The Regulation Mechanism Of Sexual Reproduction In The Human Fungal Pathogen Cryptococcus Deneoformans

The Cryptococcus species complex,an opportunistic human pathogenic fungus,generally cause fatal meningitis.In fungal pathogen,sexual reproduction is a key strategy underlying formation of hyper-virulent strains through meiotic recombination generates genetic diversity in progeny and has caused serious outbreaks of Cryptococcus infection in North America.Therefore,it is closely linked to the complex’s excellent environmental adaptation and virulence evolution.Among the Cryptococcus species complex,Cryptococcus deneoformans(formly Cryptococcus neoformans var.neoformans,serotype D),with two sexual reproduction modes and simple genetic manipulation,is considered to be the model organisms used to study sexual reproduction.C.deneoformans has two mating types(α and a)and can undergo different sexual cycles:α-a bisexual reproduction and unisexual reproduction.The bisexual and unisexual cycles share multiple developmental and molecular processes including hyphal formation,basidial maturation,meiosis,and spore production.In addition,these two sexual cycles can be discriminated by the approaches to achieve pre-meiotic diploidization:bisexual reproduction involves formation of α/a zygotes via α-a cell-cell fusion(syngamy),while unisexual reproduction is mainly accomplished by autodiploidization,which likely occurs via endoreplication.However,it remains unclear about the regulatory basis underlying developmental continuity of the multistage sexual cycles and the hallmark events specific for different reproduction modes.In fungi,transcription factors are often terminal components of various signaling cascades and are critical for a wide range of biological processes.To identify the core regulatory mechanisms that regulate sexual reproduction of C.deneoformans,we focused on transcription factors.It is identified that 111 transcription factors exhibit dynamic expression during sexual reproduction through comparative transcriptomic analysis,suggesting that these transcription factors may play an important role in the regulation of sexual reproduction.We constructed a library containing 111 transcription factor deletion mutants,and analyzed each transcription factor deletion strain for a total of eight phenotypic characteristics,which cover the previously identified morphogenesis events and pre-meiotic diploidization phases throughout two sexual life cycles.Quantitative and semiquantitative assessments unveiled a large number of novel genotype-phenotype connections related to sexual regulation,offering a comprehensive and unprecedented understanding of the functional transcription factors networks that shape cryptococcal ability to undergo the distinct sexual cycles.In addition,the transcription factor deletion mutant library constructed in this study provides valuable strain resources for studying the general biology and pathogenicity of C.deneoformans.The vast majority of identified transcription factors are pleiotropic regulators that exert their functions in no less than two stages.Velvet family regulators Cval and Vea2 can regulate multiple developmental stages of sexual reproduction in C.deneoformans,including hyphal initiation,hyphal extension,and cell fusion.Transcriptome analysis found that Cval controls the formation and development of hyphae by regulating Znf2.Futher by using transcriptomic evaluation combined with phenotype analysis,it was found that Cval controls bisexual syngamy through a novel mating-responsive protein Cfs1.During unisexual reproduction,autodiploidization is involved in the chromosomal ploidy process instead of cell fusion.However,it remains unclear about the regulatory mechanisms and genetic basis underlying sexual diploidization in fungal sexual reproduction independent of cell fusion.We identified Cqs2 is a core transcription factor that mediates unisexual self-diploidization,while it does not regulate bisexual syngamy.Further studies showed that the Qsp1-Cqs2-Pum1 regulatory cascade mediates a haploid-dependent ploidy duplication mechanism.The failure to induce ploidy increase in diploid by Puml may be attributed to its inability to sufficiently increase the expression of certain cell cycle genes in the diploid context.In addition,we constructed tetraploid strains of C.deneoformans by cell fusion to explore the effects of Cryptococcal polyploidy cells on environmental adaptability and sexual reproduction.We found that polyploid strains exhibited significant defects in sexual reproduction and the ability to infect the host or survive abotic stress.This result shows that polyploidy is unfit for the life cycle of C.deneoformans.In summary,we revealed the molecular and ploidy-based regulatory mechanisms of the sexual reproductive cycles of C.deneoformans,providing a paradigm for related studies in other pathogenic fungi.