Multiple Horizontal Mini-chromosome Transfers Drive Genome Evolution of Clonal Blast Fungus Lineages

Abstract

Crop disease pandemics are often driven by asexually reproducing clonal lineages of plant pathogens that reproduce asexually. How these clonal pathogens continuously adapt to their hosts despite harboring limited genetic variation, and in absence of sexual recombination remains elusive. Here, we reveal multiple instances of horizontal chromosome transfer within pandemic clonal lineages of the blast fungus Magnaporthe (Syn. Pyricularia) oryzae. We identified a horizontally transferred 1.2Mb accessory mini-chromosome which is remarkably conserved between M. oryzae isolates from both the rice blast fungus lineage and the lineage infecting Indian goosegrass (Eleusine indica), a wild grass that often grows in the proximity of cultivated cereal crops. Furthermore, we show that this mini-chromosome was horizontally acquired by clonal rice blast isolates through at least nine distinct transfer events over the past three centuries. These findings establish horizontal mini-chromosome transfer as a mechanism facilitating genetic exchange among different host-associated blast fungus lineages. We propose that blast fungus populations infecting wild grasses act as genetic reservoirs that drive genome evolution of pandemic clonal lineages that afflict cereal crops.

Keywords: clonal blast fungus lineages; crop disease pandemics; genetic reservoirs; horizontal mini-chromosome transfer; wild hosts.

Fig. 1.

Clonal rice blast fungus isolates display variable mChr content. a) Genome-wide SNP-based NeighborNet analysis confirms the nine rice blast fungus isolates (red dots) belong to clonal lineage II (green) (Latorre et al. 2020). b) CHEF gel karyotyping reveals variable mChr content. A conserved 1.7Mb mChr (mChrC, green) is found in eight of nine isolates. A 2Mb mChr (mChrM, blue), present in isolate AG006, is a mosaic composed of fragments from three other mChr (mChrC, mChrA, and mChrS; see panel e) from the same isolate. A third 1.2Mb mChr (mChrA, red) found in AG006, is absent from the genomes of the other isolates (see panels E and F). c) Schematic karyotype of Italian isolates. Core chromosomes are shown in white. mChr studied in detail are highlighted in colors, while the rest are in gray. d) mChrC exhibits high synteny across isolates and is also found in isolate FR13 (Langner et al. 2021). Telomeric sequences are indicated by a vertical line (purple). e) Inferred mChrM sequence composition. f) Whole-genome alignment between AG006 (green) and AG002 (brown). mChrA (AG006_Contig10) and AG006_Contig17 (in red) are absent from AG002.

Fig. 2.

mChrA-like sequences are present across multiple host-associated blast fungus lineages. a) Genome-wide SNP-based NJ tree of 413 M. oryzae and M. grisea isolates. M. oryzae isolates are color-coded by lineage and M. grisea is in black. The 126 isolates defined as mChrA-like sequence carriers (supplementary table S9, Supplementary Material online) are highlighted by a red square and belong to six different M. oryzae lineages. Arrows indicate isolates with mChrA-related karyotyping information (Peng et al. 2019; Rahnama et al. 2020), see Fig. 4). Colors of dotted lines across the rice blast lineage represent different genetic subgroups (three clonal lineages and a recombining group) (Latorre et al. 2020). Scale bar represents nucleotide substitutions per position. b) Bimodal distribution of mChrA breadth of coverage across 413 M. oryzae and M. grisea isolates. The coverage cutoff (61%) for mChrA-like sequence presence or absence is indicated by the dotted red line. Arrows as in (a). c) mChrA breadth of coverage across 276 rice blast fungus isolates. Colors represent different genetic subgroups in the Oryza lineage. Arrows and coverage cutoff as in (b).

Fig. 3.

Discordant genetic clustering between the core genome and mChrA. a-d). SNP-based NJ trees (a and c) and Principal Component Analyses (PCA, b and d) of 126 M. oryzae isolates carrying the mChrA-like sequence. Discordance between core genome (a and b) and mChrA (c and d) genetic clustering is observed (highlighted by red arrows). Scale bar represents nucleotide substitutions per position. e) Percentage of tree topologies where a monophyletic relationship was observed for 100 randomly selected 1.2Mb core-chromosomal regions. In all instances the Oryza lineage was monophyletic, and in no instance did the Eleusine and Oryza blast fungus lineages cluster together. f) F_ST between rice blast fungus isolates (n = 32) and isolates from the Eleusine lineage (Br62 and B51) both carrying mChrA-like sequences. Each dot (gray) indicates the weighted F_ST per 5 kb window using a step size of 500 bp. The number of windows per contig are at the top of each box. Core chromosome contigs >2Mb and mChrA are shown.

Fig. 4.

Eleusine isolate Br62 and Oryza isolate AG006 carry an intact and highly syntenic mChrA. a) CHEF-gel karyotyping of AG006 and Br62. Six gel lanes per isolate are shown representing a single biological replicate. Br62 carries a 1.2Mb mChr, the same size as mChrA in AG006 (in red). b) Whole-genome alignment of Br62 (orange) and AG006 (green). Br62_Contig07 aligns exclusively to mChrA (AG006_Contig10) and AG006_Contig17 (red). For both isolates a schematic karyotype is depicted. c) Alignment of mChrA in AG006 and Br62 reveal high synteny, except for a rearrangement in the central region. Alignments covering a fraction of mChrA are seen among mChrA in AG006 and the mChrA-like mChr1 in Lolium isolate LpKY97 (magenta) and the mChr in Triticum isolate B71 (blue). Telomeric sequences are indicated by vertical lines (purple/brown). The host plant of each isolate is shown on the left.

Fig. 5.

Multiple mChrA transfers occurred in clonal rice blast fungus lineages. a) D-statistics. Lines depict 99% confidence intervals and the red dot the estimated D value. Lines not encompassing D = 0 are gray and the rest black. Jack-knife blocks were 5Mb long. b) Average Kimura two-parameter (K2P) distances between homologous mChrA sequences from +mChrA isolates to Br62, and average distances of random core chromosome homologous sequences from +mChrA isolates to Br62. c) Average K2P genetic distances between two sets of homologous random core chromosome sequences from +mChrA isolates to Br62. d) Ancestral states of mChrA presence or absence along the clonal rice blast fungus phylogeny. Thick lines indicate mChrA is present. Branches are color-coded by lineage. The SA05-43 isolate from the Setaria blast fungus lineage was chosen as an outgroup. Branches with evidence for horizontal mChrA acquisition are indicated by a red diamond, the branch where there is evidence for sexual transfer is indicated by a yellow diamond. The brown bars in the nodes represent the 95% Highest Posterior Density.

Fig. 6.

Model. Horizontal mini-chromosome transfers from blast fungus lineages infecting wild grasses drive genome evolution of clonal lineages infecting crops. The recurrent acquisition of mChrA from wild grass-infecting blast fungus lineages by clonal rice blast fungus lineages enhances their evolutionary adaptability and capacity to respond to changing environments and hosts. The coexistence of infected crops and wild hosts facilitates this genetic exchange, posing a challenge to the management of crop disease pandemics.