RESUMO
In exploring the evolutionary trajectories of both pathogenesis and karyotype dynamics in fungi, we conducted a large-scale comparative genomic analysis spanning the Cryptococcus genus, encompassing both global human fungal pathogens and nonpathogenic species, and related species from the sister genus Kwoniella. Chromosome-level genome assemblies were generated for multiple species, covering virtually all known diversity within these genera. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at preadaptive pathogenic potential, our analysis found evidence of gene gain (via horizontal gene transfer) and gene loss in pathogenic Cryptococcus species, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the 2 genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5, or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion-fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus. Instead, Cryptococcus species with less than 14 chromosomes showed reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Overall, our findings advance our understanding of genetic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.
Assuntos
Cromossomos Fúngicos , Cryptococcus , Evolução Molecular , Genoma Fúngico , Genômica , Cariótipo , Cryptococcus/genética , Cryptococcus/patogenicidade , Cryptococcus/classificação , Cromossomos Fúngicos/genética , Genômica/métodos , Filogenia , Sintenia , Centrômero/genética , Criptococose/microbiologia , HumanosRESUMO
Small RNAS (sRNAs) participate in regulatory RNA interference (RNAi) mechanisms in a wide range of eukaryotic organisms, including fungi. The fungus Fusarium fujikuroi, a model for the study of secondary metabolism, contains a complete set of genes for RNAi pathways. We have analyzed by high-throughput sequencing the content of sRNAs in total RNA samples of F. fujikuroi grown in synthetic medium in the dark or after 1 h of illumination, using libraries below 150 nt, covering sRNAs and their precursors. For comparison, a parallel analysis with Fusarium oxysporum was carried out. The sRNA reads showed a higher proportion of 5' uracil in the RNA samples of the expected sizes in both species, indicating the occurrence of genuine sRNAs, and putative miRNA-like sRNAs (milRNAS) were identified with prediction software. F. fujikuroi carries at least one transcriptionally expressed Ty1/copia-like retrotransposable element, in which sRNAs were found in both sense and antisense DNA strands, while in F. oxysporum skippy-like elements also show sRNA formation. The finding of sRNA in these mobile elements indicates an active sRNA-based RNAi pathway. Targeted deletion of dcl2, the only F. fujikuroi Dicer gene with significant expression under the conditions tested, did not produce appreciable phenotypic or transcriptomic alterations.
RESUMO
We performed a functional analysis of two potential partners of ASF1, a highly conserved histone chaperone that plays a crucial role in the sexual development and DNA damage resistance in the ascomycete Sordaria macrospora. ASF1 is known to be involved in nucleosome assembly and disassembly, binding histones H3 and H4 during transcription, replication and DNA repair and has direct and indirect roles in histone recycling and modification as well as DNA methylation, acting as a chromatin modifier hub for a large network of chromatin-associated proteins. Here, we functionally characterized two of these proteins, RTT109 and CHK2. RTT109 is a fungal-specific histone acetyltransferase, while CHK2 is an ortholog to PRD-4, a checkpoint kinase of Neurospora crassa that performs similar cell cycle checkpoint functions as yeast RAD53. Through the generation and characterization of deletion mutants, we discovered striking similarities between RTT109 and ASF1 in terms of their contributions to sexual development, histone acetylation, and protection against DNA damage. Phenotypic observations revealed a developmental arrest at the same stage in Δrtt109 and Δasf1 strains, accompanied by a loss of H3K56 acetylation, as detected by western blot analysis. Deletion mutants of rtt109 and asf1 are sensitive to the DNA damaging agent methyl methanesulfonate, but not hydroxyurea. In contrast, chk2 mutants are fertile and resistant to methyl methanesulfonate, but not hydroxyurea. Our findings suggest a close functional association between ASF1 and RTT109 in the context of development, histone modification, and DNA damage response, while indicating a role for CHK2 in separate pathways of the DNA damage response.
Assuntos
Histonas , Proteínas de Saccharomyces cerevisiae , Sordariales , Histonas/metabolismo , Metanossulfonato de Metila/farmacologia , Chaperonas Moleculares/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Reparo do DNA , Dano ao DNA , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Histona Acetiltransferases/metabolismo , AcetilaçãoRESUMO
A large-scale comparative genomic analysis was conducted for the global human fungal pathogens within the Cryptococcus genus, compared to non-pathogenic Cryptococcus species, and related species from the sister genus Kwoniella. Chromosome-level genome assemblies were generated for multiple species of both genera, resulting in a dataset encompassing virtually all of their known diversity. Although Cryptococcus and Kwoniella have comparable genome sizes (about 19.2 and 22.9 Mb) and similar gene content, hinting at pre-adaptive pathogenic potential, our analysis found evidence in pathogenic Cryptococcus species of specific examples of gene gain (via horizontal gene transfer) and gene loss, which might represent evolutionary signatures of pathogenic development. Genome analysis also revealed a significant variation in chromosome number and structure between the two genera. By combining synteny analysis and experimental centromere validation, we found that most Cryptococcus species have 14 chromosomes, whereas most Kwoniella species have fewer (11, 8, 5 or even as few as 3). Reduced chromosome number in Kwoniella is associated with formation of giant chromosomes (up to 18 Mb) through repeated chromosome fusion events, each marked by a pericentric inversion and centromere loss. While similar chromosome inversion-fusion patterns were observed in all Kwoniella species with fewer than 14 chromosomes, no such pattern was detected in Cryptococcus. Instead, Cryptococcus species with less than 14 chromosomes, underwent chromosome reductions primarily through rearrangements associated with the loss of repeat-rich centromeres. Additionally, Cryptococcus genomes exhibited frequent interchromosomal translocations, including intercentromeric recombination facilitated by transposons shared between centromeres. Taken together, our findings advance our understanding of genomic changes possibly associated with pathogenicity in Cryptococcus and provide a foundation to elucidate mechanisms of centromere loss and chromosome fusion driving distinct karyotypes in closely related fungal species, including prominent global human pathogens.
RESUMO
IMPORTANCE: Histone chaperones are proteins that are involved in nucleosome assembly and disassembly and can therefore influence all DNA-dependent processes including transcription, DNA replication, and repair. ASF1 is a histone chaperone that is conserved throughout eukaryotes. In contrast to most other multicellular organisms, a deletion mutant of asf1 in the fungus Sordaria macrospora is viable; however, the mutant is sterile. In this study, we could show that the histone-binding ability of ASF1 is required for fertility in S. macrospora, whereas the function of ASF1 in maintenance of genome stability does not require histone binding. We also showed that the histone modifications H3K27me3 and H3K56ac are misregulated in the Δasf1 mutant. Furthermore, we identified a large duplication on chromosome 2 of the mutant strain that is genetically linked to the Δasf1 allele present on chromosome 6, suggesting that viability of the mutant might depend on the presence of the duplicated region.