Abstract
Profiling bacterial transcriptome in planta is challenging due to the low abundance of bacterial RNA in infected plant tissues. Here, we describe a protocol to profile transcriptome of a foliar bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, in the leaves of Arabidopsis thaliana at an early stage of infection using RNA sequencing (RNA-Seq). Bacterial cells are first physically isolated from infected leaves, followed by RNA extraction, plant rRNA depletion, cDNA library synthesis, and RNA-Seq. This protocol is likely applicable not only to the A. thaliana–P. syringae pathosystem but also to different plant-bacterial combinations.
Keywords: RNA-Seq, Bacterial pathogen, Arabidopsis, Transcriptome, Pseudomonas
Background
Plants have evolved innate immune systems to fend off pathogen attack. Molecular mechanisms of pathogen recognition and immune signaling pathways have been intensively studied over the past decades. However, how plant immunity affects pathogen metabolism to inhibit pathogen growth is scarcely understood often because of the difficulty in profiling pathogen responses in planta. In case of bacterial pathogens, transcriptome profiling inside plant leaves is difficult to study because the amount of bacterial mRNA is much lower than that of plants; and it is particularly challenging at an early stage of infection due to the low population density of bacteria in plants. To overcome this limitation, we established a method for isolating bacteria from the infected plant leaves and profiling bacterial transcriptome with RNA-Seq. This method has been successfully used for profiling the transcriptome of the model bacterial pathogen Pseudomonas syringae pv. tomato DC3000 in the model plant Arabidopsis thaliana in various conditions (Nobori et al., 2018).
Materials and Reagents
Equipment
Software
Procedure
The overview of the procedure is shown in Figure 1. Figure 1. Workflow summary of in planta bacterial transcriptome analysis. The figure is adopted from Nobori et al. (2018), PNAS. Leaves infected with P. syringae are crushed and incubated in the bacterial isolation buffer, followed by filtering out large plant tissues. Then the flow through is centrifuged to separate bacterial cells from plant cells. Bacterial cells are harvested and subjected to RNA extraction, cDNA library preparation, and RNA-seq.
Data analysis
Mapping and counting of Illumina reads for transcriptome:
Notes
Recipes
Acknowledgments
This protocol has been adapted from a previously published paper (Nobori et al., 2018). This work was supported by the Max Planck Society and Deutsche Forschungsgemeinschaft Grant SFB670 (to K.T.) and a predoctoral fellowship from the Nakajima Foundation (to T.N.).
Competing interests
The authors declare no conflicts of interest or competing interests.
References
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