PHS1 regulates meiotic recombination and homologous chromosome pairing by controlling the transport of RAD50 to the nucleus

Abstract

Recombination and pairing of homologous chromosomes are critical for bivalent formation in meiotic prophase. In many organisms, including yeast, mammals, and plants, pairing and recombination are intimately interconnected. The POOR HOMOLOGOUS SYNAPSIS1 (PHS1) gene acts in coordination of chromosome pairing and early recombination steps in plants, ensuring pairing fidelity and proper repair of meiotic DNA double-strand-breaks. In phs1 mutants, chromosomes exhibit early recombination defects and frequently associate with non-homologous partners, instead of pairing with their proper homologs. Here, we show that the product of the PHS1 gene is a cytoplasmic protein that functions by controlling transport of RAD50 from cytoplasm to the nucleus. RAD50 is a component of the MRN protein complex that processes meiotic double-strand-breaks to produce single-stranded DNA ends, which act in the homology search and recombination. We demonstrate that PHS1 plays the same role in homologous pairing in both Arabidopsis and maize, whose genomes differ dramatically in size and repetitive element content. This suggests that PHS1 affects pairing of the gene-rich fraction of the genome rather than preventing pairing between repetitive DNA elements. We propose that PHS1 is part of a system that regulates the progression of meiotic prophase by controlling entry of meiotic proteins into the nucleus. We also document that in phs1 mutants in Arabidopsis, centromeres interact before pairing commences along chromosome arms. Centromere coupling was previously observed in yeast and polyploid wheat while our data suggest that it may be a more common feature of meiosis.

Fig. 1.

The Arabidopsis thaliana PHS1 homolog. (A) The AtPHS1 gene. Blue triangle = position of the Ds insertion in the Atphs1–1 mutant. (B) Paring of 5S rRNA loci in a wild-type and Atphs1–1 mutant meiocytes. Only one 5S locus associated with a nonhomologous chromosome segment is shown in the mutant. Closeups are shown in insets. Images are flat projection of several consecutive optical sections but do not represent entire nuclei. (Scale bar, 5 μm.)

Fig. 2.

Centromere coupling in the Arabidopsis Atphs1 mutant. Each image is a flat projection across the entire nucleus. (Scale bar, 5 μm.)

Fig. 3.

Installation of chromosome axis and SC proteins on meiotic chromosomes in phs1 mutants in Arabidopsis and maize. (A) Installation of ASY1, a chromosome-axis associated protein in zygotene meiocytes. (B) Installation of ZYP1, the central element protein of the SC, and AFD1, a meiosis-specific cohesin, in pachytene. (Scale bar, 5 μm.)

Fig. 4.

The maize phs1-R148H mutation. (A) The maize Phs1 gene. Blue triangle = position of Mutator transposon insertion resulting in the original phs1-O mutation. Violet box = region used for the TILLING screen. Asterisk = positions of the phs1-R148H mutation. (B) Paring of 5S rRNA loci in a wild-type and phs1-R148H mutant meiocytes. Both loci in the mutant are associated with nonhomologous chromosome segments. Closeups of homologously paired 5S rRNA loci in the wild-type and one of the loci in the mutant are shown in insets. The other 5S locus in the mutant is visible in the lower right corner of the cell. Images are flat projection of several consecutive optical sections but do not represent entire nuclei. (Scale bar, 5 μm.)

Fig. 5.

Immunolocalization of RAD50 in wild-type and phs1 mutant meiocytes. Each image is a single optical section. (Scale bar, 5 μm.)

Fig. 6.

Immunolocalization of the PHS1 protein. The phs1 mutants pictured are Atphs1–1 in Arabidopsis and phs1-O in maize. Each image is a single optical section. (Scale bar, 10 μm.)

Fig. 7.

A model for meiotic prophase regulation by controlling transport of meiotic proteins into the nucleus. PHS1 regulates DSB resection by controlling nuclear import of RAD50. MPA1 regulates DSB repair by controlling nuclear import of RAD51 and MSH4.