Live-cell analysis of DNA methylation during sexual reproduction in Arabidopsis reveals context and sex-specific dynamics controlled by noncanonical RdDM.

Cytosine methylation is a key epigenetic mark in many organisms, important for both transcriptional control and genome integrity. While relatively stable during somatic growth, DNA methylation is reprogrammed genome-wide during mammalian reproduction. Reprogramming is essential for zygotic totipotency and to prevent transgenerational inheritance of epimutations. However, the extent of DNA methylation reprogramming in plants remains unclear. Here, we developed sensors reporting with single-cell resolution CG and non-CG methylation in Arabidopsis. Live imaging during reproduction revealed distinct and sex-specific dynamics for both contexts. We found that CHH methylation in the egg cell depends on DOMAINS REARRANGED METHYLASE 2 (DRM2) and RNA polymerase V (Pol V), two main actors of RNA-directed DNA methylation, but does not depend on Pol IV. Our sensors provide insight into global DNA methylation dynamics at the single-cell level with high temporal resolution and offer a powerful tool to track CG and non-CG methylation both during development and in response to environmental cues in all organisms with methylated DNA, as we illustrate in mouse embryonic stem cells.

Cell cycle status of male and female gametes during Arabidopsis reproduction.

Fertilization in Arabidopsis (Arabidopsis thaliana) is a highly coordinated process that begins with a pollen tube delivering the 2 sperm cells into the embryo sac. Each sperm cell can then fertilize either the egg or the central cell to initiate embryo or endosperm development, respectively. The success of this double fertilization process requires a tight cell cycle synchrony between the male and female gametes to allow karyogamy (nuclei fusion). However, the cell cycle status of the male and female gametes during fertilization remains elusive as DNA quantification and DNA replication assays have given conflicting results. Here, to reconcile these results, we quantified the DNA replication state by DNA sequencing and performed microscopic analyses of fluorescent markers covering all phases of the cell cycle. We show that male and female Arabidopsis gametes are both arrested prior to DNA replication at maturity and initiate their DNA replication only during fertilization.

TRIMETHYLGUANOSINE SYNTHASE1 mutations decanalize female germline development in Arabidopsis.

Here, we report the characterization of a plant RNA methyltransferase, orthologous to yeast trimethylguanosine synthase1 (Tgs1p) and whose downregulation was associated with apomixis in Paspalum grasses. Using phylogenetic analyses and yeast complementation, we determined that land plant genomes all encode a conserved, specific TGS1 protein. Next, we studied the role of TGS1 in female reproduction using reporter lines and loss-of-function mutants in Arabidopsis thaliana. pAtTGS1:AtTGS1 reporters showed a dynamic expression pattern. They were highly active in the placenta and ovule primordia at emergence but, subsequently, showed weak signals in the nucellus. Although expressed throughout gametophyte development, activity became restricted to the female gamete and was also detected after fertilization during embryogenesis. TGS1 depletion altered the specification of the precursor cells that give rise to the female gametophytic generation and to the sporophyte, resulting in the formation of a functional aposporous-like lineage. Our results indicate that TGS1 participates in the mechanisms restricting cell fate acquisition to a single cell at critical transitions throughout the female reproductive lineage and, thus, expand our current knowledge of the mechanisms governing female reproductive fate in plants.

Efficient but occasionally imperfect vertical transmission of gut mutualistic protists in a wood-feeding termite.

Although mutualistic associations between animals and microbial symbionts are widespread in nature, the mechanisms that have promoted their evolutionary persistence remain poorly understood. A vertical mode of symbiont transmission (from parents to offspring) is thought to ensure partner fidelity and stabilization, although the efficiency of vertical transmission has rarely been investigated, especially in cases where hosts harbour a diverse microbial community. Here we evaluated vertical transmission rates of cellulolytic gut oxymonad and parabasalid protists in the wood-feeding termite Reticulitermes grassei. We sequenced amplicons of the 18S rRNA gene of protists from 24 colonies of R. grassei collected in two populations. For each colony, the protist community was characterized from the gut of 14 swarming reproductives and from a pool of 10 worker guts. A total of 98 operational taxonomic units belonging to 13 species-level taxa were found. The vertical transmission rate was estimated for each protist present in a colony based on its frequency among the reproductives. The results revealed that transmission rates were high, with an average of 0.897 (±0.164) per protist species. Overall, the protist community did not differ between reproductive sexes, suggesting that both the queen and the king could contribute to the gut microbiota of the offspring. A positive relationship between the transmission rate of protists and their prevalence within populations was also detected. However, transmission rates alone do not explain the prevalence of protists. In conclusion, these findings reveal key forces behind a conserved, multispecies mutualism, raising further questions on the roles of horizontal transfer and negative selection in shaping symbiont prevalence.

Regulation of FUSCA3 Expression During Seed Development in Arabidopsis.

FUSCA3 (FUS3) is a master regulator of seed development important in establishing and maintaining embryonic identity whose expression is tightly regulated at genetic and epigenetic levels. Despite this prominent role, the control of FUS3 expression remains poorly understood. Promoter and functional complementation analyses provided insight into the regulation of FUS3. W-boxes present in the promoter proximal to the start of transcription are recognized by WRKY type-1 factors which are necessary for the activation of FUS3 expression. The RY motif, the binding site of B3 factors, is important for the activation of FUS3 in the embryo proper but not in the suspensor. The loss of a negative regulatory sequence (NRS) leads to preferential expression of FUS3 in the vasculature of vegetative tissues. Since the NRS includes the RY motif, mechanisms of activation and repression target adjacent or overlapping regions. These findings discriminate the regulation of FUS3 from that of LEAFY COTYLEDON2 by the control exerted by WRKY factors and by the presence of the RY motif, yet also confirm conservation of certain regulatory elements, thereby implicating potential regulation by BASIC PENTACYSTEINE (BPC) factors and POLYCOMB REPRESSIVE COMPLEX2 (PRC2).

Arabidopsis pollen tube germination and growth depend on the mitochondrial calcium uniporter complex.

The mitochondrial calcium uniporter complex (MCUc) was recently characterized in details in metazoans and consists of pore-forming units (MCUs) and regulatory factors that channel calcium (Ca2+ ) ion into the mitochondria. MCUs participate in many stress and developmentally related processes involving Ca2+ . Although multiple homologues of MCUs and one regulatory subunit are usually present in plants, the first functional characterization and contribution to Ca2+ related processes of these proteins have been reported recently. Here, we focused on two predicted Arabidopsis MCUs and studied their role in the germination and the growth of pollen tube, a tip-growing cell type highly dependent on Ca2+ homeostasis. Heterologous expression of MCU1 or MCU2 in yeast is sufficient to generate a mitochondrial Ca2+ influx. MCU1 and MCU2 fluorescent reporters are co-expressed in the vegetative cell mitochondria of the pollen grain but are undetectable in the embryo sac. We demonstrate that MCU1 and MCU2 can form a heterotypic complex. Phenotypic analyses revealed an impaired pollen tube germination and growth in vitro only for the mcu2 mutants suggesting a predominant role of MCU2. Our results show that mitochondrial Ca2+ controlled by MCUs is an additional player in Arabidopsis pollen tube germination and growth.

Isolation, characterization and PCR multiplexing of microsatellite loci for two sub-species of terrestrial isopod Porcellio dilatatus (Crustacea, Oniscidea).

Several microsatellite markers have already been developed for different terrestrial isopod species such as Armadillidium vulgare, A. nasatum and Porcellionides pruinosus. In all these species, the endosymbiont Wolbachia has a feminizing effect that generates a female bias in sex ratio and reduces the number of reproductive males. Thus this can potentially decrease the genetic diversity of host populations. However, in some other isopod species, Wolbachia induces cytoplasmic incompatibility (CI); the most commonly described effect of Wolbachia in arthropods. The CI by rendering some crossings incompatible can reduce the gene flow and strengthen genetic differentiation between isopod populations. To date, the influence of Wolbachia inducing CI on population structure of terrestrial isopods has never been investigated. In this study, we developed 10 polymorphic microsatellite markers shared by two sub-species of Porcellio dilatatus. Crossings between the two sub-species are partially incompatible due to two CI-inducing Wolbachia strains. These new microsatellite markers will allow us to investigate the effect of CI on host genetic differentiation in this species complex.

Production of viable gametes without meiosis in maize deficient for an ARGONAUTE protein.

Apomixis is a form of asexual reproduction through seeds in angiosperms. Apomictic plants bypass meiosis and fertilization, developing offspring that are genetically identical to their mother. In a genetic screen for maize (Zea mays) mutants mimicking aspects of apomixis, we identified a dominant mutation resulting in the formation of functional unreduced gametes. The mutant shows defects in chromatin condensation during meiosis and subsequent failure to segregate chromosomes. The mutated locus codes for AGO104, a member of the ARGONAUTE family of proteins. AGO104 accumulates specifically in somatic cells surrounding the female meiocyte, suggesting a mobile signal rather than cell-autonomous control. AGO104 is necessary for non-CG methylation of centromeric and knob-repeat DNA. Digital gene expression tag profiling experiments using high-throughput sequencing show that AGO104 influences the transcription of many targets in the ovaries, with a strong effect on centromeric repeats. AGO104 is related to Arabidopsis thaliana AGO9, but while AGO9 acts to repress germ cell fate in somatic tissues, AGO104 acts to repress somatic fate in germ cells. Our findings show that female germ cell development in maize is dependent upon conserved small RNA pathways acting non-cell-autonomously in the ovule. Interfering with this repression leads to apomixis-like phenotypes in maize.

Inactivation of a DNA methylation pathway in maize reproductive organs results in apomixis-like phenotypes.

Apomictic plants reproduce asexually through seeds by avoiding both meiosis and fertilization. Although apomixis is genetically regulated, its core genetic component(s) has not been determined yet. Using profiling experiments comparing sexual development in maize (Zea mays) to apomixis in maize-Tripsacum hybrids, we identified six loci that are specifically downregulated in ovules of apomictic plants. Four of them share strong homology with members of the RNA-directed DNA methylation pathway, which in Arabidopsis thaliana is involved in silencing via DNA methylation. Analyzing loss-of-function alleles for two maize DNA methyltransferase genes belonging to that subset, dmt102 and dmt103, which are downregulated in the ovules of apomictic plants and are homologous to the Arabidopsis CHROMOMETHYLASEs and DOMAINS REARRANGED METHYLTRANSFERASE families, revealed phenotypes reminiscent of apomictic development, including the production of unreduced gametes and formation of multiple embryo sacs in the ovule. Loss of DMT102 activity in ovules resulted in the establishment of a transcriptionally competent chromatin state in the archesporial tissue and in the egg cell that mimics the chromatin state found in apomicts. Interestingly, dmt102 and dmt103 expression in the ovule is found in a restricted domain in and around the germ cells, indicating that a DNA methylation pathway active during reproduction is essential for gametophyte development in maize and likely plays a critical role in the differentiation between apomictic and sexual reproduction.

Development of a primary cell model derived from porcine dorsal soft palate for foot-and-mouth disease virus research and diagnosis.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals that has a significant socio-economic impact. One concern associated with this disease is the ability of its etiological agent, the FMD virus (FMDV), to persist in its hosts through underlying mechanisms that remain to be elucidated. While persistence has been described in cattle and small ruminants, it is unlikely to occur in pigs. One of the factors limiting the progress in understanding FMDV persistence and, in particular, differential persistence is the lack of suitable in vitro models. A primary bovine cell model derived from the dorsal soft palate, which is the primary site of replication and persistence of FMDV in cattle, has been developed, and it seemed relevant to develop a similar porcine model. Cells from two sites of FMDV replication in pigs, namely, the dorsal soft palate and the oropharyngeal tonsils, were isolated and cultured. The epithelial character of the cells from the dorsal soft palate was then assessed by immunofluorescence. The FMDV-sensitivity of these cells was assessed after monolayer infection with FMDV O/FRA/1/2001 Clone 2.2. These cells were also grown in multilayers at the air-liquid interface to mimic a stratified epithelium susceptible to FMDV infection. Consistent with what has been shown in vivo in pigs, our study showed no evidence of persistence of FMDV in either the monolayer or multilayer model, with no infectious virus detected 28 days after infection. The development of such a model opens up new possibilities for the study and diagnosis of FMDV in porcine cells.

[Setting up indicators in biobanking: why and how?]. / Mise en place d'indicateurs de suivi au sein d'une tumorothèque et/ou d'un centre de ressources biologiques : pourquoi et comment ?

The biobanking area is highly complex, and its complexity is increasing along with its growth and demand. Due to the advancements in genetic research, stem cell research and regenerative medicine, biobanking has become ever more important and plays a key role in biomedical research. The robustness and the reproducibility of research results depend greatly on the quality and on the number of the samples used, and thus on the expertise of biobanks having supplied these samples. Undoubtedly, the recognition of a research biobank depends on the impact of the research projects conducted with samples obtained from tumour bank(s), but also on many other criteria. It thus seems important to determine a number of indicators within a biobank to estimate objective criteria for the performance of these structures. These indicators can allow to make some strategic decisions knowing that biobanks are expensive structures to maintain in the present hospital context. The use of these indicators could also contribute to the elaboration of an "biobank impact factor of" or so called "bioresource research impact factor" (BRIF). We describe here four major categories of indicators (quality, activity, scientific production, visibility), which seem to be useful for the evaluation of a biobank by making a proposition of allocation of coefficients for the various considered items.

Organ geometry channels reproductive cell fate in the Arabidopsis ovule primordium.

In multicellular organisms, sexual reproduction requires the separation of the germline from the soma. In flowering plants, the female germline precursor differentiates as a single spore mother cell (SMC) as the ovule primordium forms. Here, we explored how organ growth contributes to SMC differentiation. We generated 92 annotated 3D images at cellular resolution in Arabidopsis. We identified the spatio-temporal pattern of cell division that acts in a domain-specific manner as the primordium forms. Tissue growth models uncovered plausible morphogenetic principles involving a spatially confined growth signal, differential mechanical properties, and cell growth anisotropy. Our analysis revealed that SMC characteristics first arise in more than one cell but SMC fate becomes progressively restricted to a single cell during organ growth. Altered primordium geometry coincided with a delay in the fate restriction process in katanin mutants. Altogether, our study suggests that tissue geometry channels reproductive cell fate in the Arabidopsis ovule primordium.

Differential involvement of the five RNA helicases in adaptation of Bacillus cereus ATCC 14579 to low growth temperatures.

Bacillus cereus ATCC 14579 possesses five RNA helicase-encoding genes overexpressed under cold growth conditions. Out of the five corresponding mutants, only the ΔcshA, ΔcshB, and ΔcshC strains were cold sensitive. Growth of the ΔcshA strain was also reduced at 30°C but not at 37°C. The cold phenotype was restored with the cshA gene for the ΔcshA strain and partially for the ΔcshB strain but not for the ΔcshC strain, suggesting different functions at low temperature.

The YvfTU two-component system is involved in plcR expression in Bacillus cereus.

BACKGROUND: Most extracellular virulence factors produced by Bacillus cereus are regulated by the pleiotropic transcriptional activator PlcR. Among strains belonging to the B. cereus group, the plcR gene is always located in the vicinity of genes encoding the YvfTU two-component system. The putative role of YvfTU in the expression of the PlcR regulon was therefore investigated. RESULTS: Expression of the plcR gene was monitored using a transcriptional fusion with a lacZ reporter gene in a yvfTU mutant and in its B. cereus ATCC 14579 parental strain. Two hours after the onset of the stationary phase, a stage at which the PlcR regulon is highly expressed, the plcR expression in the yvfTU mutant was only 50% of that of its parental strain. In addition to the reduced plcR expression in the yvfTU mutant, a few members of the PlcR regulon showed a differential expression, as revealed by transcriptomic and proteomic analyses. The virulence of the yvfTU mutant in a Galleria mellonella insect model was slightly lower than that of the parental strain. CONCLUSION: The YvfTU two-component system is not required for the expression of most of the virulence factors belonging to the PlcR regulon. However, YvfTU is involved in expression of plcR, a major regulator of virulence in B. cereus.

Insertional mutagenesis reveals genes involved in Bacillus cereus ATCC 14579 growth at low temperature.

Transposon mutagenesis of Bacillus cereus ATCC 14579 yielded cold-sensitive mutants. Mutants of genes encoding enzymes of the central metabolism were affected by cold, but also by other stresses, such as pH or salt, whereas a mutant with transposon insertion in the promoter region of BC0259 gene, encoding a putative DEAD-box RNA helicase displaying homology with Escherichia coli CsdA and Bacillus subtilis CshA RNA helicases, was only cold-sensitive. Expression of the BC0259 gene at 10 degrees C is reduced in the mutant. Analysis of the 5' untranslated region revealed the transcriptional start and putative cold shock-responsive elements. The role of this RNA helicase in the cold-adaptive response of B. cereus is discussed.