Abstract:
In this work, we identified and characterized centromeres and studied their structural organization in a group of Cryptococcus species of the fungal phylum Basidiomycota. Further, we examined the spatio-temporal dynamics of the centromere-kinetochore complex
in one of these species, Cryptococcus neoformans, an opportunistic pathogen that infects
immunocompromised as well as immunocompetent people worldwide.
Since centromeres have been shown to be evolving rapidly, we were interested in
studying centromere evolution in closely related Cryptococcus species. Thus, we sought to
identify centromeres in four species of the Cryptococcus species complex (Table 1.2).
Chromatin immunoprecipitation (ChIP), with conserved kinetochore proteins CENP-A and
CENP-C, followed by next generation sequencing (ChlP-seq) or qPCR was used to identify
and/or validate the centromeres. This analysis led to the identification of large regional
centromeres in all four Cryptococcus species. In all cases, each centromere resides in a long
ORF-free and transcriptionally silent region on the respective chromosome. Sequence
analysis of centromere DNA sequences revealed that they are enriched with retrotransposons.
Retrotransposons present in the pathogenic species of the Cryptococcus species complex
differ from those present in the non-pathogenic species hinting that centromeres are evolving
rapidly in the Cryptococcus species complex.
Among the pathogenic species in Cryptococcus species complex, one of the species
was known to be RNAi-deficient (R265) whereas the other two are RNAi-proficient (H99
and JEC21). We discovered a correlation between the presence of the RNAi machinery in the
genome and the length of the centromere of the respective species - the centromere length in
the RNAi-deficient species is significantly reduced as compared to the RNAi proficient
strains. Comparison of retrotransposons present in centromeres of the three pathogenic
species revealed that RNAi proficient species harbour a set of full-length retroelements; the
RNAi-deficient species, on the other hand, completely lacks them. Using an experimental
evolution based approach, we showed that in the RNAi mutants of H99, that usually
possesses long retrotransposon-rich centromeres, undergo structural alterations across the centromere regions. Using in silico analysis based on a specific set of properties of
centromeres, we also identified centromeres in three Ustilago species - another
basidiomycetous species complex which are major plant pathogens. Like Cryptococcus,
RNAi is lost in one of the three species in Ustilago species complex. Comparison of putative
centromere length in the three Ustilago species revealed that the RNAi-deficient species ((/.
maydis) has shorter centromeres than those of the RNAi-proficient species (U. hordei and U.
bromivora). Taken together, we could identify centromeres in six pathogenic basidiomycetes
belonging to two different species complexes. Both species complexes consist of one RNAideficient
species with shorter centromeres compared to those of the RNAi-proficient species.
Based on these observations, we conclude that RNAi components may be shaping up the
structure of the centromere in basidiomycetous yeast species.