ABSTRACT
Meiotic drive elements like Spore killer-2 (Sk-2) in Neurospora are transmitted through sexual reproduction to the next generation in a biased manner. Sk-2 achieves this biased transmission through spore killing. Here, we identify rfk-1 as a gene required for the spore killing mechanism. The rfk-1 gene is associated with a 1,481 bp DNA interval (called AH36) near the right border of the 30 cM Sk-2 element, and its deletion eliminates the ability of Sk-2 to kill spores. The rfk-1 gene also appears to be sufficient for spore killing because its insertion into a non-Sk-2 isolate disrupts sexual reproduction after the initiation of meiosis. Although the complete rfk-1 transcript has yet to be defined, our data indicate that rfk-1 encodes a protein of at least 39 amino acids and that rfk-1 has evolved from a partial duplication of gene ncu07086. We also present evidence that rfk-1’s location near the right border of Sk-2 is critical for the success of spore killing. Increasing the distance of rfk-1 from the right border of Sk-2 causes it to be inactivated by a genome defense process called meiotic silencing by unpaired DNA (MSUD), adding to accumulating evidence that MSUD exists, at least in part, to protect genomes from meiotic drive.
INTRODUCTION
In eukaryotic organisms, genetic loci are typically transmitted through sexual reproduction to the next generation in a Mendelian manner. However, some loci possess the ability to improve their own transmission rate through meiosis at the expense of a competing locus. These “selfish” loci are often referred to as meiotic drive elements (Zimmering et al. 1970). The genomic conflict caused by meiotic drive elements may impact processes ranging from gametogenesis to speciation (Lindholm et al. 2016). Meiotic drive elements are found across the eukaryote tree of life (Burt and Trivers 2008; Bravo Núñez et al. 2018) and classic examples include SD in fruit flies (Larracuente and Presgraves 2012), the t-complex in mice (Lyon 2003; Sugimoto 2014), and Ab10 in Zea mays (Rhoades 1952; Kanizay et al. 2013). In the fungal kingdom, the known meiotic drive elements achieve biased transmission through spore killing (Raju 1994) and a handful of spore killer systems have been studied in detail. While the prion-based spore killing mechanism of het-s in Podospora anserina is the best characterized (Dalstra et al. 2003; Saupe 2011), the mechanisms by which other fungal meiotic drive elements kill spores are mostly unknown (e.g., see Grognet et al. 2014; Hu et al. 2017; Nuckolls et al. 2017).
Two fungal meiotic drive elements have been identified in the fungus Neurospora intermedia (Turner and Perkins, 1979). This species is closely related to the genetic model Neurospora crassa (Davis 2000), and the mating processes in both fungi are essentially identical. Mating begins with fertilization of an immature fruiting body called a protoperithecium by a mating partner of the opposite mating type. After fertilization, the protoperithecium develops into a mature fruiting body called a perithecium. The nuclei from each parent multiply within the developing perithecium, and a single nucleus from each parent is sequestered into a tube-like meiotic cell (Raju 1980). Meiosis begins with fusion of the parental nuclei and ends with production of four recombinant daughter nuclei. Each recombinant nucleus proceeds through a single round of mitosis, resulting in a total of eight nuclei in the meiotic cell. A process known as ascosporogenesis then constructs cell walls and membranes around each nucleus to produce sexual spores called ascospores. Maturing ascospores accumulate a dark pigment and develop the shape of a spindle; thus, at the end of ascosporogenesis, the mature meiotic cells appear to contain eight miniature black American footballs (Figure 1A). The meiotic cells also serve as ascospore sacs (asci) and a single perithecium can produce hundreds of asci, each derived from a unique meiotic event.
During an effort in the 1970s to collect and characterize Neurospora isolates from around the world, Turner and Perkins discovered pairs of compatible mating partners that did not produce asci with eight viable ascospores (Perkins 1974; Turner and Perkins 1979). This outcome was more common when crosses were performed between isolates from widely separated populations, and in some cases the abnormal asci were attributed to heterozygosity of chromosome rearrangements between mating partners. However, for a few isolates of N. intermedia, asci with atypical phenotypes were due to chromosomal factors called Spore killer-2 (Sk-2) and Spore killer-3 (Sk-3). Sk-2 and Sk-3 are not single genes; rather, they are complexes of genes that span approximately 30 cM of chromosome III, and they are transmitted through meiosis as single units due to a recombination suppression mechanism thought to be enforced by inversions (Turner and Perkins 1979; Campbell and Turner 1987; Hammond et al. 2012; Harvey et al. 2014). Unlike standard genetic elements, which display a Mendelian transmission rate of 50% through sexual reproduction, Sk-2 and Sk-3 are transmitted at levels approaching 100% (Turner and Perkins 1979). This biased transmission occurs because Sk-2 and Sk-3 kill ascospores that do not inherit resistance to spore killing (Raju 1979; Turner and Perkins 1979). For example, in Sk-2 × Spore killer-sensitive (SkS) crosses, asci with four black ascospores and four clear (“white”) ascospores are produced (Figure 1A). This phenotype can be symbolized as 4B:4W. The four black ascospores are typically viable and nearly always of the Sk-2 genotype, while the four white ascospores are inviable and presumed to be of the SkS genotype. The same phenomenon occurs in Sk-3 × SkS crosses, except the four black ascospores are of the Sk-3 genotype.
Although spore killers have not yet been detected in wild isolates of N. crassa, Sk-2 and Sk-3 have been introgressed into this species for genetic analysis. Introgression of Sk-2 and Sk-3 has allowed the discovery of resistance to spore killing in natural populations of N. crassa (Turner and Perkins 1979; Turner 2001). One of the Sk-2-resistant isolates (FGSC 2222) carries a resistant version of a gene whose function is best described by its name: resistant to Spore killer (rsk). Crosses of rskLA × Sk-2, where rskLA is the Louisiana allele of rsk carried by FGSC 2222, produce asci with an 8B:0W phenotype because ascospores inherit either rskLA or Sk-2, and both are sufficient for resistance to Sk-2-based spore killing (Hammond et al. 2012). Discovery of rskLA made identifying other rsk alleles possible, some of which do not provide resistance to the known spore killers. For example, the Oak Ridge rsk allele (rskOR), typical of most laboratory strains, is resistant to neither Sk-2 nor Sk-3. Additionally, some rsk alleles confer resistance to Sk-3 but not Sk-2. An example is rskPF5123, which exists in an N. intermedia isolate from French Polynesia. Sk-2 and Sk-3 also carry resistant versions of rsk, referred to as rskSk-2 and rskSk-3, respectively. Crosses homozygous for Sk-2 (i.e., Sk-2 × Sk-2) or Sk-3 produce asci with an 8B:0W phenotype because each ascospore inherits a resistant rsk allele. Furthermore, heterozygous crosses between different spore killers (e.g., Sk-2 × Sk-3) produce asci with a 0B:8W phenotype (Turner and Perkins 1979) because each ascospore inherits either rskSk-2 or rskSk-3 but not both (and rskSk-2 ascospores are killed by Sk-3 while rskSk-3 ascospores are killed by Sk-2).
The Killer-Neutralization (KN) model has been proposed to explain how Sk-2 and Sk-3 achieve biased transmission through sexual reproduction (Hammond et al. 2012). The KN model holds that Sk-2 and Sk-3 each use a resistance protein and a killer protein (or nucleic acid) and both proteins are active throughout meiosis and ascosporogenesis. During the early stages of meiosis, in an SkS × Sk-2 (or Sk-3) cross, both the resistance protein and the killer protein are hypothesized to diffuse throughout the meiotic cell. This unrestricted movement allows the resistance protein to neutralize the killer protein wherever the latter protein may be found. However, once ascospores are separated from the cytoplasm, the resistance protein becomes restricted to those ascospores that produce it (e.g., Sk-2 ascospores), and ascospores that do not carry a resistant version of rsk (e.g., SkS ascospores) are subsequently killed. This model requires the killer protein to move between ascospores after ascospore delimitation or to have a long half-life that allows it to remain functional in sensitive ascospores.
Evidence for the KN model is seen in the outcome of SkS × Sk-2 rskΔSk-2 crosses, where the latter strain has been deleted of its rsk allele. These crosses do not produce ascospores; instead, they produce asci that abort meiosis before ascospore production (Hammond et al. 2012). Meiotic cells of these crosses lack a resistant RSK, which likely causes the killing process to begin early in meiosis (at the ascus level) rather than during ascosporogenesis (at the ascospore level). The KN model is also supported by the existence of different rsk alleles. Previous studies have demonstrated the sequence of RSK to be the most important factor towards determining which killer it neutralizes (Hammond et al. 2012), suggesting that RSK and the killer may interact by a “lock and key” mechanism. To test this hypothesis, the killer must first be identified.
As described above, SkS × Sk-2 rskΔSk-2 crosses produce abortive asci. We recently used this characteristic to screen for mutations that disrupt spore killing (Harvey et al. 2014). Specifically, we fertilized an SkS mating partner with mutagenized Sk-2 rskΔSk-2 conidia (asexual spores that also function as fertilizing propagules). We reasoned that only an Sk-2 rskΔSk-2 conidium mutated in a gene “required for spore killing” (rfk) would produce viable ascospores when crossed with SkS. The screen allowed us to isolate six rfk mutants (ISU-3211 through ISU-3216). Complementation analysis of each mutant strain suggested all to be mutated at the same locus, which was subsequently named rfk-1 and mapped to a 45 kb region within Sk-2 on chromosome III. Here, we report the identification of rfk-1 as a gene encoding a protein of at least 39 amino acids. In addition to identifying rfk-1, we have found that the cellular process of meiotic silencing by unpaired DNA places limits on the location of rfk-1 within Sk-2. The implications of this finding with respect to meiotic drive element evolution are discussed.
MATERIALS AND METHODS
Strains, media, and crossing conditions
The strains used in this study are listed along with genotype information in Table 1. Vogel’s minimum medium (Vogel 1956), with supplements as required, was used to grow and maintain all strains. Hygromycin B and nourseothricin sulfate (Gold Biotechnology) were used at a working concentration of 200 μg / ml and 45 μg / ml, respectively. Synthetic crossing medium (pH 6.5) with 1.5% sucrose, as described by Westergaard and Mitchell (1947), was used for crosses. Crosses were unidirectional and performed on a laboratory benchtop at room temperature under ambient lighting (Samarajeewa et al. 2014). After fertilization, crosses were allowed to mature for 12-16 days before perithecial dissection in 25 or 50% glycerol and asci were examined with a standard compound light microscope and imaging system. Ascus phenotype designations were based on qualitative observations. More than 90% of the asci from a cross had to display the same phenotype to receive one of the following designations: 8B:0W, 4B:4W, or aborted.
Genetic modification of N. crassa, genotyping, and sequence confirmations
A technique called double-joint PCR was used to construct all deletion vectors (Yu et al. 2004; Hammond et al. 2011). Transgene-insertion vectors were designed to insert transgenes along with a hygromycin resistance cassette (hph) next to his-3 on chromosome I. Construction details for deletion and insertion vectors are provided in Supporting Information (Tables S1–S4). Transformations of N. crassa were performed by electroporation of conidia (Margolin et al. 1997). Homokaryons were derived from heterokaryotic transformants with a microconidium isolation technique (Ebbole and Sachs, 1990) or by crossing the transformants to standard laboratory strains (F2-23 or F2-26) to obtain homokaryotic ascospores. Site-directed mutagenesis was performed essentially as described for the QuikChange II Site-Directed Mutagenesis Kit (Revision E.01, Agilent Technologies) and details for its use are provided in Table S5. All genotypes were confirmed by polymerase chain reaction (PCR) assays on genomic DNA isolated from lyophilized (freeze-dried) mycelia with IBI Scientific’s Mini Genomic DNA Kit (Plant/Fungi). Sanger sequencing was used to confirm sequences and/or identify mutations in PCR products and plasmids.
Data availability
All strains and plasmids generated during this study are available upon request. Supplemental files available at FigShare.
RESULTS
Deletion of a DNA interval spanning most of Sk-2INS1 eliminates spore killing
The annotated 45 kb rfk-1 region contains 14 protein-coding genes, two pseudogenes (denoted with an asterisk), an inverted sequence (Sk-2INV1), an inversion breakpoint, and an 11 kb insertion sequence (Sk-2INS1; GenBank: KJ908288.1; Figure 1B). To refine the location of rfk-1 within this 45 kb region, intervals v3, v4, and v5 (Figure 1B and Table 2) were deleted and replaced with hph and the resulting deletion strains were crossed with an SkS mating partner. We found that while deletion of interval v3 or v4 had no effect on spore killing (asci are 4B:4W; Figure 1, C and D), deletion of v5 eliminated it (asci are 8B:0W; Figure 1E).
A DNA interval between ncu07838* and ncu06238 is required for spore killing
Interval v5 spans most of Sk-2INS1 (Figure 2, A and B). To further refine the position of rfk-1 within Sk-2INS1, we constructed nine additional deletion strains and crossed each one with an SkS mating partner (Figure 2B and Table 2). Surprisingly, deletion of the annotated genes and pseudogenes within Sk-2INS1 did not interfere with spore killing (Figure 3, A–D). In contrast, deletion of the intergenic region between ncu07838* and ncu06238 eliminated spore killing (Figure 3, E–I).
An ascus aborting element exists between ncu07838* and ncu06238
The above results suggest that rfk-1 is found within the intergenic region between ncu07838* and ncu06238 and that rfk-1 is required for spore killing. But, is rfk-1 also sufficient for spore killing? To answer this question, we genetically-modified eight SkS strains to carry different intervals of Sk-2INS1 (Figure 2C and Table 2) and found that each strain produced normal asci when crossed with an SkS mating partner (Figure S1). The reason for this finding can be traced to a silencing process called meiotic silencing by unpaired DNA (MSUD; Hammond 2017; Aramayo and Selker 2013). In a standard cross, where only one mating partner carries an ectopic transgene (e.g., an interval of Sk-2INS1), MSUD identifies the transgene as unpaired and silences it for the duration of meiosis. Therefore, to detect a phenotype that requires the expression of an unpaired transgene during meiosis, it is often necessary to suppress MSUD. MSUD suppression can be achieved by deleting a gene called sad-2 from one mating partner of a cross (Shiu et al. 2006). With this technique, we found that some Sk-2INS1 intervals have no effect on ascus development, while others abort it. For example, normal asci are produced by strains carrying intervals AH4Sk-2, AH6Sk-2, AH14Sk-2, or AH32Sk-2 (Figure 4, A–C and H), while aborted asci are produced by strains carrying intervals AH30Sk-2, AH31Sk-2, AH36Sk-2, or AH37Sk-2 (Figure 4, D–G). The ascus abortion phenotype can be explained by the presence of rfk-1 without the presence of a resistant version of rsk. Taken together, these findings suggest that intervals AH30Sk-2, AH31Sk-2, AH36Sk-2, and AH37Sk-2 contain rfk-1 and that rfk-1 is sufficient for spore killing.
The AH36 interval from an rfk-1 strain does not cause ascus abortion
The shortest abortion-inducing interval identified by the above experiments is AH36, located between positions 27,899 and 29,381 of the 45 kb rfk-1 region (Figure 2C and Table 2). Because the research path that led us to AH36 began with mapping the position of rfk-1 in strain ISU-3211 (Harvey et al. 2014), AH36 in ISU-3211 (referred to as AH363211) should harbor at least one mutation that disrupts rfk-1 function. To test this hypothesis, we transferred AH363211 to an SkS genetic background and crossed the resulting strain with an SkS sad-2Δ mating partner. As expected, we found that SkS sad-2Δ × SkS AH363211 crosses produce normal asci (Figure 5).
The G28326A mutation disrupts the ascus-aborting ability of AH36Sk-2
The different phenotypes associated with AH36Sk-2 and AH363211 suggest that they differ at the sequence level. Indeed, sequencing of these two alleles allowed us to identify seven guanine to adenine transition mutations in AH363211 (Figure 6A; G27904A, G27945A, G27972A, G28052A, G28104A, G28300A, and G28326A). To determine if one (or more) of these mutations is responsible for the inability of AH363211 to cause ascus abortion, we examined six of the seven mutations by site-directed mutagenesis. For each mutation, this involved mutating the base in a clone of interval AH36Sk-2, placing the mutated interval (e.g., AH36Sk-2 [G27945A]) in an SkS strain, and crossing the transgenic strain to an SkS sad-2Δ mating partner. Through this procedure, we found that only one of the six mutations examined (i.e., G28326A) eliminates the ascus-aborting ability of AH36Sk-2 (Figure 7).
We also identified a 46–48 bp tandem repeat (7.17 repeats) between positions 28,384 and 28,722 (Figure 6, A and B). The sequences of AH36Sk-2 and AH363211 are identical between these positions and thus the biological significance of the tandem repeats with respect to spore killing is currently unknown.
A putative start codon for RFK-1 is located within AH36
The G28326A mutation is 62 bp to the right of a putative start codon at position 28,264 (Figure 6). To test if this “ATG” could serve as the start codon for RFK-1, we constructed two deletion vectors: v199 and v200 (Figure 8A). Vector v199 deletes the interval between 28,131 and 28,264 and replaces it with hph and the promoter of the N. crassa ccg-1 gene, thereby inserting hph-ccg-1(P) directly upstream of the ATG at position 28,264 (Figure 8B). As a control, we used vector v200 to place hph-ccg-1(P) directly upstream of position 28,354, located 90 bases to the right of the proposed rfk-1 start codon. When inserted directly upstream of 28,264, hph-ccg-1(P) has no effect on spore killing (Figure 8, C and D). In contrast, when inserted 90 bases to the right of this position, hph-ccg-1(P) disrupts spore killing (Figure 8E). These findings demonstrate that the ATG at position 28,264 could serve as the rfk-1 start codon. Furthermore, they suggest that placement of hph-ccg-1(P) directly upstream of position 28,354 interrupts the rfk-1 coding region.
The arrangement of rfk-1 within Sk-2 protects it from MSUD
The right border of Sk-2 is found at position 29,151 (Figure 9A, dotted line; Table 2; Harvey et al. 2014). To the right of this position, the sequences of Sk-2 and SkS strains are very similar. For example, a simple ClustalW alignment (Thompson et al. 1994; Hall 1999) finds that Sk-2 positions 29,152 through 35,728 are 94.4% identical to the corresponding positions within SkS (GenBank: CM002238.1, positions 2,011,073 to 2,017,662). In contrast, the sequences to the left of the Sk-2 border are unrelated between Sk-2 and SkS strains (Figure 9A). Interestingly, most of AH36 is found to the left of the Sk-2 border, and thus most of AH36, including rfk-1, is unpaired during meiosis in SkS × Sk-2 crosses. If so, how does rfk-1 avoid inactivation by MSUD? While the molecular details of how MSUD detects unpaired DNA are unknown, we considered the possibility that the distance of rfk-1 from a “paired” sequence allows it to avoid MSUD (e.g., see the ncu06238 genes in Sk-2 and SkS, Figure 9A). To test this hypothesis, we inserted hph immediately to the right of AH36 in a standard Sk-2 strain (Figure 9A). We refer to this particular allele as v140Δ::hph. The v140Δ::hph allele increases the distance of rfk-1 from paired sequences by a length of 1391 bp (the length of hph minus the 21 bp that were deleted by v140). As predicted, we found that spore killing is absent in SkS × Sk-2 v140Δ::hph crosses (Figure 9B). To confirm that the lack of spore killing is a result of the increased distance of rfk-1 from paired DNA during meiosis, we inserted hph at the corresponding location in an SkS strain (Figure 9A). We refer to this allele as v150Δ::hph. When an SkS v150Δ::hph strain is crossed with an Sk-2 v140Δ::hph strain, spore killing is normal (Figure 9C). Thus, the proximity of rfk-1 to paired DNA helps it avoid inactivation by MSUD. As a final test of this hypothesis, we crossed SkS sad-2Δ and Sk-2 v140Δ::hph mating partners and found that spore killing is also normal in this cross (Figure 9D), most likely because sad-2Δ suppresses MSUD, which makes the distance of rfk-1 from paired sequences irrelevant to the expression of rfk-1 during meiosis.
The rfk-1 gene does not include ncu06238
To confirm that ncu06238, the gene to the right of rfk-1 (as depicted in Figure 10A), is not required for spore killing, we deleted ncu06238 from both SkS and Sk-2 and analyzed ascus phenotypes in crosses involving ncu06238 deletion strains. However, we found that SkS ncu06238Δ × SkS crosses produce asci with varying numbers of fully developed ascospores (Figure 10A). Therefore, we could not use ascus phenotype to determine if spore killing is functional in SkS ncu06238Δ × Sk-2 ncu06238Δ crosses (Figure 10B). Instead, we calculated the percentage of progeny with an Sk-2 genotype produced by a cross between SkS ncu06238Δ and Sk-2 ncu06238Δ mating partners. We found that 46 of 47 progeny had the Sk-2 genotype (data not shown). Therefore, because meiotic drive functions without ncu06238, the rfk-1 coding region does not overlap or include positions occupied by ncu06238.
Replacement of AH363211 with AH36Sk-2 restores spore killing to an rfk-1 mutant
The rfk-1 mutant strain ISU-3211 carries seven mutations within its AH36 interval (Figure 6). To confirm that at least one of these mutations (presumably G28326A) is responsible for ISU-3211’s inability to kill ascospores, we replaced AH363211 in a descendant of ISU-3211 (strain ISU-3222) with AH36Sk-2::hph (Figure 11A and Table S4). Because the presence of an hph marker to the right of AH36 disrupts spore killing in an MSUD-dependent manner (Figure 9B), we performed our test crosses with both a standard SkS mating partner and an SkS v150Δ::hph mating partner. As expected, we found that replacing AH363211 with AH36Sk-2 restores spore killing to a spore killing-deficient strain (Figure 11, B–G). These results demonstrate that the AH363211 interval is responsible for the loss of spore killing in ISU-3211 and its rfk-1 descendants.
The RFK-1 protein contains (at least) 39 amino acids
Assuming that the start codon for RFK-1 begins at position 28,264, and that the pre-mRNA for rfk-1 includes no introns (see discussion), we can propose the following hypothesis: RFK-1 is a 39 amino acid protein encoded by DNA located between positions 28,263 and 28,384 (Table 2 and Figure 12A). We found support for this hypothesis by sequencing the AH36 intervals in strains ISU-3211 through ISU-3216 (Figure 12A), which are the six Sk-2 rfk-1 isolates obtained by our initial screen for spore killing-deficient mutants (Harvey et al. 2014). Specifically, we found that AH363211 contains the previously discussed G28326A mutation, which changes the 21st codon from a tryptophan codon to a stop codon; AH363212 contains an extra thymine within a run of six thymines between positions 28,281 and 28,288, which causes a frameshift mutation in the 9th rfk-1 codon; and AH363213 contains a G28348A mutation, which changes the 29th codon from an alanine codon to a threonine codon. In addition, we found that the sequences of AH363214, AH363215, and AH363216, are all identical to the sequence of AH363211, suggesting that ISU-3211, ISU-3214, ISU-3215, and ISU-3216 were all “fathered” by the same mutagenized conidium. In all, we identified at least one potential codon-altering mutation between positions 28,263 and 28,384 in each of the six known rfk-1 mutants. This strongly suggests that the interval between positions 28,263 and 28,384 contains at least part, if not all, of the RFK-1 coding sequence.
RFK-1 is related to NCU07086
To investigate the origin of rfk-1, we downloaded a list of predicted N. crassa proteins from the National Center for Biotechnology Information (NCBI)’s Genome Database (Accession No. GCA_000182925.2) and performed a BLASTP search (Camacho et al. 2009) on the list with the hypothetical 39 aa RFK-1 sequence as query (Figure 12A). We found that the most significant match (Expect = 2e-7) to RFK-1 is a hypothetical 362-aa protein called NCU07086 (NCBI Protein Database: XP_960351.1). NCU07086 is encoded by the ncu07086 gene on N. crassa chromosome VI and is predicted to contain four introns (Figure 12B, I1 through I4; NCBI Gene Database, 3876500). A search of NCBI’s conserved domain database (CDD v3.16; Marchler-Bauer et al. 2015) with the predicted sequence of NCU07086 identified a region with a low-scoring match to the AtpF Superfamily (Expect=2.32e-3; Figure 12B). Interestingly, RFK-1 is highly similar to the first 39 amino acids of NCU07086 (Figure 12C), and it appears that the 46–48 bp repeat within AH36 (Figure 6) expanded from a single 47 bp sequence within ncu07086’s first intron (Figure 12D). These findings suggest that rfk-1 evolved from a partial duplication of the ncu07086 gene.
DISCUSSION
The biological mechanism used by the Neurospora Spore killers to achieve biased transmission is believed to require the action of a resistance protein and a killer protein. In a previous work, we isolated six rfk mutants (ISU-3211 through ISU-3216) and provided evidence that each is mutated at the same locus, subsequently named rfk-1 (Harvey et al. 2014). The rfk-1 locus in ISU-3211 was mapped to a 45 kb region of Sk-2. We began this study with the goal of identifying rfk-1. At first, we intended to use three point crossing assays to further refine the position of rfk-1 within the 45 kb rfk-1 region. These assays were to be performed with hph markers inserted between genes ncu06192 and ncu06191 (with vector v3) and between genes ncu06239 and ncu06240 (with vector v4); therefore, deletion vectors v3 and v4 were designed to delete relatively small intervals from the rfk-1 region (25 bp and 261 bp, respectively; Table 2) and they were not expected to influence spore killing. Accordingly, they had no effect on spore killing (Figure 1, C and D). In contrast, v5 was designed to delete a 10,718 bp interval, spanning most of the Sk-2INS1 sequence, in hopes that rfk-1 would be found somewhere within it (Table 2). Fortunately, deletion of interval v5 (intervals are named after the deletion vectors designed to delete them) was successful and its removal from Sk-2 eliminated Sk-2’s ability to kill ascospores (Figure 1E). We were thus able to focus our efforts on deleting subintervals of v5, which allowed us to track rfk-1 to the intergenic region between ncu07238* and ncu06238 (Figures 2, 3, and 10).
We also tested various subintervals of v5 for the presence of rfk-1 by transferring them to an SkS strain and performing test crosses with an SkS sad-2Δ mating partner (Figure 4). For this assay to yield positive results, rfk-1 must be sufficient for spore killing. Indeed, we found this to be the case when we identified four intervals (AH30, AH31, AH37, and AH36) that trigger ascus abortion. These four intervals all have the 1481 bp of AH36 in common, and the ascus abortion phenotype associated with each interval is likely due to the presence of rfk-1 without a compatible resistance gene. For example, the KN model holds that the resistance protein (RSK) and the killer are both active during early stages of meiosis (Hammond et al. 2012). Lack of a resistant version of RSK, along with expression of the killer, may cause asci to abort meiosis before ascospore delimitation. This phenomenon explains the abortion phenotypes of AH30, AH31, and AH37. However, for succinctness, we also referred to the phenotype associated with AH36 as ascus abortion, although it may be more accurate to refer to it as a “bubble” phenotype. The bubble phenotype was originally described by Raju et al. (1987), and it is thought to arise when asci and/or ascospores abort shortly after ascospore delimitation. Therefore, one explanation for the existence of the two phenotypic classes is that ascus development progresses a bit further with AH36 than it does with AH30, AH31, and AH37. Asci could progress further with AH36 if rfk-1 expression is lower from AH36 than it is from AH30, AH31, and AH37. In line with this reasoning, AH36 is the shortest of the abortion-inducing intervals, and, as a result, it may lack some of the regulatory sequences needed for full expression of rfk-1. It should be possible to address this hypothesis once the complete transcriptional unit of rfk-1 is identified.
Although we have yet to identify rfk-1’s transcriptional start (+1) site and termination site, or confirm the presence/absence of introns, we have provided strong evidence that the rfk-1 coding region includes the DNA interval between positions 28,263 and 28,384 (Figure 6). For example, a putative nonsense mutation at position 28,326 disrupts the ascus-aborting ability of interval AH36 (Figure 7); spore killing functions when a non-native promoter is attached to the putative RFK-1 start codon at position 28,264 (Figure 8); all six of the known rfk-1 mutants carry putative codon-altering mutations between positions 28,263 and 28,384 (Figure 12A), and insertion of a non-native promoter in the middle of this region disrupts spore killing (Figure 8). However, while our data indicate that the positions between 28,263 and 28,384 are part of the rfk-1 coding region, they do not eliminate the possibility that the coding sequences for RFK-1 include additional positions upstream and/or downstream of 28,263 and 28,384, respectively. Indeed, our preliminary analysis of RNAseq data from SkS × Sk-2 crosses (unpublished data) strongly suggests that an intron may exist between positions 28,379 and 28,775. The 5’ splice site of this hypothetical intron is related to the 5’ splice site of the first intron of ncu07086 (Figure 12D). If this intron does exist within the rfk-1 pre-mRNA, the RFK-1 stop codon would shift downstream and the length of RFK-1 would increase to 101 aa (assuming position 28,264 is the start codon and no other introns influence the stop codon position). Future work will seek to fully characterize the rfk-1 coding region by identifying the transcriptional start site, termination site, and any introns that may exist for the primary rfk-1 transcript, as well as for any biologically significant variants, if they were to exist.
While this work represents a significant step towards understanding the mechanism of Sk-2-based spore killing, many questions remain unanswered. For example, although it appears that RFK-1 evolved from NCU07086, does RFK-1 interfere with NCU07086 function as part of the spore killing mechanism? NCU07086 contains a region with slight homology to the AtpF Superfamily (Figure 12D). Interestingly, the atpF gene in E. coli (also known as uncF; NCBI Gene ID 948247) encodes subunit b of the F-type ATP synthase complex (Walker et al. 1984; Dunn 1992; McLachlin and Dunn 1997; Revington et al. 1999). This hints that RFK-1 could mediate spore killing by targeting eukaryotic F-type ATP synthases, which are associated with mitochondrial membranes in eukaryotes (Stewart et al. 2014). However, NCU07086 in N. crassa has not been investigated and a much more likely candidate for the b subunit of N. crassa’s F- type ATP synthase is found in NCU00502 (KEGG oxidative phosphorylation pathway: ncr00190, release 87.0, Kanehisa and Goto 2000; Kanehisa et al. 2016). Thus, at this point in time, a role for RFK-1 in disrupting mitochondrial function as part of the spore killing process is purely speculative.
Although the primary goal of this work was to identify rfk-1, the identity of which has been of interest to meiotic drive researchers since the discovery of Sk-2 nearly four decades ago, we unexpectedly discovered the strongest evidence to date that genomes in some, if not all, lineages of eukaryotic organisms possess elaborate defense processes to protect themselves from meiotic drive. With respect to Neurospora genomes, this defense process appears to be MSUD. The first hint that MSUD defends Neurospora genomes from meiotic drive appeared in 2007, when it was discovered that Sk-2 and Sk-3 are weak MSUD suppressors (Raju et al. 2007). Next, in 2012, it was found that the position of rsk within Sk-2 allows it to pair with rsk in the SkS genome during Sk-2 × SkS crosses. If rsk is not paired during these crosses (e.g., if it is deleted from the SkS mating partner), it is silenced by MSUD and the entire ascus is killed by the killer protein, which we now know to be RFK-1. In the current work, we found that the position of rfk-1 within Sk-2 is also critical for the success of meiotic drive because it allows rfk-1 to escape inactivation by MSUD. However, unlike rsk, rfk-1 is only found in Sk-2 strains and it cannot be paired in Sk-2 × SkS crosses. Evolution appears to have found a way to circumvent this problem by positioning rfk-1 close to sequences that are paired during meiosis (i.e. close to ncu06238 in Figure 10). Our data indicate that the proximity of rfk-1 to paired sequences allows it to escape inactivation by MSUD, which is critical for the success of spore killing. Overall, our findings add to accumulating evidence that MSUD antagonizes the evolution of meiotic drive elements by placing significant constraints on the arrangement of critical genes within the elements. Furthermore, our findings suggest that eukaryotic genomes like those of Neurospora fungi have evolved elaborate defense mechanisms to protect themselves from meiotic drive.
ACKNOWLEDGEMENTS
We are grateful to members of the Brown, Hammond, Johannesson, and Shiu laboratories for assistance with various technical aspects of this work. We are pleased to acknowledge use of materials generated by P01 GM068087 “Functional Analysis of a Model Filamentous Fungus”. We are also grateful to the Fungal Genetics Stock Center, whose preservation and distribution of Neurospora isolates helped make this work possible (FGSC; McCluskey et al. 2010). This project was supported by a grant from the National Science Foundation (MCB# 1615626) (T.M.H.). P.K.T.S was supported by the National Science Foundation (MCB# 1715534) and the University of Missouri Research Board and Research Council. H.J. was supported by the European Research Council grant under the program H2020, ERC-2014-CoG, project 648143, and the Swedish Research Council. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.