AtPRD1 is required for meiotic double strand break formation in Arabidopsis thaliana

Abstract

The initiation of meiotic recombination by the formation of DNA double-strand breaks (DSBs) catalysed by the Spo11 protein is strongly evolutionary conserved. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation, but, unlike Spo11, few of these proteins seem to be conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we have isolated a new gene, AtPRD1, whose mutation affects meiosis in Arabidopsis thaliana. In Atprd1 mutants, meiotic recombination rates fall dramatically, early recombination markers (e.g., DMC1 foci) are absent, but meiosis progresses until achiasmatic univalents are formed. Besides, Atprd1 mutants suppress DSB repair defects of a large range of meiotic mutants, showing that AtPRD1 is involved in meiotic recombination and is required for meiotic DSB formation. Furthermore, we showed that AtPRD1 and AtSPO11-1 interact in a yeast two-hybrid assay, suggesting that AtPRD1 could be a partner of AtSPO11-1. Moreover, our study reveals similarity between AtPRD1 and the mammalian protein Mei1, suggesting that AtPRD1 could be a Mei1 functional homologue.

Figure 1

Sequence alignment of AtPRD1 and HsMei1 homologues. OsPRD1 for Oriza sativa; MtPRD1 for Medicago truncatula, PtPRD1 for Populus tricocarpa, PpPRD1 for Physcomitrella patens; HsMei1 for Homo sapiens; XtPRD1 for Xenopus tropicalis, and MmMei1 for Mus musculus homologues. The numbers indicate amino-acid position, identical amino acids are boxed in black, whereas similar amino acids are boxed in grey. The sequence interruption provoked by T-DNA insertions in the mutant alleles is indicated.

Figure 2

Male sporogenesis and meiosis of wild-type and the Atprd1-1 plants. (A–D) DIC microscopy of male meiocytes (A, C) or male meiosis products (tetrads of microspores, (B, D)). (A, B): wild type. (C, D) Atprd1-1. Scale bar, 10 μm. (E–J) DAPI staining of wild-type PMCs during meiosis. (E) Zygotene, (F) pachytene, (G) diakinesis, (H) metaphase I, (I) metaphase II, (J) anaphase II. (K–P) DAPI staining of mutant PMCs during meiosis. (K) Pachytene-like stage: homologues remain unsynapsed. (L) Diakinesis stage shows 10 univalents (bright dots correspond to centromeres). (M) Metaphase I with 10 univalents instead of five bivalents as seen in panel H. (N) Anaphase I shows a random segregation of the 10 univalents. (O) Some metaphase/anaphase I transitions show such stretched chromosomes. (P) Anaphase II showing six instead of four batches of chromosomes. (Q–V) Co-immunolocalisation of ASY1 (red) and ZYP1 (green) in wild-type (Q–S) and Atprd1-1 mutant (T–V) PMC. For each cell, each single labelling is shown as well as the overlay of both signals (merge). Scale bar, 10 μm.

Figure 3

Immunolocalisation of ASY1 (anti-ASY1) and AtDMC1 (anti-DMC1) in wild type and asynaptic mutants. (A–L) Anti-AtDMC1 labelling (green) in wild type during early leptotene (A–C), late leptotene/early zygotene (D–F), late zygotene (G–I) and pachytene (J–L) stages. No AtDMC1 foci can be seen above background during prophase I of Atdmc1 (M–O), Atspo11-1 (P–R), Atprd1-1 (S–U) and Atprd1-2 (V–X) PMC. For a single antibody, all photographs were taken with the same exposition, but enhanced anti-DMC1 signal in the different mutant backgrounds is provided as Supplementary data. Scale bar, 10 μm.

Figure 4

DAPI staining of male meiocytes during anaphase I. Wild-type PMC show an equal repartition of chromosomes during anaphase I (A). In the asynaptic mutants Atdmc1 (C), Atspo11-1-1 (E) and Atprd1-1 (G), random segregation of the 10 univalents is observed at the same stage. An Atrad51-1 mutant shows a severe chromosome fragmentation in the first meiotic division (B), which is not abolished in Atdmc1Atrad51 (D). By contrast, fragmentation is abolished in Atspo11-1Atrad51-1 (F) and Atprd1-1Atrad51-1 (H). Scale bar, 10 μm.

Figure 5

AtPRD1 interacts with AtSPO11-1 in a yeast two-hybrid assay. AtPRD1 (1–802) and AtPRD1 (755–1330) correspond to the N-terminal and the C-terminal part of AtPRD1, respectively. AgT encodes for the SV40 large T-antigen. The AH109 strain was co-transformed with the constructs indicated, carrying a binding domain (BD) and an activation domain (AD), and grown on synthetic drop-out (SD) media lacking the aa leucine and tryptophan (SD-LW) or leucine, tryptophan, adenine and histidine (SD-LWAH). Serial dilutions of diploid strains were performed before spotting on media. Yeast containing both vectors grew on SD-LW; positive interactions appear as white spot on SD-LWAH.