Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSWednesday, March 13 3:00 PM–6:00 PMFred Farr ForumGenome Defense, Epigenetics and RNAiCo-chairs: Patrick Shiu and Sven SaupeMeiotic silencing by unpaired DNA in Neurospora. Thomas M. Hammond 1 , Hua Xiao 2 , Erin C. Boone 2 , Logan M. Decker 2 , David G. Rehard 2 , Seung A. Lee 2 ,Tony D. Perdue 3 , Patricia J. Pukkila 3 , Patrick K. T. Shiu 2 . 1) School of Biological Sciences, Illinois State University, Normal, IL; 2) Division of BiologicalSciences, University of Missouri, Columbia, MO; 3) Department of Biology, University of North Carolina, Chapel Hill, NC.Neurospora crassa has a cytoplasm that is shared by the entire hyphal network, making it particularly vulnerable to attack by repetitive elements.Accordingly, several surveillance mechanisms are in place to protect the genome integrity of the fungus. For example, if a gene is lacking a partner duringhomolog pairing in meiosis, all copies of this gene are silenced by a process known as meiotic silencing by unpaired DNA (MSUD). MSUD requires commonRNAi proteins (e.g., RNA-directed RNA polymerase, Dicer, and Argonaute) and may work as follows: an unpaired gene triggers the production of anaberrant RNA molecule, which is made double-stranded (by SAD-1) and processed into siRNAs (by DCL-1). These siRNAs are subsequently used to destroycomplementary mRNAs (by SMS-2). The aforementioned proteins colocalize in the perinuclear region, possibly forming a silencing complex that inspectsand processes RNA molecules as they exit the nucleus. We will discuss the recent advances in our understanding of this unique silencing pathway.Mechanism of quelling, a small RNA-mediated gene silencing pathway. Zhenyu Zhang, Shwu-Shin Chang, Yi Liu. Dept Physiology, Univ Texas SW Med Ctr,Dallas, TX.RNAi is a conserved gene silencing mechanism from fungi to mammals. Quelling is an RNAi-related phenomenon that post-transcriptionally silencesrepetitive DNA and transposon in Neurospora. We previously identified a type of DNA damage-induced small RNA called qiRNAs that originate fromribosomal DNA. To understand how small RNAs are generated from repetitive DNA, we carried out a genetic screen to identify genes required for qiRNAbiogenesis. Factors directly involved in homologous recombination (HR) and chromatin remodeling factors required for HR are essential for qiRNAproduction. HR is also required for quelling, and quelling is also the result of DNA damage, indicating that quelling and qiRNA production share a commonmechanism. Together, our results suggest that DNA damage triggered HR-based recombination allows the RNAi pathway to recognize repetitive DNA toproduce small RNA.SIS, a sex genome defense mechanism operating in Cryptococcus neoformans. Xuying Wang, Sabrina Darwiche, Joseph Heitman. Department ofMolecular Genetics and Microbiology, Duke University Medical Center, Durham, NC.Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from yeast to hyphae during a-a opposite-sex mating and a-a unisexual reproduction (same-sex mating). Infectious spores are generated during both processes. We previously identified a sex induced silencing (SIS)pathway in the C. neoformans serotype A var. grubii lineage, in which tandem transgene arrays trigger RNAi-dependent gene silencing at a high frequencyduring a-a opposite-sex mating, but at an ~250-fold lower frequency during asexual mitotic vegetative growth. Here we report that SIS also operatesduring a-a unisexual reproduction. A self-fertile strain containing either SXI2a-URA5 or NEO-URA5 transgene arrays exhibited an elevated silencingfrequency during solo and unisexual mating compared with mitotic vegetative growth. We also found that SIS operates at a similar efficiency on transgenearrays of the same copy number during either a-a unisexual reproduction or a-a opposite-sex mating. URA5-derived small RNAs were detected in thesilenced progeny of a-a unisexual reproduction and RNAi core components were required, providing evidence that SIS induced by same-sex mating is alsomediated by RNAi via sequence-specific small RNAs. This study, together with our previous finding of SIS in a-a opposite-sex mating of the C. neoformansserotype A var. grubii lineage, demonstrates that SIS is a conserved process between the divergent C. neoformans serotype A and serotype D siblingspecies. In each case, our data show that the SIS RNAi pathway operates to defend the genome via squelching transposon activity during the sexual cycles.Thus, our discovery of SIS brings a fresh perspective to meiotic silencing involving the upregulation of RNAi pathways as a strategy to guard genomicintegrity during sex. More importantly, the presence of SIS in both a-a unisexual reproduction and a-a opposite-sex mating indicate that SIS may betriggered by the shared pheromone sensing Cpk1 MAPK signal transduction cascade. Ongoing studies focus on defining at a mechanistic level how the SISRNAi pathway is initiated, including identifying new components involved in SIS.Fungi use prion folds for signal transduction processes involving STAND proteins. Asen Daskalov, Khalid Salamat, Sven J. Saupe. CNRS, IBGC UMR5095,BORDEAUX, AQUITAINE, France.Prions are proteins embedding genetic information into their structural state. Generally, those proteins exist in a soluble state and sporadically asinfectious amyloid aggregates. Podospora anserina’s [Het-s] is one of the best characterized fungal prions with a remarkably high prevalence in wildpopulations. [Het-s] functions in vegetative incompatibility - a biological process occurring during anastomosis between two genetically incompatiblestrains. The HET-s protein exists in a soluble state - [Het-s*] - or can switch to an aggregated amyloid state - [Het-s] - the prion form. When an [Het-s] prioninfected strain fuses with a strain expressing the alternative allelic variant of the het-s locus - het-S - a cell death reaction of the heterokaryon occurs.Recent studies shed light on the mechanism of [Het-s]/HET-S incompatibility reaction. Differing by 13 amino acids both proteins shares a two domainarchitecture; a globular N-terminal domain called HeLo and a C-terminal Prion Forming Domain (PFD). The latter is able to adopt a b-sheet richconformation with a specific b-solenoid fold. It has been demonstrated that in presence of [Het-s] amyloid fibers HET-S turns into a pore-forming toxin:transconformation of the HET-S PFD by [Het-s] fibers triggers the refolding of the HET-S HeLo domain, inducing the cell death reaction. In an attempt tobetter characterize the conserved features of the [Het-s] b-solenoid fold and identify new distant homologues of HET-S/s, we have generated a minimalconsensus sequence motif of it. Surprisingly, the second best hit in a BLASTp search is in the N-terminal region (3-23) of the product encoded by nwd2, theimmediately adjacent gene to het-S. NWD2 is a STAND protein. STAND proteins form signal transducing hubs through oligomerization upon ligandrecognition. That in mind and several bioinformatics observations led us to propose that HET-S and NWD2 are functional partners in various filamentousfungal species using the amyloid fold in a signal transducing pathway. We will present experimental evidence that NWD2 is able to trigger HET-S toxicity in27th Fungal Genetics Conference | 37