A spontaneous mutation in MutL-Homolog 3 (HvMLH3) affects synapsis and crossover resolution in the barley desynaptic mutant des10

Although meiosis is evolutionarily conserved, many of the underlying mechanisms show species-specific differences. These are poorly understood in large genome plant species such as barley (Hordeum vulgare) where meiotic recombination is very heavily skewed to the ends of chromosomes. The characterization of mutant lines can help elucidate how recombination is controlled. We used a combination of genetic segregation analysis, cytogenetics, immunocytology and 3D imaging to genetically map and characterize the barley meiotic mutant DESYNAPTIC 10 (des10). We identified a spontaneous exonic deletion in the orthologue of MutL-Homolog 3 (HvMlh3) as the causal lesion. Compared with wild-type, des10 mutants exhibit reduced recombination and fewer chiasmata, resulting in the loss of obligate crossovers and leading to chromosome mis-segregation. Using 3D structured illumination microscopy (3D-SIM), we observed that normal synapsis progression was also disrupted in des10, a phenotype that was not evident with standard confocal microscopy and that has not been reported with Mlh3 knockout mutants in Arabidopsis. Our data provide new insights on the interplay between synapsis and recombination in barley and highlight the need for detailed studies of meiosis in nonmodel species. This study also confirms the importance of early stages of prophase I for the control of recombination in large genome cereals.Isabelle Colas, Malcolm Macaulay, James D. Higgins, Dylan Phillips, Abdellah Barakate ... Robbie Waugh ... et al

The plant Rad50-Mre11 protein complex.

International audienceThe Rad50-Mre11-Xrs2/Nbs1 protein complex plays critical roles in cellular processes involving DNA ends. This complex is implicated in DNA recombination and replication, meiosis, telomere maintenance and cellular DNA damage responses. The Rad50 and Mre11 proteins are essential for viability in animals, although not in yeast. We have prepared antibodies to the Rad50 protein of the model plant Arabidopsis thaliana which recognize a 175 kDa protein in wild-type Arabidopsis protein extracts. Furthermore, we report here demonstration of the existence of the Rad50-Mre11 complex by co-immunoprecipitation of the Rad50 and Mre11 proteins from the plant cell extracts

Ku80 plays a role in non-homologous recombination but is not required for T-DNA integration in Arabidopsis.

Chromosomal breaks are repaired by homologous recombination (HR) or non-homologous end joining (NHEJ) mechanisms. The Ku70/Ku80 heterodimer binds DNA ends and plays roles in NHEJ and telomere maintenance in organisms ranging from yeast to humans. We have previously identified a ku80 mutant of the model plant Arabidopsis thaliana and shown the role of Ku80 in telomere homeostasis in plant cells. We show here that this mutant is hypersensitive to the DNA-damaging agent methyl methane sulphonate and has a reduced capacity to carry out NHEJ recombination. To understand the interplay between HR and NHEJ in plants, we measured HR in the absence of Ku80. We find that the frequency of intrachromosomal HR is not affected by the absence of Ku80. Previous work has clearly implicated the Ku heterodimer in Agrobacterium-mediated T-DNA transformation of yeast. Surprisingly, ku80 mutant plants show no defect in the efficiency of T-DNA transformation of plants with Agrobacterium, showing that an alternative pathway must exist in plants

Ku80 plays a role in non-homologous recombination but is not required for T-DNA integration in Arabidopsis.

International audienceChromosomal breaks are repaired by homologous recombination (HR) or non-homologous end joining (NHEJ) mechanisms. The Ku70/Ku80 heterodimer binds DNA ends and plays roles in NHEJ and telomere maintenance in organisms ranging from yeast to humans. We have previously identified a ku80 mutant of the model plant Arabidopsis thaliana and shown the role of Ku80 in telomere homeostasis in plant cells. We show here that this mutant is hypersensitive to the DNA-damaging agent methyl methane sulphonate and has a reduced capacity to carry out NHEJ recombination. To understand the interplay between HR and NHEJ in plants, we measured HR in the absence of Ku80. We find that the frequency of intrachromosomal HR is not affected by the absence of Ku80. Previous work has clearly implicated the Ku heterodimer in Agrobacterium-mediated T-DNA transformation of yeast. Surprisingly, ku80 mutant plants show no defect in the efficiency of T-DNA transformation of plants with Agrobacterium, showing that an alternative pathway must exist in plants

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

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