Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSBck1 from Saccharomyces cerevisiae. Colony morphology of the Cpbck1-null mutants differed dramatically from the wild type that mutants showed theinvasive growth pattern characterized by slower growth rate, absence of distinctive aerial hyphae resulting in almost absence of conidia-bearing structureand conidia, sparse mycelial growth on the surface of agar plate with abnormal pigmentation, and irregular mycelial mat within the restricted area.Feeding hyphae growing under the plate showed less branched and relatively slower growth pattern. Interestingly, the Cpbck1-null mutant producedsectors appeared as thick rubbery patches of matted growth without pigmentation and sporulation. Complementation of the Cpbck1-null mutant with awild-type allele rescued mutant phenotypes indicating that the mutant phenotypes were due to the absence of the Cpbck1 gene. Intracellular structureobserved by electron microscope revealed both invasive growth-type and sectored-type showed the occurrence of hypertrophy of cell wall, multiple nucleiwithin swollen cells and intrahyphal hyphae. DNA methylation, an indicative of epigenetic marker, examined by Southern blot analysis and bisulfite DNAmodification of putative target genes revealed that there was difference in the DNA methylation pattern between original Cpbck1-null mutant andsectored isolate. This study suggests that epigenetic changes are predisposed by the loss of function mutation of a specific gene Cpbck1 and it will be ofinterest to determine what decide the transition of the mycelia growth pattern from the invasive and very-sick hyphal growth type to compact-mat type.The Cpbck1-null mutant showed the sectored phenotype accompanied with the changes in DNA methylation demonstrated that the fungal signalingpathway implicated in the control of epigenetic processes, without which abnormal degeneration such as sectoring occurred.440. NUP-6 (Importin a) is required for DNA methylation in Neurospora crassa. Andrew D. Klocko 1 , Michael R. Rountree 1 , Paula L. Grisafi 1 , Shan M. Hays 2 ,Eric U. Selker 1 . 1) Institute of Molecular Biology, University of Oregon, Eugene, OR 97448; 2) Department of Natural and Environmental Sciences, WesternState College of Colorado, Gunnison, CO 81231.Heterochromatic regions on chromosomes are essential for numerous cellular processes, including centromere function and gene silencing. Therepetitive DNA found in heterochromatin is highly compacted, frequently A:T rich, and in some species such as Neurospora crassa, methylated atcytosines. This DNA methylation can effectively silence genes. Interestingly, unlike the situation in some eukaryotes, loss of DNA methylation is notrequired for viability in Neurospora. The dispensability of DNA methylation in Neurospora allows for the identification of defective in methylation (dim)genes that have critical roles in the establishment, maintenance, and/or regulation of DNA methylation. This approach established that, at least inNeurospora, DNA methylation is initiated by the histone methyltransferase activity of a multi-subunit complex, DCDC (DIM-5/-7/-9 CUL-4 DDB-1 dim-8Complex), which catalyzes tri-methylation of lysine 9 on histone H3 (H3K9me3). While the identification of the components of the DCDC was an importantstep to understanding heterochromatin formation, much is still unknown about the DCDC, including its detailed function, regulation, and assembly. Here,we characterize the action of a previously unidentified dim mutant, dim-3. We found that dim-3 corresponds to the nup-6 gene, which encodes theImportin a subunit (NUP-6) for classical nuclear transport. NUP-6 dim-3 virtually abolishes H3K9me3 and significantly reduces DNA methylation, and causesDIM-5 and DIM-7 to be mislocalized from heterochromatin, suggesting DCDC activity is impacted in a dim-3 strain. Curiously, nuclear transport of DCDCcomponents in a dim-3 strain background appears to be equal to or greater than in a wild type background. The possibility exists that NUP-6 could beimportant in directing the DCDC to heterochromatin or in assembly of the DCDC, and we will address these hypotheses. In addition, the mutations found indim-3 could prevent its gene product, NUP-6, from facilitating DCDC action.441. Identification and characterization of a Blastomyces dermatitidis mutant with a bidirectional defect in the phase transition. Amber J. Marty,Gregory M. Gauthier. University of Wisconsin - Madison, 1550 Linden Drive, Microbial Sciences Building, Madison, WI, 53706.Collectively, the dimorphic fungi are the most common cause of invasive fungal disease worldwide. The ability of these fungi to undergo a shift betweenmold and yeast is critical for pathogenesis. In the soil (22°C), these fungi grow as mold, which produce infectious conidia. Following soil disruption,aerosolized conidia inhaled into the lungs of a host (37°C) convert into yeast to cause pneumonia. Knowledge of the mechanisms used to regulate thisphase transition is limited. To uncover genes that control the phase transition, Agrobacterium tumefaciens-mediated DNA transfer was used tomutagenize Blastomyces dermatitidis conidia. We generated and screened 22,000 insertional mutants for defects in the phase transition. We identified amutant, 11-9-75, with a single site of T-DNA insertion that grew as pseudohyphae at 37°C and 22°C, rather than yeast or mold. Adaptor PCR, DNAsequencing, and reverse transcription PCR (RT-PCR) revealed the T-DNA was located in the 5’ UTR of an uncharacterized gene (referred to as BKY1) thatwas not annotated in the B. dermatitidis genome. Analysis of cDNA indicated BKY1 was 1546 bp in length, lacked introns, and the ORF was predicted toencode a 156 amino acid protein. BLAST analyses against the NCBI database failed to reveal homologs of BKY1 in other fungi. The T-DNA insert alteredtranscription of BKY1 in mutant 11-9-75. BKY1 transcript in the mutant was 35-fold higher versus wild type (WT) by quantitative real-time PCR (qPCR) andtruncated at the 5’ UTR when analyzed by rapid amplification of cDNA ends (RACE). RT-PCR supported the qPCR and RACE analyses, and indicated theGAPDH promoter, which is upstream of a hygromycin resistance cassette in the T-DNA was driving increased transcription of truncated BKY1. The T-DNAinsert also altered alternative splicing of a gene with unknown function, Bd594, which was less than 1.2 kb downstream of BKY1. Although transcriptabundance of Bd594 in mutant 11-9-75 was similar to WT, the frequency of intron excision was reduced. In conclusion, we have identified an insertionalmutant with a bidirectional defect on the phase transition; it grows as pseudohyphae instead of yeast at 37°C or mold at 22°C. The T-DNA insert alterstranscription of adjacent genes, BKY1 and Bd594, in a poorly characterized region in the B. dermatitidis genome.27th Fungal Genetics Conference | 229