Management practices for control of ragwort species

The ragwort species common or tansy ragwort (Jacobaea vulgaris, formerly Senecio jacobaea), marsh ragwort (S. aquaticus), Oxford ragwort (S. squalidus) and hoary ragwort (S. erucifolius) are native in Europe, but invaded North America, Australia and New Zealand as weeds. The abundance of ragwort species is increasing in west-and central Europe. Ragwort species contain different groups of secondary plant compounds defending them against generalist herbivores, contributing to their success as weeds. They are mainly known for containing pyrrolizidine alkaloids, which are toxic to grazing cattle and other livestock causing considerable losses to agricultural revenue. Consequently, control of ragwort is obligatory by law in the UK, Ireland and Australia. Commonly used management practices to control ragwort include mechanical removal, grazing, pasture management, biological control and chemical control. In this review the biology of ragwort species is shortly described and the different management practices are discussed

An overview of NMR-based metabolomics to identify secondary plant compounds involved in host plant resistance

Secondary metabolites provide a potential source for the generation of host plant resistance and development of biopesticides. This is especially important in view of the rapid and vast spread of agricultural and horticultural pests worldwide. Multiple pests control tactics in the framework of an integrated pest management (IPM) programme are necessary. One important strategy of IPM is the use of chemical host plant resistance. Up to now the study of chemical host plant resistance has, for technical reasons, been restricted to the identification of single compounds applying specific chemical analyses adapted to the compound in question. In biological processes however, usually more than one compound is involved. Metabolomics allows the simultaneous detection of a wide range of compounds, providing an immediate image of the metabolome of a plant. One of the most universally used metabolomic approaches comprises nuclear magnetic resonance spectroscopy (NMR). It has been NMR which has been applied as a proof of principle to show that metabolomics can constitute a major advancement in the study of host plant resistance. Here we give an overview on the application of NMR to identify candidate compounds for host plant resistance. We focus on host plant resistance to western flower thrips (Frankliniella occidentalis) which has been used as a model for different plant species

Towards eco-friendly crop protection: natural deep eutectic solvents and defensive secondary metabolites

Plant science

Vasodilator-Stimulated Phosphoprotein Activity Is Required for <i>Coxiella burnetii</i> Growth in Human Macrophages

<div><p><i>Coxiella burnetii</i> is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and <i>C</i>. <i>burnetii</i> initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). <i>C</i>. <i>burnetii</i> manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during <i>C</i>. <i>burnetii</i> infection. Avirulent and virulent <i>C</i>. <i>burnetii</i> triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in <i>C</i>. <i>burnetii</i>-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages.</p></div

VASP S239E expression interferes with optimal PV formation.

<p>THP-1 cells were transfected with constructs encoding GFP-tagged wild type VASP (GFP-VASP), GFP-VASP (S157E), or GFP-VASP (S239E). At 72 hpi, cells were processed for fluorescence microscopy and DNA was stained with DAPI (blue). Actin was labeled with phalloidin (red). Bar, 10 μm. N = nucleus and * indicates the PV. GFP-VASP and GFP-VASP (S157E) localized around the PV with actin, while GFP-VASP (S239E) expression interfered with vacuole expansion and actin co-localization.</p

VASP is differentially phosphorylated during <i>C</i>. <i>burnetii</i> infection.

<p>THP-1 cells infected for 24 or 96 h and uninfected control cells were harvested and total protein immunoprecipitated with a PKA phospho substrate-specific antibody. (A) Immunoprecipitated proteins were subjected to Coomassie Blue staining and the arrow indicates a ~ 47 kDa protein only in infected cell samples. UI = Uninfected Cells. (B) Schematic of VASP showing five phosphorylation sites (Y39, S157, S239, T278, and S322) and EVH protein-protein interaction domains. PRO = proline rich region. (C) Immunoprecipitated proteins were subjected to immunoblot analysis using antibodies directed against VASP, phosphorylated VASP (S157), or PKA phospho substrates. Lysates were analyzed for β-tubulin to confirm equal protein loading. Agarose bead-bound PKA phospho substrate-specific antibody was eluted, immunoblotted, and probed with anti-IgG antibody to confirm equal amounts of antibody for immunoprecipitations. Densitometry analysis is shown in the graph and represents an average of three independent experiments. Error bars indicate the standard error of the mean. Control = uninfected cells. Increased levels of total and phosphorylated VASP (S157) are present in <i>C</i>. <i>burnetii</i>-infected cells.</p

VASP (S239E) expression prevents phagosome trafficking to the PV.

<p>THP-1 cells were transfected with wild type (WT) GFP-VASP (GFP-VASP), GFP-VASP (S157E), or GFP-VASP (S239E), then infected with <i>C</i>. <i>burnetii</i>. Cells were incubated with fluorescent beads (violet) overnight and processed for confocal microscopy at 72 hpi. DNA was stained with DAPI (blue). Bar, 10 μm. N = nucleus and * indicates the PV in micrographs. The scatter plot displays the percentage of beads present within PV in GFP-VASP-, GFP-VASP (S157E)-, or GFP-VASP (S239E)-expressing cells. At least 20 cells from randomly selected fields were used for quantification. The horizontal bar indicates average percentage. <b>*</b> indicates p < 0.0001 according to a Student’s <i>t</i> test comparing wild type- and mutant VASP-expressing cells. Bead-containing phagosomes were directed to the PV in GFP-VASP- and GFP-VASP (S157E)-expressing cells. However, GFP-VASP (S239E) expression impaired bead trafficking to the PV.</p

VASP activity is required for virulent <i>C</i>. <i>burnetii</i> PV formation.

<p>THP-1 cells (A) or primary hAMs (B) were infected with virulent <i>C</i>. <i>burnetii</i> and lysates collected at the indicated times. Immunoblotting was performed using antibodies directed against phosphorylated (S157 and S239) or total VASP (left panels). β-tubulin was used as a loading control. UI = Uninfected cells. Virulent <i>C</i>. <i>burnetii</i> triggered increased levels of phosphorylated VASP from 24–96 hpi in THP-1 cells and hAMs. In the middle and right panels, THP-1 cells (A) or hAMs (B) were transfected with non-targeting (NT) or VASP-specific siRNA. Cells were processed for confocal microscopy at 72 hpi. DNA was labeled with DAPI (blue), and CD63 (green) and <i>C</i>. <i>burnetii</i> (red) were detected with antibodies. Bar, 10 μm. VASP-depleted macrophages contain smaller PV than NT siRNA-transfected cells.</p