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1. The clock in growing hyphae and their synchronization in Neurospora crassa

   Cheong, Jia H; Qiu, Xiao; Liu, Yang; Krach, Emily; Guo, Yinping; Bhusal, Shishir; Schuttler, Heinz-Bernd; Arnold, J; Mao, L (June 2024, Communications biology)

   Riquelme, Meritxell; Akhtar, Andam; Rosenthal, Christina (Ed.)

   Utilizing a microfluidic chip with serpentine channels, we inoculated the chip with an agar plug with Neurospora crassa mycelium and successfully captured individual hyphae in channels. For the first time, we report the presence of an autonomous clock in hyphae. Fluorescence of a mCherry reporter gene driven by a clock-controlled gene-2 promoter (ccg-2p) was measured simultaneously along hyphae every half an hour for at least 6 days. We entrained single hyphae to light over a wide range of day lengths, including 6,12, 24, and 36 h days. Hyphae tracked in individual serpentine channels were highly synchronized (K = 0.60-0.78). Furthermore, hyphae also displayed temperature compensation properties, where the oscillation period was stable over a physiological range of temperatures from 24 °C to 30 °C (Q10 = 1.00-1.10). A Clock Tube Model developed could mimic hyphal growth observed in the serpentine chip and provides a mechanism for the stable banding patterns seen in race tubes at the macroscopic scale and synchronization through molecules riding the growth wave in the device. 

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2. Common origin of sterol biosynthesis points to a feeding strategy shift in Neoproterozoic animals

   https://doi.org/10.1038/s41467-023-43545-z  

   Brunoir, T.; Mulligan, C.; Sistiaga, A.; Vuu, K. M.; Shih, P. M.; O’Reilly, S. S.; Summons, R. E.; Gold, D. A. (December 2023, Nature Communications)

   Abstract Steranes preserved in sedimentary rocks serve as molecular fossils, which are thought to record the expansion of eukaryote life through the Neoproterozoic Era ( ~ 1000-541 Ma). Scientists hypothesize that ancient C₂₇steranes originated from cholesterol, the major sterol produced by living red algae and animals. Similarly, C₂₈and C₂₉steranes are thought to be derived from the sterols of prehistoric fungi, green algae, and other microbial eukaryotes. However, recent work on annelid worms–an advanced group of eumetazoan animals–shows that they are also capable of producing C₂₈and C₂₉sterols. In this paper, we explore the evolutionary history of the24-C sterol methyltransferase(smt) gene in animals, which is required to make C₂₈₊sterols. We find evidence that thesmtgene was vertically inherited through animals, suggesting early eumetazoans were capable of C₂₈₊sterol synthesis. Our molecular clock of the animalsmtgene demonstrates that its diversification coincides with the rise of C₂₈and C₂₉steranes in the Neoproterozoic. This study supports the hypothesis that early eumetazoans were capable of making C₂₈₊sterols and that many animal lineages independently abandoned its biosynthesis around the end-Neoproterozoic, coinciding with the rise of abundant eukaryotic prey. 

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3. Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, β- and γ- TLO gene function

   https://doi.org/10.1371/journal.pgen.1011082  

   Fletcher, Jessica; O’Connor-Moneley, James; Frawley, Dean; Flanagan, Peter R.; Alaalm, Leenah; Menendez-Manjon, Pilar; Estevez, Samuel Vega; Hendricks, Shane; Woodruff, Andrew L.; Buscaino, Alessia; et al (December 2023, PLOS Genetics)

   Forche, Anja (Ed.)

   TheCandida albicansgenome contains between ten and fifteen distinctTLOgenes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo inC.albicanswe deleted all fourteenTLOgenes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in thetloΔ mutant strain compared to the parent, while RNA-seq analysis showed that thetloΔ mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white-opaque switching and filamentous growth. Consequently, thetloΔ mutant grows poorly in glucose- and galactose-containing media, is unable to grow as true hyphae, is more sensitive to oxidative stress and is less virulent in the wax worm infection model. Reintegration of genes representative of the α-, β- and γ-TLOclades resulted in the complementation of the mutant phenotypes, but to different degrees.TLOα1could restore phenotypes and gene expression patterns similar to wild-type and was the strongest activator of glycolytic and Tye7-regulated gene expression. In contrast, the two γ-TLOgenes examined (i.e.,TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression ofTLOβ2in thetloΔmutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tloβ2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegratedTLOgenes in atloΔ/med3Δdouble mutant background failed to restore any of the phenotypes tested, suggesting that complementation of these Tlo-regulated processes requires a functional Mediator tail module. Together, these data confirm the importance of Med2/Tlo in a wide range ofC.albicanscellular activities and demonstrate functional diversity within the gene family which may contribute to the success of this yeast as a coloniser and pathogen of humans. 

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4. Membrane fluidity control by the Magnaporthe oryzae acyl-CoA binding protein sets the thermal range for host rice cell colonization

   https://doi.org/10.1371/journal.ppat.1012738  

   Richter, Michael; Segal, Lauren M; Rocha, Raquel O; Rokaya, Nisha; de_Queiroz, Aline R; Riekhof, Wayne R; Roston, Rebecca L; Wilson, Richard A (November 2024, PLOS Pathogens)

   Thomma, Bart PHJ (Ed.)

   Following leaf cuticle penetration by specialized appressorial cells, the devastating blast fungusMagnaporthe oryzaegrows as invasive hyphae (IH) in living rice cells. IH are separated from host cytoplasm by plant-derived membranes forming an apoplastic compartment and a punctate biotrophic interfacial complex (BIC) that mediate the molecular host-pathogen interaction. What molecular and cellular processes determine the temperature range for this biotrophic growth stage is an unanswered question pertinent to a broader understanding of how phytopathogens may cope with environmental stresses arising under climate change. Here, we shed light on thermal adaptation inM.oryzaeby disrupting theACB1gene encoding the single acyl-CoA-binding protein, an intracellular transporter of long-chain acyl-CoA esters. Loss ofACB1affected fatty acid desaturation levels and abolished pathogenicity at optimal (26°C) and low (22°C) but not elevated (29°C) infection temperatures (the latter following post-penetration shifts from 26°C). Relative to wild type, the Δacb1mutant strain exhibited poor vegetative growth and impaired membrane trafficking at 22°C and 26°C, but not at 29°C.In planta, Δacb1biotrophic growth was inhibited at 26°C–which was accompanied by a multi-BIC phenotype—but not at 29°C, where BIC formation was normal. Underpinning the Δacb1phenotype was impaired membrane fluidity at 22°C and 26°C but not at elevated temperatures, indicating Acb1 suppresses membrane rigidity at optimal- and suboptimal- but not supraoptimal temperatures. Deducing a temperature-dependent role for Acb1 in maintaining membrane fluidity homeostasis reveals how the thermal range for rice blast disease is both mechanistically determined and wider than hitherto appreciated. 

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5. Rhizosphere microbial community composition shifts diurnally and in response to natural variation in host clock phenotype

   https://doi.org/10.1128/msystems.01487-21  

   Hubbard, Charley J.; Harrison, Joshua G.; McMinn, Robby; Bennett Ponsford, Julian C.; Maignien, Lois; Ewers, Brent; Weinig, Cynthia (June 2023, mSystems)

   Heck, Michelle (Ed.)

   ABSTRACT Plant-associated microbial assemblages are known to shift at time scales aligned with plant phenology, as influenced by the changes in plant-derived nutrient concentrations and abiotic conditions observed over a growing season. But these same factors can change dramatically in a sub-24-hour period, and it is poorly understood how such diel cycling may influence plant-associated microbiomes. Plants respond to the change from day to night via mechanisms collectively referred to as the internal “clock,” and clock phenotypes are associated with shifts in rhizosphere exudates and other changes that we hypothesize could affect rhizosphere microbes. The mustardBoechera strictahas wild populations that contain multiple clock phenotypes of either a 21- or a 24-hour cycle. We grew plants of both phenotypes (two genotypes per phenotype) in incubators that simulated natural diel cycling or that maintained constant light and temperature. Under both cycling and constant conditions, the extracted DNA concentration and the composition of rhizosphere microbial assemblages differed between time points, with daytime DNA concentrations often triple what were observed at night and microbial community composition differing by, for instance, up to 17%. While we found that plants of different genotypes were associated with variation in rhizosphere assemblages, we did not see an effect on soil conditioned by a particular host plant circadian phenotype on subsequent generations of plants. Our results suggest that rhizosphere microbiomes are dynamic at sub-24-hour periods, and those dynamics are shaped by diel cycling in host plant phenotype. IMPORTANCEWe find that the rhizosphere microbiome shifts in composition and extractable DNA concentration in sub-24-hour periods as influenced by the plant host’s internal clock. These results suggest that host plant clock phenotypes could be an important determinant of variation in rhizosphere microbiomes. 

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