5 research outputs found
The 124202 candidate effector of <i>Melampsora larici-populina</i> interacts with membranes in <i>Nicotiana</i> and <i>Arabidopsis</i>
No full text<p><i>Melampsora larici-populina</i> (<i>Mlp</i>) is a parasitic fungus causing poplar leaf rust, a disease that threatens poplar plantations worldwide. Like other phytopathogens, <i>Mlp</i> translocates specialized proteins, called effectors, into host tissues and cells to eventually divert host resources. 124202 is a hypothetical <i>Mlp</i> effector selected through an <i>in silico</i> secretome analysis. It shares about 30% identity with the <i>M. lini</i> AvrM effector. Using heterologous systems, the objectives of this work were to assess if 124202 could mitigate <i>Arabidopsis</i> defence or potentiate <i>Pseudomonas</i> virulence and investigate its putative interaction partners in plants. Yeast two-hybrid screens identified three potential 124202 interactors in plants: lipoxygenase 2, synaptotagmin A and quinolinate synthase. Expression of a fluorescently tagged 124202 protein in <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i> showed that it could be associated with membranes but may also be found in the cytoplasm of host cells. Bacterial infection assays in wild-type and 124202-expressing <i>Arabidopsis</i> lines indicate that 124202 does not alter the susceptibility of <i>Arabidopsis</i> to <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000. Overall, our results suggest that the function of 124202 might involve vesicle-mediated trafficking but is unlikely to quantifiably contribute to the suppression of plant immunity.</p
Video_1_Preparing Arabidopsis thaliana root protoplasts for cryo electron tomography.mp4
No full textThe use of protoplasts in plant biology has become a convenient tool for the application of transient gene expression. This model system has allowed the study of plant responses to biotic and abiotic stresses, protein location and trafficking, cell wall dynamics, and single-cell transcriptomics, among others. Although well-established protocols for isolating protoplasts from different plant tissues are available, they have never been used for studying plant cells using cryo electron microscopy (cryo-EM) and cryo electron tomography (cryo-ET). Here we describe a workflow to prepare root protoplasts from Arabidopsis thaliana plants for cryo-ET. The process includes protoplast isolation and vitrification on EM grids, and cryo-focused ion beam milling (cryo-FIB), with the aim of tilt series acquisition. The whole workflow, from growing the plants to the acquisition of the tilt series, may take a few months. Our protocol provides a novel application to use plant protoplasts as a tool for cryo-ET.</p
