Recombinant b alleles of Ustilago maydis

The fungal plant pathogen Ustilago maydis has a multiallelic sexual incompatibility system governed by an estimated 25 alleles at the b locus. Haploid strains must have different b alleles in order to be sexually compatible and pathogenic. Existing sequence data from several b alleles show a variable domain in the first 110 codons and a constant domain through the rest of the open reading frame. To delineate the region responsible for allelic specificity, a series of recombinant alleles containing the variable region of b1 and the constant region of b2 were constructed using an in vivo recombination strategy. A haploid strain of U. maydis, carrying the b2 allele, was transformed with linear DNA containing progressive 3’ end deletions of the b1 variable region. Homologous integration of the transforming DNA resulted in replacement of the resident b2 variable region with part or all of the b1 variable region, producing a b1-b2 recombinant. A map of alleles containing recombination points throughout the variable region delineated a 48 codon domain responsible for allelic specificity. Recombination between b1 and b2 within this region generated alleles with different specificities from either b1 or b2. Analysis of nucleotide sequences of recombinant alleles derived from specific plasmids showed that homologous integration can occur anywhere from the end of the linear transforming DNA to about 100 by from the end. A comparison of amino acid sequences of recombinant alleles with recombination points near the left and right borders of the specificity domain showed that nonconservative amino acid substitutions can alter allelic specificity of the b locus. These results suggest that new alleles of the multiallelic b locus may have evolved by a combination of meiotic crossing over and mutation within the specificity region.Land and Food Systems, Faculty ofGraduat