Genomic rearrangements generate hypervariable mini-chromosomes in host-specific isolates of the blast fungus

Abstract

Supernumerary mini-chromosomes-a unique type of genomic structural variation-have been implicated in the emergence of virulence traits in plant pathogenic fungi. However, the mechanisms that facilitate the emergence and maintenance of mini-chromosomes across fungi remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), mini-chromosomes have been first described in the early 1990s but, until very recently, have been overlooked in genomic studies. Here we investigated structural variation in four isolates of the blast fungus M. oryzae from different grass hosts and analyzed the sequences of mini-chromosomes in the rice, foxtail millet and goosegrass isolates. The mini-chromosomes of these isolates turned out to be highly diverse with distinct sequence composition. They are enriched in repetitive elements and have lower gene density than core-chromosomes. We identified several virulence-related genes in the mini-chromosome of the rice isolate, including the virulence-related polyketide synthase Ace1 and two variants of the effector gene AVR-Pik. Macrosynteny analyses around these loci revealed structural rearrangements, including inter-chromosomal translocations between core- and mini-chromosomes. Our findings provide evidence that mini-chromosomes emerge from structural rearrangements and segmental duplication of core-chromosomes and might contribute to adaptive evolution of the blast fungus.

Fig 1. Host-specific isolates of M. oryzae contain mini-chromosomes of various sizes.

Contour-clamped homogenous electric field (CHEF) gel electrophoresis of intact M. oryzae chromosomes. Chromosome size variation between M. oryzae isolates is present in both, mini-chromosomes and core chromosomes. Strains FR13, US71, and CD156 contain mini-chromosomes ranging in size between approximately 850 kb and 1.5 Mb. Left lane: Saccharomyces cerevisiae chromosomes as size marker.

Fig 2. M. oryzae strain FR13 contains a 1.7 Mb mini-chromosome assembled into 2 scaffolds.

A) Circos plot of mini-chromosome isolation sequencing (MCIS) read coverage and repeat content across FR13 scaffolds < 2 Mb. Outer ring (rainbow colors): FR13 scaffolds and scaffold sizes. Outer track (Red/Black): MCIS coverage per sliding window. Window size = 1000 bp; Slide distance: 500 bp. Y-axes: average coverage per 1 kb window; axis limits set to zero to maximum coverage. Inner track (Blue/Black): Repeat content per sliding window. Window size = 10 kbp; Slide distance: 5 kbp. Y-axes: repeat content in bp per 10 kb window; axis limits set to zero to maximum. B) Circos plot of MCIS read coverage and repeat content for scaffolds <200 kb (enlarged from A).

Fig 3. Mini-chromosomes of M. oryzae are isolate specific.

A) Circos plots depicting alignments between mini-chromosomes (left) and core-chromosomes (right). Outer ring: Mini-chromosome scaffolds and sizes. Alignments > 10 kb are plotted as genetic links in the center. B) Relative fraction of mini- and core-chromosomes that generate pairwise alignments. Relative fraction of the genomic compartment that form alignments > 10 kb are shown for each pairwise alignment and each individual isolate. Approximately 75% of the core-chromosomes generate alignments under these parameters, whereas only 2.61–28.39% of mini-chromosomes do.

Fig 4. Mini-chromosomes are less conserved than core-chromosomes across lineages of M. oryzae.

A) Conservation of core- and mini-chromosomes across host-specific lineages of M. oryzae based on breadth of coverage of non-redundant whole genome alignments against the three mini- and selected core-chromosome scaffolds. Heatmap values show the relative breadth of coverage of alignments for each mini-chromosome or core-chromosome scaffolds. Left panel: Schematic representation of a coalescent species tree representing host-specific genetic lineages of M. oryzae. Host genera are indicated in colors. Yellow = eragrostis lineage; Brown = brachiaria lineage 1 (isolate Bm88324) and 2 (Br2); Red = stenotaphrum lineage. B) Scatterplots showing the average breadth of coverage of alignments of core- over mini- chromosomes per host-specific lineage. C) Boxplots summarizing the breadth of coverage by genomic compartment across host-specific lineages.

Fig 5. Mini-chromosomes have lower gene and higher repeat content than core-chromosomes.

A) Bar plots of gene and repeat content of mini- (red) and core-chromosomes (grey) of isolates FR13, US71, and CD156. B) Density plots resulting from a bootstrapping analysis of gene and repeat content of isolates FR13, US71, and CD156. 10000 core-genomic fragments were randomly sampled for each mini-chromosome scaffold and distribution of genes and basepairs of repeats are shown as densities. Number of genes and repeat content of mini-chromosome scaffolds are indicated by arrowheads. The size of mini-chromosome scaffolds is given in parentheses. Lower and upper 2.5% frequency intervals are shown in red and blue. X-axis: number of genes and basepairs of repeats. Y-axis: Frequency in 10000 fragments. Y-axis limits set to min/max.

Fig 6. Genomic rearrangements around virulence-related loci on mini-chromosomes.

A) Synteny analysis around the AVR-Pik loci on the FR13 mini-chromosome and corresponding chromosomes and contigs in the related isolates Guy11, FJ81278, and the reference strain 70–15. The 761 kb region around the variant AVR-PikA on the mini-chromosome with high similarity to the end of chromosome 2 of the reference strain 70–15 is shown in purple. The makrosyntenic region between 70–15 and FR13 is disrupted on the mini-chromosome (synteny break between region 1 and 2). The synteny is disrupted earlier in Guy11 and there are 2 inversions around the AVR-Pik locus. Another syntenic region (82 kb) is present between the end of the mini-chromosome encoding the AVR-PikD variant and contig 21 of isolate FJ81278. Syntenic regions larger than 10 kb are shown. Illustration of supercontig 8.2 starts at 6 Mb for better visualization. B) Synteny analysis around the Ace1-containing secondary metabolite cluster on the FR13 mini-chromosome and corresponding contigs of isolates Guy11, FJ81278, 70–15, US71, and CD156. The Ace1 cluster is located at the end of the 761 kb syntenic region between the FR13 mini-chromosome and supercontig 8.2 of isolate 70–15. The synteny around the Ace1 locus is highly conserved in closely related isolates, including the foxtail millet isolate US71. In strains Guy11, US71, and 70–15 the cluster is located on core-chromosomes. The macro-synteny around the Ace1 cluster is disrupted by a 392 kb insertion on the FR13 mini-chromosome. The Ace1 cluster is shown in red and the two syntenic regions (761 kb and 164 kb) are shown in purple and yellow, respectively. Mini-chromosome scaffolds are highlighted in blue.

Fig 7. Core-genomic rearrangements are associated with mini-chromosome emergence.

A) Synteny analysis of 70–15 chromosome 2 and syntenic scaffolds and contigs in FR13, Guy11, and FJ81278. Syntenic regions between isolates are shown as genetic links in blue (alignments in forward direction) and red (reverse direction). Aligning regions are indicated as red and blue boxes. Scaffold and contig breaks are indicated as grey tones. The mini-chromosome scaffold 9 is shown in bright blue. Regions around the major rearrangement are numbered and indicated by colored boxes. B) Analysis of GC-content, gene-content, and repeat-content in regions i-iv surrounding the major rearrangement. Colors correspond to colors in (A). Axis limits for GC-content was set to 0.5–0.7. Axis limits for gene- and repeat-content was set to 0 / max and the center line represents the average of the contig. Gene- and repeat content is plotted as bp per 1kb sliding window. Gene and repeat models are plotted below in bright blue and bright red, respectively.