Neurospora fungi harbor a group of meiotic drive elements known as Spore killers (Sk). Spore killer-2 (Sk-2) and Spore killer-3 (Sk-3) are two Sk elements that map to a region of suppressed recombination. Although this recombination block is limited to crosses between Sk and Sk-sensitive (Sk S ) strains, its existence has hindered Sk characterization. Here we report the circumvention of this obstacle by combining a classical genetic screen with next-generation sequencing technology and three-point crossing assays. This approach has allowed us to identify a novel locus called rfk-1, mutation of which disrupts spore killing by Sk-2. We have mapped rfk-1 to a 45-kb region near the right border of the Sk-2 element, a location that also harbors an 11-kb insertion (Sk-2 INS1 ) and part of a .220-kb inversion (Sk-2 INV1 ). These are the first two chromosome rearrangements to be formally identified in a Neurospora Sk element, providing evidence that they are at least partially responsible for Sk-based recombination suppression. Additionally, the proximity of these chromosome rearrangements to rfk-1 (a critical component of the spore-killing mechanism) suggests that they have played a key role in the evolution of meiotic drive in Neurospora.G ENOMES typically consist of large groups of cooperating genes that encode information needed to maintain and reproduce themselves as well as their cellular environments. However, individual genes within genomes do not always cooperate (Dawkins 2006;Burt and Trivers 2008). For example, ranGAP in fruit flies and tcd1 in mice are selfish genes that collude with others to bias meiotic transmission ratios to their advantage (Kusano et al. 2003;Lyon 2003). While ranGAP works within a gene complex known as Segregation Distorter, tcd1 works within another called the t haplotype. Because these selfish gene complexes "drive" through meiosis, they are referred to as meiotic drive elements. Such elements are found throughout sexual organisms, with other well-known examples being X d in stalkeyed flies, Ab10 in maize, and the Spore killers in various fungi (Rhoades 1942;Raju 1994;Presgraves et al. 1997;Burt and Trivers 2008).Neurospora fungi spend most of their life cycle in a vegetative haploid phase. However, specific environmental cues (e.g., nitrogen limitation) can trigger initiation of the sexual cycle, which includes a brief diploid phase that allows meiotic drive to function in this group of fungi. Sexual reproduction in Neurospora begins when an asexual spore (conidium) from a male parent donates a nucleus to an immature fruiting body (protoperithecium) of a female parent. Pairs of nuclei, one from each parent, fuse within the meiotic cells (asci) of the fertilized fruiting body (perithecium) to form a diploid nucleus that passes through the standard stages of meiosis (Raju 1980). The four haploid meiotic products then undergo a postmeiotic mitosis to produce eight nuclei, each of which is incorporated into an American football-shaped ascospore (Raju 1980). Ascospores darken as...

Sk-2 is a meiotic drive element that was discovered in wild populations of Neurospora fungi over 40 years ago. While early studies quickly determined that Sk-2 transmits itself through sexual reproduction in a biased manner via spore killing, the genetic factors responsible for this phenomenon have remained mostly unknown. Here, we identify and characterize rfk-1, a gene required for Sk-2-based spore killing. The rfk-1 gene contains four exons, three introns, and two stop codons, the first of which undergoes RNA editing to a tryptophan codon during sexual development. Translation of an unedited rfk-1 transcript in vegetative tissue is expected to produce a 102-amino acid protein, whereas translation of an edited rfk-1 transcript in sexual tissue is expected to produce a protein with 130 amino acids. These findings indicate that unedited and edited rfk-1 transcripts exist and that these transcripts could have different roles with respect to the mechanism of meiotic drive by spore killing. Regardless of RNA editing, spore killing only succeeds if rfk-1 transcripts avoid silencing caused by a genome defense process called meiotic silencing by unpaired DNA (MSUD). We show that rfk-1's MSUD avoidance mechanism is linked to the genomic landscape surrounding the rfk-1 gene, which is located near the Sk-2 border on the right arm of chromosome III. In addition to demonstrating that the location of rfk-1 is critical to spore-killing success, our results add to accumulating evidence that MSUD helps protect Neurospora genomes from complex meiotic drive elements.

The conserved two-component XMAP215/TACC modulator of microtubule stability is required in multiple animal phyla for acentrosomal spindle assembly during oocyte meiotic cell division. In C. elegans, XMAP215/zyg-9 and TACC/tac-1 mutant oocytes exhibit multiple and indistinguishable oocyte spindle assembly defects beginning early in meiosis I. To determine if these defects represent one or more early requirements with additional later and indirect consequences, or multiple temporally distinct and more direct requirements, we have used live cell imaging and fast-acting temperature-sensitive zyg-9 and tac-1 alleles to dissect their requirements at high temporal resolution. Temperature upshift and downshift experiments indicate that the ZYG-9/TAC-1 complex has multiple temporally distinct and separable requirements throughout oocyte meiotic cell division. First, we show that during prometaphase ZYG-9 and TAC-1 promote the coalescence of early pole foci into a bipolar structure, stabilizing pole foci as they grow and limiting their growth rate, with these requirements being independent of an earlier defect in microtubule organization that occurs upon nuclear envelope breakdown. Second, during metaphase, ZYG-9 and TAC-1 maintain spindle bipolarity by suppressing ectopic pole formation. Third, we show that ZYG-9 and TAC-1 also are required for spindle assembly during meiosis II, independently of their meiosis I requirements. The metaphase pole stability requirement appears to be important for maintaining chromosome congression, and we discuss how negative regulation of microtubule stability by ZYG-9/TAC-1 during oocyte meiotic cell division might account for the observed defects in spindle pole coalescence and stability.

The conserved two-component XMAP215/TACC modulator of microtubule stability is required in multiple animal phyla for acentrosomal spindle assembly during oocyte meiotic cell division, with C. elegans XMAP215/zyg-9 and TACC/tac-1 mutant oocytes exhibiting multiple and indistinguishable defects beginning early in meiosis I. To determine if these defects represent one or more early requirements with additional later and indirect consequences, or multiple temporally distinct and more direct requirements, we have used live cell imaging and fast-acting temperature-sensitive zyg-9 and tac-1 alleles to dissect at high temporal resolution their oocyte meiotic spindle assembly requirements. Our results from temperature upshift and downshift experiments indicate that the ZYG-9/TAC-1 complex has multiple temporally distinct and separable requirements throughout oocyte meiotic cell division. First, we show that during prometaphase ZYG-9 and TAC-1 promote the coalescence of early pole foci into a bipolar structure both by stabilizing pole foci as they grow and by limiting their growth rate, with these requirements being independent of an earlier defect in microtubule organization. Second, during metaphase, ZYG-9 and TAC-1 maintain spindle bipolarity by suppressing ectopic pole formation, and this pole stability is important for maintaining chromosome congression. Finally, we show that ZYG-9 and TAC-1 also are required for the proper coalescence of pole foci during meiosis II, independently of their requirements during meiosis I. Our findings highlight the value of fast-acting temperature-sensitive alleles for high resolution temporal dissection of gene requirements, and we discuss how negative regulation of microtubule stability by ZYG-9/TAC-1 during oocyte meiotic cell division might account for the observed defects in spindle pole coalescence and stability.

The CRISPR-associated Cas9 enzyme is used in molecular biology to engineer the genomes of a wide range of organisms. While Cas9 can be injected or transfected into a target cell to achieve the desired goal, there are situations where stable expression of Cas9 within a target organism is preferable. Here, we show that the model filamentous fungus Neurospora crassa is recalcitrant to heterologous expression of a human-optimized version of Streptococcus pyogenes cas9. Furthermore, partial optimization of cas9 by synonymous codon exchange failed to improve its expression in the fungus. Finally, we show that transgene expression can be detected when cas9Hs sequences are placed in the 3’ UTR regions of transgene-derived mRNAs, but not when the same sequences are in the translated part of the transgene-derived mRNA. This finding suggests that the primary obstacle to high cas9Hs expression levels in N. crassa is translational in nature.