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

Nature Microbiology volume 8, pages 2142–2153 (2023)Cite this article

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An Author Correction to this article was published on 21 November 2023

This article has been updated

Abstract

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.

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Fig. 1: The 32 phased parental chromosomes of the AMF heterokaryotic strain A4.
Fig. 2: Phylogenetic tree constructed with 65 R. irregularis strains.
Fig. 3: Population analyses of R. irregularis.
Fig. 4: Collinearity and variation between co-existing genomes.
Fig. 5: Relative nuclear abundance and expression.

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Data availability

All genome and RNA-seq data and reads newly obtained are available in GenBank under the BioProject PRJNA922099. All genome assemblies, annotations and data used to generate analyses and figures are available at https://zenodo.org/record/8292462.

Code availability

All codes used in this study are available at https://zenodo.org/record/8292462.

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Acknowledgements

We thank J. Oliveira for helping some RNA-seq confirmatory mapping analyses, and P.-M. Delaux, T. James, A. MacLean and V. Kokkoris for their helpful comments on an earlier version of this manuscript, and five anonymous referees for constructive comments. Our research is funded by the Discovery programme of the Natural Sciences and Engineering Research Council (RGPIN2020-05643), a Discovery Accelerator Supplements Program (RGPAS-2020-00033). N.C. is a University of Ottawa Research Chair in Microbial Genomics. J.S. was supported by an Australian Research Council (ARC) Discovery Early Career Researcher Award (DE190100066). Research in the C.G. lab was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant ‘RECEIVE’, no. 759731).

Author information

Author notes

  1. Yanina S. Rizzi & Caroline Gutjahr

    Present address: Max-Planck-Institute of Molecular Plant Physiology, Potsdam-Golm, Germany

  2. These authors contributed equally: Jana Sperschneider, Gokalp Yildirir.

Authors and Affiliations

  1. Black Mountain Science and Innovation Park, CSIRO Agriculture and Food, Canberra, Australian Capital Territory, Australia

    Jana Sperschneider

  2. Department of Biology, University of Ottawa, Ottawa, Ontario, Canada

    Gokalp Yildirir, Mathu Malar C, Ariane Mayrand Nicol, Essam Sorwar, Matthew Villeneuve-Laroche, Elizabeth K. Brauer & Nicolas Corradi

  3. Plant Genetics, TUM School of Life Sciences, Technical University of Munich, Freising, Germany

    Yanina S. Rizzi & Caroline Gutjahr

  4. Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan

    Eric C. H. Chen & Wataru Iwasaki

  5. Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada

    Elizabeth K. Brauer & Whynn Bosnich

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Contributions

J.S., G.Y., Y.S.R. and C.G. conducted investigations and acquired resources. A.M.N., M.C.M., E.C.H.C., W.B., E.S., M.V.-L., W.I. and E.K.B. conducted investigations and provided intellectual input. N.C. conceptualized and supervised the project, wrote the paper and performed visualizations with help from J.S. and G.Y.

Corresponding author

Correspondence to Nicolas Corradi.

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Nature Microbiology thanks Michael F. Seidl, Antonio Di Pietro and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The phased parental chromosomes of the AMF heterokaryotic strain A5.

(a) The karyoplots represent the chromosomes of the haplotypes, represented in blue and brown. The A/B compartments are shown in darker and lighter color, respectively. (b) The histograms of allele frequency distribution are based on bi-allelic SNPs filtered based on the relative genome coverage of each haplotype to exclude contaminants and are overlapped by density curves (black). The X and Y axes represent the SNP frequency (ratio) and density, respectively. Analyses are based 1-1 aligned regions (excluding repetitive regions), representing between 64,331,134 to 69,274,838 bp of each haplotype. (c) Hi-C contact maps showing the compartmentalization in chromosomes. In these heat maps, genome coordinates are represented on both axes. These bright squares highlight increased contact frequency within and between chromosomes and are further analyzed to group them into A/B compartments.

Extended Data Fig. 2 The phased parental chromosomes of the AMF heterokaryotic strain G1.

(a) The karyoplots represent the chromosomes of the haplotypes, represented in blue and brown. The A/B compartments are shown in darker and lighter color, respectively. (b) The histograms of allele frequency distribution are based on bi-allelic SNPs filtered based on the relative genome coverage of each haplotype to exclude contaminants and are overlapped by density curves (black). The X and Y axes represent the SNP frequency (ratio) and density, respectively. Analyses are based 1-1 aligned regions (excluding repetitive regions), representing between 64,331,134 to 69,274,838 bp of each haplotype. (c) Hi-C contact maps showing the compartmentalization in chromosomes. In these heat maps, genome coordinates are represented on both axes. These bright squares highlight increased contact frequency within and between chromosomes and are further analyzed to group them into A/B compartments.

Extended Data Fig. 3 The phased parental chromosomes of the AMF heterokaryotic strain SL1.

(a) The karyoplots represent the chromosomes of the haplotypes, represented in blue and brown. The A/B compartments are shown in darker and lighter color, respectively. (b) The histograms of allele frequency distribution are based on bi-allelic SNPs filtered based on the relative genome coverage of each haplotype to exclude contaminants and are overlapped by density curves (black). The X and Y axes represent the SNP frequency (ratio) and density, respectively. Analyses are based 1-1 aligned regions (excluding repetitive regions), representing between 64,331,134 to 69,274,838 bp of each haplotype. (c) Hi-C contact maps showing the compartmentalization in chromosomes. In these heat maps, genome coordinates are represented on both axes. These bright squares highlight increased contact frequency within and between chromosomes and are further analyzed to group them into A/B compartments.

Extended Data Fig. 4 Percentage of Hi-C contacts that link within and between nuclear-separated haplotypes for all AMF heterokaryons.

The assemblies have a strong dikaryotic Hi-C phasing signal, with over 90% of inter-chromosomal (trans) contacts occurring within the nucleus. The chromosomes are fully phased, with only weak Hi-C trans contact to the other nucleus in some regions. The plots represent the inter- and intra-chromosomal Hi-C contacts of heterokaryon strains A4, A5, G1 and SL1 (from top to bottom), for each haplotype they contain (left to right). The bars represent the percentage of Hi-C contacts while the colors indicate the identity of the contact.

Extended Data Fig. 5 A/B compartments of heterokaryotic strains show difference in gene and repeat concentrations and overall gene expression levels.

P-value of two-tailed t test is 2.22e-16 for all plots. n for Compartment A = 34687 10 kb fragments, for Compartment B, n = 48734. a−b) Boxplots illustrating the difference in gene (Gene/10 kb) and repeat densities (Repeat/10 kb) respectively between A/B compartments of all heterokaryotic genomes analyzed in this study. n for Compartment A = 40468 10 kb fragments, for Compartment B, n = 57260. c) Boxplots showing gene expression level (logTPM + 1) of A/B compartments of all heterokaryotic genomes. n for Compartment A = 35754 10 kb fragments, for Compartment B, n = 33826.d). Collinearity scores, measured by the gene order conservation of blocks of 5 genes (Orthoscore) between haplotypes, is significantly higher in the gene rich A-compartment in all AMF heterokaryons. n for Compartment A = 16501 fragments, for Compartment B, n = 11322.The third quartile (edge of box), first quartile (edge of box), median (middle black line), outliers (dots) and range of data (whiskers) are shown. Compartment A is shown in red and Compartment B is shown in blue color. Asterisks indicate a statistically significant difference determined by two-tailed t-test: * = P < 0.05; ** = P < 0.01; *** = P < 0.001; **** = P < 0.0001. TPM; Transcript per million.

Extended Data Fig. 6 Relative abundance of GO-Terms for genes specific to each haplotype in all AMF heterokaryons.

Data based on genes differentially expressed with a significance level of at least Padj<0.01 (P-values produced using Wald test and are adjusted by Benjamini and Hochberg method). Barplots represent the ratio of genes belonging to each haplotype for each GO-term. Dashed line indicates equal number of genes, ratio = 1.

Extended Data Fig. 7 Examples of structural variation among co-existing genomes in AMF heterokaryons.

D-Genies dot plots show overall synteny and reveal few structural variations between coexisting haplotypes in heterokaryotic strains. Both haplotypes from the same strains are located in x- and y-axes. The aligned regions are represented as dots, and the colors represent identity. The diagonal lines represent synteny between two haplotypes. Breaks indicate rearrangement events.

Extended Data Fig. 8 Differences in 5mC methylation between co-existing genome in AMF heterokaryons.

(a) Violin-plots illustrating the relative density and differences in the degree of methylation among haplotypes. (b) Boxplots illustrating the difference in repeat 5mC methylation (left) and gene 5mC methylation (right) between A/B compartments in A4 and A5. The third quartile (edge of box), first quartile (edge of box), median (middle black line), outliers (dots) and range of data (whiskers) are shown. Two-tailed t test p-values for A4 MAT1 are 6e-13 and 0.0021 for repeats and genes, respectively. n = 8837 repeats and n = 10563 genes in A compartment, n = 13451 repeats and n = 9594 genes in B compartment. For A4 MAT2, p-values are 8.9e-07 and 2e-05 for repeats and genes, respectively. n = 8904 repeats and n = 10597 genes in A compartment, n = 13336 repeats and n = 9450 genes in B compartment. P-values for A5 MAT3 are 1.9e-10 and 0.35 for repeats and genes, respectively. n = 8251 repeats and n = 9350 genes in A compartment, n = 11367 repeats and n = 8009 genes in B compartment. For A5 MAT6, p-values are 1.1e-14 and 0.0002 for repeats and genes, respectively. n = 8389 repeats and n = 9447 genes in A compartment, n = 11676 repeats and n = 8112 genes in B compartment. (c) Boxplots illustrating the difference in methylation frequency between haplotypes in A4, A5 and G1(left), and the percentage of methylation per base within and among haplotypes in A4, A5 and G1(right).

Extended Data Fig. 9 Principal component analyses (PCA) of RNA-seq datasets.

We used clustering and PCA analysis from the package DESeq2 to infer the similarity expression among replicates and conditions in all AMF heterokaryons and lineages. The analyses shows both low inter-replicate variance and significant differences in expression among conditions.

Extended Data Fig. 10 Genome specific Go-term enrichment comparisons for 1-1 orthologues.

GoTerm enrichment comparisons of 1-1 orthologues between haplotype specific genes upregulated in brachypodium vs lotus and chicory vs carrot comparisons in each strain. Brachypodium vs lotus comparisons are shown in the upper plots, whereas chicory vs carrot comparisons are shown in lower plots. Each bar represents percentage of upregulated genes for the same GoTerm in each haplotype. Only GoTerms shared among all haplotype-specific upregulated genes are shown to highlight the respective functional contribution of each haplotype across hosts and strains. Data based on genes differentially expressed with a significance level of at least Padj < 0.01 (P-values produced using Wald test and are adjusted by Benjamini and Hochberg method).

Supplementary information

Reporting Summary

Supplementary Table 1

List and location of secreted proteins and candidate effectors present on each co-existing genomes in AMF heterokaryons.

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Sperschneider, J., Yildirir, G., Rizzi, Y.S. et al. Arbuscular mycorrhizal fungi heterokaryons have two nuclear populations with distinct roles in host–plant interactions. Nat Microbiol 8, 2142–2153 (2023). https://doi.org/10.1038/s41564-023-01495-8

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