A large transposable element mediates metal resistance in the fungus Paecilomyces variotiiHighlights d A gene cluster in the fungus Paecilomyces variotii confers metal resistance d The gene cluster displays hallmarks of coordinated movement as a mobile element d The cluster is variably located in different genomic locations in different strains d HEPHAESTUS is an example of transposons carrying beneficial traits in eukaryotes Authors

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Blackleg is a worldwide disease of canola (Brassica napus), caused by a complex of fungal species in the genus Leptosphaeria, that impacts canola production and seed quality. Demethylation inhibitor (DMI) fungicides that target sterol 14α-demethylase are an integral part of disease control. Here, we report six DMI-resistant isolates of Leptosphaeria maculans and two different types of genetic modification related to the resistance. Analysis of the regulatory region of the DMI target gene ERG11 (also known as CYP51) revealed a 275-bp insertion in two of the isolates and three long terminal repeat retrotransposons (5,263, 5,267, and 5,248 bp) inserted in the promoter region of three resistant isolates. Genetic approaches confirmed that these elements are responsible for DMI resistance in L. maculans and crosses show segregation consistent with a single locus. Reverse-transcription quantitative PCR assays demonstrated that the 275-bp insertion increases ERG11 gene expression, conferring DMI fungicide resistance both in vitro and in planta. Moreover, transformation of a susceptible isolate of L. maculans with ERG11 driven by a promoter containing the 275-bp insertion increased resistance to tebuconazole. A minimal shift of the values of concentration whereby 50% of the mycelial growth is inhibited in vitro was observed in resistant isolates containing long terminal repeat retrotransposons; nevertheless, these isolates were able to develop significant lesions on cotyledons from fungicide-treated seedlings. This is the first report of genetic modifications in L. maculans relating to DMI fungicide resistance.

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Leptin is a pro-inflammatory cytokine secreted by the adipose tissue. Dopamine D 2 receptors (D 2 R) have anti-inflammatory effects in the brain and kidney tissues. Mouse and human adipocytes express the (D 2 R); D 2 R protein was 10-fold greater in adipocytes from human visceral than subcutaneous tissue. However, the function of D 2 R in adipocytes is not well understood. The D 2-like receptor agonist Quinpirole increased the protein expression of leptin and IL-6, not adiponectin and Visfatin, along with their mRNA expression (24 hr). It also increased the mRNA expression of TNF-alpha, MCP1 and NFkB-p50 (24 hr). An acute increase in the protein expression of leptin (15 min, 30min, 2 hr) and TNF-alpha (30 min) was also found in the cells treated with quinpirole. The leptin concentration in the culture media was increased by quinpirole bathing the 3T3-L1 adipocytes. These effects were prevented by the D 2 R antagonist L741,626. Similarly, siRNA-mediated silencing of Drd2 decreased the leptin and IL-6 and mRNA and protein expressions. The D 2 R-mediated increase in leptin expression was prevented by the PI 3 K inhibitor LY294002. The quinpirole treatment in C57Bl/6J mice increased serum leptin concentration and leptin mRNA in visceral adipocyte tissue but not in subcutaneous adippocytes confirming the stimulatory effect of D 2 R on leptin in vivo. Our results suggest that the stimulation of D 2 R increases leptin production and may have tissue specific pro-inflammatory effect in adipocytes.

29The horizontal transfer of large gene clusters by mobile elements is a key driver of prokaryotic 30 adaptation in response to environmental stresses. Eukaryotic microbes face similar 31 environmental stresses yet a parallel role for mobile elements has not yet been established. A 32 stress faced by all microorganisms is the prevalence of toxic metals in their environment. In 33 fungi, identified mechanisms for protection against metals generally rely on genes that are 34 dispersed within an organism's genome. Here we have discovered a large (~85 kb) region that 35 confers resistance to several metals in the genomes of some, but not all, strains of a fungus, 36Paecilomyces variotii. We name this region HEPHAESTUS (Hϕ) and present evidence that this 37 region is mobile within the P. variotii genome with features highly characteristic of a 38 transposable element. While large gene clusters including those for the synthesis of secondary 39 metabolites have been widely reported in fungi, these are not mobile within fungal genomes. 40 HEPHAESTUS contains the greatest complement of host-beneficial genes carried by a 41 transposable element in eukaryotes. This suggests that eukaryotic transposable elements might 42 play a role analogous to their bacterial counterparts in the horizontal transfer of large regions of 43 host-beneficial DNA. Genes within HEPHAESTUS responsible for individual metal resistances 44 include those encoding a P-type ATPase transporter, PcaA, required for cadmium and lead 45 resistance, a transporter, ZrcA, providing resistance to zinc, and a multicopper oxidase, McoA, 46 conferring resistance to copper. Additionally, a subregion of Hϕ conferring resistance to 47 arsenate was identified. The presence of a strikingly similar cluster in the genome of another 48 fungus, Penicillium fuscoglaucum, suggests that HEPHAESTUS arrived in P. variotii via horizontal 49 gene transfer.50 51 KEYWORDS 52 53 Eurotiales, gene cluster, metal homeostasis, horizontal gene transfer, transposon 54 3 INTRODUCTION 55 56Metals are used within diverse fields such as agriculture, construction, electronics and 57 pharmaceutical science. A number of these, such as cadmium, lead and arsenic, are toxic even 58 at low levels of exposure and thus environmental contamination resulting from their use is an 59 increasing concern 1 . The prevalence of toxic metals in the environment is of particular 60 relevance to fungal biology. Some fungi are able to accumulate metals to high concentrations, 61 making such fungi dangerous for human consumption 2,3 , allowing bioremediation of 62 contaminated sites 4 or concentrating valuable metals for extraction 5 . Zinc and copper play 63 essential roles in many cellular functions and are often important in fungal virulence 6 . However, 64 at high concentrations these metals can also be toxic; for example, copper-based molecules are 65 frequently used as fungicides in agriculture. 66 67 Compared to prokaryotes, our understanding of the evolutionary mechanisms through which 68 eukaryotes have ada...

Background The daily cycling of plant physiological processes is speculated to arise from the coordinated rhythms of gene expression. However, the dynamics of diurnal 3D genome architecture and their potential functions underlying the rhythmic gene expression remain unclear. Results Here, we reveal the genome-wide rhythmic occupancy of RNA polymerase II (RNAPII), which precedes mRNA accumulation by approximately 2 h. Rhythmic RNAPII binding dynamically correlates with RNAPII-mediated chromatin architecture remodeling at the genomic level of chromatin interactions, spatial clusters, and chromatin connectivity maps, which are associated with the circadian rhythm of gene expression. Rhythmically expressed genes within the same peak phases of expression are preferentially tethered by RNAPII for coordinated transcription. RNAPII-associated chromatin spatial clusters (CSCs) show high plasticity during the circadian cycle, and rhythmically expressed genes in the morning phase and non-rhythmically expressed genes in the evening phase tend to be enriched in RNAPII-associated CSCs to orchestrate expression. Core circadian clock genes are associated with RNAPII-mediated highly connected chromatin connectivity networks in the morning in contrast to the scattered, sporadic spatial chromatin connectivity in the evening; this indicates that they are transcribed within physical proximity to each other during the AM circadian window and are located in discrete “transcriptional factory” foci in the evening, linking chromatin architecture to coordinated transcription outputs. Conclusion Our findings uncover fundamental diurnal genome folding principles in plants and reveal a distinct higher-order chromosome organization that is crucial for coordinating diurnal dynamics of transcriptional regulation.

lipolysis is closely associated with obesity and insulin resistance. Berberine (BBr), a natural alkaloid derived from Coptis chinensis, has been shown to regulate lipolysis and improve insulin resistance. However, the underlying mechanism remains unclear. The present results suggested that BBr stimulated lipolysis in porcine adipocytes in a dose-and time-dependent manner, which was independent of the caMP/protein kinase a pathway. Further experimental results indicated that BBr increased phosphorylation levels of aMP-activated protein kinase (aMPK) and adipose triglyceride lipase (aTGl), along with downregulation of Perilipin a. The aMPK inhibitor compound c significantly reversed the effect of BBr on lipolysis, Perilipin a expression and aTGl phosphorylation. Furthermore, BBr promoted expression levels of genes related to fatty acid oxidation, such as peroxisome proliferator-activated receptor γ coactivator-1α, mitochondrial transcription factor a, carnitine palmitoyl-transferase-1 and uncoupling protein 2, which were abrogated by aMPKα1 knockdown. Moreover, it was found that BBr-induced lipolysis did not elevate serine phosphorylation of insulin receptor substrate-1 to block insulin signaling. collectively, the present results suggested that BBr induced lipolysis in porcine adipocytes via a pathway that involves aMPK activation, but does not cause insulin resistance.