Arbuscular mycorrhizal fungi heterokaryons have two nuclear populations with distinct roles in host-plant interactions.

Arbuscular mycorrhizal fungi (AMF) are prominent root symbionts that can carry thousands of nuclei deriving from two parental strains in a large syncytium. These co-existing genomes can also vary in abundance with changing environmental conditions. Here we assemble the nuclear genomes of all four publicly available AMF heterokaryons using PacBio high-fidelity and Hi-C sequencing. We find that the two co-existing genomes of these strains are phylogenetically related but differ in structure, content and epigenetics. We confirm that AMF heterokaryon genomes vary in relative abundance across conditions and show this can lead to nucleus-specific differences in expression during interactions with plants. Population analyses also reveal signatures of genetic exchange indicative of past events of sexual reproduction in these strains. This work uncovers the origin and contribution of two nuclear genomes in AMF heterokaryons and opens avenues for the improvement and environmental application of these strains.

Long reads and Hi-C sequencing illuminate the two-compartment genome of the model arbuscular mycorrhizal symbiont Rhizophagus irregularis.

Chromosome folding links genome structure with gene function by generating distinct nuclear compartments and topologically associating domains. In mammals, these undergo preferential interactions and regulate gene expression. However, their role in fungal genome biology is unclear. Here, we combine Nanopore (ONT) sequencing with chromatin conformation capture sequencing (Hi-C) to reveal chromosome and epigenetic diversity in a group of obligate plant symbionts: the arbuscular mycorrhizal fungi (AMF). We find that five phylogenetically distinct strains of the model AMF Rhizophagus irregularis carry 33 chromosomes with substantial within-species variability in size, as well as in gene and repeat content. Strain-specific Hi-C contact maps reveal a 'checkerboard' pattern that underline two dominant euchromatin (A) and heterochromatin (B) compartments. Each compartment differs in the level of gene transcription, regulation of candidate effectors and methylation frequencies. The A-compartment is more gene-dense and contains most core genes, while the B-compartment is more repeat-rich and has higher rates of chromosomal rearrangement. While the B-compartment is transcriptionally repressed, it has significantly more secreted proteins and in planta upregulated candidate effectors, suggesting a possible host-induced change in chromosome conformation. Overall, this study provides a fine-scale view into the genome biology and evolution of model plant symbionts, and opens avenues to study the epigenetic mechanisms that modify chromosome folding during host-microbe interactions.

The genome of Geosiphon pyriformis reveals ancestral traits linked to the emergence of the arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal fungi (AMF) (subphylum Glomeromycotina)1 are among the most prominent symbionts and form the Arbuscular Mycorrhizal symbiosis (AMS) with over 70% of known land plants.2,3 AMS allows plants to efficiently acquire poorly soluble soil nutrients4 and AMF to receive photosynthetically fixed carbohydrates. This plant-fungus symbiosis dates back more than 400 million years5 and is thought to be one of the key innovations that allowed the colonization of lands by plants.6 Genomic and genetic analyses of diverse plant species started to reveal the molecular mechanisms that allowed the evolution of this symbiosis on the host side, but how and when AMS abilities emerged in AMF remain elusive. Comparative phylogenomics could be used to understand the evolution of AMS.7,8 However, the availability of genome data covering basal AMF phylogenetic nodes (Archaeosporales, Paraglomerales) is presently based on fragmentary protein coding datasets.9Geosiphon pyriformis (Archaeosporales) is the only fungus known to produce endosymbiosis with nitrogen-fixing cyanobacteria (Nostoc punctiforme) presumably representing the ancestral AMF state.10-12 Unlike other AMF, it forms long fungal cells ("bladders") that enclose cyanobacteria. Once in the bladder, the cyanobacteria are photosynthetically active and fix nitrogen, receiving inorganic nutrients and water from the fungus. Arguably, G. pyriformis represents an ideal candidate to investigate the origin of AMS and the emergence of a unique endosymbiosis. Here, we aimed to advance knowledge in these questions by sequencing the genome of G. pyriformis, using a re-discovered isolate.

More Filtering on SNP Calling Does Not Remove Evidence of Inter-Nucleus Recombination in Dikaryotic Arbuscular Mycorrhizal Fungi.

Evidence for the existence of dikaryote-like strains, low nuclear sequence diversity and inter-nuclear recombination in arbuscular mycorrhizal fungi has been recently reported based on single nucleus sequencing data. Here, we aimed to support evidence of inter-nuclear recombination using an approach that filters SNP calls more conservatively, keeping only positions that are exclusively single copy and homozygous, and with at least five reads supporting a given SNP. This methodology recovers hundreds of putative inter-nucleus recombination events across publicly available sequence data from individual nuclei. Challenges related to the acquisition and analysis of sequence data from individual nuclei are highlighted and discussed, and ways to address these issues in future studies are presented.

Comparative genomics of Rhizophagus irregularis, R. cerebriforme, R. diaphanus and Gigaspora rosea highlights specific genetic features in Glomeromycotina.

Glomeromycotina is a lineage of early diverging fungi that establish arbuscular mycorrhizal (AM) symbiosis with land plants. Despite their major ecological role, the genetic basis of their obligate mutualism remains largely unknown, hindering our understanding of their evolution and biology. We compared the genomes of Glomerales (Rhizophagus irregularis, Rhizophagus diaphanus, Rhizophagus cerebriforme) and Diversisporales (Gigaspora rosea) species, together with those of saprotrophic Mucoromycota, to identify gene families and processes associated with these lineages and to understand the molecular underpinning of their symbiotic lifestyle. Genomic features in Glomeromycotina appear to be very similar with a very high content in transposons and protein-coding genes, extensive duplications of protein kinase genes, and loss of genes coding for lignocellulose degradation, thiamin biosynthesis and cytosolic fatty acid synthase. Most symbiosis-related genes in R. irregularis and G. rosea are specific to Glomeromycotina. We also confirmed that the present species have a homokaryotic genome organisation. The high interspecific diversity of Glomeromycotina gene repertoires, affecting all known protein domains, as well as symbiosis-related orphan genes, may explain the known adaptation of Glomeromycotina to a wide range of environmental settings. Our findings contribute to an increasingly detailed portrait of genomic features defining the biology of AM fungi.

High intraspecific genome diversity in the model arbuscular mycorrhizal symbiont Rhizophagus irregularis.

Arbuscular mycorrhizal fungi (AMF) are known to improve plant fitness through the establishment of mycorrhizal symbioses. Genetic and phenotypic variations among closely related AMF isolates can significantly affect plant growth, but the genomic changes underlying this variability are unclear. To address this issue, we improved the genome assembly and gene annotation of the model strain Rhizophagus irregularis DAOM197198, and compared its gene content with five isolates of R. irregularis sampled in the same field. All isolates harbor striking genome variations, with large numbers of isolate-specific genes, gene family expansions, and evidence of interisolate genetic exchange. The observed variability affects all gene ontology terms and PFAM protein domains, as well as putative mycorrhiza-induced small secreted effector-like proteins and other symbiosis differentially expressed genes. High variability is also found in active transposable elements. Overall, these findings indicate a substantial divergence in the functioning capacity of isolates harvested from the same field, and thus their genetic potential for adaptation to biotic and abiotic changes. Our data also provide a first glimpse into the genome diversity that resides within natural populations of these symbionts, and open avenues for future analyses of plant-AMF interactions that link AMF genome variation with plant phenotype and fitness.

Ultra-low input transcriptomics reveal the spore functional content and phylogenetic affiliations of poorly studied arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are a group of soil microorganisms that establish symbioses with the vast majority of land plants. To date, generation of AMF coding information has been limited to model genera that grow well axenically; Rhizoglomus and Gigaspora. Meanwhile, data on the functional gene repertoire of most AMF families is non-existent. Here, we provide primary large-scale transcriptome data from eight poorly studied AMF species (Acaulospora morrowiae, Diversispora versiforme, Scutellospora calospora, Racocetra castanea, Paraglomus brasilianum, Ambispora leptoticha, Claroideoglomus claroideum and Funneliformis mosseae) using ultra-low input ribonucleic acid (RNA)-seq approaches. Our analyses reveals that quiescent spores of many AMF species harbour a diverse functional diversity and solidify known evolutionary relationships within the group. Our findings demonstrate that RNA-seq data obtained from low-input RNA are reliable in comparison to conventional RNA-seq experiments. Thus, our methodology can potentially be used to deepen our understanding of fungal microbial function and phylogeny using minute amounts of RNA material.

Statistical analysis of fractionation resistance by functional category and expression.

BACKGROUND: The current literature establishes the importance of gene functional category and expression in promoting or suppressing duplicate gene loss after whole genome doubling in plants, a process known as fractionation. Inspired by studies that have reported gene expression to be the dominating factor in preventing duplicate gene loss, we analyzed the relative effect of functional category and expression. METHODS: We use multivariate methods to study data sets on gene retention, function and expression in rosids and asterids to estimate effects and assess their interaction. RESULTS: Our results suggest that the effect on duplicate gene retention fractionation by functional category and expression are independent and have no statistical interaction. CONCLUSION: In plants, functional category is the more dominant factor in explaining duplicate gene loss.

An ethnobotany of the Lukomir Highlanders of Bosnia & Herzegovina.

BACKGROUND: This aim of this study is to report upon traditional knowledge and use of wild medicinal plants by the Highlanders of Lukomir, Bjelasnica, Bosnia and Herzegovina (B&H). The Highlanders are an indigenous community of approximately 60 transhumant pastoralist families who speak Bosnian (Bosanski) and inhabit a highly biodiverse region of Europe. This paper adds to the growing record of traditional use of wild plants within isolated communities in the Balkans. METHODS: An ethnobotanical study using consensus methodology was conducted in Lukomir in Bjelasnica's mountains and canyons. Field work involved individual semi-structured interviews during which informants described plants, natural product remedies, and preparation methods on field trips, garden tours, while shepherding, or in settings of their choice. Plant use categories were ranked with informant consensus factor and incorporated into a phylogenetic tree. Plants cited were compared to other ethnobotanical surveys of the country. RESULTS: Twenty five people were interviewed, resulting in identification of 58 species (including two subspecies) from 35 families, which were cited in 307 medicinal, 40 food, and seven material use reports. Individual plant uses had an average consensus of five and a maximum consensus of 15 out of 25. There were a number of rare and endangered species used as poisons or medicine that are endemic to Flora Europaea and found in Lukomir. Ten species (including subspecies) cited in our research have not previously been reported in the systematic ethnobotanical surveys of medicinal plant use in B&H: (Elymus repens (L.) Gould, Euphorbia myrsinites L., Jovibarba hirta (L.) Opiz, Lilium bosniacum (Beck) Fritsch, Matricaria matricarioides (Less.) Porter ex Britton, Phyllitis scolopendrium (L.) Newman, Rubus saxatilis L., Silene uniflora Roth ssp. glareosa (Jord.) Chater & Walters, Silene uniflora Roth ssp. prostrata (Gaudin) Chater & Walters, Smyrnium perfoliatum L.). New uses not reported in any of the aforementioned systematic surveys were cited for a total of 28 species. Thirteen percent of medicinal plants cited are endemic: Helleborus odorus Waldst. et Kit., Gentiana lutea L., Lilium bosniacum (Beck) Fritsch, Silene uniflora Roth ssp. glareosa (Jord.) Chater & Walters., Silene uniflora Roth ssp. prostrata (Gaudin) Chater & Walters, Salvia officinalis L., Jovibarba hirta (L.) Opiz, and Satureja montana L. CONCLUSIONS: These results report on the cohesive tradition of medicinal plant use among healers in Lukomir, Bosnia and Herzegovina. This work facilitates the community's development by facilitating local and international conversations about their traditional medicine and sharing insight for conservation in one of Europe's most diverse endemic floristic regions, stewarded by one of Europe's last traditional Highland peoples.

Gene expression and fractionation resistance.

BACKGROUND: Previous work on whole genome doubling in plants established the importance of gene functional category in provoking or suppressing duplicate gene loss, or fractionation. Other studies, particularly in Paramecium have correlated levels of gene expression with vulnerability or resistance to duplicate loss. RESULTS: Here we analyze the simultaneous effect of function category and expression in two plant data sets, rosids and asterids. CONCLUSION: We demonstrate function category and expression level have independent effects, though expression does not play the dominant role it does in Paramecium.

The dynamics of functional classes of plant genes in rediploidized ancient polyploids.

RESULTS: We measure the simultaneous dynamics of duplicate orthologous gene loss in rosids, in asterids, and in monocots, as influenced by biological functional class. This pan-angiosperm view confirms common tendencies and consistency through time for both ancient and more recent whole genome polyploidization events. CONCLUSIONS: The gene loss analysis represents an assessment of post-polyploidization evolution, at the level of individual gene families within and across sister genomes. Functional analysis confirms universal trends previously reported for more recent plant polyploidy events: genes involved with regulation and responses were retained in multiple copies, while genes involved with metabolic and catalytic processes tended to lose copies, across all three groups of plants.To understand the particular evolutionary patterns of plant genomes, there is a need to systematically survey the fate of the subgenomes of polyploids fixed as whole genome duplicates, including patterns of retention of duplicate, triplicate, etc. genes.