The dietary specialist fruit fly Drosophila sechellia has evolved to specialize on the toxic fruit of its host plant Morinda citrifolia. Toxicity of Morinda fruit is primarily due to high levels of octanoic acid (OA). Using RNA interference (RNAi), prior work found that knockdown of Osiris family genes Osiris 6 (Osi6), Osi7, and Osi8 led to increased susceptibility to OA in adult D. melanogaster flies, likely representing genes underlying a Quantitative Trait Locus (QTL) for OA resistance in D. sechellia. While genes in this major effect locus are beginning to be revealed, prior work has shown at least five regions of the genome contribute to OA resistance. Here, we identify new candidate OA resistance genes by performing differential gene expression analysis using RNA-sequencing (RNA-seq) on control and OA-exposed D. sechellia flies. We found 104 significantly differentially expressed genes with annotated orthologs in D. melanogaster, including six Osiris gene family members, consistent with previous functional studies and gene expression analyses. Gene ontology (GO) term enrichment showed significant enrichment for cuticle development in upregulated genes and significant enrichment of immune and defense responses in downregulated genes, suggesting important aspects of the physiology of D. sechellia that may play a role in OA resistance. In addition, we identified five candidate OA resistance genes that potentially underlie QTL peaks outside of the major effect region, representing promising new candidate genes for future functional studies.

Accurate chromosome segregation during meiosis relies on the prior establishment of at least one crossover recombination event between homologous chromosomes. Most meiotic recombination intermediates that give rise to interhomolog crossovers are embedded within a hallmark chromosomal structure called the synaptonemal complex (SC), but the mechanisms that coordinate the processes of SC assembly (synapsis) and crossover recombination remain poorly understood. Among known structural components of the budding yeast SC, the Zip1 protein is unique for its independent role in promoting crossover recombination; Zip1 is specifically required for the large subset of crossovers that also rely on the meiosis-specific MutSγ complex. Here we report that adjacent regions within Zip1’s N terminus encompass its crossover and synapsis functions. We previously showed that deletion of Zip1 residues 21–163 abolishes tripartite SC assembly and prevents robust SUMOylation of the SC central element component, Ecm11, but allows excess MutSγ crossover recombination. We find the reciprocal phenotype when Zip1 residues 2–9 or 10–14 are deleted; in these mutants SC assembles and Ecm11 is hyperSUMOylated, but MutSγ crossovers are strongly diminished. Interestingly, Zip1 residues 2–9 or 2–14 are required for the normal localization of Zip3, a putative E3 SUMO ligase and pro-MutSγ crossover factor, to Zip1 polycomplex structures and to recombination initiation sites. By contrast, deletion of Zip1 residues 15–20 does not detectably prevent Zip3’s localization at Zip1 polycomplex and supports some MutSγ crossing over but prevents normal SC assembly and Ecm11 SUMOylation. Our results highlight distinct N terminal regions that are differentially critical for Zip1’s roles in crossing over and SC assembly; we speculate that the adjacency of these regions enables Zip1 to serve as a liaison, facilitating crosstalk between the two processes by bringing crossover recombination and synapsis factors within close proximity of one another.

During meiosis a large subset of interhomolog recombination repair intermediates form within the physical context of the synaptonemal complex (SC), a protein-rich structure assembled at the interface of aligned homologous chromosomes. However, the functional relationship between SC structure and homologous recombination remains poorly defined. In prior work we determined that tripartite SC is dispensable for recombination in S. cerevisiae; SC central element proteins Ecm11 and Gmc2 instead limit the number of recombination events. Here we report that while dispensable for recombination per se, SC central element proteins influence the processing of interhomolog recombination intermediates in a manner that minimizes errors in mismatch correction. Failure to correct mis-paired bases within heteroduplex at meiotic recombination sites leads to genotypically sectored colonies (post meiotic segregation events) arising from mitotic proliferation of mismatch-containing spores. We discovered an increase in post-meiotic segregation at the THR1 locus in cells lacking Ecm11 or Gmc2, or in the SC-deficient but crossover recombination-proficient zip1[Δ21-163] mutant. High-throughput sequencing of octad meiotic products revealed a genome-wide increase in recombination events with uncorrected mismatches in ecm11 mutants relative to wild type. Meiotic cells missing Ecm11 also display longer gene conversion tracts, but tract length alone does not account for the higher frequency of uncorrected mismatches. Interestingly, the per-nucleotide mismatch frequency is elevated in ecm11 mutants when analyzing all gene conversion tracts, but is similar between wild type and ecm11 if one considers only those events with uncorrected mismatches. Our data suggest that a subset of recombination events is similarly susceptible to mismatch repair errors in both wild type and ecm11 strains, but in ecm11 mutants many more recombination events fall into this inefficient repair category. Finally, we observe elevated post-meiotic segregation at THR1 in mutants with a dual deficiency in MutSγ-mediated crossover recombination and SC assembly, but not in the mlh3 mutant, which lacks MutSγ crossovers but has abundant SC. We propose that SC structure promotes efficient mismatch repair of joint molecule recombination intermediates resolved via both MutSγ-associated and MutSγ-independent pathways, and is the molecular basis for elevated post-meiotic segregation in both MutSγ crossover-proficient (ecm11, gmc2) and MutSγ crossover-deficient (msh4, zip3) strains.

Accurate chromosome segregation during meiosis relies on the prior establishment of at least one crossover recombination event between homologous chromosomes, which is often associated with the meiosis-specific MutSg complex. The recombination intermediates that give rise to MutSg interhomolog crossovers are embedded within a hallmark meiotic prophase structure called the synaptonemal complex (SC), but the mechanisms that coordinate the processes of SC assembly (synapsis) and crossover recombination remain poorly understood. Among known central region building blocks of the budding yeast SC, the Zip1 protein is unique for its SCindependent role in promoting MutSg crossovers. Here we report that adjacent regions within Zip1's unstructured N terminus encompass its crossover and SC assembly functions. We previously showed that deletion of Zip1 residues 21-163 abolishes tripartite SC assembly and prevents the robust SUMOylation of the SC central element component, Ecm11, but allows excess MutSg crossover recombination. We find the reciprocal phenotype when Zip1 residues 2-9 or 10-14 are deleted; in these mutants SC assembles and Ecm11 is hyperSUMOylated, but MutSg crossovers are strongly diminished. Interestingly, Zip1 residues 2-9 or 2-14 are required for the normal localization of Zip3, a putative E3 SUMO ligase and pro-MutSg crossover factor, to Zip1 polycomplex structures and to recombination initiation sites. By contrast, deletion of Zip1 residues 15-20 does not detectably prevent Zip3's localization at Zip1 polycomplex and supports some MutSg crossing over but prevents normal SC assembly and robust Ecm11SUMOylation. These results highlight distinct N terminal regions that are differentially critical for Zip1's roles in crossover recombination and SC assembly; we speculate that the adjacency of these regions enables Zip1 to serve as a liaison, facilitating crosstalk between the two processes by bringing crossover recombination and synapsis factors in close proximity to one another.

A large subset of meiotic recombination intermediates form within the physical context of synaptonemal complex (SC), but the functional relationship between SC structure and homologous recombination remains obscure. Our prior analysis of strains deficient for SC central element proteins demonstrated that tripartite SC is dispensable for interhomolog recombination in S. cerevisiae. Here we report that while dispensable for recombination per se, SC proteins promote efficient mismatch repair at interhomolog recombination sites. Failure to repair mismatches within heteroduplex-containing meiotic recombination intermediates leads to genotypically sectored colonies (post meiotic segregation events). We discovered increased post-meiotic segregation at THR1 in cells lacking Ecm11 or Gmc2, or in the SC-deficient but recombination-proficient zip1[Δ21-163] mutant. High-throughput sequencing of octad meiotic products furthermore revealed a genome-wide increase in recombination events with unrepaired mismatches in ecm11 mutants relative to wild type. Meiotic cells missing Ecm11 display longer gene conversion tracts, but tract length alone does not account for the higher frequency of unrepaired mismatches. Interestingly, the per-nucleotide mismatch frequency is elevated in ecm11 when analyzing all gene conversion tracts, but is similar between wild type and ecm11 if considering only those events with unrepaired mismatches. Thus, in both wild type and ecm11 strains a subset of recombination events is susceptible to a similar degree of inefficient mismatch repair, but in ecm11 mutants a larger fraction of events fall into this inefficient repair category. Finally, we observe elevated post-meiotic segregation at THR1 in mutants with a dual deficiency in MutSγ crossover recombination and SC assembly, but not in the mlh3 mutant, which lacks MutSγ crossovers but has abundant SC. We propose that SC structure promotes efficient mismatch repair of joint molecule recombination intermediates, and that absence of SC is the molecular basis for elevated post-meiotic segregation in both MutSγ crossover-proficient (ecm11, gmc2) and MutSγ crossover-deficient (msh4, zip3) strains.