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1. Genetic Manipulation of Vibrio fischeri

   https://doi.org/10.1002/cpmc.115  

   Christensen, David G. ; Tepavčević, Jovanka ; Visick, Karen L. ( September 2020 , Current Protocols in Microbiology)

   Vibrio fischeriis a nonpathogenic organism related to pathogenicVibriospecies. The bacterium has been used as a model organism to study symbiosis in the context of its association with its host, the Hawaiian bobtail squidEuprymna scolopes. The genetic tractability of this bacterium has facilitated the mapping of pathways that mediate interactions between these organisms. The protocols included here describe methods for genetic manipulation ofV. fischeri. Following these protocols, the researcher will be able to introduce linear DNA via transformation to make chromosomal mutations, to introduce plasmid DNA via conjugation and subsequently eliminate unstable plasmids, to eliminate antibiotic resistance cassettes from the chromosome, and to randomly or specifically mutagenizeV. fischeriwith transposons. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Transformation ofV. fischeriwith linear DNA

   Basic Protocol 2: Plasmid transfer intoV. fischerivia conjugation

   Support Protocol 1: Removing FRT‐flanked antibiotic resistance cassettes from theV. fischerigenome

   Support Protocol 2: Eliminating unstable plasmids fromV. fischeri

   Alternate Protocol 1: Introduction of exogenous DNA using a suicide plasmid

   Alternate Protocol 2: Site‐specific transposon insertion using a suicide plasmid

   Alternate Protocol 3: Random transposon mutagenesis using a suicide plasmid

    

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2. Proper Care and Feeding of Coccidioides : A Laboratorian's Guide to Cultivating the Dimorphic Stages of C. immitis and C. posadasii

   https://doi.org/10.1002/cpmc.113  

   Mead, Heather L. ; Van Dyke, Marley C. Caballero ; Barker, Bridget M. ( September 2020 , Current Protocols in Microbiology)

   Coccidioidomycosis (“Valley fever”) is caused byCoccidioides immitisandC. posadasii. These fungi are thermally dimorphic, cycling between mycelia and arthroconidia in the environment and converting into spherules and endospores within a host.Coccidioidescan cause a broad spectrum of disease that can be difficult to treat. There has been a steady increase in disease, with an estimated 350,000 new infections per year in the United States. With the increase in disease and difficulty in treatment, there is an unmet need to increase research in basic biology and identify new treatments, diagnostics, and vaccine candidates. Here, we describe protocols required in anyCoccidioideslaboratory, such as growing, harvesting, and storing the different stages of this dimorphic fungal pathogen. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Growth and harvest of liquid mycelia cultures for extractions

   Alternate Protocol 1: Large‐volume growth and harvest of liquid mycelia cultures

   Basic Protocol 2: Mycelial growth on solid medium

   Alternate Protocol 2: Maintaining mycelial growth on solid medium

   Basic Protocol 3: Harvesting and quantification of arthroconidia

   Alternate Protocol 3: Long‐term storage of arthroconidia

   Basic Protocol 4: Parasitic spherule growth and harvest

   Alternate Protocol 4: Obtaining endospores from spherules

   Basic Protocol 5: Intranasal infection of murine models

    

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3. Targeted Genetic Changes in Candida albicans Using Transient CRISPR‐Cas9 Expression

   https://doi.org/10.1002/cpz1.19  

   Huang, Manning Y. ; Cravener, Max V. ; Mitchell, Aaron P. ( January 2021 , Current Protocols)

   Candida albicansis an opportunistic fungal pathogen responsible for significant disease and mortality. Absent complete mating and other convenient methods, dissection of its virulence factors relies on robust tools to delete, complement, and otherwise modify genes of interest in this diploid organism. Here we describe the design principles and use of CRISPR associated nuclease 9 (Cas9) and single‐guide RNAs transiently expressed from PCR cassettes to modify genes of interest, generating homozygous mutants in a single transformation step. © 2021 Wiley Periodicals LLC.

   This article was corrected on 27 February 2023. See the end of the full text for details.

   Basic Protocol 1: PCR amplification of CRISPR components

   Basic Protocol 2: Transformation ofCandida albicans

   Basic Protocol 3: Selecting and genotyping transformants

   Alternate Protocol 1: Deletion with recyclable markers by CRISPR induced marker excision (CRIME)

   Alternate Protocol 2: Knock‐in and combining multiple cassettes with overlapping homology

    

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4. Soybean Hairy Root Transformation: A Rapid and Highly Efficient Method

   https://doi.org/10.1002/cpz1.195  

   Song, Jaehyo ; Tóth, Katalin ; Montes‐Luz, Bruna ; Stacey, Gary ( July 2021 , Current Protocols)

   New genetic engineering techniques have advanced the field of plant molecular biology, andAgrobacterium‐mediated transformation has enabled the discovery of numerous molecular and genetic functions. It has been widely used in many plants, including the economically important crop, soybean. Large‐scale genetic analyses are needed to comprehend the molecular mechanisms that underlie the agronomic traits of soybean, and the generation of stable transgenic plants involves a lengthy and laborious process.Agrobacterium rhizogenes‐mediated hairy root transformation is a quick and efficient method for investigations of root‐specific processes and interactions. Generation of composite plants with transgenic roots and wild‐type shoots allows for the study of the genetic mechanisms involved in root biology, such as theBradyrhizobium‐soybean interaction. Here, we provide an updated protocol for generating hairy soybean roots in as little as 18 days in a cost‐ and space‐effective manner and demonstrate possible uses of composite plants with soybean nodulation assays and gene expression analysis as examples. © 2021 Wiley Periodicals LLC.

   Basic Protocol 1: Soybean hairy root transformation

   Basic Protocol 2: Soybean nodulation assay

   Alternate Protocol: Soybean nodulation assay in germination pouches

   Support Protocol:Bradyrhizobium japonicumculture preparation for inoculation

   Basic Protocol 3: Histochemical GUS staining for promoter analysis

    

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5. Corynebacterium diphtheriae Virulence Analyses Using a Caenorhabditis elegans Model

   https://doi.org/10.1002/cpmc.109  

   Chen, Yi‐Wei ; Ton‐That, Hung ( July 2020 , Current Protocols in Microbiology)

   Corynebacterium diphtheriaeis the leading cause of pharyngeal diphtheria, a respiratory disease characterized by formation of a pseudomembrane at the site of infection. Although outbreaks ofC. diphtheriaeinfections are rare nowadays, the emergence of multidrug‐resistantC. diphtheriaestrains is one of the most significant public health concerns worldwide. AlthoughC. diphtheriaehas been studied for more than a century and diphtheria toxin and pili have been identified as major virulence factors, little is known about factors involved in bacterial colonization and development of disease. Here, we describe the utilization ofCaenorhabditis elegansas a cost‐effective, versatile model of infection to evaluateC. diphtheriaevirulence. We provide detailed protocols for nematode synchronization and for evaluation of nematode survival and formation of a deformed anal region induced byC. diphtheriaeinfection. These protocols will permit future high‐throughput screenings of virulence factors inC. diphtheriaeand advance our knowledge ofC. diphtheriaepathogenesis. © 2020 Wiley Periodicals LLC.

   Basic Protocol 1: Synchronization of nematodes

   Basic Protocol 2: Assay for nematode survival followingC. diphtheriaeinfection

   Basic Protocol 3: Assays for bacterial colonization and formation of deformed anal region

    

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