Generation of a ToxA knockout strain of the wheat tan spot pathogen Pyrenophora tritici-repentis

The necrotrophic fungal pathogen Pyrenophora tritici-repentis causes tan spot, a major disease of wheat throughout the world. The proteinaceous effector ToxA is responsible for foliar necrosis on ToxA sensitive wheat genotypes. The single copy ToxA gene was deleted from a wild-type race 1 P. tritici-repentis isolate via homologous recombination of a knockout construct. Expression of the ToxA transcript was found to be absent in transformants (toxa), as was ToxA protein production in fungal culture filtrates. Plant bioassays were conducted to test transformant pathogenicity. The toxa strains were unable to induce necrosis on ToxA sensitivity wheat genotypes. To our knowledge, this is the first demonstration of a targeted gene knockout in P. tritici-repentis. The ability to undertake gene deletions will facilitate the characterisation of other pathogenicity effectors of this economically significant necrotroph

Genomic distribution of a novel Pyrenophora tritici-repentis ToxA insertion element

© 2018 Moolhuijzen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The ToxA effector is a major virulence gene of Pyrenophora tritici-repentis (Ptr), a necrotrophic fungus and the causal agent of tan spot disease of wheat. ToxA and co-located genes are believed to be the result of a recent horizontally transferred highly conserved 14kb region a major pathogenic event for Ptr. Since this event, monitoring isolates for pathogenic changes has become important to help understand the underlying mechanisms in play. Here we examined ToxA in 100 Ptr isolates from Australia, Europe, North and South America and the Middle East, and uncovered in isolates from Denmark, Germany and New Zealand a new variation, a novel 166 bp insertion element (PtrHp1) which can form a perfectly matched 59 bp inverted repeat hairpin structure located downstream of the ToxA coding sequence in the 3' UTR exon. A wider examination revealed PtrHp1 elements to be distributed throughout the genome. Analysis of genomes from Australia and North America had 50-112 perfect copies that often overlap other genes. The hairpin element appears to be unique to Ptr and the lack of ancient origins in other species suggests that PtrHp1 emerged after Ptr speciation. Furthermore, the ToxA UTR insertion site is identical for different isolates, which suggests a single insertion event occurred after the ToxA horizontal transfer. In vitro and in planta-detached leaf assays found that the PtrHp1 element insertion had no effect on ToxA expression. However, variation in the expression of ToxA was detected between the Ptr isolates from different demographic locations, which appears to be unrelated to the presence of the element. We envision that this discovery may contribute towards future understanding of the possible role of hairpin elements in Ptr

Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative Luciferase-Vpr packaging-based assay

The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity

Heterologous Expression of the Pyrenophora tritici-repentis Effector Proteins ToxA and ToxB, and the Prevalence of Effector Sensitivity in Australian Cereal Crops

Here, we evaluate the expression of the proteinaceous effectors ToxA and ToxB, produced by the necrotrophic fungal pathogen Pyrenophora tritici-repentis, which confer tan spot disease susceptibility on wheat. These necrotrophic effectors were expressed in two heterologous systems: Escherichia coli and Pichia pastoris. The E. coli SHuffle system was demonstrated to be superior to P. pastoris in generating high-levels of recombinant proteins that were soluble and stable. In addition, protein extracts from P. pastoris induced non-specific chlorosis on wheat, postulated to be caused by co-purified glucanases secreted by the host. Up to 79.6 μg/ml of ToxB was obtained using the SHuffle system in the absence of the native signal peptide, whilst the ToxA yield was considerably lower at 3.2 μg/ml. Results indicated that a histidine tag at the ToxA C-terminus interfered with effector functionality. Heterologously expressed ToxA and ToxB were tested on a panel of Australian cereals, including 122 varieties of bread wheat, 16 durum, 20 triticale and 5 barley varieties, as well as common plant model species including tobacco and Arabidopsis thaliana. A varying degree of effector sensitivities was observed, with a higher ToxB sensitivity and prevalence in the durum and triticale varieties. ToxB-induced chlorosis was also detected on barley. The heterologous expression of effectors that are easily scalable, will facilitate effector-assisted selection of varieties in wheat breeding programs as well as the investigation of P. tritici-repentis effectors in host and non-host interactions

Fractal Spacetime Structure in Asymptotically Safe Gravity

Four-dimensional Quantum Einstein Gravity (QEG) is likely to be an asymptotically safe theory which is applicable at arbitrarily small distance scales. On sub-Planckian distances it predicts that spacetime is a fractal with an effective dimensionality of 2. The original argument leading to this result was based upon the anomalous dimension of Newton's constant. In the present paper we demonstrate that also the spectral dimension equals 2 microscopically, while it is equal to 4 on macroscopic scales. This result is an exact consequence of asymptotic safety and does not rely on any truncation. Contact is made with recent Monte Carlo simulations.Comment: 20 pages, late

Mapping citizen science contributions to the UN Sustainable Development Goals

The UN Sustainable Development Goals (SDGs) are a vision for achieving a sustainable future. Reliable, timely, comprehensive, and consistent data are critical for measuring progress towards, and ultimately achieving, the SDGs. Data from citizen science represent one new source of data that could be used for SDG reporting and monitoring. However, information is still lacking regarding the current and potential contributions of citizen science to the SDG indicator framework. Through a systematic review of the metadata and work plans of the 244 SDG indicators, as well as the identification of past and ongoing citizen science initiatives that could directly or indirectly provide data for these indicators, this paper presents an overview of where citizen science is already contributing and could contribute data to the SDG indicator framework. The results demonstrate that citizen science is “already contributing” to the monitoring of 5 SDG indicators, and that citizen science “could contribute” to 76 indicators, which, together, equates to around 33%. Our analysis also shows that the greatest inputs from citizen science to the SDG framework relate to SDG 15 Life on Land, SDG 11 Sustainable Cities and Communities, SDG 3 Good Health and Wellbeing, and SDG 6 Clean Water and Sanitation. Realizing the full potential of citizen science requires demonstrating its value in the global data ecosystem, building partnerships around citizen science data to accelerate SDG progress, and leveraging investments to enhance its use and impact