An introgressed gene causes meiotic drive in Neurospora sitophila.

Meiotic drive elements cause their own preferential transmission following meiosis. In fungi, this phenomenon takes the shape of spore killing, and in the filamentous ascomycete Neurospora sitophila, the Sk-1 spore killer element is found in many natural populations. In this study, we identify the gene responsible for spore killing in Sk-1 by generating both long- and short-read genomic data and by using these data to perform a genome-wide association test. We name this gene Spk-1 Through molecular dissection, we show that a single 405-nt-long open reading frame generates a product that both acts as a poison capable of killing sibling spores and as an antidote that rescues spores that produce it. By phylogenetic analysis, we demonstrate that the gene has likely been introgressed from the closely related species Neurospora hispaniola, and we identify three subclades of N. sitophila, one where Sk-1 is fixed, another where Sk-1 is absent, and a third where both killer and sensitive strain are found. Finally, we show that spore killing can be suppressed through an RNA interference-based genome defense pathway known as meiotic silencing by unpaired DNA. Spk-1 is not related to other known meiotic drive genes, and similar sequences are only found within Neurospora These results shed light on the diversity of genes capable of causing meiotic drive, their origin and evolution, and their interaction with the host genome.

Gene drive by Fusarium SKC1 is dependent on its competing allele.

The Fusarium verticillioides SKC1 gene driver is transmitted to offspring in a biased manner through spore killing. The mechanism that allows SKC1 to kill non-SKC1 offspring while sparing others is poorly understood. Here we report that gene drive by SKC1 is dependent on SKC1's competing allele. We propose that SKC1's competing allele influences the ability of a genome defense process to detect SKC1, and we provide evidence that this genome defense process is meiotic silencing by unpaired DNA (MSUD). Our findings suggest that the successful deployment of gene drivers to control pathogenic fungi will require researchers to consider how competing alleles influence the ability of gene drivers to be detected by genome defense processes.

Molecular dissection of Neurospora Spore killer meiotic drive elements.

Meiotic drive is a non-Mendelian inheritance phenomenon in which certain selfish genetic elements skew sexual transmission in their own favor. In some cases, progeny or gametes carrying a meiotic drive element can survive preferentially because it causes the death or malfunctioning of those that do not carry it. In Neurospora, meiotic drive can be observed in fungal spore killing. In a cross of Spore killer (Sk) × WT (Sk-sensitive), the ascospores containing the Spore killer allele survive, whereas the ones with the sensitive allele degenerate. Sk-2 and Sk-3 are the most studied meiotic drive elements in Neurospora, and they each theoretically contain two essential components: a killer element and a resistance gene. Here we report the identification and characterization of the Sk resistance gene, rsk (resistant to Spore killer). rsk seems to be a fungal-specific gene, and its deletion in a killer strain leads to self-killing. Sk-2, Sk-3, and naturally resistant isolates all use rsk for resistance. In each killer system, rsk sequences from an Sk strain and a resistant isolate are highly similar, suggesting that they share the same origin. Sk-2, Sk-3, and sensitive rsk alleles differ from each other by their unique indel patterns. Contrary to long-held belief, the killer targets not only late but also early ascospore development. The WT RSK protein is dispensable for ascospore production and is not a target of the spore-killing mechanism. Rather, a resistant version of RSK likely neutralizes the killer element and prevents it from interfering with ascospore development.

A DEAD-box RNA helicase mediates meiotic silencing by unpaired DNA.

During the sexual phase of Neurospora crassa, unpaired genes are subject to a silencing mechanism known as meiotic silencing by unpaired DNA (MSUD). MSUD targets the transcripts of an unpaired gene and utilizes typical RNA interference factors for its process. Using a reverse genetic screen, we have identified a meiotic silencing gene called sad-9, which encodes a DEAD-box RNA helicase. While not essential for vegetative growth, SAD-9 plays a crucial role in both sexual development and MSUD. Our results suggest that SAD-9, with the help of the SAD-2 scaffold protein, recruits the SMS-2 Argonaute to the perinuclear region, the center of MSUD activity.

Fine-scale mapping in Neurospora crassa by using genome-wide knockout strains.

Fine-scale genetic mapping is often hindered by the lack of adequate markers surrounding the locus of interest. In the filamentous ascomycete Neurospora crassa, the genome has been sequenced and an effort has been made to generate genome-wide deletion strains for the entire gene set. Accordingly, the hygromycin-resistant marker in each deletion strain can be used as a mapping locus in a classical three-point cross, along with the mapping target and a standard marker. We have demonstrated the feasibility of this fine-scale mapping approach in N. crassa by refining the location of r(Sk-2).

A-to-I mRNA editing controls spore death induced by a fungal meiotic drive gene in homologous and heterologous expression systems.

Spore killers are meiotic drive elements that can block the development of sexual spores in fungi. In the maize ear rot and mycotoxin-producing fungus Fusarium verticillioides, a spore killer called SkK has been mapped to a 102-kb interval of chromosome V. Here, we show that a gene within this interval, SKC1, is required for SkK-mediated spore killing and meiotic drive. We also demonstrate that SKC1 is associated with at least 4 transcripts, 2 sense (sense-SKC1a and sense-SKC1b) and 2 antisense (antisense-SKC1a and antisense-SKC1b). Both antisense SKC1 transcripts lack obvious protein-coding sequences and thus appear to be noncoding RNAs. In contrast, sense-SKC1a is a protein-coding transcript that undergoes A-to-I editing to sense-SKC1b in sexual tissue. Translation of sense-SKC1a produces a 70-amino-acid protein (Skc1a), whereas the translation of sense-SKC1b produces an 84-amino-acid protein (Skc1b). Heterologous expression analysis of SKC1 transcripts shows that sense-SKC1a also undergoes A-to-I editing to sense-SKC1b during the Neurospora crassa sexual cycle. Site-directed mutagenesis studies indicate that Skc1b is responsible for spore killing in Fusarium verticillioides and that it induces most meiotic cells to die in Neurospora crassa. Finally, we report that SKC1 homologs are present in over 20 Fusarium species. Overall, our results demonstrate that fungal meiotic drive elements like SKC1 can influence the outcome of meiosis by hijacking a cell's A-to-I editing machinery and that the involvement of A-to-I editing in a fungal meiotic drive system does not preclude its horizontal transfer to a distantly related species.

Fluorescent and bimolecular-fluorescent protein tagging of genes at their native loci in Neurospora crassa using specialized double-joint PCR plasmids.

The double-joint polymerase chain reaction (DJ-PCR) is a technique that can be used to construct vectors for targeted genome integration without laborious subcloning steps. Here we report the availability of plasmids that facilitate DJ-PCR-based construction of Neurospora crassa tagging vectors. These plasmids allow the creation of green or red fluorescent protein (GFP or RFP) tagging vectors for protein localization studies, as well as split-yellow fluorescent protein (YFP) tagging vectors for bimolecular fluorescence complementation (BiFC) analyses. We have demonstrated the utility of each plasmid with the tagging of known meiotic silencing proteins. Microscopic analysis of the tagged strains indicates that SMS-2 and QIP form macromolecular complexes in the perinuclear region during meiosis.

An NCBP3-Domain Protein Mediates Meiotic Silencing by Unpaired DNA.

In the filamentous fungus Neurospora crassa, genes unpaired during meiosis are silenced by a process known as meiotic silencing by unpaired DNA (MSUD). MSUD utilizes common RNA interference (RNAi) proteins, such as Dicer and Argonaute, to target homologous mRNAs for silencing. Previously, we demonstrated that nuclear cap-binding proteins NCBP1 and NCBP2 are involved in MSUD. We report here that SAD-8, a protein similar to human NCBP3, also mediates silencing. Although SAD-8 is not essential for either vegetative or sexual development, it is required for MSUD. SAD-8 localizes predominantly in the nucleus and interacts with both NCBP1 and NCBP2. Similar to NCBP1 and NCBP2, SAD-8 interacts with a component (Argonaute) of the perinuclear meiotic silencing complex (MSC), further implicating the involvement of cap-binding proteins in silencing.

Suppressors of Meiotic Silencing by Unpaired DNA.

Meiotic silencing by unpaired DNA (MSUD) is a gene silencing process that occurs within meiotic cells of Neurospora crassa and other fungi. We have previously developed a high-throughput screen to identify suppressors of this silencing pathway. Here, a list of MSUD suppressor candidates from a single pass of the first 84 plates of the Neurospora knockout library is provided.

Identification of rfk-1, a Meiotic Driver Undergoing RNA Editing in Neurospora.

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.

DCL-1 colocalizes with other components of the MSUD machinery and is required for silencing.

In Neurospora, a gene present in an abnormal number of copies is usually a red flag for mischief. One way to deal with these potential intruders is by destroying their transcripts. Widely known as RNA interference (RNAi), this mechanism depends on the "dicing" of a double-stranded RNA intermediate into small-interfering RNA, which in turn guide the degradation of mRNA from the target gene. Quelling is a vegetative silencing system in Neurospora that utilizes such a mechanism. Quelling depends on the redundant activity of two Dicer-like ribonucleases, DCL-1 and DCL-2. Here, we show that Meiotic Silencing by Unpaired DNA (MSUD), a mechanism that silences expression from unpaired DNA during meiosis, requires the dcl-1 (but not the dcl-2) gene for its function. This result suggests that MSUD operates in a similar manner to Quelling and other RNAi systems. DCL-1 colocalizes with SAD-1 (an RdRP), SAD-2, and SMS-2 (an Argonaute) in the perinuclear region.

Convergent evolution of complex genomic rearrangements in two fungal meiotic drive elements.

Meiotic drive is widespread in nature. The conflict it generates is expected to be an important motor for evolutionary change and innovation. In this study, we investigated the genomic consequences of two large multi-gene meiotic drive elements, Sk-2 and Sk-3, found in the filamentous ascomycete Neurospora intermedia. Using long-read sequencing, we generated the first complete and well-annotated genome assemblies of large, highly diverged, non-recombining regions associated with meiotic drive elements. Phylogenetic analysis shows that, even though Sk-2 and Sk-3 are located in the same chromosomal region, they do not form sister clades, suggesting independent origins or at least a long evolutionary separation. We conclude that they have in a convergent manner accumulated similar patterns of tandem inversions and dense repeat clusters, presumably in response to similar needs to create linkage between genes causing drive and resistance.

An RNA Recognition Motif-Containing Protein Functions in Meiotic Silencing by Unpaired DNA.

Meiotic silencing by unpaired DNA (MSUD) is a biological process that searches pairs of homologous chromosomes (homologs) for segments of DNA that are unpaired. Genes found within unpaired segments are silenced for the duration of meiosis. In this report, we describe the identification and characterization of Neurospora crassa sad-7, a gene that encodes a protein with an RNA recognition motif (RRM). Orthologs of sad-7 are found in a wide range of ascomycete fungi. In N. crassa, sad-7 is required for a fully efficient MSUD response to unpaired genes. Additionally, at least one parent must have a functional sad-7 allele for a cross to produce ascospores. Although sad-7-null crosses are barren, sad-7Δ strains grow at a wild-type (wt) rate and appear normal under vegetative growth conditions. With respect to expression, sad-7 is transcribed at baseline levels in early vegetative cultures, at slightly higher levels in mating-competent cultures, and is at its highest level during mating. These findings suggest that SAD-7 is specific to mating-competent and sexual cultures. Although the role of SAD-7 in MSUD remains elusive, green fluorescent protein (GFP)-based tagging studies place SAD-7 within nuclei, perinuclear regions, and cytoplasmic foci of meiotic cells. This localization pattern is unique among known MSUD proteins and raises the possibility that SAD-7 coordinates nuclear, perinuclear, and cytoplasmic aspects of MSUD.

A mus-51 RIP allele for transformation of Neurospora crassa.

This report describes the construction and characterization of mus-51RIP70 , an allele for high-efficiency targeted integration of transgenes into the genome of the model eukaryote Neurospora crassa. Two of the mus-51RIP70 strains investigated in this work (RZS27.10 and RZS27.18) can be obtained from the Fungal Genetics Stock Center. The two deposited strains are, to our knowledge, genetically identical and neither one is preferred over the other for use in Neurospora research.

A Meiotic Drive Element in the Maize Pathogen Fusarium verticillioides Is Located Within a 102 kb Region of Chromosome V.

Fusarium verticillioides is an agriculturally important fungus because of its association with maize and its propensity to contaminate grain with toxic compounds. Some isolates of the fungus harbor a meiotic drive element known as Spore killer (Sk(K)) that causes nearly all surviving meiotic progeny from an Sk(K) × Spore killer-susceptible (Sk(S)) cross to inherit the Sk(K) allele. Sk(K) has been mapped to chromosome V but the genetic element responsible for meiotic drive has yet to be identified. In this study, we used cleaved amplified polymorphic sequence markers to genotype individual progeny from an Sk(K) × Sk(S) mapping population. We also sequenced the genomes of three progeny from the mapping population to determine their single nucleotide polymorphisms. These techniques allowed us to refine the location of Sk(K) to a contiguous 102 kb interval of chromosome V, herein referred to as the Sk region. Relative to Sk(S) genotypes, Sk(K) genotypes have one extra gene within this region for a total of 42 genes. The additional gene in Sk(K) genotypes, herein named SKC1 for Spore Killer Candidate 1, is the most highly expressed gene from the Sk region during early stages of sexual development. The Sk region also has three hyper-variable regions, the longest of which includes SKC1 The possibility that SKC1, or another gene from the Sk region, is an essential component of meiotic drive and spore killing is discussed.

RNA silencing of mycotoxin production in Aspergillus and Fusarium species.

Mycotoxins are natural fungal products that are defined by their harmful effects on humans and animals. Aflatoxin contamination of maize by Aspergillus species and trichothecene contamination of small grains by Fusarium species are two of the most severe mycotoxin problems in the United States. We are investigating RNA silencing in an effort to identify novel ways to control mycotoxin contamination of crops. Transformation of two Aspergilli (A. flavus and A. parasiticus) and a Fusarium (F. graminearum) with inverted repeat transgenes (IRT) containing sequences of mycotoxin-specific regulatory genes suppressed mycotoxin production in all three plant-pathogenic fungi. This atoxigenic phenotype was stable during infection on corn and wheat, and importantly, F. graminearum IRT strains were less virulent on wheat than were wild type. The IRT did not alter physiological characteristics of the fungi, such as spore production and growth rate on solid media. These results indicate that RNA silencing exists in Aspergillus and Fusarium plant pathogens and suggest that RNA silencing technology may be a useful tool for eliminating mycotoxin contamination of agricultural products.

Complex formation of RNA silencing proteins in the perinuclear region of Neurospora crassa.

In Neurospora, genes not paired during meiosis are targeted by meiotic silencing by unpaired DNA (MSUD). Here, our bimolecular fluorescence complementation (BiFC) study suggests that RNA-directed RNA polymerase, Dicer, Argonaute, and others form a silencing complex in the perinuclear region, with intimate interactions among the majority of them. We have also shown that SAD-2 is likely the anchor for this assembly.

A critical component of meiotic drive in Neurospora is located near a chromosome rearrangement.

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.

Efficient detection of unpaired DNA requires a member of the rad54-like family of homologous recombination proteins.

Meiotic silencing by unpaired DNA (MSUD) is a process that detects unpaired regions between homologous chromosomes and silences them for the duration of sexual development. While the phenomenon of MSUD is well recognized, the process that detects unpaired DNA is poorly understood. In this report, we provide two lines of evidence linking unpaired DNA detection to a physical search for DNA homology. First, we have found that a putative SNF2-family protein (SAD-6) is required for efficient MSUD in Neurospora crassa. SAD-6 is closely related to Rad54, a protein known to facilitate key steps in the repair of double-strand breaks by homologous recombination. Second, we have successfully masked unpaired DNA by placing identical transgenes at slightly different locations on homologous chromosomes. This masking falls apart when the distance between the transgenes is increased. We propose a model where unpaired DNA detection during MSUD is achieved through a spatially constrained search for DNA homology. The identity of SAD-6 as a Rad54 paralog suggests that this process may be similar to the searching mechanism used during homologous recombination.

Identification of small RNAs associated with meiotic silencing by unpaired DNA.

In Neurospora crassa, unpaired genes are silenced by a mechanism called meiotic silencing by unpaired DNA (MSUD). Although some RNA interference proteins are necessary for this process, its requirement of small RNAs has yet to be formally established. Here we report the characterization of small RNAs targeting an unpaired region, using Illumina sequencing.