Execution of the meiotic noncoding RNA expressionprogram and the onset of gametogenesis in yeastrequire the conserved exosome subunit Rrp6Aurélie Lardenoisa,1, Yuchen Liua,1, Thomas Waltherb, Frédéric Chalmela, Bertrand Evrarda, Marina Granovskaiac,Angela Chud, Ronald W. Davisd,e,2, Lars M. Steinmetzc, and Michael Primiga,2

aInstitut National de la Santé et de la Recherche Médicale, Unité 625, Institut Fédératif de Recherche 140, Université de Rennes 1, F-35042 Rennes, France;bUnité Mixte de Recherche (UMR) Institut National des Sciences Appliquées (INSA)/Centre National de la Recherche Scientifique 5504 and UMR INSA/InstitutNational de la Recherche Agronomique 792, F-31077 Toulouse, France; cEuropean Molecular Biology Laboratory, 69117 Heidelberg, Germany; dStanfordGenome Technology Center, Palo Alto, CA 94304; and eDepartment of Biochemistry, Stanford University, Stanford, CA 94305

Contributed by Ronald W. Davis, November 8, 2010 (sent for review September 22, 2010)

Budding yeast noncoding RNAs (ncRNAs) are pervasively tran-scribed during mitosis, and some regulate mitotic protein-codinggenes. However, little is known about ncRNA expression duringmeiotic development. Using high-resolution profilingwe identifiedan extensive meiotic ncRNA expression program interlaced withthe protein-coding transcriptome via sense/antisense transcriptpairs, bidirectional promoters, and ncRNAs that overlap the regu-latory regions of genes.Meiotic unannotated transcripts (MUTs) aremitotic targets of the conserved exosome component Rrp6, whichitself is degraded after the onset of meiosis when MUTs and otherncRNAs accumulate in successive waves. Diploid cells lacking Rrp6fail to initiate premeiotic DNA replication normally and cannotundergo efficient meiotic development. The present study demon-strates a unique function for budding yeast Rrp6 in degradingdistinct classes of meiotically induced ncRNAs during vegetativegrowth and the onset of meiosis and thus points to a critical role ofdifferential ncRNA expression in the execution of a conserveddevelopmental program.

Saccharomyces cerevisiae | sporulation | tiling arrays

Meiosis is a conserved developmental pathway during whichcells replicate and recombine their DNA before they prog-

ress through two successive divisions to produce haploid gametes.In simple eukaryotes such as yeasts, the process is partially con-trolled by a complex expression program coordinating the activityof several hundred protein-coding genes (1–3).Although most work on meiotic development has focused on

protein-coding genes, some evidence for the expression of non-coding RNAs (ncRNAs) in sporulating budding yeast and fissionyeast has been reported (4–7). The presence of noncodingtranscripts of various sizes during the reproductive stages of theeukaryotic life cycle has been observed in many multicellularorganisms. Moreover, the exosome, which includes the RNaseD-type exoribonuclease Rrp6 involved in mitotic ncRNA turn-over, is conserved from yeasts to mammals (8–11). Importantly,the fission yeast ortholog of Rrp6 was shown to be involved notonly in the mitotic degradation of meiotic mRNAs but also of atleast one meiotic noncoding transcript encoded by sme2 (12, 13).There is a rapidly growing body of evidence that ncRNAs

regulate many biological processes via mechanisms that involveeither their synthesis or their posttranscriptional activity (14, 15).However, very little is known about the abundance, transcriptboundaries, genomic localization, and possible roles of meioti-cally induced ncRNAs in the key experimental model organismSaccharomyces cerevisiae. Furthermore, the role of the buddingyeast exosome component Rrp6 in coordinating the transitionfrom mitosis to meiosis has not been investigated (16).Here, we used high-resolution oligonucleotide tiling arrays to

study the protein-coding and noncoding expression programunderlying vegetative and reproductive phases of the haploid anddiploid budding yeast life cycle, and we integrated the resultswith genomic, genetic, and biochemical analyses of Rrp6. Our

data reveal extensively interleaved meiotic mRNA and ncRNAtranscriptomes, and they identify meiosis-specific noncoding tran-scripts. Moreover, the present study provides clues for the im-portance of staggered ncRNA accumulation during the exit frommitotic growth and the transition through meiotic developmentvia Rrp6’s role and the protein’s posttranslational down-regula-tion after the onset of meiosis.

ResultsHigh-Resolution Expression Profiling of the Budding Yeast Life Cycle.We analyzed vegetatively growing and sporulating diploidMATa/α cells, a sporulation-deficientMATα/α control strain, andsynchronized mitotic haploid MATa cells (SI Materials andMethods and Fig. S1 A and B) (17) using high-resolution oligo-nucleotide tiling arrays (18) as well as modified normalizationand segmentation methods (Figs. S1C and S2A) (19). The goal ofthese experiments was to identify and characterize ncRNAs thatare preferentially or specifically expressed in diploid cells un-dergoing meiotic development.Previous work identified stable unannotated transcripts (SUTs)

as ncRNAs being detectable during mitosis (20) and cryptic un-stable transcripts (CUTs) as ncRNAs accumulating during vege-tative growth only in the absence of Rrp6 (9). Following thisnomenclature, we designated ncRNAs found in meiotic but notin fermenting or respiring cells as meiotic unannotated transcripts(MUTs), and we refer to transcripts typically showing peak ex-pression in respiring or sporulating MATa/α cells as rsSUTs.Detailed information about molecular biological and compu-

tationalmethods is available inSIMaterials andMethods.Genome-wide normalized and log2-transformed expression data at thesingle oligonucleotide probe level are accessible via GermOnline’sSaccharomyces Genomics Viewer (www.germonline.org) (21).

Genome-Wide Identification of Meiotically Induced ncRNAs and TheirSense/Antisense Configuration. In this report we focus on 1,452differentially expressed ncRNAs, which we organized into threecategories: those that overlap with sense mRNAs on the opposite

Author contributions: L.S. and M.P. designed research; Y.L., T.W., and B.E. performedresearch; F.C., M.G., A.C., R.W.D., and L.S. contributed new reagents/analytic tools; A.L.and F.C. analyzed data; and A.L. and M.P. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

Data deposition: Raw data are available via the European Bioinformatics Institute’srepository ArrayExpress (http://www.ebi.ac.uk/arrayexpress) [accession no. E-TABM-915(MATa/α and MATα/α)].

See Commentary on page 891.1A.L. and Y.L. contributed equally to this work.2To whom correspondence may be addressed. E-mail: michael.primig@inserm.fr ordbowe@stanford.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1016459108/-/DCSupplemental.

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DNA strand, a second group that overlaps with other known ornewly identified sense ncRNAs, and those that are not associatedwith any transcripts on the opposite strand (Fig. S2 B and C). Atotal of 878 ncRNAs (363 MUTs) were found to overlap withmRNAs. To explore the functional relevance of sense/antisensepairs, we selected rsSUTs and MUTs that overlapped with dif-ferentially expressed mRNAs and determined how their expres-sion profiles correlated with each other (ncRNAs are “antisense”and mRNAs are “sense” transcripts; Dataset S1). Thirty-threersSUTs (Fig. 1A) and six MUTs (Fig. 1B) showed significantlyopposed patterns, whereas 11 rsSUTs (Fig. 1C) and 32MUTs (Fig.1D) displayed significantly similar profiles compared with the dif-ferentially expressed mRNAs. The group of protein-coding genesthat showed opposed patterns to antisense rsSUTs was signifi-cantly enriched for Gene Ontology terms (22) such as sexual re-production (GO: 0019953; five observed/one expected by chancep= 9.1 × 10−3) and cellular cell wall organization (0007047; sevenobserved/one expected by chance; p=3.4× 10−4). It is noteworthythat genes involved in cell division (CHS2, CLN3) or cell cycleprogression (HUG1), which are down- or up-regulated during

meiosis, respectively, overlap with meiotically induced antisenseMUTs (Fig. 2 A–C).Subsequently, we searched for 421 noncoding transcripts not

associated with mRNAs to explore functions not directly relatedto the expression of protein-coding genes. Indeed, 120 tran-scripts (43 rsSUTs, Fig. 1E; 77 MUTs, Fig. 1F) overlapped withknown antisense ncRNAs such as CUTs, SUTs, and small nu-cleolar RNAs (snoRNAs), whereas 301 (132 rsSUTs, Fig. 1G;169 MUTs, Fig. 1H) overlapped with other antisense ncRNAs onthe opposite strand. The remaining 202 ncRNAs (78 rsSUTs,Fig. 1I; 124 MUTs, Fig. 1J) were not associated with transcriptsbut in some cases with genome features such as promoter regionsand DNA replication elements. For example, the highly inducedMUT1465 covers the promoter region of CLN2 (Fig. 2D). OtherMUTs—including some that are expressed from bidirectionalpromoters that control expression of ncRNAs and mRNAs in allpossible combinations (Fig. S3 A and B)—cover autonomouslyreplicating sequences (ARSs) such asARS220 andARS607 (Fig. 2E and F). Finally, we identified numerous ncRNAs not associatedwith any of the genome features we investigated, including manythat seem to be controlled by bidirectional promoters (Fig. S3C).These results reveal a complex meiotic ncRNA expression

program interlaced with the mRNA transcriptome via partiallyor totally overlapping sense/antisense transcript pairs, bidirec-tional promoters, and ncRNAs that cover developmentally reg-ulated promoters of protein-coding genes.

Rrp6 Controls MUTs and rsSUTs During Vegetative Growth. To de-termine whether Rrp6 controls the expression of meiotic ncRNAsduring mitosis we compared tiling array expression data fromMATaRRP6 vs. rrp6 strains and identified 22MUTsand59 rsSUTsthat strongly accumulated in a pattern similar to 106 CUTs in themutant (greater than threefold change), as shown in Fig. 3A (20).We conclude that Rrp6 is required for mitotic degradation ofa subset of rsSUTs and MUTs detectable in haploid cells.We next confirmed and extended these findings in two dif-

ferent diploid strains by RT-PCR: whereas MUT1290, -100,-523, and -1465 showed the expected pattern in fermenting(YPD), respiring (YPA), and sporulating (SPII 2–12 h) wild-typecells, respectively (Fig. 3B), we found that MATa/α rrp6 cellsaccumulated MUTs during vegetative growth in the presence ofdifferent carbon sources (fermentation, respiration) and sporu-lation (Fig. 3B). Whereas diploid JHY222 rrp6 cells displayedthe temperature-sensitive phenotype previously reported (16),the diploid SK1 rrp6 mutant already failed to grow normally atthe permissive temperatures (25 °C/30 °C; Fig. S4 A and B) andwas therefore not further investigated.We next examined the poly-A polymerase Pap2, a component

of the TRAMP complex, which enhances Rrp6’s activity (23, 24).Although Pap2 was suggested to act in the same pathway as Rrp6(9), an RT-PCR analysis of MUTs inMATa/α pap2 cells revealedno mitotic accumulation and showed their meiotic expression tobe indistinguishable from the one observed in wild-type strains(Fig. 3B, Bottom). The ACT1 control revealed no fluctuations inRNA concentration throughout the sample sets.These findings show that Rrp6 prevents MUTs from accu-

mulating during mitosis. Pap2 is not essential for this process inthe cases we examined, most probably because it is partially re-dundant with Trf5, a TRAMP subunit homologous to and syn-thetically lethal with Pap2 (25, 26).

Rrp6 Is Developmentally Regulated at the Posttranslational Level.Further analysis of CUTs during meiosis revealed their staggeredaccumulation similar to SUTs and MUTs in sporulating MATa/αbut typically not in starving MATα/α cells (Fig. 3C). This resultprompted us to hypothesize that Rrp6might be inactivated duringmeiosis and spore formation. Tiling array data and RT-PCRresults shown in Fig. 4 A and B, respectively, reveal fairly constantRRP6 mRNA levels and transcript boundaries during fermenta-tion, respiration, sporulation, and starvation (MATα/α in SPII);a transient drop in transcript concentration is detectable around

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Fig. 1. Global expression profile of differentially expressed ncRNAs. Eachline corresponds to an annotated segment, and each column represents theaveraged values (computed for each segment) observed in duplicate sam-ples. (A) Antisense rsSUTs or (B) MUTs and differentially expressed sensemRNAs showing opposed expression patterns. (C) rsSUTs or (D) MUTs anddifferentially expressed antisense mRNAs showing similar patterns. (E and F)Sense/antisense pairs of rsSUTs and MUTs and known ncRNAs. (G and H)Sense/antisense pairs of rsSUTs and MUTs. (I and J) rsSUTs or MUTs notoverlapping with any transcript. MATa/α cells were cultured in YPD, YPA(black bar), and sporulation medium (SPII, hourly samples from 1 to 12 h;light green bar). TheMATα/α strain was incubated in YPA (black bar) and SPII(6 and 10 h; dark green bar). MATa cells were grown in rich medium (YPD,samples were taken every 5 min from 0 to 135 min covering two cycles; blackbars). Right: Scale for log2-transformed values, containing 20 colors corre-sponding to increments of 5 percentiles each.

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4–6 h into sporulation, but overall the data argue against a majortranscriptional control mechanism acting on RRP6 expression.We next investigated Rrp6’s stability and observed a ≈2.5-fold

decrease in Rrp6 concentration as cells switched from fermen-tation to respiration. Interestingly, we also detected a furtherdecrease of the Rrp6 level by a factor of 2 at the onset of meiosis(SPII 2–6 h) until it approached the threshold level of detectionby Western blotting during postmeiotic spore formation (SPII 8–10 h). As opposed to that, protein levels remained essentiallyconstant in all samples in the case of the Pgk1 control (Fig. 4C).We conclude that RRP6 is constitutively transcribed (showing

moderate mRNA fluctuations during the mitotic and meiotic cellcycles) while the Rrp6 protein is down-regulated as cells progressthrough meiotic development. It is remarkable that Rrp6’s pro-gressive destruction coincides with increasing pervasive accumu-lationofmeiotic ncRNAs (compareFig. 1 andFig. 3CwithFig. 4C).

Rrp6 Is Essential for Premeiotic DNA Replication, the Meiotic Di-visions, and Spore Formation. We next asked whether Rrp6 wasessential for the transition between vegetative growth and meioticdifferentiation. To this end, rrp6mutant cells were sporulated, andtheir ability to progress through S-phase to form bi- and tetranu-cleate cells duringM-phase and to package nuclei into four-sporedasci was examined. Indeed, the MATa/α rrp6 strain failed to un-dergo normal premeiotic DNA replication because most cellscould not initiate the process, and only a small fraction completed

it after a substantial delay. As opposed to that,MATa/α pap2 cellsreplicated theirDNAnormally, albeit slightly earlier than thewild-type strain (Fig. 4D). We next determined that cells lacking Rrp6could not transit throughMI,MII, and undergo spore formation atnormal levels (Fig. 4E), whereas the MATa/α pap2 mutant initi-ated M-phase faster than the wild-type background and formedspores as efficiently as theMATa/α PAP2 strain (Fig. S4C).Taken together, these results confirm that Rrp6 is important

for robust mitotic growth (or any growth at all in the SK1 strainbackground) and suggest that its mitotic and premeiotic presenceis required for normal premeiotic DNA replication, execution ofthe meiotic divisions, and spore formation.

DiscussionThe present study addresses the important question of how themRNA and ncRNA transcriptomes are interleaved during thevegetative and reproductive phases of the budding yeast life cycle.Moreover, it reveals Rrp6—a highly conserved exoribonucleaseassociated with the nuclear exosome—to be posttranslationallydown-regulated during meiotic M-phase and spore formationwhen pervasive ncRNA accumulation occurs. Finally, it demon-strates Rrp6 to be important for a normal transition from themitotic to the meiotic cell cycle and efficient gamete formation.

ncRNA Expression and Function During Sporulation. Exit from mi-tosis and proper execution of the meiotic developmental path-way requires reprogramming the transcriptome from a cyclical toa linear process. This includes repression of mitotic genes in-compatible with meiotic development, activation of genes spe-cifically needed for meiosis and gametogenesis, and adaptationof cell cycle-regulated loci to the meiotic divisions. Althoughsuch regulatory functions have typically been attributed to DNAbinding transcriptional activators and repressors (27, 28),a number of mitotic ncRNAs were recently shown to positivelyor negatively influence gene expression via changes of histonemodification or transcriptional interference (17, 29–32). It istherefore conceivable that developmentally regulated ncRNAssuch as MUTs contribute to the control of genes required for, forexample, cytokinesis (CHS2) and the mitotic cell cycle (CLN3,HUG1) through similar mechanisms.Some of the noncoding RNAsmay also regulate protein-coding

genes by inhibiting the activity of their promoters, as shown earlierfor loci involved in metabolic functions (33; for review see ref. 34).In such a case, it is not the ncRNA itself but its synthesis and thepausing of RNA polymerase during elongation that prevents ac-tivator binding (35). For example, the mRNA concentration ofCLN2 declines during sporulation, and its 5′-regulatory region iscovered by MUT1465; CLN2 is a repressor of IME1 (the inducerof meiosis), and therefore CLN2’s down-regulation is importantfor the onset of meiosis (36). Another interesting example isCDC6, which is essential for DNA replication (37). The gene’smRNAdeclines as cells transit through themeiotic divisions, whileat least one strongly meiotically induced ncRNA (SUT200) coversits entire upstream region precisely at the onset of meiotic M-phase (www.germonline.org).Other MUTs could regulate ARS elements during sporulation

via transcription interference (38). For example, the mitoticallyactive ARS605 was shown to be inhibited during meiotic pro-phase by the expression of the early meiotic gene MSH4, whichprecludes Orc1 binding to its target motif (39). It is thus tempt-ing to speculate that ncRNAs directly contribute to the regula-tion of ARS activity, either alone or together with mRNAs (seefor example ECM23/MUT1498, which cover ARS1621; www.germonline.org), thereby influencing the efficiency of DNA rep-lication at different stages of growth and development.

Meiotic ncRNAs and Rrp6 Show Inverse Patterns of DevelopmentalRegulation. A striking outcome of our study is that ncRNAs notdetected during mitosis in wild-type cells accumulate to high levelsduring meiosis and spore formation (9, 20, 40). Contrary to thispattern, the Rrp6 protein is stable during mitotic growth (fermen-

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tation) but diminishes as cells switch to respiration and sporulation.A simple interpretation of our findings is that Rrp6 directly targetsMUTs,CUTs, and rsSUTsduring vegetative growth in thepresenceof glucose and that these ncRNAs are able to exert their functionswhen Rrp6 levels decline during respiration and sporulation.Two explanations for the decreasing Rrp6 protein concen-

trations are conceivable: MUT1312 might inhibit Rrp6 trans-lation via an RNA interference-independent mechanism (Fig. 4A and B) (41). Alternatively, the Rrp6 protein might be targetedby a protease that is partially induced during respiration (when

the Rrp6 protein level drops by 50%) and that reaches its fullactivity after the induction of meiosis. Intriguingly, this is thecase for Ume6, which is destroyed by the anaphase promotingcomplex/cyclosome during early meiosis (42). Such a mechanismwould imply that early meiotic mRNAs and meiotically inducedncRNAs are orchestrated via simultaneous destruction of theDNA-binding repressor Ume6 and the exoribonuclease Rrp6.

Rrp6 Function Is Important for the Transition from Mitosis to Meiosis.Rrp6 contains three domains required for nuclear localization,exoribonuclease activity, and RNA substrate specificity (43). Al-though a number of mRNAs are deregulated in the absence ofRrp6 (Dataset S2) it seems that protein-coding transcripts arerecognized by the protein as substrates only in mutants that fail toexport them from the nucleus (44). The protein is thus pre-dominantly required for efficient 5.8S rRNA3′-end formation andmitotic processing of snRNAs, snoRNAs, and ncRNAs, the latterin cooperation with Pap2 (9, 16, 20, 40, 45). Importantly, fissionyeast Rrp6 was recently shown to target meiotic mRNAs untimelyexpressed during mitotic growth and the meiotic ncRNA encodedby the sme2 gene, which is essential for Meiosis I (13, 46, 47). Thisis in keeping with the finding that Rrp6 targets MUTs and othermeiotically induced ncRNAs during vegetative growth in the dis-tantly related yeast S. cerevisiae. Given the opposed profiles ofmeiotic ncRNA and Rrp6 stabilities during growth and de-velopment and the phenotypes of rrp6 cells, we speculate thatRrp6is a negative regulator of meiotic development and that the pro-tein’s progressive down-regulation during the transition fromfermentation to respiration and sporulation is important for effi-cient gametogenesis by allowing ncRNAs to accumulate.The outcome of our analysis, together with reports on meiotic

ncRNAs in fission yeast (6, 7), increasing evidence for ncRNAs inthe mammalian germline (8), and the finding that fly Rrp6 is im-portant for cell cycle progression (48), raise the intriguing possi-bility that conserved mechanisms involving noncoding transcriptsand RNase D-type ribonucleases contribute to the control of ga-metogenesis during germ cell development in higher eukaryotes.

Materials and MethodsYeast Strains, Media, and Sporulation Conditions. We studied wild-type andmutant derivatives of S. cerevisiae SK1 and JHY222 strains (SI Materialsand Methods; Table S1).

Expression Profiling. Total RNA from diploid cells was quality controlled witha BioAnalyzer (Agilent) and used to enrich for poly-A+ transcripts with the Oli-gotex kit (Qiagen). PurifiedpolyA+ RNAwas reverse transcribed in the presenceof actinomycin D to prevent spurious antisense RNA synthesis (49). Proprietaryhigh-density oligonucleotide tiling microarrays (Sc_tlg GeneChips) were usedfor raw data production using the GCS3OOO TG system (Affymetrix) (17).

Tiling Array Data Analysis. Data quality control was carried out using R scripts(50). A modified version of the tilingArray Bioconductor package was usedfor data processing and normalization (18). Segments were identified byfirst applying the segmentation algorithm to five sample groups, followedby merging similarly expressed segments located within <50 base pairs ofeach other. Segments corresponding to differentially expressed transcriptsidentified using AMEN were inspected in a genome-wide manual curationstep (50). A detailed description of our approach is available in SI Materialsand Methods. Raw data are available via the European BioinformaticsInstitute’s repository ArrayExpress (http://www.ebi.ac.uk/arrayexpress) at E-TABM-915 (MATa/α and MATα/α).

ACKNOWLEDGMENTS. We thank R. Strich for a critical reading of themanuscript; O. Collin, A. Roult, and R. Fabretti for information technologysupport; P. Demougin, J. Dyczkowski, Z. Xu, and M. Ritchie for technicalsupport; and J. Horecka (Stanford Genome Technology Center, Palo Alto,CA) for JHY222. The Rrp6 antibody was a gift from M. Schmid (University ofAarhus, Aarhus C, Denmark). This work was supported by fellowships fromInstitut National de la Santé et de la Recherche Médicale (INSERM)/InstitutNational de l’Environnement Industriel et des Risques (INERIS) and theFrench Ministry of Education (to A.L. and Y.L., respectively); by INSERMAvenir Grant R07216NS and Fondation pour la Recherche Médicale GrantINE20071111109 (to M.P.); and by National Institutes of Health Grants 5R01GM068717 and 5P01 HG000205 (to R.W.D. and L.S.).

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Fig. 4. Rrp6 regulation and function. (A) Genomic heatmap of expressiondata for RRP6/MUT1312 from strains and samples as given. The layout is as inFig. 2, and the scale is as in Fig. 1. (B) RT-PCR expression data for RRP6/MUT1312 using samples as in Fig. 3B. (C) Western blot data for Rrp6 andPgk1 detected in extracts prepared from a diploid rrp6 mutant (rrp6) anddiploid growing (YPD, YPA) and sporulating wild-type cells (time pointstaken every 2 h from 2 to 10 h). Histogram below the bands shows relativeRrp6 protein amounts on the y-axis detected in the samples given on the x-axis. (D) DNA replication dynamics analyzed by fluorescence-activated cellsorting in diploid strains containing wild-type (RRP6) and mutant (rrp6,pap2) alleles. Respiring cells (YPA) were compared with hourly samples fromsporulating cells (SPII, 1–12 h). DNA content is given at the bottom. (E) Wild-type (RRP6) and mutant (rrp6) strains undergoing MI, MI+II, and ascus for-mation. The x-axis shows the time of incubation in SPII sporulation mediumin hours, and the y-axis displays the percentage of cells within the pop-ulation that have completed landmark events. The average of three in-dependent sporulation assays is shown.

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