The double-stranded break-forming activity of plant SPO11s and a novel rice SPO11 revealed by a Drosophila bioassay

Abstract

Background: SPO11 is a key protein for promoting meiotic recombination, by generating chromatin locus- and timing-specific DNA double-strand breaks (DSBs). The DSB activity of SPO11 was shown by genetic analyses, but whether SPO11 exerts DSB-forming activity by itself is still an unanswered question. DSB formation by SPO11 has not been detected by biochemical means, probably because of a lack of proper protein-folding, posttranslational modifications, and/or specific SPO11-interacting proteins required for this activity. In addition, plants have multiple SPO11-homologues.

Results: To determine whether SPO11 can cleave DNA by itself, and to identify which plant SPO11 homologue cleaves DNA, we developed a Drosophila bioassay system that detects the DSB signals generated by a plant SPO11 homologue expressed ectopically. We cytologically and genetically demonstrated the DSB activities of Arabidopsis AtSPO11-1 and AtSPO11-2, which are required for meiosis, in the absence of other plant proteins. Using this bioassay, we further found that a novel SPO11-homologue, OsSPO11D, which has no counterpart in Arabidopsis, displays prominent DSB-forming activity. Quantitative analyses of the rice SPO11 transcripts revealed the specific increase in OsSPO11D mRNA in the anthers containing meiotic pollen mother cells.

Conclusions: The Drosophila bioassay system successfully demonstrated that some plant SPO11 orthologues have intrinsic DSB activities. Furthermore, we identified a novel SPO11 homologue, OsSPO11D, with robust DSB activity and a possible meiotic function.

Figure 1

Confocal images of DSB signals generated by plant SPO11 expression in the mei-W68 mus301 double mutant background (mei-W68¹/mei-W68¹; mus301^D4/Df(3L)66C-G28). The DNA is green. The γ-H2Av is red, and shows the DSB signals. When the two signals overlap, the signal is yellow. Each inset shows an enlarged view of the oocyte nucleus. All images are single confocal sections. Each scale bar shows 20 μm. (A) An egg chamber of mei-W68⁺ /mei-W68¹; mus301^D4/Df(3L)66C-G28. (B) An egg chamber of mei-W68¹/mei-W68¹; mus301^D4/Df(3L)66C-G28. The frequencies of the oocyte nuclei with DSB signals for the total oocyte nuclei of stage 2-8 egg chambers were 100% (344/345, 3 ovaries) in the mei-W68¹ heterozygote, and 7.4% (16/220, 2 ovaries) in the mei-W68¹ homozygote (see Figure 2A). The frequencies of the oocyte nuclei with a karyosome morphological defect (Figure 1) for the total oocyte nuclei of stage 3-8 egg chambers were 91% (254/278, 3 ovaries) in the mei-W68 heterozygote, and 1.3% (2/177, 2 ovaries) in the mei-W68 homozygote (see Figure 2B). (C) An egg chamber of mei-W68¹ /mei-W68¹; mus301^D4 /Df(3L)66C-G28 with transgene P{hsp83-mei-W68⁺cDNA, M53-3} (indicated as Vector name{gene, insertion #}). The frequency of the oocyte nuclei with DSB signals for the total oocyte nuclei of stage 2-8 egg chambers was 100% (180/180, 2 ovaries) in the mei-W68 homozygote with P{hsp83-mei-W68⁺cDNA, M53-3}, similar to the mei-W68¹ heterozygote. The frequency of the oocyte nuclei with defective karyosome morphology for the total oocyte nuclei of stage 3-8 egg chambers was 70% (93/132) in the mei-W68 homozygote with P{hsp83-mei-W68⁺cDNA, M53-3}, similar to the mei-W68¹ heterozygote. The karyosomes in the insets of panels A and C (SPO11 positive oocyte with DSB repair deficiency, as positive controls) have defective morphology (non-disc shape), while the karyosome in the inset of panel B (DSB-repair defective oocyte without functional SPO11 as negative controls) has normal morphology (disc shape). (D-G) Egg chambers of mei-W68¹ /mei-W68¹; mus301^D4 /Df(3L)66C-G28 with transgene: P{hsp83-AtSPO11-1 cDNA, A5-1} (D); P{hsp83-AtSPO11-2 cDNA, 2M3-1} (E); P{hsp83-OsSPO11A cDNA, 3M2-3} (F); P{hsp83-OsSPO11D cDNA, F2-1} (G). Quantitative data with respect to the DSB signals and the defective karyosome morphology in the oocyte nuclei of these transgenic flies are shown in Figure 2.

Figure 2

DSB formation by plant SPO11 expression in the mei-W68 mus301 double mutant background (mei-W68¹ /mei-W68¹; mus301^D4 /Df(3L)66C-G28). (A) Oocyte nuclei with DSB signals, which were detected by staining γ-H2Av, were scored in stage 2-8 egg chambers. The number of oocyte nuclei with DSB signals was divided by the total number of oocyte nuclei observed. (B) Oocyte nuclei with defective karyosome morphology, which was observed as a non-disc shape, were scored in stage 3-8 egg chambers. The number of oocyte nuclei with defective karyosome morphology was divided by the total number of oocyte nuclei observed. The values of the positive control (bearing the hsp83-mei-W68⁺ transgene) for panels A and B were described in the legend to Figure 1C. Transgene (Vector name{gene, insertion #}) [Number of ovaries used for observations]: P{hsp83-AtSPO11-1 cDNA, A5} [7]; P{hsp83-AtSPO11-2 cDNA, M3-1} [3]; P{hsp83-OsSPO11A cDNA, 3M2-3} [3]; P{hsp83-OsSPO11B cDNA, M6-2} [2] and P{hsp83-OsSPO11B cDNA, M7-1} [1]; P{hsp83-OsSPO11A cDNA, 3M2-3} with P{hsp83-OsSPO11B cDNA, F3-2} [2]; P{hsp83-OsSPO11D cDNA, F2-1} [2]; P{hsp83-OsSPO11D^Y213F, M33-F1} [2] and P{hsp83-OsSPO11D^Y213F, F22-F2} [1]. n indicates the number of oocyte nuclei observed. P values from a chi test between the mei-W68 mus301 double mutants without any transgene and those with a plant SPO11 transgene. ***: P < 1E-03; **: 1E-03 <P < 1E-02; *: 1E-02 <P < 5E-02.

Figure 3

Aberrant meiotic disjunction of the X-chromosome in mei-W68 homozygous (dmspo11-deficient) Drosophila oocytes induced by plant SPO11s. (A) Normal chromosome segregation in meiosis. Black and white bold lines represent homologous chromosomes, such as X-chromosomes. Each pair of black or white bold lines indicates a parental chromosome duplicated by premeiotic DNA replication, a milestone of the start of meiosis, and each pair remains together until meiosis II. Black and white ovals represent their centromeres. All pairs of duplicated homologous chromosomes (left; indicated as two black and two white lines) are physically connected by at least a chiasma (indicated by a cross) formed by meiotic recombination, and then are segregated into sister cells (middle). After meiosis II, each recombinant or nonrecombinant chromosome is received by each of the four meiotic products (Egg). (B) In nondisjunction at meiosis I, one of the sister cells receives both X-chromosomes and the other receives no X-chromosome. In meiosis II, the former sister cell generates two diplo-X eggs, and the latter yields two nullo-X eggs. Thus, each non-disjunction event of the X-chromosome in meiosis I generates two diplo-X eggs and two nullo-X eggs. (C) If a DSB(s) is introduced into one of the X-chromosomes, but is not repaired at prophase of meiosis I, then the broken X-chromosome is lost. When random segregation follows in meiosis I, one of the sister cells receives the single X-chromosome and the other receives the duplicated X-chromosome. In meiosis II, the sister cell that received the single X-chromosome generates one nullo-X egg and one normal egg, and the other sister cell that received the duplicated X-chromosome generates two normal eggs. Thus, the X-chromosome aberration in meiosis generates more nullo-X eggs than diplo-X eggs.

Figure 4

Comparative analysis of SPO11-homologues. (A) Multiple sequence alignment of motifs I to V of SPO11. The bold tyrosine (Y) residue with an asterisk in motif I is the putative active center for covalent bonding to the 5' termini of cleaved DNA upon meiotic double-stranded cleavage. The triple asterisk in motif V indicates the conserved DXD sequence (see text). Underlined sequences were used for phylogenetic analyses. (B) Schematic diagrams of the genomic structures of OsSPO11 genes. Black boxes, coding regions; grey boxes, untranslated regions; solid lines, introns. Arrows indicate primers for real-time RT-PCR analyses. (C) Phylogenetic analyses of the amino acid sequences of motifs I-V. A neighbor-joining (NJ) tree was constructed using ClustalX ver. 2.0. Aga, Anopheles gambiae (EAA05541); Ago, Ashbya gossypii (AAS51945); Am, Apis mellifera (XP_001122679); Ap, Aeropyrum pernix (BAA79679); At1, Arabidopsis thaliana (CAB81544); At2, A. thaliana (CAB81545); At3, A. thaliana (ABI54341); Ca, Candida albicans (EAK95423); Dm, Drosophila melanogaster (AAC61735); Dr, Danio rerio (NP_991245); Dv, Drosophila virilis (EDW61133); Gg, Gallus gallus (XP_001232076); Gz, Gibberella zeae (XP_386125); Hs, Homo sapiens (AAD52562); Hw, Haloquadratum walsbyi (CAJ52765); Mj, Methanocaldococcus jannaschii (Q57815); Mm, Mus musculus (AAD52563); Nc, Neurospora crassa (CAB88597); Nv, Nematostella vectensis (EDO44118); Ol2, Ostreococcus lucimarinus (ABO99188); Ol3, O. lucimarinus (ABO95960); OsA, OsSPO11A; OsB, OsSPO11B; OsC, OsSPO11C; OsD, OsSPO11D; Pa, Podospora anserina (CAP73361); Pp1, Physcomitrella patens (EDQ80601); Pp2, P. patens (EDQ56207); Pp3, P. patens (EDQ71569); Ps, Pichia stipitis (XP_001384151); Pt1, Populus trichocarpa (EEE85181); Pt3, P. trichocarpa (EEE71999); Rc1, Ricinus communis (EEF39612); Rc3, R. communis (EEF37163); Sb1, Sorghum bicolor (EER93438); Sb2, S. bicolor (EES14573); Sb3, S. bicolor (EER95053); Sc, Saccharomyces cerevisiae (AAA65532); Sp, Schizosaccharomyces pombe (CAB11511); Ss, Sulfolobus shibatae (CAA71605); Tg, Taeniopygia guttata (XP_002195778); Tn, Thermoproteus neutrophilus (ACB40317); Xt, Xenopus tropicalis (AAH80352). The entire sequences of these SPO11 homologues are shown in Additional File 1 Figure S1.

Figure 5

Quantitative real-time RT-PCR analyses of the amounts of rice SPO11 mRNAs in cells during pollen-development and in organs. The data shown in the table, representing the accuracy of the data in this figure, were plotted in a 3-dimensional manner for a comparison of their expression profiles. Root, seven day-old root after germination; A0, the anthers in 0 cm DALL (stage A0); A3, the anthers in 3 cm DALL (stage A3); A5, the anthers in 5 cm DALL (stage A5); A10, the anthers in 10 cm DALL (stage A10); BB, before blooming. All data obtained were normalized by the amount of actin gene (AK072796) mRNA, and are expressed as relative mRNA levels, which are the average values with SD obtained from three independent samples.