Centromeres are chromosomal regions that serve as platforms for kinetochore assembly and spindle attachments, ensuring accurate chromosome segregation during cell division. Despite functional ...conservation, centromere DNA sequences are diverse and often repetitive, making them challenging to assemble and identify. Here, we describe centromeres in an oomycete Phytophthora sojae by combining long-read sequencing-based genome assembly and chromatin immunoprecipitation for the centromeric histone CENP-A followed by high-throughput sequencing (ChIP-seq). P. sojae centromeres cluster at a single focus at different life stages and during nuclear division. We report an improved genome assembly of the P. sojae reference strain, which enabled identification of 15 enriched CENP-A binding regions as putative centromeres. By focusing on a subset of these regions, we demonstrate that centromeres in P. sojae are regional, spanning 211 to 356 kb. Most of these regions are transposon-rich, poorly transcribed, and lack the histone modification H3K4me2 but are embedded within regions with the heterochromatin marks H3K9me3 and H3K27me3. Strikingly, we discovered a Copia-like transposon (CoLT) that is highly enriched in the CENP-A chromatin. Similar clustered elements are also found in oomycete relatives of P. sojae, and may be applied as a criterion for prediction of oomycete centromeres. This work reveals a divergence of centromere features in oomycetes as compared to other organisms in the Stramenopila-Alveolata-Rhizaria (SAR) supergroup including diatoms and Plasmodium falciparum that have relatively short and simple regional centromeres. Identification of P. sojae centromeres in turn also advances the genome assembly.
The centromere DNA locus on a eukaryotic chromosome facilitates faithful chromosome segregation. Despite performing such a conserved function, centromere DNA sequence as well as the organization of ...sequence elements is rapidly evolving in all forms of eukaryotes. The driving force that facilitates centromere evolution remains an enigma. Here, we studied the evolution of centromeres in closely related species in the fungal phylum of Basidiomycota. Using ChIP-seq analysis of conserved inner kinetochore proteins, we identified centromeres in three closely related Cryptococcus species: two of which are RNAi-proficient, while the other lost functional RNAi. We find that the centromeres in the RNAi-deficient species are significantly shorter than those of the two RNAi-proficient species. While centromeres are LTR retrotransposon-rich in all cases, the RNAi-deficient species lost all full-length retroelements from its centromeres. In addition, centromeres in RNAi-proficient species are associated with a significantly higher level of cytosine DNA modifications compared with those of RNAi-deficient species. Furthermore, when an RNAi-proficient Cryptococcus species and its RNAi-deficient mutants were passaged under similar conditions, the centromere length was found to be occasionally shortened in RNAi mutants. In silico analysis of predicted centromeres in a group of closely related Ustilago species, also belonging to the Basidiomycota, were found to have undergone a similar transition in the centromere length in an RNAi-dependent fashion. Based on the correlation found in two independent basidiomycetous species complexes, we present evidence suggesting that the loss of RNAi and cytosine DNA methylation triggered transposon attrition, which resulted in shortening of centromere length during evolution.
The diploid budding yeast
harbors unique CENPA-rich 3- to 5-kb regions that form the centromere (CEN) core on each of its eight chromosomes. The epigenetic nature of these CENs does not permit the ...stabilization of a functional kinetochore on an exogenously introduced CEN plasmid. The flexible nature of such centromeric chromatin is exemplified by the reversible silencing of a transgene upon its integration into the CENPA-bound region. The lack of a conventional heterochromatin machinery and the absence of defined boundaries of CENPA chromatin makes the process of CEN specification in this organism elusive. Additionally, upon native CEN deletion,
can efficiently activate neocentromeres proximal to the native CEN locus, hinting at the importance of CEN-proximal regions. In this study, we examine this CEN-proximity effect and identify factors for CEN specification in
We exploit a counterselection assay to isolate cells that can silence a transgene when integrated into the CEN-flanking regions. We show that the frequency of reversible silencing of the transgene decreases from the central core of
to its peripheral regions. Using publicly available
high-throughput chromosome conformation capture data, we identify a 25-kb region centering on the CENPA-bound core that acts as CEN-flanking compact chromatin (CFCC).
- and
-chromosomal interactions associated with the CFCC spatially segregates it from bulk chromatin. We further show that neocentromere activation on chromosome 7 occurs within this specified region. Hence, this study identifies a specialized CEN-proximal domain that specifies and restricts the centromeric activity to a unique region.
Fungi colonizing plants are gaining attention because of their ability to promote plant growth and suppress pathogens. While most studies focus on endosymbionts from grasses and legumes, the large ...and diverse group of ericaceous plants has been much neglected. We recently described one of the very few fungal endophytes promoting the growth of the Ericaceae
(American cranberry), notably the
isolate EC4. Here, we show that EC4 also suppresses fungal pathogens, which makes it a promising endophyte for sustainable cranberry cultivation. By dual-culture assays on agar plates, we tested the potential growth suppression (or biocontrol) of EC4 on other microbes, notably 12 pathogenic fungi and one oomycete reported to infect not only cranberry but also blueberry, strawberry, tomato plants, rose bushes and olive trees. Under greenhouse conditions, EC4 protects cranberry plantlets infected with one of the most notorious cranberry-plant pathogens,
, known to cause upright dieback and berry rot. The nuclear genome sequence of EC4 revealed a large arsenal of genes potentially involved in biocontrol. About ∼60 distinct clusters of genes are homologs of secondary metabolite gene clusters, some of which were shown in other fungi to synthesize nonribosomal peptides and polyketides, but in most cases, the exact compounds these clusters may produce are unknown. The EC4 genome also encodes numerous homologs of hydrolytic enzymes known to degrade fungal cell walls. About half of the nearly 250 distinct glucanases and chitinases are likely involved in biocontrol because they are predicted to be secreted outside the cell. Transcriptome analysis shows that the expression of about a quarter of the predicted secondary-metabolite gene clusters and glucan and chitin-degrading genes of EC4 is stimulated when it is co-cultured with
. Some of the differentially expressed EC4 genes are alternatively spliced exclusively in the presence of the pathogen, altering the proteins' domain content and subcellular localization signal, thus adding a second level of proteome adaptation in response to habitat competition. To our knowledge, this is the first report of
-induced alternative splicing of biocontrol genes.
Genomic rearrangements associated with speciation often result in variation in chromosome number among closely related species.
species show variable karyotypes ranging between six and nine ...chromosomes. Here, we experimentally identified all eight centromeres in
as 3-5-kb long kinetochore-bound regions that span an AT-rich core and are depleted of the canonical histone H3. Centromeres of similar sequence features were identified as CENP-A-rich regions in
, which has seven chromosomes, and histone H3 depleted regions in
and
with nine chromosomes each. Analysis of synteny conservation across centromeres with newly generated chromosome-level genome assemblies suggests two distinct mechanisms of chromosome number reduction from an inferred nine-chromosome ancestral state: (a) chromosome breakage followed by loss of centromere DNA and (b) centromere inactivation accompanied by changes in DNA sequence following chromosome-chromosome fusion. We propose that AT-rich centromeres drive karyotype diversity in the
species complex through breakage and inactivation.
Centromeres of
form on unique and different DNA sequences but a closely related species,
, possesses homogenized inverted repeat (HIR)-associated centromeres. To investigate the mechanism of ...centromere type transition, we improved the fragmented genome assembly and constructed a chromosome-level genome assembly of
by employing PacBio sequencing, chromosome conformation capture sequencing (3C-seq), chromoblot, and genetic analysis of engineered aneuploid strains. Further, we analyzed the 3D genome organization using 3C-seq data, which revealed spatial proximity among the centromeres as well as telomeres of seven chromosomes in
. Intriguingly, we observed evidence of inter-centromeric translocations in the common ancestor of
and
. Identification of putative centromeres in closely related
,
and
indicates loss of ancestral HIR-associated centromeres and establishment of evolutionary new centromeres (ENCs) in
. We propose that spatial proximity of the homologous centromere DNA sequences facilitated karyotype rearrangements and centromere type transitions in human pathogenic yeasts of the CUG-Ser1 clade.
The establishment of centromeric chromatin and its propagation by the centromere-specific histone CENPA is mediated by epigenetic mechanisms in most eukaryotes. DNA replication origins, origin ...binding proteins, and replication timing of centromere DNA are important determinants of centromere function. The epigenetically regulated regional centromeres in the budding yeast
have unique DNA sequences that replicate earliest in every chromosome and are clustered throughout the cell cycle. In this study, the genome-wide occupancy of the replication initiation protein Orc4 reveals its abundance at all centromeres in
Orc4 is associated with four different DNA sequence motifs, one of which coincides with tRNA genes (tDNA) that replicate early and cluster together in space. Hi-C combined with genome-wide replication timing analyses identify that early replicating Orc4-bound regions interact with themselves stronger than with late replicating Orc4-bound regions. We simulate a polymer model of chromosomes of
and propose that the early replicating and highly enriched Orc4-bound sites preferentially localize around the clustered kinetochores. We also observe that Orc4 is constitutively localized to centromeres, and both Orc4 and the helicase Mcm2 are essential for cell viability and CENPA stability in
Finally, we show that new molecules of CENPA are recruited to centromeres during late anaphase/telophase, which coincides with the stage at which the CENPA-specific chaperone Scm3 localizes to the kinetochore. We propose that the spatiotemporal localization of Orc4 within the nucleus, in collaboration with Mcm2 and Scm3, maintains centromeric chromatin stability and CENPA recruitment in
.
Ericaceae thrive in poor soil, which we postulate is facilitated by microbes living inside those plants. Here, we investigate the growth stimulation of the American cranberry (
) by one of its fungal ...endosymbionts, EC4. We show that the symbiont resides inside the epidermal root cells of the host but extends into the rhizosphere via its hyphae. Morphological classification of this fungus is ambiguous, but phylogenetic inference based on 28S rRNA identifies EC4 as a
species (Chaetosphaeriaceae, Sordariomycetes, Ascomycetes). We sequenced the genome and transcriptome of EC4, providing the first 'Omics' information of a Chaetosphaeriaceae fungus. The 55.3-Mbp nuclear genome contains 17,582 potential protein-coding genes, of which nearly 500 have the capacity to promote plant growth. For comparing gene sets involved in biofertilization, we annotated the published genome assembly of the plant-growth-promoting
. The number of proteins involved in phosphate transport and solubilization is similar in the two fungi. In contrast, EC4 has ~50% more genes associated with ammonium, nitrate/nitrite transport, and phytohormone synthesis. The expression of 36 presumed plant-growth-promoting EC4 genes is stimulated when the fungus is in contact with the plant. Thus, Omics and
tests make EC4 a promising candidate for cranberry biofertilization on nutrient-poor soils.
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