A Homokaryotic Derivative of a Phanerochaete chrysosporium Strain and Its Use in Genomic Analysis of Repetitive Elements

Abstract

Analysis of complex gene families in the lignin-degrading basidiomycete Phanerochaete chrysosporium has been hampered by the dikaryotic nuclear condition. To facilitate genetic investigations in P. chrysosporium strain BKM-F-1767, we isolated a homokaryon from regenerated protoplasts. The nuclear condition was established by PCR amplification of five unlinked genes followed by probing with allele-specific oligonucleotides. Under standard nitrogen-limited culture conditions, lignin peroxidase, manganese peroxidase, and glyoxal oxidase activities of the homokaryon were equivalent to those of the parental dikaryon. We used the homokaryon to determine the genomic organization and to assess transcriptional effects of a family of repetitive elements. Previous studies had identified an insertional mutation, Pce1, within lignin peroxidase allele lipI2. The element resembled nonautonomous class II transposons and was present in multiple copies in strain BKM-F-1767. In the present study, three additional copies of the Pce1-like element were cloned and sequenced. The distribution of elements was nonrandom; all localized to the same 3.7-Mb chromosome, as assessed by segregation analysis and Southern blot analysis of the homokaryon. Reverse transcription-PCR (RT-PCR) showed that Pce1 was not spliced from the lipI2 transcript in either the homokaryon or the parental dikaryon. However, both strains had equivalent lignin peroxidase activity, suggesting that some lip genes may be redundant.

Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78

“…Genomic DNA was purified from a homokaryotic strain RP-78 (ref. 39; available from USDA Forest Mycology Center) and was sheared to give an approximate fragment size of 3 kbp. The DNA fragments were bluntend repaired and then size selected on an agarose gel.…”

A Single Mating-Type Locus Composed of Homeodomain Genes Promotes Nuclear Migration and Heterokaryosis in the White-Rot Fungus Phanerochaete chrysosporium

“…Segregation and mating analyses utilized single basidiospore and single conidiospore isolates derived primarily from heterokaryon BKM-F-1767 and secondarily from ME-OC-11. One strain used in the experiments (RP-78) is the protoplastregenerated homokaryon derived from heterokaryon BKM-F-1767, which was used for genome sequencing (77). Cultures were routinely propagated on malt extract agar (MEA) containing 1.5% malt extract and 1.5% agar.…”

Characterization of a multicopper oxidase gene cluster in Phanerochaete chrysosporium and evidence of altered splicing of the mco transcripts

“…P. chrysosporium homokaryotic strain RP-78 (Stewart et al, 2000) and dikaryotic strain BKM-F-1767 were obtained from the Center for Mycology Research, Forest Products Laboratory, Madison, WI. The fungus was grown in defined media containing wood-derived crystalline cellulose (Avicel) as sole carbon source for six days, as previously described (Wymelenberg et al, 2002).…”