Molecular dissection of Neurospora Spore killer meiotic drive elements

Abstract

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.

In fungi, plants, and animals, not all genes follow the Mendelian pattern of inheritance. Meiotic drive, sometimes referred to as segregation distortion, describes the phenomenon in which certain “cheating” alleles are recovered in more than half of the progeny (1). Two well-known examples are the segregation distorter (SD) in Drosophila and the t haplotype in Mus. In these cases, sperm not carrying the aggressive allele (the drive element) either degenerate (in flies) or become functionally impaired (in mice) (2, 3). In fungi, meiotic drive can be observed as spore killing, in which ascospores (sexual spores) that carry the “Spore killer” element survive preferentially (4). Examples of spore killing can be found in Neurospora sitophila, Neurospora intermedia, Podospora anserina, Gibberella fujikuroi, and Cochliobolus heterostrophus.

Spore killer-2 (Sk-2) and Spore killer-3 (Sk-3), which behave similarly, are the most studied distortion elements in Neurospora (5). Originally discovered in N. intermedia, the two spore-killing factors have been introgressed into Neurospora crassa for extensive genetic studies. In a Spore killer (Sk) × WT (Sk-sensitive or Sk^S) cross, regardless of which acts as the female, the four Sk-containing spores are black (B) and viable, whereas the four Sk-sensitive spores are white (W) and inviable. Manifestation of killing does not occur until late ascospore development: Degeneration of sensitive spores takes place after the normal progression of meiosis, spore delimitation, and the second postmeiotic mitosis (6). Homozygous killer × killer and sensitive × sensitive crosses are normal and yield 8B:0W asci (spore sacs). When sensitive nuclei are enclosed in the same spore with the killer ones, as seen in a giant-spored N. crassa mutant and in the four-spored (heterokaryotic) Neurospora tetrasperma, they can escape the elimination process (7, 8). These observations suggest that the killer produces a resistance factor that shelters all nuclei residing in the same spore. The Sk-2 and Sk-3 killers do not encode resistance to each other, and all eight progeny are inviable when the two mate.

The Sk-2 and Sk-3 loci, as defined by their killing ability and self-resistance, have been mapped to the same region on chromosome III. Their loci can only be defined as a 30-map unit region because recombination is blocked between Spore killers and WT within this interval (9). Nonkiller strains that are resistant to spore killing have been found in nature (5, 9–11). Because recombination block is not observed between WT and a resistant strain, the associated resistance gene can be mapped accurately. For example, the r(Sk-2) gene, which confers resistance to Sk-2 killing, is located within the recombination block region (9).

Other than the killing and the resistance factors, Sk-2 and Sk-3 haplotypes also contain a suppressor of meiotic silencing by unpaired DNA (MSUD) (12). MSUD, a process that silences expression from unpaired genes during meiosis (13–15), requires common RNAi proteins, such as an RNA-directed RNA polymerase, a Dicer, and an Argonaute slicer (16–18). The MSUD suppressors found in Sk-2 and Sk-3 are not as strong as the classic sad-1^Δ and sad-2^Δ suppressors, and they do not suppress the silencing of all unpaired loci (12).

Although widely distributed in nature, meiotic drive elements have not been extensively characterized at the molecular level, owing to their complex genetic organization (1, 19). To begin the molecular dissection of the Sk elements, we first took aim at cloning the r(Sk-2) gene and determining how it is related to the overall mechanism of Neurospora spore killing.

Results

Characteristics of Sk-2 and r(Sk-2).

Spanning the centromere of chromosome III, the Sk-2 and Sk-3 killer haplotypes are defined as a 30-map unit (recombination block) region (Fig. 1A; see ref. 20). r(Sk-2) is located at the left end of this region. A normal perithecium (fruiting body) produces roughly 200 asci, each containing eight progeny that mature into melanized ascospores (Fig. 2A). In an Sk-2 × WT cross, the Sk-2–containing progeny mature normally, whereas the Sk-2–sensitive progeny degenerate (Fig. 2B). r(Sk-2), not itself a killer (Fig. 2C), confers resistance to Sk-2 such that all eight ascospores develop normally in an Sk-2 × r(Sk-2) ascus (Fig. 2D).

Fig. 1.

Genetic locations of Sk-2, Sk-3, and r(Sk-2). (A) The Spore killer region on chromosome III. Sk-2 and Sk-3 are located in a 30-map unit region within which recombination is blocked in heterozygous (Sk × WT) crosses. (B) The r(Sk-2) locus was bracketed by genetic crossovers. Hygromycin resistance genes were placed in a sensitive (OR) background to generate strains with markers between genes 09149 and 09150 (hph^49/50; P17-13) or between genes 09155 and 09156 (hph^55/56; P17-12). These strains were then crossed to a cum r(Sk-2) acr-7 strain (LA; P8-11) to create two mapping populations. The genotypes of the recombinant progeny indicate that r(Sk-2)^LA should lie between the two hph markers (Tables S1 and S2). Amplified fragment length polymorphism (AFLP) and amplified sequence polymorphism (ASP) markers were then used to determine the approximate crossover point in two progeny (#34 and #119). For example, progeny #34 has the LA pattern for AFLP1 and the OR pattern for the other three markers (Fig. S1). This analysis placed r(Sk-2) to the right of AFLP1 and to the left of ASP1, eliminating all genes except 09151, 09152, 09153, and 09154.

Fig. 2.

Phenotypes of Sk^S (sensitive), Sk-2, and r(Sk-2) crosses. (A) Sk^S × Sk^S (F2-26 × P6-07). In a typical (sensitive × sensitive) cross, each mature ascus contains eight spindle-shaped (American football-like) ascospores. (B) Sk-2 × Sk^S (F1-16 × P6-07). In a heterozygous Spore killer cross, the four Sk-2 (black/melanized) progeny develop normally, whereas the four non–Sk-2 (white and small) progeny abort. The 4B:4W asci demonstrate a first-division segregation pattern and show that no crossing-over has occurred between Sk-2 and the centromere. (C) Sk^S × r(Sk-2) (F2-26 × P15-52). r(Sk-2), a resistant strain isolated from LA, does not encode the killing element and is not aggressive toward a sensitive strain (8B:0W). (D) Sk-2 × r(Sk-2) (F1-16 × P15-52). All eight progeny survive when a killer is crossed to a resistant strain. Because crossing-over is prohibited within the recombination block region, each progeny inherits a resistance gene [either the self-resistance factor within the Sk-2 haplotype or the resistance factor from a nonkilling r(Sk-2) strain].

Refinement of the r(Sk-2)^LA Locus.

The r(Sk-2) allele used in this study was isolated from a naturally resistant N. crassa strain found in Louisiana (LA) [hereafter r(Sk-2)^LA] (5). Because this strain does not contain the killer haplotype and the associated recombination block, the r(Sk-2) location can be determined by conventional mapping. Using the hygromycin resistance marker (hph) present in each deletion strain of the N. crassa knockout library, we have mapped r(Sk-2) to between genes 09145 and 09159 (21). This region contains 55 kb of DNA with 13 predicted genes, including 09145 and 09159. To further refine the location of r(Sk-2), we placed hph markers between 09149 and 09150 and between 09155 and 09156 (Fig. 1B). We then performed three-point crosses to determine the gene order of the two hph markers with respect to r(Sk-2) and acr-7 (acriflavine resistant). Our analysis of 1,000 progeny from two crosses indicated that r(Sk-2) should lie between (but not including) 09149 and 09156 (Tables S1 and S2). To eliminate some of this region’s six predicted genes as candidates for r(Sk-2), we set out to bracket its location by determining the approximate crossover points in some of the recombinants from our mapping populations. This was accomplished by identifying molecular markers between the r(Sk-2)^LA strain and its three-point cross partner [a strain with the standard Oak Ridge (OR) background] and analyzing these markers in the progeny (Fig. S1). Results from these analyses place r(Sk-2) to the right of 09150 and left of 09155 (Fig. 1B).

Deletion of 09151 Correlates with the Loss of Resistance to Sk-2.

The above mapping results narrow r(Sk-2) to a region spanning four genes: 09151, 09152, 09153, and 09154. To determine which, if any, of these genes is required for Sk-2 resistance, three of the four genes were individually deleted from an r(Sk-2)^LA strain (attempts to obtain a deletion strain for 09152 were unsuccessful). The deletion strains were subsequently crossed to Sk-2. Whereas 09153^ΔLA and 09154^ΔLA deletions had no adverse effect on Sk-2 resistance, a 09151^ΔLA deletion correlated with the production of empty asci (Fig. 3A). Importantly, this ascus abortion is specific to Sk-2 crosses: The 09151^ΔLA deletion strain produced normal asci in crosses to a sensitive strain.

Fig. 3.

09151 is necessary for resistance to Sk-2. The coding regions for 09151, 09153, and 09154 were individually deleted from an r(Sk-2)^LA strain. The deletion strains (P15-54, P17-01, and P17-02) were then crossed to Sk-2 (F1-16) and Sk-2 sad-2^Δ (F5-18) strains. (A) An Sk-2 × 09151^ΔLA cross produced predominantly aborted asci (containing granulated cytoplasm and no spores). This result suggests that the deletion of 09151^LA and the unpairing of 09151^Sk-2 eliminate resistance to spore killing for the entire ascus during early sexual development. (B) When a strong MSUD suppressor (sad-2^Δ) was present, 09151^ΔLA acted like a normal Sk^S strain in a cross (see text for details). (C–F) Deletions of 09153 and 09154 had no effect on Sk-2 resistance.

This ascus abortion phenotype can be explained by the following: (i) both 09151^LA and the corresponding allele in Sk-2 (09151^Sk-2) are involved in killer resistance, (ii) the 09151^ΔLA deletion results in the unpairing of the 09151^Sk-2 allele during meiosis, and (iii) this unpairing leads to MSUD-based silencing of 09151^Sk-2, causing the death of the entire ascus. If the above hypotheses are true, then including a strong MSUD suppressor such as sad-2^Δ (22) in the cross should relieve 09151^Sk-2 silencing and produce a normal killing phenotype (4B:4W). Indeed, asci from such a cross displayed normal spore killing (Fig. 3B), suggesting that 09151 is necessary for Sk-2 resistance in an r(Sk-2)^LA strain.

Expression of 09151^LA in a Sensitive Strain Allows It to Gain Sk-2 Resistance.

To determine whether 09151^LA alone is sufficient to confer Sk-2 resistance, we tested whether it can grant resistance to a sensitive (OR) strain. To achieve this, we inserted the 09151^LA gene into the his-3 locus [a standard gene placement locus located on linkage group (LG) IR] of an OR (sad-2^Δ) strain. Because we were unable to determine how a 09152^ΔLA deletion would affect Sk-2 resistance, we also created a 09152^LA insertion strain in a similar manner. Whereas 09152^LA did not confer Sk-2 resistance in a cross (Fig. 4A), 09151^LA did. In the latter cross (Sk-2 × his-3⁺::09151^LA sad-2^Δ), an interesting partial resistant ascus combination could be observed; because 09151^LA (located on LG I) and Sk-2 (LG III) segregate independently, three different types of asci were seen: 8B:0W (four 09151^LA and four Sk-2, all survived), 4B:4W (four 09151^LA Sk-2 survived and four 09151^OR aborted), and 6B:2W (two 09151^LA, two Sk-2, and two 09151^LA Sk-2 survived, whereas two 09151^OR aborted) (Fig. 4B). Note that this cross produced mostly aborted asci if sad-2^Δ was not included (Fig. 4C).

Fig. 4.

An ectopic 09151^LA gene confers partial Sk-2 resistance in a sensitive background. To determine whether the 09151^LA and 09152^LA alleles confer Sk-2 resistance, they were placed at an ectopic site (the his-3 locus on chromosome IR) in an Sk^S sad-2^Δ background. The sad-2^Δ allele was included to prevent the possibility of the ectopic alleles or their homologs being silenced by MSUD. (A) 09152^LA does not confer Sk-2 resistance, and susceptible 4B:4W asci were observed. F1-16 × P17-05. (B) The ectopic 09151^LA gene allowed a sensitive strain to gain Sk-2 resistance. Because the two resistance genes (the ectopic 09151^LA in the transformant and the native resistance gene in the Sk-2 killer) are located on different chromosomes, three possible segregation patterns can be observed (see text for details): 8B:0W (long arrow), 6B:2W (short arrow), and 4B:4W (arrowhead). MSUD-deficient (sad-2^Δ) background. F1-16 × P17-04. (C) In an MSUD-proficient (sad-2⁺) background, the above cross produced mostly aborted asci. This observation demonstrates the importance of proper 09151 pairing. F2-19 × P17-06.

To avoid the segregation patterns seen in the above cross, we replaced 09151^OR with 09151^LA in a sensitive strain (with a sad-2⁺, MSUD-proficient background). 09151^LA, when placed at its native locus, allowed the transformant to behave exactly like a resistant r(Sk-2)^LA strain and give 8B:0W asci when crossed to Sk-2 (Fig. 5A). Collectively, these data demonstrate that 09151^LA indeed corresponds to the r(Sk-2)^LA gene activity, and when it is introduced into a sensitive N. crassa strain, it allows the recipient to confer resistance to Sk-2 killing.

Fig. 5.

Replacement of the 09151^OR allele with a resistant allele [from r(Sk-2), r(Sk-3), Sk-2, or Sk-3] transformed the recipient fungus into a strain fully resistant to either Sk-2 or Sk-3. These observations suggest that both the Sk-2 and Sk-3 systems rely on 09151 for their resistance specificity. Resistant 8B:0W asci are seen in A (F1-16 × P15-56), D (F3-16 × P17-15), E (F2-19 × P17-16), and H (F3-14 × P17-17), whereas susceptible 4B:4W asci are seen in B (F3-16 × P15-56), C (F1-16 × P17-15), F (F3-14 × P17-16), and G (F2-19 × P17-17).

To determine whether the 09151^LA gene must be placed at its native locus to function properly, or whether it can be placed anywhere within the Sk-2 recombination block, we inserted 09151^LA into a sensitive strain at a location ∼600 kb inside the right border of this block (denoted IIIR). Such insertion strain gave 8B:0W asci when crossed to Sk-2, as long as sad-2^Δ was part of the genetic background (Fig. S2A). When sad-2^Δ was absent, ascus abortion occurred (Fig. S2B). Evidently, the placement of 09151^LA within the recombination block anywhere other than its native locus will make an otherwise sensitive strain fully Sk-2–resistant, providing that a strong dominant MSUD suppressor (e.g., sad-2^Δ) is present in the cross.

Sk-2 Killer Uses 09151^Sk-2 to Confer Resistance.

The evidence provided thus far reveals an MSUD-enforced constraint on the genomic location of 09151^LA. The existence of this constraint suggests that a 09151^LA-like sequence (i.e., 09151^Sk-2) is required by Sk-2 for resistance to its own killer. To test this hypothesis, the 09151^Sk-2 allele was deleted from an Sk-2 strain. As predicted, crosses of the 09151^ΔSk-2 deletion strain to a sensitive strain resulted in self-killing and mostly aborted asci (Fig. 6A). To test whether 09151^Sk-2 is sufficient for resistance to killing, it was used to replace the OR allele at the native locus in a sensitive strain. As expected, 09151^Sk-2 confers complete resistance to Sk-2 in an MSUD-proficient background (Fig. 5E). These results suggest that the hypothetical resistance gene in Sk-2 is allelic to the resistance gene (09151^LA) found in the naturally resistant r(Sk-2)^LA strain.

Fig. 6.

Deletion of the 09151 gene from the Sk-2 and Sk-3 killers led to self-killing. The 09151 gene was deleted from an Sk-2 and an Sk-3 strain, and the subsequent deletion mutants were crossed to a sensitive (Sk^S) strain. (A) After the 09151 gene was deleted, an Sk-2 strain became self-destructive in a cross. Most asci were aborted, containing granulated cytoplasm and no spores. F2-26 × P15-57. (B) The 09151 deletion had a similar effect on Sk-3. However, the abortive cross in this case had a less severe phenotype (i.e., inviable bubble spores could be seen). It is possible that the killer element in Sk-3 is less potent than the one found in Sk-2 (at least during early ascus development). F2-26 × P17-03.

09151 Is also Involved in Sk-3 Resistance.

The finding that 09151 provides resistance to both Sk-2 and r(Sk-2) strains raises an obvious question: does the Sk-3 system also use the same gene to prevent killing? Initial findings on 09151^LA suggest that this is the case. As expected, when an unpaired ectopic copy of 09151^LA was introduced to a sensitive strain (at IIIR), spore killing occurred in a cross between the transformant and Sk-3 sad-2^Δ [because an r(Sk-2)^LA strain is resistant to Sk-2 and not Sk-3] (Fig. S2C). However, when the same transformant was crossed to Sk-3 sad-2⁺ (an MSUD-proficient strain), 0B:8W asci were obtained, suggesting that the silencing of the Sk-3 resistance gene had occurred (Fig. S2D). The most plausible interpretation is that 09151^LA has sequence similarity with the hypothetical Sk-3 resistance gene.

To directly test whether the 09151 alleles from r(Sk-3) and Sk-3 strains are involved in resistance to the Sk-3 killer, experiments similar to those described for 09151^LA and 09151^Sk-2 were performed. These included replacement of the 09151^OR allele in a sensitive strain with either 09151^r(Sk-3) or 09151^Sk-3. These replacements created strains that produced fully resistant asci when crossed to Sk-3 (Fig. 5 D and H), indicating that these alleles confer Sk-3 resistance. Additionally, deletion of the 09151^Sk-3 allele from an Sk-3 genetic background resulted in a loss of self-resistance in crosses to a sensitive strain (Fig. 6B). Together, our results indicate that 09151 is responsible for resistance in r(Sk-3) and Sk-3 strains.

Because the two resistance genes in naturally resistant isolates [r(Sk-2) and r(Sk-3)], the two resistance genes in Sk-2 and Sk-3 killers, and the sensitive gene are all allelic with each other (i.e., different versions of the 09151 gene), we are regrouping them under one name: rsk (resistant to Spore killer). In this system, the aforementioned 09151 (rsk) alleles would be referred to as rsk^r(Sk-2), rsk^r(Sk-3), rsk^Sk-2, rsk^Sk-3, and rsk^S. For occasions when the origin of the allele is of interest, we propose to add that information at the end; for example, rsk^{r(Sk-2)-LA2222} (or rsk^LA2222 in short).

Sequence Comparison Among Sensitive, Sk-2–Resistant, and Sk-3–Resistant RSK Proteins.

The WT OR rsk gene encodes a 486-aa polypeptide, with no currently recognizable motifs. RSK seems to be a fungal-specific protein—RSK-like sequences are only found in N. crassa and closely related fungi (e.g., Sordaria macrospora) as hypothetical proteins.

The aforementioned experiments demonstrate that different rsk alleles provide resistance to different Spore killers. This aspect of rsk-based killer resistance is perhaps most clearly depicted in Fig. 5, where replacement of the sensitive rsk^OR allele with any one of the four resistant rsk alleles created a strain that is resistant to either Sk-2 or Sk-3, but not both. To determine how these resistant alleles are related to each other, we performed a series of sequence analyses. Protein alignments and sequence identity comparisons indicate that the distinction between RSK^r(Sk-2) and RSK^Sk-2 proteins is minor (94% identical) compared with their differences from the sensitive RSK^S protein (70–71% identical; Fig. S3 and Table S3). Most noticeably, the two Sk-2–resistant alleles (LA2222 and BN7401) contain the same insertion/deletion (indel) pattern compared with the sensitive allele (Fig. 7), suggesting that they share the same origin.

Fig. 7.

Topography of RSK proteins. Sk-2 and Sk-3 use different alleles of the rsk gene to confer resistance. The Sk-2 killer and strains naturally resistant to Sk-2 have near-identical RSK proteins. The same is true for the Sk-3 killer and its naturally resistant strains, except that their proteins have different deletion regions (shown in red). Fig. S3 shows a detailed protein alignment.

The Sk-3–resistant RSK^r(Sk-3) and RSK^Sk-3 proteins are 97% identical to each other but are only 61–64% identical to the sensitive protein and the Sk-2–resistant proteins (Table S3). The identity scores can be explained by the notion that the two Sk-3–resistant alleles are evolutionarily related and the fact that they have their own characteristic indel pattern (Fig. 7).

Resistance in Global Strains Correlates with Their rsk Sequences.

Perkins and coworkers have assembled a collection of Neurospora strains from around the globe, some of which have been tested for resistance to spore killing (10). We obtained several N. crassa strains from Brazil, Ivory Coast, and Haiti, and asked whether their resistance or sensitivity is related to their particular rsk sequences (Fig. S4). In each case, the Sk-2–resistant strain encodes a protein >99% identical to the LA resistant protein (Table S4). The sensitive protein in each case is >94% identical to the N. crassa OR sensitive protein, with the exception of the one from Ivory Coast (CI4821), which resembles a truncated RSK^Sk-3.

rsk Is Not Required for Ascospore Development.

One possible model for killing is that RSK is required for ascospore development. Under such a scenario, the killer element can target a sensitive RSK protein for inactivation, whereas it does not recognize a resistant one. To test this hypothesis, we examined a cross homozygous for rsk^Δ and showed that it made normal, eight black-spored asci, suggesting that the normal function of RSK is not related to ascospore production.

Interestingly, an RSK paralog is found in N. crassa (E-value = 2e-20), whose coding sequence (09148) is separated from rsk by only two genes (Fig. 1B). It is possible that rsk and its paralog are functionally redundant and that one would have to delete both genes to observe a malfunction in ascospore production. In crosses homozygous for both gene deletions, ascospore production seemed normal (Fig. S5). This suggests that neither gene is important for ascospore production and that they are not targets of the killer.

Model for Sk Killing and Resistance.

A possible model for spore killing is as follows: the killer element targets one or multiple molecules (e.g., proteins, nucleic acids, and metabolites). These molecules are important for two meiotic functions, namely ascospore formation and ascospore maturation. (It is possible that only one type of molecule is being targeted and that the two ascospore phenotypes are manifestations of the same biochemical pathway.) When a resistant RSK is absent from the very beginning (e.g., in an Sk-2 rsk^Δ × WT cross), the killer targets early ascospore development, resulting in the production of empty asci. On the other hand, in an Sk-2 × WT cross, RSK^Sk-2 is present during early ascus development. The resistant RSK somehow neutralizes the killer's activity and allows ascospores to develop (Fig. 8A). After spore delimitation, the expression of the resistant RSK^Sk-2 protein is spore-autonomous, and it protects the Sk-2 progeny. There are no resistant proteins in the sensitive spores, allowing the killer element (which either has a long half-life or is not spore-autonomous) to disrupt their maturation (Fig. 8B).

Fig. 8.

RSK killer-neutralization model. The available evidence supports a model whereby different versions of RSK neutralize different killers. (A) Recognition and neutralization of specific killer elements depends on the RSK structure. (B) Resistance and killer elements are expressed in the ascus before spore delimitation. RSK neutralizes the killer, allowing proper ascus development. After spore delimitation, RSK becomes spore-autonomous, and only the progeny carrying a resistant version of rsk can continue to neutralize the killer. The killer element presumably has a long half-life or is not spore-autonomous.

Discussion

Although meiotic drive elements are widely represented in fungi, plants, insects, and mammals, the identities of their genetic components remain mostly elusive. The Podospora [Het-s] prion, the mouse t locus, and the Drosophila Dox and SD loci are among the few examples of molecularly characterized distortion systems (23–28). Our molecular characterization of the rsk resistance gene has set the foundation for delineating the Neurospora Spore killer system.

Most autosomal gamete killers contain a distorter (killer, driver) and a responder (resistance, target) (1). For the Drosophila SD system, it has been proposed that the distorter affects the nuclear transport of small RNA derived from a sensitive responder allele, which ultimately disrupts chromatin condensation and spermatid maturation (25, 29). A priori, it seems possible that the Neurospora killer targets a sensitive RSK protein, whose normal function is required for spore maturation. However, our results suggest that the RSK protein is not the target of the killer. Instead, a resistant version of the RSK protein neutralizes the killing action, possibly by antagonizing the killer element (directly or indirectly).

The process of Neurospora spore killing has been cytologically defined for more than three decades (6). Ascus development in a heterozygous Sk cross is normal until after spore delimitation and the second postmeiotic mitosis. At this point, development of the non-Sk spores arrests, the characteristic vacuolate cytoplasm becomes amorphous, and the nuclei slowly degenerate. Accordingly, the killer has been thought to target only late ascospore development. However, our data indicate that this is untrue because even ascospore formation was affected when an rsk-less killer was crossed to a sensitive strain. This suggests that the killer element is active at or before initial ascus development and that it can affect both early (spore formation) and late (spore maturation) sporulation events. The ascus abortion phenotype is not observed in normal Sk × WT crosses because the resistant RSK protein protects the entire ascus up until spore delimitation. After that, RSK presumably becomes spore-autonomous, and only Sk spores can survive.

Before this study, little was known about the genetic nature of Sk resistance. It was unclear whether the resistance element is encoded by a single gene, and if so, whether it is allelic to the resistance gene found in naturally resistant (but nonkilling) strains. Also unknown was whether Sk-2 and Sk-3 share the same resistance component. Our results indicate that Sk-2, Sk-3, r(Sk-2), and r(Sk-3) strains all use the same gene (rsk) for resistance. As a matter of fact, rsk alleles from Sk-2 and r(Sk-2) strains have highly similar indel changes, and they are different from those seen in Sk-3/r(Sk-3) isolates (which share their own unique indel pattern). It is possible that rsk^Sk-2 and rsk^Sk-3 alleles evolved independently and that each sequence type is shared by both the killer and a naturally resistant strain.

The two elements (killer and resistance) in an autosomal gamete killer are usually linked, with the recombination between them blocked (1). The impaired recombination, often induced by chromosomal rearrangements (e.g., inversions), prevents the killer element from segregating with a sensitive element (a self-destructive combination). In Neurospora, chromosomal rearrangements that lead to extensive unpairing could be potentially detrimental, as unpaired genes are silenced during meiosis by MSUD (13). If MSUD exists in the species concerned, and the recombination block region occupied by the killer/resistance elements contains meiotically important genes, a killer × sensitive cross could become barren, thus hampering the spread of the meiotic drive complex. Alternatively, the killer gene and/or the resistance gene could be unpaired and MSUD could prevent their expression. Perhaps for one or both of the scenarios described above, Spore killers in Neurospora (Sk-2 and Sk-3) each contain a semidominant suppressor of MSUD (12). These suppressors are not as strong as other classic MSUD suppressors, and they do not suppress (or only partially suppress) the silencing of certain unpaired genes. Our study suggests that rsk is paired between a killer and a sensitive strain and that the Sk-encoded MSUD suppressor is not strong enough to prevent the silencing of an unpaired rsk gene. These findings suggest that there is a biological constraint on the placement of rsk and the evolution of Sk rearrangement. It remains to be seen whether the killer gene is normally paired in a heterozygous Sk cross and whether there is a true advantage for a killer haplotype to encode an MSUD suppressor.

The identification of the Sk resistance gene provides the first step of delineation of this complex meiotic drive haplotype, an accomplishment that will aid the future characterization of the killer and the MSUD-suppressing components. Sk provides yet another example of diverse cellular mechanisms that selfish genetic elements can exploit to advance their own survival. Further studies of this and other meiotic drive systems will no doubt shed light on the evolution of aggressive DNA elements, as well as the intragenomic conflicts they inflict.

Materials and Methods

Neurospora Manipulation, Genetic Loci, and Transformation.

Strains used in this study are listed in Table S5. Genetic markers and knockouts used in this study are originated from the Fungal Genetics Stock Center (30) and the Neurospora Functional Genomics group (31), and their descriptions can be found at http://bmbpcu36.leeds.ac.uk/∼gen6ar/newgenelist/genes/gene_list.htm. N. crassa sequences can be downloaded from http://www.broadinstitute.org/annotation/genome/neurospora/MultiHome.html. Some of the gene names, such as “NCU09151,” are abbreviated in this report (as in “09151” or “51”). Standard Neurospora techniques were used throughout (http://www.fgsc.net/Neurospora/NeurosporaProtocolGuide.htm). Gene insertion and deletion vectors for this study were constructed using conventional restriction fragment ligation and/or double-joint PCR (DJ-PCR) (32, 33). Transformation was performed as previously described (34), using hygromycin resistance (hph), histidine prototrophy (his-3⁺), or uridine prototrophy (pyr-4⁺) as selection.

Examination of Asci and Ascospore Production.

Rosettes of asci were dissected from 10- to 14-d-old fruiting bodies in 25% glycerol with the aid of a VanGuard 1231CM microscope. A VanGuard 1274ZH microscope (equipped with a Canon Power Shot S3 IS digital camera) was used to examine and photograph the collected asci under magnification. Visual examination of ascospore production, using fluffy (fl) strains as designated females, was performed as previously described (35).