The insertion of a mitochondrial selfish element into the nuclear genome and its consequences

Abstract

Homing endonucleases (HE) are enzymes capable of cutting DNA at highly specific target sequences, the repair of the generated double-strand break resulting in the insertion of the HE-encoding gene ("homing" mechanism). HEs are present in all three domains of life and viruses; in eukaryotes, they are mostly found in the genomes of mitochondria and chloroplasts, as well as nuclear ribosomal RNAs. We here report the case of a HE that accidentally integrated into a telomeric region of the nuclear genome of the fungal maize pathogen Ustilago maydis. We show that the gene has a mitochondrial origin, but its original copy is absent from the U. maydis mitochondrial genome, suggesting a subsequent loss or a horizontal transfer from a different species. The telomeric HE underwent mutations in its active site and lost its original start codon. A potential other start codon was retained downstream, but we did not detect any significant transcription of the newly created open reading frame, suggesting that the inserted gene is not functional. Besides, the insertion site is located in a putative RecQ helicase gene, truncating the C-terminal domain of the protein. The truncated helicase is expressed during infection of the host, together with other homologous telomeric helicases. This unusual mutational event altered two genes: The integrated HE gene subsequently lost its homing activity, while its insertion created a truncated version of an existing gene, possibly altering its function. As the insertion is absent in other field isolates, suggesting that it is recent, the U. maydis 521 reference strain offers a snapshot of this singular mutational event.

Keywords: gene birth; gene transfer; homing endonuclease; intron; mitochondrion.

Figure 1

Identification of the UMAG_11064 gene. (a) Within‐group correspondence analysis of Ustilago maydis codon usage. Each gene is represented according to its coordinates along the first two principal factors. The genomic origin of each gene is indicated by a cross for nuclear genes and a dot for mitochondrial genes. (b) Genomic context of the gene UMAG_11064. GC content in 300 bp windows sliding by 1 bp, and distribution of GC content in 300 bp windows of mitochondrial genome of U. maydis. The dash line represents the median of the distribution

Figure 2

Phylogeny of UMAG_11064 and its homologous sequences. Maximum likelihood tree inferred from nucleotide sequences. Node labels show bootstrap support values. Discontinuous branches indicate that the corresponding sequence is pseudogenized, with multiple frameshifts and insertions/deletions. Branch annotations show the minimum and maximum dN/dS ratio (ω) estimated from 10 independent runs of the codeml program. The yellow triangle indicates the supposed branch where the frameshift within the active domain of the ancestral HE occurred (see Figure 3)

Figure 3

Alignment of UMAG_11064 and its upstream sequence with intron 1 from the cox1 gene of Sporisorium reilianum, Tilletia indica, and Tilletia walkeri, as well as the coding sequence of the Agaricus bisporus HE. Shading indicates the level of amino‐acid conservation, showing conserved residues (in black) and residues with similar biochemical properties (grayscale). Amino‐acids noted as “X” have incomplete codons due to frameshifts. Highlighted exclamation marks denote inferred frameshifts and “*” characters stop codons. The location of the active site of the HE (GVY‐YVG) is highlighted

Figure 4

Intron structure of the cox1 gene in Ustilago maydis and Sporisorium reilianum. Annotated HEs are indicated. Red boxes depict cox1 exons, numbered from e1 to e9. Introns are represented by connecting lines and numbered i1 to i8. Arrows within introns show LAGLIDADG (light blue) and GIY‐YIG HEs (pink). Dashed arrows correspond to HEGs inferred by blast search, while solid arrows correspond to the annotation from the GenBank files. Piecewise sequence similarity between U. maydis and S. reilianum is displayed with a color gradient

Figure 5

Maximum likelihood phylogeny of UMAG_11065 Ustilago maydis paralogs together with the closest homolog from F. oxysporum (see Table 1). Node labels indicate support values as percentage. Nodes with support values lower than 60% have been collapsed

Figure 6

Presence of the UMAG_11064 gene and structure of the cox1 gene in several Ustilago maydis and Sporisorium reilianum strains, as assessed by PCR, together with their phylogeny. The UMAG_11072 gene, located 90 kb downstream the UMAG_11064 gene on chromosome 9, was used as a positive control. Strains 521 and 518 are two strains resulting from the same spore from a field isolate from the United States. SG200 is a genetically engineered strain derived from a cross between the 518 and 521 strains. Strain 10‐1 is another field isolate from the United States. Strains I2, O2, P2, S5, and T6 from field isolates from Mexico

Figure 7

Patterns of gene expression for UMAG_11064 and UMAG_11065, together with neighboring and homologous genes. (a) Gene expression profiles for genes in the chromosome 9 telomeric region (as depicted on Figure 1b). Straight lines represent three independent replicates, while the blue curve depicts the smoothed conditional mean computed using the LOESS method. (b) Gene expression profiles for the UMAG_11065 homologs (Figure 5). Legends as in (a). (c) Clustering of the UMAG_11065 homologs based on their averaged expression profile (see Methods). Hpi, hours postinfection; Dpi, days postinfection

Figure 8

Phenotype assessment of the double deletion strain. (a) The simultaneous deletion of UMAG_11064 and UMAG_11065 does not affect virulence. Maize seedlings were infected with the indicated strains. Disease symptoms were scored at 12 dpi according to Kämper et al. (2006) using the color code depicted on the right. Colors reflect the degree of severity, from brown‐red (severe) to light yellow (mild). Data represent mean of n = 3 biologically independent experiments. Total numbers of infected plants are indicated above the respective columns. (b) Stress assay of the double deletion strain (Δ11064Δ11065), lacking both genes UMAG_11064 and UMAG_11065, compared to the parental SG200 strain. Assays were repeated at least three times with comparable results

Figure 9

Possible evolutionary scenario recapitulating the events leading to the formation of the UMAG_11064 and UMAG_11065 Ustilago maydis genes. Importantly, each step in this model may have occurred by genetic drift alone. Positive selection may have favored—but is not required to explain—the spread of the nuclear and/or the loss of the mitochondrial HEGs