Wolbachia utilize host actin for efficient maternal transmission in Drosophila melanogaster.

Wolbachia pipientis is a ubiquitous, maternally transmitted bacterium that infects the germline of insect hosts. Estimates are that Wolbachia infect nearly 40% of insect species on the planet, making it the most prevalent infection on Earth. The bacterium, infamous for the reproductive phenotypes it induces in arthropod hosts, has risen to recent prominence due to its use in vector control. Wolbachia infection prevents the colonization of vectors by RNA viruses, including Drosophila C virus and important human pathogens such as Dengue and Chikungunya. Here we present data indicating that Wolbachia utilize the host actin cytoskeleton during oogenesis for persistence within and transmission between Drosophila melanogaster generations. We show that phenotypically wild type flies heterozygous for cytoskeletal mutations in Drosophila profilin (chic(221/+) and chic(1320/+)) or villin (qua(6-396/+)) either clear a Wolbachia infection, or result in significantly reduced infection levels. This reduction of Wolbachia is supported by PCR evidence, Western blot results and cytological examination. This phenotype is unlikely to be the result of maternal loading defects, defects in oocyte polarization, or germline stem cell proliferation, as the flies are phenotypically wild type in egg size, shape, and number. Importantly, however, heterozygous mutant flies exhibit decreased total G-actin in the ovary, compared to control flies and chic(221) heterozygous mutants exhibit decreased expression of profilin. Additionally, RNAi knockdown of profilin during development decreases Wolbachia titers. We analyze evidence in support of alternative theories to explain this Wolbachia phenotype and conclude that our results support the hypothesis that Wolbachia utilize the actin skeleton for efficient transmission and maintenance within Drosophila.

A mutation in the FHA domain of Coprinus cinereus Nbs1 Leads to Spo11-independent meiotic recombination and chromosome segregation.

Nbs1, a core component of the Mre11-Rad50-Nbs1 complex, plays an essential role in the cellular response to DNA double-strand breaks (DSBs) and poorly understood roles in meiosis. We used the basidiomycete Coprinus cinereus to examine the meiotic roles of Nbs1. We identified the C. cinereus nbs1 gene and demonstrated that it corresponds to a complementation group previously known as rad3. One allele, nbs1-2, harbors a point mutation in the Nbs1 FHA domain and has a mild spore viability defect, increased frequency of meiosis I nondisjunction, and an altered crossover distribution. The nbs1-2 strain enters meiosis with increased levels of phosphorylated H2AX, which we hypothesize represent unrepaired DSBs formed during premeiotic replication. In nbs1-2, there is no apparent induction of Spo11-dependent DSBs during prophase. We propose that replication-dependent DSBs, resulting from defective replication fork protection and processing by the Mre11-Rad50-Nbs1 complex, are competent to form meiotic crossovers in C. cinereus, and that these crossovers lead to high levels of faithful chromosome segregation. In addition, although crossover distribution is altered in nbs1-2, the majority of crossovers were found in subtelomeric regions, as in wild-type. Therefore, the location of crossovers in C. cinereus is maintained when DSBs are induced via a Spo11-independent mechanism.

Analysis of the Basidiomycete Coprinopsis cinerea reveals conservation of the core meiotic expression program over half a billion years of evolution.

Coprinopsis cinerea (also known as Coprinus cinereus) is a multicellular basidiomycete mushroom particularly suited to the study of meiosis due to its synchronous meiotic development and prolonged prophase. We examined the 15-hour meiotic transcriptional program of C. cinerea, encompassing time points prior to haploid nuclear fusion though tetrad formation, using a 70-mer oligonucleotide microarray. As with other organisms, a large proportion (∼20%) of genes are differentially regulated during this developmental process, with successive waves of transcription apparent in nine transcriptional clusters, including one enriched for meiotic functions. C. cinerea and the fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe diverged ∼500-900 million years ago, permitting a comparison of transcriptional programs across a broad evolutionary time scale. Previous studies of S. cerevisiae and S. pombe compared genes that were induced upon entry into meiosis; inclusion of C. cinerea data indicates that meiotic genes are more conserved in their patterns of induction across species than genes not known to be meiotic. In addition, we found that meiotic genes are significantly more conserved in their transcript profiles than genes not known to be meiotic, which indicates a remarkable conservation of the meiotic process across evolutionarily distant organisms. Overall, meiotic function genes are more conserved in both induction and transcript profile than genes not known to be meiotic. However, of 50 meiotic function genes that were co-induced in all three species, 41 transcript profiles were well-correlated in at least two of the three species, but only a single gene (rad50) exhibited coordinated induction and well-correlated transcript profiles in all three species, indicating that co-induction does not necessarily predict correlated expression or vice versa. Differences may reflect differences in meiotic mechanisms or new roles for paralogs. Similarities in induction, transcript profiles, or both, should contribute to gene discovery for orthologs without currently characterized meiotic roles.

Meiotic localization of Mre11 and Rad50 in wild type, spo11-1, and MRN complex mutants of Coprinus cinereus.

The Mre11-Rad50-Nbs1 (MRN) complex is required for numerous cellular processes that involve interactions with DNA double-strand breaks. For the majority of these processes, the MRN complex is thought to act as a unit, with each protein aiding the activity of the others. We have examined the relationship between Mre11 and Rad50 during meiosis in the basidiomycete Coprinus cinereus (Coprinopsis cinerea), investigating to what extent activities of Mre11 and Rad50 are interdependent. We showed that mre11-1 is epistatic to rad50-1 with respect to the time of meiotic arrest, indicating that Mre11 activity facilitates the diffuse diplotene arrest of rad50 mutants. Anti-Mre11 and anti-Rad50 antibodies were used to examine MRN complex localization in a wild-type strain and in spo11, mre11, and rad50 mutants. In wild type, numbers of Mre11 and Rad50 foci peaked at time points corresponding to leptotene and early zygotene. In the spo11-1 mutant, which is defective in meiotic double-strand break formation, foci accumulated throughout prophase I. Of seven MRN mutants examined, only two rad50 strains exhibited Mre11 and Rad50 foci that localized to chromatin, although Mre11 protein was found in the cell for all of them. Analysis of predicted mutant structures showed that stable localization of Mre11 and Rad50 does not depend upon a wild-type hook-proximal coiled coil, but does require the presence of the Rad50 ATPase/adenylate cyclase domains. We found that Mre11 and Rad50 were interdependent for binding to meiotic chromosomes. However, the majority of foci observed apparently contained only one of the two proteins. Independent Mre11 and Rad50 foci might indicate disassociation of the complex during meiosis or could reflect independent structural roles for the two proteins in meiotic chromatin.

Coprinus cinereus rad50 mutants reveal an essential structural role for Rad50 in axial element and synaptonemal complex formation, homolog pairing and meiotic recombination.

The Mre11/Rad50/Nbs1 (MRN) complex is required for eukaryotic DNA double-strand break (DSB) repair and meiotic recombination. We cloned the Coprinus cinereus rad50 gene and showed that it corresponds to the complementation group previously named rad12, identified mutations in 15 rad50 alleles, and mapped two of the mutations onto molecular models of Rad50 structure. We found that C. cinereus rad50 and mre11 mutants arrest in meiosis and that this arrest is Spo11 dependent. In addition, some rad50 alleles form inducible, Spo11-dependent Rad51 foci and therefore must be forming meiotic DSBs. Thus, we think it likely that arrest in both mre11-1 and the collection of rad50 mutants is the result of unrepaired or improperly processed DSBs in the genome and that Rad50 and Mre11 are dispensable in C. cinereus for DSB formation, but required for appropriate DSB processing. We found that the ability of rad50 mutant strains to form Rad51 foci correlates with their ability to promote synaptonemal complex formation and with levels of stable meiotic pairing and that partial pairing, recombination initiation, and synapsis occur in the absence of wild-type Rad50 catalytic domains. Examination of single- and double-mutant strains showed that a spo11 mutation that prevents DSB formation enhances axial element (AE) formation for rad50-4, an allele predicted to encode a protein with intact hook region and hook-proximal coiled coils, but not for rad50-1, an allele predicted to encode a severely truncated protein, or for rad50-5, which encodes a protein whose hook-proximal coiled-coil region is disrupted. Therefore, Rad50 has an essential structural role in the formation of AEs, separate from the DSB-processing activity of the MRN complex.