TRV Expression Vector Was Used To Analyze The Function Of Secretory Protein In Magnaporthe Oryzae

Magnaporthe oryzae is one of the most important pathogens of rice blast.From seedling to adult plant stage,rice is susceptible to this pathogen.In this study,TRV vector?Tobacco rattle virus?was used to express the secreted protein of Magnaporthe oryzae in Nicotiana benthamiana,and the secreted protein causing necrosis of N.benthamiana leaves was screened and its function was analyzed.The secreted proteins that can inhibit the necrosis of cells by pro-apoptotic mouse protein?Bax?are screened and their functions are initially analyzed.The main results are as follows:1.Construction of TRV expression vector for screening for secreted proteins from Magnaporthe oryzaeThe TRV expression vector system was established,and the genes of Lys domain,Cutinase domain,Chitin domain and CFEM domain were cloned into TRV expression vector,and the function of secreted protein of Magnaporthe oryzae was analyzed.Screening for secreted proteins that cause necrotic in N.benthamiana cells,and the secreted proteins of inhibit the necrosis of N.benthamiana leaves caused by Bax.The results of viral infiltration of N.benthamiana results showed that TRV-MGG₀0734,TRV-MGG₀3792,TRV-MGG₀5083,TRV-MGG₀5798,TRV-MGG₀5871,TRV-MGG₀7553,TRV-MGG₀7565 and TRV-MGG₁0473 caused necrotic phenotype.The TRV-Bax secreted proteins were inhibited as TRV-MGG₀1872,TRV-MGG₀3468,TRV-MGG₀6771,TRV-MGG₀7153,TRV-MGG₀1943,and TRV-MGG 05023.2.Functional analysis of secreted proteins of Magnaporthe oryzae causing cell necrosisSignal peptide prediction of eight secreted proteins causing necrosis of N.benthamiana leaves revealed that the N-terminus contained a signal peptide of approximately 16-22 amino acids in size.In this study,three secreted proteins,MGG 00734,MGG₀3792 and MGG₀5798 were selected as the research objects.According to the predicted results,the TRV expression vector without signal peptide sequence was constructed by PCR,and the effect of signal peptide on three secreted proteins was analyzed.TRV-MGG₀0734?sp and TRV-MGG₀3792?sp did not cause necrosis of cells.TRV-MGG₀5798?sp caused N.benthamiana leaf necrosis to be more serious than TRV-MGG₀5798.The TRV expression vector with three secreted proteins and Myc tag was constructed,and specific bands were detected by Western blot,The results showed that they could all be expressed in N.benthamiana.Bioinformatics analysis of the MGG₀5798 protein sequence revealed the presence of the Cutinase domain and construction of a domain-deficient TRV expression vector.Tobacco infiltration results indicated that the domain is essential for cell necrosis.The transient expression vector of 05798 was constructed,and the results of Agrobacterium infiltration inoculation further confirmed that the 05798?sp aggravates cell necrosis,and the green fluorescence of protein aggregation in the leaves of 35S-MGG₀5798 and 35S-MGG₀5798?sp was observed by laser confocal.3.Preliminaiy functional analysis of secreted proteins inhibiting TRV-Bax-induced necrosis of N.benthamiana cellsThree MGG₀7153,MGG₀6771 and MGG₀3468 of 6 secreted proteins that completely inhibited TRV-Bax necrosis were selected for bioinformatics analysis of their physical and chemical properties,and the effects of TRV-XEG1,TRV-INF1 and TRV-MGG₀3792 on cell necrosis were analyzed.Agrobacterium tumefaciens infiltration results showed that none of the three secreted proteins could inhibit plant cell death induced by TRV-INF1,TRV.MGG₀3468 and TRV-MGG₀7153 are able to inhibit plant cell death induced by TRV-MGG.03792,TRV-MGG₀3468 and TRV-MGG₀6771 can inhibit the cell death induced by TRV-X:EG1;when the three secreted proteins are coOinjectcd with TRV-Bax,Western blot analysis indicates that these three proteins can be expressed by TRV vector in N.benthamiana,and secreted proteins can inhibitccllnecrosiscausedby Bax.TRV expression vector to screen 8 secreted proteins causing cell necrosis,6 secreted proteins inhibiting TRV-Bax necrosis,and preliminary functional analysis,laid the foundation for further research on the pathogenicity of rice blast.