Assembly and comparative analyses of the Geosiphon pyriformis metagenome.

Geosiphon pyriformis, a representative of the fungal sub-phylum Glomeromycotina, is unique in its endosymbiosis with cyanobacteria within a fungal cell. This symbiotic relationship occurs in bladders containing nuclei of G. pyriformis, Mollicutes-like bacterial endosymbionts (MRE), and photosynthetically active and dividing cells of Nostoc punctiforme. Recent genome analyses have shed light on the biology of G. pyriformis, but the genome content and biology of its endosymbionts remain unexplored. To fill this gap, we gathered and examined metagenomic data from the bladders of G. pyriformis, where N. punctiforme and MRE are located. This ensures that our analyses are focused on the organs directly involved in the symbiosis. By comparing this data with the genetic information of related cyanobacteria and MREs from other species of Arbuscular Mycorrhizal Fungi, we aimed to reveal the genetic content of these organisms and understand how they interact at a genetic level to establish a symbiotic relationship. Our analyses uncovered significant gene expansions in the Nostoc endosymbiont, particularly in mobile elements and genes potentially involved in xenobiotic degradation. We also confirmed that the MRE of Glomeromycotina are monophyletic and possess a highly streamlined genome. These genomes show dramatic differences in both structure and content, including the presence of enzymes involved in environmental sensing and stress response.

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.

Updated Assembly of Phytophthora ramorum pr102 Isolate Incorporating Long Reads from PacBio Sequencing.

The NA1 clonal lineage of Phytophthora ramorum is responsible for sudden oak death, an epidemic that has devastated California coastal forest ecosystems. An NA1 isolate, Pr102, derived from coast live oak in California, was previously sequenced and reported with a 65-Mb assembly containing 12 Mb of gaps in 2,576 scaffolds. Here, we report an improved 70-Mb genome in 1,512 scaffolds with 6,752 bp of gaps after incorporating PacBio P5-C3 long reads. This assembly contains 19,494 gene models (average gene length of 2,515 bp) compared with 16,134 genes (average gene length of 1,673 bp) in the previous version. We predicted 29 new RXLR genes and 76 new paralogs of a total 392 RXLR genes from this assembly. We predicted 35 CRN genes compared with 19 in an earlier version with six paralogs. Our long non-coding RNA prediction identified 255 candidates. This new resource will be invaluable for future evolution studies on the invasive plant pathogen.

Haplotype-Phased Genome Assembly of Virulent Phytophthora ramorum Isolate ND886 Facilitated by Long-Read Sequencing Reveals Effector Polymorphisms and Copy Number Variation.

Phytophthora ramorum is a destructive pathogen that causes sudden oak death disease. The genome sequence of P. ramorum isolate Pr102 was previously produced, using Sanger reads, and contained 12 Mb of gaps. However, isolate Pr102 had shown reduced aggressiveness and genome abnormalities. In order to produce an improved genome assembly for P. ramorum, we performed long-read sequencing of highly aggressive P. ramorum isolate CDFA1418886 (abbreviated as ND886). We generated a 60.5-Mb assembly of the ND886 genome using the Pacific Biosciences (PacBio) sequencing platform. The assembly includes 302 primary contigs (60.2 Mb) and nine unplaced contigs (265 kb). Additionally, we found a 'highly repetitive' component from the PacBio unassembled unmapped reads containing tandem repeats that are not part of the 60.5-Mb genome. The overall repeat content in the primary assembly was much higher than the Pr102 Sanger version (48 versus 29%), indicating that the long reads have captured repetitive regions effectively. The 302 primary contigs were phased into 345 haplotype blocks and 222,892 phased variants, of which the longest phased block was 1,513,201 bp with 7,265 phased variants. The improved phased assembly facilitated identification of 21 and 25 Crinkler effectors and 393 and 394 RXLR effector genes from two haplotypes. Of these, 24 and 25 RXLR effectors were newly predicted from haplotypes A and B, respectively. In addition, seven new paralogs of effector Avh207 were found in contig 54, not reported earlier. Comparison of the ND886 assembly with Pr102 V1 assembly suggests that several repeat-rich smaller scaffolds within the Pr102 V1 assembly were possibly misassembled; these regions are fully encompassed now in ND886 contigs. Our analysis further reveals that Pr102 is a heterokaryon with multiple nuclear types in the sequences corresponding to contig 10 of ND886 assembly.

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.

Early branching arbuscular mycorrhizal fungus Paraglomus occultum carries a small and repeat-poor genome compared to relatives in the Glomeromycotina.

The arbuscular mycorrhizal fungi (AMFs) are obligate root symbionts in the subphylum Glomeromycotina that can benefit land plants by increasing their soil nutrient uptake in exchange for photosynthetically fixed carbon sources. To date, annotated genome data from representatives of the AMF orders Glomerales, Diversisporales and Archaeosporales have shown that these organisms have large and highly repeated genomes, and no genes to produce sugars and fatty acids. This led to the hypothesis that the most recent common ancestor (MRCA) of Glomeromycotina was fully dependent on plants for nutrition. Here, we aimed to further test this hypothesis by obtaining annotated genome data from a member of the early diverging order Paraglomerales (Paraglomus occultum). Genome analyses showed this species carries a 39.6 Mb genome and considerably fewer genes and repeats compared to most AMF relatives with annotated genomes. Consistent with phylogenies based on ribosomal genes, our phylogenetic analyses suggest P. occultum as the earliest diverged branch within Glomeromycotina. Overall, our analyses support the view that the MRCA of Glomeromycotina carried hallmarks of obligate plant biotrophy. The small genome size and content of P. occultum could either reflect adaptive reductive processes affecting some early AMF lineages, or indicate that the high gene and repeat family diversity thought to drive AMF adaptability to host and environmental change was not an ancestral feature of these prominent plant symbionts.

Regulation of mating genes during arbuscular mycorrhizal isolate co-existence-where is the evidence?

A recent study published by Mateus et al. [1] claimed that 18 "mating-related" genes are differentially expressed in the model arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis when genetically distinct fungal strains co-colonize a host plant. To clarify the level of evidence for this interesting conclusion, we first aimed to validate the functional annotation of these 18 R. irregularis genes using orthology predictions. These analyses revealed that, although sequence relationship exists, only 2 of the claimed 18 R. irregularis mating genes are potential orthologues to validated fungal mating genes. We also investigated the RNA-seq data from Mateus et al. [1] using classical RNA-seq methods and statistics. This analysis found that the over-expression during strain co-existence was not significant at the typical cut-off of the R. irregularis strains DAOM197198 and B1 in plants. Overall, we do not find convincing evidence that the genes involved have functions in mating, or that they are reproducibly up or down regulated during co-existence in plants.

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.

Parasexual and Sexual Reproduction in Arbuscular Mycorrhizal Fungi: Room for Both.

Arbuscular mycorrhizal fungi (AMF) harbor thousands of nuclei in a large syncytium at all times. Although mating processes have not been observed in AMF, their cells and genomes show many signatures of sexual reproduction. Here, we describe how some of these signatures could also arise from parasexual processes in these widespread plant symbionts. As such, parasexual and sexual evolution could both be at play in generating nuclear diversity in AMF.

Asexual Evolution and Forest Conditions Drive Genetic Parallelism in Phytophthora ramorum.

It is commonly assumed that asexual lineages are short-lived evolutionarily, yet many asexual organisms can generate genetic and phenotypic variation, providing an avenue for further evolution. Previous work on the asexual plant pathogen Phytophthora ramorum NA1 revealed considerable genetic variation in the form of Structural Variants (SVs). To better understand how SVs arise and their significance to the California NA1 population, we studied the evolutionary histories of SVs and the forest conditions associated with their emergence. Ancestral state reconstruction suggests that SVs arose by somatic mutations among multiple independent lineages, rather than by recombination. We asked if this unusual phenomenon of parallel evolution between isolated populations is transmitted to extant lineages and found that SVs persist longer in a population if their genetic background had a lower mutation load. Genetic parallelism was also found in geographically distant demes where forest conditions such as host density, solar radiation, and temperature, were similar. Parallel SVs overlap with genes involved in pathogenicity such as RXLRs and have the potential to change the course of an epidemic. By combining genomics and environmental data, we identified an unexpected pattern of repeated evolution in an asexual population and identified environmental factors potentially driving this phenomenon.

Draft Genome Sequence for the Tree Pathogen Phytophthora plurivora.

Species from the genus Phytophthora are well represented among organisms causing serious diseases on trees. Phytophthora plurivora has been implicated in long-term decline of woodland trees across Europe. Here we present a draft genome sequence of P. plurivora, originally isolated from diseased European beech (Fagus sylvatica) in Malmö, Sweden. When compared with other sequenced Phytophthora species, the P. plurivora genome assembly is relatively compact, spanning 41 Mb. This is organized in 1,919 contigs and 1,898 scaffolds, encompassing 11,741 predicted genes, and has a repeat content of approximately 15%. Comparison of allele frequencies revealed evidence for tetraploidy in the sequenced isolate. As in other sequenced Phytophthora species, P. plurivora possesses genes for pathogenicity-associated RXLR and Crinkle and Necrosis effectors, predominantly located in gene-sparse genomic regions. Comparison of the P. plurivora RXLR effectors with orthologs in other sequenced species in the same clade (Phytophthora multivora and Phytophthora capsici) revealed that the orthologs were likely to be under neutral or purifying selection.