Study On Molecular Mechanism Of Host Defense Reaction Regulated Bypuccinia Striiformis F.sp.Tritici Effector Hasp172

Stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst.),is an important fungal disease in wheat production.The utilization of disease-resistant varieties is the main method of controlling stripe rust.However,the resistance of wheat cultivar will be lost due to the constant pathogenicity variation of Pst.Stripe rust continues to threaten global wheat production and global food security.The study for molecular mechanism of Pst pathogenicity and its variation is of great significance to explore the new strategies for durably controlling the disease.A large number of studies have shown that pathogens inhibit the defense response of hosts by secretory effectors to promote pathogen infection and pathogenesis.In the preliminary work,it was found that Hasp172 could inhibit cell necrosis induced by Bax and we speculated that Hasp172 could play a key role in the pathogenesis of pathogen.However,the molecular mechanism of Hasp172 regulation host defense response was unclear.Therefore,the study was used to identify the secreted protein Hasp172 and the yeast two-hybrid technique was used to screen the target of its interaction.In this study,we have obtained the following results:1?Functional analysis of effector genesThe effector gene Hasp172 which was 303 bp was amplified by RT-PCR.Through the sequence analysis and yeast secretion system confirmed that Hasp172 was secretory protein.With the Agrobacterium-mediated PVX viral expression system,overexpression of Hasp172 in the model plant-Nicotiana benthamiana can inhibited the cell death induced by Bax.qRT-PCR analysis showed that Hasp172 was induced in the 6-24 h process of infection,suggesting that it may play a role in early infection.Host-induced gene silencing BSMV-HIGS showed that the number of haustoriums in pathogen was significantly reduced after silencing Hasp172 gene.But due to the functional redundancy of the effectors,there is no significant change in the amount of Sporulation.Subcellular localization of Hasp172 mediated by Agrobacterium showed that the it was located in the mitochondria.2?Screening and Functional Analysis of the target interacting with Hasp172A interaction protein,TaVDAC1,was identified by uisng a yeast two-hybrid(Y2H)screen.Further experiments confirmed the interaction between Hasp172 and TaVDAC1 in the mitochondria by using yeast two-hybrid methods and bimolecular fluorescent complementary(BIFC)experiments.The transcriptional abundance of TaVDAC1 in compatible and incompatible interactions was determined by qRT-PCR.The results showed that the expression of TaVDAC1 in incompatible system was significantly changed.The expression of TaVDAC1 in the early stage of pathogen infection was induced.It indicated that TaVDAC1 playeda importent role in plant disease resistance.And TaVDAC1 is indeed located in mitochondria.3?Screening and Functional Analysis of the target interacting with TaVDAC1The interaction between wheat RING type E3 ubiquitin ligase and TaVDAC1 was confirmed by yeast two-hybrid to analyze the downstream pathways of TaVDAC1.Overexpression of TaRING1 in the model plant-Nicotiana benthamiana can inhibited the cell death.It is defined that the effector Hasp172 played a role in the process of pathogen infection and the regulatory mechanism was clarified,which laid the foundation for revealing the molecular mechanism of interaction between stripe rust and wheat.And it provided a theoretical basis and technical support for the development of a new strategy for the sustainable control of wheat stripe rust simultaneously.