Distribution of legume arabinogalactan protein-extensin (AGPE) glycoproteins in symbiotically defective pea mutants with abnormal infection threads

The interface between the host cell and the microsymbiont is an important zone for development and differentiation during consecutive stages of Rhizobium-legume symbiosis. Legume root nodule extensins, otherwise known as arabinogalactan protein-extensins (AGPEs) are abundant components of infection thread matrix. We have characterized the origin and distribution of these glycoproteins at the symbiotic interface of root nodules of symbiotically defective mutants of pea (Pisum sativum L.) by using immunogold localization with MAC265 an anti-AGPE monoclonal antibody. For mutants with defective growth of infection threads, the AGPE epitope was abundant in the extracellular matrix surrounding infected host cells in the central infected tissue of the nodule, as well as in the lumen of Rhizobiuminduced infection threads. This seems to indicate a mistargeting of AGPE as a consequence of abnormal growth of the infection threads. Furthermore, mutants in the gene sym33 showed reduced labeling with MAC265 and, in some infection threads and droplets, the label was completely absent, a phenomenon that is not observed in wild-type nodules. This suggests an alteration in the composition of the infection thread matrix for sym33 mutants, which may be correlated to the absence of endocytosis of rhizobia into the host cytoplasm.     

Identification of COS markers in the Pinaceae

Conserved ortholog set (COS) markers are evolutionary conserved, single-copy genes, identified from large databases of express sequence tags (ESTs). They are of particular use for constructing syntenic genetic maps among species. In this study, we identified a set of 1,813 putative single-copy COS markers between spruce and loblolly pine, then designed primers for 931 of these markers and tested these primers with DNA from spruce, pine, and Douglas fir. Of these 931 primers, 56% (524) amplified a product in both spruce and pine, and 71% (373) of these were single-banded; 224 amplicons were single-banded in all three species. Even though these COS markers were selected from large EST databases, a substantial proportion (20–30%) of amplicons displayed multiple bands or smears, suggesting significant paralogy. Sequencing of three single-banded amplicons showed high nucleotide similarities among 29 conifer species, suggesting orthology of single-banded amplicons. Screening for COS marker polymorphism in two pedigrees of white spruce and two pedigrees of loblolly pine revealed an average informativeness of 36% for spruce and 24% for pine (e.g., at least one parent was heterozygous for a single-nucleotide polymorphism within the entire amplified product). This corresponds to an average nucleotide heterozygosity of 0.05% and 0.03%, respectively, which is considerably lower than that found in other studies of spruce and pine. Thus, the advantages of COS markers for constructing syntenic maps are offset by their lower polymorphism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Improving consensus structure by eliminating averaging artifacts

Background  

Common structural biology methods (i.e., NMR and molecular dynamics) often produce ensembles of molecular structures. Consequently, averaging of 3D coordinates of molecular structures (proteins and RNA) is a frequent approach to obtain a consensus structure that is representative of the ensemble. However, when the structures are averaged, artifacts can result in unrealistic local geometries, including unphysical bond lengths and angles.     

Impact of urban development on aquatic macroinvertebrates in south eastern Australia: degradation of in-stream habitats and comparison with non-urban streams

Internationally, waterways within urban areas are subject to broad-scale environmental impairment from urban land uses. In this study, we used in-stream macroinvertebrates as surrogates to measure the aquatic health of urban streams in the established suburbs of northern Sydney, in temperate south eastern Australia. We compared these with samples collected from streams flowing in adjacent naturally vegetated catchments. Macroinvertebrates were collected over a 30-month period from riffle, edge and pool rock habitats and were identified to the family level. Macroinvertebrate assemblages were assessed against the influence of imperviousness and other catchment and water quality variables. The study revealed that urban streams were significantly impaired compared with those that flowed through naturally vegetated non-urban catchments. Urban streams had consistently lower family richness, and sensitive guilds were rare or missing. We found that variation in community assemblages among the in-stream habitats (pool edges, riffles and pool rocks) were more pronounced within streams in naturally vegetated catchments than in urban waterways.     

An expanded and flexible form of the vacuolar ATPase membrane sector

The vacuolar ATPase integral membrane c-ring from Nephrops norvegicus occurs in paired complexes in a double membrane. Using cryo-electron microscopy and single particle image processing of 2D crystals, we have obtained a projection structure of the c-ring of N. norvegicus. The c-ring was found to be very flexible, most likely as a result of an expanded conformation of the c subunits. This structure may support a role for the vacuolar ATPase c-rings in membrane fusion.     

A putative de novo evolved gene required for spermatid chromatin condensation in Drosophila melanogaster

Comparative genomics has enabled the identification of genes that potentially evolved de novo from non-coding sequences. Many such genes are expressed in male reproductive tissues, but their functions remain poorly understood. To address this, we conducted a functional genetic screen of over 40 putative de novo genes with testis-enriched expression in Drosophila melanogaster and identified one gene, atlas, required for male fertility. Detailed genetic and cytological analyses showed that atlas is required for proper chromatin condensation during the final stages of spermatogenesis. Atlas protein is expressed in spermatid nuclei and facilitates the transition from histone- to protamine-based chromatin packaging. Complementary evolutionary analyses revealed the complex evolutionary history of atlas. The protein-coding portion of the gene likely arose at the base of the Drosophila genus on the X chromosome but was unlikely to be essential, as it was then lost in several independent lineages. Within the last ~15 million years, however, the gene moved to an autosome, where it fused with a conserved non-coding RNA and evolved a non-redundant role in male fertility. Altogether, this study provides insight into the integration of novel genes into biological processes, the links between genomic innovation and functional evolution, and the genetic control of a fundamental developmental process, gametogenesis.     

Phytoplasma effector SAP54 induces indeterminate leaf-like flower development in Arabidopsis plants

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches' Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches' broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host.     

Cisplatin treatment of primary and metastatic epithelial ovarian carcinomas generates residual cells with mesenchymal stem cell-like profile

Epithelial mesenchymal transition (EMT) and cancer stem cells (CSC) have been associated with resistance to chemotherapy. Eighty percent of ovarian cancer patients initially respond to platinum-based combination therapy but most return with recurrence and ultimate demise. To better understand such chemoresistance we have assessed the potential role of EMT in tumor cells collected from advanced-stage ovarian cancer patients and the ovarian cancer cell line OVCA 433 in response to cisplatin in vitro. We demonstrate that cisplatin-induced transition from epithelial to mesenchymal morphology in residual cancer cells correlated with reduced E-cadherin, and increased N-cadherin and vimentin expression. The mRNA expression of Snail, Slug, Twist, and MMP-2 were significantly enhanced in response to cisplatin and correlated with increased migration. This coincided with increased cell surface expression of CSC-like markers such as CD44, α2 integrin subunit, CD117, CD133, EpCAM, and the expression of stem cell factors Nanog and Oct-4. EMT and CSC-like changes in response to cisplatin correlated with enhanced activation of extracellular signal-regulated kinase (ERK)1/2. The selective MEK inhibitor U0126 inhibited ERK2 activation and partially suppressed cisplatin-induced EMT and CSC markers. In vivo xenotransplantation of cisplatin-treated OVCA 433 cells in zebrafish embryos demonstrated significantly enhanced migration of cells compared to control untreated cells. U0126 inhibited cisplatin-induced migration of cells in vivo, suggesting that ERK2 signaling is critical to cisplatin-induced EMT and CSC phenotypes, and that targeting ERK2 in the presence of cisplatin may reduce the burden of residual tumor, the ultimate cause of recurrence in ovarian cancer patients.     

Longitudinal patterns in carbon and nitrogen fluxes and stream metabolism along an urban watershed continuum

An urban watershed continuum framework hypothesizes that there are coupled changes in (1) carbon and nitrogen cycling, (2) groundwater-surface water interactions, and (3) ecosystem metabolism along broader hydrologic flowpaths. It expands our understanding of urban streams beyond a reach scale. We evaluated this framework by analyzing longitudinal patterns in: C and N concentrations and mass balances, groundwater-surface interactions, and stream metabolism and carbon quality from headwaters to larger order streams. 52 monitoring sites were sampled seasonally and monthly along the Gwynns Falls watershed, which drains 170 km² of the Baltimore Long-Term Ecological Research site. Regarding our first hypothesis of coupled C and N cycles, there were significant inverse linear relationships between nitrate and dissolved organic carbon (DOC) and nitrogen longitudinally (P < 0.05). Regarding our second hypothesis of coupled groundwater-surface water interactions, groundwater seepage and leaky piped infrastructure contributed significant inputs of water and N to stream reaches based on mass balance and chloride/fluoride tracer data. Regarding our third hypothesis of coupled ecosystem metabolism and carbon quality, stream metabolism increased downstream and showed potential to enhance DOC lability (e.g., ~4 times higher mean monthly primary production in urban streams than forest streams). DOC lability also increased with distance downstream and watershed urbanization based on protein and humic-like fractions, with major implications for ecosystem metabolism, biological oxygen demand, and CO₂ production and alkalinity. Overall, our results showed significant in-stream retention and release (0–100 %) of watershed C and N loads over the scale of kilometers, seldom considered when evaluating monitoring, management, and restoration effectiveness. Given dynamic transport and retention across evolving spatial scales, there is a strong need to longitudinally and synoptically expand studies of hydrologic and biogeochemical processes beyond a stream reach scale along the urban watershed continuum.     

The Plasmodesmal Protein PDLP1 Localises to Haustoria-Associated Membranes during Downy Mildew Infection and Regulates Callose Deposition

The downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) is a filamentous oomycete that invades plant cells via sophisticated but poorly understood structures called haustoria. Haustoria are separated from the host cell cytoplasm and surrounded by an extrahaustorial membrane (EHM) of unknown origin. In some interactions, including Hpa-Arabidopsis, haustoria are progressively encased by host-derived, callose-rich materials but the molecular mechanisms by which callose accumulates around haustoria remain unclear. Here, we report that PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1) is expressed at high levels in Hpa infected cells. Unlike other plasma membrane proteins, which are often excluded from the EHM, PDLP1 is located at the EHM in Hpa-infected cells prior to encasement. The transmembrane domain and cytoplasmic tail of PDLP1 are sufficient to convey this localization. PDLP1 also associates with the developing encasement but this association is lost when encasements are fully mature. We found that the pdlp1,2,3 triple mutant is more susceptible to Hpa while overexpression of PDLP1 enhances plant resistance, suggesting that PDLPs enhance basal immunity against Hpa. Haustorial encasements are depleted in callose in pdlp1,2,3 mutant plants whereas PDLP1 over-expression elevates callose deposition around haustoria and across the cell surface. These data indicate that PDLPs contribute to callose encasement of Hpa haustoria and suggests that the deposition of callose at haustoria may involve similar mechanisms to callose deposition at plasmodesmata.