Biology subjects

Richards, T. A.

Publications and source records attributed to Richards, T. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Concanavalin A as a pan-eukaryotic nuclear envelope marker for expansion microscopy

Across eukaryotes, the nuclear envelope exhibits distinct remodelling strategies during mitosis: complete breakdown (open mitosis), partial disruption (intermediate), or full retention (closed). However, both light and electron microscopy of the nuclear envelope, at sufficient resolution to determine integrity and mode of remodelling, have proven technically challenging. Here, Concanavalin A (ConA), a glycan-binding plant lectin, provides discernible nuclear envelope/endoplasmic reticulum (NE/ER) labelling in Ultrastructure Expansion Microscopy (U-ExM). Applied post-expansion, ConA circumvents antibody optimisation and labels the NE across mammalian cells and diverse microbial eukaryotes. By imaging four opisthokont and amoebozoan species with established mitotic strategies, C. perkinsii, D. discoideum, S. pombe, and S. arctica, we distinguished the full spectrum of NE remodelling. Applied to species with poorly characterised mitotic strategies, ConA revealed intermediate mitosis with polar fenestrae in the multinucleate stramenopile A. limacinum and a life cycle-coupled switch between open and closed mitosis in the amoebozoan P. polycephalum. These observations support the hypothesis that multinucleated life cycles favour closed or intermediate mitosis, protecting chromosomes from capture by neighbouring spindles during synchronous divisions. Together, these results establish ConA in combination with expansion microscopy as a broadly applicable tool for uncovering the diversity and evolution of NE remodelling across eukaryotes.

cell biology↗

bioRxiv2026

Long-read single-cell genomics: resolving chimeras in multiple displacement amplification

Multiple displacement amplification (MDA) enables whole-genome amplification from single cells, but introduces chimeric artifacts that severely compromise downstream analyses, particularly with long-read sequencing. Here, we systematically evaluate long-read PacBio HiFi sequencing of MDA amplified DNA from single cells using the model green alga Chlamydomonas reinhardtii. We show that MDA-derived libraries exhibit highly uneven coverage and extreme chimera rates impacting up to 70% of reads, leading to thousands of artefactual structural variants and misassemblies when assembled using algorithms designed for bulk sequencing. To overcome these challenges, we developed lrSAGA (long-read Single Amplified Genome Assembly), a novel tool to assemble long-read MDA sequencing datasets. Assemblies generated using lrSAGA are more complete, more contiguous, and have 75-95% fewer misassemblies compared to conventional assembly algorithms. Although overall contiguity is limited by MDA coverage dropouts, we demonstrate that up to 68% of the C. reinhardtii genome can be accurately assembled from just a single haploid cell. We further validated lrSAGA using published Oxford Nanopore and PacBio HiFi data from single or half Caenorhabditis elegans worms, generating accurate and highly complete assemblies. Applying our approach to single protist cells isolated from environmental water samples, we performed PacBio HiFi single-cell genome sequencing of four uncultivated microbial eukaryotes: an amoeboflagellate from the Naegleria genus, a flagellate from the Bodo genus, and two deep-branching flagellates from the enigmatic CRuMs supergroup, Collodictyon triciliatum and Diphylleia rotans. From single cells, we generated high-quality draft genome assemblies estimated to be 70-84% complete, demonstrating the potential of long-read single-cell genomics to unlock genome diversity from uncultivated microbial eukaryotes.

genomics↗

bioRxiv2026

Adaptation to free-living drives loss of beneficial endosymbiosis through metabolic trade-offs

Symbioses are widespread (1) and underpin the function of diverse ecosystems (2-6), but their evolutionary stability is challenging to explain (7,8). Fitness trade-offs between contrasting intracellular and extracellular niches could act to stabilise endosymbioses because adaptation to either niche is predicted to reduce fitness in the alternate niche, thus reinforcing symbiosis (8,9). Here, we experimentally evolved four diverse Chlorella green algal endosymbionts of Paramecium bursaria to free-living conditions supplying either an amino acid, as provisioned by hosts (10,11), or nitrate, as available in freshwater (12), as the sole nitrogen source. Experimental algal populations adapted to free-living environments, generally increasing in population density and cellular chlorophyll content over time. In one of the four endosymbiont strains, adaptation to the nitrate free-living environment, but not the amino acid environments, drove the loss of fitness benefits to the host in reconstituted symbioses. This loss was not associated with reduced ability to grow on host-provisioned amino acids, nor lost ability to release the sugars provisioned to the host (10,13). Genome sequencing of evolved algal lines revealed genomic divergence between nitrate-adapted and amino acid-adapted lines, affecting genes involved with metabolic organisation and intracellular resource transport. Untargeted metabolomic profiling further showed extensive changes to membrane remodelling and turnover in N-evolved lines. Together, our data support a role for metabolic trade-offs driving divergence between contrasting intracellular and extracellular niches, with nitrogen as a key environmental axis driving divergence. Fitness trade-offs may, therefore, be a general, simple mechanism acting to reinforce symbiosis, contributing to evolutionary stability.

evolutionary biology↗

bioRxiv2026

Stable transformation of Micractinium conductrix SAG 241.80: New tools for exploring photosymbiotic interactions

Endosymbiosis played a key role in the evolution of cellular complexity, but mechanisms underpinning establishment and maintenance remain unclear. Paramecium bursaria harbours Chlorellaceae algal endosymbionts and is a valuable model system for understanding photosymbiosis. However, there are a lack of molecular tools available for exploring the association. Here we report stable transformation of the P. bursaria endosymbiont Micractinium conductrix SAG 241.80. Bioluminescent reporter assays were used to identify endogenous regulatory sequences for transgene expression and to refine a protocol for delivery of exogenous DNA. The bleomycin resistance gene Shble was then identified as an effective selectable marker for isolating transformed cell lines. We demonstrate that endogenous introns enhance transgene expression and that isolation of transformants with desirable characteristics can be achieved without extensive screening by using a 2A peptide to couple expression of an upstream non-selectable transgene to expression of the Shble selectable marker. Finally, we demonstrate that M. conductrix SAG 241.80 transformants expressing fluorescent proteins can be introduced into host cells and observed in the absence of selection, enabling distinct endosymbiont genotypes to be unambiguously identified, compared and monitored within the host environment. The development of molecular tools reported here opens new avenues for addressing unresolved questions in photosymbiosis. Significance StatementO_LIParamecium bursaria is a single-celled, mixotrophic ciliate which forms photosymbiotic interactions with Chlorellaceae green algae and is a popular model system for symbiosis research. C_LIO_LIWe established a stable transformation protocol for the P. bursaria algal endosymbiont M. conductrix SAG 241.80 and identified properties of synthetic gene constructs which facilitate isolation of transformants with robust transgene expression that can be detected within the host environment. C_LIO_LIThis marks significant progress in model system development and opens new avenues for exploring molecular and cellular mechanisms underpinning photosymbiotic interactions. C_LI

evolutionary biology↗

bioRxiv2025

De novo genome sequence assembly of the model algal endosymbiont Micractinium conductrix derived from its host Paramecium bursaria 186b

Endosymbiosis is a major driver of evolutionary innovation and underpins the function of diverse ecosystems. The origins and evolution of endosymbiosis are challenging to study experimentally due to the short-lived culturability of many microbial strains derived from endosymbiotic interactions. The facultative endosymbiosis between the ciliate, Paramecium bursaria, and the green alga, Micractinium conductrix (Chlorellaceae, Trebouxiophyceae), is ecologically widespread and has emerged as a powerful lab-tractable model system. This endosymbiosis is founded upon a reciprocal nutrient exchange, but each of the species can be cultured independently enabling quantification of symbiotic fitness effects, new partnerships to be generated in the lab, and co-associations to be subject to experimental evolution. To date, evolve-and-resequence approaches have been limited due to a lack of high-quality genome assemblies enabling gene variants to be identified. Here, we report a near telomere-to-telomere genome assembly for M. conductrix 186b, using a range of sequencing technologies. Comparative analysis shows that this is one of the most complete Chlorellaceae algal genome assemblies available to date. To aid accurate gene calling and annotation we conducted both RNAseq and Iso-Seq transcriptome sequencing experiments. Collectively these omics datasets will facilitate: i) comparative genomics studies of endosymbiont evolution, ii) evolve-and-resequence experiments, iii) genome-scale metabolic modelling studies, and iv) identification of targets for genetic modification experiments and biotechnological applications. Significance statementEndosymbiosis, where one species, the endosymbiont, lives inside the cell of another species, the host, has played a key role in evolution of complex life. However, the origins and evolution of obligate endosymbioses are often challenging to study because the key events are hidden deep in evolutionary time. Facultative microbial endosymbioses, such as between the ciliate Paramecium bursaria and the green alga Micractinium conductrix, offer experimentally tractable model systems where interacting species can be grown independently or in association allowing studies of the origin, evolution and fitness effects of symbiosis. Here we report the genome sequence for M. conductrix from P. bursaria 186b, enabling genomic studies of the evolution of endosymbiosis and simplifying gene target acquisition for microbe engineering and biotechnological applications.

genomics↗

bioRxiv2024

Immune-like glycan-sensing and horizontally-acquired glycan-processing orchestrate host control in a microbial endosymbiosis

Endosymbiosis was a key factor in the evolution of eukaryotic cellular complexity. Yet the mechanisms that allow host regulation of intracellular symbionts, a pre-requisite for stable endosymbiosis and subsequent organelle evolution, are largely unknown. Here, we describe an immune-like glycan-sensing/processing network, partly assembled through horizontal gene-transfers (HGTs), that enables Paramecium bursaria to control its algal endosymbionts. Using phylogenetics, RNA-interference (RNAi), and metabolite exposure experiments, we show that P. bursaria regulates endosymbiont destruction using glycan-sensing/processing - a system that includes a eukaryotic-wide chitin-binding chitinase-like protein (CLP) localized to the host phago-lysosome. RNAi of CLP alters expression of eight glycan-processing genes, including two prokaryote-derived HGTs, during endosymbiont destruction. Furthermore, glycan-sensing/processing dynamically regulates endosymbiont number in P. bursaria, plasticity crucial to maximize host fitness across ecological conditions. CLP is homologous to a human phagocyte-associated innate immune factor, revealing how immune functions can be alternatively adapted and expanded, partly through HGT, enabling endosymbiotic control. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/613017v2_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@13aeaaorg.highwire.dtl.DTLVardef@302e6dorg.highwire.dtl.DTLVardef@138224forg.highwire.dtl.DTLVardef@54aca8_HPS_FORMAT_FIGEXP M_FIG C_FIG

evolutionary biology↗

bioRxiv2024

De novo genome sequence assembly of the RNAi-tractable endosymbiosis model system Paramecium bursaria 186b reveals factors shaping intron repertoire

How two species engage in stable endosymbiosis is a biological quandary. The study of facultative endosymbiotic interactions has emerged as a useful approach to understand how endosymbiotic functions can arise. The ciliate protist Paramecium bursaria hosts green algae of the order Chlorellales in a facultative photo-endosymbiosis. We have recently reported RNAi as a tool for understanding gene function in Paramecium bursaria 186b, CCAP strain 1660/18 [1]. To complement this work, here we report a highly complete host genome and transcriptome sequence dataset, using both Illumina and PacBio sequencing methods to aid genome analysis and to enable the design of RNAi experiments. Our analyses demonstrate Paramecium bursaria, like other ciliates such as diverse species of Paramecia, possess numerous tiny introns. These data, combined with the alternative genetic code common to ciliates, makes gene identification and annotation challenging. To explore intron evolutionary dynamics further we show that alternative splicing leading to intron retention occurs at a higher frequency among the smaller number of longer introns, identifying a source of selection against longer introns. These data will aid the investigation of genome evolution in the Paramecia and provide additional source data for the exploration of endosymbiotic functions.

genomics↗

bioRxiv2024

Multiple Independent Genetic Code Reassignments of the UAG Stop Codon in Phyllopharyngean Ciliates

The translation of nucleotide sequences into amino acid sequences, governed by the genetic code, is one of the most conserved features of molecular biology. The standard genetic code, which uses 61 sense codons to encode one of the 20 standard amino acids and 3 stop codons (UAA, UAG, and UGA) to terminate translation, is used by most extant organisms. The protistan phyla Ciliophora (the ciliates) are an unusual exception to this norm, exhibiting the greatest diversity of non-canonical nuclear genetic code variants and evidence of repeated changes in code. In this study, we report the discovery of multiple independent genetic code changes within the Phyllopharyngea class of ciliates. By mining publicly available ciliate genome datasets, we discovered that three ciliate species from the TARA Oceans eukaryotic metagenome dataset use the UAG codon to putatively encode leucine. We identified novel suppressor tRNA genes in two of these genomes. Phylogenomics analysis revealed that these three uncultivated taxa form a monophyletic lineage within the Phyllopharyngea class. Expanding our analysis by reassembling published phyllopharyngean genome datasets led to the discovery that the UAG codon had also been reassigned to putatively code for glutamine in Hartmannula sinica and Trochilia petrani. Phylogenomics analysis suggests that this occurred via two independent genetic code change events. These data demonstrate that the reassigned UAG codons have widespread usage as sense codons within the phyllopharyngean ciliates. Furthermore, we show that the function of UAA is firmly fixed as the preferred stop codon. These findings shed light on the evolvability of the genetic code in understudied microbial eukaryotes.

evolutionary biology↗

bioRxiv2023

Duplication and neofunctionalization of a horizontally-transferred xyloglucanase as a facet of the red queen co-evolutionary dynamic

Oomycetes are heterotrophic protists that share phenotypic similarities with fungi, including the ability to cause plant diseases, but branch in a separate and distant region of the eukaryotic tree of life. It has been suggested that multiple horizontal gene transfers (HGTs) from fungi-to-oomycetes contributed to the evolution of plant-pathogenic traits. These HGTs are predicted to include secreted proteins that degrade plant cell walls. This is a key trait in the pathology of many oomycetes, as the plant cell wall represents a primary barrier to pathogen invasion and a rich source of carbohydrates. Many of the HGT gene families identified have undergone multiple rounds of duplication. Using a combination of phylogenomic analysis and functional assays, we investigate the diversification of a horizontally-transferred xyloglucanase gene family in the model oomycete species Phytophthora sojae. Our analyses detect 11 genes retained in P. sojae among a complex pattern of gene duplications and losses. Using a phenotype assay, based on heterologous expression in yeast, we show that eight of these paralogs have xyloglucanase function, including variants with distinct protein characteristics, such as a long-disordered C-terminal extension that can increase xyloglucanase activity. The functional xyloglucanase variants analysed subtend an ancestral node close to the fungi-oomycetes gene transfer, suggesting the horizontally-transferred gene was a bona fide xyloglucanase. Expression of xyloglucanase paralogs in Nicotiana benthamiana triggers distinct patterns of reactive oxygen species (ROS) generation, demonstrating that enzyme variants differentially stimulate pattern-triggered immunity in plants. Mass spectrometry of detectable enzymatic products demonstrates that some paralogs catalyze production of variant breakdown profiles, suggesting that secretion of multiple xyloglucanase variants increases efficiency of xyloglucan breakdown, as well as potentially diversifying the range of Damage-Associated Molecular Patterns (DAMPs) released during pathogen attack. We suggest that such patterns of protein neofunctionalization, and variant host responses, represent an aspect of the Red Queen host-pathogen co-evolutionary dynamic. Significance StatementThe oomycetes are a diverse group of eukaryotic microbes that include some of the most devastating pathogens of plants. Oomycetes perceive, invade, and colonize plants in similar ways to fungi, in part because they acquired the genes to attack and feed on plants from fungi. These genes are predicted to be useful to oomycete plant pathogens because they have undergone multiple rounds of gene duplication. One key enzyme for attacking plant cell wall structures is called xyloglucanase. Xyloglucanase in the oomycetes has undergone multiple rounds of gene duplication, leading to variants including an enzyme with a C-terminal extension that increases activity. Some xyloglucanase variants trigger unique patterns of reactive oxygen species (ROS) in planta, and generate different profiles of cell wall breakdown products - such outcomes could act to mystify and increase the workload of the plant immune system, allowing successful pathogens to proliferate.

evolutionary biology↗

bioRxiv2023

Self-assembling viral histones unravel early nucleosome evolution

Nucleosomes are a core-component of eukaryotic nuclei, forming the structural basis of chromatin and co-ordinating processes from gene expression to chromosome segregation. Composed of a DNA-protein complex consisting of the four individual histones, H2A, H2B, H3, and H4, the nucleosome and its associated functions were key innovations during eukaryotic evolution1,2. However, functional constraints and the extinction of stem-eukaryotes have concealed how these dynamic systems evolved from simpler histone homologues in Archaea3-5. Viral histones have also previously been identified and are thought to reflect an ancestral state as they often comprise multiple histone paralogues arranged within a single protein, termed histone repeats6-11. Here, using viruses as an alternative source of variation, we expand the known diversity of histones and develop an empirical hypothesis for the origin of the nucleosome. Our analysis identified hundreds of histones with variable domain repeat configurations including histone singlets, doublets, triplets, and quadruplets, the latter comprising the four core histones arranged in series. Viral histone repeats consistently branch between Archaea and eukaryotes in phylogenetic trees and display intermediate functions, self-assembling into eukaryotic-like nucleosomes that stack into archaeal-like oligomers capable of impacting genomic activity and condensing DNA. The linkers conjoining the histone repeats also facilitate nucleosome formation and can promote the assembly of eukaryotic nucleosomes in the bacterium, Escherichia coli. Combining these data, we hypothesize that viral histone repeats represent molecular relics acquired by viruses from stem-eukaryotes during eukaryogenesis and suggest that nucleosome evolution may have proceeded through histone repeat intermediates.

evolutionary biology↗

bioRxiv2023

Fungal zoospores show contrasting swimming patterns specific to phylum and cytology

Zoosporic fungi, also called chytrids, produce motile spores with flagellar swimming tails (zoospores)1,2. These fungi are key components of aquatic food webs, acting as pathogens, saprotrophs and prey3-8. Little is known about the swimming behaviour of fungal zoospores, a crucial factor governing dispersal, biogeographical range, ecological function and infection dynamics6,9. Here, we track the swimming patterns of zoospores from 12 evolutionary divergent species of zoosporic fungi across seven orders of the Chytridiomycota and the Blastocladiomycota phyla. We report two major swimming patterns which correlate with the cytoskeletal ultrastructure of these zoospores. Specifically, we show that species without major cytoplasmic tubulin components swim in a circular fashion, while species that harbour prominent cytoplasmic tubulin structures swim in a pattern akin to a random walk (move-stop-redirect-move). We confirm cytoskeleton architecture by performing fluorescence confocal microscopy of the zoospores across all 12 species. We then treat representative species with variant swimming behaviours and cytoplasmic-cytoskeletal arrangements with tubulin stabilizing (Taxol) and depolymerizing (Nocodazole) pharmacological-compounds. We observed that when treating the random-walk species with Nocodazole their swimming behaviour changes to a circular swimming pattern. Confocal imaging of the nocodazole-treated zoospores demonstrates these cells maintain flagellum tubulin structures but lack their characteristic cytoplasmatic tubulin arrangement. These data confirm that the capability of zoospores to perform complex movements as a random walk is linked to the presence of prominent cytoplasmatic tubulin structures. We discuss the link between cytology, sensation, and swimming behaviour manifest in zoosporic fungi.

evolutionary biology↗

bioRxiv2022

Identification of a Non-Canonical Ciliate Nuclear Genetic Code Where UAA and UAG Code for Different Amino Acids

The genetic code is one of the most highly conserved features across life. Only a few lineages have deviated from the "universal" genetic code. Amongst the few variants of the genetic code reported to date, the codons UAA and UAG virtually always have the same translation, suggesting that their evolution is coupled. Here, we report the genome and transcriptome sequencing of a novel ciliate, belonging to the Oligohymenophorea class, where the translation of the UAA and UAG stop codons have changed to specify different amino acids. Genomic and transcriptomic analyses revealed that UAA has been reassigned to encode lysine, while UAG has been reassigned to encode glutamic acid. We identified multiple suppressor tRNA genes with anticodons complementary to the reassigned codons. We show that the retained UGA stop codon is enriched in the 3UTR immediately downstream of the coding region of genes, suggesting that there is functional drive to maintain tandem stop codons. Using a phylogenomics approach, we reconstructed the ciliate phylogeny and mapped genetic code changes, highlighting the remarkable number of independent genetic code changes within the Ciliophora group of protists. According to our knowledge, this is the first report of a genetic code variant where UAA and UAG encode different amino acids.

genomics↗

bioRxiv2022

Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and C53-mediated autophagy

UFMylation mediates the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and regulates the selective degradation of endoplasmic reticulum (ER) via autophagy (ER-phagy) to maintain ER homeostasis. Specifically, collisions of the ER-bound ribosomes trigger ribosome UFMylation, which in turn activates C53-mediated autophagy that clears the toxic incomplete polypeptides. C53 has evolved non-canonical shuffled ATG8 interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. Why these non-canonical motifs were selected during evolution, instead of canonical ATG8 interacting motifs remains unknown. Here, using a phylogenomics approach, we show that UFMylation is conserved across the eukaryotes and secondarily lost in fungi and some other species. Further biochemical assays have confirmed those results and showed that the unicellular algae, Chlamydomonas reinhardtii has a functional UFMylation machinery, overturning the assumption that this process is linked to multicellularity. Our conservation analysis also revealed that UFM1 co-evolves with the sAIMs in C53, reflecting a functional link between UFM1 and the sAIMs. Using biochemical and structural approaches, we confirmed the interaction of UFM1 with the C53 sAIMs and found that UFM1 and ATG8 bound to the sAIMs in a different mode. Conversion of sAIMs into canonical AIMs prevented binding of UFM1 to C53, while strengthening ATG8 interaction. This led to the autoactivation of the C53 pathway and sensitized Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral toggle switch embodied in the sAIMs that regulates C53-mediated autophagy to maintain ER homeostasis.

plant biology↗

bioRxiv2021

Membrane permeability differentiation at the lipid divide

One of the deepest branches in the tree of life separates the Archaea from the Bacteria. These prokaryotic groups have distinct cellular systems including fundamentally different phospholipid membrane bilayers. This dichotomy has been termed the lipid divide and possibly bestows different biophysical and biochemical characteristics on each cell type. Classic experiments suggest that bacterial membranes (formed from lipids extracted from Escherichia coli for example) show permeability to key metabolites comparable to archaeal membranes (formed from lipids extracted from Halobacterium salinarum), yet systematic analyses based on direct measurements of membrane permeability are absent. Here we develop a new approach for assessing the membrane permeability of ~10 m unilamellar vesicles, consisting of an aqueous medium enclosed by a single lipid bilayer. Comparing the permeability of eighteen metabolites demonstrates that diether glycerol-1-phosphate lipids with methyl branches, often the most abundant membrane lipids of known archaea, are permeable to a wide range of compounds useful for core metabolic networks, including amino acids, sugars, and nucleobases. Permeability is significantly lower in diester glycerol-3-phosphate lipids without methyl branches, the common building block of bacterial membranes. To identify the membrane characteristics that determine permeability we use this experimental platform to test a variety of lipid forms bearing a diversity of intermediate characteristics. We found that increased membrane permeability is dependent on both the methyl branches present on the archaeal phospholipid tails and the ether bond between the tails and the head group. These permeability differences must have had profound effects on the cell physiology and proteome evolution of early prokaryotic forms. To explore this further, we compare the abundance and distribution of transmembrane transporter-encoding protein families present on genomes sampled from across the prokaryotic tree of life. Archaea have a reduced repertoire of transporter gene families, consistent with increased membrane permeation. These results demonstrate that the lipid divide demarcates a clear difference in permeability function with implications for understanding some of the earliest transitions in cell evolution.

evolutionary biology↗

bioRxiv2021

Emergent RNA-RNA interactions can promote stability in a nascent phototrophic endosymbiosis

Eukaryote-eukaryote endosymbiosis was responsible for the spread of chloroplast (plastid) organelles. Stability is required for the metabolic and genetic integration that drives the establishment of new organelles, yet the mechanisms which act to stabilise nascent endosymbioses - between two fundamentally selfish biological organisms - are unclear. Theory suggests that enforcement mechanisms, which punish misbehaviour, may act to stabilise such interactions by resolving conflict. However, how such mechanisms can emerge in a nascent endosymbiosis has yet to be explored. Here, we propose that endosymbiont-host RNA-RNA interactions, arising from digestion of the endosymbiont population, can result in a cost to host growth for breakdown of the endosymbiosis. Using the model nascent endosymbiosis, Paramecium bursaria - Chlorella spp., we demonstrate that this mechanism is dependent on the host RNA-interference (RNAi) system. We reveal through small RNA (sRNA) sequencing that endosymbiont-derived mRNA released upon endosymbiont digestion can be processed by the host RNAi system into 23-nt sRNA. We predict multiple regions of shared sequence identity between endosymbiont and host mRNA, and demonstrate through delivery of synthetic endosymbiont sRNA that exposure to these regions can knock-down expression of complementary host genes, resulting in a cost to host growth. This process of host gene knock-down in response to endosymbiont-derived RNA processing by host RNAi factors, which we term RNAi-collisions, represents a mechanism which can promote stability in a nascent eukaryote-eukaryote endosymbiosis. By imposing a cost for breakdown of the endosymbiosis, endosymbiont-host RNA-RNA interactions may drive maintenance of the symbiosis across fluctuating ecological conditions and symbiotic status. SIGNIFICANCE STATEMENTStable endosymbiosis between eukaryotic microbes has driven the evolution of further cellular complexity. Yet the mechanisms which can act to stabilise a nascent eukaryote-eukaryote endosymbiosis are unclear. Using the model nascent endosymbiotic system, Paramecium bursaria-Chlorella, we demonstrate that endosymbiont-host RNA-RNA interactions can drive a cost to host growth upon endosymbiont digestion, punishing the host for misbehaviour. These RNA-RNA interactions are facilitated by the host RNA-interference system. For endosymbiont mRNA sharing a high-level of sequence identity with host transcripts, this process can result in host gene knock-down. We propose that these endosymbiont-host RNA-RNA interactions- RNAi collisions-represent a viable enforcement mechanism to sanction the host for breakdown of the endosymbiosis, promoting the stability of a nascent endosymbiotic interaction.

evolutionary biology↗

bioRxiv2021

A functional bacterial-derived restriction modification system in the mitochondrion of a heterotrophic protist

The overarching trend in mitochondrial evolution is functional streamlining coupled with gene loss; therefore, gene acquisition by mitochondria is considered to be exceedingly rare. Selfish elements in the form of self-splicing introns occur in many organellar genomes, but the wider diversity of selfish elements, and how they persist in organellar genomes, has not been explored. In the mitochondrial genome of a marine heterotrophic katablepharid protist, we identify a functional type II restriction modification system originating from a horizontal gene transfer event involving bacteria related to flavobacteria. This restriction modification system consists of an HpaII-like endonuclease and a cognate cytosine methyltransferase. We demonstrate that these proteins are functional by heterologous expression in both bacterial and eukaryotic cells. These results suggest that toxin-antitoxin selfish elements, such as restriction modification systems, could be co-opted by eukaryotic genomes to drive uniparental organellar inheritance.

evolutionary biology↗

bioRxiv2020

Validation of a Core RNA-interference Pathway as a Tool for Reverse Genetics in the Emergent Model Phototrophic Endosymbiosis, Paramecium bursaria

Endosymbiosis was fundamental for the evolution of eukaryotic complexity. Endosymbiotic interactions can be dissected through forward and reverse-genetic experiments, such as RNA-interference (RNAi). However, distinguishing small (s)RNA pathways in a eukaryote-eukaryote endosymbiotic interaction is challenging. Here, we investigate the repertoire of RNAi pathway protein-encoding genes in the model nascent endosymbiotic system, Paramecium bursaria-Chlorella spp. Using comparative genomics and transcriptomics supported by phylogentics, we identify essential proteome components of the small interfering (si)RNA, scan (scn)RNA, and internal eliminated sequence (ies)RNA pathways. Our analyses reveal that copies of these components have been retained throughout successive whole genome duplication (WGD) events in the Paramecium clade. We then validate feeding-induced siRNA-based RNAi in P. bursaria via knock-down of the splicing factor, u2af1, which we show to be crucial to host growth. Finally, using simultaneous knock-down paradox controls to rescue the effect u2af1 knock-down, we demonstrate that feeding-induced RNAi in P. bursaria is dependent upon a core pathway of host-encoded Dcr1, Piwi and Pds1 components. Our experiments confirm the presence of a functional, host-derived RNAi pathway in P. bursaria that generates 23-nt siRNA, validating use of the P. bursaria-Chlorella spp. system to investigate the genetic basis of a nascent endosymbiosis.

molecular biology↗

bioRxiv2018

A new framework for the study of apicomplexan diversity across environments

Apicomplexans are a group of microbial eukaryotes that contain some of the most well-studied parasites, including widespread intracellular pathogens of mammals such as Toxoplasma and Plasmodium (the agent of malaria), and emergent pathogens like Cryptosporidium and Babesia. Decades of research have illuminated the pathogenic mechanisms, molecular biology, and genomics of model apicomplexans, but we know surprisingly little about their diversity and distribution in natural environments. In this study we analyze the distribution of apicomplexans across a range of both host-associated and free-living environments, covering animal hosts from cnidarians to mammals, and ecosystems from soils to fresh and marine waters. Using publicly available small subunit (SSU) rRNA gene databases, high-throughput environmental sequencing (HTES) surveys such as Tara Oceans and VAMPS, as well as our own generated HTES data, we developed an apicomplexan reference database, which includes the largest apicomplexan SSU rRNA tree available to date and encompasses comprehensive sampling of this group and their closest relatives. This tree allowed us to identify and correct incongruences in the molecular identification of sequences, particularly within the hematozoans and the gregarines. Analyzing the diversity and distribution of apicomplexans in HTES studies with this curated reference database also showed a widespread, and quantitatively important, presence of apicomplexans across a variety of free-living environments. These data allow us to describe a remarkable molecular diversity of this group compared with our current knowledge, especially when compared with that identified from described apicomplexan species. This revision is most striking in marine environments, where potentially the most diverse apicomplexans apparently exist, but have not yet been formally recognized. The new database will be useful for both microbial ecology and epidemiological studies, and provide valuable reference for medical and veterinary diagnosis especially in cases of emerging, zoonotic, and cryptic infections.\n\nAuthor SummaryApicomplexans are important animal and human parasites, but little is known about their distribution and diversity in the natural environment. We have developed a phylogenetically informed and manually curated reference database for the SSU rRNA barcode gene, and analyzed all publicly available sequences from a broad range of environments, providing a needed framework to analyze high-throughput environmental sequencing (HTES) data. The reference database and the amplicon sequences identified a striking diversity of apicomplexans in the environment, including habitats not usually associated with this group (e.g. open ocean). The phylogenetic framework will underpin microbial ecology studies and provide valuable resources for medical and veterinary biology.

microbiology↗