Double Knockout And Mutant Phenotype Analysis Of The Peroxisome Peroxidase Binding Enzyme PEX4 And Its Anchored Protein PEX22 Of Magnaporthe Oryzae

Rice blast caused Magnaporthe oryzae,is one of the fungal diseases which threaten yield and quality of rice,resulting in serious losses.As a model organism for studying the pathogenic mechanism of plant pathogenic fungi,rice blast fungus has a large number of genes involved in regulation during the disease development,involving conidial germination,appressorial cell formation,and glycerol synthesis.Therefore,the study of its related genes and pathogenic mechanism can be used as an important theoretical basis for the control of rice blast.Peroxisomes are important organelles in eukaryotic cells and participate in a variety of physiological and metabolic processes in organisms,such as glyoxylate cycle,?-oxidation of fatty acids,and regulation of reactive oxygen species,peroxidases.The peroxisomal matrix protein is synthesized in the cytoplasm and is recognized by the transport system by its own PTS signal.Pex5 and Pex7 are receptors for PTS1 and PTS2,respectively.After PTS is recognized by the receptors Pex5 and Pex7,the receptor complex transports the matrix protein to the inside of the peroxisomes by the docking complexes(Pex13,Pexl45 and Pexl7).Pex5 and Pex7 were then returned to the cytoplasm through the ubiquitin system for the next cycle.lt has been reported that Pex4-encoded ubiquitin-conj ugating enzyme and its anchored protein Pex22 participate in the ubiquitination and recycling of Pex5 and Pex7,thereby affecting peroxisomal formation.In the absence of Pex4 and Pex22,cell characteristics do not have typical peroxisomes and influence the localization of peroxisomal matrix proteins.In the previous work,we knocked out the and genes of M.oryzae and analyzed the phenotypes of the mutants.The results showed that the loss of both MoPEX4 and MoPEX22 affected the groMtth and pathogenicity of M.oryzae.In order to further study the function and relationship of MoPEX4 and MoPEK22 in the formation of peroxisomes and the growth and development of M.oryzae,we performed a single double-knockout study on the knock-out and double-knock mutants of MoPEX4 and MoPEX22.In this study,the phenotypes of MoPEX4 and MoPEX22 single knockout mutants and double knockout mutants were analyzed by double knockout,and the relationship between MoPEX4 and MoPEX4 was explored through gene expression analysis.The result is as follows:The knockout vector pKO-MoPEX4 containing the hygromycin(HPH)resistance gene was introduced into the Mopex22(SUR)mutant strain of M.oryzae through Agrobacterium-mediated transformation(AtMT)to obtain the double knock mutants Mopex4/pex22;Real-time PCR showed that the deletion of MoPEX22 gene increased the expression of MoPEX4 gene in Mopex22 strain,the deletion of MoPEX4 gene increased the expression level of MoPEX22 gene in Mopex4 strain;The localization of PTS1 and PTS2 on peroxisomes was affected by the deletion of MoPEX4?MoPEX22 and double knockout genes;The constructed mCherry-Hex1 fusion protein vector was introduced into the wild-type and mutants,and it was found that the deletion of MoPEX4?MoPEX22 and double knockout genes all affected the localization of the Hexl;The constructed GFP-MoPEX5 fusion protein vector was introduced into the wild type and the mutants respectively.The deletion of MoPEX4?MoPEX22 and double knockout genes did not affect the localization of MoPEX5;In cultured for 7 days on CM medium,compared with wild-type Guy-11,the deletion of MoPEX4,MoPEX22 and double knockout gene resulted in a decrease in the growth rate and sporulation of M.oryzae,and the most severe decrease the deletion of double-knockout gene;Melanin production assays showed tha the production of melanin was significantly reduced after knockout of MoPEX4?MoPEX22 and double knockout genes,and the most severe decrease of double knockout genes;To determining the pathogenicity of the fungus;MoPEX4,MoPEX22 and double knockout genes were involved in the pathogenicity of M.oryzae,and deletion of these genes lead to a significant reduction of pathogenicity of M.oryzae in barley and rice;MoPEX4?MoPEX22 and double knockout genes are involved in the process of spore germination,appressorium formation,and cell swelling pressure accumulation of M.oryzae.Deletion of these genes affected the fungal spore germination and appressorial cell formation;After 7 days cultivation on CM medium containing 200?g/mL Congo red and 100 ?g/mL Calcolflour white showed that MoPEX4?MoPEX22 and double knockout genes play an important role in maintaining the cell wall integrity of M.oryzae,and the loss of MoPEX22 gene had the significant influence;After 7 days cultivation on CM medium containing 5mM H2O2,1mM Methyl viologen revealed that the MoPEX4?MoPEX22 and double knockout genes attenuated the active oxygen tolerance of M.oryzae,and the loss of double knockout genes had the greatest effect.