SummaryCellular life emerged ~3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles due to the Earth’s rotation. The advantage conferred upon organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation-reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterising their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription-translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular time-keeping with redox homeostatic mechanisms following the Great Oxidation Event ~2.5 billion years ago.

Circadian rhythms are ubiquitous in eukaryotes, and co-ordinate numerous aspects of behaviour, physiology and metabolism, from sleep/wake cycles in mammals to growth and photosynthesis in plants1,2. This daily timekeeping is thought to be driven by transcriptional/translational feedback loops, whereby rhythmic expression of clock gene products regulates expression of associated genes in approximately 24-hour cycles. The specific transcriptional components differ between phylogenetic kingdoms3. The unicellular pico-eukaryotic alga, Ostreococcus tauri, possesses a naturally minimised clock, which includes many features that are shared with higher eukaryotes (plants), such as a central negative feedback loop that involves the morning-expressed CCA1 and evening-expressed TOC1 genes4. Given that recent observations in animals and plants have revealed prominent post-translational contributions to timekeeping5, a reappraisal of the transcriptional contribution to oscillator function is overdue. Here we show that non-transcriptional mechanisms are sufficient to sustain circadian timekeeping in the eukaryotic lineage, though they normally function in conjunction with transcriptional components. We identify oxidation of peroxiredoxin proteins as a transcription-independent rhythmic biomarker, which is also rhythmic in mammals6. Moreover we show that pharmacological modulators of the mammalian clockwork have the same effects on rhythms in Ostreococcus. Post-translational mechanisms, and at least one rhythmic marker, appear to be better conserved than transcriptional clock regulators. It is plausible that the oldest oscillator components are non-transcriptional in nature, as in cyanobacteria7, and are conserved across kingdoms.

SUMMARYTo promote host colonization, many plant pathogens secrete effector proteins that either suppress or counteract host defences. However, when these effectors are recognized by the host's innate immune system, they trigger resistance rather than promoting virulence. Effectors are therefore key molecules in determining disease susceptibility or resistance. We show here that Avr2, secreted by the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol), shows both activities: it is required for full virulence in a susceptible host and also triggers resistance in tomato plants carrying the resistance gene I-2. Point mutations in AVR2, causing single amino acid changes, are associated with I-2-breaking Fol strains. These point mutations prevent recognition by I-2, both in tomato and when both genes are co-expressed in leaves of Nicotiana benthamiana. Fol strains carrying the Avr2 variants are equally virulent, showing that virulence and avirulence functions can be uncoupled. Although Avr2 is secreted into the xylem sap when Fol colonizes tomato, the Avr2 protein can be recognized intracellularly by I-2, implying uptake by host cells.

Resistance (R) proteins in plants are involved in pathogen recognition and subsequent activation of innate immune responses. Most resistance proteins contain a central nucleotide-binding domain. This so-called NB-ARC domain consists of three subdomains: NB, ARC1, and ARC2. The NB-ARC domain is a functional ATPase domain, and its nucleotide-binding state is proposed to regulate activity of the R protein. A highly conserved methionine-histidine-aspartate (MHD) motif is present at the carboxy-terminus of ARC2. An extensive mutational analysis of the MHD motif in the R proteins I-2 and Mi-1 is reported. Several novel autoactivating mutations of the MHD invariant histidine and conserved aspartate were identified. The combination of MHD mutants with autoactivating hydrolysis mutants in the NB subdomain showed that the autoactivation phenotypes are not additive. This finding indicates an important regulatory role for the MHD motif in the control of R protein activity. To explain these observations, a three-dimensional model of the NB-ARC domain of I-2 was built, based on the APAF-1 template structure. The model was used to identify residues important for I-2 function. Substitution of the selected residues resulted in the expected distinct phenotypes. Based on the model, it is proposed that the MHD motif fulfils the same function as the sensor II motif found in AAA+ proteins (ATPases associated with diverse cellular activities)-co-ordination of the nucleotide and control of subdomain interactions. The presented 3D model provides a framework for the formulation of hypotheses on how mutations in the NB-ARC exert their effects.

Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behavior and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes1-3. A fundamental knowledge gap exists however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology4,5. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg 2+ ] i , which act as a cell-autonomous timekeeping component to determine key clock properties in both a human cell line and a unicellular alga that diverged from metazoans more than 1 billion years ago6. Given the essential role of Mg 2+ as a cofactor for ATP, a functional consequence of [Mg 2+ ] i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg 2+ availability has potential to impact upon many of the cell's >600 MgATP-dependent enzymes7 and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR8 is regulated through [Mg 2+ ] i oscillations. It will now be important to identify which additional biological # To whom correspondence should be sent: Gerben.vanOoijen@ed.ac.uk, oneillj@mrc-lmb.cam.ac.uk. Author contributions GvO and JSO conceived the approach and designed the study. LFL and COY generated the Neurospora result. JD and LE performed ICP analyses. GvO and LLH performed Ostreococcus experiments. Human U2OS cell experiments were performed by KAF. MP performed mouse fibroblast experiments. NPH provided analytical and intellectual contributions. GvO and JSO wrote the manuscript.

SummaryTomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-4/Avr4-induced HR in Nicotiana benthamiana as demonstrated by virus-induced gene silencing (VIGS). The gene encodes a CC-NB-LRR type resistance (R) protein analogue that we have designated NRC1 (NB-LRR protein required for HR-associated cell death 1). Here we describe that knock-down of NRC1 in tomato not only affects the Cf-4/Avr4-induced HR but also compromises Cf-4-mediated resistance to C. fulvum. In addition, VIGS using NRC1 in N. benthamiana revealed that this protein is also required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx and Mi. Transient expression of NRC1 D481V , which encodes a constitutively active NRC1 mutant protein, triggers an elicitor-independent HR. Subsequently, we transiently expressed this auto-activating protein in N. benthamiana silenced for genes known to be involved in HR signalling, thereby allowing NRC1 to be positioned in an HR signalling pathway. We found that NRC1 requires RAR1 and SGT1 to be functional, whereas it does not require NDR1 and EDS1. As the Cf-4 protein requires EDS1 for its function, we hypothesize that NRC1 functions downstream of EDS1. We also found that NRC1 acts upstream of a MAP kinase pathway. We conclude that Cf-mediated resistance signalling requires a downstream NB-LRR protein that also functions in cell death signalling pathways triggered by other R proteins.

Gene-for-gene resistance in plants is based on the presence of a resistance (R) gene in the host and a matching Avirulence (Avr) gene in the pathogen. Many R genes have been cloned over the past two decades, mostly from the Solanaceae. The gene products, called R proteins, display modular domain structures. R protein function has recently been shown to require dynamic interactions between the various domains. In addition to these intramolecular interactions, R proteins interact with other proteins to form signaling complexes that are able to activate an innate immune response that arrests proliferation of the invading pathogen, thereby conferring disease resistance. In this review, we summarize current understanding of R protein structure and function, as well as the molecular mechanisms underlying the activation of defense signaling processes. As well as being a rich source for R genes, Solanaceae are a leading model system in which to study inter- and intramolecular interactions of R proteins.

Circadian clocks are biological timekeepers that allow living cells to time their activity in anticipation of predictable environmental changes. Detailed understanding of the circadian network of higher plants, such as Arabidopsis thaliana, is hampered by the high number of partially redundant genes. However, the picoeukaryotic alga Ostreococcus tauri, which was recently shown to possess a small number of non-redundant clock genes, presents an attractive alternative target for detailed modelling of circadian clocks in the green lineage. Based on extensive time-series data from in vivo reporter gene assays, we developed a model of the Ostreococcus clock as a feedback loop between the genes TOC1 and CCA1. The model reproduces the dynamics of the transcriptional and translational reporters over a range of photoperiods. Surprisingly, the model is also able to predict the transient behaviour of the clock when the light conditions are altered. Despite the apparent simplicity of the clock circuit, it displays considerable complexity in its response to changing light conditions. Systematic screening of the effects of altered day length revealed a complex relationship between phase and photoperiod, which is also captured by the model. The complex light response is shown to stem from circadian gating of light-dependent mechanisms. This study provides insights into the contributions of light inputs to the Ostreococcus clock. The model suggests that a high number of light-dependent reactions are important for flexible timing in a circadian clock with only one feedback loop.