FIGL1 and its novel partner FLIP form a conserved complex that regulates homologous recombination.

Homologous recombination is central to repair DNA double-strand breaks, either accidently arising in mitotic cells or in a programed manner at meiosis. Crossovers resulting from the repair of meiotic breaks are essential for proper chromosome segregation and increase genetic diversity of the progeny. However, mechanisms regulating crossover formation remain elusive. Here, we identified through genetic and protein-protein interaction screens FIDGETIN-LIKE-1 INTERACTING PROTEIN (FLIP) as a new partner of the previously characterized anti-crossover factor FIDGETIN-LIKE-1 (FIGL1) in Arabidopsis thaliana. We showed that FLIP limits meiotic crossover together with FIGL1. Further, FLIP and FIGL1 form a protein complex conserved from Arabidopsis to human. FIGL1 interacts with the recombinases RAD51 and DMC1, the enzymes that catalyze the DNA strand exchange step of homologous recombination. Arabidopsis flip mutants recapitulate the figl1 phenotype, with enhanced meiotic recombination associated with change in counts of DMC1 and RAD51 foci. Our data thus suggests that FLIP and FIGL1 form a conserved complex that regulates the crucial step of strand invasion in homologous recombination

FIGL1 and its novel partner FLIP form a conserved complex that regulates homologous recombination - Fig 4

<p>Mutation in <i>FLIP</i> restores crossover formation in <i>zmm</i> mutants: A. Schematic representation of the <i>FLIP</i> gene (Fidgetin-Like-1 Interacting Protein). Exons appear as blue boxes. The red line and red triangle indicate the missense mutation in <i>flip-1</i> and the <i>flip-2</i> T-DNA insertion, respectively. B. Average number of bivalents (blue) and pairs of univalents (red) per male meiocyte at metaphase I (Fig 4C). Light blue represents rod shaped bivalents indicating that one chromosome arm has at least one CO, and one arm has no CO. Dark blue represents ring shaped bivalent indicating the presence of at least one CO on both chromosome arms. The number of cells analyzed for each genotype is indicated in brackets. C. DAPI staining of Chromosome spreads of male meiocytes at metaphase I. Scale bars 10μm. D. Fertility measured as number of seeds per fruit. Each dot represents a plant; at least 10 fruits per plant were analyzed.</p

DMC1 foci in wild type <i>figl1</i>, <i>flip</i> and <i>figl1 flip</i>.

<p>A. Triple immunolocalization of ASY1 (red), ZYP1 (blue) and DMC1 (green) on meiotic chromosome spreads. Merged pictures are shown. Partial and full ZYP1 polymerization defines the zygotene and pachytene stages, respectively. Scale bars 10μm. B. Quantification of DMC1 foci at leptotene, zygotene and pachytene in wild type, <i>figl1</i>, <i>flip</i> and <i>figl1 flip</i>. Each dot represents an individual cell and bars indicate the mean. P values are the results of Fisher's LSD tests.</p

<i>FLIP</i> limits Class II COs.

<p>A. Interference ratio is the ratio of the genetic size in an interval with CO in an adjacent interval divided by the genetic size of the same interval without CO in the adjacent interval. This ratio provides an estimate of the strength of CO interference. IR close to 0 means strong interference; Interference ratio = 1 (purple line) indicates that interference is absent. The test of absence of interference is shown in purple (n.s <i>p</i> > 0.05; ** <i>p</i> < 0.01; *** <i>p</i> < 0.001). Comparison of Interference ratio between the genotypes wild type and mutants is indicated in black (n.s <i>p</i> > 0.05; * <i>p</i> < 0.05 ** <i>p</i> < 0.01; *** <i>p</i> < 0.001). B. Chromosome spreads of male meiocytes at metaphase I and anaphase I. Scale bars 10μm.</p

Yeast-two-hybrid experiments testing interactions between Arabidopsis FIGL1, FLIP, RAD51 and DMC1 proteins.

<p>Proteins of interest were fused with Gal4 DNA binding domain (BD, left) and with Gal4 activation domain (AD, top), respectively, and co-expressed in yeast cells. Full-length and truncated protein are schematically represented. For each combination, serial dilutions of yeast cells were spotted on non-selective medium (-LW), moderately selective media (-LWH) and more selective media (-LWHA). ++: Growth on both LWH and LWHA, interpreted as strong interaction. +: Growth on LWH and not on LWHA, interpreted as weak interaction. +*: Growth on LWH but cannot be interpreted as positive interaction because of auto-activation of one of the construct.—: Growth on neither LWH nor LWHA. n.d. Not determined. Pictures of yeasts are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007317#pgen.1007317.s001" target="_blank">S1 Fig</a>.</p

<i>FLIP</i> genetically interacts with <i>SDS</i>.

<p>A. Immunostaining of DMC1 (green) and the chromosome axis protein ASY1 (red) on leptotene/zygotene meiotic chromosome spreads. B. Quantification of DMC1 foci at leptotene/zygotene in <i>sds</i>, <i>sds figl1</i> and <i>sds flip</i>. Each dot represents an individual cell and bars indicate the mean. C. Co-immunolocalization of ASY1 (red) and ZYP1 (green), which mark respectively chromosome axes and synapsed regions. Synapsis was partially restored in <i>sds flip</i> compared to single mutant <i>sds</i>. Scale bars 10μm. D. DAPI staining of chromosome spreads of male meiocytes at metaphase I and anaphase I. Scale bars 10μm. E. Fertility measured as number of seeds per fruit. Each dot represents a plant; at least 12 fruits per plant were analyzed. P values are the results of Fisher's LSD tests.</p

Phylogenetic tree depicting the evolutionary conservation of FLIP, FIGL1, RAD51 and DMC1 orthologs in a range of eukaryotic species.

<p>FLIP, FIGL1, DMC1 and RAD51 are presented as dots in green, red, blue and turquoise color, respectively. Gene accession numbers are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007317#pgen.1007317.s005" target="_blank">S2 Table</a>. A version of this figure with a larger number of species can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007317#pgen.1007317.s006" target="_blank">S3 Fig</a> and as an interactive tree at <a href="http://itol.embl.de/tree/132166555992271498216301" target="_blank">http://itol.embl.de/tree/132166555992271498216301</a>.</p

Tandem affinity purification using FIGL1 and FLIP as baits.

<p>Two replicates of Tandem affinity purifications (TAP1 and TAP2) followed by mass spectrometry were performed using either FIGL1 (A) or FLIP (B) as a bait over-expressed in cultured cells. For filtering specific and false positive interactors, refer to Materials and Methods and [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007317#pgen.1007317.ref036" target="_blank">36</a>]. The number of peptides and the fraction of the protein covered are indicated for each hit. Raw data are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007317#pgen.1007317.s004" target="_blank">S1 Table</a>.</p

Yeast-two-hybrid experiments testing interactions between human FIGNL1, FLIP, RAD51 and DMC1 proteins.

<p>Proteins of interest were fused with Gal4 DNA binding domain (BD, left) and with Gal4 activation domain (AD, top), respectively, and co-expressed in yeast cells. Full-length and truncated protein are schematically represented. For each combination, serial dilutions of yeast cells were spotted on non-selective medium (-LW), moderately selective media (-LWH) and more selective media (-LWHA). ++: Growth on both LWH and LWHA, interpreted as strong interaction. +: Growth on LWH and not on LWHA, interpreted as weak interaction. +*: Growth on LWH but cannot be interpreted as positive interaction because of auto-activation of one of the construct. Growth on neither LWH nor LWHA. n.d. Not determined. Pictures of yeasts are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007317#pgen.1007317.s002" target="_blank">S2 Fig</a>.</p

Unleashing meiotic crossovers in crops

Improved plant varieties are important in our attempts to face the challenges of a growing human population and limited planet resources. Plant breeding relies on meiotic crossovers to combine favourable alleles into elite varieties1. However, meiotic crossovers are relatively rare, typically one to three per chromosome2, limiting the efficiency of the breeding process and related activities such as genetic mapping. Several genes that limit meiotic recombination were identified in the model species Arabidopsis thaliana2. Mutation of these genes in Arabidopsis induces a large increase in crossover frequency. However, it remained to be demonstrated whether crossovers could also be increased in crop species hybrids. We explored the effects of mutating the orthologues of FANCM3, RECQ44 or FIGL15 on recombination in three distant crop species, rice (Oryza sativa), pea (Pisum sativum) and tomato (Solanum lycopersicum). We found that the single recq4 mutation increases crossovers about three-fold in these crops, suggesting that manipulating RECQ4 may be a universal tool for increasing recombination in plants. Enhanced recombination could be used with other state-of-the-art technologies such as genomic selection, genome editing or speed breeding6 to enhance the pace and efficiency of plant improvement.Université Fédérale de Toulous