BackgroundMolecular clock methodologies allow for the estimation of divergence times across a variety of organisms; this can be particularly useful for groups lacking robust fossil histories, such as microbial eukaryotes with few distinguishing morphological traits. Here we have used a Bayesian molecular clock method under three distinct clock models to estimate divergence times within oomycetes, a group of fungal-like eukaryotes that are ubiquitous in the environment and include a number of devastating pathogenic species. The earliest fossil evidence for oomycetes comes from the Lower Devonian (~400 Ma), however the taxonomic affinities of these fossils are unclear.ResultsComplete genome sequences were used to identify orthologous proteins among oomycetes, diatoms, and a brown alga, with a focus on conserved regulators of gene expression such as DNA and histone modifiers and transcription factors. Our molecular clock estimates place the origin of oomycetes by at least the mid-Paleozoic (~430-400 Ma), with the divergence between two major lineages, the peronosporaleans and saprolegnialeans, in the early Mesozoic (~225-190 Ma). Divergence times estimated under the three clock models were similar, although only the strict and random local clock models produced reliable estimates for most parameters.ConclusionsOur molecular timescale suggests that modern pathogenic oomycetes diverged well after the origin of their respective hosts, indicating that environmental conditions or perhaps horizontal gene transfer events, rather than host availability, may have driven lineage diversification. Our findings also suggest that the last common ancestor of oomycetes possessed a full complement of eukaryotic regulatory proteins, including those involved in histone modification, RNA interference, and tRNA and rRNA methylation; interestingly no match to canonical DNA methyltransferases could be identified in the oomycete genomes studied here.

Cullin-RING E3 ligases (CRLs) regulate different aspects of plant development and are activated by modification of their cullin subunit with the ubiquitin-like protein NEDD8 (NEural precursor cell expressed Developmentally Down-regulated 8) (neddylation) and deactivated by NEDD8 removal (deneddylation). The constitutively photomorphogenic9 (COP9) signalosome (CSN) acts as a molecular switch of CRLs activity by reverting their neddylation status, but its contribution to embryonic and early seedling development remains poorly characterized. Here, we analyzed the phenotypic defects of csn mutants and monitored the cullin deneddylation/neddylation ratio during embryonic and early seedling development. We show that while csn mutants can complete embryogenesis (albeit at a slower pace than wild-type) and are able to germinate (albeit at a reduced rate), they progressively lose meristem activity upon germination until they become unable to sustain growth. We also show that the majority of cullin proteins are progressively neddylated during the late stages of seed maturation and become deneddylated upon seed germination. This developmentally regulated shift in the cullin neddylation status is absent in csn mutants. We conclude that the CSN and its cullin deneddylation activity are required to sustain postembryonic meristem function in Arabidopsis.

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