Discovery of a fourth evolutionary lineage of Phytophthora ramorum: EU2

Phytophthora ramorum is a recently introduced, aggressive Phytophthora species that has caused extensive mortality of oak and tanoak trees in the western USA and Japanese larch trees in the UK. P. ramorum is also present on Rhododendron, Camellia, and Viburnum in the nursery industry, which is thought to have been the pathway for its spread into new geographic regions including forests and natural ecosystems. Three lineages of P. ramorum have been described, informally designated EU1, NA1, and NA2, and each lineage is believed to originate from an as yet unknown exotic centre of origin. Preliminary SSR and sequence analysis of isolates from a UK P. ramorum survey revealed seven isolates with profiles that did not match the previously known lineages. Detailed SSR and multilocus sequence analysis of these isolates are presented, allowing us to assign these isolates to a new P. ramorum lineage, designated EU2. Although the known geographical origin of these isolates is currently limited to Northern Ireland and western Scotland, the EU2 lineage isolates have been obtained from four different host plants, including Japanese larch. All isolates are of A1 compatibility type, which implies that this finding does not increase the risk of outcrossing with the EU1 lineage isolates already present in the UK. The oldest EU2 strain was isolated in 2007 but no SSR-based intraEU2 lineage genotypic diversity was detected. The combination of these elements points to a recent introduction, despite emergency phytosanitary measures to control introduction and spread. A PCR-RFLP method for the rapid identification of EU2 lineage isolates is presented.     

Nuclear heat shock response and novel nuclear domain 10 reorganization in respiratory syncytial virus-infected a549 cells identified by high-resolution two-dimensional gel electrophoresis

The pneumovirus respiratory syncytial virus (RSV) is a leading cause of epidemic respiratory tract infection. Upon entry, RSV replicates in the epithelial cytoplasm, initiating compensatory changes in cellular gene expression. In this study, we have investigated RSV-induced changes in the nuclear proteome of A549 alveolar type II-like epithelial cells by high-resolution two-dimensional gel electrophoresis (2DE). Replicate 2D gels from uninfected and RSV-infected nuclei were compared for changes in protein expression. We identified 24 different proteins by peptide mass fingerprinting after matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS), whose average normalized spot intensity was statistically significant and differed by +/-2-fold. Notable among the proteins identified were the cytoskeletal cytokeratins, RNA helicases, oxidant-antioxidant enzymes, the TAR DNA binding protein (a protein that associates with nuclear domain 10 [ND10] structures), and heat shock protein 70- and 60-kDa isoforms (Hsp70 and Hsp60, respectively). The identification of Hsp70 was also validated by liquid chromatography quadropole-TOF tandem MS (LC-MS/MS). Separate experiments using immunofluorescence microscopy revealed that RSV induced cytoplasmic Hsp70 aggregation and nuclear accumulation. Data mining of a genomic database showed that RSV replication induced coordinate changes in Hsp family proteins, including the 70, 70-2, 90, 40, and 40-3 isoforms. Because the TAR DNA binding protein associates with ND10s, we examined the effect of RSV infection on ND10 organization. RSV induced a striking dissolution of ND10 structures with redistribution of the component promyelocytic leukemia (PML) and speckled 100-kDa (Sp100) proteins into the cytoplasm, as well as inducing their synthesis. Our findings suggest that cytoplasmic RSV replication induces a nuclear heat shock response, causes ND10 disruption, and redistributes PML and Sp100 to the cytoplasm. Thus, a high-resolution proteomics approach, combined with immunofluorescence localization and coupled with genomic response data, yielded unexpected novel insights into compensatory nuclear responses to RSV infection.     

Two-dimensional peptide mapping of fibronectins from bovine aortic endothelial cells and bovine plasma

We have made a comparison between plasma and endothelial cell fibronectin, since these cells are in intimate contact with plasma $\text{in vivo}$. Cellular and secreted fibronectins were purified from cloned lines of adult bovine aortic endothelial cells, and compared to purified bovine plasma fibronectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional peptide mapping. When unreduced, all three fibronectins migrated on gels as single bands with M_r 440,000. After reduction, cellular and secreted fibronectins migrated on gels as single bands with M_r 220,000, but plasma fibronectin migrated as two bands with M_r 220,000 and 210,000. All three fibronectins, including the two subunits of plasma fibronectin, had identical structures by peptide mapping analysis.     

A microbial role in the construction of Mono Lake carbonate chimneys?

Lacustrine carbonate chimneys are striking, metre‐scale constructions. If these were bioinfluenced constructions, they could be priority targets in the search for early and extraterrestrial microbial life. However, there are questions over whether such chimneys are built on a geobiological framework or are solely abiotic geomorphological features produced by mixing of lake and spring waters. Here, we use correlative microscopy to show that microbes were living around Pleistocene Mono Lake carbonate chimneys during their growth. A plausible interpretation, in line with some recent works by others on other lacustrine carbonates, is that benthic cyanobacteria and their associated extracellular organic material (EOM) formed tubular biofilms around rising sublacustrine spring vent waters, binding calcium ions and trapping and binding detrital silicate sediment. Decay of these biofilms would locally have increased calcium and carbonate ion activity, inducing calcite precipitation on and around the biofilms. Early manganese carbonate mineralisation was directly associated with cell walls, potentially related to microbial activity though the precise mechanism remains to be elucidated. Much of the calcite crystal growth was likely abiotic, and no strong evidence for either authigenic silicate growth or a clay mineral precursor framework was observed. Nevertheless, it seems likely that the biofilms provided initial sites for calcite nucleation and encouraged the primary organised crystal growth. We suggest that the nano‐, micro‐ and macroscale fabrics of these Pleistocene Mono Lake chimneys were affected by the presence of centimetre‐thick tubular and vertically stacked calcifying microbial mats. Such carbonate chimneys represent a promising macroscale target in the exploration for ancient or extraterrestrial life.