Performance and long-term stability of the barley hordothionin gene in multiple transgenic apple lines

Introduction of sustainable scab resistance in elite apple cultivars is of high importance for apple cultivation when aiming at reducing the use of chemical crop protectants. Genetic modification (GM) allows the rapid introduction of resistance genes directly into high quality apple cultivars. Resistance genes can be derived from apple itself but genetic modification also opens up the possibility to use other, non-host resistance genes. A prerequisite for application is the long-term performance and stability of the gene annex trait in the field. For this study, we produced and selected a series of transgenic apple lines of two cultivars, i.e. ‘Elstar’ and ‘Gala’ in which the barley hordothionin gene (hth) was introduced. After multiplication, the GM hth-lines, non-GM susceptible and resistant controls and GM non-hth controls were planted in a random block design in a field trial in 40 replicates. Scab resistance was monitored after artificial inoculation (first year) and after natural infection (subsequent years). After the trial period, the level of expression of the hth gene was checked by quantitative RT-PCR. Four of the six GM hth apple lines proved to be significantly less susceptible to apple scab and this trait was found to be stable for the entire 4-year period. Hth expression at the mRNA level was also stable

The Nuclear Protein Sge1 of Fusarium oxysporum Is Required for Parasitic Growth

Dimorphism or morphogenic conversion is exploited by several pathogenic fungi and is required for tissue invasion and/or survival in the host. We have identified a homolog of a master regulator of this morphological switch in the plant pathogenic fungus Fusarium oxysporum f. sp. lycopersici. This non-dimorphic fungus causes vascular wilt disease in tomato by penetrating the plant roots and colonizing the vascular tissue. Gene knock-out and complementation studies established that the gene for this putative regulator, SGE1 (SIX Gene Expression 1), is essential for pathogenicity. In addition, microscopic analysis using fluorescent proteins revealed that Sge1 is localized in the nucleus, is not required for root colonization and penetration, but is required for parasitic growth. Furthermore, Sge1 is required for expression of genes encoding effectors that are secreted during infection. We propose that Sge1 is required in F. oxysporum and other non-dimorphic (plant) pathogenic fungi for parasitic growth

Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays

Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays

Utilizing Targeted Gene Therapy with Nanoparticles Binding Alpha v Beta 3 for Imaging and Treating Choroidal Neovascularization

Purpose: The integrin αvβ3 is differentially expressed on neovascular endothelial cells. We investigated whether a novel intravenously injectable αvβ3 integrin-ligand coupled nanoparticle (NP) can target choroidal neovascular membranes (CNV) for imaging and targeted gene therapy. Methods: CNV lesions were induced in rats using laser photocoagulation. The utility of NP for in vivo imaging and gene delivery was evaluated by coupling the NP with a green fluorescing protein plasmid (NP-GFPg). Rhodamine labeling (Rd-NP) was used to localize NP in choroidal flatmounts. Rd-NP-GFPg particles were injected intravenously on weeks 1, 2, or 3. In the treatment arm, rats received NP containing a dominant negative Raf mutant gene (NP-ATPμ-Raf) on days 1, 3, and 5. The change in CNV size and leakage, and TUNEL positive cells were quantified. Results: GFP plasmid expression was seen in vivo up to 3 days after injection of Rd-NP-GFPg. Choroidal flatmounts confirmed the localization of the NP and the expression of GFP plasmid in the CNV. Treating the CNV with NP-ATPμ-Raf decreased the CNV size by 42% (P<0.001). OCT analysis revealed that the reduction of CNV size started on day 5 and reached statistical significance by day 7. Fluorescein angiography grading showed significantly less leakage in the treated CNV (P<0.001). There were significantly more apoptotic (TUNEL-positive) nuclei in the treated CNV. Conclusion: Systemic administration of αvβ3 targeted NP can be used to label the abnormal blood vessels of CNV for imaging. Targeted gene delivery with NP-ATPμ-Raf leads to a reduction in size and leakage of the CNV by induction of apoptosis in the CNV

Survival in amoeba: a major selection pressure on the presence of bacterial copper and zinc resistance determinants?: identification of a "copper pathogenicity island"

The presence of metal resistance determinants in bacteria usually is attributed to geological or anthropogenic metal contamination in different environments or associated with the use of antimicrobial metals in human healthcare or in agriculture. While this is certainly true, we hypothesize that protozoan predation and macrophage killing are also responsible for selection of copper/zinc resistance genes in bacteria. In this review, we outline evidence supporting this hypothesis, as well as highlight the correlation between metal resistance and pathogenicity in bacteria. In addition, we introduce and characterize the "copper pathogenicity island" identified in Escherichia coli and Salmonella strains isolated from copper- and zinc-fed Danish pigs

The Wor1-like Protein Fgp1 Regulates Pathogenicity, Toxin Synthesis and Reproduction in the Phytopathogenic Fungus Fusarium graminearum

WOR1 is a gene for a conserved fungal regulatory protein controlling the dimorphic switch and pathogenicity determents in Candida albicans and its ortholog in the plant pathogen Fusarium oxysporum, called SGE1, is required for pathogenicity and expression of key plant effector proteins. F. graminearum, an important pathogen of cereals, is not known to employ switching and no effector proteins from F. graminearum have been found to date that are required for infection. In this study, the potential role of the WOR1-like gene in pathogenesis was tested in this toxigenic fungus. Deletion of the WOR1 ortholog (called FGP1) in F. graminearum results in greatly reduced pathogenicity and loss of trichothecene toxin accumulation in infected wheat plants and in vitro. The loss of toxin accumulation alone may be sufficient to explain the loss of pathogenicity to wheat. Under toxin-inducing conditions, expression of genes for trichothecene biosynthesis and many other genes are not detected or detected at lower levels in Δfgp1 strains. FGP1 is also involved in the developmental processes of conidium formation and sexual reproduction and modulates a morphological change that accompanies mycotoxin production in vitro. The Wor1-like proteins in Fusarium species have highly conserved N-terminal regions and remarkably divergent C-termini. Interchanging the N- and C- terminal portions of proteins from F. oxysporum and F. graminearum resulted in partial to complete loss of function. Wor1-like proteins are conserved but have evolved to regulate pathogenicity in a range of fungi, likely by adaptations to the C-terminal portion of the protein

Reduction spheroids preserve a uranium isotope record of the ancient deep continental biosphere

S.M. acknowledges the support of the NASA Astrobiology Institute grant NNA13AA90A, Foundations of Complex Life, Evolution, Preservation and Detection on Earth and Beyond, and the European Union’s Horizon 2020 Research and Innovation Programme under Marie Skłodowska-Curie grant agreement 747877. Av.S.H. was supported by a NASA Astrobiology Institute Postdoctoral Fellowship and acknowledges the support of Xiangli Wang and Devon Cole for lab assistance. S.M. and Av.S.H. thank Noah Planavsky for technical advice, lab support, and comments on an early draft. J.P. was supported by NERC under grant number NE/L001764/1. The isotope facility at SUERC is supported by NERC. The authors thank the two anonymous referees for constructive criticisms that improved the manuscript.Peer reviewedPublisher PD