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Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH).

ABSTRACT: Background:Plasmopara obducens is the biotrophic oomycete responsible for impatiens downy mildew, a destructive disease of Impatiens that causes high crop loss. Currently, there are no available methods for the microscopic detection of P. obducens from leaves of impatiens, which may be contributing to the spread of the disease. Fluorescence in situ hybridization (FISH) is a sensitive and robust method that uses sequence-specific, fluorescence-labeled oligonucleotide probes to detect target organisms from the environment. To study this important pathogen, we developed and standardized a FISH technique for the visualization of P. obducens from Impatiens walleriana tissues using a species-specific 24-mer oligonucleotide probe designed to target a region of the rRNA internal transcribed spacer 2 (ITS2). Results:Since P. obducens cannot be propagated in vitro, we developed a custom E. coli expression vector that transcribes the P. obducens rRNA-ITS target sequence (clone-FISH) for use as a control and to optimize hybridization conditions. The FISH assay could detect P. obducens sporangiophores, sporangia and oospores, and hyphae from naturally infected I. walleriana leaves and stems. Cross-reactivity was not observed from plant tissue, and the assay did not react when applied to E. coli with self-ligated plasmids and non-target oomycete species. Conclusions:This FISH protocol may provide a valuable tool for the study of this disease and could potentially be used to improve early monitoring of P. obducens, substantially reducing the persistence and spread of this destructive plant pathogen.

SUBMITTER: Salgado-Salazar C

PROVIDER: S-EPMC6199785 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH).

Salgado-Salazar Catalina C Bauchan Gary R GR Wallace Emma C EC Crouch Jo Anne JA

Plant methods 20181024

<h4>Background</h4><i>Plasmopara obducens</i> is the biotrophic oomycete responsible for impatiens downy mildew, a destructive disease of <i>Impatiens</i> that causes high crop loss. Currently, there are no available methods for the microscopic detection of <i>P. obducens</i> from leaves of impatiens, which may be contributing to the spread of the disease. Fluorescence in situ hybridization (FISH) is a sensitive and robust method that uses sequence-specific, fluorescence-labeled oligonucleotide ...[more]

PMID: 30386410

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Project description:Changes in gene expression form a crucial part of the plant response to pathogen infection. Whole-leaf expression profiling has played a valuable role in identifying genes and processes that contribute to the interactions between the model plant Arabidopsis thaliana and a diverse range of pathogens. However, for highly localised infections, such as downy mildew caused by the biotrophic oomycete pathogen Hyaloperonospora arabidopsidis (Hpa), whole-leaf profiling may fail to capture the complete Arabidopsis response. Highly localised expression changes may be diluted by the comparative abundance of non-responding leaf cells or the Hpa oomycete evading detection by cells. Furthermore, local and systemic Hpa responses of a differing nature may become convoluted. To address this we applied the technique of Fluorescence Activated Cell Sorting (FACS), typically used for analyzing plant abiotic responses, to the study of plant-pathogen interactions. Using the promoter of Downy Mildew Resistant 6 (DMR6) linked to GFP as a fluorescent marker of pathogen-contacting cells, we isolated Hpa-proximal and Hpa-distal cells from infected leaf samples using FACS, and measured global gene expression. The whole experiment was carried out in triplicate, with two time points (5 and 7 days post-inoculation) and three cell types (pathogen-proximal, pathogen-distal and uninfected control), totalling 18 microarrays. Transgenic Arabidopsis thaliana Col-0 with the transgene pDMR6:GFP was used for all experiments. Seedling populations were inoculated with 30,000-60,000 spores/ml of Hyaloperonospora arabidopsis strain Noks1 at 7 days old, and overground tissue sampled at 5 and 7 days post-inoculation. Protoplasts were generated from these samples according to Grønlund et al. 2012 JOVE, and sorted by fluorescence activated cell sorting into a GFP-positive (pathogen-proximal) and GFP-negative (pathogen-distal) population. As a control, uninfected seedlings were also sampled at 12 and 14 days old (equivalent age of 5 and 7 days post-inoculation), protoplasts generated and sorted to yield a GFP-negative population of uninfected control cells. Gene expression was analysed using NimbleGen 12 x 135K microarrays, designed for the TAIR10 genome. Data was normalised using the Robust Multichip Averaging algorithm.

Dataset Information

Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH).

Publications

Visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH).

Similar Datasets

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