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    Protocols in Current Issue
    In-vitro and in-planta Botrytis cinerea Inoculation Assays for Tomato
    Authors:  Jiajie Lian, Hongyu Han, Jiuhai Zhao and Chuanyou Li, date: 04/20/2018, view: 117, Q&A: 0
    Botrytis cinerea (B. cinerea) attacks many crops of economic importance, represents one of the most extensively studied necrotrophic pathogens. Inoculation of B. cinerea and phenotypic analysis of plant resistance are key ...
    Metal-tagging Transmission Electron Microscopy for Localisation of Tombusvirus Replication Compartments in Yeast
    Authors:  Isabel Fernández de Castro and Cristina Risco, date: 04/20/2018, view: 69, Q&A: 0
    Positive-stranded (+) RNA viruses are intracellular pathogens in humans, animals and plants. To build viral replicase complexes (VRCs) viruses manipulate lipid flows and reorganize subcellular membranes. Redesigned membranes concentrate viral and ...
    Method for CRISPR/Cas9 Mutagenesis in Candida albicans
    Authors:  Neta Dean and Henry Ng, date: 04/20/2018, view: 116, Q&A: 0
    Candida albicans is the most prevalent and important human fungal pathogen. The advent of CRISPR as a means of gene editing has greatly facilitated genetic analysis in C. albicans. Here, we describe a detailed step-by-step ...
    In-vitro and in-planta Botrytis cinerea Inoculation Assays for Tomato
    Authors:  Jiajie Lian, Hongyu Han, Jiuhai Zhao and Chuanyou Li, date: 04/20/2018, view: 117, Q&A: 0
    [Abstract] Botrytis cinerea (B. cinerea) attacks many crops of economic importance, represents one of the most extensively studied necrotrophic pathogens. Inoculation of B. cinerea and phenotypic analysis of plant resistance are key procedures to investigate the mechanism of plant immunity. Here we describe a protocol for B. ...
    Metal-tagging Transmission Electron Microscopy for Localisation of Tombusvirus Replication Compartments in Yeast
    Authors:  Isabel Fernández de Castro and Cristina Risco, date: 04/20/2018, view: 69, Q&A: 0
    [Abstract] Positive-stranded (+) RNA viruses are intracellular pathogens in humans, animals and plants. To build viral replicase complexes (VRCs) viruses manipulate lipid flows and reorganize subcellular membranes. Redesigned membranes concentrate viral and host factors and create an environment that facilitates the formation of VRCs within replication ...
    Method for CRISPR/Cas9 Mutagenesis in Candida albicans
    Authors:  Neta Dean and Henry Ng, date: 04/20/2018, view: 116, Q&A: 0
    [Abstract] Candida albicans is the most prevalent and important human fungal pathogen. The advent of CRISPR as a means of gene editing has greatly facilitated genetic analysis in C. albicans. Here, we describe a detailed step-by-step procedure to construct and analyze C. albicans deletion mutants. This protocol uses plasmids that ...
    A Microfluidic Device for Massively Parallel, Whole-lifespan Imaging of Single Fission Yeast Cells
    Authors:  Stephen K Jones Jr, Eric C Spivey, James R Rybarski and Ilya J Finkelstein, date: 04/05/2018, view: 233, Q&A: 0
    [Abstract] Whole-lifespan single-cell analysis has greatly increased our understanding of fundamental cellular processes such as cellular aging. To observe individual cells across their entire lifespan, all progeny must be removed from the growth medium, typically via manual microdissection. However, manual microdissection is laborious, low-throughput, and ...
    Tracking Lipid Transfer by Fatty Acid Isotopolog Profiling from Host Plants to Arbuscular Mycorrhiza Fungi
    Authors:  Andreas Keymer, Claudia Huber, Wolfgang Eisenreich and Caroline Gutjahr, date: 04/05/2018, view: 181, Q&A: 0
    [Abstract] Lipid transfer from host plants to arbuscular mycorrhiza fungi was hypothesized for several years because sequenced arbuscular mycorrhiza fungal genomes lack genes encoding cytosolic fatty acid synthase (Wewer et al., 2014; Rich et al., 2017). It was finally shown by two independent experimental approaches (Jiang et al., ...
    Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes
    Authors:  Yuichi Ichikawa and Paul D. Kaufman, date: 03/20/2018, view: 338, Q&A: 0
    [Abstract] Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the ...
    Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9
    [Abstract] Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended ...
    Design of Hybrid RNA Polymerase III Promoters for Efficient CRISPR-Cas9 Function
    Authors:  Joshua Misa, Cory Schwartz and Ian Wheeldon, date: 03/20/2018, view: 359, Q&A: 0
    [Abstract] The discovery of the CRISPR-Cas9 system from Streptococcus pyogenes has allowed the development of genome engineering tools in a variety of organisms. A frequent limitation in CRISPR-Cas9 function is adequate expression levels of sgRNA. This protocol provides a strategy to construct hybrid RNA polymerase III (Pol III) promoters that ...
    Large Scale Field Inoculation and Scoring of Maize Southern Leaf Blight and Other Maize Foliar Fungal Diseases
    Authors:  Shannon M. Sermons and Peter J. Balint-Kurti, date: 03/05/2018, view: 249, Q&A: 0
    [Abstract] Field-grown maize is inoculated with Cochliobolus heterostrophus, causal agent of southern leaf blight disease, by dropping sorghum grains infested with the fungus into the whorl of each maize plant at an early stage of growth. The initial lesions produce secondary inoculum that is dispersed by wind and rain, causing multiple cycles of ...
    An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking
    [Abstract] Formaldehyde crosslinking is widely used in combination with chromatin immunoprecipitation (ChIP) to measure the locations along DNA and relative levels of transcription factor (TF)-DNA interactions in vivo. However, the measurements that are typically made do not provide unambiguous information about the dynamic properties of these ...