Giant Starship Elements Mobilize Accessory Genes in Fungal Genomes.

Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a newly discovered group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27 to 393 kb and last shared a common ancestor ca. 400 Ma. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize four additional Starships whose activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyager's activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first dedicated agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum.

Frequency, composition and mobility of Escherichia coli-derived transposable elements in holdings of plasmid repositories.

By providing the scientific community with uniform and standardized resources of consistent quality, plasmid repositories play an important role in enabling scientific reproducibility. Plasmids containing insertion sequence elements (IS elements) represent a challenge from this perspective, as they can change the plasmid structure and function. In this study, we conducted a systematic analysis of a subset of plasmid stocks distributed by plasmid repositories (The Arabidopsis Biological Resource Center and Addgene) which carry unintended integrations of bacterial mobile genetic elements. The integration of insertion sequences was most often found in, but not limited to, pBR322-derived vectors, and did not affect the function of the specific plasmids. In certain cases, the entire stock was affected, but the majority of the stocks tested contained a mixture of the wild-type and the mutated plasmids, suggesting that the acquisition of IS elements likely occurred after the plasmids were acquired by the repositories. However, comparison of the sequencing results of the original samples revealed that some plasmids already carried insertion mutations at the time of donation. While an extensive BLAST analysis of 47 877 plasmids sequenced from the Addgene repository uncovered IS elements in only 1.12%, suggesting that IS contamination is not widespread, further tests showed that plasmid integration of IS elements can propagate in conventional Escherichia coli hosts over a few tens of generations. Use of IS-free E. coli hosts prevented the emergence of IS insertions as well as that of small indels, suggesting that the use of IS-free hosts by donors and repositories could help limit unexpected and unwanted IS integrations into plasmids.

Phlox Species Show Quantitative and Qualitative Resistance to a Population of Powdery Mildew Isolates from the Eastern United States.

Ornamental plants in the genus Phlox are extensively planted in landscapes and home gardens around the world. A major limitation to a more widespread use of these plants is their susceptibility to powdery mildew (PM). In this study, we used multilocus sequence typing (MLST) analysis to gain insights into the population diversity of 32 Phlox PM pathogen (Golovinomyces magnicellulatus and Podosphaera sp.) isolates collected from the eastern United States and relate it to the ability to overcome host resistance. Low genetic diversity and a lack of structure were found within our population. Whole genome comparison of two isolates was used to support low genetic diversity evidence found with the MLST analysis. Recombination was suggested by the incongruences observed in the six phylogenetic trees generated from the housekeeping genes TEF-1α, CSI, ITS, IGS, H3, and TUB. Contrasting with low genetic diversity, we found high phenotypic diversity when using 10 of the 32 isolates to evaluate host resistance in four different Phlox species (P. paniculata 'Dunbar Creek', P. amoena OPGC 3598, P. glaberrima OPGC 3594, and P. subulata OPGC 4185) using in vitro bioassays. We observed quantitative and qualitative resistance in all Phlox species and a consistent low disease severity in our control, P. paniculata 'Dunbar Creek'. Taken together, the results generated in this study constitute a robust screening of popular Phlox germplasm that can be incorporated into breeding programs for PM resistance and provides significant information on the evolution of PM pathogens.

FgHtf1 Regulates Global Gene Expression towards Aerial Mycelium and Conidiophore Formation in the Cereal Fungal Pathogen Fusarium graminearum.

The homeobox gene family of transcription factors (HTF) controls many developmental pathways and physiological processes in eukaryotes. We previously showed that a conserved HTF in the plant-pathogenic fungus Fusarium graminearum, Htf1 (FgHtf1), regulates conidium morphology in that organism. This study investigated the mechanism of FgHtf1-mediated regulation and identified putative FgHtf1 target genes by a chromatin immunoprecipitation assay combined with parallel DNA sequencing (ChIP-seq) and RNA sequencing. A total of 186 potential binding peaks, including 142 genes directly regulated by FgHtf1, were identified. Subsequent motif prediction analysis identified two DNA-binding motifs, TAAT and CTTGT. Among the FgHtf1 target genes were FgHTF1 itself and several important conidiation-related genes (e.g., FgCON7), the chitin synthase pathway genes, and the aurofusarin biosynthetic pathway genes. In addition, FgHtf1 may regulate the cAMP-protein kinase A (PKA)-Msn2/4 and Ca2+-calcineurin-Crz1 pathways. Taken together, these results suggest that, in addition to autoregulation, FgHtf1 also controls global gene expression and promotes a shift to aerial growth and conidiation in F. graminearum by activation of FgCON7 or other conidiation-related genes.IMPORTANCE The homeobox gene family of transcription factors is known to be involved in the development and conidiation of filamentous fungi. However, the regulatory mechanisms and downstream targets of homeobox genes remain unclear. FgHtf1 is a homeobox transcription factor that is required for phialide development and conidiogenesis in the plant pathogen F. graminearum In this study, we identified FgHtf1-controlled target genes and binding motifs. We found that, besides autoregulation, FgHtf1 also controls global gene expression and promotes conidiation in F. graminearum by activation of genes necessary for aerial growth, FgCON7, and other conidiation-related genes.

Genotyping-by-Sequencing-Based Genetic Analysis of African Rice Cultivars and Association Mapping of Blast Resistance Genes Against Magnaporthe oryzae Populations in Africa.

Understanding the genetic diversity of rice germplasm is important for the sustainable use of genetic materials in rice breeding and production. Africa is rich in rice genetic resources that can be utilized to boost rice productivity on the continent. A major constraint to rice production in Africa is rice blast, caused by the hemibiotrophic fungal pathogen Magnaporthe oryzae. In this report, we present the results of a genotyping-by-sequencing (GBS)-based diversity analysis of 190 African rice cultivars and an association mapping of blast resistance (R) genes and quantitative trait loci (QTLs). The 190 African cultivars were clustered into three groups based on the 184K single nucleotide polymorphisms generated by GBS. We inoculated the rice cultivars with six African M. oryzae isolates. Association mapping identified 25 genomic regions associated with blast resistance (RABRs) in the rice genome. Moreover, PCR analysis indicated that RABR_23 is associated with the Pi-ta gene on chromosome 12. Our study demonstrates that the combination of GBS-based genetic diversity population analysis and association mapping is effective in identifying rice blast R genes/QTLs that contribute to resistance against African populations of M. oryzae. The identified markers linked to the RABRs and 14 highly resistant cultivars in this study will be useful for rice breeding in Africa.

Epigallocatechin-3-gallate enhances clearance of phosphorylated tau in primary neurons.

OBJECTIVES: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by intracellular accumulations of phosphorylated forms of the microtubule binding protein tau. This study aimed to explore a novel mechanism for enhancing the clearance of these pathological tau species using the green tea flavonoid epigallocatechin-3-gallate (EGCG). EGCG is a potent antioxidant and an activator of the Nrf2 transcriptional pathway. Nrf2 activators including EGCG have shown promise in mitigating amyloid pathology in vitro and in vivo. This study assessed whether EGCG could also alter tau clearance. METHODS: Rat primary cortical neuron cultures were treated on day in vitro 8 with EGCG and analyzed for changes in gene and protein expression using luciferase assay, q-PCR, and western blotting. RESULTS: EGCG treatment led to a significant decrease in the protein levels of three AD-relevant phospho-tau epitopes. Unexpectedly, EGCG does not appear to be facilitating this effect through the Nrf2 pathway or by increasing autophagy in general. However, EGCG did significantly increase mRNA expression of the key autophagy adaptor proteins NDP52 and p62. DISCUSSION: In this study, we show that EGCG enhances the clearance of AD-relevant phosphorylated tau species in primary neurons. Interestingly, this result appears to be independent of both Nrf2 activation and enhanced autophagy - two previously reported mechanisms of phytochemical-induced tau clearance. EGCG did significantly increase expression of two autophagy adaptor proteins. Taken together, these results demonstrate that EGCG has the ability to increase the clearance of phosphorylated tau species in a highly specific manner, likely through increasing adaptor protein expression.

p75 neurotrophin receptor and fenretinide-induced signaling in neuroblastoma.

PURPOSE: Neuroblastoma is the most common extracranial solid tumor of childhood. The retinoic acid analogue, fenretinide (4-hydroxyphenyl retinamide; 4-HPR), induces apoptosis in neuroblastoma cells in vitro and is currently in clinical trials for children with refractory neuroblastoma. We have previously shown that expression of the p75 neurotrophin receptor (p75NTR) enhances apoptosis induction and mitochondrial accumulation of reactive oxygen species by 4-HPR in neuroblastoma cells. We now examine the signaling events that underlie this effect. METHODS: Systematic examination of pro- and anti-apoptotic signaling effectors was performed by Western blot. Specific inhibitors of JNK phosphorylation and scavengers of mitochondrial reactive oxygen species were used to demonstrate the roles of these phenomena in the enhancement of fenretinide efficacy. RESULTS: The present studies demonstrate that enhancement of 4-HPR-induced apoptosis by p75NTR is dependent upon p38MAPK phosphorylation, JNK phosphorylation, caspase 3 activation, Akt cleavage, and decreased Akt phosphorylation. In addition, treatment with 4-HPR results in upregulation of MKK4 and MEKK1, and phosphorylation of MKK3/6. Efforts to enhance the efficacy of 4-HPR and to identify those tumors most likely to respond to it might exploit these effectors of 4-HPR-induced apoptosis. CONCLUSIONS: Pharmacological agents that enhance MKK4 or MEKK1 expression or JNK expression or phosphorylation may enhance efficacy of 4-HPR in neuroblastomas that do not express high levels of p75NTR.

p75NTR: an enhancer of fenretinide toxicity in neuroblastoma.

OBJECTIVE: Neuroblastoma is a common, frequently fatal, neural crest tumor of childhood. Chemotherapy-resistant neuroblastoma cells typically have Schwann cell-like ("S-type") morphology and express the p75 neurotrophin receptor (p75NTR). p75NTR has been previously shown to modulate the redox state of neural crest tumor cells. We, therefore, hypothesized that p75NTR expression level would influence the effects of the redox-active chemotherapeutic drug fenretinide on neuroblastoma cells. METHODS: Transfection and lentiviral transduction were used to manipulate p75NTR expression in these cell lines. Sensitivity to fenretinide was determined by concentration- and time-cell survival studies. Apoptosis incidence was determined by morphological assessment and examination of cleavage of poly-ADP ribose polymerase and caspase-3. Generation and subcellular localization of reactive oxygen species were quantified using species- and site-specific stains and by examining the effects of site-selective antioxidants on cell survival after fenretinide treatment. Studies of mitochondrial electron transport employed specific inhibitors of individual proteins in the electron transport chain. RESULTS: Knockdown of p75NTR attenuates fenretinide-induced accumulation of mitochondrial superoxide and apoptosis. Overexpression of p75NTR has the opposite effects. Pretreatment of cells with 2-thenoyltrifluoroacetone or dehydroascorbic acid uniquely prevents mitochondrial superoxide accumulation and cell death after fenretinide treatment, indicating that mitochondrial complex II is the likely site of fenretinide-induced superoxide generation and p75NTR-induced potentiation of these phenomena. CONCLUSION: Modification of expression of p75NTR in a particular neuroblastoma cell line modifies its susceptibility to fenretinide. Enhancers of p75NTR expression or signaling could be potential drugs for use as adjuncts to chemotherapy of neural tumors.

Pharmacologic management of high-risk neuroblastoma in children.

Neuroblastoma is the most common extracranial solid tumor of childhood. It accounts for 15% of pediatric cancer deaths. Children with high-risk disease have a 3-year event-free survival rate of only 20%. Chemotherapy is the mainstay of treatment in children with advanced neuroblastoma. The aim of this article was to review and critically evaluate the pharmacotherapy of neuroblastoma, using peer reviewed and review literature from 2000-11. All peer reviewed, published human subject studies of therapy for neuroblastoma in children were included. Animal model and in vitro studies were included only if they added to the understanding of the mechanism of a proposed or existing human neuroblastoma therapy. Current therapeutic options for neuroblastoma involve insufficient differentiation of normal from neoplastic tissue. Critically needed new approaches will increasingly exploit targeting of therapy for unique characteristics of the neuroblastoma cell. Pharmacotherapy for neuroblastoma still suffers from an inadequate therapeutic window. Enhancement of toxicity for tumor and safety for normal tissues will entail innovation in targeting neuroblastoma cells and rescuing or protecting normal tissue elements.