Transgenerational Maintenance Research Articles

Immune priming in plants: from the onset to transgenerational maintenance.

Enhancing plant resistance against pests and diseases by priming plant immunity is an attractive concept for crop protection because it provides long-lasting broad-spectrum protection against pests and diseases. This review provides a selected overview of the latest advances in research on the molecular, biochemical and epigenetic drivers of plant immune priming. We review recent findings about the perception and signalling mechanisms controlling the onset of priming by the plant stress metabolite β-aminobutyric acid. In addition, we review the evidence for epigenetic regulation of long-term maintenance of priming and discuss how stress-induced reductions in DNA hypomethylation at transposable elements can prime defence genes. Finally, we examine how priming can be exploited in crop protection and articulate the opportunities and challenges of translating research results from the Arabidopsis model system to crops.

Single mutation makes Escherichia coli an insect mutualist

Microorganisms often live in symbiosis with their hosts, and some are considered mutualists, where all species involved benefit from the interaction. How free-living microorganisms have evolved to become mutualists is unclear. Here we report an experimental system in which non-symbiotic Escherichia coli evolves into an insect mutualist. The stinkbug Plautia stali is typically associated with its essential gut symbiont, Pantoea sp., which colonizes a specialized symbiotic organ. When sterilized newborn nymphs were infected with E. coli rather than Pantoea sp., only a few insects survived, in which E. coli exhibited specific localization to the symbiotic organ and vertical transmission to the offspring. Through transgenerational maintenance with P. stali, several hypermutating E. coli lines independently evolved to support the host’s high adult emergence and improved body colour; these were called ‘mutualistic’ E. coli. These mutants exhibited slower bacterial growth, smaller size, loss of flagellar motility and lack of an extracellular matrix. Transcriptomic and genomic analyses of ‘mutualistic’ E. coli lines revealed independent mutations that disrupted the carbon catabolite repression global transcriptional regulator system. Each mutation reproduced the mutualistic phenotypes when introduced into wild-type E. coli, confirming that single carbon catabolite repression mutations can make E. coli an insect mutualist. These findings provide an experimental system for future work on host–microbe symbioses and may explain why microbial mutualisms are omnipresent in nature.

Genome-Wide Distribution of Nascent Transcripts in Sperm DNA, Products of a Late Wave of General Transcription.

Mature spermatozoa contain a whole repertoire of the various classes of cellular RNAs, both coding and non-coding. It was hypothesized that after fertilization they might impact development, a claim supported by experimental evidence in various systems. Despite the current increasing interest in the transgenerational maintenance of epigenetic traits and their possible determination by RNAs, little remains known about conservation in sperm and across generations and the specificities and mechanisms involved in transgenerational maintenance. We identified two distinct fractions of RNAs in mature mouse sperm, one readily extracted in the aqueous phase of the classical TRIzol procedure and a distinct fraction hybridized with homologous DNA in DNA-RNA complexes recovered from the interface, purified after DNase hydrolysis and analyzed by RNA-seq methodology. This DNA-associated RNA (D RNA) was found to represent as much as half of the cell contents in differentiated sperm, in which a major part of the cytoplasmic material has been discarded. Stable complexes were purified free of proteins and identified as hybrids (R-loops) on the basis of their sensitivity to RNase H hydrolysis. Further analysis by RNA-seq identified transcripts from all the coding and non-coding regions of the genome, thus revealing an extensive wave of transcription, prior to or concomitant with the terminal compaction of the chromatin.

A Bidimensional Segregation Mode Maintains Symbiont Chromosome Orientation toward Its Host.

All living organisms require accurate segregation of their genetic material. However, in microbes, chromosome segregation is less understood than replication and cell division, which makes its decipherment a compelling research frontier. Furthermore, it has only been studied in free-living microbes so far. Here, we investigated this fundamental process in arod-shaped symbiont, Candidatus Thiosymbion oneisti. This gammaproteobacterium divides longitudinally as to form a columnar epithelium ensheathing its nematode host. We hypothesized that uninterrupted host attachment would affect bacterial chromosome dynamics and set out to localize specific chromosomal loci and putative DNA-segregating proteins by fluorescence in situ hybridization and immunostaining, respectively. First, DNA replication origins (ori) number per cell demonstrated symbiont monoploidy. Second, we showed that sister ori segregate diagonally prior to septation onset. Moreover, the localization pattern of the centromere-binding protein ParB recapitulates that of ori, and consistently, we showed recombinant ParB to specifically bind an ori-proximal site (parS) invitro. Third, chromosome replication ends prior to cell fission, and as the poles start to invaginate, termination of replication (ter) sites localize medially, at the leading edges of the growing septum. They then migrate to midcell, concomitantly with septation progression and until this is completed. In conclusion, we propose that symbiont ParB might drive chromosome segregation along the short axis and that tethering of sister ter regions to the growing septum mediates their migration along the long axis. Crucially, active bidimensional segregation of the chromosome allows transgenerational maintenance of its configuration, and therefore, it may represent an adaptation to symbiosis. VIDEO ABSTRACT.

The meiotic phosphatase GSP-2/PP1 promotes germline immortality and small RNA-mediated genome silencing.

Germ cell immortality, or transgenerational maintenance of the germ line, could be promoted by mechanisms that could occur in either mitotic or meiotic germ cells. Here we report for the first time that the GSP-2 PP1/Glc7 phosphatase promotes germ cell immortality. Small RNA-induced genome silencing is known to promote germ cell immortality, and we identified a separation-of-function allele of C. elegans gsp-2 that is compromised for germ cell immortality and is also defective for small RNA-induced genome silencing and meiotic but not mitotic chromosome segregation. Previous work has shown that GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. At the generation of sterility, gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents, histone methylation and histone phosphorylation defects in oocytes, phenotypes that mirror those observed in sterile small RNA-mediated genome silencing mutants. Our data suggest that a meiosis-specific function of GSP-2 ties small RNA-mediated silencing of the epigenome to germ cell immortality. We also show that transgenerational epigenomic silencing at hemizygous genetic elements requires the GSP-2 phosphatase, suggesting a functional link to small RNAs. Given that LAB-1 localizes to the interface between homologous chromosomes during pachytene, we hypothesize that small localized discontinuities at this interface could promote genomic silencing in a manner that depends on small RNAs and the GSP-2 phosphatase.

Role of coprophagy in the cecal microbiome development of an herbivorous bird Japanese rock ptarmigan.

The transgenerational maintenance of symbiotic microbes that benefit host nutrition and health is evolutionarily advantageous. In some vertebrate lineages, coprophagy is used as a strategy for effectively transmitting microbes across generations. However, this strategy has still not been studied in birds. Accordingly, the aim of the present study was to evaluate the role of maternal cecal feces consumption by Japanese rock ptarmigan (Lagopus muta japonica) chicks as a strategy for acquiring essential gut microbes. Both the duration of coprophagy behavior by the chicks and the development process of the chick cecal microbiome (n=20 one- to three-week-old chicks, from three broods) were investigated. In all three broods, coprophagy behavior was only observed from 3 to 18 days of age. Furthermore, there was no significant difference in the number of bacterial operational taxonomic units (OTUs) in 1-week-old chicks (n=651) and adults (n=609), and most of the main OTUs observed in the adults were already present in the 1-week-old chicks. These results indicate that, in this precocial bird species, coprophagy may contribute to the early establishment of cecal bacteria that are essential for food digestion and, thus, chick survival. In fact, Japanese rock ptarmigan chicks consume the same food as their hens from the time of hatching. This behavior may have applications to ex-situ conservation.

Spontaneous gain of susceptibility suggests a novel mechanism of resistance to hybrid dysgenesis in Drosophila virilis.

Syndromes of hybrid dysgenesis (HD) have been critical for our understanding of the transgenerational maintenance of genome stability by piRNA. HD in D. virilis represents a special case of HD since it includes simultaneous mobilization of a set of TEs that belong to different classes. The standard explanation for HD is that eggs of the responder strains lack an abundant pool of piRNAs corresponding to the asymmetric TE families transmitted solely by sperm. However, there are several strains of D. virilis that lack asymmetric TEs, but exhibit a “neutral” cytotype that confers resistance to HD. To characterize the mechanism of resistance to HD, we performed a comparative analysis of the landscape of ovarian small RNAs in strains that vary in their resistance to HD mediated sterility. We demonstrate that resistance to HD cannot be solely explained by a maternal piRNA pool that matches the assemblage of TEs that likely cause HD. In support of this, we have witnessed a cytotype shift from neutral (N) to susceptible (M) in a strain devoid of all major TEs implicated in HD. This shift occurred in the absence of significant change in TE copy number and expression of piRNAs homologous to asymmetric TEs. Instead, this shift is associated with a change in the chromatin profile of repeat sequences unlikely to be causative of paternal induction. Overall, our data suggest that resistance to TE-mediated sterility during HD may be achieved by mechanisms that are distinct from the canonical syndromes of HD.

The C. elegans SET-2/SET1 histone H3 Lys4 (H3K4) methyltransferase preserves genome stability in the germline

Maintaining the integrity of genetic information across generations is essential for both cell survival and reproduction, and requires the timely repair of DNA damage. Histone-modifying enzymes play a central role in the DNA repair process through the deposition and removal of post-translational modifications on the histone tails. Specific histone modification act in the DNA repair process through the recruitment of proteins and complexes with specific enzymatic activities, or by altering the chromatin state at the site of DNA lesions. The conserved SET1/MLL family of histone methyltransferases (HMT) catalyzes methylation of histone H3 on Lysine 4 (H3K4), a histone modification universally associated with actively transcribed genes. Studies have focused on the role of SET1/MLL proteins in epigenetic regulation of gene expression. Much less is known on their role in the DNA repair process in a developmental context. Here we show that SET-2, the Caenorhabditis elegans orthologue of SET1, is required to preserve germline genome integrity over subsequent generations. Animals lacking the SET-2 catalytic subunit show a transgenerational increase in sensitivity to DNA damage-inducing agents that is accompanied by a defect in double-strand break (DSB) repair and chromosome fragmentation. These defects are not due to a failure to activate the DNA damage response (DDR) that allows detection, signaling and repair of DNA lesions, because cell cycle arrest and apoptosis, key components of this pathway, are efficiently induced in set-2 mutant animal. Rather, our results suggest that SET-2 plays a role in the transgenerational maintenance of genome stability by acting in DNA repair downstream of DDR signaling.

Reduced Insulin/IGF-1 Signaling Restores Germ Cell Immortality to Caenorhabditis elegans Piwi Mutants

Defects in the Piwi/piRNA pathway lead to transposon desilencing and immediate sterility in many organisms. We found that the C.elegans Piwi mutant prg-1 became sterile after growth for many generations. This phenotype did not occur for RNAi mutants with strong transposon-silencing defects and was separable from the role of PRG-1 in transgene silencing. Brief periods of starvation extended the transgenerational lifespan of prg-1 mutants by stimulating the DAF-16/FOXO longevity transcription factor. Constitutive activation of DAF-16 via reduced daf-2 insulin/IGF-1 signaling immortalized prg-1 strains via RNAi proteins and histone H3 lysine 4 demethylases. In late-generation prg-1 mutants, desilencing of repetitive segments of the genome occurred, and silencing of repetitive loci was restored in prg-1; daf-2 mutants. This study reveals an unexpected interface between aging and transgenerational maintenance of germ cells, where somatic longevity is coupled to a genome-silencing pathway that promotes germ cell immortality in parallel to the Piwi/piRNA system.

Transgenerational maintenance of transgene body CG but not CHG and CHH methylation

In plants, RNA-directed DNA methylation (RdDM) can target both transgene promoters and coding regions/gene bodies. RdDM leads to methylation of cytosines in all sequence contexts: CG, CHG and CHH. Upon segregation of the RdDM trigger, at least CG methylation can be maintained at promoter regions in the progeny. So far, it is not clear whether coding region methylation can be also maintained. We showed that the body of Potato spindle tuber viroid (PSTVd) transgene constructs became densely de novo methylated at CG, CHG and CHH sites upon PSTVd infection. In this study, we demonstrate that in viroid-free progeny plants, asymmetric CHH and CHG methylation was completely lost. However, symmetric CG methylation was stably maintained for at least two generations. Importantly, the presence of transgene body methylation did not lead to an increase of dimethylation of histone H3 lysine 9 or a decrease of acetylation of H3. Our data supports the view that CG methylation can be maintained not only in promoters but also in the body of transgenes. They further suggest that maintenance of methylation may occur independently of tested chromatin modifications.

Reprogramming the Epigenome in Arabidopsis Pollen

Epigenetic reprogramming in Arabidopsis thaliana occurs in developing pollen. The male gametophyte is derived from haploid microspores via two postmeiotic cell divisions to give rise to the gametes (sperm cells, SC) and the vegetative cell (VC). The purification of individual cell types during pollen development coupled with genome-wide DNA methylation analysis and small RNA sequencing has revealed a dynamic regulation of the epigenome during gametogenesis. Interestingly, imprinted loci and previously identified variable epialleles are hypermethylated in the germline; however, their stability after fertilization appears to require targeted demethylation in the neighboring vegetative cell nucleus, possibly by releasing mobile small RNAs that reinforce transcriptional gene silencing and DNA methylation in the gametes. These results have led to a new model for the establishment and transgenerational maintenance of epigenetic marks in flowering plants.

Épigénétique du spermatozoïde : un rôle inattendu de l’ARN

Familial clustering of a disease in the absence of a definite Mendelian determination is not an uncommon observation. Complex multigenic systems, variable penetrance, environmental factors, as well as other ad hoc explanations are commonly invoked. On the other hand, experimental models recently generated in the mouse lead to the alternative, non-exclusive concept of the transgenerational maintenance of a pathogenic epigenetic variation. Thus, we recently identified a new mode of epigenetic inheritance mediated by RNA and micro-RNA which is released by sperm. This discovery, namely that RNA molecules present in the spermatozoon head may be possible vectors for the hereditary transfer of epigenetic modifications, may have both fundamental and medical interests.

Inheritance of an Epigenetic Mark: The CpG DNA Methyltransferase 1 Is Required for De Novo Establishment of a Complex Pattern of Non-CpG Methylation

Site-specific methylation of cytosines is a key epigenetic mark of vertebrate DNA. While a majority of the methylated residues are in the symmetrical (meC)pG:Gp(meC) configuration, a smaller, but significant fraction is found in the CpA, CpT and CpC asymmetric (non-CpG) dinucleotides. CpG methylation is reproducibly maintained by the activity of the DNA methyltransferase 1 (Dnmt1) on the newly replicated hemimethylated substrates (meC)pG:GpC. On the other hand, establishment and hereditary maintenance of non-CpG methylation patterns have not been analyzed in detail. We previously reported the occurrence of site- and allele-specific methylation at both CpG and non-CpG sites. Here we characterize a hereditary complex of non-CpG methylation, with the transgenerational maintenance of three distinct profiles in a constant ratio, associated with extensive CpG methylation. These observations raised the question of the signal leading to the maintenance of the pattern of asymmetric methylation. The complete non-CpG pattern was reinstated at each generation in spite of the fact that the majority of the sperm genomes contained either none or only one methylated non-CpG site. This observation led us to the hypothesis that the stable CpG patterns might act as blueprints for the maintenance of non-CpG DNA methylation. As predicted, non-CpG DNA methylation profiles were abrogated in a mutant lacking Dnmt1, the enzymes responsible for CpG methylation, but not in mutants defective for either Dnmt3a or Dnmt2.

Hérédité épigénétique chez la souris : implication d'ARN et de microARN

By contrast with a wide definition of the 'epigenetic variation', including all changes in gene expression that do not result from alteration of the gene structure, a more restricted class had been defined, initially in plants, under the name 'paramutation'. It corresponds to epigenetic modifications distinct from the regulatory interactions of the cell differentiation pathways, mitotically stable and sexually transmitted with non-Mendelian patterns. This class of epigenetic changes appeared for some time restricted to the plant world, but examples progressively accumulated of epigenetic inheritance in organisms ranging from mice to humans. Occurrence of paramutation in the mouse and possible mechanisms were then established in the paradigmatic case of a mutant phenotype maintained and hereditarily transmitted by wild type homozygotes. Together with recent findings in plants indicative of a necessary step of RNA amplification in the reference maize paramutation, the mouse studies point to a new role of RNA, as an inducer and hereditary determinant of epigenetic variation. Given the known presence of a wide range of RNAs in human spermatozoa, as well as a number of unexplained cases of familial disease predisposition and transgenerational maintenance, speculations can be extended to possible roles of RNA-mediated inheritance in human biology and pathology.