Genetic and morphological characterization of Cladobotryum species causing cobweb disease of mushrooms

Cladobotryum dendroides (= Dactylium dendroides) has hitherto been regarded as the major causal agent of cobweb disease of the cultivated mushroom, Agaricus bisporus. Nucleotide sequence data for the internal transcribed spacer (ITS) regions of four Cladobotryum/Hypomyces species reported to be associated with cobweb disease, however, indicate that the most common pathogen is now C. mycophilum. This cobweb pathogen varies somewhat in conidial septation from published descriptions of C. mycophilum and lacks the distinctive colony odor. ITS sequencing revealed minor nucleotide variation which split isolates of the pathogen into three subgroups, two comprising isolates that were sensitive to methylbenzimidazole carbamate (MBC) fungicides and one comprising MBC-resistant isolates. The MBC-resistant isolates, which were only obtained from Ireland and Great Britain, clustered together strongly in randomly amplified polymorphic DNA (RAPD) PCR analysis, suggesting that they may be clonal. The MBC-sensitive isolates were more diverse. A RAPD fragment of 800 to 900 bp, containing a microsatellite and found in the MBC-resistant isolates, also indicated their clonal nature; the microsatellites of these isolates contained the same number of GA repeats. Smaller, polymorphic microsatellites, similarly comprising GA repeats, in the MBC-sensitive isolates in general correlated with their geographic origin.

NSUN4 Is a Dual Function Mitochondrial Protein Required for Both Methylation of 12S rRNA and Coordination of Mitoribosomal Assembly

Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m(5)C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.

A sting in the spit: widespread cross-infection of multiple RNA viruses across wild and managed bees

Declining populations of bee pollinators are a cause of concern, with major repercussions for biodiversity loss and food security. RNA viruses associated with honeybees represent a potential threat to other insect pollinators, but the extent of this threat is poorly understood. This study aims to attain a detailed understanding of the current and ongoing risk of emerging infectious disease (EID) transmission between managed and wild pollinator species across a wide range of RNA viruses. Within a structured large-scale national survey across 26 independent sites, we quantify the prevalence and pathogen loads of multiple RNA viruses in co-occurring managed honeybee (Apis mellifera) and wild bumblebee (Bombus spp.) populations. We then construct models that compare virus prevalence between wild and managed pollinators. Multiple RNA viruses associated with honeybees are widespread in sympatric wild bumblebee populations. Virus prevalence in honeybees is a significant predictor of virus prevalence in bumblebees, but we remain cautious in speculating over the principle direction of pathogen transmission. We demonstrate species-specific differences in prevalence, indicating significant variation in disease susceptibility or tolerance. Pathogen loads within individual bumblebees may be high and in the case of at least one RNA virus, prevalence is higher in wild bumblebees than in managed honeybee populations. Our findings indicate widespread transmission of RNA viruses between managed and wild bee pollinators, pointing to an interconnected network of potential disease pressures within and among pollinator species. In the context of the biodiversity crisis, our study emphasizes the importance of targeting a wide range of pathogens and defining host associations when considering potential drivers of population decline.

RNA interference in adult Ascaris suum - an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite

The sustainable control of animal parasitic nematodes requires the development of efficient functional genomics platforms to facilitate target validation and enhance anthelmintic discovery. Unfortunately, the utility of RNA interference (RNAi) for the validation of novel drug targets in nematode parasites remains problematic. Ascaris suum is an important veterinary parasite and a zoonotic pathogen. Here we show that adult A. suum is RNAi competent, and highlight the induction, spread and consistency of RNAi across multiple tissue types. This platform provides a new opportunity to undertake whole organism-, tissue- and cell-level gene function studies to enhance target validation processes for nematode parasites of veterinary/medical significance.

Short-chain fatty acids cause an IL-8 response in cystic fibrosis airways via increased GPR41

RATIONALE: Anaerobic bacteria are present in large numbers in the airways of people with cystic fibrosis (PWCF). In the gut, anaerobes produce short-chain fatty acids (SCFAs) that modulate immune/inflammatory processes.

OBJECTIVES: To investigate the capacity of anaerobes to contribute to CF airway pathogenesis via SCFAs.

METHODS: Samples from 109 PWCF were processed using anaerobic microbiological culture with bacteria present identified by 16S RNA sequencing. SCFAs levels in anaerobe supernatants and bronchoalveolar lavage (BAL) were determined by gas chromatography. The mRNA and/or protein expression of SCFAs receptors, GPR41 and GPR43, in CF and non-CF bronchial brushings, and 16HBE14o- and CFBE41o- cells were evaluated using RT-PCR, western blot, laser scanning cytometry and confocal microscopy. SCFAs-induced IL-8 secretion was monitored by ELISA.

MEASUREMENTS AND MAIN RESULTS: Fifty seven of 109 (52.3%) PWCF were anaerobe-positive. Prevalence increased with age, from 33.3% to 57.7% in PWCF under (n=24) and over 6 years (n=85). All evaluated anaerobes produced millimolar concentrations of SCFAs, including acetic, propionic and butyric acid. SCFAs levels were higher in BAL samples from adults than children. GPR41 levels were elevated in; CFBE41o- versus 16HBE14o- cells; CF versus non-CF bronchial brushings; 16HBE14o- cells after treatment with CFTR inhibitor CFTR(inh)-172, CF BAL, or inducers of endoplasmic reticulum stress. SCFAs induced a dose-dependent and pertussis toxin-sensitive IL-8 response in bronchial epithelial cells with a higher production of IL-8 in CFBE41o- than 16HBE14o- cells.

CONCLUSIONS: This study illustrates that SCFAs contribute to excessive production of IL-8 in CF airways colonized with anaerobes via upregulated GPR41.

PTEN mRNA detection by chromogenic, RNA in situ technologies: a reliable alternative to PTEN immunohistochemistry

Immunohistochemical staining for phosphatase and tensin homolog (PTEN) does not have either an acceptable standard protocol or concordance of scoring between pathologists. Evaluation of PTEN mRNA with a unique and verified sequence probe may offer a realistic alternative providing a robust and reproducible protocol. In this study, we have evaluated an in situ hybridization (ISH) protocol for PTEN mRNA using RNAScope technology and compared it with a standard protocol for PTEN immunohistochemistry (IHC). PTEN mRNA expression by ISH was consistently more sensitive than PTEN IHC, with 56% of samples on a mixed-tumor tissue microarray (TMA) showing high expression by ISH compared with 42% by IHC. On a prostate TMA, 49% of cases showed high expression by ISH compared with 43% by IHC. Variations in PTEN mRNA expression within malignant epithelium were quantifiable using image analysis on the prostate TMAs. Within tumors, clear overexpression of PTEN mRNA on malignant epithelium compared with benign epithelium was frequently observed and quantified. The use of SpotStudio software in the mixed-tumor TMA allowed for clear demonstration of varying levels of PTEN mRNA between tumor samples by the mRNA methodology. This was evident by the quantifiable differences between distinct oropharyngeal tumors (up to 3-fold increase in average number of spots per cell between 2 cases). mRNA detection of PTEN or other biomarkers, for which optimal or standardized immunohistochemical techniques are not available, represents a means by which heterogeneity of expression within focal regions of tumor can be explored with more confidence.

Exogenous RNA interference exposes contrasting roles for sugar exudation in host-finding by plant pathogens

Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNA interference (RNAi)-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, STP1 and STP2, in tomato seedlings triggered corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponded directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observed no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato and potentially other crop plants.

Oil hydrocarbon-bacteria interactions in coastal environments

The ability of microorganisms to use oil hydrocarbons as a source of carbon and energy is crucial for environmental oil detoxification. However, there is still a lack of knowledge on fundamental aspects of this process on specific habitats and under different climate scenarios. In the first phase of this work, the culturable fraction of the oil hydrocarbon (OH) degrading bacteria from the sea surface microlayer (SML) of the estuarine system Ria de Aveiro was characterized. In the second phase, the impact of oil contamination on the active bacterial community was studied under climate change scenarios. Pseudomonas emerged as the prevailing genera among OH degrading bacteria in the SML. Moreover, culture-independent methods revealed that the relative abundance and diversity of Gammaproteobacteria, in which Pseudomonas is included, varies along an estuarine gradient of contamination. In order to access the impact of oil contamination on microbial communities under climate change scenarios, an experimental life support system for microcosm experiments (ELLS) was developed and validated for simulation of climate change effects on microbial communities. With the ELSS it is possible to simulate, in controlled conditions, fundamental parameters of the dynamics of coastal and estuarine systems while maintaining community structure in terms of the abundance of the most relevant members of the indigenous bacterial community. A microcosm experiment in which the independent and combined impact of ultraviolet radiation, ocean acidification and oil contamination on microbial communities was conducted. The impact on bacterial communities was accessed with a 16S RNA (cDNA) based barcode pyrosequencing approach. There was a drastic decrease of Desulfobacterales relative abundance after oil contamination under the reduced pH value estimated for 2100, when compared to present values. Since members of this order are known OH degraders, such a significant decrease may have consequences on OH detoxification of contaminated environments under the pH levels of the ocean expected for the future. Metagenome predictions based on the 16S RNA database indicated that several degradation pathways of OH could be affected under oil contamination and reduced water pH. Taken together, the results from this work bring new information on the dynamics of OH degrading bacteria in coastal and estuarine environments under present and future climate scenarios.

Analysis of RNA-seq data from the interaction of Coffea spp. - Colletotrichum kahawae

Tese de mestrado em Bioinformática e Biologia Computacional (Bioinformática), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2014