Biology subjects

Martin, F.

Publications and source records attributed to Martin, F..

32 records · Page 2Linked to original sources

bioRxiv2022

The genome of the oomycete Peronosclerospora sorghi, a cosmopolitan pathogen of maize and sorghum, is inflated with dispersed pseudogenes

Several species in the oomycete genus Peronosclerospora cause downy mildew on maize and can result in significant yield losses in Asia. Biosurveillance of these pathogens is a high priority to prevent epidemics on maize in the US and consequent damage to the US economy. The unresolved taxonomy and dearth of molecular resources for Peronosclerospora spp. hinder these efforts. P. sorghi is a pathogen of sorghum and maize with a global distribution, for which limited diversity has been detected in the southern USA. We characterized the genome, transcriptome, and mitogenome of an isolate, representing the US pathotype 6. The highly homozygous genome was assembled using 10x Genomics linked reads and scaffolded using Hi-C into 13 chromosomes. The total assembled length was 319.6 Mb--larger than any other oomycete previously assembled. The mitogenome was 38 kb, similar in size to other oomycetes, although it had a unique gene order. Nearly 20,000 genes were annotated in the nuclear genome, more than described for other downy mildew causing oomycetes. The 13 chromosomes of P. sorghi were highly syntenic with the 17 chromosomes of Peronospora effusa with conserved centromeric regions and distinct chromosomal fusions. The increased assembly size and gene count of P. sorghi is due to extensive retrotransposition, resulting in putative pseudogenization. Ancestral genes had higher transcript abundance and were enriched for differential expression. This study provides foundational resources for analysis of Peronosclerospora and comparisons to other oomycete genera. Further genomic studies of global Peronosclerospora spp. will determine the suitability of the mitogenome, ancestral genes, and putative pseudogenes for marker development and taxonomic relationships.

genomics↗

bioRxiv2021

LRH-1/NR5A2 regulates the PTGS2-PGE2-PTGER1 signalling axis contributing to islet survival and antidiabetic actions of the agonist BL001

We have previously described a role of LRH-1/NR5A2 in islet morphogenesis during postnatal development and reported that the treatment with BL001, an agonist of LRH-1/NR5A2, protects islets against-stress induced apoptosis and reverts hyperglycemia in 3 mouse models of Type 1 Diabetes Mellitus (T1DM). Islet transcriptome profiling revealed that most differentially expressed genes after BL001 treatment are involved in immunomodulation, among them, the increase in PTGS2/COX2 expression. Herein, we dissected the cellular and molecular branches of the BL001/LRH-1/NR5A2 signalling axis in order to chart the mode of action confering beta cell protection and hyperglycaemia reversion. We found that constitutive LRH-1/NR5A2 ablation within the insulin expression domain (RIP-Cre mouse model) caused a significant beta cell mass reduction characterized by blunted proliferation correlating with animal growth retardation, weight loss and hypoglycemia, leading to lethality before weaning. Using an inducible approach (pdx1PBCreER mouse model), specific deletion of LRH-1/NR5A2 in adult beta cells abolished the anti diabetic effect of BL001 in streptozotocin treated mice, correlating with complete beta-cell mass destruction. Additionally, BL001 induced Ptgs2 expression, was blunted in islets lacking LRH-1/NR5A2. The combined BL001/cytokine treatment did not further stimulate Ptgs2 expression above levels detected with cytokine alone yet secreted PGE2 levels were increased 5-fold. Inactivation of PTGS2 blunted induction of the target and its product PGE2 in islets treated with cytokines alone or with BL001. Importantly, PTGS2 inactivated islets were refractory to the BL001 protective effect under cytokine attack as evidenced by increased Bax expression levels, cytochrome C release and cleaved PARP. The PTGER1 antagonist ONO-8130, but not the PTGER4 antagonist L-161,982, negated BL001-mediated islet survival. Our results establish that the beneficial properties of BL001 against stress-induced cell death are specifically conveyed by LRH-1/NR5A2 activation in beta cells and downstream stimulation of the PTGS2-PGE2/PTGER1 signalling axis.

physiology↗

bioRxiv2021

The ectomycorrhizal basidiomycete Laccaria bicolor releases a GH28 polygalacturonase that plays a key role in symbiosis establishment

O_LIIn ectomycorrhiza, root penetration and colonization of the intercellular space by symbiotic hyphae is thought to rely on the mechanical force that results from hyphal tip growth, enhanced by the activity of secreted cell-wall-degrading enzymes. C_LIO_LIHere, we characterize the biochemical properties of the symbiosis-induced polygalacturonase LbGH28A from the ectomycorrhizal fungus Laccaria bicolor. The transcriptional regulation of LbGH28A was measured by qPCR. The biological relevance of LbGH28A was confirmed by generating RNAi-silenced LbGH28A mutants. We localized the LbGH28A protein by immunofluorescence confocal and immunogold cytochemical microscopy in poplar ectomycorrhizal roots. C_LIO_LIqPCR confirmed the induced expression of LbGH28A during ectomycorrhiza formation. L. bicolor RNAi mutants have a lower ability to establish ectomycorrhiza confirming the key role of this enzyme in symbiosis. The purified recombinant LbGH28A has its highest activity towards pectin and polygalacturonic acid. In situ localization of LbGH28A indicates that this endopolygalacturonase is located in both fungal and plant cell walls at the symbiotic hyphal front. C_LIO_LIThe present findings suggest that the symbiosis-induced pectinase LbGH28A is involved in the Hartig net formation and is an important determinant for successful symbiotic colonization. C_LI

plant biology↗

bioRxiv2021

Molecular basis of differential adventitious rooting competence in poplar genotypes

O_LIRecalcitrant adventitious root (AR) development is a major hurdle in propagating commercially important woody plants. Although significant progress has been made to identify genes involved in subsequent steps of AR development, the molecular basis of differences in apparent recalcitrance to form AR between easy-to-root and difficult-to-root genotypes remains unknown. C_LIO_LITo address this, we generated cambium tissue-specific transcriptomic data from stem cuttings of hybrid aspen, T89 (difficult-to-root) and hybrid poplar OP42 (easy-to-root) and used transgenic approaches to verify the role of several transcription factors (TF) in the control of adventitious rooting. C_LIO_LIIncreased peroxidase activity is positively correlated with better rooting. We found differentially expressed genes encoding Reactive Oxygen Species (ROS) scavenging proteins to be enriched in OP42 compared to T89. A higher number of differentially expressed TF in OP42 compared to T89 cambium cells was revealed by a more intense transcriptional reprograming in the former. PtMYC2, a potential negative regulator, was less expressed in OP42 compared to T89. Using transgenic approaches, we have demonstrated that PttARF17.1 and PttMYC2.1 negatively regulate adventitious rooting. C_LIO_LIOur results provide insights into the molecular basis of genotypic differences in AR and implicate differential expression of the master regulator MYC2 as a critical player in this process. C_LI

plant biology↗

bioRxiv2021

Cryptic genetic structure and copy-number variation in the ubiquitous forest symbiotic fungus Cenococcum geophilum

Ectomycorrhizal (ECM) fungi associated with plants constitute one of the most successful symbiotic interactions in forest ecosystems. ECM support trophic exchanges with host plants and are important factors for the survival and stress resilience of trees. However, ECM clades often harbour morpho-species and cryptic lineages, with weak morphological differentiation. How this relates to intraspecific genome variability and ecological functioning is poorly known. Here, we analysed 16 European isolates of the ascomycete Cenococcum geophilum, an extremely ubiquitous forest symbiotic fungus with no known sexual or asexual spore forming structures but with a massively enlarged genome. We carried out whole-genome sequencing to identify single-nucleotide polymorphisms. We found no geographic structure at the European scale but divergent lineages within sampling sites. Evidence for recombination was restricted to specific cryptic lineages. Lineage differentiation was supported by extensive copy-number variation. Finally, we confirmed heterothallism with a single MAT1 idiomorph per genome. Synteny analyses of the MAT1 locus revealed substantial rearrangements and a pseudogene of the opposite MAT1 idiomorph. Our study provides the first evidence for substantial genome-wide structural variation, lineage-specific recombination and low continent-wide genetic differentiation in C. geophilum. Our study provides a foundation for targeted analyses of intra-specific functional variation in this major symbiosis. Originality-Significance StatementWe provide the first report on the genetic structure and copy-number variation of the globally ubiquitous and key forest symbiotic fungus Cenococcum geophilum using whole-genome sequencing data. We found divergent lineages within sampling sites, while closely related lineages appear over large geographic distances on a continental scale. Even though no sexual spore forming structures have been reported to date, we provide evidence of recombination in a specific lineage suggesting mating activity. Our findings help explain the high genetic diversity occurring within populations and their resilience to changing and adverse environmental conditions. Furthermore, we identify a single MAT1 idiomorph per genome, confirming heterothallism, and discover that major genomic rearrangements are found in their flanking regions based on chromosomal synteny analysis. Intriguingly, a pseudogene of the opposite functional idiomorph has been characterised in each genome, suggesting a common homothallic ancestor to the species. As Cenococcum geophilum is a pivotal mycorrhizal associate of a broad range of trees and shrubs providing nutrition and water supply to their hosts, we highlight and discuss the potential role of the large genome-wide structural variations in environmental selection.

genomics↗

bioRxiv2021

The Metabesity Factor HMG20A Potentiates Astrocyte Survival and Reactivity Preserving Neuronal Integrity

RationaleWe recently demonstrated that the Metabesity factor HMG20A regulates islet beta-cell functional maturity and adaptation to physiological stress such as pregnancy and pre-diabetes. HMG20A also dictates central nervous system (CNS) development via inhibition of the LSD1/CoREST complex but its expression pattern and function in adult brain remains unknown. Herein we sought to determine whether HMG20A is expressed in the adult CNS, specifically in hypothalamic astrocytes that are key in glucose homeostasis and whether similar to islets, HMG20A potentiates astrocyte function in response to environmental cues. MethodsHMG20A expression profile was assessed by quantitative PCR (RT-PCR) and/or immunofluorescence in: 1) the hypothalamus of mice exposed or not to a high-fat diet, 2) human blood leukocytes and adipose tissue obtained from healthy or diabetic individuals 3) primary mouse hypothalamic astrocytes exposed to either high glucose or palmitate. To investigate the function and regulatory mechanism of HMG20A, RNA-seq and cell metabolic parameters were performed on astrocytes treated or not with a siHMG20A. The regulatory function of HMG20A on astrogliosis was also assessed pharmacologically using ORY1001. Astrocyte-mediated neuronal survival was evaluated using conditioned media from siHMG20A-treated astrocytes. ResultsWe show that Hmg20a is predominantly expressed in hypothalamic astrocytes, the main nutrient-sensing cell type of the brain. Hmg20A expression was upregulated in diet-induced obesity and glucose intolerant mice, correlating with increased transcript levels of Gfap and Il1b indicative of inflammation and astrogliosis. Expression levels were also increased in adipose tissue of obese non-diabetic individuals as compared to obese diabetic patients. HMG20A silencing in astrocytes resulted in repression of inflammatory, cholesterol biogenesis and epithelial-to-mesenchymal transition pathways with a concomitant increase in apoptosis and reduced mitochondrial bioenergetics. Motoneuron viability was also hindered in HMG20A-depleted astrocyte-derived conditioned media. Astrogliosis was induced using ORY1001, a pharmacological inhibitor of the LSD1/CoREST complex, mimicking the effect of HMG20A. ConclusionHMG20A coordinates the astrocyte polarization state. Under physiological pressure such as obesity and insulin resistance that induces low grade inflammation, HMG20A expression is increased to induce astrogliosis in an attempt to preserve the neuronal network and glucose homeostasis. Nonetheless, a chronic metabesity state or functional mutations will result in lower levels of HMG20A, failure to promote astrogliosis and increase susceptibility of neurons to stress-mediated apoptosis. Such effects could be therapeutically reversed by ORY1001-induced astrogliosis.

cell biology↗

bioRxiv2021

Translation Inhibitory Elements from Hox a3 and a11 mRNAs use uORFs for translation inhibition

During embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5UTR. First, an Internal Ribosome Entry Site (IRES) enables cap-independent translation. The second regulon is a Translation Inhibitory Element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of Hox a3 and a11 TIE elements. Both TIEs possess an upstream Open Reading Frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In TIE a3, we identify a uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of TIE a11 is different, it also contains a uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a start-stop. The a11 start-stop sequence is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hox a11 main ORF.

biochemistry↗

bioRxiv2020

Colonization of naïve roots from Populus tremula x alba involves successive waves of fungi andbacteria with different trophic abilities

Through their roots, trees interact with a highly complex community of microorganisms belonging to various trophic guilds and contributing to tree nutrition, development and protection against stresses. Tree roots select for specific microbial species from the bulk soil communities. The root microbiome formation is a dynamic process but little is known on how the different microorganisms colonize the roots and how the selection occurs. To decipher if the final composition of the root microbiome is the product of several waves of colonization by different guilds of microorganisms, we planted sterile rooted cuttings of Gray Poplar obtained from plantlets propagated in axenic conditions in natural soil taken from a poplar stand. We analyzed the root microbiome at different time points between 2 and 50 days of culture by combining high-throughput Illumina MiSeq sequencing of fungal rDNA ITS and bacterial 16S rRNA amplicons with Confocal Laser Scanning Microscope observations. The microbial colonisation of poplar roots took place in three stages but the dynamic was different between bacteria and fungi. Root bacterial communities were clearly different from the soil after two days of culture. By contrast, if fungi were also already colonizing roots after two days, the initial communities were very close to the one of the soil and were dominated by saprotrophs. Those were slowly replaced by endophytes and ectomycorhizal fungi. The replacement of the most abundant fungal and bacterial community members observed in poplar roots along time suggest potential competition effect between microorganisms and/or a selection by the host. ImportanceThe tree root microbiome is composed of a very diverse set of bacterial and fungal communities. These microorganisms have a profound impact on tree growth, development and protection against different types of stress. They mainly originate from the bulk soil and colonize the root system which provides a unique nutrient rich-environment for a diverse assemblage of microbial communities. In order to better understand how the tree root microbiome is shaped along time, we observed the composition of root-associated microbial communities of naive plantlets of poplar transferred in natural soil. The composition of the final root microbiome rely on a series of colonization stages characterized by the dominance of different fungal guilds and bacterial community members along time. Our observations suggest an early stabilization of bacterial communities, whereas fungal communities are established following a more gradual pattern.

microbiology↗

bioRxiv2020

The viral protein NSP1 acts as a ribosome gatekeeper for shutting down host translation and fostering SARS-CoV-2 translation

SARS-CoV-2 coronavirus is responsible for Covid-19 pandemic. In the early phase of infection, the single-strand positive RNA genome is translated into non-structural proteins (NSP). One of the first proteins produced during viral infection, NSP1, binds to the host ribosome and blocks the mRNA entry channel. This triggers translation inhibition of cellular translation. In spite of the presence of NSP1 on the ribosome, viral translation proceeds however. The molecular mechanism of the so-called viral evasion to NSP1 inhibition remains elusive. Here, we confirm that viral translation is maintained in the presence of NSP1. The evasion to NSP1-inhibition is mediated by the cis-acting RNA hairpin SL1 in the 5UTR of SARS-CoV-2. NSP1-evasion can be transferred on a reporter transcript by SL1 transplantation. The apical part of SL1 is only required for viral translation. We show that NSP1 remains bound on the ribosome during viral translation. We suggest that the interaction between NSP1 and SL1 frees the mRNA accommodation channel while maintaining NSP1 bound to the ribosome. Thus, NSP1 acts as a ribosome gatekeeper, shutting down host translation or fostering SARS-CoV-2 translation depending on the presence of the SL1 5UTR hairpin. SL1 is also present and necessary for translation of sub-genomic RNAs in the late phase of the infectious program. Consequently, therapeutic strategies targeting SL1 should affect viral translation at early and late stages of infection. Therefore, SL1 might be seen as a genuine Achille heel of the virus.

molecular biology↗

bioRxiv2020

Spelling changes and fluorescent tagging with prime editing vectors for plants

Prime editing (PE) is a recent adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase (RT) fusion and a guide RNA (pegRNA) amended with template and primer binding site (PBS) sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of PE in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for PE in dicots and monocots, their validation in Nicotiana benthamiana, rice and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.

plant biology↗

bioRxiv2020

LINE elements are a reservoir of regulatory potential in mammalian genomes

To investigate the mechanisms driving regulatory evolution across tissues, we experimentally mapped promoters, enhancers, and gene expression in liver, brain, muscle, and testis from ten diverse mammals. The regulatory landscape around genes included both tissue-shared and tissue-specific regulatory regions, where tissue-specific promoters and enhancers evolved most rapidly. Genomic regions switching between promoters and enhancers were more common across species, and less common across tissues within a single species. Long Interspersed Nuclear Elements (LINEs) played recurrent evolutionary roles: LINE L1s were associated with tissue-specific regulatory regions, whereas more ancient LINE L2s were associated with tissue-shared regulatory regions and with those switching between promoter and enhancer signatures across species. Our analyses of the tissue-specificity and evolutionary stability among promoters and enhancers reveal how specific LINE families have helped shape the dynamic mammalian regulome. HIGHLIGHTSO_LITissue-specific regulatory regions evolve faster than tissue-shared C_LIO_LISwitching promoter and enhancer regulatory roles is frequent in evolution C_LIO_LILINE L1s contribute to the evolution of tissue-specific regulatory regions C_LIO_LILINE L2s are associated with broad tissue activity and dynamic regulatory signatures C_LI

genomics↗

bioRxiv2020

RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors

Plant diseases are often diagnosed by the method of DNA extraction followed by PCR. DNA extraction from plant tissue can be a recalcitrant and lengthy process, and sometimes ends up with inhibitors that reduce PCR amplification efficiency. Here we present a unique approach, RPA-PCR couple, to exclude the DNA extraction step from the standard plant diagnostic process. The process crudely macerates plant tissue in water for a few minutes, and then transfers the macerate supernatant to a Recombinase Polymerase Amplification (RPA) reaction. Following an incubation of 20 minutes at 39 {degrees}C, the RPA reaction can be directly utilized in PCR amplification. In RPA-PCR couple, the RPA reaction is run at slower reaction kinetics to promotes amplification of long amplicons and the slower reaction kinetics are achieved by lowering RPA components concentrations. In this proof of concept study, we targeted Phytophthora intergenic mitochondrial spacer between atp9 and nad9 genes and the two common Phytophthora pathogens of strawberry: P. fragariae and P. cactorum. We presented coupling of RPA with real time TaqMan and SYBR Green PCR assays, and conventional PCR amplification aimed at Sanger sequencing. We found the RPA-PCR couple specific and capable of detecting as low as 10 fg of Phytophthora genomic DNA. Moreover, comparing RPA-PCR couple with the routine method of DNA extraction followed by PCR generated comparable results for the field samples. The idea of RPA-PCR couple to exclude DNA extraction may have vast application in different fields such as clinical diagnostics, food inspection and soil sciences.

molecular biology↗

bioRxiv2018

RNA Binding Motif Protein 48 is required for U12 splicing and maize endosperm differentiation

The last eukaryotic common ancestor had two classes of introns that are still found in most eukaryotic lineages. Common U2-type and rare U12-type introns are spliced by the major and minor spliceosomes, respectively. Relatively few splicing factors have been shown to be specific to the minor spliceosome. We found that the maize RNA Binding Motif Protein48 (RBM48) is a U12 splicing factor that functions to promote cell differentiation and repress cell proliferation. RBM48 is coselected with the U12 splicing factor, ZRSR2/RGH3. Protein-protein interactions between RBM48, RGH3, and U2 Auxiliary Factor (U2AF) subunits suggest major and minor spliceosome factors may form complexes during intron recognition. Human RBM48 interacts with ARMC7. Maize RBM48 and ARMC7 have a conserved protein-protein interaction. These data predict that RBM48 is likely to function in U12 splicing throughout eukaryotes and that U12 splicing promotes endosperm cell differentiation in maize.

plant biology↗

bioRxiv2017

Repeat associated mechanisms of genome evolution and function revealed by the Mus caroli and Mus pahari genomes

Understanding the mechanisms driving lineage-specific evolution in both primates and rodents has been hindered by the lack of sister clades with a similar phylogenetic structure having high-quality genome assemblies. Here, we have created chromosome-level assemblies of the Mus caroli and Mus pahari genomes. Together with the Mus musculus and Rattus norvegicus genomes, this set of rodent genomes is similar in divergence times to the Hominidae (human-chimpanzee-gorilla-orangutan). By comparing the evolutionary dynamics between the Muridae and Hominidae, we identified punctate events of chromosome reshuffling that shaped the ancestral karyotype of Mus musculus and Mus caroli between 3 to 6 MYA, but that are absent in the Hominidae. In fact, Hominidae show between four-and seven-fold lower rates of nucleotide change and feature turnover in both neutral and functional sequences suggesting an underlying coherence to the Muridae acceleration. Our system of matched, high-quality genome assemblies revealed how specific classes of repeats can play lineage-specific roles in related species. For example, recent LINE activity has remodeled protein-coding loci to a greater extent across the Muridae than the Hominidae, with functional consequences at the species level such as reproductive isolation. Furthermore, we charted a Muridae-specific retrotransposon expansion at unprecedented resolution, revealing how a single nucleotide mutation transformed a specific SINE element into an active CTCF binding site carrier specifically in Mus caroli. This process resulted in thousands of novel, species-specific CTCF binding sites. Our results demonstrate that the comparison of matched phylogenetic sets of genomes will be an increasingly powerful strategy for understanding mammalian biology.

genomics↗