Evolutionary dynamics of mating-type loci of Mycosphaerella spp. occurring on banana

Abstract

The devastating Sigatoka disease complex of banana is primarily caused by three closely related heterothallic fungi belonging to the genus Mycosphaerella: M. fijiensis, M. musicola, and M. eumusae. Previous phylogenetic work showing common ancestry led us to analyze the mating-type loci of these Mycosphaerella species occurring on banana. We reasoned that this might provide better insight into the evolutionary history of these species. PCR and chromosome-walking approaches were used to clone the mating-type loci of M. musicola and M. eumusae. Sequences were compared to the published mating-type loci of M. fijiensis and other Mycosphaerella spp., and a novel organization of the MAT loci was found. The mating-type loci of the examined Mycosphaerella species are expanded, containing two additional Mycosphaerella-specific genes in a unique genomic organization. The proteins encoded by these novel genes show a higher interspecies than intraspecies homology. Moreover, M. fijiensis, M. musicola, and M. eumusae contain two additional mating-type-like loci, containing parts of both MAT1-1-1 and MAT1-2-1. The data indicate that M. fijiensis, M. musicola, and M. eumusae share an ancestor in which a fusion event occurred between MAT1-1-1 and MAT1-2-1 sequences and in which additional genes became incorporated into the idiomorph. The new genes incorporated have since then evolved independently in the MAT1-1 and MAT1-2 loci. Thus, these data are an example of the evolutionary dynamics of fungal MAT loci in general and show the great flexibility of the MAT loci of Mycosphaerella species in particular.

FIG. 1.

Organization of mating-type loci of Mycosphaerella graminicola, M. fijiensis, M. musicola, and M. eumusae. Dissimilar sequences (idiomorphs) are indicated by white boxes, and identical sequences by black boxes. Predicted gene models are indicated by arrows. The scale at the bottom shows size in kilobases.

FIG. 2.

Pairwise comparison of mating-type loci of Mycosphaerella graminicola, M. fijiensis, M. musicola, and M. eumusae. MAT1-1 and MAT1-2 and flanking regions of M. graminicola, M. fijiensis, M. musicola, and M. eumusae were analyzed by BLAST2. Plots show the presence of inverted regions with high levels of identity within the idiomorphs of the banana pathogens. The MAT1-1 sequences are plotted on the x axis, and the MAT1-2 sequences are plotted on the y axis. Numbers indicate the size of the analyzed fragments in kilobases.

FIG. 3.

Phylogram of the predicted amino acid sequences of MATORF1 and MATORF2. The phylograms are based upon the maximum likelihood method implemented in the PhyML program. Analyses were performed for MATORF1 (a) and MATORF2 (b) from both idiomorphs of Mycosphaerella eumusae, M. fijiensis, M. musicola, M. graminicola, and Septoria passerinii. Bootstrap support values from 500 replicates are shown as percentages at the nodes. Scale bars indicate the number of substitutions per site.

FIG. 4.

RT-PCR analysis of MATORF1 and MATORF2 expression in Mycosphaerella fijiensis. The isolates used were the MAT1-1 isolate CIRAD86 and the M. fijiensis MAT1-2 isolate CIRAD251. PCR with primer combinations ORF1F/ORF1R (lanes 1 to 4) and ORF2F/ORF2R (lanes 5 to 8) was performed on cDNA derived from CIRAD251 (lanes 1 and 5), cDNA derived from CIRAD86 (lanes 2 and 6), genomic DNA from CIRAD251 (lanes 3 and 7), and genomic DNA from CIRAD86 (lanes 4 and 8). The lane labeled M contains a DNA marker. The numbers to left and right show nucleotide sizes of amplified fragments.

FIG. 5.

Schematic overview of the organization and composition of the Mycosphaerella fijiensis MAT1/2A and MAT1/2B fusion loci. Indicated are the areas sharing >90% nucleotide identity between the Mycosphaerella fijiensis MAT1/2A (a) and MAT1/2B (b) fusion loci and the M. fijiensis mating-type genes MAT1-1-1 and MAT1-2-1. MAT1-1-1 sequences and their corresponding positions in MAT1/2A and MAT1/2B are marked in black. MAT1-2-1 sequences and corresponding locations in MAT1/2A and MAT1/2B are marked in gray. Double-ended arrows mark the positions of corresponding homologous stretches. The positions and names of the primers used to amplify the fusion loci from M. eumusae and M. musicola are marked with carets.