL2 to prevent it from promoting immunity.PstGSRE1-m9 is the functional domain of PstGSRE1 and PstGSRE1will lose its function without PstGSRE1-m9 domain.However,only the PstGSRE1-m9domain exhibited a weaker function than the PstGSRE1,which may suggest that the other part of PstGSRE1 can enhance the function of PstGSRE1-m9.Furthermore,we use the NLS and NES signal for the PstGSRE1 and found that PstGSRE1 interacted with TaLOL2 in cytoplasm to prevent TaLOL2 tranfer into nucleus to regular the plant denfence response.Finally,we identified an unconventionally secreted effector,which contained an HMG domain,named as PstHMG1.Relative expression analysis of this gene found that PstHMG1has two peaks during the early infection stage.One is 448-fold up-regulated at the beginning of urediniospores germinating,and the second is 744-fold up-regulated at 48 hpi.Later,we found that PstHMG1 can induce cell death in both N.benthamiana and wheat,and it can also digest DNA of N.benthamiana,wheat and Pst.PstHMG1 do not contain a N-terminal signal peptide,but it can be detected in the Intercellular fluid.This result indicated that PstHMG1 is an unconventionally secreted effector.Furthermore,we performed the immunocolloidal gold technique and found that PstHMG1 was in both infection hypha and haustoria in Pst and also in both chloroplast and nucleus in wheat.Knockdown the PstHMG1 demonstrated that PstHMG1 contributes to Pst infection.Overall,our result suggested that PstHMG1 may secret into wheat cells and induce DNA damage to facilitate infection of Pst.