Molecular dissection of Neurospora Spore killermeiotic drive elementsThomas M. Hammond1,2, David G. Rehard1, Hua Xiao, and Patrick K. T. Shiu2

Division of Biological Sciences, University of Missouri, Columbia, MO 65211

Edited by Reed B. Wickner, National Institutes of Health, Bethesda, MD, and approved May 26, 2012 (received for review February 24, 2012)

Meiotic drive is a non-Mendelian inheritance phenomenon inwhich certain selfish genetic elements skew sexual transmissionin their own favor. In some cases, progeny or gametes carryinga meiotic drive element can survive preferentially because itcauses the death or malfunctioning of those that do not carry it.In Neurospora, meiotic drive can be observed in fungal spore kill-ing. In a cross of Spore killer (Sk) × WT (Sk-sensitive), the asco-spores containing the Spore killer allele survive, whereas the oneswith the sensitive allele degenerate. Sk-2 and Sk-3 are the moststudied meiotic drive elements in Neurospora, and they each the-oretically contain two essential components: a killer element anda resistance gene. Here we report the identification and character-ization of the Sk resistance gene, rsk (resistant to Spore killer). rskseems to be a fungal-specific gene, and its deletion in a killer strainleads to self-killing. Sk-2, Sk-3, and naturally resistant isolates alluse rsk for resistance. In each killer system, rsk sequences from anSk strain and a resistant isolate are highly similar, suggesting thatthey share the same origin. Sk-2, Sk-3, and sensitive rsk allelesdiffer from each other by their unique indel patterns. Contraryto long-held belief, the killer targets not only late but also earlyascospore development. The WT RSK protein is dispensable forascospore production and is not a target of the spore-killing mech-anism. Rather, a resistant version of RSK likely neutralizes thekiller element and prevents it from interfering with ascosporedevelopment.

intragenomic conflict | segregation distortion | selfish elements

In fungi, plants, and animals, not all genes follow the Mendelianpattern of inheritance. Meiotic drive, sometimes referred to as

segregation distortion, describes the phenomenon in which cer-tain “cheating” alleles are recovered in more than half of theprogeny (1). Two well-known examples are the segregationdistorter (SD) in Drosophila and the t haplotype in Mus. Inthese cases, sperm not carrying the aggressive allele (the driveelement) either degenerate (in flies) or become functionallyimpaired (in mice) (2, 3). In fungi, meiotic drive can be ob-served as spore killing, in which ascospores (sexual spores) thatcarry 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-killingfactors have been introgressed into Neurospora crassa for ex-tensive genetic studies. In a Spore killer (Sk) × WT (Sk-sensitiveor SkS) 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 ofkilling does not occur until late ascospore development: De-generation of sensitive spores takes place after the normal pro-gression of meiosis, spore delimitation, and the secondpostmeiotic 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 thekiller ones, as seen in a giant-spored N. crassa mutant and in the

four-spored (heterokaryotic) Neurospora tetrasperma, they canescape the elimination process (7, 8). These observations suggestthat the killer produces a resistance factor that shelters all nucleiresiding in the same spore. The Sk-2 and Sk-3 killers do notencode resistance to each other, and all eight progeny are in-viable 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 chro-mosome III. Their loci can only be defined as a 30-map unit re-gion because recombination is blocked between Spore killers andWT within this interval (9). Nonkiller strains that are resistant tospore killing have been found in nature (5, 9–11). Because re-combination block is not observed between WT and a resistantstrain, the associated resistance gene can be mapped accurately.For example, the r(Sk-2) gene, which confers resistance to Sk-2killing, 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 byunpaired DNA (MSUD) (12). MSUD, a process that silencesexpression from unpaired genes during meiosis (13–15), requirescommon RNAi proteins, such as an RNA-directed RNA poly-merase, a Dicer, and an Argonaute slicer (16–18). The MSUDsuppressors found in Sk-2 and Sk-3 are not as strong as theclassic sad-1Δ and sad-2Δ suppressors, and they do not suppressthe 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 themolecular dissection of the Sk elements, we first took aim atcloning the r(Sk-2) gene and determining how it is related to theoverall mechanism of Neurospora spore killing.

ResultsCharacteristics of Sk-2 and r(Sk-2). Spanning the centromere ofchromosome III, the Sk-2 and Sk-3 killer haplotypes are definedas 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 normalperithecium (fruiting body) produces roughly 200 asci, eachcontaining eight progeny that mature into melanized ascospores(Fig. 2A). In an Sk-2 × WT cross, the Sk-2–containing progenymature normally, whereas the Sk-2–sensitive progeny degenerate

Author contributions: T.M.H. and P.K.T.S. designed research; T.M.H., D.G.R., and H.X.performed research; T.M.H. and P.K.T.S. contributed new reagents/analytic tools; T.M.H.,D.G.R., and P.K.T.S. analyzed data; and T.M.H. and P.K.T.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The sequences reported in this paper have been deposited in the Gen-Bank database (accession nos. JX065596–JX065606).

See Commentary on page 11900.1T.M.H. and D.G.R. contributed equally to this work.2To whom correspondence may be addressed. E-mail: tmhammo@ilstu.edu or shiup@missouri.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1203267109/-/DCSupplemental.

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(Fig. 2B). r(Sk-2), not itself a killer (Fig. 2C), confers resistanceto Sk-2 such that all eight ascospores develop normally in anSk-2 × r(Sk-2) ascus (Fig. 2D).

Refinement of the r(Sk-2)LA Locus. The r(Sk-2) allele used in thisstudy was isolated from a naturally resistant N. crassa strainfound in Louisiana (LA) [hereafter r(Sk-2)LA] (5). Because thisstrain does not contain the killer haplotype and the associatedrecombination block, the r(Sk-2) location can be determined byconventional mapping. Using the hygromycin resistance marker(hph) present in each deletion strain of the N. crassa knockoutlibrary, we have mapped r(Sk-2) to between genes 09145 and09159 (21). This region contains 55 kb of DNA with 13 predictedgenes, including 09145 and 09159. To further refine the locationof r(Sk-2), we placed hph markers between 09149 and 09150 andbetween 09155 and 09156 (Fig. 1B). We then performed three-point crosses to determine the gene order of the two hphmarkerswith respect to r(Sk-2) and acr-7 (acriflavine resistant). Ouranalysis of 1,000 progeny from two crosses indicated that r(Sk-2)should lie between (but not including) 09149 and 09156 (TablesS1 and S2). To eliminate some of this region’s six predictedgenes as candidates for r(Sk-2), we set out to bracket its locationby determining the approximate crossover points in some of therecombinants from our mapping populations. This was accom-plished by identifying molecular markers between the r(Sk-2)LA

strain and its three-point cross partner [a strain with the standardOak Ridge (OR) background] and analyzing these markers in theprogeny (Fig. S1). Results from these analyses place r(Sk-2) tothe right of 09150 and left of 09155 (Fig. 1B).

Deletion of 09151 Correlates with the Loss of Resistance to Sk-2. Theabove mapping results narrow r(Sk-2) to a region spanning four

genes: 09151, 09152, 09153, and 09154. To determine which, ifany, of these genes is required for Sk-2 resistance, three of thefour genes were individually deleted from an r(Sk-2)LA strain(attempts to obtain a deletion strain for 09152 were unsuccess-ful). The deletion strains were subsequently crossed to Sk-2.Whereas 09153ΔLA and 09154ΔLA deletions had no adverse effecton Sk-2 resistance, a 09151ΔLA deletion correlated with theproduction of empty asci (Fig. 3A). Importantly, this ascusabortion is specific to Sk-2 crosses: The 09151ΔLA deletion strainproduced normal asci in crosses to a sensitive strain.This ascus abortion phenotype can be explained by the

following: (i) both 09151LA and the corresponding allelein Sk-2 (09151Sk-2) are involved in killer resistance, (ii) the09151ΔLA deletion results in the unpairing of the 09151Sk-2

allele during meiosis, and (iii) this unpairing leads to MSUD-based silencing of 09151Sk-2, causing the death of the entireascus. If the above hypotheses are true, then including a strongMSUD suppressor such as sad-2Δ (22) in the cross shouldrelieve 09151Sk-2 silencing and produce a normal killing phe-notype (4B:4W). Indeed, asci from such a cross displayednormal spore killing (Fig. 3B), suggesting that 09151 is neces-sary for Sk-2 resistance in an r(Sk-2)LA strain.

Expression of 09151LA in a Sensitive Strain Allows It to Gain Sk-2Resistance. To determine whether 09151LA alone is sufficient toconfer Sk-2 resistance, we tested whether it can grant resistanceto a sensitive (OR) strain. To achieve this, we inserted the09151LA gene into the his-3 locus [a standard gene placementlocus located on linkage group (LG) IR] of an OR (sad-2Δ)strain. Because we were unable to determine how a 09152ΔLA

deletion would affect Sk-2 resistance, we also created a 09152LA

insertion strain in a similar manner. Whereas 09152LA did notconfer Sk-2 resistance in a cross (Fig. 4A), 09151LA did. In thelatter cross (Sk-2 × his-3+::09151LA sad-2Δ), an interesting par-tial resistant ascus combination could be observed; because09151LA (located on LG I) and Sk-2 (LG III) segregate

48 49 50 51 52 53 54 55 56

hph49/50 hph55/56

AFLP1

AFLP2

A

FLP3

A

SP1

1 kb

(51 paralog)

ORProgeny #34 LA

ORProgeny #119LA

...cum r(Sk-2) acr-7 acr-2 sc ser-1 pro-1 ad-4 leu-1 his-7 ad-2 trp-1

Sk-2, Sk-3 A

B

Fig. 1. Genetic locations of Sk-2, Sk-3, and r(Sk-2). (A) The Spore killer re-gion on chromosome III. Sk-2 and Sk-3 are located in a 30-map unit regionwithin which recombination is blocked in heterozygous (Sk ×WT) crosses. (B)The r(Sk-2) locus was bracketed by genetic crossovers. Hygromycin resistancegenes were placed in a sensitive (OR) background to generate strains withmarkers between genes 09149 and 09150 (hph49/50; P17-13) or betweengenes 09155 and 09156 (hph55/56; P17-12). These strains were then crossed toa cum r(Sk-2) acr-7 strain (LA; P8-11) to create two mapping populations. Thegenotypes of the recombinant progeny indicate that r(Sk-2)LA should liebetween the two hph markers (Tables S1 and S2). Amplified fragmentlength polymorphism (AFLP) and amplified sequence polymorphism (ASP)markers were then used to determine the approximate crossover point intwo progeny (#34 and #119). For example, progeny #34 has the LA patternfor AFLP1 and the OR pattern for the other three markers (Fig. S1). Thisanalysis placed r(Sk-2) to the right of AFLP1 and to the left of ASP1, elimi-nating all genes except 09151, 09152, 09153, and 09154.

A B

C D

SkS Sk-2

SkS

r(S

k-2)

Fig. 2. Phenotypes of SkS (sensitive), Sk-2, and r(Sk-2) crosses. (A) SkS × SkS

(F2-26 × P6-07). In a typical (sensitive × sensitive) cross, each mature ascuscontains eight spindle-shaped (American football-like) ascospores. (B) Sk-2 ×SkS (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 (whiteand small) progeny abort. The 4B:4W asci demonstrate a first-divisionsegregation pattern and show that no crossing-over has occurred betweenSk-2 and the centromere. (C) SkS × r(Sk-2) (F2-26 × P15-52). r(Sk-2), a re-sistant strain isolated from LA, does not encode the killing element and isnot 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 resistantstrain. Because crossing-over is prohibited within the recombination blockregion, each progeny inherits a resistance gene [either the self-resistancefactor within the Sk-2 haplotype or the resistance factor from a nonkillingr(Sk-2) strain].

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independently, three different types of asci were seen: 8B:0W(four 09151LA and four Sk-2, all survived), 4B:4W (four 09151LA

Sk-2 survived and four 09151OR aborted), and 6B:2W (two09151LA, two Sk-2, and two 09151LA Sk-2 survived, whereas two09151OR aborted) (Fig. 4B). Note that this cross produced mostlyaborted asci if sad-2Δ was not included (Fig. 4C).To avoid the segregation patterns seen in the above cross, we

replaced 09151OR with 09151LA in a sensitive strain (with a sad-2+, MSUD-proficient background). 09151LA, when placed at itsnative locus, allowed the transformant to behave exactly likea resistant r(Sk-2)LA strain and give 8B:0W asci when crossed toSk-2 (Fig. 5A). Collectively, these data demonstrate that 09151LA

indeed corresponds to the r(Sk-2)LA gene activity, and when it isintroduced into a sensitive N. crassa strain, it allows the recipientto confer resistance to Sk-2 killing.To determine whether the 09151LA gene must be placed at its

native locus to function properly, or whether it can be placedanywhere within the Sk-2 recombination block, we inserted09151LA into a sensitive strain at a location ∼600 kb inside theright border of this block (denoted IIIR). Such insertion straingave 8B:0W asci when crossed to Sk-2, as long as sad-2Δ was partof the genetic background (Fig. S2A). When sad-2Δ was absent,ascus abortion occurred (Fig. S2B). Evidently, the placement of09151LA within the recombination block anywhere other than itsnative locus will make an otherwise sensitive strain fully Sk-2–resistant, providing that a strong dominant MSUD suppressor(e.g., sad-2Δ) is present in the cross.

Sk-2 Killer Uses 09151Sk-2 to Confer Resistance. The evidence pro-vided thus far reveals an MSUD-enforced constraint on the ge-nomic location of 09151LA. The existence of this constraintsuggests that a 09151LA-like sequence (i.e., 09151Sk-2) is re-quired by Sk-2 for resistance to its own killer. To test this

hypothesis, the 09151Sk-2 allele was deleted from an Sk-2strain. As predicted, crosses of the 09151ΔSk-2 deletion strain toa sensitive strain resulted in self-killing and mostly aborted asci(Fig. 6A). To test whether 09151Sk-2 is sufficient for resistanceto killing, it was used to replace the OR allele at the nativelocus in a sensitive strain. As expected, 09151Sk-2 conferscomplete resistance to Sk-2 in an MSUD-proficient background(Fig. 5E). These results suggest that the hypothetical resistancegene in Sk-2 is allelic to the resistance gene (09151LA) found inthe naturally resistant r(Sk-2)LA strain.

09151 Is also Involved in Sk-3 Resistance. The finding that 09151provides resistance to both Sk-2 and r(Sk-2) strains raises anobvious question: does the Sk-3 system also use the same gene toprevent killing? Initial findings on 09151LA suggest that this is thecase. As expected, when an unpaired ectopic copy of 09151LA

was introduced to a sensitive strain (at IIIR), spore killing oc-curred in a cross between the transformant and Sk-3 sad-2Δ

[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 toSk-3 sad-2+ (an MSUD-proficient strain), 0B:8W asci wereobtained, suggesting that the silencing of the Sk-3 resistancegene had occurred (Fig. S2D). The most plausible interpretationis that 09151LA has sequence similarity with the hypothetical Sk-3resistance gene.

A B

C D

E F

Sk-2 Sk-2 sad-2Δ

0915

4ΔLA

0915

3ΔLA

0915

1ΔLA

Fig. 3. 09151 is necessary for resistance to Sk-2. The coding regions for09151, 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-2Δ (F5-18) strains. (A) An Sk-2 × 09151ΔLA cross producedpredominantly aborted asci (containing granulated cytoplasm and nospores). This result suggests that the deletion of 09151LA and the unpairingof 09151Sk-2 eliminate resistance to spore killing for the entire ascus duringearly sexual development. (B) When a strong MSUD suppressor (sad-2Δ) waspresent, 09151ΔLA acted like a normal SkS strain in a cross (see text fordetails). (C–F) Deletions of 09153 and 09154 had no effect on Sk-2 resistance.

Sk-2

his-

3+::0

9151

LA

his-

3+::0

9152

LA

sad-

2Δhi

s-3+

::091

51LA

sad -

2Δ

A

B

C

Fig. 4. An ectopic 09151LA gene confers partial Sk-2 resistance in a sensitivebackground. To determine whether the 09151LA and 09152LA alleles conferSk-2 resistance, they were placed at an ectopic site (the his-3 locus onchromosome IR) in an SkS sad-2Δ background. The sad-2Δ allele was includedto prevent the possibility of the ectopic alleles or their homologs being si-lenced by MSUD. (A) 09152LA does not confer Sk-2 resistance, and susceptible4B:4W asci were observed. F1-16 × P17-05. (B) The ectopic 09151LA geneallowed a sensitive strain to gain Sk-2 resistance. Because the two resistancegenes (the ectopic 09151LA in the transformant and the native resistancegene in the Sk-2 killer) are located on different chromosomes, three possiblesegregation patterns can be observed (see text for details): 8B:0W (longarrow), 6B:2W (short arrow), and 4B:4W (arrowhead). MSUD-deficient (sad-2Δ) background. F1-16 × P17-04. (C) In an MSUD-proficient (sad-2+) back-ground, the above cross produced mostly aborted asci. This observationdemonstrates the importance of proper 09151 pairing. F2-19 × P17-06.

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To directly test whether the 09151 alleles from r(Sk-3) and Sk-3 strains are involved in resistance to the Sk-3 killer, experimentssimilar to those described for 09151LA and 09151Sk-2 were per-formed. These included replacement of the 09151OR allele ina sensitive strain with either 09151r(Sk-3) or 09151Sk-3. Thesereplacements created strains that produced fully resistant asciwhen crossed to Sk-3 (Fig. 5 D and H), indicating that thesealleles confer Sk-3 resistance. Additionally, deletion of the09151Sk-3 allele from an Sk-3 genetic background resulted ina loss of self-resistance in crosses to a sensitive strain (Fig. 6B).Together, our results indicate that 09151 is responsible for re-sistance in r(Sk-3) and Sk-3 strains.Because the two resistance genes in naturally resistant iso-

lates [r(Sk-2) and r(Sk-3)], the two resistance genes in Sk-2 andSk-3 killers, and the sensitive gene are all allelic with eachother (i.e., different versions of the 09151 gene), we areregrouping them under one name: rsk (resistant to Sporekiller). In this system, the aforementioned 09151 (rsk) alleleswould be referred to as rskr(Sk-2), rskr(Sk-3), rskSk-2, rskSk-3, andrskS. For occasions when the origin of the allele is of interest,we propose to add that information at the end; for exam-ple, rskr(Sk-2)-LA2222 (or rskLA2222 in short).

Sequence Comparison Among Sensitive, Sk-2–Resistant, and Sk-3–Resistant RSK Proteins. The WT OR rsk gene encodes a 486-aapolypeptide, with no currently recognizable motifs. RSK seemsto be a fungal-specific protein—RSK-like sequences are onlyfound in N. crassa and closely related fungi (e.g., Sordariamacrospora) as hypothetical proteins.

The aforementioned experiments demonstrate that differentrsk alleles provide resistance to different Spore killers. This as-pect of rsk-based killer resistance is perhaps most clearly depic-ted in Fig. 5, where replacement of the sensitive rskOR allele withany one of the four resistant rsk alleles created a strain that isresistant to either Sk-2 or Sk-3, but not both. To determine howthese resistant alleles are related to each other, we performeda series of sequence analyses. Protein alignments and sequenceidentity comparisons indicate that the distinction betweenRSKr(Sk-2) and RSKSk-2 proteins is minor (94% identical) com-pared with their differences from the sensitive RSKS protein(70–71% identical; Fig. S3 and Table S3). Most noticeably, thetwo Sk-2–resistant alleles (LA2222 and BN7401) contain thesame insertion/deletion (indel) pattern compared with the sen-sitive allele (Fig. 7), suggesting that they share the same origin.The Sk-3–resistant RSKr(Sk-3) and RSKSk-3 proteins are 97%

identical to each other but are only 61–64% identical to thesensitive protein and the Sk-2–resistant proteins (Table S3). Theidentity scores can be explained by the notion that the two Sk-3–resistant alleles are evolutionarily related and the fact that theyhave their own characteristic indel pattern (Fig. 7).

Resistance in Global Strains Correlates with Their rsk Sequences.Perkins and coworkers have assembled a collection of Neu-rospora strains from around the globe, some of which havebeen tested for resistance to spore killing (10). We obtainedseveral N. crassa strains from Brazil, Ivory Coast, and Haiti,and asked whether their resistance or sensitivity is related totheir particular rsk sequences (Fig. S4). In each case, the Sk-2–resistant strain encodes a protein >99% identical to the LA

Sk-209

151Δ

OR

::Sk-

309

151Δ

OR

::Sk-

209

151Δ

OR

::r(S

k-3)

0915

1ΔO

R::r

(Sk-

2) A B

C D

E F

G H

Sk-3

Fig. 5. Replacement of the 09151OR allele with a resistant allele [from r(Sk-2),r(Sk-3), Sk-2, or Sk-3] transformed the recipient fungus into a strain fullyresistant to either Sk-2 or Sk-3. These observations suggest that both the Sk-2and Sk-3 systems rely on 09151 for their resistance specificity. Resistant 8B:0Wasci are seen inA (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).

0915

1ΔS

k-3

SkS

A

B

0915

1ΔS

k-2

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 thesubsequent deletion mutants were crossed to a sensitive (SkS) strain. (A)After the 09151 gene was deleted, an Sk-2 strain became self-destructive ina cross. Most asci were aborted, containing granulated cytoplasm and nospores. 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 inSk-3 is less potent than the one found in Sk-2 (at least during early ascusdevelopment). F2-26 × P17-03.

RSK

RSK

RSKSk-3

Sk-2

WT

Fig. 7. Topography of RSK proteins. Sk-2 and Sk-3 use different alleles of thersk gene to confer resistance. The Sk-2 killer and strains naturally resistant toSk-2 have near-identical RSK proteins. The same is true for the Sk-3 killer andits naturally resistant strains, except that their proteins have different deletionregions (shown in red). Fig. S3 shows a detailed protein alignment.

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resistant protein (Table S4). The sensitive protein in each caseis >94% identical to the N. crassa OR sensitive protein, withthe exception of the one from Ivory Coast (CI4821), whichresembles a truncated RSKSk-3.

rsk Is Not Required for Ascospore Development.One possible modelfor killing is that RSK is required for ascospore development.Under such a scenario, the killer element can target a sensitiveRSK protein for inactivation, whereas it does not recognizea resistant one. To test this hypothesis, we examined a crosshomozygous for rskΔ and showed that it made normal, eightblack-spored asci, suggesting that the normal function of RSK isnot 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 byonly two genes (Fig. 1B). It is possible that rsk and its paralog arefunctionally redundant and that one would have to delete bothgenes to observe a malfunction in ascospore production. Incrosses homozygous for both gene deletions, ascospore pro-duction seemed normal (Fig. S5). This suggests that neither geneis important for ascospore production and that they are nottargets of the killer.

Model for Sk Killing and Resistance. A possible model for sporekilling is as follows: the killer element targets one or multiplemolecules (e.g., proteins, nucleic acids, and metabolites). Thesemolecules are important for two meiotic functions, namely asco-spore formation and ascosporematuration. (It is possible that onlyone type of molecule is being targeted and that the two ascosporephenotypes are manifestations of the same biochemical pathway.)When a resistant RSK is absent from the very beginning (e.g., in anSk-2 rskΔ × WT cross), the killer targets early ascospore de-velopment, resulting in the production of empty asci. On the otherhand, in an Sk-2 ×WT cross, RSKSk-2 is present during early ascusdevelopment. The resistant RSK somehow neutralizes the killer’sactivity and allows ascospores to develop (Fig. 8A). After sporedelimitation, the expression of the resistant RSKSk-2 protein isspore-autonomous, and it protects the Sk-2 progeny. There are noresistant proteins in the sensitive spores, allowing the killer ele-ment (which either has a long half-life or is not spore-autonomous)to disrupt their maturation (Fig. 8B).

DiscussionAlthough meiotic drive elements are widely represented in fungi,plants, insects, and mammals, the identities of their genetic com-ponents remain mostly elusive. The Podospora [Het-s] prion, themouse t locus, and the Drosophila Dox and SD loci are among thefew examples of molecularly characterized distortion systems (23–28).Ourmolecular characterization of the rsk resistance gene has setthe 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 Dro-sophila SD system, it has been proposed that the distorter affectsthe nuclear transport of small RNA derived from a sensitiveresponder allele, which ultimately disrupts chromatin conden-sation and spermatid maturation (25, 29). A priori, it seemspossible that the Neurospora killer targets a sensitive RSK pro-tein, whose normal function is required for spore maturation.However, our results suggest that the RSK protein is not thetarget of the killer. Instead, a resistant version of the RSK pro-tein neutralizes the killing action, possibly by antagonizing thekiller element (directly or indirectly).The process of Neurospora spore killing has been cytolo-

gically defined for more than three decades (6). Ascus develop-ment in a heterozygous Sk cross is normal until after sporedelimitation and the second postmeiotic mitosis. At this point,development of the non-Sk spores arrests, the characteristicvacuolate cytoplasm becomes amorphous, and the nuclei slowly

degenerate. Accordingly, the killer has been thought to targetonly late ascospore development. However, our data indicatethat this is untrue because even ascospore formation was af-fected when an rsk-less killer was crossed to a sensitive strain.This suggests that the killer element is active at or before initialascus development and that it can affect both early (spore for-mation) and late (spore maturation) sporulation events. Theascus abortion phenotype is not observed in normal Sk × WTcrosses because the resistant RSK protein protects the entireascus up until spore delimitation. After that, RSK presumablybecomes 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 isencoded by a single gene, and if so, whether it is allelic to theresistance gene found in naturally resistant (but nonkilling)strains. Also unknown was whether Sk-2 and Sk-3 share the sameresistance 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. Asa matter of fact, rsk alleles from Sk-2 and r(Sk-2) strains havehighly similar indel changes, and they are different from thoseseen in Sk-3/r(Sk-3) isolates (which share their own unique indelpattern). It is possible that rskSk-2 and rskSk-3 alleles evolved in-dependently and that each sequence type is shared by both thekiller and a naturally resistant strain.The two elements (killer and resistance) in an autosomal

gamete killer are usually linked, with the recombination betweenthem blocked (1). The impaired recombination, often induced bychromosomal rearrangements (e.g., inversions), prevents thekiller element from segregating with a sensitive element (a self-destructive combination). In Neurospora, chromosomal rear-rangements that lead to extensive unpairing could be potentiallydetrimental, as unpaired genes are silenced during meiosis byMSUD (13). If MSUD exists in the species concerned, and therecombination block region occupied by the killer/resistanceelements contains meiotically important genes, a killer × sensitivecross could become barren, thus hampering the spread of the

Killer RSK Protein Neutralization

Sk-3

Sk-2

A

B

Fig. 8. RSK killer-neutralization model. The available evidence supportsa model whereby different versions of RSK neutralize different killers. (A)Recognition and neutralization of specific killer elements depends on the RSKstructure. (B) Resistance and killer elements are expressed in the ascus beforespore delimitation. RSK neutralizes the killer, allowing proper ascus de-velopment. After spore delimitation, RSK becomes spore-autonomous, andonly the progeny carrying a resistant version of rsk can continue to neutralizethe killer. The killer element presumably has a long half-life or is not spore-autonomous.

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meiotic drive complex. Alternatively, the killer gene and/or theresistance gene could be unpaired and MSUD could prevent theirexpression. Perhaps for one or both of the scenarios describedabove, Spore killers in Neurospora (Sk-2 and Sk-3) each containa semidominant suppressor of MSUD (12). These suppressors arenot as strong as other classic MSUD suppressors, and they do notsuppress (or only partially suppress) the silencing of certain un-paired genes. Our study suggests that rsk is paired between a killerand a sensitive strain and that the Sk-encoded MSUD suppressoris not strong enough to prevent the silencing of an unpaired rskgene. These findings suggest that there is a biological constraint onthe placement of rsk and the evolution of Sk rearrangement. Itremains to be seen whether the killer gene is normally paired ina heterozygous Sk cross and whether there is a true advantage fora 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, anaccomplishment that will aid the future characterization of thekiller and the MSUD-suppressing components. Sk provides yetanother example of diverse cellular mechanisms that selfish ge-netic elements can exploit to advance their own survival. Furtherstudies of this and other meiotic drive systems will no doubt shedlight on the evolution of aggressive DNA elements, as well as theintragenomic conflicts they inflict.

Materials and MethodsNeurospora Manipulation, Genetic Loci, and Transformation. Strains used inthis study are listed in Table S5. Genetic markers and knockouts used in

this study are originated from the Fungal Genetics Stock Center (30) andthe Neurospora Functional Genomics group (31), and their descriptionscan 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 ofthe 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 inser-tion and deletion vectors for this study were constructed using conventionalrestriction fragment ligation and/or double-joint PCR (DJ-PCR) (32, 33).Transformation was performed as previously described (34), using hygromycinresistance (hph), histidine prototrophy (his-3+), or uridine prototrophy (pyr-4+)as selection.

Examination of Asci and Ascospore Production. Rosettes of asci were dissectedfrom 10- to 14-d-old fruiting bodies in 25% glycerol with the aid of a Van-Guard 1231CM microscope. A VanGuard 1274ZH microscope (equipped witha Canon Power Shot S3 IS digital camera) was used to examine and photo-graph the collected asci under magnification. Visual examination of asco-spore production, using fluffy (fl) strains as designated females, wasperformed as previously described (35).

ACKNOWLEDGMENTS. This study would not be possible without the pioneer-ing work of David Perkins, Namboori Raju, Barbara Turner, and others. Wethank Bob Metzenberg, James Birchler, members of the P.K.T.S. laboratory,the Fungal Genetics Stock Center, the Neurospora Functional Genomics group,and colleagues from our community for their help. This work was supportedby National Science Foundation Grants MCB0918937/1157942 (to P.K.T.S.).T.M.H. was supported by a Life Science fellowship from the University ofMissouri and a Ruth L. Kirschstein National Research Service Award from theNational Institute of General Medical Sciences.

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