Functional Analysis Of P5CDH Gene Pst25505 Of Stripe Rust

Wheat(Triticum aestivum L.)is one of the most important food crops.Stripe rust is a fungal disease that severely reduces the yield of wheat.The pathogen of stripe rust,Puccinia striiformis f.sp.tritici(Pst),can produce yellow blister on wheat leaves,causing damages by disturbing the normal physiological processes of wheat.To prevent and control stripe rust,planting resistant wheat varieties and applying fungicide are mainly ways.However,the latter can easily induce the resistance to fungicide and cause great damages to the environment.Therefore,the application of resistant wheat varieties is the most economical and effective approach for stripe rust prevention and control.Nevertheless,the application of resistant varieties nowadays is also facing with the "loss" of resistance problem,the sexual reproduction and other reasons resulted in virulent variation of Pst rapidly,causing the original resistant wheat varieties losing resistance,new dominant race thereby generated and consequently leads to the epidemics of stripe rust.Based on the above reasons,it is important to study how Pst regulates the plant immune system to achieve the infection and the related molecular mechanisms,which is significant for effective and continuous prevention and control of the stripe rust.Previously,we screened out a gene Pst25505 from transcriptome database,which was highly expressed in the wheat and Pst affinity interaction,preliminary functional characterization toward Pst25505.There are following mianly results:1.In silico analysis revealed that Pst25505 encodes a δ-(1)-pyrrolin-5-carboxylic acid dehydrogenase(P5CDH),which belongs to the aldehyde dehydrogenase superfamily.The Secretome P 2.0 Server predicted that it might be an unconventionally secreted protein.Real-time PCR analysis of wheat leaves inoculated with CYR31 confirmed that the expression of this gene was upregulated at the penetration and early parasitic stages,peaked at36 hours.The subcellular localization conducted on Nicotiana benthamiana showed that the protein in located in the cytoplasm.The result was further confirmed in wheat protoplast.2.Agrobacterium tumefaciens mediated transient expression of Pst25505 inhibited programmed cell death induced by BAX and INF1 at N.benthamiana.Real-time PCR analysis to N.benthamiana sampled 12 hours after the transient expression of Pst25505 showed the reduced expression of the marker genes of salicylic acid and jamsonic acid dependent immunity.3.Using BSMV mediated gene silencing(BSMV-HIGS)technology,I temporarily silenced Pst25505 in the affinity strain CYR31,comparing to control which not silenced target genes Pst25505,the interaction of silenced CYR31 with the wheat resulted in increased accumulation of reactive oxygen species(ROS),restricted hyphae area and the mycelia growth,inferring the reduced pathogenicity of Pst.4.Host proteins that potentially interacted with Pst25505 were screened by immune precipitation mass spectrometry(IP-MS).Catalase(CAT)and ascorbate peroxidase(APX)were speculated as candidate targets for Pst25505.