Phenotypical Anylisis Of MoPEX7 And MoPEX20 Double Mutants In Development And Pathogenicity Of Magnaporthe Oryzae

Rice blast,caused by Magnaporthe oryzae,is one of the three major diseases on rice.M.oryzae is an important filamentous fungus with typical pathogenesis and infection cycle,which made the fungus to be a good model for the research on the interaction between plants and fungal pathogens.The peroxisome has already been demonstrated to play a vital role in the pathogenicity of M.oryzae.Peroxisome is a ubiquitous eukaryotic organelle with monolayer membrane and abundant in enzymes involved in a variety of biochemical metabolic processes,such as glyoxylate cycle,regulation of reactive oxygen species and fatty acid ?-oxidation.Peroxisome is origin from the endoplasmic reticulum.The peroxisomal matrix proteins and membrane proteins are encoded by nuclear genes,synthesized in cytoplasm,and by peroxisome targeting signal(PTS),recognized and imported into the organelles.Genes involved in peroxisomal formation were assigned as PEX,and the proteins encoded were called Peroxins.The import of PTS1-containing matrix proteins depends on PEX5 encoded receptor,and PTS2-containing proteins depend on the receptor PEX7 and its co-receptor PEX20.We previously investigated PEX7 and PEX20 separately in the rice blast fungus and found that the two genes are both required in the growth and pathogenicity of the fungus.To further reveal the differences and similarities between the roles of the two genes in peroxisomal biogenesis and fungal development,in the present work,we constructed the double mutants of the two genes and phenotypical compared the single mutants and double mutant.The results are as follows:1 The replacement vector of MoPEX7 carrying NEO gene(G418 resistance)was constructed and introduced into the ?pex20 mutant to obtain the double mutant?pex20?pex7.2 On the complete medium,?pex7,?pex20 and ?pex20?pex7 mutants were found no significant difference in the growth rate to the wild type.The colony appearance of ?pex7 did not differ from the wild type,but its sporulation decreased.The aerial hyphae of ?pex20 and ?pex20?pex7 were less generated,and the sporulation decreased,than the wild type.3 The subcellular localization of PTS1 was normal in ?pex7,?pex20 and ?pex20?pex7 as that in the wild type,while the positioning of PTS2 was all disordered in the three mutants.4 On rice and barley leaves,the pathogenicity of ?pex7,?pex20 and ?pex20?pex7 were decreased than that of the wild type,meanwhile the virulence ?pex20?pex7 was less than?pex7.5 ?pex7,?pex20 and ?pex20?pex7 were unable to develop on the media with Tween80,Olive oil or Oleic acid as the sole carbon source,indicating their unviability to utilize the long-chain fatty acids.However,the three mutants can use the CH3COONa as carbon source,they grow normally on MM-C + 50mM CH3 COON with decreased growth rates.6 On the media containing 100g/ml Congo red and 150g/ml Calcofluor white,the growth of the three mutants were inhibited.On Congo red medium,the inhibition of ?pex7 and?pex20 was no difference,on Calcofluor white medium,the situation is the same,.while the inhibition of ?pex20?pex7 was significantly reduced.7 On the media containing methyl viologen,the growth of ?pex7,?pex20 and?pex20?pex7 was decreased,and the decrease levels of ?pex20 and ?pex20?pex7 were higher than that of ?pex7.8 Induced on Terylene film,the spore germination mutants was unchanged,but the appressorium formation of ?pex7??pex20??pex20?pex7 was significant reduced,compared with the wild type.Meanwhile,the appressorium formation rate of ?pex7 and?pex20.was no difference.but ?pex20?pex7 was significantly reduced.9 On barley leaves,?pex7,?pex20 and ?pex20?pex7 penetrated the host cuticle successfully at 36 h post inoculation,and the infection hyphae were visible,but the penetration rate of ?pex7 was less than that of ?pex20.10 The ability of ?pex7,?pex20 and ?pex20?pex7 mutants to produce melanin were measured and found that the melanin production of the mutants was all reduced than the wild type,meanwhile,?pex7 produced relatively more melanin than ?pex20 and?pex20?pex7.11 The Nile red staining for spores and appressoria indicated that undegraded lipid bodies were partially residual in the spores and germ tubes of the mutants.In summary,the ?pex7 and ?pex20 show differences in colony morphology,sporulation,pathogenicity,cell wall integrity,melanin yield and tolerance to active oxygen.This may imply that the Pex20 play more roles than the involvement in PTS2 containing proteins as the co-receptor of Pex7.