Identification And Functional Analysis Of The Key Gene PsPKA Of Camp Signalling Pathway In The Wheat Stripe Rust Fungus

Wheat stripe rust,caused by Pucciniastiiformis f.sp.tritici(Pst),is one of the most important diseases threatening to global wheat production.At present,cultivation of disease-resistant varieties is the most effective,economic and environment security method to control wheat stripe rust.However,most of named resistance genes from wheat have been overcomed by new virulent Pst races,and conventional cross-breeding often lags behind the variation of wheat strip rust.Therefore,original strategies must be changed,and new rust resistance gene resource must be explored to ensure food safety in production and achieve durable disease control of wheat stripe rust in our country.Cellular signal transduction in organisms include molecular pathways of cell sensation and transduction environmental stimuli,the expression of regulated genes,and regulation of physiological responses.Plant-pathogen interaction involves a series of processes of signal recognition,signal transduction and defense response activation.The growth,development and pathogenicity of pathogenic fungi are also dependent on regulation of extracellular signal transduction pathways.Previous studies showed that the second messenger cAMP signaling pathway play an important role in activation of protein kinase A(PKA)for regulating the growth of fungal hyphae,virulence,pathogenicity and melanin formation.To further explore the mechanism of Pst pathogenicity during the interaction between wheat and stripe rust fungus,we performed functional ananlysis of the key gene PsPKA of cAMP signaling pathway in Pst.1.A length of 1443 bp of PsPKA was obtained,which encoded 360 amino acid residues.qRT-PCR assays showed that the transcriptional levels of PsPKA was gradually increased in the early infection stage,and peaked at 18 h after inoculation,and then decreased.2.Ater complemented Magnaporthe oryzae CPKA mutant with PsPKA,the pathogenicity of △CPKA can be partially recovered.3.Yeast two-hybridization assays indicated that the presence of interactions between PsPKA and PsPrfl.4.Knocking down PsPKA gene with BSMV-HIGS showed that temporary silencing of PsPKA would decrease the number of urediosorus of Pst significantly.Histological observation with fluorescence microscopy found that the number of haustorium and hyphae length in the 48 h was repressed,the hyphae length and colony area in the 120 h were reduced significantly.These results suggest that PsPKA gene might play an important role in regulating Pst development and pathogenicity.5.Using particle bombardment 2 RNAi vectors,PKA-pMCG161 and pMCG161 were transformed into wheat varieties 1376.In total 23 transgenic plants of PKA-pMCG161 and 15 transgenic plants of pMCG161 were obtained,respectively.The transgenic rate of the PKA-pMCG161 was 1.04%and 1.09%,respectively.