Abstract
Current and emerging plant diseases caused by obligate parasitic microbes such as rusts, downy mildews, and powdery mildews threaten worldwide crop production and food safety. These obligate parasites are typically unculturable in the laboratory, posing technical challenges to characterize them at the genetic and genomic level. Here we have developed a data analysis pipeline integrating several bioinformatic software programs. This pipeline facilitates rapid gene discovery and expression analysis of a plant host and its obligate parasite simultaneously by next generation sequencing of mixed host and pathogen RNA (i.e., metatranscriptomics). We applied this pipeline to metatranscriptomic sequencing data of sweet basil (Ocimum basilicum) and its obligate downy mildew parasite Peronospora belbahrii, both lacking a sequenced genome. Even with a single data point, we were able to identify both candidate host defense genes and pathogen virulence genes that are highly expressed during infection. This demonstrates the power of this pipeline for identifying genes important in host–pathogen interactions without prior genomic information for either the plant host or the obligate biotrophic pathogen. The simplicity of this pipeline makes it accessible to researchers with limited computational skills and applicable to metatranscriptomic data analysis in a wide range of plant-obligate-parasite systems.
Highlights
Many devastating agricultural plant diseases are caused by obligate parasitic microbes
To demonstrate the application of this pipeline, we generated a test set of RNA-seq data from sweet basil infected with P. belbahrii (Figure 2)
We have developed a computational pipeline composed of freely available software for analyzing metatranscriptomic data
Summary
Many devastating agricultural plant diseases are caused by obligate parasitic microbes. These parasites include fungi, such as rusts (Hulbert and Pumphrey, 2014) and powdery mildews (Glawe, 2008), and oomycetes such as downy mildews (Yarwood, 1956; Perfect and Green, 2001). Obligate parasites are typically recalcitrant to axenic culture, resistant to genetic manipulation, and require living host plants to survive and propagate (Glazebrook, 2005; Bindschedler et al, 2016). These characteristics make it challenge to study the pathogenesis using conventional genetics and molecular biology, impeding the development of effective control strategies. Numerous transcriptomic studies have applied RNA-seq to plants, plant pathogens, or mixed host–pathogen samples (metatranscriptomics)
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