The stealth episome: suppression of gene expression on the excised genomic island PPHGI-1 from Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1), which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR) leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1), revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS) of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC) was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state

FISH-eyed and genome-wide views on the spatial organisation of gene expression

AbstractEukaryotic cells store their genome inside a nucleus, a dedicated organelle shielded by a double lipid membrane. Pores in these membranes allow the exchange of molecules between the nucleus and cytoplasm. Inside the mammalian cell nucleus, roughly 2 m of DNA, divided over several tens of chromosomes is packed. In addition, protein and RNA molecules functioning in DNA-metabolic processes such as transcription, replication, repair and the processing of RNA fill the nuclear space. While many of the nuclear proteins freely diffuse and display a more or less homogeneous distribution across the nuclear interior, some appear to preferentially cluster and form foci or bodies. A non-random structure is also observed for DNA: increasing evidence shows that selected parts of the genome preferentially contact each other, sometimes even at specific sites in the nucleus. Currently a lot of research is dedicated to understanding the functional significance of nuclear architecture, in particular with respect to the regulation of gene expression. Here we will evaluate evidence implying that the folding of DNA is important for transcriptional control in mammals and we will discuss novel high-throughput techniques expected to further boost our knowledge on nuclear organisation

DNA nanostructures for biotechnological applications

Deoxyribonucleic acid (DNA) is a versatile biomolecule which can be used for the rational design and assembly of nanoscale structures. This thesis explores the use of functional DNA- enzyme nanostructures for applications in biocatalysis and for directed motion on the nanoscale. In the first part of this thesis, a DNA scaffold was outlined for the display and immobilization of enzyme cascades. Confinement or spatial organization of enzyme cascades is adopted in biological systems to prevent loss of reactive intermediates and to facilitate substrate conversion in chemically complex and crowded intracellular environments. We adopt a strategy to create concentrated enzyme assemblies directed by a DNA structure generated by F29 rolling circle amplification (RCA). These DNA assemblies, DNA nanoballs, were investigated for the display of two bi-enzyme systems. Firstly, a horseradish peroxidase and glucose oxidase enzyme pair, and secondly, a transaminase and norcoclaurine synthase bi- enzyme system for the synthesis of biotechnologically relevant benzylisoquinoline (BIA) precursors. The second part of this thesis concerns the use of enzymatic catalysis as a means of affecting the motion of a nanoscale DNA structure. Molecular movement on the micro and nanoscales is a fundamental feature of biological systems, and recreating this functionality represents an important step in the realization of intelligent synthetic devices for directed transport and chemotaxis in response to stimuli. While directed motion has been shown for DNA structures on predefined tracks to which they are hybridized, enzymatic catalysis has not been investigated as an approach to controlling the motion of DNA nanostructures. We show that the motion of a DNA structure tethered to multiple lysine decarboxylase molecules is enhanced by its substrate, L-lysine the ‘fuel’, in a concentration dependent manner, based on nanoparticle tracking analysis (NTA) and DLS analyses

Dissecting molecular mechanisms of disease in the wheat pathogen, Parastagonospora nodorum

Dissecting molecular mechanisms of disease in the wheat pathogen, Parastagonospora nodorum Parastagonospora nodorum is a wheat specific pathogen that causes annual losses to the Australian wheat industry in excess of $100 million AUD. This necrotrophic fungus kills the host tissue generating necrotic lesions within which fruiting bodies develop, spreading spores and continuing the disease cycle. This polycyclic infection cycle leads to field epidemics resulting in the losses to growers. Sporulation and virulence are the two crucial aspects for disease development in the P. nodorum-wheat pathosystem and form the basis of this project. A forward genetics approach was employed to discover novel mechanisms by which P. nodorum facilitates infection on wheat. A library of random P. nodorum insertion mutants was generated, and subsequently screened for gain and loss of virulence phenotypes on non-susceptible and susceptible wheat cultivars. From a library of 950 transformants seven displayed a consistent avirulent phenotype on the susceptible wheat cultivar. To identify the disrupted loci leading to avirulence, genomes of the seven avirulent P. nodorum strains were sequenced elucidating a Catechol-1,2-dioxygenase and a Copper dependent amine oxidase. To complement the virulence investigation, a combined transcriptomics and metabolomics approach was employed to decipher sporulation in this pathogen. This is of particular interest as the canonical sporulation pathways in other, model fungi, were previously shown to be not applicable in P. nodorum. A differential gene analysis of fungal material collected at critical developmental time points identified several key genes involved in initiating a sporulation cascade. Notably, a WetA homolog was identified, along with an uncharacterised Aquaporin-like protein and a Pr1-like protein. Metabolomics and subsequent sporulation assays revealed a polyamine pathway plays an integral role in initiating and coordinating asexual development of P. nodorum

Different Domains of the RNA Polymerase of Infectious Bursal Disease Virus Contribute to Virulence

BACKGROUND: Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. IBDV has a bi-segmented double-stranded RNA genome. Segments A and B encode the capsid, ribonucleoprotein and non-structural proteins, or the virus polymerase (RdRp), respectively. Since the late eighties, very virulent (vv) IBDV strains have emerged in Europe inducing up to 60% mortality. Although some progress has been made in understanding the molecular biology of IBDV, the molecular basis for the pathogenicity of vvIBDV is still not fully understood. METHODOLOGY, PRINCIPAL FINDINGS: Strain 88180 belongs to a lineage of pathogenic IBDV phylogenetically related to vvIBDV. By reverse genetics, we rescued a molecular clone (mc88180), as pathogenic as its parent strain. To study the molecular basis for 88180 pathogenicity, we constructed and characterized in vivo reassortant or mosaic recombinant viruses derived from the 88180 and the attenuated Cu-1 IBDV strains. The reassortant virus rescued from segments A of 88180 (A88) and B of Cu-1 (BCU1) was milder than mc88180 showing that segment B is involved in 88180 pathogenicity. Next, the exchange of different regions of BCU1 with their counterparts in B88 in association with A88 did not fully restore a virulence equivalent to mc88180. This demonstrated that several regions if not the whole B88 are essential for the in vivo pathogenicity of 88180. CONCLUSION, SIGNIFICANCE: The present results show that different domains of the RdRp, are essential for the in vivo pathogenicity of IBDV, independently of the replication efficiency of the mosaic viruses

Characterisation of rhizobia for the new annual pasture legume Scorpiurus muricatus targeted for medium-to-low rainfall areas of southern Australia

Legumes play an integral role in increasing agricultural productivity, particularly in low input agricultural systems in Australia, due to their ability to form symbiotic interactions with a group of soil bacteria called rhizobia. However, in medium-to-low rainfall areas of southern Australia, there is a lack of suitable annual pasture legumes, which is limiting agricultural productivity and profitability in these farming systems. Scorpiurus muricatus is an annual legume from the Mediterranean which possesses high nutritive value and palatability for livestock, is high yielding, capable of self-seeding and is well-adapted to hot and dry summers. As such, S. muricatus is currently being evaluated as a new pasture legume for southern Australia. Crucial to the success of introducing this legume will be the availability of a highly effective rhizobial inoculant strain. This thesis therefore sought to characterise the phylogeny, free-living and symbiotic phenotype of a range of bacteria isolated from Scorpiurus spp. A total of 19 strains were investigated, with 16s rRNA sequencing demonstrating that 18 of these strains belonged to the genus Mesorhizobium, with the remaining strain (WSM1184) most closely related to Agrobacterium tumefaciens. Analysis of nifH and nodC symbiosis genes further showed that the characterised Mesorhizobium strains generally shared highly similar sequences for these loci, indicating a comparatively high degree of genetic similarity. In particular, WSM1343 (isolated from Scorpiurus sulcatus growing in Morocco) and WSM1386 (isolated from S. sulcatus in Manjimup, Western Australia) were shown to share highly similar symbiosis genes, but divergent 16S rRNA genes, suggesting the possibility that these strains may contain symbiosis genes on mobile Integrative and Conjugative Elements (ICEs). While the temperature tolerance and apparent optimum growth temperature of the test strains of 28°C was consistent with that commonly reported for Mesorhizobium spp., their growth rate was atypical for this genus, with 15 of the 18 strains having a growth rate on YMA at 28°C slower than that generally described for Mesorhizobium. This slower growth rate may be a common feature of rhizobia from S. muricatus nodules and therefore should be considered when isolating organisms from this legume. Symbiotic effectiveness experiments showed all Mesorhizobium strains nodulated S. muricatus and fixed N2 on this host, with the most effective strain producing 67.5% of the mean shoot dry weight of the N-fed control plants. Host range experiments demonstrated a subset of the Mesorhizobium strains nodulate existing Australian commercial pasture legumes Biserrula pelecinus and Lotus corniculatus, with the effectiveness data suggesting these strains fix N2 poorly on both hosts. In contrast, none of the strains tested were able to nodulate the grain legume Cicer arietinum. While this thesis has characterised the phylogeny, free-living and symbiotic phenotype of a range of S. muricatus microsymbionts, further work is required before a suitable commercial inoculant strain can be recommended for this pasture legume. First, all the strains tested in this thesis were isolated from S. sulcatus plants or soils with Scorpiurus spp. present, rather than S. muricatus and it is not known whether strains from either species are cross-compatible for effective N2 fixation. Future studies may therefore locate more effective N2-fixing rhizobia for S. muricatus by isolating microsymbionts from this host in the field. Second, experiments testing the ability of commercial inoculants for already-established pasture legumes B. pelecinus (WSM1497), Lotus sp. (SU343, CC829) and the grain legume C. arietinum (CC1192) to nodulate and fix N2 on S. muricatus need to be conducted to determine whether these inoculants will interact with this legume. Finally, the data strongly suggest that S. muricatus-nodulating Mesorhizobium spp. may contain symbiosis genes on mobile symbiosis ICEs. Given that the phenomenon of ICE transfer has led to the evolution of poorly effective microsymbionts for B. pelecinus, it is imperative that these S. muricatus strains be interrogated for the presence and transfer of symbiosis ICEs, in order to manage this mobility in any future commercial inoculant strain that is released for this pasture legume species

Developing mitoribosomal profiling to investigate quality control of human mitrochondrial protein synthesis

PhD ThesisMitochondria are essential organelles of nucleated cells that have the ability to conduct intra-organellar protein synthesis. Many aspects of this process are poorly understood especially the quality control and the rescue of stalled mitoribosomes. My project was focused on investigating potential candidates involved in quality control and ribosome rescue, including the release factor protein C12orf65. The importance of this protein in mt-translation was confirmed in a patient harbouring a mutation in C12orf65 gene, which displayed a general decrease in de novo mt-translation with subsequent disruption in assembly of OXPHOS complexes I, IV and V. I investigated the consequences on mitochondrial homeostasis in such a cell line. However, for a molecular understanding of the mechanism of this protein’s function a different, more focused approach was needed. To this end I applied ribosome profiling to mitochondria. Ribosome profiling provides a genome-wide analysis of protein synthesis by deep-sequencing of the mRNA fragments protected by ribosomes. It allows monitoring of progression of translation in vivo and can be used to identify contributions by regulating factors. I optimized the protocol for use in human mitochondria, initially on a cell line with a mutation in mt-tRNAVal. The decrease in stability of the uncharged tRNAVal resulted in an increase in mitoribosomal density over the valine codons, consistent with ribosomal stalling. I then used the final protocol, proven effective to study defects in mitoribosome progression, on cells with siRNA depleted C12orf65. Reducing transcript levels to 30% of control gave modest differences in mitoribosomal profiles. The control profiles, however, allowed normal features of mitochondrial translation to be identified. Although the exact function of C12orf65 remains unknown its involvement in mitochondrial protein translation is clear. Further applications of mitoribosome profiling to depleted or mutant cell lines should elucidate mechanisms that rescue stalled mitoribosomes and the potential role of C12orf65