Submitted to: Fungal Genetics Conference Proceedings
Publication Type: Abstract only
Publication Acceptance Date: 3/30/2001
Publication Date: 4/29/2001
Citation: CHURCHILL, A.C., MCLANE, H., MUNGEKAR, S.S., SIRVENT, T., KRASNOFF, S.B., HERFORTH, A., GIBSON, D.M. CLONING POLYKETIDE PIGMENT BIOSYNTHETIC GENES FROM THE CHESTNUT BLIGHT FUNGUS CRYPHONECTRIA PARASITICA AND CHEMICAL ANALYSES OF PIGMENT MUTANTS. FUNGAL GENETICS CONFERENCE PROCEEDINGS. 2001. Interpretive Summary:
Technical Abstract: The orange and yellow pigments of Cryphonectria parasitica consist of a family of aromatic polyketides of anthraquinone derivation. Emodin and the other pigments of C. parasitica exhibit numerous and diverse biological activities; however, their roles in the biology of the fungus are unknown. We are using insertional mutagenesis (REMI) and PCR amplification of conserved domains of polyketide synthases (PKSs) to clone genes involved i polyketide pigment production in C. parasitica. Using REMI, we have isolated thirteen mitotically stable pigment mutants, several of which were isolated as fast-growing sectors from primary transformants. Altered pigment production in many of the REMI mutants generally correlates with significantly reduced asexual sporulation and impaired fertility in sexual crosses. Five of ten pigment mutants failed to produce perithecia in crosses; each of the remaining crosses produced only a few perithecia in comparison with control transformants, which produced numbers of peritheci and levels of pigment comparable to wild type strains. Chemical profiles of several pigment mutants, analyzed via HPLC, show significant alterations in amounts and ratios of some of the known pigments; several unknown compounds are being further characterized by HPLC-MS. We have also used PCR amplification, with primers designed to conserved domains of PKSs, to clone putative pigment biosynthetic genes. We have isolated fragments of seven unique genes from C. parasitica that show significant sequence similarity to other fungal and bacterial PKS and fatty acid synthase genes. We are conducting complementation analyses of pigment mutants with hybridizing cosmid clones to assess their roles in pigment biosynthesis.