A Mechanically Transmitted DNA Mycovirus Is Targeted by the Defence Machinery of Its Host, Botrytis cinerea

Eukaryotic circular single-stranded DNA (ssDNA) viruses were known only to infect plants and vertebrates until the discovery of the isolated DNA mycovirus from the fungus Sclerotinia sclerotiorum. Similar viral sequences were reported from several other sources and classified in ten genera within the Genomoviridae family. The current study reports two circular ssDNA mycoviruses isolated from the phytopathogen Botrytis cinerea, and their assignment to a newly created genus tentatively named Gemydayirivirus. The mycoviruses, tentatively named botrytis gemydayirivirus 1 (BGDaV1) and BGDaV2, are 1701 and 1693 nt long and encode three and two open reading frames (ORFs), respectively. Of the predicted ORFs, only ORF I, which codes for a replication initiation protein (Rep), shared identity with other proteins in GenBank. BGDaV1 is infective as cell-free purified particles and confers hypovirulence on its natural host. Investigation revealed that BGDaV1 is a target for RNA silencing and genomic DNA methylation, keeping the virus at very low titre. The discovery of BGDaV1 expands our knowledge of the diversity of genomoviruses and their interaction with fungal hosts.

Orc4 spatiotemporally stabilizes centromeric chromatin

Spatiotemporal regulation in DNA replication maintains kinetochore stability. The epigenetically regulated centromeres (CENs) in the budding yeast Candida albicans have unique DNA sequences, replicate early and are clustered throughout the cell cycle. In this study, the genome-wide occupancy of replication initiation protein Orc4 reveals its abundance at all CENs in C. albicans. Orc4 associates with four different DNA motifs, one of which coincides with tRNA genes. Hi-C combined with genome-wide replication timing analyses identify enriched interactions among early or late replicating Orc4-bound regions. A simulated polymer model of chromosomes reveals that early replicating and strongly enriched Orc4-bound sites localize towards the kinetochores. Orc4 is constitutively localized to CENs, and both Orc4 and Mcm2 stabilize CENPA. CENPA chaperone Scm3 localizes at the kinetochore during anaphase, coinciding with the loading time of CENPA. We propose that this spatiotemporal nuclear localization of Orc4, with Mcm2 and Scm3, recruits CENPA and stabilizes centromeric chromatin.