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.

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].

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.

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.

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).

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.

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.

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.