Cup is essential for oskar mRNA translational repression during early Drosophila oogenesis.

Study of the timing and location for mRNA translation across model systems has begun to shed light on molecular events fundamental to such processes as intercellular communication, morphogenesis, and body pattern formation. In D. melanogaster, the posterior mRNA determinant, oskar, is transcribed maternally but translated only when properly localized at the oocyte's posterior cortex. Two effector proteins, Bruno1 and Cup, mediate steps of oskar mRNA regulation. The current model in the field identifies Bruno1 as necessary for Cup's recruitment to oskar mRNA and indispensable for oskar's translational repression. We now report that this Bruno1-Cup interaction leads to precise oskar mRNA regulation during early oogenesis and, importantly, the two proteins mutually influence each other's mRNA expression and protein distribution in the egg chamber. We show that these factors stably associate with oskar mRNA in vivo. Cup associates with oskar mRNA without Bruno1, while surprisingly Bruno1's stable association with oskar mRNA depends on Cup. We demonstrate that the essential factor for oskar mRNA repression in early oogenesis is Cup, not Bruno1. Furthermore, we find that Cup is a key P-body component that maintains functional P-body morphology during oogenesis and is necessary for oskar mRNA's association with P-bodies. Therefore, Cup drives the translational repression and stability of oskar mRNA. These experimental results point to a regulatory feedback loop between Bruno 1 and Cup in early oogenesis that appears crucial for oskar mRNA to reach the posterior pole and its expression in the egg chamber for accurate embryo development.

Alterations of endophytic microbial community function in Spartina alterniflora as a result of crude oil exposure.

The 2010 Deepwater Horizon disaster remains one of the largest oil spills in history. This event caused significant damage to coastal ecosystems, the full extent of which has yet to be fully determined. Crude oil contains toxic heavy metals and substances such as polycyclic aromatic hydrocarbons that are detrimental to some microbial species and may be used as food and energy resources by others. As a result, oil spills have the potential to cause significant shifts in microbial communities. This study assessed the impact of oil contamination on the function of endophytic microbial communities associated with saltmarsh cordgrass (Spartina alterniflora). Soil samples were collected from two locations in coastal Louisiana, USA: one severely affected by the Deepwater Horizon oil spill and one relatively unaffected location. Spartina alterniflora seedlings were grown in both soil samples in greenhouses, and GeoChip 5.0 was used to evaluate the endophytic microbial metatranscriptome shifts in response to host plant oil exposure. Oil exposure was associated with significant shifts in microbial gene expression in functional categories related to carbon cycling, virulence, metal homeostasis, organic remediation, and phosphorus utilization. Notably, significant increases in expression were observed in genes related to metal detoxification with the exception of chromium, and both significant increases and decreases in expression were observed in functional gene subcategories related to hydrocarbon metabolism. These findings show that host oil exposure elicits multiple changes in gene expression from their endophytic microbial communities, producing effects that may potentially impact host plant fitness.

Rhizosphere microbial communities reflect genotypic and trait variation in a salt marsh ecosystem engineer.

PREMISE: There is growing recognition that intraspecific genetic variation in plants can influence associated soil microbial communities, but the functional bridges linking plant genotype with microbial community structure are not well understood. This deficit is due in part to a prevailing focus on characterizing relationships between microbial communities and functional trait variation among plant species or across plant communities, rather than within a single species. METHODS: We examined whether and how spatiotemporal variation in salt marsh rhizosphere microbial communities reflect plant provenance (genotypic variation) and associated trait variation within an ecosystem engineer, Spartina alterniflora. We planted S. alterniflora from four genetically distinct source populations in replicate sets of experimental plots across a shoreline in southeastern Louisiana, USA. After 2 years, we measured functional plant traits and profiled microbial communities. RESULTS: Bacterial and fungal α-diversity and richness were significantly higher in winter than in summer and corresponded to plant trait variation associated with provenance. Notably, 20% of the variation in fungal community composition was explained by trait differences while bacterial community structure did not reflect plant provenance or trait variation. However, evidence was found suggesting that bacterial communities are indirectly shaped by the influence of plant provenance on soil physicochemical properties. CONCLUSIONS: This study illustrates that intraspecific genetic and corresponding trait variation in an ecosystem engineer can shape rhizosphere microbial communities, with fungal communities being more responsive than bacteria to the influence of plant provenance and associated trait variation. Our results highlight the potential relevance of plant intraspecific variation in plant-microbe-soil feedbacks shaping naturally depauperate ecosystems like salt marshes.

Persisting responses of salt marsh fungal communities to the Deepwater Horizon oil spill.

The plant microbiome, composed of diverse interacting microorganisms, is thought to undergird host integrity and well-being. Though it is well understood that environmental perturbations like oil pollution can alter the diversity and composition of microbiomes, remarkably little is known about how disturbance alters plant-fungal associations. Using Next-Generation sequencing of the 18S rDNA internal transcribed spacer (ITS1) region, we examined outcomes of enduring oil exposure on aboveground leaf and belowground endophytic root and rhizosphere fungal communities of Spartina alterniflora, a highly valued ecosystem engineer in southeastern Louisiana marshes affected by the 2010 Deepwater Horizon accident. We found that aboveground foliar fungal communities exhibited site-dependent compositional turnover with consequent loss in diversity according to oiling history. Rhizosphere soil communities also exhibited shifts in community composition associated with oiling history, whereas root endophytic communities did not. Oiling did not increase or decrease similarities among aboveground and belowground communities within an individual host, indicating that host plant characteristics exert stronger control than external factors on fungal community composition. These results show that fungal community responses to oiling vary within tissues of the same host plant, and that differences in the local environment, or alternatively, site-specific differences in residual oil constrain the magnitude of exposure responses. Our study offers novel perspectives on how environmental contaminants and perturbations can influence plant microbiomes, highlighting the importance of assessing long-term ecological outcomes of oil pollution to better understand how shifts in microbial communities influence plant performance and ecosystem function. Our findings are relevant to coastal management programs tasked with responding to oil spills and increasing pressures arising from intensifying development and climate change. Understanding how modification of plant-microbiome associations influences plant performance, particularly of ecosystem engineers like S. alterniflora, can help guide efforts to protect and restore at-risk coastal ecosystems.

Single-Molecule RNA In Situ Hybridization (smFISH) and Immunofluorescence (IF) in the Drosophila Egg Chamber.

Detection of nucleic acids in whole tissues has become key in our understanding of gene expression during development. In situ hybridization (ISH) has been an invaluable technique in the making of numerous discoveries. Most recently, the technical advance of using short, fluorescently labeled probes has allowed for the detection of single-mRNA molecules. Thus, quantification of RNA levels in single cells or even within subcellular regions is now possible without RNA isolation. In combination with the immunofluorescence (IF) technique, visualization of nucleic acids and associating proteins is achieved with higher resolution than ever before using light microscopy. Here we describe the steps implemented to achieve the visualization of individual messenger RNAs (mRNA) using single-molecule FISH (smFISH) probes, as well as detection of mRNA/protein (mRNP) complexes via smFISH in combination with IF.