Screening Of Haustorium Specific Effectors Of Puccinia Striiformis F.sp. Tritici And The Wheat Targets Identification Of HASP268 Effector

Wheat stripe rust,caused by Puccinia striiformis f.sp tritici(Pst),was one of most destructive disease worldwide,it could cause a remarkable reduction in agricultural production.The pathogenicity mechanism study of Pst was hindered as no effective transformation system have been established.Haustoria are special infection structure of Pst during infection,and exercise the power to maintain interests of biotrophic pathogen,such as nutrients uptake,carbohydrate biosynthesis,and host defense rejection in the host tissue.Based on the transcriptome of isolated Pst haustoria,we analyzed the function of 65 candidate effectors which had much higher transcript level in haustoria stage.The detail results are as below:1.By using an Agrobacterium tumefaciens mediated transient expression assay in Nicotiana benthamiana,we identified 29 candidate effectors which could suppress Bax-triggered PCD.To confirm HASP candidates were expressed,the protein BAX was examined with anti-BAX using western blot.These results indicated that 29 candidate effectors could be involved in defense response.2.We further applied type ? secretion system and found that 18 HASP candidates could suppress the callose deposit,a critical symbol of PTI,which indicated that 18 candidates could suppress PTI response.About 13 candidates could weaken the production of reactive oxygen burst,and 3 effectors inhibit hypersensitive reaction of host.To study whether these HASP effectors could promote the development of fungal,we investigate fungal hyphae,the number of haustoria and infected areas at 24 hpi and 48 hpi,especially fungal biomass at 120 hpi.Histological observation data reveals that some effectors(14 effectors)could be in favour of the extension of hyphae and the formation of haustoria.Furthermore,fungal biomass is significantly higher 2-12 fold at 120 hpi.3.To find out the subcellular localization of these candidates,HASP268 and HASP241 were located in chloroplast.HASP221 was accumulated in discrete cytoplasmic body.HASP213 and HASP219 were located in nuleus,but HASP213 might present on cytoplasmicmembrane in some transiently expressed plant cells.Using yeast two-hybrid system,we verified three targets of HASP268: ISP(iron-sulfur protein),NF-YC(the submit C of transcription factor NF-Y)and CNR8(cell number regulator 8).