Maintenance of sex-related genes and the co-occurrence of both mating types in Verticillium dahliae

Abstract

Verticillium dahliae is a cosmopolitan, soilborne fungus that causes a significant wilt disease on a wide variety of plant hosts including economically important crops, ornamentals, and timber species. Clonal expansion through asexual reproduction plays a vital role in recurring plant epidemics caused by this pathogen. The recent discovery of recombination between clonal lineages and preliminary investigations of the meiotic gene inventory of V. dahliae suggest that cryptic sex appears to be rare in this species. Here we expanded on previous findings on the sexual nature of V. dahliae. Only 1% of isolates in a global collection of 1120 phytopathogenic V. dahliae isolates contained the MAT1-1 idiomorph, whereas 99% contained MAT1-2. Nine unique multilocus microsatellite types comprised isolates of both mating types, eight of which were collected from the same substrate at the same time. Orthologs of 88 previously characterized sex-related genes from fungal model systems in the Ascoymycota were identified in the genome of V. dahliae, out of 93 genes investigated. Results of RT-PCR experiments using both mating types revealed that 10 arbitrarily chosen sex-related genes, including MAT1-1-1 and MAT1-2-1, were constitutively expressed in V. dahliae cultures grown under laboratory conditions. Ratios of non-synonymous (amino-acid altering) to synonymous (silent) substitutions in V. dahliae MAT1-1-1 and MAT1-2-1 sequences were indistinguishable from the ratios observed in the MAT genes of sexual fungi in the Pezizomycotina. Patterns consistent with strong purifying selection were also observed in 18 other arbitrarily chosen V. dahliae sex-related genes, relative to the patterns in orthologs from fungi with known sexual stages. This study builds upon recent findings from other laboratories and mounts further evidence for an ancestral or cryptic sexual stage in V. dahliae.

Figure 1. Color-coded results of SELECTON analyses of Verticillium dahliae MAT1-1-1, compared to sequences from nine different sexual fungi in the Pezizomycotina.

Shades of yellow (colors 1 and 2) indicate a Ka/Ks ratio>1 (positive selection), and shades of purple (colors 3 through 7) indicate a Ka/Ks ratio<1 (purifying selection); A) results from the M8 model; B) results of the MEC model; amino acid sequence of the α domain is indicated by black border.

Figure 2. Color-coded results of SELECTON analyses of Verticillium dahliae MAT1-2-1, compared to sequences from nine different sexual fungi in the Pezizomycotina.

Shades of yellow (colors 1 and 2) indicate a Ka/Ks ratio>1 (positive selection) and shades of purple (colors 3 through 7) indicate a Ka/Ks ratio<1 (purifying selection); A) results from the M8 model; B) results of the MEC model; amino acid sequence of the HMG domain is indicated by black border.

Figure 3. Reverse-transcriptase PCR results of 10 Verticillium dahliae orthologs of genes associated with the sexual cycle in model fungal systems; gene names are provided for each lane; A) RT-PCR results from V. dahliae strain 58 (MAT1-1).

B) RT-PCR results from V. dahliae strain Ls 17 (MAT1-2);