| Circadian rhythm organizes inner physiology with respect to the external world,providing life with the ability to anticipate and thereby better prepare for major fluctuations in its environment.To determine the importance of the circadian rhythm genein the rice blast fungus Magnaporthe oryzae,here we functionally characterized the MoFRQ gene,an ortholog of Neurospora crassafrqwhich is the central component of the oscillator.Our results showed the abundance of MoFRQ transcripts haveno oscillated under constant darkness.However,the expression level of MoFRQ was higher under light conditions than in the darkness.Light not only stimulated the expression of MoFRQ,but also accumulated MoFrq-GFP into the nucleus.The Mofrq deletion mutant is defective in aerial hyphal growth under constant light.The conidiation of the mutant was significantly reduced in comparison with the wild-type Guy11.We further verified the interaction between MoFrq and Htf1 by yeast-two-hybrid assay.These two genes might form a heterodimer to regulate the conidia formation from the conidiophores.The Mofrq mutant rarely caused typical lesions on rice or barely leaves,indicated a significant reduction in virulence.Defects of the Mofrq mutant in appressorium formation and penetration were detected as well,further confirmed the crucial role of MoFrq in plant infection.The mobilization and degradation of glycogen from conidia to appressoria was negatively impacted by deletion of MoFRQ,which might be responsible for the defect of Mofrq mutant inpenetration.The Mofrq mutant is hypersensitive to oxidative stress but not hyperosmotic stress.The PKA activity in the Mofrq mutant was also reduced.Therefore,Mofrq mutant was defective in vegetative growth,conidiation,appressoria penetration,oxidative stress responses and signaling activation.On the other hand,we also deleted the ortholog of MoFRQ inFusarium graminearum,but found FgFRQ was dispensable for both vegetative and invasive growth.We then expressed FgFRQ in the Mofrq mutant,but failed tocomplemented the defects of Mofrq mutant in growth and plant infection.Although N.crassa was not a pathogenic fungus,FRQ gene fully complemented the function of its ortholog inM.oryzae.Interstingly,FRQ and MoFRQ but not FgFRQ has a strong bais for non-optimized codons,suggested this codon usage was important for the function of FRQ genes.For plant pathogenic fungi,autophagic processes result in the transport of cellular contents to vacuoles for degradation,thereby contributing to the balance between synthesis and degradation in cells.Autophagy processes also be extremely useful in pathogenicity of plant pathogenic fungi.To determine the role of autophagy in the F.graminearum,we identified 26 ATG genesand deleted all of them for functional analysis in this study.In different growth stages of F.graminearum,most ATG genes have higher expression levels in hypha or conidium.Among those deletion mutants,15 of them were defective in aerial hyphal growth but only 12 of them were significantly reduced in growth rates.10 atg deletion mutants werereduced in conidiation.13 mutants were blocked in perithecia formation,and 7 mutnts were defective in ascospore formation or discharge.There were 17 atg mutantshad a significantly reduction in pathogenicity,7 of them werechangedon DON production.Overall,ATG genes were important for various aspects of growth,aerial hyphal growth,sexual reproduction,and infection processes in F.graminearum. |
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