Analysis Of The Role Of Transcription Factor ADA-6 In Conidiation Of Neurospora Crassa

Conidiation is a biological process important for filamentous fungi to reproduce,disperse and ultimately form infection.The production of fungal asexual spore is also regulated by the internal molecular mechanism.The elucidation of the molecular mechanism of fungal asexual spore has important significance for the utilization of the beneficial fungi and the prevention and control of the harmful fungi.Neurospora crassa is easy to cultivate and not easy to be polluted.The gene sequence of Neurospora crassa has been released,gene information is detailed and complete,and more than 70% of the genes have targeted knockout mutants,and also Neurospora crassa has the largest mutant library,can be used as a good research model material of filamentous fungi conidiation.In order to comprehensively analyze the genes regulated by ADA-6,transcriptomic profiles generated by RNA-seq from the samples of Neurospora crassa wild-type strain before and after induction of conidiation(before induction 0 h,induced 12 h,induced 24 h)were compared.Gene ontology analysis of differentially expressed genes indicated that oxidation-reduction process and metabolic process are mainly influenced during the induction of conidiation.Among these,genes related to ROS(reactive oxygen species)are enriched,indicating the ROS production during conidiation.Moreover,genes associated with conidiation(including regulatory genes and spores structural genes)expressed specifically.To understand the roles of these induced genes in conidiation,we analyzed the phenotype of the mutants for up-regulated genes and found 6 new genes(NCU09792,NCU05159,NCU06112,NCU05079,NCU00461,NCU07521(fwd-2))that positively contribute to conidiation.The conidial production of knockout mutants of these genes are much lower than that of the wild type strain,and the increased expression of these genes in conidiation indicate that they positively regulate conidiation.Transcriptomic profiles generated by digital gene expression profiling from the ada-6 deletion mutant and the wild-type strain were compared.Gene ontology analysis of differentially expressed genes between WT and ada-6 deletion mutant indicated that oxidation-reduction process,organic acids,carboxylic acids,amino acid transport processes,cell wall structure and membrane modification process are mainly influenced during the induction of conidiation,gene related to redox enzyme activity and hydrolase also expressed specifically.Among the down-regulated genes,two which were already known to be involved in conidiation – fluffy,eas were examined further in a time course experiment.Transcription of two genes in the ada-6 deletion mutant was lower than in the wild-type strain during conidial development.Transcription factor ADA-6 positively regulated fl and eas.poi-2 and nop-1 were previously reported to be associated with conidiation,RNA-seq and qRT-PCR results show that ADA-6 regulate poi-2 and nop-1 with another transcription factor.Enzyme encoding gene NCU04865,NCU09209,NCU08755,NCU08856 involved in the oxidation reduction process,transcription of four genes in the ada-6 deletion mutant was lower than in the wild-type strain during conidial development.indicating that ADA-6 may coregulation of these genes with another transcription factor.Our work further deepen the understanding of conidiation and its regulatory networks in N.crassa.It has important significance for our knowledge of conidiation of filamentous fungi.