Many bacteria live as intracellular symbionts, causing persistent infections within insects. One extraordinarily common infection is that of Wolbachia pipientis, which infects 40% of insect species and induces reproductive effects. The bacteria are passed from generation to generation both vertically (through the oocyte) and horizontally (by environmental transmission). Maintenance of the infection within Drosophila melanogaster is sensitive to the regulation of actin, as Wolbachia inefficiently colonizes strains hemizygous for the profilin or villin genes. Therefore, we hypothesized that Wolbachia must depend on the host actin cytoskeleton. In this study, we identify and characterize a Wolbachia protein (WD0830) that is predicted to be secreted by the bacterial parasite. Expression of WD0830 in a model eukaryote (the yeast Saccharomyces cerevisiae) induces a growth defect associated with the appearance of aberrant, filamentous structures which colocalize with rhodamine-phalloidin-stained actin. Purified WD0830 bundles actin in vitro and cosediments with actin filaments, suggesting a direct interaction of the two proteins. We characterized the expression of WD0830 throughout Drosophila development and found it to be upregulated in third-instar larvae, peaking in early pupation, during the critical formation of adult tissues, including the reproductive system. In transgenic flies, heterologously expressed WD0830 localizes to the developing oocyte. Additionally, overexpression of WD0830 results in increased Wolbachia titers in whole flies, in stage 9 and 10 oocytes, and in embryos, compared to controls, suggesting that the protein may facilitate Wolbachia’s replication or transmission. Therefore, this candidate secreted effector may play a role in Wolbachia’s infection of and persistence within host niches.

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 (chic221/+ and chic1320/+) or villin (qua6-396/+) 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 chic221 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.

Only limited information is available concerning the effects of low-shear modeled microgravity (LSMMG) on cell function and morphology. We examined the behavior of Saccharomyces cerevisiae grown in a high-aspectratio vessel, which simulates the low-shear and microgravity conditions encountered in spaceflight. With the exception of a shortened lag phase (90 min less than controls; P < 0.05), yeast cells grown under LSMMG conditions did not differ in growth rate, size, shape, or viability from the controls but did differ in the establishment of polarity as exhibited by aberrant (random) budding compared to the usual bipolar pattern of controls. The aberrant budding was accompanied by an increased tendency of cells to clump, as indicated by aggregates containing five or more cells. We also found significant changes (greater than or equal to twofold) in the expression of genes associated with the establishment of polarity (BUD5), bipolar budding (RAX1, RAX2, and BUD25), and cell separation (DSE1, DSE2, and EGT2). Thus, low-shear environments may significantly alter yeast gene expression and phenotype as well as evolutionary conserved cellular functions such as polarization. The results provide a paradigm for understanding polarity-dependent cell responses to microgravity ranging from pathogenesis in fungi to the immune response in mammals.Understanding the effects of microgravity on the functions of eukaryotic and prokaryotic cells is important for the safety and health of astronauts during spaceflight. Mechanisms through which cells sense gravity and adapt in its absence address fundamental questions regarding the evolution of terrestrial life and general mechanosensory responses in living organisms. However, spaceflight studies are technically difficult and expensive. Fortunately, low-shear fluid dynamics (referred to as simulated microgravity or low-shear modeled microgravity [LSMMG]), a key aspect of the microgravity environment, can be modeled in specialized ground-based bioreactors. One such bioreactor called the high-aspect-ratio vessel (HARV) was used in this study (49). The HARV is a constantly rotating culture vessel that is typically used for suspension culture and tissue growth (21,29,41). Yeast cells are inoculated into the vessel, and all air bubbles are removed. The cells suspended within the culture medium do not settle but, rather, revolve around a horizontal axis, continuously falling through the fluid at 1 ϫ g under terminal velocity conditions. The cells are not agitated but move sufficiently enough in the HARV to allow for the ample exchange of dissolved gases through a permeable membrane in the device and the exchange of nutrients and wastes in the medium within the vessel. In addition, the system randomizes the unidirectional pull of gravity and minimizes turbulence (shear), altering the net effect of gravity on the cells by creating a state of "functional weightlessness" (21, 29, 41). The bioreactor does not remove the force due to gravity, but, rather, the gravitational vector present in the...

All plants studied in natural ecosystems are symbiotic with fungi (1), which obtain nutrients while either positively, negatively, or neutrally affecting host fitness (2). Plant adaptation to selective pressures is considered to be regulated by the plant genome (3). To test whether mutualistic fungi contribute to plant adaptation, we collected 200 Dichanthelium lanuginosum plants from geothermal soils at 10 sites in Lassen Volcanic (LVNP) and Yellowstone ( YNP) National Parks. These soils have annual temperature fluctuations ranging from about 20°to 50°C (4 ).Plants and their roots were removed and assessed for fungal colonization (5). A fungal endophyte was isolated from the roots, crowns, leaves, and seed coats of all plants collected. Cultures established from single

Wolbachia pipientis is an intracellular symbiont of arthropods well known for the reproductive manipulations induced in the host and, more recently, for the ability of Wolbachia to block virus replication in insect vectors. Since Wolbachia cannot yet be genetically manipulated, and due to the constraints imposed when working with an intracellular symbiont, little is known about mechanisms used by Wolbachia for host interaction. Here we employed a bioinformatics pipeline and identified 163 candidate effectors, potentially secreted by Wolbachia into the host cell. A total of 84 of these candidates were then subjected to a screen of growth defects induced in yeast upon heterologous expression which identified 14 top candidates likely secreted by Wolbachia. These predicted secreted effectors may function in concert as we find that their native expression is correlated and is highly upregulated at specific time points during Drosophila development. In addition, the evolutionary histories of some of these predicted effectors are also correlated, suggesting they may function together, or in the same pathway, during host infection. Similarly, most of these predicted effectors are limited to one or two Wolbachia strains—perhaps reflecting shared evolutionary history and strain specific functions in host manipulation. Identification of these Wolbachia candidate effectors is the first step in dissecting the mechanisms of symbiont–host interaction in this important system.