Molecular Mechanism Of An Effector Protein Hasp190 From Stripe Rust Fungus In Regulating Host Immunity

Wheat stripe rust caused by Puccinia striiformis f.sp.tritici?Pst?is a serious fungal disease of wheat.The disease occurs in a wide range,spreads quickly,and the virulence changes frequently,resulting in the rust resistance loss of the main cultivars in wheat production,and the prevention of wheat stripe rust are in a passive state now.Therefore,intensive analysis of infection and pathogenicity mechanism of wheat stripe rust is significant for controling this disease persistently.Studies have shown that pathogens regulate host's immune defense by secreting effector proteins to promote pathogen infection.This study screened the candidate effector gene Hasp190 based on the preliminary study of transcriptome sequencing of Pst haustoria.The interaction target of Hasp190 was defined and the mechanism of the target in pathogen infection were analyzed by various molecular biological methods.The results lay a foundation for further revealing the molecular mechanism of wheat and stripe rust interaction,and also provided a theoretical basis for the development of new disease prevention and control strategies.The key results are as below:1.Characterizations of Hasp190The ORF of Hasp190 consists of 834 bp,which encoding 277 amino acids.The first 23amino acids at the N-terminal are signal peptide.There are not trans-membrane structure and known conserved domain by Pfam analysis.Its isoelectric point?pI?is 5.74 and molecular weight is 27.98 kDa.The signal peptide of Hasp190 was confirmed to have a secretory function by the yeast invertase-deficient system.qRT-PCR was used to analyze the expression level of Hasp190 in wheat infected by stripe rust.The expression level of Hasp190 was significantly up-regulated at 6-48 h after pathogen infection,reaching a peak at48 h,and the expression level was increased by about 540 times,indicating that Hasp190might play an important role in the early Pst infection stage.2.Hasp190 interactes with a wheat transcription factor TaASR2The candidate interaction target wheat transcription factor TaASR2?Abscisic acid-,stress-,and ripening-induced?of Hasp190 was screened by yeast two-hybrid technique,and the physical interaction between Hasp190 and TaASR2 was further confirmed by pull down,bimolecular fluorescence complementation?BiFC?and co-immunoprecipitation?CoIP?.The transient expressio n system of tobacco showed that both TaASR2 and Hasp190 were localized in the nucleus,but when both were expressed in tobacco,most of the two proteins enter the nucleus,and only a small amount remains in the cytoplasm,indicating that the interaction between Hasp190 and TaASR2 might mainly occur in the nucleus.3.Functional analysis of TaASR2ASR protein is a family of proteins widely existing in plants,most of which function as transcription factors.The expression characteristics of TaASR2 were analyzed by qRT-PCR,and it showed that the transcription level of TaASR2 was increased by 5 times at 24 hours after inoculation,indicating that TaASR2 might be involved in the defense response of wheat in the early Pst infection stage.Through the verification of transcriptional activation function,it was found that TaASR2 had transcriptional activation function and transcriptional activation domain was located at the N-terminal.The TaASR2 gene was silenced by virus-mediated gene silencing technique?VIGS?,and the necrotic area of wheat leaves were significantly reduced after inoculation of an avirulent race of Pst,CYR23,in the silent segment.Statistical analysis of the tissue showed that the necrotic area and H₂O₂ accumulation caused by stripe rust infection were significantly reduced at 48 h and 120 h,indicating that TaASR2 may be a disease-resistant gene.