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.

The genome of an intranuclear parasite, Paramicrosporidium saccamoebae, reveals alternative adaptations to obligate intracellular parasitism.

Intracellular parasitism often results in gene loss, genome reduction, and dependence upon the host for cellular functioning. Rozellomycota is a clade comprising many such parasites and is related to the diverse, highly reduced, animal parasites, Microsporidia. We sequenced the nuclear and mitochondrial genomes of Paramicrosporidium saccamoebae [Rozellomycota], an intranuclear parasite of amoebae. A canonical fungal mitochondrial genome was recovered from P. saccamoebae that encodes genes necessary for the complete oxidative phosphorylation pathway including Complex I, differentiating it from most endoparasites including its sequenced relatives in Rozellomycota and Microsporidia. Comparative analysis revealed that P. saccamoebae shares more gene content with distantly related Fungi than with its closest relatives, suggesting that genome evolution in Rozellomycota and Microsporidia has been affected by repeated and independent gene losses, possibly as a result of variation in parasitic strategies (e.g. host and subcellular localization) or due to multiple transitions to parasitism.

Molecular diagnostic toolkit for Rhizophagus irregularis isolate DAOM-197198 using quantitative PCR assay targeting the mitochondrial genome.

Rhizophagus irregularis (previously named Glomus irregulare) is one of the most widespread and common arbuscular mycorrhizal fungal (AMF) species. It has been recovered worldwide in agricultural and natural soils, and the isolate DAOM-197198 has been utilized as a commercial inoculant for two decades. Despite the ecological and economical importance of this taxon, specific markers for quantification of propagules by quantitative real-time PCR (qPCR) are extremely limited and none have been rigorously validated for quality control of manufactured products such as biofertilizers. From the sequencing of 14 complete AMF mitochondrial (mt) genomes, a qPCR assay using a hydrolysis probe designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of R. irregularis isolate DAOM-197198. Specificity tests were performed using standard PCR and qPCR, and results clearly showed that the primers specifically amplified the isolate DAOM-197198, yielding a PCR product of 106 bp. According to the qPCR analyses on spores produced in vitro, the average copy number of mt genomes per spore was 3172 ± 304 SE (n = 6). Quantification assays were successfully undertaken on known and unknown samples in liquid suspensions and commercial dry formulations to show the accuracy, precision, robustness, and reproducibility of the qPCR assay. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate DAOM-197198. The approach of molecular toolkit used in our study could be applied to other AMF taxa and will be useful to research institutions and governmental and industrial laboratories running routine quality control of AMF-based products.

Independent mitochondrial and nuclear exchanges arising in Rhizophagus irregularis crossed-isolates support the presence of a mitochondrial segregation mechanism.

BACKGROUND: Arbuscular mycorrhizal fungi (AMF) are members of the phylum Glomeromycota, an early divergent fungal lineage that forms symbiotic associations with the large majority of land plants. These organisms are asexual obligate biotrophs, meaning that they cannot complete their life cycle in the absence of a suitable host. These fungi can exchange genetic information through hyphal fusions (i.e. anastomosis) with genetically compatible isolates belonging to the same species. The occurrence of transient mitochondrial length-heteroplasmy through anastomosis between geographically distant Rhizophagus irregularis isolates was previously demonstrated in single spores resulting from crossing experiments. However, (1) the persistence of this phenomenon in monosporal culture lines from crossed parental isolates, (2) its correlation with nuclear exchanges and (3) the potential mechanisms responsible for mitochondrial inheritance are still unknown. Using the AMF model organism R. irregularis, we tested whether the presence of a heteroplasmic state in progeny spores was linked to the occurrence of nuclear exchanges and whether the previously observed heteroplasmic state persisted in monosporal in vitro crossed-culture lines. We also investigated the presence of a putative mitochondrial segregation apparatus in Glomeromycota by identifying proteins similar to those found in other fungal groups. RESULTS: We observed the occurrence of biparental inheritance both for mitochondrial and nuclear markers tested in single spores obtained from crossed-isolates. However, only one parental mitochondrial DNA and nuclear genotype were recovered in each monosporal crossed-cultures, with an overrepresentation of certain mitochondrial haplotypes. These results strongly support the presence of a nuclear-independent mitochondrial segregation mechanism in R. irregularis. Furthermore, a nearly complete set of genes was identified with putative orthology to those found in other fungi and known to be associated with the mitochondrial segregation in Saccharomyces cerevisiae and filamentous fungi. CONCLUSIONS: Our findings suggest that mitochondrial segregation might take place either during spore formation or colony development and that it might be independent of the nuclear segregation machinery. We present the basic building blocks for a better understanding of the mitochondrial inheritance process and segregation in these important symbiotic fungi. The comprehension of these processes is of great importance since it has been shown that different segregated lines of the same isolate can have variable effects on the host plant.

Intraisolate mitochondrial genetic polymorphism and gene variants coexpression in arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are multinucleated and coenocytic organisms, in which the extent of the intraisolate nuclear genetic variation has been a source of debate. Conversely, their mitochondrial genomes (mtDNAs) have appeared to be homogeneous within isolates in all next generation sequencing (NGS)-based studies. Although several lines of evidence have challenged mtDNA homogeneity in AMF, extensive survey to investigate intraisolate allelic diversity has not previously been undertaken. In this study, we used a conventional polymerase chain reaction -based approach on selected mitochondrial regions with a high-fidelity DNA polymerase, followed by cloning and Sanger sequencing. Two isolates of Rhizophagus irregularis were used, one cultivated in vitro for several generations (DAOM-197198) and the other recently isolated from the field (DAOM-242422). At different loci in both isolates, we found intraisolate allelic variation within the mtDNA and in a single copy nuclear marker, which highlighted the presence of several nonsynonymous mutations in protein coding genes. We confirmed that some of this variation persisted in the transcriptome, giving rise to at least four distinct nad4 transcripts in DAOM-197198. We also detected the presence of numerous mitochondrial DNA copies within nuclear genomes (numts), providing insights to understand this important evolutionary process in AMF. Our study reveals that genetic variation in Glomeromycota is higher than what had been previously assumed and also suggests that it could have been grossly underestimated in most NGS-based AMF studies, both in mitochondrial and nuclear genomes, due to the presence of low-level mutations.

Mitochondrial genome rearrangements in glomus species triggered by homologous recombination between distinct mtDNA haplotypes.

Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms.AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes (dpo), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp.) with substantial mtDNA synteny divergence,were sequenced and compared with available glomeromycotan mitochondrial genomes. We used an extensive nucleotide/protein similarity network-based approach to investigated podiversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo-induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity.We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants.

Detection of a transient mitochondrial DNA heteroplasmy in the progeny of crossed genetically divergent isolates of arbuscular mycorrhizal fungi.

Nonself fusion and nuclear genetic exchange have been documented in arbuscular mycorrhizal fungi (AMF), particularly in Rhizophagus irregularis. However, mitochondrial transmission accompanying nonself fusion of genetically divergent isolates remains unknown. Here, we tested the hypothesis that mitochondrial DNA (mtDNA) heteroplasmy occurs in the progeny of spores, obtained by crossing genetically divergent mtDNAs in R. irregularis isolates. Three isolates of geographically distant locations were used to investigate nonself fusions and mtDNA transmission to the progeny. We sequenced two additional mtDNAs of two R. irregularis isolates and developed isolate-specific size-variable markers in intergenic regions of these isolates and those of DAOM-197198. We achieved three crossing combinations in pre-symbiotic and symbiotic phases. Progeny spores per crossing combination were genotyped using isolate-specific markers. We found evidence that nonself recognition occurs between isolates originating from different continents both in pre-symbiotic and symbiotic phases. Genotyping patterns of individual spores from the progeny clearly showed the presence of markers of the two parental mtDNA haplotypes. Our results demonstrate that mtDNA heteroplasmy occurs in the progeny of the crossed isolates. However, this heteroplasmy appears to be a transient stage because all the live progeny spores that were able to germinate showed only one mtDNA haplotype.

Rapid mitochondrial genome evolution through invasion of mobile elements in two closely related species of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are common and important plant symbionts. They have coenocytic hyphae and form multinucleated spores. The nuclear genome of AMF is polymorphic and its organization is not well understood, which makes the development of reliable molecular markers challenging. In stark contrast, their mitochondrial genome (mtDNA) is homogeneous. To assess the intra- and inter-specific mitochondrial variability in closely related Glomus species, we performed 454 sequencing on total genomic DNA of Glomus sp. isolate DAOM-229456 and we compared its mtDNA with two G. irregulare isolates. We found that the mtDNA of Glomus sp. is homogeneous, identical in gene order and, with respect to the sequences of coding regions, almost identical to G. irregulare. However, certain genomic regions vary substantially, due to insertions/deletions of elements such as introns, mitochondrial plasmid-like DNA polymerase genes and mobile open reading frames. We found no evidence of mitochondrial or cytoplasmic plasmids in Glomus species, and mobile ORFs in Glomus are responsible for the formation of four gene hybrids in atp6, atp9, cox2, and nad3, which are most probably the result of horizontal gene transfer and are expressed at the mRNA level. We found evidence for substantial sequence variation in defined regions of mtDNA, even among closely related isolates with otherwise identical coding gene sequences. This variation makes it possible to design reliable intra- and inter-specific markers.

Group I intron-mediated trans-splicing in mitochondria of Gigaspora rosea and a robust phylogenetic affiliation of arbuscular mycorrhizal fungi with Mortierellales.

Gigaspora rosea is a member of the arbuscular mycorrhizal fungi (AMF; Glomeromycota) and a distant relative of Glomus species that are beneficial to plant growth. To allow for a better understanding of Glomeromycota, we have sequenced the mitochondrial DNA of G. rosea. A comparison with Glomus mitochondrial genomes reveals that Glomeromycota undergo insertion and loss of mitochondrial plasmid-related sequences and exhibit considerable variation in introns. The gene order between the two species is almost completely reshuffled. Furthermore, Gigaspora has fragmented cox1 and rns genes, and an unorthodox initiator tRNA that is tailored to decoding frequent UUG initiation codons. For the fragmented cox1 gene, we provide evidence that its RNA is joined via group I-mediated trans-splicing, whereas rns RNA remains in pieces. According to our model, the two cox1 precursor RNA pieces are brought together by flanking cox1 exon sequences that form a group I intron structure, potentially in conjunction with the nad5 intron 3 sequence. Finally, we present analyses that address the controversial phylogenetic association of Glomeromycota within fungi. According to our results, Glomeromycota are not a separate group of paraphyletic zygomycetes but branch together with Mortierellales, potentially also Harpellales.