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.ico2024-03-19T07:39:05ZAltered 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 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 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 estrogen-related receptor (ERR), 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, via 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, trans effect hotspots were also identified that were based on the autosomes but affected the Z, and also that were based on the Z chromosome but that affected autosomal DNA methylation regulation. 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.New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesisYonglin RuanXuehui LiXinyi WangGang ZhaiQiyong LouXia JinJiangyan HeJie MeiWuhan XiaoJianfang GuiZhan Yin10.1371/journal.pgen.10111702024-03-07T14:00:00Z2024-03-07T14:00:00Z<p>by Yonglin Ruan, Xuehui Li, Xinyi Wang, Gang Zhai, Qiyong Lou, Xia Jin, Jiangyan He, Jie Mei, Wuhan Xiao, Jianfang Gui, Zhan Yin</p>
The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in <i>cytochrome P450 17a1</i> (<i>cyp17a1</i>)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher <i>Fanconi anemia complementation group L</i> (<i>fancl</i>) expression in the gonads of <i>doublesex and mab-3 related transcription factor 1</i> (<i>dmrt1</i>)-/- and <i>cyp17a1</i>-/-;<i>dmrt1</i>-/- fish. The additional depletion of <i>fancl</i> in <i>cyp17a1</i>-/-;<i>dmrt1</i>-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in <i>cyp17a1</i>-/-;<i>dmrt1</i>-/-;<i>fancl</i>-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on <i>fancl</i> transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found <i>in vitro</i>. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in <i>cyp17a1</i>-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting <i>fancl</i> transcription during the critical stage of sexual fate determination in zebrafish.An epigenetic timer regulates the transition from cell division to cell expansion during Arabidopsis petal organogenesisRuirui HuangVivian F. Irish10.1371/journal.pgen.10112032024-03-05T14:00:00Z2024-03-05T14:00:00Z<p>by Ruirui Huang, Vivian F. Irish</p>
A number of studies have demonstrated that epigenetic factors regulate plant developmental timing in response to environmental changes. However, we still have an incomplete view of how epigenetic factors can regulate developmental events such as organogenesis, and the transition from cell division to cell expansion, in plants. The small number of cell types and the relatively simple developmental progression required to form the Arabidopsis petal makes it a good model to investigate the molecular mechanisms driving plant organogenesis. In this study, we investigated how the RABBIT EARS (RBE) transcriptional repressor maintains the downregulation of its downstream direct target, <i>TCP5</i>, long after RBE expression dissipates. We showed that RBE recruits the Groucho/Tup1-like corepressor TOPLESS (TPL) to repress <i>TCP5</i> transcription in petal primordia. This process involves multiple layers of changes such as remodeling of chromatin accessibility, alteration of RNA polymerase activity, and histone modifications, resulting in an epigenetic memory that is maintained through multiple cell divisions. This memory functions to maintain cell divisions during the early phase of petal development, and its attenuation in a cell division-dependent fashion later in development enables the transition from cell division to cell expansion. Overall, this study unveils a novel mechanism by which the memory of an epigenetic state, and its cell-cycle regulated decay, acts as a timer to precisely control organogenesis.RNA cis-regulators are important for <i>Streptococcus pneumoniae in vivo</i> successIndu WarrierAriana PerrySara M. HubbellMatthew EichelmanTim van OpijnenMichelle M. Meyer10.1371/journal.pgen.10111882024-03-05T14:00:00Z2024-03-05T14:00:00Z<p>by Indu Warrier, Ariana Perry, Sara M. Hubbell, Matthew Eichelman, Tim van Opijnen, Michelle M. Meyer</p>
Bacteria have evolved complex transcriptional regulatory networks, as well as many diverse regulatory strategies at the RNA level, to enable more efficient use of metabolic resources and a rapid response to changing conditions. However, most RNA-based regulatory mechanisms are not well conserved across different bacterial species despite controlling genes important for virulence or essential biosynthetic processes. Here, we characterize the activity of, and assess the fitness benefit conferred by, twelve cis-acting regulatory RNAs (including several riboswitches and a T-box), in the opportunistic pathogen <i>Streptococcus pneumoniae</i> TIGR4. By evaluating native locus mutants of each regulator that result in constitutively active or repressed expression, we establish that growth defects in planktonic culture are associated with constitutive repression of gene expression, while constitutive activation of gene expression is rarely deleterious. In contrast, in mouse nasal carriage and pneumonia models, strains with either constitutively active and repressed gene expression are significantly less fit than matched control strains. Furthermore, two RNA-regulated pathways, FMN synthesis/transport and pyrimidine synthesis/transport display exceptional sensitivity to mis-regulation or constitutive gene repression in both planktonic culture and <i>in vivo</i> environments. Thus, despite lack of obvious phenotypes associated with constitutive gene expression <i>in vitro</i>, the fitness benefit conferred on bacteria via fine-tuned metabolic regulation through cis-acting regulatory RNAs is substantial <i>in vivo</i>, and therefore easily sufficient to drive the evolution and maintenance of diverse RNA regulatory mechanisms.Genome evolution and divergence in <i>cis-</i>regulatory architecture is associated with condition-responsive development in horned dung beetlesPhillip L. DavidsonArmin P. Moczek10.1371/journal.pgen.10111652024-03-05T14:00:00Z2024-03-05T14:00:00Z<p>by Phillip L. Davidson, Armin P. Moczek</p>
Phenotypic plasticity is thought to be an important driver of diversification and adaptation to environmental variation, yet the genomic mechanisms mediating plastic trait development and evolution remain poorly understood. The Scarabaeinae, or true dung beetles, are a species-rich clade of insects recognized for their highly diversified nutrition-responsive development including that of cephalic horns—evolutionarily novel, secondary sexual weapons that exhibit remarkable intra- and interspecific variation. Here, we investigate the evolutionary basis for horns as well as other key dung beetle traits via comparative genomic and developmental assays. We begin by presenting chromosome-level genome assemblies of three dung beetle species in the tribe Onthophagini (> 2500 extant species) including <i>Onthophagus taurus</i>, <i>O</i>. <i>sagittarius</i>, and <i>Digitonthophagus gazella</i>. Comparing these assemblies to those of seven other species across the order Coleoptera identifies evolutionary changes in coding sequence associated with metabolic regulation of plasticity and metamorphosis. We then contrast chromatin accessibility in developing head horn tissues of high- and low-nutrition <i>O</i>. <i>taurus</i> males and females and identify distinct <i>cis</i>-regulatory architectures underlying nutrition- compared to sex-responsive development, including a large proportion of recently evolved regulatory elements sensitive to horn morph determination. Binding motifs of known and new candidate transcription factors are enriched in these nutrition-responsive open chromatin regions. Our work highlights the importance of chromatin state regulation in mediating the development and evolution of plastic traits, demonstrates gene networks are highly evolvable transducers of environmental and genetic signals, and provides new reference-quality genomes for three species that will bolster future developmental, ecological, and evolutionary studies of this insect group.Protein subcellular relocalization and function of duplicated flagellar calcium binding protein genes in honey bee trypanosomatid parasiteXuye YuanTatsuhiko Kadowaki10.1371/journal.pgen.10111952024-03-04T14:00:00Z2024-03-04T14:00:00Z<p>by Xuye Yuan, Tatsuhiko Kadowaki</p>
The honey bee trypanosomatid parasite, <i>Lotmaria passim</i>, contains two genes that encode the flagellar calcium binding protein (FCaBP) through tandem duplication in its genome. FCaBPs localize in the flagellum and entire body membrane of <i>L</i>. <i>passim</i> through specific N-terminal sorting sequences. This finding suggests that this is an example of protein subcellular relocalization resulting from gene duplication, altering the intracellular localization of FCaBP. However, this phenomenon may not have occurred in <i>Leishmania</i>, as one or both of the duplicated genes have become pseudogenes. Multiple copies of the <i>FCaBP</i> gene are present in several <i>Trypanosoma</i> species and <i>Leptomonas pyrrhocoris</i>, indicating rapid evolution of this gene in trypanosomatid parasites. The N-terminal flagellar sorting sequence of <i>L</i>. <i>passim</i> FCaBP1 is in close proximity to the BBSome complex, while that of <i>Trypanosoma brucei</i> FCaBP does not direct GFP to the flagellum in <i>L</i>. <i>passim</i>. Deletion of the two <i>FCaBP</i> genes in <i>L</i>. <i>passim</i> affected growth and impaired flagellar morphogenesis and motility, but it did not impact host infection. Therefore, <i>FCaBP</i> represents a duplicated gene with a rapid evolutionary history that is essential for flagellar structure and function in a trypanosomatid parasite.Divergent role of Mitochondrial Amidoxime Reducing Component 1 (MARC1) in human and mouseEriks SmagrisLisa M. ShihanianIvory J. MintahParnian BigdelouYuliya LivsonHeather BrownNiek VerweijCharleen HuntReid O’Brien JohnsonTyler J. GreerSuzanne A. HartfordGeorge HindyLuanluan SunJonas B. NielsenGabor HalaszLuca A. LottaAndrew J. MurphyMark W. SleemanViktoria Gusarova10.1371/journal.pgen.10111792024-03-04T14:00:00Z2024-03-04T14:00:00Z<p>by Eriks Smagris, Lisa M. Shihanian, Ivory J. Mintah, Parnian Bigdelou, Yuliya Livson, Heather Brown, Niek Verweij, Charleen Hunt, Reid O’Brien Johnson, Tyler J. Greer, Suzanne A. Hartford, George Hindy, Luanluan Sun, Jonas B. Nielsen, Gabor Halasz, Luca A. Lotta, Andrew J. Murphy, Mark W. Sleeman, Viktoria Gusarova</p>
Recent human genome-wide association studies have identified common missense variants in <i>MARC1</i>, p.Ala165Thr and p.Met187Lys, associated with lower hepatic fat, reduction in liver enzymes and protection from most causes of cirrhosis. Using an exome-wide association study we recapitulated earlier <i>MARC1</i> p.Ala165Thr and p.Met187Lys findings in 540,000 individuals from five ancestry groups. We also discovered novel rare putative loss of function variants in <i>MARC1</i> with a phenotype similar to <i>MARC1</i> p.Ala165Thr/p.Met187Lys variants. In vitro studies of recombinant human MARC1 protein revealed Ala165Thr substitution causes protein instability and aberrant localization in hepatic cells, suggesting MARC1 inhibition or deletion may lead to hepatoprotection. Following this hypothesis, we generated <i>Marc1</i> knockout mice and evaluated the effect of <i>Marc1</i> deletion on liver phenotype. Unexpectedly, our study found that whole-body <i>Marc1</i> deficiency in mouse is not protective against hepatic triglyceride accumulation, liver inflammation or fibrosis. In attempts to explain the lack of the observed phenotype, we discovered that Marc1 plays only a minor role in mouse liver while its paralogue Marc2 is the main Marc family enzyme in mice. Our findings highlight the major difference in MARC1 physiological function between human and mouse.Extreme restructuring of <i>cis</i>-regulatory regions controlling a deeply conserved plant stem cell regulatorDanielle CirenSophia ZebellZachary B. Lippman10.1371/journal.pgen.10111742024-03-04T14:00:00Z2024-03-04T14:00:00Z<p>by Danielle Ciren, Sophia Zebell, Zachary B. Lippman</p>
A striking paradox is that genes with conserved protein sequence, function and expression pattern over deep time often exhibit extremely divergent <i>cis</i>-regulatory sequences. It remains unclear how such drastic <i>cis</i>-regulatory evolution across species allows preservation of gene function, and to what extent these differences influence how <i>cis-</i>regulatory variation arising within species impacts phenotypic change. Here, we investigated these questions using a plant stem cell regulator conserved in expression pattern and function over ~125 million years. Using <i>in-vivo</i> genome editing in two distantly related models, <i>Arabidopsis thaliana</i> (Arabidopsis) and <i>Solanum lycopersicum</i> (tomato), we generated over 70 deletion alleles in the upstream and downstream regions of the stem cell repressor gene <i>CLAVATA3</i> (<i>CLV3</i>) and compared their individual and combined effects on a shared phenotype, the number of carpels that make fruits. We found that sequences upstream of tomato <i>CLV3</i> are highly sensitive to even small perturbations compared to its downstream region. In contrast, Arabidopsis <i>CLV3</i> function is tolerant to severe disruptions both upstream and downstream of the coding sequence. Combining upstream and downstream deletions also revealed a different regulatory outcome. Whereas phenotypic enhancement from adding downstream mutations was predominantly weak and additive in tomato, mutating both regions of Arabidopsis <i>CLV3</i> caused substantial and synergistic effects, demonstrating distinct distribution and redundancy of functional <i>cis</i>-regulatory sequences. Our results demonstrate remarkable malleability in <i>cis</i>-regulatory structural organization of a deeply conserved plant stem cell regulator and suggest that major reconfiguration of <i>cis</i>-regulatory sequence space is a common yet cryptic evolutionary force altering genotype-to-phenotype relationships from regulatory variation in conserved genes. Finally, our findings underscore the need for lineage-specific dissection of the spatial architecture of <i>cis</i>-regulation to effectively engineer trait variation from conserved productivity genes in crops.Basement membrane diversification relies on two competitive secretory routes defined by Rab10 and Rab8 and modulated by dystrophin and the exocyst complexCynthia DennisPierre PouchinGraziella RichardVincent Mirouse10.1371/journal.pgen.10111692024-03-04T14:00:00Z2024-03-04T14:00:00Z<p>by Cynthia Dennis, Pierre Pouchin, Graziella Richard, Vincent Mirouse</p>
The basement membrane (BM) is an essential structural element of tissues, and its diversification participates in organ morphogenesis. However, the traffic routes associated with BM formation and the mechanistic modulations explaining its diversification are still poorly understood. <i>Drosophila melanogaster</i> follicular epithelium relies on a BM composed of oriented BM fibrils and a more homogenous matrix. Here, we determined the specific molecular identity and cell exit sites of BM protein secretory routes. First, we found that Rab10 and Rab8 define two parallel routes for BM protein secretion. When both routes were abolished, BM production was fully blocked; however, genetic interactions revealed that these two routes competed. Rab10 promoted lateral and planar-polarized secretion, whereas Rab8 promoted basal secretion, leading to the formation of BM fibrils and homogenous BM, respectively. We also found that the dystrophin-associated protein complex (DAPC) and Rab10 were both present in a planar-polarized tubular compartment containing BM proteins. DAPC was essential for fibril formation and sufficient to reorient secretion towards the Rab10 route. Moreover, we identified a dual function for the exocyst complex in this context. First, the Exo70 subunit directly interacted with dystrophin to limit its planar polarization. Second, the exocyst complex was also required for the Rab8 route. Altogether, these results highlight important mechanistic aspects of BM protein secretion and illustrate how BM diversity can emerge from the spatial control of distinct traffic routes.Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machinesKouhei KishidaYang Grace LiNatsumi Ogawa-KishidaPratick KharaAbu Amar M. Al MamunRachel E. BossermanPeter J. Christie10.1371/journal.pgen.10110882024-03-04T14:00:00Z2024-03-04T14:00:00Z<p>by Kouhei Kishida, Yang Grace Li, Natsumi Ogawa-Kishida, Pratick Khara, Abu Amar M. Al Mamun, Rachel E. Bosserman, Peter J. Christie</p>
Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate—TraD and TraD—T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.Meiotic prophase length modulates Tel1-dependent DNA double-strand break interferenceLuz María López RuizDominic JohnsonWilliam H. GittensGeorge G. B. BrownRachal M. AllisonMatthew J. Neale10.1371/journal.pgen.10111402024-03-01T14:00:00Z2024-03-01T14:00:00Z<p>by Luz María López Ruiz, Dominic Johnson, William H. Gittens, George G. B. Brown, Rachal M. Allison, Matthew J. Neale</p>
During meiosis, genetic recombination is initiated by the formation of many DNA double-strand breaks (DSBs) catalysed by the evolutionarily conserved topoisomerase-like enzyme, Spo11, in preferred genomic sites known as hotspots. DSB formation activates the Tel1/ATM DNA damage responsive (DDR) kinase, locally inhibiting Spo11 activity in adjacent hotspots via a process known as DSB interference. Intriguingly, in <i>S</i>. <i>cerevisiae</i>, over short genomic distances (<15 kb), Spo11 activity displays characteristics of concerted activity or clustering, wherein the frequency of DSB formation in adjacent hotspots is greater than expected by chance. We have proposed that clustering is caused by a limited number of sub-chromosomal domains becoming primed for DSB formation. Here, we provide evidence that DSB clustering is abolished when meiotic prophase timing is extended via deletion of the <i>NDT80</i> transcription factor. We propose that extension of meiotic prophase enables most cells, and therefore most chromosomal domains within them, to reach an equilibrium state of similar Spo11-DSB potential, reducing the impact that priming has on estimates of coincident DSB formation. Consistent with this view, when Tel1 is absent but Ndt80 is present and thus cells are able to rapidly exit meiotic prophase, genome-wide maps of Spo11-DSB formation are skewed towards pericentromeric regions and regions that load pro-DSB factors early—revealing regions of preferential priming—but this effect is abolished when <i>NDT80</i> is deleted. Our work highlights how the stochastic nature of Spo11-DSB formation in individual cells within the limited temporal window of meiotic prophase can cause localised DSB clustering—a phenomenon that is exacerbated in <i>tel1</i>Δ cells due to the dual roles that Tel1 has in DSB interference and meiotic prophase checkpoint control.Sequestrase chaperones protect against oxidative stress-induced protein aggregation and [<i>PSI</i><sup>+</sup>] prion formationZorana CarterDeclan CreamerAikaterini KouvidiChris M. Grant10.1371/journal.pgen.10111942024-02-29T14:00:00Z2024-02-29T14:00:00Z<p>by Zorana Carter, Declan Creamer, Aikaterini Kouvidi, Chris M. Grant</p>
Misfolded proteins are usually refolded to their functional conformations or degraded by quality control mechanisms. When misfolded proteins evade quality control, they can be sequestered to specific sites within cells to prevent the potential dysfunction and toxicity that arises from protein aggregation. Btn2 and Hsp42 are compartment-specific sequestrases that play key roles in the assembly of these deposition sites. Their exact intracellular functions and substrates are not well defined, particularly since heat stress sensitivity is not observed in deletion mutants. We show here that Btn2 and Hsp42 are required for tolerance to oxidative stress conditions induced by exposure to hydrogen peroxide. Btn2 and Hsp42 act to sequester oxidized proteins into defined PQC sites following ROS exposure and their absence leads to an accumulation of protein aggregates. The toxicity of protein aggregate accumulation causes oxidant sensitivity in <i>btn2 hsp42</i> sequestrase mutants since overexpression of the Hsp104 disaggregase rescues oxidant tolerance. We have identified the Sup35 translation termination factor as an <i>in vivo</i> sequestrase substrate and show that Btn2 and Hsp42 act to suppress oxidant-induced formation of the yeast [<i>PSI</i><sup>+</sup>] prion, which is the amyloid form of Sup35. [<i>PSI</i><sup>+</sup>] prion formation in sequestrase mutants does not require IPOD (insoluble protein deposit) localization which is the site where amyloids are thought to undergo fragmentation and seeding to propagate their heritable prion form. Instead, both amorphous and amyloid Sup35 aggregates are increased in <i>btn2 hsp42</i> mutants consistent with the idea that prion formation occurs at multiple intracellular sites during oxidative stress conditions in the absence of sequestrase activity. Taken together, our data identify protein sequestration as a key antioxidant defence mechanism that functions to mitigate the damaging consequences of protein oxidation-induced aggregation.Glycan strand cleavage by a lytic transglycosylase, MltD contributes to the expansion of peptidoglycan in <i>Escherichia coli</i>Moneca KaulSuraj Kumar MeherKrishna Chaitanya NallamotuManjula Reddy10.1371/journal.pgen.10111612024-02-29T14:00:00Z2024-02-29T14:00:00Z<p>by Moneca Kaul, Suraj Kumar Meher, Krishna Chaitanya Nallamotu, Manjula Reddy</p>
Peptidoglycan (PG) is a protective sac-like exoskeleton present in most bacterial cell walls. It is a large, covalently crosslinked mesh-like polymer made up of many glycan strands cross-bridged to each other by short peptide chains. Because PG forms a continuous mesh around the bacterial cytoplasmic membrane, opening the mesh is critical to generate space for the incorporation of new material during its expansion. In <i>Escherichia coli</i>, the ‘space-making activity’ is known to be achieved by cleavage of crosslinks between the glycan strands by a set of redundant PG endopeptidases whose absence leads to rapid lysis and cell death. Here, we demonstrate a hitherto unknown role of glycan strand cleavage in cell wall expansion in <i>E</i>. <i>coli</i>. We find that overexpression of a membrane-bound lytic transglycosylase, MltD that cuts the glycan polymers of the PG sacculus rescues the cell lysis caused by the absence of essential crosslink-specific endopeptidases, MepS, MepM and MepH. We find that cellular MltD levels are stringently controlled by two independent regulatory pathways; at the step of post-translational stability by a periplasmic adaptor-protease complex, NlpI-Prc, and post-transcriptionally by RpoS, a stationary-phase specific sigma factor. Further detailed genetic and biochemical analysis implicated a role for MltD in cleaving the nascent uncrosslinked glycan strands generated during the expansion of PG. Overall, our results show that the combined activity of PG endopeptidases and lytic transglycosylases is necessary for successful expansion of the cell wall during growth of a bacterium.