PLOS Genetics: New ArticlesPLOShttps://journals.plos.org/plosgenetics/webmaster@plos.orghttps://journals.plos.org/plosgenetics/feed/atomAll PLOS articles are Open Access.https://journals.plos.org/plosgenetics/resource/img/favicon.icohttps://journals.plos.org/plosgenetics/resource/img/favicon.ico2024-03-28T17:44:43ZA natural bacterial pathogen of <i>C</i>. <i>elegans</i> uses a small RNA to induce transgenerational inheritance of learned avoidanceTitas SenguptaJonathan St. AngeRachel KaletskyRebecca S. MooreRenee J. SetoJacob MarogiCameron MyhrvoldZemer GitaiColeen T. Murphy10.1371/journal.pgen.10111782024-03-28T14:00:00Z2024-03-28T14:00:00Z<p>by Titas Sengupta, Jonathan St. Ange, Rachel Kaletsky, Rebecca S. Moore, Renee J. Seto, Jacob Marogi, Cameron Myhrvold, Zemer Gitai, Coleen T. Murphy</p>
<i>C</i>. <i>elegans</i> can learn to avoid pathogenic bacteria through several mechanisms, including bacterial small RNA-induced learned avoidance behavior, which can be inherited transgenerationally. Previously, we discovered that a small RNA from a clinical isolate of <i>Pseudomonas aeruginosa</i>, PA14, induces learned avoidance and transgenerational inheritance of that avoidance in <i>C</i>. <i>elegans</i>. <i>Pseudomonas aeruginosa</i> is an important human pathogen, and there are other <i>Pseudomonads</i> in <i>C</i>. <i>elegans’</i> natural habitat, but it is unclear whether <i>C</i>. <i>elegans</i> ever encounters PA14-like bacteria in the wild. Thus, it is not known if small RNAs from bacteria found in <i>C</i>. <i>elegans’</i> natural habitat can also regulate host behavior and produce heritable behavioral effects. Here we screened a set of wild habitat bacteria, and found that a pathogenic <i>Pseudomonas vranovensis</i> strain isolated from the <i>C</i>. <i>elegans</i> microbiota, GRb0427, regulates worm behavior: worms learn to avoid this pathogenic bacterium following exposure, and this learned avoidance is inherited for four generations. The learned response is entirely mediated by bacterially-produced small RNAs, which induce avoidance and transgenerational inheritance, providing further support that such mechanisms of learning and inheritance exist in the wild. We identified Pv1, a small RNA expressed in <i>P</i>. <i>vranovensis</i>, that has a 16-nucleotide match to an exon of the <i>C</i>. <i>elegans</i> gene <i>maco-1</i>. Pv1 is both necessary and sufficient to induce learned avoidance of Grb0427. However, Pv1 also results in avoidance of a beneficial microbiome strain, <i>P</i>. <i>mendocina</i>. Our findings suggest that bacterial small RNA-mediated regulation of host behavior and its transgenerational inheritance may be functional in <i>C</i>. <i>elegans’</i> natural environment, and that this potentially maladaptive response may favor reversal of the transgenerational memory after a few generations. Our data also suggest that different bacterial small RNA-mediated regulation systems evolved independently, but define shared molecular features of bacterial small RNAs that produce transgenerationally-inherited effects.Kombucha Tea-associated microbes remodel host metabolic pathways to suppress lipid accumulationRachel N. DuMez-KornegayLillian S. BakerAlexis J. MorrisWhitney L. M. DeLoachRobert H. Dowen10.1371/journal.pgen.10110032024-03-28T14:00:00Z2024-03-28T14:00:00Z<p>by Rachel N. DuMez-Kornegay, Lillian S. Baker, Alexis J. Morris, Whitney L. M. DeLoach, Robert H. Dowen</p>
The popularity of the ancient, probiotic-rich beverage Kombucha Tea (KT) has surged in part due to its purported health benefits, which include protection against metabolic diseases; however, these claims have not been rigorously tested and the mechanisms underlying host response to the probiotics in KT are unknown. Here, we establish a reproducible method to maintain <i>C</i>. <i>elegans</i> on a diet exclusively consisting of Kombucha Tea-associated microbes (KTM), which mirrors the microbial community found in the fermenting culture. KT microbes robustly colonize the gut of KTM-fed animals and confer normal development and fecundity. Intriguingly, animals consuming KTMs display a marked reduction in total lipid stores and lipid droplet size. We find that the reduced fat accumulation phenotype is not due to impaired nutrient absorption, but rather it is sustained by a programed metabolic response in the intestine of the host. KTM consumption triggers widespread transcriptional changes within core lipid metabolism pathways, including upregulation of a suite of lysosomal lipase genes that are induced during lipophagy. The elevated lysosomal lipase activity, coupled with a decrease in lipid droplet biogenesis, is partially required for the reduction in host lipid content. We propose that KTM consumption stimulates a fasting-like response in the <i>C</i>. <i>elegans</i> intestine by rewiring transcriptional programs to promote lipid utilization. Our results provide mechanistic insight into how the probiotics in Kombucha Tea reshape host metabolism and how this popular beverage may impact human metabolism.Correction: Tissue-specific and <i>cis</i>-regulatory changes underlie parallel, adaptive gene expression evolution in house miceThe PLOS Genetics Staff10.1371/journal.pgen.10112132024-03-27T14:00:00Z2024-03-27T14:00:00Z<p>by The PLOS Genetics Staff </p><i>Dmrt1</i> is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapiaShuangshuang QiShengfei DaiXin ZhouXueyan WeiPing ChenYuanyuan HeThomas D. KocherDeshou WangMinghui Li10.1371/journal.pgen.10112102024-03-27T14:00:00Z2024-03-27T14:00:00Z<p>by Shuangshuang Qi, Shengfei Dai, Xin Zhou, Xueyan Wei, Ping Chen, Yuanyuan He, Thomas D. Kocher, Deshou Wang, Minghui Li</p>
Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identified <i>amhy</i>, <i>dmrt1</i>, <i>gsdf</i> as male and <i>foxl2</i>, <i>foxl3</i>, <i>cyp19a1a</i> as female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads of <i>dmrt1</i>;<i>cyp19a1a</i> double mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads of <i>dmrt1</i>;<i>cyp19a1a</i>;<i>cyp19a1b</i> triple mutants still developed as ovaries. The gonads of <i>foxl3</i>;<i>cyp19a1a</i> double mutants developed as testes, while the gonads of <i>dmrt1</i>;<i>cyp19a1a</i>;<i>foxl3</i> triple mutants eventually developed as ovaries. In contrast, the gonads of <i>amhy</i>;<i>cyp19a1a</i>, <i>gsdf</i>;<i>cyp19a1a</i>, <i>amhy</i>;<i>foxl2</i>, <i>gsdf</i>;<i>foxl2</i> double and <i>amhy</i>;<i>cyp19a1a</i>;<i>cyp19a1b</i>, <i>gsdf</i>;<i>cyp19a1a</i>;<i>cyp19a1b</i> triple mutants developed as testes with spermatogenesis via up-regulation of <i>dmrt1</i> in both somatic and germ cells. The gonads of <i>amhy</i>;<i>foxl3</i> and <i>gsdf</i>;<i>foxl3</i> double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation of <i>dmrt1</i>. Taking the respective ovary and underdeveloped testis of <i>dmrt1</i>;<i>foxl3</i> and <i>dmrt1</i>;<i>foxl2</i> double mutants reported previously into consideration, we demonstrated that once <i>dmrt1</i> mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other than <i>dmrt1</i>, including its upstream <i>amhy</i> and downstream <i>gsdf</i>, could be rescued by mutating female pathway gene. Overall, our data suggested that <i>dmrt1</i> is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.<i>Spoink</i>, a LTR retrotransposon, invaded <i>D. melanogaster</i> populations in the 1990sRiccardo PianezzaAlmorò ScarpaPrakash NarayananSarah SignorRobert Kofler10.1371/journal.pgen.10112012024-03-26T14:00:00Z2024-03-26T14:00:00Z<p>by Riccardo Pianezza, Almorò Scarpa, Prakash Narayanan, Sarah Signor, Robert Kofler</p>
During the last few centuries <i>D. melanogaster</i> populations were invaded by several transposable elements, the most recent of which was thought to be the <i>P</i>-element between 1950 and 1980. Here we describe a novel TE, which we named <i>Spoink</i>, that has invaded <i>D. melanogaster</i>. It is a 5216nt LTR retrotransposon of the Ty3/gypsy superfamily. Relying on strains sampled at different times during the last century we show that <i>Spoink</i> invaded worldwide <i>D. melanogaster</i> populations after the <i>P</i>-element between 1983 and 1993. This invasion was likely triggered by a horizontal transfer from the <i>D. willistoni</i> group, much as the <i>P</i>-element. <i>Spoink</i> is probably silenced by the piRNA pathway in natural populations and about 1/3 of the examined strains have an insertion into a canonical piRNA cluster such as <i>42AB</i>. Given the degree of genetic investigation of <i>D. melanogaster</i> it is perhaps surprising that <i>Spoink</i> was able to invade unnoticed.Recombination, admixture and genome instability shape the genomic landscape of <i>Saccharomyces cerevisiae</i> derived from spontaneous grape fermentsChris M. WardCristobal A. OnettoSteven Van Den HeuvelKathleen M. CuijversLaura J. HaleAnthony R. Borneman10.1371/journal.pgen.10112232024-03-22T14:00:00Z2024-03-22T14:00:00Z<p>by Chris M. Ward, Cristobal A. Onetto, Steven Van Den Heuvel, Kathleen M. Cuijvers, Laura J. Hale, Anthony R. Borneman</p>
Cultural exchange of fermentation techniques has driven the spread of <i>Saccharomyces cerevisiae</i> across the globe, establishing natural populations in many countries. Despite this, Oceania is thought to lack native populations of <i>S</i>. <i>cerevisiae</i>, only being introduced after colonisation. Here we investigate the genomic landscape of 411 <i>S</i>. <i>cerevisiae</i> isolated from spontaneous grape fermentations in Australia across multiple locations, years, and grape cultivars. Spontaneous fermentations contained highly recombined mosaic strains that exhibited high levels of genome instability. Assigning genomic windows to putative ancestral origin revealed that few closely related starter lineages have come to dominate the genetic landscape, contributing most of the genetic variation. Fine-scale phylogenetic analysis of loci not observed in strains of commercial wine origin identified widespread admixture with European derived beer yeast along with three independent admixture events from potentially endemic Oceanic lineages that was associated with genome instability. Finally, we investigated Australian ecological niches for basal isolates, identifying phylogenetically distinct <i>S</i>. <i>cerevisiae</i> of non-European, non-domesticated origin associated with admixture loci. Our results illustrate the effect commercial use of microbes may have on local microorganism genetic diversity and demonstrates the presence of non-domesticated, potentially endemic lineages of <i>S</i>. <i>cerevisiae</i> in Australian niches that are actively admixing.Opticool: Cutting-edge transgenic optical toolsKelli D. FenelonJulia KrauseTheodora Koromila10.1371/journal.pgen.10112082024-03-22T14:00:00Z2024-03-22T14:00:00Z<p>by Kelli D. Fenelon, Julia Krause, Theodora Koromila</p>
Only a few short decades have passed since the sequencing of GFP, yet the modern repertoire of transgenically encoded optical tools implies an exponential proliferation of ever improving constructions to interrogate the subcellular environment. A myriad of tags for labeling proteins, RNA, or DNA have arisen in the last few decades, facilitating unprecedented visualization of subcellular components and processes. Development of a broad array of modern genetically encoded sensors allows real-time, in vivo detection of molecule levels, pH, forces, enzyme activity, and other subcellular and extracellular phenomena in ever expanding contexts. Optogenetic, genetically encoded optically controlled manipulation systems have gained traction in the biological research community and facilitate single-cell, real-time modulation of protein function in vivo in ever broadening, novel applications. While this field continues to explosively expand, references are needed to assist scientists seeking to use and improve these transgenic devices in new and exciting ways to interrogate development and disease. In this review, we endeavor to highlight the state and trajectory of the field of in vivo transgenic optical tools.Canadian COVID-19 host genetics cohort replicates known severity associationsElika GargPaola Arguello-PascualliOlga VishnyakovaAnat R. HalevySamantha YooJennifer D. BrooksShelley B. BullFrance GagnonCelia M. T. GreenwoodRayjean J. HungJerald F. LawlessJordan Lerner-EllisJessica K. DennisRohan J. S. AbrahamJean-Michel GarantBhooma ThiruvahindrapuramSteven J. M. JonesCGEn HostSeq InitiativeLisa J. StrugAndrew D. PatersonLei SunLloyd T. Elliott10.1371/journal.pgen.10111922024-03-22T14:00:00Z2024-03-22T14:00:00Z<p>by Elika Garg, Paola Arguello-Pascualli, Olga Vishnyakova, Anat R. Halevy, Samantha Yoo, Jennifer D. Brooks, Shelley B. Bull, France Gagnon, Celia M. T. Greenwood, Rayjean J. Hung, Jerald F. Lawless, Jordan Lerner-Ellis, Jessica K. Dennis, Rohan J. S. Abraham, Jean-Michel Garant, Bhooma Thiruvahindrapuram, Steven J. M. Jones, CGEn HostSeq Initiative , Lisa J. Strug, Andrew D. Paterson, Lei Sun, Lloyd T. Elliott</p>
The HostSeq initiative recruited 10,059 Canadians infected with SARS-CoV-2 between March 2020 and March 2023, obtained clinical information on their disease experience and whole genome sequenced (WGS) their DNA. We analyzed the WGS data for genetic contributors to severe COVID-19 (considering 3,499 hospitalized cases and 4,975 non-hospitalized after quality control). We investigated the evidence for replication of loci reported by the International Host Genetics Initiative (HGI); analyzed the X chromosome; conducted rare variant gene-based analysis and polygenic risk score testing. Population stratification was adjusted for using meta-analysis across ancestry groups. We replicated two loci identified by the HGI for COVID-19 severity: the <i>LZTFL1/SLC6A20</i> locus on chromosome 3 and the <i>FOXP4</i> locus on chromosome 6 (the latter with a variant significant at P < 5E-8). We found novel significant associations with <i>MRAS</i> and <i>WDR89</i> in gene-based analyses, and constructed a polygenic risk score that explained 1.01% of the variance in severe COVID-19. This study provides independent evidence confirming the robustness of previously identified COVID-19 severity loci by the HGI and identifies novel genes for further investigation.Compartment specific responses to contractility in the small intestinal epitheliumTaylor HinnantWenxiu NingTerry Lechler10.1371/journal.pgen.10108992024-03-22T14:00:00Z2024-03-22T14:00:00Z<p>by Taylor Hinnant, Wenxiu Ning, Terry Lechler</p>
Tissues are subject to multiple mechanical inputs at the cellular level that influence their overall shape and function. In the small intestine, actomyosin contractility can be induced by many physiological and pathological inputs. However, we have little understanding of how contractility impacts the intestinal epithelium on a cellular and tissue level. In this study, we probed the cell and tissue-level effects of contractility by using mouse models to genetically increase the level of myosin activity in the two distinct morphologic compartments of the intestinal epithelium, the crypts and villi. We found that increased contractility in the villar compartment caused shape changes in the cells that expressed the transgene and their immediate neighbors. While there were no discernable effects on villar architecture or cell polarity, even low levels of transgene induction in the villi caused non-cell autonomous hyperproliferation of the transit amplifying cells in the crypt, driving increased cell flux through the crypt-villar axis. In contrast, induction of increased contractility in the proliferating cells of the crypts resulted in nuclear deformations, DNA damage, and apoptosis. This study reveals the complex and diverse responses of different intestinal epithelial cells to contractility and provides important insight into mechanical regulation of intestinal physiology.DNAJB1-PRKACA fusion protein-regulated LINC00473 promotes tumor growth and alters mitochondrial fitness in fibrolamellar carcinomaRosanna K. MaPei-Yin TsaiAlaa R. FarghliAlexandria ShumwayMatt KankeJohn D. GordanTaranjit S. GujralKhashayar VakiliManabu NukayaLeila NoetzliSean Ronnekleiv-KellyWendy BroomJoeva BarrowPraveen Sethupathy10.1371/journal.pgen.10112162024-03-21T14:00:00Z2024-03-21T14:00:00Z<p>by Rosanna K. Ma, Pei-Yin Tsai, Alaa R. Farghli, Alexandria Shumway, Matt Kanke, John D. Gordan, Taranjit S. Gujral, Khashayar Vakili, Manabu Nukaya, Leila Noetzli, Sean Ronnekleiv-Kelly, Wendy Broom, Joeva Barrow, Praveen Sethupathy</p>
Fibrolamellar carcinoma (FLC) is a rare liver cancer that disproportionately affects adolescents and young adults. Currently, no standard of care is available and there remains a dire need for new therapeutics. Most patients harbor the fusion oncogene <i>DNAJB1-PRKACA</i> (DP fusion), but clinical inhibitors are not yet developed and it is critical to identify downstream mediators of FLC pathogenesis. Here, we identify long noncoding RNA LINC00473 among the most highly upregulated genes in FLC tumors and determine that it is strongly suppressed by RNAi-mediated inhibition of the DP fusion in FLC tumor epithelial cells. We show by loss- and gain-of-function studies that LINC00473 suppresses apoptosis, increases the expression of FLC marker genes, and promotes FLC growth in cell-based and <i>in vivo</i> disease models. Mechanistically, LINC00473 plays an important role in promoting glycolysis and altering mitochondrial activity. Specifically, LINC00473 knockdown leads to increased spare respiratory capacity, which indicates mitochondrial fitness. Overall, we propose that LINC00473 could be a viable target for this devastating disease. Schematic was created with BioRender.com.Reciprocal regulation of enterococcal cephalosporin resistance by products of the autoregulated <i>yvcJ-glmR-yvcL</i> operon enhances fitness during cephalosporin exposureDušanka DjorićSamantha N. AtkinsonChristopher J. Kristich10.1371/journal.pgen.10112152024-03-21T14:00:00Z2024-03-21T14:00:00Z<p>by Dušanka Djorić, Samantha N. Atkinson, Christopher J. Kristich</p>
Enterococci are commensal members of the gastrointestinal tract and also major nosocomial pathogens. They possess both intrinsic and acquired resistance to many antibiotics, including intrinsic resistance to cephalosporins that target bacterial cell wall synthesis. These antimicrobial resistance traits make enterococcal infections challenging to treat. Moreover, prior therapy with antibiotics, including broad-spectrum cephalosporins, promotes enterococcal proliferation in the gut, resulting in dissemination to other sites of the body and subsequent infection. As a result, a better understanding of mechanisms of cephalosporin resistance is needed to enable development of new therapies to treat or prevent enterococcal infections. We previously reported that flow of metabolites through the peptidoglycan biosynthesis pathway is one determinant of enterococcal cephalosporin resistance. One factor that has been implicated in regulating flow of metabolites into cell wall biosynthesis pathways of other Gram-positive bacteria is GlmR. In enterococci, GlmR is encoded as the middle gene of a predicted 3-gene operon along with YvcJ and YvcL, whose functions are poorly understood. Here we use genetics and biochemistry to investigate the function of the enterococcal <i>yvcJ-glmR-yvcL</i> gene cluster. Our results reveal that YvcL is a DNA-binding protein that regulates expression of the <i>yvcJ-glmR-yvcL</i> operon in response to cell wall stress. YvcJ and GlmR bind UDP-GlcNAc and reciprocally regulate cephalosporin resistance in <i>E</i>. <i>faecalis</i>, and binding of UDP-GlcNAc by YvcJ appears essential for its activity. Reciprocal regulation by YvcJ/GlmR is essential for fitness during exposure to cephalosporin stress. Additionally, our results indicate that enterococcal GlmR likely acts by a different mechanism than the previously studied GlmR of <i>Bacillus subtilis</i>, suggesting that the YvcJ/GlmR regulatory module has evolved unique targets in different species of bacteria.A quantitative genetic model of background selection in humansVince BuffaloAndrew D. Kern10.1371/journal.pgen.10111442024-03-20T14:00:00Z2024-03-20T14:00:00Z<p>by Vince Buffalo, Andrew D. Kern</p>
Across the human genome, there are large-scale fluctuations in genetic diversity caused by the indirect effects of selection. This “linked selection signal” reflects the impact of selection according to the physical placement of functional regions and recombination rates along chromosomes. Previous work has shown that purifying selection acting against the steady influx of new deleterious mutations at functional portions of the genome shapes patterns of genomic variation. To date, statistical efforts to estimate purifying selection parameters from linked selection models have relied on classic Background Selection theory, which is only applicable when new mutations are so deleterious that they cannot fix in the population. Here, we develop a statistical method based on a quantitative genetics view of linked selection, that models how polygenic additive fitness variance distributed along the genome increases the rate of stochastic allele frequency change. By jointly predicting the equilibrium fitness variance and substitution rate due to both strong and weakly deleterious mutations, we estimate the distribution of fitness effects (DFE) and mutation rate across three geographically distinct human samples. While our model can accommodate weaker selection, we find evidence of strong selection operating similarly across all human samples. Although our quantitative genetic model of linked selection fits better than previous models, substitution rates of the most constrained sites disagree with observed divergence levels. We find that a model incorporating selective interference better predicts observed divergence in conserved regions, but overall our results suggest uncertainty remains about the processes generating fitness variation in humans.Altered Fhod3 expression involved in progressive high-frequency hearing loss via dysregulation of actin polymerization stoichiometry in the cuticular plateEly Cheikh BoussatyYuzuru NinoyuLeonardo R. AndradeQingzhong LiRyu TakeyaHideki SumimotoTakahiro OhyamaKarl J. WahlinUri ManorRick A. Friedman10.1371/journal.pgen.10112112024-03-18T14:00:00Z2024-03-18T14:00:00Z<p>by Ely Cheikh Boussaty, Yuzuru Ninoyu, Leonardo R. Andrade, Qingzhong Li, Ryu Takeya, Hideki Sumimoto, Takahiro Ohyama, Karl J. Wahlin, Uri Manor, Rick A. Friedman</p>
Age-related hearing loss (ARHL) is a common sensory impairment with complex underlying mechanisms. In our previous study, we performed a meta-analysis of genome-wide association studies (GWAS) in mice and identified a novel locus on chromosome 18 associated with ARHL specifically linked to a 32 kHz tone burst stimulus. Consequently, we investigated the role of Formin Homology 2 Domain Containing 3 (Fhod3), a newly discovered candidate gene for ARHL based on the GWAS results. We observed Fhod3 expression in auditory hair cells (HCs) primarily localized at the cuticular plate (CP). To understand the functional implications of Fhod3 in the cochlea, we generated Fhod3 overexpression mice (<i>Pax2-Cre</i><sup>+/-</sup><i>; Fhod3</i><sup>Tg/+</sup>) (TG) and HC-specific conditional knockout mice (<i>Atoh1-Cre</i><sup>+/-</sup><i>; Fhod3</i><sup>fl/fl</sup>) (KO). Audiological assessments in TG mice demonstrated progressive high-frequency hearing loss, characterized by predominant loss of outer hair cells, and a decreased phalloidin intensities of CP. Ultrastructural analysis revealed loss of the shortest row of stereocilia in the basal turn of the cochlea, and alterations in the cuticular plate surrounding stereocilia rootlets. Importantly, the hearing and HC phenotype in TG mice phenocopied that of the KO mice. These findings suggest that balanced expression of Fhod3 is critical for proper CP and stereocilia structure and function. Further investigation of Fhod3 related hearing impairment mechanisms may lend new insight towards the myriad mechanisms underlying ARHL, which in turn could facilitate the development of therapeutic strategies for ARHL.Genome biology and evolution of mating-type loci in four cereal rust fungiZhenyan LuoAlistair McTaggartBenjamin Schwessinger10.1371/journal.pgen.10112072024-03-18T14:00:00Z2024-03-18T14:00:00Z<p>by Zhenyan Luo, Alistair McTaggart, Benjamin Schwessinger</p>
Permanent heterozygous loci, such as sex- or mating-compatibility regions, often display suppression of recombination and signals of genomic degeneration. In Basidiomycota, two distinct loci confer mating compatibility. These loci encode homeodomain (<i>HD</i>) transcription factors and pheromone receptor (<i>Pra</i>)-ligand allele pairs. To date, an analysis of genome level mating-type (MAT) loci is lacking for obligate biotrophic basidiomycetes in the <i>Pucciniales</i>, an order containing serious agricultural plant pathogens. Here, we focus on four species of <i>Puccinia</i> that infect oat and wheat, including <i>P</i>. <i>coronata</i> f. sp. <i>avenae</i>, <i>P</i>. <i>graminis</i> f. sp. <i>tritici</i>, <i>P</i>. <i>triticina</i> and <i>P</i>. <i>striiformis</i> f. sp. <i>tritici</i>. MAT loci are located on two separate chromosomes supporting previous hypotheses of a tetrapolar mating compatibility system in the <i>Pucciniales</i>. The <i>HD</i> genes are multiallelic in all four species while the PR locus appears biallelic, except for <i>P</i>. <i>graminis</i> f. sp. <i>tritici</i>, which potentially has multiple alleles. HD loci are largely conserved in their macrosynteny, both within and between species, without strong signals of recombination suppression. Regions proximal to the PR locus, however, displayed signs of recombination suppression and genomic degeneration in the three species with a biallelic PR locus. Our observations support a link between recombination suppression, genomic degeneration, and allele diversity of MAT loci that is consistent with recent mathematical modelling and simulations. Finally, we confirm that <i>MAT</i> genes are expressed during the asexual infection cycle, and we propose that this may support regulating nuclear maintenance and pairing during infection and spore formation. Our study provides insights into the evolution of MAT loci of key pathogenic <i>Puccinia</i> species. Understanding mating compatibility can help predict possible combinations of nuclear pairs, generated by sexual reproduction or somatic recombination, and the potential evolution of new virulent isolates of these important plant pathogens.Gene dosage of independent dynein arm motor preassembly factors influences cilia assembly in <i>Chlamydomonas reinhardtii</i>Gervette M. PennySusan K. Dutcher10.1371/journal.pgen.10110382024-03-18T14:00:00Z2024-03-18T14:00:00Z<p>by Gervette M. Penny, Susan K. Dutcher</p>
Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene <i>DNAAF4</i>. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In <i>Chlamydomonas reinhardtii</i>, a ciliated unicellular alga, the <i>DNAAF4</i> ortholog is called <i>PF23</i>. The <i>pf23-1</i> mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (<i>pf23-4</i>) completely lack ODAs and IDAs and are even shorter than cilia from <i>pf23-1</i>. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant <i>Chlamydomonas</i> strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type <i>Chlamydomonas</i> cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with <i>pf23</i> and other <i>DNAAF</i> mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for <i>pf23</i> and <i>wdr92</i> or <i>oda8</i> show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.Subscaling of a cytosolic RNA binding protein governs cell size homeostasis in the multiple fission alga ChlamydomonasDianyi LiuCristina Lopez-PazYubing LiXiaohong ZhuangJames Umen10.1371/journal.pgen.10105032024-03-18T14:00:00Z2024-03-18T14:00:00Z<p>by Dianyi Liu, Cristina Lopez-Paz, Yubing Li, Xiaohong Zhuang, James Umen</p>
Coordination of growth and division in eukaryotic cells is essential for populations of proliferating cells to maintain size homeostasis, but the underlying mechanisms that govern cell size have only been investigated in a few taxa. The green alga <i>Chlamydomonas reinhardtii</i> (Chlamydomonas) proliferates using a multiple fission cell cycle that involves a long G1 phase followed by a rapid series of successive S and M phases (S/M) that produces 2<sup>n</sup> daughter cells. Two control points show cell-size dependence: the Commitment control point in mid-G1 phase requires the attainment of a minimum size to enable at least one mitotic division during S/M, and the S/M control point where mother cell size governs cell division number (n), ensuring that daughter distributions are uniform. <i>tny1</i> mutants pass Commitment at a smaller size than wild type and undergo extra divisions during S/M phase to produce small daughters, indicating that TNY1 functions to inhibit size-dependent cell cycle progression. <i>TNY1</i> encodes a cytosolic hnRNP A-related RNA binding protein and is produced once per cell cycle during S/M phase where it is apportioned to daughter cells, and then remains at constant absolute abundance as cells grow, a property known as subscaling. Altering the dosage of <i>TNY1</i> in heterozygous diploids or through mis-expression increased Commitment cell size and daughter cell size, indicating that TNY1 is a limiting factor for both size control points. Epistasis placed <i>TNY1</i> function upstream of the retinoblastoma tumor suppressor complex (RBC) and one of its regulators, Cyclin-Dependent Kinase G1 (CDKG1). Moreover, CDKG1 protein and mRNA were found to over-accumulate in <i>tny1</i> cells suggesting that CDKG1 may be a direct target of repression by TNY1. Our data expand the potential roles of subscaling proteins outside the nucleus and imply a control mechanism that ties TNY1 accumulation to pre-division mother cell size.P53 and BCL-2 family proteins PUMA and NOXA define competitive fitness in pluripotent cell competitionJose A. Valverde-LopezLin Li-BaoRocío SierraElisa SantosGiovanna GiovinazzoCovadonga Díaz-DíazMiguel Torres10.1371/journal.pgen.10111932024-03-15T14:00:00Z2024-03-15T14:00:00Z<p>by Jose A. Valverde-Lopez, Lin Li-Bao, Rocío Sierra, Elisa Santos, Giovanna Giovinazzo, Covadonga Díaz-Díaz, Miguel Torres</p>
Cell Competition is a process by which neighboring cells compare their fitness. As a result, viable but suboptimal cells are selectively eliminated in the presence of fitter cells. In the early mammalian embryo, epiblast pluripotent cells undergo extensive Cell Competition, which prevents suboptimal cells from contributing to the newly forming organism. While competitive ability is regulated by MYC in the epiblast, the mechanisms that contribute to competitive fitness in this context are largely unknown. Here, we report that P53 and its pro-apoptotic targets PUMA and NOXA regulate apoptosis susceptibility and competitive fitness in pluripotent cells. PUMA is widely expressed specifically in pluripotent cells <i>in vitro</i> and <i>in vivo</i>. We found that P53 regulates MYC levels in pluripotent cells, which connects these two Cell Competition pathways, however, MYC and PUMA/NOXA levels are independently regulated by P53. We propose a model that integrates a bifurcated P53 pathway regulating both MYC and PUMA/NOXA levels and determines competitive fitness.Uncharted territories: Solving the mysteries of male meiosis in fliesLingSze LeeLeah F. Rosin10.1371/journal.pgen.10111852024-03-15T14:00:00Z2024-03-15T14:00:00Z<p>by LingSze Lee, Leah F. Rosin</p>
The segregation of homologous chromosomes during meiosis typically requires tight end-to-end chromosome pairing. However, in <i>Drosophila</i> spermatogenesis, male flies segregate their chromosomes without classic synaptonemal complex formation and without recombination, instead compartmentalizing homologs into subnuclear domains known as chromosome territories (CTs). How homologs find each other in the nucleus and are separated into CTs has been one of the biggest riddles in chromosome biology. Here, we discuss our current understanding of pairing and CT formation in flies and review recent data on how homologs are linked and partitioned during meiosis in male flies.Functional labeling of individualized postsynaptic neurons using optogenetics and <i>trans-</i>Tango in <i>Drosophila</i> (FLIPSOT)Allison N. CastanedaAinul HudaIona B. M. WhitakerJulianne E. ReillyGrace S. ShelbyHua BaiLina Ni10.1371/journal.pgen.10111902024-03-14T14:00:00Z2024-03-14T14:00:00Z<p>by Allison N. Castaneda, Ainul Huda, Iona B. M. Whitaker, Julianne E. Reilly, Grace S. Shelby, Hua Bai, Lina Ni</p>
A population of neurons interconnected by synapses constitutes a neural circuit, which performs specific functions upon activation. It is essential to identify both anatomical and functional entities of neural circuits to comprehend the components and processes necessary for healthy brain function and the changes that characterize brain disorders. To date, few methods are available to study these two aspects of a neural circuit simultaneously. In this study, we developed FLIPSOT, or functional labeling of individualized postsynaptic neurons using optogenetics and <i>trans-</i>Tango. FLIPSOT uses (1) <i>trans-</i>Tango to access postsynaptic neurons genetically, (2) optogenetic approaches to activate (FLIPSOTa) or inhibit (FLIPSOTi) postsynaptic neurons in a random and sparse manner, and (3) fluorescence markers tagged with optogenetic genes to visualize these neurons. Therefore, FLIPSOT allows using a presynaptic driver to identify the behavioral function of individual postsynaptic neurons. It is readily applied to identify functions of individual postsynaptic neurons and has the potential to be adapted for use in mammalian circuits.INSIDER: Interpretable sparse matrix decomposition for RNA expression data analysisKai ZhaoSen HuangCuichan LinPak Chung ShamHon-Cheong SoZhixiang Lin10.1371/journal.pgen.10111892024-03-14T14:00:00Z2024-03-14T14:00:00Z<p>by Kai Zhao, Sen Huang, Cuichan Lin, Pak Chung Sham, Hon-Cheong So, Zhixiang Lin</p>
RNA sequencing (RNA-Seq) is widely used to capture transcriptome dynamics across tissues, biological entities, and conditions. Currently, few or no methods can handle multiple biological variables (e.g., tissues/ phenotypes) and their interactions simultaneously, while also achieving dimension reduction (DR).
We propose INSIDER, a general and flexible statistical framework based on matrix factorization, which is freely available at https://github.com/kai0511/insider. INSIDER decomposes variation from different biological variables and their interactions into a shared low-rank latent space. Particularly, it introduces the elastic net penalty to induce sparsity while considering the grouping effects of genes. It can achieve DR of high-dimensional data (of > = 3 dimensions), as opposed to conventional methods (e.g., PCA/NMF) which generally only handle 2D data (e.g., sample × expression). Besides, it enables computing ’adjusted’ expression profiles for specific biological variables while controlling variation from other variables. INSIDER is computationally efficient and accommodates missing data. INSIDER also performed similarly or outperformed a close competing method, SDA, as shown in simulations and can handle complex missing data in RNA-Seq data. Moreover, unlike SDA, it can be used when the data cannot be structured into a tensor. Lastly, we demonstrate its usefulness via real data analysis, including clustering donors for disease subtyping, revealing neuro-development trajectory using the BrainSpan data, and uncovering biological processes contributing to variables of interest (e.g., disease status and tissue) and their interactions.A fatty acid anabolic pathway in specialized-cells sustains a remote signal that controls egg activation in <i>Drosophila</i>Mickael PoidevinNicolas MazurasGwénaëlle BontonouPierre DelamotteBéatrice DenisMaëlle DevilliersPerla AkikiDelphine PetitLaura de LucaPriscilla SoulieCynthia GilletClaude Wicker-ThomasJacques Montagne10.1371/journal.pgen.10111862024-03-14T14:00:00Z2024-03-14T14:00:00Z<p>by Mickael Poidevin, Nicolas Mazuras, Gwénaëlle Bontonou, Pierre Delamotte, Béatrice Denis, Maëlle Devilliers, Perla Akiki, Delphine Petit, Laura de Luca, Priscilla Soulie, Cynthia Gillet, Claude Wicker-Thomas, Jacques Montagne</p>
Egg activation, representing the critical oocyte-to-embryo transition, provokes meiosis completion, modification of the vitelline membrane to prevent polyspermy, and translation of maternally provided mRNAs. This transition is triggered by a calcium signal induced by spermatozoon fertilization in most animal species, but not in insects. In <i>Drosophila melanogaster</i>, mature oocytes remain arrested at metaphase-I of meiosis and the calcium-dependent activation occurs while the oocyte moves through the genital tract. Here, we discovered that the oenocytes of fruitfly females are required for egg activation. Oenocytes, cells specialized in lipid-metabolism, are located beneath the abdominal cuticle. In adult flies, they synthesize the fatty acids (FAs) that are the precursors of cuticular hydrocarbons (CHCs), including pheromones. The oenocyte-targeted knockdown of a set of FA-anabolic enzymes, involved in very-long-chain fatty acid (VLCFA) synthesis, leads to a defect in egg activation. Given that some but not all of the identified enzymes are required for CHC/pheromone biogenesis, this putative VLCFA-dependent remote control may rely on an as-yet unidentified CHC or may function in parallel to CHC biogenesis. Additionally, we discovered that the most posterior ventral oenocyte cluster is in close proximity to the uterus. Since oocytes dissected from females deficient in this FA-anabolic pathway can be activated <i>in vitro</i>, this regulatory loop likely operates upstream of the calcium trigger. To our knowledge, our findings provide the first evidence that a physiological extra-genital signal remotely controls egg activation. Moreover, our study highlights a potential metabolic link between pheromone-mediated partner recognition and egg activation.Transposition of <i>HOPPLA</i> in siRNA-deficient plants suggests a limited effect of the environment on retrotransposon mobility in <i>Brachypodium distachyon</i>Michael ThiemeNikolaos MinadakisChristophe HimberBettina KellerWenbo XuKinga RutowiczCalvin MatteoliMarcel BöhrerBart RymenDebbie Laudencia-ChingcuancoJohn P. VogelRichard SiboutChristoph StrittTodd BlevinsAnne C. Roulin10.1371/journal.pgen.10112002024-03-12T14:00:00Z2024-03-12T14:00:00Z<p>by Michael Thieme, Nikolaos Minadakis, Christophe Himber, Bettina Keller, Wenbo Xu, Kinga Rutowicz, Calvin Matteoli, Marcel Böhrer, Bart Rymen, Debbie Laudencia-Chingcuanco, John P. Vogel, Richard Sibout, Christoph Stritt, Todd Blevins, Anne C. Roulin</p>
Long terminal repeat retrotransposons (LTR-RTs) are powerful mutagens regarded as a major source of genetic novelty and important drivers of evolution. Yet, the uncontrolled and potentially selfish proliferation of LTR-RTs can lead to deleterious mutations and genome instability, with large fitness costs for their host. While population genomics data suggest that an ongoing LTR-RT mobility is common in many species, the understanding of their dual role in evolution is limited. Here, we harness the genetic diversity of 320 sequenced natural accessions of the Mediterranean grass <i>Brachypodium distachyon</i> to characterize how genetic and environmental factors influence plant LTR-RT dynamics in the wild. When combining a coverage-based approach to estimate global LTR-RT copy number variations with mobilome-sequencing of nine accessions exposed to eight different stresses, we find little evidence for a major role of environmental factors in LTR-RT accumulations in <i>B</i>. <i>distachyon</i> natural accessions. Instead, we show that loss of RNA polymerase IV (Pol IV), which mediates RNA-directed DNA methylation in plants, results in high transcriptional and transpositional activities of RLC_BdisC024 (<i>HOPPLA</i>) LTR-RT family elements, and that these effects are not stress-specific. This work supports findings indicating an ongoing mobility in <i>B</i>. <i>distachyon</i> and reveals that host RNA-directed DNA methylation rather than environmental factors controls their mobility in this wild grass model.Ecdysone-controlled nuclear receptor ERR regulates metabolic homeostasis in the disease vector mosquito <i>Aedes aegypti</i>Dan-Qian GengXue-Li WangXiang-Yang LyuAlexander S. RaikhelZhen Zou10.1371/journal.pgen.10111962024-03-11T14:00:00Z2024-03-11T14:00:00Z<p>by Dan-Qian Geng, Xue-Li Wang, Xiang-Yang Lyu, Alexander S. Raikhel, Zhen Zou</p>
Hematophagous mosquitoes require vertebrate blood for their reproductive cycles, making them effective vectors for transmitting dangerous human diseases. Thus, high-intensity metabolism is needed to support reproductive events of female mosquitoes. However, the regulatory mechanism linking metabolism and reproduction in mosquitoes remains largely unclear. In this study, we found that the expression of <i>estrogen-related receptor (ERR)</i>, a nuclear receptor, is activated by the direct binding of 20-hydroxyecdysone (20E) and ecdysone receptor (EcR) to the ecdysone response element (EcRE) in the <i>ERR</i> promoter region during the gonadotropic cycle of <i>Aedes aegypti</i> (named AaERR). RNA interference (RNAi) of <i>AaERR</i> in female mosquitoes led to delayed development of ovaries. mRNA abundance of genes encoding key enzymes involved in carbohydrate metabolism (CM)—<i>glucose-6-phosphate isomerase</i> (<i>GPI</i>) and <i>pyruvate kinase</i> (<i>PYK</i>)—was significantly decreased in <i>AaERR</i> knockdown mosquitoes, while the levels of metabolites, such as glycogen, glucose, and trehalose, were elevated. The expression of <i>fatty acid synthase</i> (<i>FAS</i>) was notably downregulated, and lipid accumulation was reduced in response to <i>AaERR</i> depletion. Dual luciferase reporter assays and electrophoretic mobility shift assays (EMSA) determined that AaERR directly activated the expression of metabolic genes, such as <i>GPI</i>, <i>PYK</i>, and <i>FAS</i>, by binding to the corresponding AaERR-responsive motif in the promoter region of these genes. Our results have revealed an important role of AaERR in the regulation of metabolism during mosquito reproduction and offer a novel target for mosquito control.A single amino acid polymorphism in natural Metchnikowin alleles of <i>Drosophila</i> results in systemic immunity and life history tradeoffsJessamyn I. PerlmutterJoanne R. ChapmanMason C. WilkinsonIsaac Nevarez-SaenzRobert L. Unckless10.1371/journal.pgen.10111552024-03-11T14:00:00Z2024-03-11T14:00:00Z<p>by Jessamyn I. Perlmutter, Joanne R. Chapman, Mason C. Wilkinson, Isaac Nevarez-Saenz, Robert L. Unckless</p>
Antimicrobial peptides (AMPs) are at the interface of interactions between hosts and microbes and are therefore expected to be rapidly evolving in a coevolutionary arms race with pathogens. In contrast, previous work demonstrated that insect AMPs tend to evolve more slowly than the genome average. Metchikowin (Mtk) is a <i>Drosophila</i> AMP that has a single amino acid residue that segregates as either proline (P) or arginine (R) in populations of four different species, some of which diverged more than 10 million years ago. These results suggest that there is a distinct functional importance to each allele. The most likely hypotheses are driven by two main questions: does each allele have a different efficacy against different specific pathogens (specificity hypothesis)? Or, is one allele a more potent antimicrobial, but with a host fitness cost (autoimmune hypothesis)? To assess their functional differences, we created <i>D</i>. <i>melanogaster</i> lines with the P allele, R allele, or <i>Mtk</i> null mutation using CRISPR/Cas9 genome editing and performed a series of life history and infection assays to assess them. In males, testing of systemic immune responses to a repertoire of bacteria and fungi demonstrated that the R allele performs as well or better than the P and null alleles with most infections. Females show some results that contrast with males, with <i>Mtk</i> alleles either not contributing to survival or with the P allele outperforming the R allele. In addition, measurements of life history traits demonstrate that the R allele is more costly in the absence of infection for both sexes. These results are consistent with both the specificity hypothesis (either allele can perform better against certain pathogens depending on context), and the autoimmune hypothesis (the R allele is generally the more potent antimicrobial in males, and carries a fitness cost). These results provide strong <i>in vivo</i> evidence that differential fitness with or without infection and sex-based functional differences in alleles may be adaptive mechanisms of maintaining immune gene polymorphisms in contrast with expectations of rapid evolution. Therefore, a complex interplay of forces including pathogen species and host sex may lead to balancing selection for immune genotypes. Strikingly, this selection may act on even a single amino acid polymorphism in an AMP.A negative feedback loop is critical for recovery of RpoS after stress in <i>Escherichia coli</i>Sophie BouilletIssam HamdallahNadim MajdalaniArti TripathiSusan Gottesman10.1371/journal.pgen.10110592024-03-11T14:00:00Z2024-03-11T14:00:00Z<p>by Sophie Bouillet, Issam Hamdallah, Nadim Majdalani, Arti Tripathi, Susan Gottesman</p>
RpoS is an alternative sigma factor needed for the induction of the general stress response in many gammaproteobacteria. Tight regulation of RpoS levels and activity is required for bacterial growth and survival under stress. In <i>Escherichia coli</i>, various stresses lead to higher levels of RpoS due to increased translation and decreased degradation. During non-stress conditions, RpoS is unstable, because the adaptor protein RssB delivers RpoS to the ClpXP protease. RpoS degradation is prevented during stress by the sequestration of RssB by anti-adaptors, each of which is induced in response to specific stresses. Here, we examined how the stabilization of RpoS is reversed during recovery of the cell from stress. We found that RpoS degradation quickly resumes after recovery from phosphate starvation, carbon starvation, and when transitioning from stationary phase back to exponential phase. This process is in part mediated by the anti-adaptor IraP, known to promote RpoS stabilization during phosphate starvation via the sequestration of adaptor RssB. The rapid recovery from phosphate starvation is dependent upon a feedback loop in which RpoS transcription of <i>rssB</i>, encoding the adaptor protein, plays a critical role. Crl, an activator of RpoS that specifically binds to and stabilizes the complex between the RNA polymerase and RpoS, is also required for the feedback loop to function efficiently, highlighting a critical role for Crl in restoring RpoS basal levels.Long-read sequencing for fast and robust identification of correct genome-edited alleles: PCR-based and Cas9 capture methodsChristopher V. McCabePeter D. PriceGemma F. CodnerAlasdair J. AllanAdam CaulderSkevoulla ChristouJorik LoefflerMatthew MackenzieElke MalzerJoffrey MiannéKrystian J. NowickiEdward J. O’NeillFran J. PikeMarie HutchisonBenoit Petit-DemoulièreMichelle E. StewartHilary GatesSara WellsNicholas D. SandersonLydia Teboul10.1371/journal.pgen.10111872024-03-08T14:00:00Z2024-03-08T14:00:00Z<p>by Christopher V. McCabe, Peter D. Price, Gemma F. Codner, Alasdair J. Allan, Adam Caulder, Skevoulla Christou, Jorik Loeffler, Matthew Mackenzie, Elke Malzer, Joffrey Mianné, Krystian J. Nowicki, Edward J. O’Neill, Fran J. Pike, Marie Hutchison, Benoit Petit-Demoulière, Michelle E. Stewart, Hilary Gates, Sara Wells, Nicholas D. Sanderson, Lydia Teboul</p>
Background <p>Recent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, <i>via</i> homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders.</p> Methodology/Principal findings <p>In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles.</p> Conclusion <p>Targeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis.</p>Succinate utilisation by <i>Salmonella</i> is inhibited by multiple regulatory systemsNicolas WennerXiaojun ZhuWill P. M. RoweKristian HändlerJay C. D. Hinton10.1371/journal.pgen.10111422024-03-08T14:00:00Z2024-03-08T14:00:00Z<p>by Nicolas Wenner, Xiaojun Zhu, Will P. M. Rowe, Kristian Händler, Jay C. D. Hinton</p>
Succinate is a potent immune signalling molecule that is present in the mammalian gut and within macrophages. Both of these infection niches are colonised by the pathogenic bacterium <i>Salmonella enterica</i> serovar Typhimurium during infection. Succinate is a C<sub>4</sub>-dicarboyxlate that can serve as a source of carbon for bacteria. When succinate is provided as the sole carbon source for <i>in vitro</i> cultivation, <i>Salmonella</i> and other enteric bacteria exhibit a slow growth rate and a long lag phase. This growth inhibition phenomenon was known to involve the sigma factor RpoS, but the genetic basis of the repression of bacterial succinate utilisation was poorly understood. Here, we use an experimental evolution approach to isolate fast-growing mutants during growth of <i>S</i>. Typhimurium on succinate containing minimal medium. Our approach reveals novel RpoS-independent systems that inhibit succinate utilisation. The CspC RNA binding protein restricts succinate utilisation, an inhibition that is antagonised by high levels of the small regulatory RNA (sRNA) OxyS. We discovered that the Fe-S cluster regulatory protein IscR inhibits succinate utilisation by repressing the C<sub>4</sub>-dicarboyxlate transporter DctA. Furthermore, the ribose operon repressor RbsR is required for the complete RpoS-driven repression of succinate utilisation, suggesting a novel mechanism of RpoS regulation. Our discoveries shed light on the redundant regulatory systems that tightly regulate the utilisation of succinate. We speculate that the control of central carbon metabolism by multiple regulatory systems in <i>Salmonella</i> governs the infection niche-specific utilisation of succinate.The regulation of methylation on the Z chromosome and the identification of multiple novel Male Hyper-Methylated regions in the chickenAndrey HöglundRie HenriksenAllison M. ChurcherCarlos M. Guerrero-BosagnaAlvaro Martinez-BarrioMartin JohnssonPer JensenDominic Wright10.1371/journal.pgen.10107192024-03-08T14:00:00Z2024-03-08T14:00:00Z<p>by Andrey Höglund, Rie Henriksen, Allison M. Churcher, Carlos M. Guerrero-Bosagna, Alvaro Martinez-Barrio, Martin Johnsson, Per Jensen, Dominic Wright</p>
DNA methylation is a key regulator of eukaryote genomes, and is of particular relevance in the regulation of gene expression on the sex chromosomes, with a key role in dosage compensation in mammalian XY systems. In the case of birds, dosage compensation is largely absent, with it being restricted to two small Male Hyper-Methylated (MHM) regions on the Z chromosome. To investigate how variation in DNA methylation is regulated on the Z chromosome we utilised a wild x domestic advanced intercross in the chicken, with both hypothalamic methylomes and transcriptomes assayed in 124 individuals. The relatively large numbers of individuals allowed us to identify additional genomic MHM regions on the Z chromosome that were significantly differentially methylated between the sexes. These regions appear to down-regulate local gene expression in males, but not remove it entirely (unlike the lncRNAs identified in the initial MHM regions). These MHM regions were further tested and the most balanced genes appear to show decreased expression in males, whilst methylation appeared to be far more correlated with gene expression in the less balanced, as compared to the most balanced genes. In addition, quantitative trait loci (QTL) that regulate variation in methylation on the Z chromosome, and those loci that regulate methylation on the autosomes that derive from the Z chromosome were mapped. Trans-effect hotspots were also identified that were based on the autosomes but affected the Z, and also one that was based on the Z chromosome but that affected both autosomal and sex chromosome DNA methylation regulation. We show that both cis and trans loci that originate from the Z chromosome never exhibit an interaction with sex, whereas trans loci originating from the autosomes but affecting the Z chromosome always display such an interaction. Our results highlight how additional MHM regions are actually present on the Z chromosome, and they appear to have smaller-scale effects on gene expression in males. Quantitative variation in methylation is also regulated both from the autosomes to the Z chromosome, and from the Z chromosome to the autosomes.<i>Cis</i>-regulatory polymorphism at <i>fiz</i> ecdysone oxidase contributes to polygenic evolutionary response to malnutrition in <i>Drosophila</i>Fanny CavigliassoMikhail SavitskyAlexey KovalBerra ErkosarLoriane SavaryHector Gallart-AyalaJulijana IvanisevicVladimir L. KatanaevTadeusz J. Kawecki10.1371/journal.pgen.10112042024-03-07T14:00:00Z2024-03-07T14:00:00Z<p>by Fanny Cavigliasso, Mikhail Savitsky, Alexey Koval, Berra Erkosar, Loriane Savary, Hector Gallart-Ayala, Julijana Ivanisevic, Vladimir L. Katanaev, Tadeusz J. Kawecki</p>
We investigate the contribution of a candidate gene, <i>fiz</i> (<i>fezzik</i>), to complex polygenic adaptation to juvenile malnutrition in <i>Drosophila melanogaster</i>. Experimental populations maintained for >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower <i>fiz</i> expression compared to unselected Control populations. Here we show that this divergence in <i>fiz</i> expression is mediated by a <i>cis</i>-regulatory polymorphism. This polymorphism, originally sampled from a natural population in Switzerland, is distinct from a second <i>cis</i>-regulatory SNP previously identified in non-African <i>D</i>. <i>melanogaster</i> populations, implying that two independent <i>cis</i>-regulatory variants promoting high <i>fiz</i> expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in <i>E</i>. <i>coli</i> demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five <i>fiz</i> paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, implying that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of <i>fiz</i> in poor diet adaptation, we show that <i>fiz</i> knockdown by RNAi results in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of <i>fiz</i> in Selected populations was favored by selection on the nutritionally poor diet because of its role in suppressing growth in response to nutrient shortage. However, they suggest that <i>fiz</i> downregulation is only adaptive in combination with other changes evolved by Selected populations, which ensure that the organism can sustain the faster growth promoted by <i>fiz</i> downregulation.The PP2A-like phosphatase Ppg1 mediates assembly of the Far complex to balance gluconeogenic outputs and enables adaptation to glucose depletionShreyas NiphadkarLavanya KarinjeSunil Laxman10.1371/journal.pgen.10112022024-03-07T14:00:00Z2024-03-07T14:00:00Z<p>by Shreyas Niphadkar, Lavanya Karinje, Sunil Laxman</p>
To sustain growth in changing nutrient conditions, cells reorganize outputs of metabolic networks and appropriately reallocate resources. Signaling by reversible protein phosphorylation can control such metabolic adaptations. In contrast to kinases, the functions of phosphatases that enable metabolic adaptation as glucose depletes are poorly studied. Using a <i>Saccharomyces cerevisiae</i> deletion screen, we identified the PP2A-like phosphatase Ppg1 as required for appropriate carbon allocations towards gluconeogenic outputs—trehalose, glycogen, UDP-glucose, UDP-GlcNAc—after glucose depletion. This Ppg1 function is mediated via regulation of the assembly of the Far complex—a multi-subunit complex that tethers to the ER and mitochondrial outer membranes forming localized signaling hubs. The Far complex assembly is Ppg1 catalytic activity-dependent. Ppg1 regulates the phosphorylation status of multiple ser/thr residues on Far11 to enable the proper assembly of the Far complex. The assembled Far complex is required to maintain gluconeogenic outputs after glucose depletion. Glucose in turn regulates Far complex amounts. This Ppg1-mediated Far complex assembly, and Ppg1-Far complex dependent control of gluconeogenic outputs enables adaptive growth under glucose depletion. Our study illustrates how protein dephosphorylation is required for the assembly of a multi-protein scaffold present in localized cytosolic pools, to thereby alter gluconeogenic flux and enable cells to metabolically adapt to nutrient fluctuations.