| Rice is one of the major food crops in the world.Rice blast caused by the Magnaporthe oryzae occurrs in various rice cultivation areas in the world,causing a serious decline in rice yield.Studies on the pathogenic mechanism of rice blast fungi can provide a convincing theoretical basis for disease prevention and control as well as resistance research.Up to now,a large number of genes related to the growth and pathogenesis of M.oryzae have been identified,involving conidia generation,appressorium formation,melanin formation and glycerol synthesis.The formation of appressorium and the accumulation of glycerol depend on the storage of lipids in conidia.Peroxisomes are the main sites of lipid metabolism.Previous studies have shown that peroxisomes are necessary for pathogenicity of M.oryzae.During the formation of peroxisomes,the matrix proteins are synthesized in the cytoplasm.Pex5 and Pex7 respectively encode the receptors of peroxisome targeting signal 1(PTS1)and PTS2,binding to the matrix protein,and transfering them to the peroxisomal membrane.The matrix protein is then transported into the peroxisomes by the interaction with the docking complex(consisting of Pex13,Pex14 and Pex17)of peroxisome and the action of the Znic ring proteins(Consisting of Pex2,Pex10 and Pex12).Then PTS1 and PTS2 are recycled throggh the ubiquitin conjμgation complex from the peroxisomes to the cytoplasm and into the next cycle.The recycling of the PTS receptor involves Pex4,a ubiquitin conjμgate enzyme together with its membrane docking protein Pex22,and a complex containing two ATPases,Pex 1 and Pex6,together with their membrane anchors Pex15 or Pex26.At present,the recycling mechanism of matrix protein receptor was less well investigated in M.oryzae.In present work,MoPEX22L,the poteintial Pex22 encoding gene in M.oryzae was functianlly characterized by using the gene replacement strategies.Firstly,the delection mutant of MoPEX22L gene was obtained by gene knockout strategy.And then the localization of peroxisomal matrix proteins in the mutant was detected by fluorescent protein localization.It was found that PTS1 and PTS2 could not be correctly located in the MoPEX22L knockout mutant.On MM,MM-C,MM-C+0.5%Tween80,CH3COONa and other fat metabolism mediums,MoPEX22L knockout mutant could not use long chain fatty acid,but can use CH3COONa as carbon source.On the medium containing Congo red,Calcolflour white or Rose-bengal,the growth of the MoPEX22L gene knockout mutant was inhibited,indicating that the cell wall was defective.The MoPEX22L gene knockout mutant were more severely inhibited than the wild-type strain on the medium containing Methyl viologen or H2O2,which indicated that the MoPEX22L gene disruption could decrease the tolerance of the strains to the reactive oxygen species.In the process of spore germination and appressorium formation,we found that the germination rate and the appressorium formation rate of cells in MoPEX22L knockout mutant were significantly decreased.On the complete medium,the colony size of the MoPEX22L gene knockout mutant was significantly lower than that of the wild type strain,and the thickness of the aerial mycelium was significantly thinner.In the process of infection on barley leaves,the infection hyphae of MoPEX22L mutant were significantly reduced.The results of pathogenicity test showed that the pathogenicity of the mutant was significantly decreased.The above results indicated that MoPEX22L gene deletion blocked the formation of peroxisome in the mutant,which caused the degradation of metabolic function,including the ability of degradation of active oxygen,the utilization of fatty acid,the decrease of cell wall integrity,and a series of develpment defects,such as the vegetative growth,the spore germination and the formation of appressorium.These defects resulted finally in the significant pathogenicity decrease. |
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