A cis ‐acting bidirectional transcription switch controls sexual dimorphism in the liverwort
2019; Springer Nature; Volume: 38; Issue: 6 Linguagem: Inglês
10.15252/embj.2018100240
ISSN1460-2075
AutoresTetsuya Hisanaga, Keitaro Okahashi, Shohei Yamaoka, Tomoaki Kajiwara, Ryuichi Nishihama, Masaki Shimamura, Katsuyuki T. Yamato, John L. Bowman, Takayuki Kohchi, Keiji Nakajima,
Tópico(s)Yeasts and Rust Fungi Studies
ResumoArticle4 January 2019Open Access Source DataTransparent process A cis-acting bidirectional transcription switch controls sexual dimorphism in the liverwort Tetsuya Hisanaga Tetsuya Hisanaga orcid.org/0000-0002-2834-7044 Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Keitaro Okahashi Keitaro Okahashi Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Shohei Yamaoka Shohei Yamaoka orcid.org/0000-0003-4154-9967 Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Tomoaki Kajiwara Tomoaki Kajiwara Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Ryuichi Nishihama Ryuichi Nishihama orcid.org/0000-0002-7032-732X Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Masaki Shimamura Masaki Shimamura orcid.org/0000-0002-5665-5116 Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima, Japan Search for more papers by this author Katsuyuki T Yamato Katsuyuki T Yamato orcid.org/0000-0002-7130-0911 Faculty of Biology-Oriented Science and Technology, Kindai University, Kinokawa, Wakayama, Japan Search for more papers by this author John L Bowman John L Bowman orcid.org/0000-0001-7347-3691 School of Biological Sciences, Monash University, Melbourne, Vic., Australia Search for more papers by this author Takayuki Kohchi Corresponding Author Takayuki Kohchi [email protected] orcid.org/0000-0002-9712-4872 Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Keiji Nakajima Corresponding Author Keiji Nakajima [email protected] orcid.org/0000-0002-1580-3354 Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Tetsuya Hisanaga Tetsuya Hisanaga orcid.org/0000-0002-2834-7044 Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Keitaro Okahashi Keitaro Okahashi Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Shohei Yamaoka Shohei Yamaoka orcid.org/0000-0003-4154-9967 Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Tomoaki Kajiwara Tomoaki Kajiwara Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Ryuichi Nishihama Ryuichi Nishihama orcid.org/0000-0002-7032-732X Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Masaki Shimamura Masaki Shimamura orcid.org/0000-0002-5665-5116 Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima, Japan Search for more papers by this author Katsuyuki T Yamato Katsuyuki T Yamato orcid.org/0000-0002-7130-0911 Faculty of Biology-Oriented Science and Technology, Kindai University, Kinokawa, Wakayama, Japan Search for more papers by this author John L Bowman John L Bowman orcid.org/0000-0001-7347-3691 School of Biological Sciences, Monash University, Melbourne, Vic., Australia Search for more papers by this author Takayuki Kohchi Corresponding Author Takayuki Kohchi [email protected] orcid.org/0000-0002-9712-4872 Graduate School of Biostudies, Kyoto University, Kyoto, Japan Search for more papers by this author Keiji Nakajima Corresponding Author Keiji Nakajima [email protected] orcid.org/0000-0002-1580-3354 Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Author Information Tetsuya Hisanaga1,‡, Keitaro Okahashi2,‡, Shohei Yamaoka2, Tomoaki Kajiwara2, Ryuichi Nishihama2, Masaki Shimamura3, Katsuyuki T Yamato4, John L Bowman5, Takayuki Kohchi *,2 and Keiji Nakajima *,1 1Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Nara, Japan 2Graduate School of Biostudies, Kyoto University, Kyoto, Japan 3Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima, Japan 4Faculty of Biology-Oriented Science and Technology, Kindai University, Kinokawa, Wakayama, Japan 5School of Biological Sciences, Monash University, Melbourne, Vic., Australia ‡These authors contributed equally to this work *Corresponding author. Tel: +81-75-753-6389; Fax: +81-75-753-6127; E-mail: [email protected] *Corresponding author. Tel: +81-743-72-5560; Fax: +81-743-72-5569; E-mail: [email protected] The EMBO Journal (2019)38:e100240https://doi.org/10.15252/embj.2018100240 See also: F Berger (March 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Plant life cycles alternate between haploid gametophytes and diploid sporophytes. While regulatory factors determining male and female sexual morphologies have been identified for sporophytic reproductive organs, such as stamens and pistils of angiosperms, those regulating sex-specific traits in the haploid gametophytes that produce male and female gametes and hence are central to plant sexual reproduction are poorly understood. Here, we identified a MYB-type transcription factor, MpFGMYB, as a key regulator of female sexual differentiation in the haploid-dominant dioicous liverwort, Marchantia polymorpha. MpFGMYB is specifically expressed in females and its loss resulted in female-to-male sex conversion. Strikingly, MpFGMYB expression is suppressed in males by a cis-acting antisense gene SUF at the same locus, and loss-of-function suf mutations resulted in male-to-female sex conversion. Thus, the bidirectional transcription module at the MpFGMYB/SUF locus acts as a toggle between female and male sexual differentiation in M. polymorpha gametophytes. Arabidopsis thaliana MpFGMYB orthologs are known to be expressed in embryo sacs and promote their development. Thus, phylogenetically related MYB transcription factors regulate female gametophyte development across land plants. Synopsis The basal land plant liverwort spends most of its life cycle in a haploid state with distinct male and female morphology. A conserved MYB-type transcription factor regulates the sexual differentiation process, suggesting a shared origin for gamete formation in plants. The autosomal MpFGMYB gene promotes female sexual differentiation in the liverwort Marchantia polymorpha. MpFGMYB belongs to a MYB subfamily whose members are known to regulate female gametophyte development in Arabidopsis thaliana. Expression of MpFGMYB is suppressed in males by its antisense gene SUFin cis. Loss-of-function mutations of either MpFGMYB in females or SUF in males leads to near-complete sex conversion phenotypes. Introduction Life cycles in land plants alternate between a haploid gametophyte, in which male and female gametes are produced, and a diploid sporophyte, which produces haploid spores via meiosis. Sexual differentiation in land plants is best characterized in flowering plants, where male- and female-specific organs of the sporophyte produce male and female gametophytes, respectively. Thus, in flowering plants, sexual development occurs sequentially in sporophyte and gametophyte generations. In contrast, in earlier diverging lineages of land plants, i.e., non-seed plants, little or no sexual differentiation is evident in the sporophytic generation, with sexual differentiation occurring essentially exclusively in the gametophyte generation. In the context of land plants, sexual differentiation has been investigated in flowering plants wherein the specification of male and female sporophytic floral organs, i.e., stamens and pistils, by the ABC genes has been elucidated (Schwarz-Sommer et al, 1990; Bowman et al, 1991). One plausible hypothesis is that the sexual differentiation of the sporophyte is imposed upon the retained gametophytes developing within the sporophytic tissues. Thus, during land plant evolution, sexual differentiation has shifted from a purely gametophytic program to a situation where the sporophyte controls sexual differentiation of gametophytes. However, it is an open question whether there exist regulators of gametophytic sexual differentiation that are conserved across land plants. To decipher the mechanisms by which gametophytic sexual differentiation is established in land plants, it is essential to study sexual differentiation in basal lineages. The liverwort Marchantia polymorpha, a recently revived model bryophyte, provides a unique opportunity to study sexual differentiation in gametophytes (Bowman, 2016; Shimamura, 2016; Bowman et al, 2017). Marchantia polymorpha has several attributes facilitating investigation of the genetic regulation of sexual reproduction such as clear sexual dimorphism in the dominant gametophyte phase and asexual propagation through gemmae formation that allows maintenance of gamete-lethal mutants, in addition to general advantages as a model plant species such as available genome sequence and efficient genetic manipulation techniques (Ishizaki et al, 2016; Bowman et al, 2017; Sugano et al, 2018). Taking advantage of these attributes, recent studies utilizing M. polymorpha have revealed several key factors controlling critical steps of sexual plant reproduction (Koi et al, 2016; Rövekamp et al, 2016; Nakajima, 2017; Yamaoka et al, 2018). In the reproductive phase of their haploid-dominant life cycle, M. polymorpha plants exhibit sexual dimorphism depending on the presence of either female (X) or male (Y) sex chromosomes (Fig 1; Bowman et al, 2017; Shimamura, 2016; Yamato et al, 2007; here, we use X and Y, not U and V, according to the convention of liverwort researchers). Female gametophytes form sexual branches with finger-like rays (archegoniophores) at the apical notch region (meristem) of a vegetative structure called the thallus. Female sexual organs (archegonia) develop at the base of each ray, and a single egg cell differentiates in each archegonium (Fig 1A). In a similar manner, male gametophytes form sexual branches with a disk-shaped morphology (antheridiophores), in which male sexual organs (antheridia) develop and eventually produce motile sperm (Fig 1B). While classical genetic studies predict the existence of a dominant "Feminizer" on the X chromosome (Haupt, 1932), mechanisms controlling sexual differentiation of M. polymorpha are largely unknown. In contrast, in angiosperms with a diploid-dominant life cycle, female and male gametophytes are highly reduced to seven-celled embryo sacs and three-celled pollen grains, respectively, and their sex-specific differentiation is dependent upon the sporophytic generation (Fig 1C). Figure 1. Schematic representations of reproductive development in Marchantia polymorpha and Arabidopsis thaliana A, B. Development of X chromosome-containing female (A) and Y chromosome-containing male (B) M. polymorpha plants. C. Development of embryo sac and pollen, in bisexual flowers of A. thaliana. Data information: In all schemes, gametophytes (haploid) are shown in green, and germline cells in orange. Sporophytic organs (diploid) are shown in gray. Download figure Download PowerPoint In this study, we identified a cis-acting bidirectional transcription module as a toggle switch between female and male differentiation in M. polymorpha. This module consists of MpFGMYB, encoding an ortholog of previously identified regulators of female gametophyte development in Arabidopsis thaliana, and its antisense gene SUF producing a long non-coding RNA (lncRNA). Our study suggests that members of this MYB subfamily regulate female sexual differentiation in the haploid growth phase of land plants, while their sex-specific expression is likely regulated by divergent inputs. Results Conserved MYB transcription factors are specifically expressed in the female gametophytes of Marchantia polymorpha and Arabidopsis thaliana To identify evolutionarily conserved regulators of female sexual differentiation in land plants, we compared transcriptome datasets of archegonia and thalli of M. polymorpha (see Data availability). Genes preferentially expressed in archegonia were screened and further selected for enrichment of related genes in the published transcriptome datasets of A. thaliana female gametophytes (embryo sacs; Yu et al, 2005; Steffen et al, 2007; Wuest et al, 2010). Among the 23 genes thus identified (Table EV1), we focused on Mapoly0001s0061 encoding a MYB-type transcription factor. In a phylogenetic tree constructed from the amino acid sequences of MYB domains, Mapoly0001s0061 was found to be closely related to three Arabidopsis genes, AtMYB64, AtMYB119, and AtMYB98, as well as two homologs in the moss Physcomitrella patens (Fig 2A and B). Because our genetic analyses indicated a role of Mapoly0001s0061 in female gametophyte development in M. polymorpha (see below) as do the three Arabidopsis homologs in the embryo sac, the female gametophytes of flowering plants (Kasahara et al, 2005; Punwani et al, 2007; Rabiger & Drews, 2013), we named Mapoly0001s0061 FEMALE GAMETOPHYTE MYB (MpFGMYB), following the Marchantia nomenclatural guidelines (Bowman et al, 2016), and hereafter refer to the clade including these genes as the FGMYB subfamily (Fig 2A). Figure 2. FGMYB genes are phylogenetically closely related to each other and expressed in female gametophytes Phylogenetic tree of R2R3-MYB proteins of clades 11, 12, and 14–16, as described by Bowman et al (2017), from representative land plant species constructed using the maximum-likelihood method based on conserved MYB domain sequences. See Source Data for the sequences used and accession numbers. Numbers at nodes indicate bootstrap values calculated from 1,000 replicates. The tree is drawn to scale, with branch lengths reflecting the number of substitutions per site. Scale bar, 0.5 substitutions per site. Arrows indicate FGMYB orthologs involved in embryo sac development in Arabidopsis thaliana, MpFGMYB of Marchantia polymorpha (this study), and the most similar Physcomitrella patens genes shown in (B). The FGMYB clade is shaded in pink, and a distantly related FOUR LIPS (FLP) clade is in blue. Schematic representations of the FGMYB polypeptide structures. R2R3 MYB domains are shown in red and a conserved amino-terminal motif of PpFGMYBs and MpFGMYB in orange. Genomic PCR analysis indicating the existence of MpFGMYB in both male [Y] and female [X] genomes of M. polymorpha. Two biological replicates were analyzed. Autosomal MpEF1α was used as a control. Real-time RT–PCR analyses indicating preferential accumulation of MpFGMYB transcripts in female sexual organs and the sporophytes. MpEF1α was used for normalization. Measurements of six biological replicates for thalli and sporophyte, and three biological replicates for gametangiophore are plotted. Bars represent mean ± SD. Symbols above the bars indicate grouping by P < 0.05 in a Tukey–Kramer test. A transcriptional reporter with 5′- and 3′-flanking sequences revealed transcription of MpFGMYB throughout mature archegonia. Scale bar, 10 μm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence. MpFGMYB-Citrine fusion proteins expressed using the 5′- and 3′-flanking sequences rescued the Mpfgmybge-1 mutant and accumulated in the nuclei of the egg and the ventral canal cell (VCC; Shimamura, 2016). Scale bar, 10 μm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence. A transcriptional AtMYB64 reporter (AtMYB64-NLS-YFP-GUS (NYG)) is specifically expressed in all four cell types of the A. thaliana embryo sac (Rabiger & Drews, 2013; Waki et al, 2013). Scale bar, 25 μm. Green, YFP fluorescence; white, cell walls. Expression of AtMYB64-Citrine fusion proteins under the AtMYB64 promoter was detected in the central cells (CC) and egg cells (EC) of the A. thaliana embryo sac (Rabiger & Drews, 2013). Scale bar, 25 μm. Green, Citrine fluorescence; white, cell walls. Source data are available online for this figure. Source Data for Figure 2 [embj2018100240-sup-0005-SDataFig2.zip] Download figure Download PowerPoint PCR analyses detected MpFGMYB in both male and female genomic DNAs, indicating the autosomal localization of MpFGMYB (Fig 2C). As expected from the transcriptome data (Bowman et al, 2017), MpFGMYB was predominantly expressed in the archegoniophores of female plants and the sporophytes (Fig 2D). Plants harboring a transcriptional MpFGMYB reporter construct (MpFGMYBpro:Citrine-NLS:MpFGMYB3′) exhibited reporter fluorescence in the archegonia (Fig 2E). In a functionally complemented translational reporter line (gMpFGMYBresist-Citrine, see below), Citrine fluorescence was localized in the nuclei of rescued archegonia (Fig 2F). As reported previously (Rabiger & Drews, 2013; Waki et al, 2013), our transcriptional and translational reporters for AtMYB64 were specifically expressed in embryo sacs of A. thaliana (Fig 2G and H; Rabiger & Drews, 2013; Waki et al, 2013). Loss-of-function MpFGMYB alleles confer a male morphology to female liverworts Previous studies demonstrated that AtMYB64 and AtMYB119 have critical roles in the development of the embryo sac (Rabiger & Drews, 2013). Similarly, another gene, AtMYB98, is known to be required for the differentiation and function of the synergids, two of the seven cells constituting the embryo sac (Kasahara et al, 2005; Punwani et al, 2007). Thus, the preferential expression of MpFGMYB in the liverwort archegonia suggests that the FGMYB genes have evolutionarily conserved roles in the development of female gametophytes in land plants. To explore this possibility, we generated knockout mutants of MpFGMYB using clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9 (CRISPR/Cas9) technology (Sugano et al, 2018). We obtained four independent loss-of-function Mpfgmyb lines with insertions or deletions that created premature stop codons in the MYB domain-coding region (Fig 3A and Appendix Fig S1A), of which three (Mpfgmyb-1ge, Mpfgmyb-2ge, Mpfgmyb-6ge) were genetically female, while the other (Mpfgmyb-4ge) was genetically male (diagnosed by sex chromosome-linked markers; Fig 3B and Appendix Fig S1C). Figure 3. Loss of MpFGMYB function results in female-to-male conversion A. MpFGMYB gene structure and locations of Mpfgmyb mutations. Gray line, 5′- and 3′-flanking sequences; light purple box, UTR; dark purple box, coding region; red box, MYB domain-coding region; arrowheads, mutation positions; black arrow, transcriptional direction; dotted line, splice patterns. B. Diagnosis of genetic sex using Y chromosome-linked and X chromosome-linked rbm27 and rhf73 markers, respectively. Two biological replicates were analyzed for each genotype. C–L. Gametangiophore morphology (C–G) and gamete development (H–L) of wild-type and mutant plants. Scale bars, 5 mm (C–G), 10 μm (H and J), 100 μm (I, K, and L). Source data are available online for this figure. Source Data for Figure 3 [embj2018100240-sup-0006-SDataFig3.pdf] Download figure Download PowerPoint The Mpfgmyb mutants were morphologically indistinguishable from the wild-type plants during the vegetative growth period (Appendix Fig S2). Genetically male Mpfgmyb mutants (hereafter designated Mpfgmyb [Y]) were also indistinguishable from wild-type males during reproductive growth (Fig 3F, G, K and L). By contrast, genetically female Mpfgmyb mutants (hereafter designated Mpfgmyb [X]) exhibited a striking sex conversion phenotype; antheridiophores developed in place of archegoniophores (Figs 3C and D, and EV2A). Furthermore, the antheridiophores of Mpfgmyb [X] contained antheridia (Fig 3I, compare with 3H, and Fig EV2B). These phenotypes were rescued by introducing a MpFGMYB genomic fragment containing synonymous mutations to resist the remaining CRISPR/Cas9 activity (gMpFGMYBresist) in the mutants (Fig 3E and J, and Appendix Fig S1B), confirming a causal relationship between the female-to-male sex conversion phenotype of Mpfgmyb [X] and the loss of MpFGMYB function. The sex conversion phenotype of Mpfgmyb [X] could also be rescued by expressing MpFGMYB-Citrine fusion proteins under the same regulatory sequences as those used in the transcriptional reporter lines (gMpFGMYBresist-Citrine; Fig 2F). This line exhibited no Citrine florescence in the apical notch region of vegetative thalli (Fig EV1A and B). After induction of reproductive growth by far-red (FR) light treatment (Ishizaki et al, 2016), MpFGMYB-Citrine proteins accumulate in ventral apical notch regions where archegoniophores will develop (Fig EV1A and C), and later in developing archegoniophores (Fig EV1A and D), consistent with the requirement of MpFGMYB in female sexual differentiation of M. polymorpha. Click here to expand this figure. Figure EV1. MpFGMYB expression precedes archegoniophore morphogenesis A. A schematic representation of the process of archegoniophore morphogenesis after far-red irradiation. Longitudinal sections of apical notch regions are presented. Around 14 days after induction, dome-shaped archegoniophore primordia developed at the ventral side of apical notch area. Regions corresponding to the images shown in (B–D) are boxed. B–D. Confocal microscopic images of the apical notch region of gMpFGMYBresist-Citrine plants without (B) or with far-red irradiation (C, D). MpFGMYB-Citrine does not accumulate in the apical notch region of vegetative thalli (B). After 10 days of FR irradiation, MpFGMYB-Citrine accumulates in the ventral side of the apical notch region (C). Expression domain of MpFGMYB-Citrine expands when the morphology of archegoniophore primordia becomes evident (D). Yellow dotted lines delineate the edges of thalli and a developing archegoniophore. Top, bright field images; middle, fluorescent images; bottom, merged images. Scale bar, 25 μm. Download figure Download PowerPoint Mpfgmyb mutant females produce sperm with nearly normal morphology but lacking motility The results presented so far indicate a key role for MpFGMYB in the female sexual differentiation of M. polymorpha. In its absence, male sexual differentiation proceeds as the default program. This female-dominant mode of sex differentiation is consistent with the classical observation that rare diploid gametophytes of M. polymorpha carrying both X and Y chromosomes exhibit a female morphology (Haupt, 1932); however, it was still unclear whether the loss of MpFGMYB function alone was sufficient to generate functional sperm in the absence of the Y chromosome. To address this question, we performed a histological analysis and found that spermiogenesis proceeds in Mpfgmyb [X] essentially as it does in the wild-type and Mpfgmyb [Y] antheridia (Fig EV2C–J). Moreover, sperm collected from Mpfgmyb [X] antheridia exhibited nuclear condensation and flagella formation (Fig 4A–C). Consistently, two autosomal genes implicated in sperm morphogenesis and known to be specifically expressed in the antheridiophores, PROTAMINE-LIKE (MpRPM) and DYNINE LIGHT CHAIN7 (MpLC7; Higo et al, 2016), were expressed in the antheridiophores of Mpfgmyb [X] (Fig 4D). Furthermore, while the expression of the archegoniophore-specific autosomal genes was suppressed in Mpfgmyb [X], X chromosome-linked genes expressed in vegetative thalli (Bowman et al, 2017) were still expressed in Mpfgmyb [X] antheridia (Fig EV3A). These data indicate that the feminization capacity of MpFGMYB is primarily associated with the sex-specific expression of autosomal genes involved in sexual differentiation, and not with the expression of sex chromosome-linked genes. Click here to expand this figure. Figure EV2. Multiple Mpfgmyb [X] alleles consistently exhibit the female-to-male sex conversion phenotype A. Gross morphology of antheridiophores developed in Mpfgmyb-6ge [X]. B–J. Histological analyses indicating antheridium formation (B), diagonal cell division of spermatogenous cells (C–F), and subsequent spermiogenesis (G–J) in the wild-type (C, G), two independent Mpfgmyb [X] mutants (D, E, H, and I), and one Mpfgmyb [Y] mutant (F, J). Data information: Scale bars, 5 mm (A), 100 μm (B), 10 μm (C–J). Download figure Download PowerPoint Figure 4. Loss of MpFGMYB function results in sperm formation in genetically female plants A–C. DAPI-staining visualization of sperm formation in wild-type male (A), Mpfgmyb [X] (B), and Mpfgmyb [Y] (C) plants. Note that background DAPI staining visualizes flagella (arrows) in addition to nuclei (arrowheads). (B′) is an enlarged image of the boxed region in (B), visualizing an incompletely condensed nucleus. Scale bar, 5 μm. D. RT–PCR analysis indicating acquisition of male-like autosomal gene expression patterns in Mpfgmyb [X] antheridiophores. Two independent Mpfgmyb [X] mutant alleles were analyzed. E, F. TEM analyses visualizing the abnormal arrangement of axonemal microtubules in Mpfgmyb [X] sperm (F), as compared with those of wild-type males (E). Scale bar, 100 nm. Source data are available online for this figure. Source Data for Figure 4 [embj2018100240-sup-0007-SDataFig4.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Characterization of the sex conversion phenotypes of Mpfgmyb [X] A. RT–PCR analysis demonstrating loss of expression of female-specific autosomal genes in the antheridiophores of Mpfgmyb [X]. Note that X chromosome-linked genes are still expressed in Mpfgmyb [X] antheridiophores as in the wild-type females, despite their male-like sexual morphologies. Biological duplicates were analyzed for each genotype. B–D. Hoechst-stained wild-type archegonia treated with sperm from wild-type (B), Mpfgmyb [X] (C), and Mpfgmyb [Y] (D) plants, indicating the inability of Mpfgmyb [X] sperm to enter wild-type archegonia. Arrowheads, sperm in archegonial cavity. Dotted lines, egg cells. Scale bars, 10 μm (B′–D′, B″–D″), 50 μm (B–D). Source data are available online for this figure. Download figure Download PowerPoint The sperm of Mpfgmyb [X] plants exhibited abnormal morphologies, such as incompletely condensed nuclei and short flagella (Fig 4B, compare with 4A and C). Transmission electron microscopy revealed that most Mpfgmyb [X] flagella had irregular axonemes, lacking the "9 + 2" arrangement of microtubules (Carothers & Kreitner, 1968) typically seen in wild-type sperm (Fig 4E and F). Consistently, sperm produced in Mpfgmyb [X] antheridia were immotile and did not enter wild-type archegonia (Fig EV3B–D). Thus, while the loss of MpFGMYB function resulted in an almost complete female-to-male sex conversion, the formation of functional sperm requires additional factors that are likely encoded by the Y chromosome (Bowman et al, 2017). Expression of MpFGMYB is suppressed by its cis-acting antisense gene SUF in males The striking sex conversion phenotype caused by the autosomal Mpfgmyb mutations raised the question as to how MpFGMYB expression is tightly suppressed in males. A close inspection of our RNA sequencing data revealed the male-specific accumulation of antisense lncRNAs derived from the MpFGMYB locus (Fig 5A and B), which we named SUPPRESSOR OF FEMINIZATION (SUF). Real-time RT–PCR analyses revealed that SUF transcripts accumulated in male gametophytes and in sporophytes, while negligible accumulation was detected in female gametophytes. In male gametophytes, antheridiophores accumulated a significantly higher amount of SUF transcripts than thalli (Fig EV4A). Importantly, 5′ and 3′ RACE PCR revealed an invariable 5′ end and a polyadenylation site of SUF transcripts, indicating that SUF constitutes a strictly defined transcription unit of RNA polymerase II (Appendix Fig S3). Strand-specific RT–PCR analyses confirmed SUF transcript accumulation in wild-type males both before and after the induction of sexual reproductive growth by far-red irradiation (Fig EV4B). Figure 5. Antisense SUF suppresses MpFGMYB expression in males A. RNA-seq analysis showing male-specific accumulation of lncRNAs derived from the MpFGMYB 3′ region (top), and diagrams illustrating wild-type and mutant MpFGMYB/SUF loci (bottom). Folded lines with a ∆ symbol indicate a deletion. B. RT–PCR analysis of wild-type and genetically male suf mutants revealed loss of SUF expression and gain of MpFGMYB expression in suf mutants after induction of reproductive growth by far-red irradiation. Two independent suf mutant alleles were analyzed. The SUF primer pair used here flanked an intron and the duplicated bands of SUF likely represent spliced and unspliced forms. C–H. Gametangiophore morphology (C, E and G) and gametangium development (D, F and H) of plants with the designated genotypes. Scale bars, 1 mm (C, E and G), 20 μm (D), 50 μm (F), 100 μm (H). Source data are available online for this figure. Source Data for Figure 5 [embj2018100240-sup-0008-SDataFig5.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Expression analyses of SUF Real-time RT–PCR analyses indicating preferential accumulation of SUF transcripts in male reproductive organs. Constitutively expressed MpEF1α was used for normalization
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