Max Planck Institute for Biology

The green alga Pleodorina japonica is an interesting volvocine species that harbors abundant rickettsial endosymbionts ("MIDORIKO") within its cytoplasm. However, the diversity and transmission of these endosymbionts within the species remain unclear. In this study, we examined the presence or absence of "MIDORIKO" and the genetic diversity in 21 culture strains of the host P. japonica population from various localities in Japan. Genomic polymerase chain reactions using "MIDORIKO"-specific primers and 4',6-diamidino-2-phenylindole-staining demonstrated that only five of the 21 strains harbored "MIDORIKO." The 16S ribosomal DNA sequences of "MIDORIKO" from these five strains (1148 bp) were identical to each other and distinct from the sequences of the rickettsial endosymbionts harbored by other algal species and protists, suggesting that "MIDORIKO" from P. japonica is specific to P. japonica. The phylogenetic results for the 21 host strains, which were resolved based on three nuclear genes encoding oxygen-evolving enhancer protein 1, F1F0 ATP synthase subunit beta and actin disagreed significantly. None of the three gene phylogenies supported the close relationship of the five "MIDORIKO"-harboring strains. A recombination test using the three concatenated genes provided strong evidence of recombination. Therefore, gene flow by sexual reproduction has likely occurred in the natural habitats of P. japonica. The transmission of "MIDORIKO" among different P. japonica genotypes could also be considered to occur via sexual reproduction, although it is likely infrequent via that method given the sporadic nature of "MIDORIKO" within the P. japonica population. Although P. japonica exhibits homothallic sexual reproduction, the present genetic data demonstrate that it is undoubtedly a biological species.

Chromatin structure plays a central role in regulating transcription, genome stability and epigenetic inheritance in eukaryotes. Much of our understanding of chromatin architecture and histone post-translational modifications (hPTMs) comes from a narrow set of animal and plant models, but emerging data from non-model lineages are challenging canonical views of how chromatin functions across the tree of life. Brown algae are complex multicellular eukaryotes that provide a unique perspective on chromatin evolution given their independent origin of complex multicellularity. Here we compile the chromatin toolkit of brown algae and show that canonical silencing systems involving DNA cytosine methylation and Polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 (H3K27) methylation were lost early in their evolution. By generating hPTM profiles from diverse brown algal clades, we resolve the nature and regulatory roles of chromatin states in this lineage and show how H3 lysine 79 (H3K79) methylation emerged and diversified as a repressive system. We further uncover sex-specific reconfigurations in species with varying degrees of sexual dimorphism and reconstruct the ancestral regulatory landscape that probably preceded the emergence of brown algae. Together, our findings illuminate the dynamic evolution of chromatin regulation in a distinct multicellular lineage and challenge assumptions about the universality of chromatin-based mechanisms across eukaryotes.

Developmental plasticity is increasingly recognised as facilitator of evolutionary novelty. However, how plasticity itself evolves and how variation in plastic trait expression is structured in populations remain unknown. The predatory nematode Pristionchus pacificus exhibits mouth-form plasticity with underlying molecular mechanisms being increasingly identified. We investigate the temporal scale of natural variation of mouth-form plasticity. An 11-year survey characterised Adoretus beetle-derived isolates from Colorado, La Réunion Island and revealed a gradual shift in mouth-form preference. Quantitative trait locus mapping of mouth-form preferences identified a single peak harbouring the developmental switch gene eud-1. Through CRISPR-engineering and biochemical assays, we show that plasticity in nonsense-mediated decay coupled with alternative start codon selection resulting in different N-terminal proteoforms of EUD-1 are associated with natural variation of mouth-form preference. This work provides molecular explanations for variation in plastic trait expression and links nonsense variants in the major developmental switch locus to ecological and evolutionary processes.

Relating billions of proteins across the tree of life remains a challenging task for comparative biosphere genomics and artificial intelligence-driven structure prediction. Here we present DIAMOND DeepClust, a cascaded, ultra-fast clustering method enabling planetary-scale organization of protein space, scaling to trillions of sequences while retaining sensitivity at low identity. Aggregating 19 billion biosphere proteins into 544 million nonsingleton clusters, we show that using our DeepClust database, available for download, can enhance structure prediction with AlphaFold2.

Abstract Extensive laboratory experimentation has revealed conserved molecular pathways controlling growth and stress responses in plants, yet how these programs operate in natural settings remains poorly understood. We investigated transcriptome organization in wild populations of Arabidopsis thaliana by sampling plants from 60 natural sites in Europe and North America across two seasons. Transcriptomes varied extensively among individuals and showed largely continuous rather than discrete structure across geography and season. Although disease and microbial colonization were common in the wild, wild transcriptomes did not simply recapitulate canonical laboratory stress signatures. Measured microbial infection, environmental, and phenotypic variables explained only a modest fraction of total expression variation, but infection-associated signals accounted for the largest share of the explainable component. Consistent with this, biotic-response networks defined in controlled laboratory experiments were well conserved in wild transcriptomes, whereas control and abiotic-response networks were substantially reorganized. Together, these results suggest that while core transcriptional modules remain recognizable across environments, regulatory relationships among modules differ markedly between laboratory and natural contexts. Graphical Abstract

Ribosome hibernation helps cells survive stress by reversibly silencing translation and limiting degradation of ribosomal complexes. Although well characterized in bacteria and eukaryotes, archaeal hibernation remains poorly understood. Using cryoEM to analyze lysates from a model archaeon, we identified AHA (AMPKγ-HPF from Archaea), a broadly conserved ribosome-associated protein factor composed of two distinct modules. Structural analyses showed that AHA’s C-terminal domain binds the small subunit, while its N-terminal region recognizes the large subunit, occluding the mRNA channel and the A- and P-tRNA binding sites and thereby enforcing translational silencing. Consistent with this proposed function, ΔAHA cells displayed reduced viability, depletion of ribosomal proteins during stationary phase, and impaired recovery upon return to growth. Phylogenetic analyses revealed that AHA’s C-terminal domain shares homology with the bacterial Hibernation Promoting Factor (HPF), indicating an origin in the last universal common ancestor (LUCA) and thereby identifying HPF as a universal hibernation module. Strikingly, we observed two AMP molecues bound to AHA’s N-terminal CBS-tetrad, which we found was structurally and evolutionary related to the eukaryotic energy sensor AMPKγ, thus linking energy sensing between archaea and eukaryotes. Together, these findings uncover a widespread archaeal ribosome hibernation factor and establish a direct evolutionary link between prokaryotic translational silencing and eukaryotic energy sensing.

Abstract Continuous developments in sequencing technologies have led to the generation of chromosome-scale genome assemblies across the whole tree of life, but our ability to annotate genomes has lacked behind. One major problem consists in the fact that typically not all genes are expressed at detectable levels at any given life stage or environment. Therefore, available transcriptome data needs to be complemented by gene prediction programs and protein homology evidence. However, how to optimally combine these different data types is not well understood. Here, we present a case study, where we community curated gene annotations of the Pristionchus pacificus strain RSC011. By incorporation of new Iso-seq and RNA-seq data and genome-wide screening, we identified and corrected more than 7,500 (∼24%) gene models. While the improved gene annotation for the RSC011 strain will be useful for the P. pacificus community, our study reveals several gene annotation problems that may affect data from other species. Among these, we identified assembly errors, artificial transcript fusions resulting from overlapping genes and polycistronic RNAs, falsely called open reading frames, and error propagation based on homology data as frequent sources of gene annotation errors. Thus, our findings may be helpful in guiding future efforts to annotate genomes across different taxonomic groups.

In plants, multiple cell types contribute to immunity, but what division of labor exists among cell types when immunity is activated? We compared, at single-cell resolution, the response of Arabidopsis thaliana leaf cells during pattern-triggered and effector-triggered immunity (PTI/ETI), sampled at 3 and 5 h after infection with Pseudomonas syringae DC3000. Core defense modules were broadly shared across cell clusters, but their activation varied in timing and intensity, with key immune receptors also showing cell type-specific expression dynamics. Mesophyll cell populations could be distinguished based on their resilience patterns: after the initial response, some populations continue to express defense genes at high levels during both PTI and ETI, while others quickly reinitiate growth-related gene expression programs but only during PTI. Gene regulatory network inference revealed WRKY-regulated modules enriched in cells sensing effectors, while salicylic acid biosynthesis regulators were activated in complementary clusters. Analysis of cue1 mutants demonstrated that core immune responses are robust to altered leaf architecture. In addition, we uncovered cryptic defense pathways, including sucrose-responsive modules, in this mutant. By capturing early immune responses at high resolution, our study reveals cell type-specific coordination of plant immunity and provides a framework for decoding immune signaling networks.

Sex determination is fundamental to eukaryotic life, yet its molecular basis varies widely across the tree of life. In most animal clades, sex-determining mechanisms are highly diverse and evolve rapidly. Here, we identify an exception in aculeate Hymenoptera, an ancient and diverse clade of haplodiploid insects that includes ants, bees, and stinging wasps. By integrating comparative genomics across Hymenoptera with genetic mapping in bumblebees and hornets, we reveal that the ANTSR locus, a multiallelic noncoding locus, has been maintained for over 150 My as the primary instructive signal for female development. This locus is located in a conserved synteny block that originated at the base of Aculeata and functions as a highly polymorphic, zygosity-based sex determiner, with only heterozygous individuals developing as females across lineages. Despite its deep evolutionary conservation, this sex locus shows no detectable sequence homology among lineages. These findings demonstrate that an essential noncoding locus can retain its function over deep evolutionary time without sequence conservation. More broadly, our results highlight haplodiploid insects as a powerful model for studying the evolution of sex determination mechanisms beyond those linked to sex chromosomes.

Continuous developments in sequencing technologies have led to the generation of chromosome-scale genome assemblies across the whole tree of life, but our ability to annotate genomes has lacked behind. One major problem consists in the fact that typically not all genes are expressed at detectable levels at any given life stage or environment. Therefore, available transcriptome data needs to be complemented by gene prediction programs and protein homology evidence. However, how to optimally combine these different data types is not well understood. Here, we present a case study, where we community curated gene annotations of the Pristionchus pacificus strain RSC011. By incorporation of new Iso-seq and RNA-seq data and genome-wide screening, we identified and corrected more than 7,500 (~24%) gene models. While the improved gene annotation for the RSC011 strain will be useful for the P. pacificus community, our study reveals several gene annotation problems that may affect data from other species. Among these, we identified assembly errors, artificial transcript fusions resulting from overlapping genes and polycistronic RNAs, falsely called open reading frames, and error propagation based on homology data as frequent sources of gene annotation errors. Thus, our findings may be helpful in guiding future efforts to annotate genomes across different taxonomic groups.