Methylxanthines Modulate Circadian Period Length Independently of the Action of Phosphodiesterase.

In Neurospora crassa, caffeine and other methylxanthines are known to inhibit phosphodiesterase (PDE) activity, leading to augmented cAMP levels. In this organism, it has also been shown that the addition of these drugs significantly lengthens the circadian period, as seen by conidiation rhythms. Utilizing in vivo bioluminescence reporters, pharmacological inhibitors, and cAMP analogs, we revisited the effect of methylxanthines and the role of cAMP signaling in the Neurospora clockworks. We observed that caffeine, like all tested methylxanthines, led to significant period lengthening, visualized with both core-clock transcriptional and translational reporters. Remarkably, this phenotype is still observed when phosphodiesterase (PDE) activity is genetically or chemically (via 3-isobutyl-1-methylxanthine) abrogated. Likewise, methylxanthines still exert a period effect in several cAMP signaling pathway mutants, including adenylate cyclase (cr-1) and protein kinase A (PKA) (Δpkac-1) mutants, suggesting that these drugs lead to circadian phenotypes through mechanisms different from the canonical PDE-cAMP-PKA signaling axis. Thus, this study highlights the strong impact of methylxanthines on circadian period in Neurospora, albeit the exact mechanisms somehow remain elusive. IMPORTANCE Evidence from diverse organisms show that caffeine causes changes in the circadian clock, causing period lengthening. The fungus Neurospora crassa is no exception; here, several methylxanthines such as caffeine, theophylline, and aminophylline cause period lengthening in a concentration-dependent manner. Although methylxanthines are expected to inhibit phosphodiesterase activity, we were able to show by genetic and pharmacological means that these drugs exert their effects through a different mechanism. Moreover, our results indicate that increases in cAMP levels and changes in PKA activity do not impact the circadian period and therefore are not part of underlying effects of methylxanthine. These results set the stage for future analyses dissecting the molecular mechanisms by which these drugs dramatically modify the circadian period.

Defects in the Ferroxidase That Participates in the Reductive Iron Assimilation System Results in Hypervirulence in Botrytis Cinerea.

The plant pathogen Botrytis cinerea is responsible for gray-mold disease, which infects a wide variety of species. The outcome of this host-pathogen interaction, a result of the interplay between plant defense and fungal virulence pathways, can be modulated by various environmental factors. Among these, iron availability and acquisition play a crucial role in diverse biological functions. How B. cinerea obtains iron, an essential micronutrient, during infection is unknown. We set out to determine the role of the reductive iron assimilation (RIA) system during B. cinerea infection. This system comprises the BcFET1 ferroxidase, which belongs to the multicopper oxidase (MCO) family of proteins, and the BcFTR1 membrane-bound iron permease. Gene knockout and complementation studies revealed that, compared to the wild type, the bcfet1 mutant displays delayed conidiation, iron-dependent sclerotium production, and significantly reduced whole-cell iron content. Remarkably, this mutant exhibited a hypervirulence phenotype, whereas the bcftr1 mutant presents normal virulence and unaffected whole-cell iron levels and developmental programs. Interestingly, while in iron-starved plants wild-type B. cinerea produced slightly reduced necrotic lesions, the hypervirulence phenotype of the bcfet1 mutant is no longer observed in iron-deprived plants. This suggests that B. cinerea bcfet1 knockout mutants require plant-derived iron to achieve larger necrotic lesions, whereas in planta analyses of reactive oxygen species (ROS) revealed increased ROS levels only for infections caused by the bcfet1 mutant. These results suggest that increased ROS production, under an iron sufficiency environment, at least partly underlie the observed infection phenotype in this mutant.IMPORTANCE The plant-pathogenic fungus B. cinerea causes enormous economic losses, estimated at anywhere between $10 billion and $100 billion worldwide, under both pre- and postharvest conditions. Here, we present the characterization of a loss-of-function mutant in a component involved in iron acquisition that displays hypervirulence. While in different microbial systems iron uptake mechanisms appear to be critical to achieve full pathogenic potential, we found that the absence of the ferroxidase that is part of the reductive iron assimilation system leads to hypervirulence in this fungus. This is an unusual and rather underrepresented phenotype, which can be modulated by iron levels in the plant and provides an unexpected link between iron acquisition, reactive oxygen species (ROS) production, and pathogenesis in the Botrytis-plant interaction.

Daily magnesium fluxes regulate cellular timekeeping and energy balance.

Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behaviour and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes. A fundamental knowledge gap exists, however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology. 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 both in a human cell line and in a unicellular alga that diverged from each other more than 1 billion years ago. 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 more than 600 MgATP-dependent enzymes and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR is regulated through [Mg(2+)]i oscillations. It will now be important to identify which additional biological processes are subject to this form of regulation in tissues of multicellular organisms such as plants and humans, in the context of health and disease.