Characterization of the multigene family encoding endopolygalacturonase in the basidiomycete Chondrostereum purpureum
Date
2001
Authors
Williams, Holly Louise
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Abstract
Chondrostereum purpureum is a white-rot basidiomycete fungus which is being developed as a biocontrol agent of hardwoods for use in reforestation sites and hydroelectric rights-of-ways. It produces several plant cell wall-degrading enzymes that are suspected virulence factors, including endopolygalacturonase (endoPG). To search for new endoPG genes in C. purpureum, degenerate oligonucleotide primers were designed based on conserved regions of seventeen ascomycete endoPG genes and two published endoPG sequences from C. purpureum. These primers were used to amplify endoPG gene fragments from C. purpureum 2128u genomic DNA. Fifty-six of the amplification products were analyzed by sequence determination or restriction digestion to reveal segments of three previously uncharacterized endoPG genes, as well as epgA, an endoPG gene sequenced from C. purpureum 2128u. The three new genes were named epgB, epgC, and epgD, and the full length sequences of two copies of epgB (epgB1 and epgB2) were retrieved from a genomic library of C. purpureum 2128u. The identities between the deduced polypeptide sequences for epgA , epgB1, epgC and epgD ranged from 61.8% to 80.0%. The two epgB genes shared 97.6% nucleotide identity and 97.1 % amino acid identity, with the majority of differences between them near the 3' ends of the genes. Phylogenetic analysis of these five basidiomycete endoPGs, seventeen ascomycete endoPGs, four ascomycete exoPGs, two bacterial endoPGs, and two bacterial exoPGs separated the C. purpureum endoPGs into their own monophyletic clade; whereas the endoPGs from ascomycete species having multiple endoPGs were divided between different monophyletic groups within the ascomycete endoPG clade. This split between the ascomycete and basidiomycete endoPGs suggests the two fungal phyla diverged before the duplication of existing endoPG genes. Mechanisms regulating the expression of the endoPG genes in C. purpureum were examined by northern analyses using mycelia grown in media with different carbohydrate sources. The epgA gene was expressed at high levels when glucose, sucrose or pectin was used as the carbon source; whereas both epgB genes, epgC, and epgD were expressed at low or non-detectable levels on all media types. The expression of epgA appeared to be constitutive and not repressed by simple sugars, suggesting EPGA may be the most important of the five endoPGs in the plant cell wall degradation caused by C. purpureum.