In planta bacterial RNA-Seq with bacterial isolation.

Plant pathogens can cause serious diseases that impact global agriculture1. Understanding how the plant immune system naturally restricts pathogen infection holds a key to sustainable disease control in modern agricultural practices. However, despite extensive studies into the molecular and genetic basis of plant defense against pathogens since the 1950s2,3, one of the most fundamental questions in plant pathology remains unanswered: how resistant plants halt pathogen growth during immune activation. In the case of bacterial infections, a major bottleneck is an inability to determine the global bacterial transcriptome and metabolic responses in planta. Here, we developed an innovative pipeline that allows for in planta high-resolution bacterial transcriptome analysis with RNA-Seq, using the model plant Arabidopsis thaliana and the foliar bacterial pathogen Pseudomonas syringae. We examined a total of 27 combinations of plant immunity and bacterial virulence mutants to gain an unprecedented insight into the bacterial transcriptomic responses during plant immunity. We were able to identify specific bacterial transcriptomic signatures that are linked to bacterial inhibition during two major forms of plant immunity: pattern-triggered immunity and effector-triggered immunity. Among them, regulation of a P. syringae sigma factor gene, involved in iron regulation and an unknown process(es), was found to play a causative role in bacterial restriction during plant immunity. This study unlocked the enigmatic mechanisms of bacterial growth inhibition during plant immunity; results have broad basic and practical implications for future study of plant diseases. In planta bacterial RNA-Seq was profiled in 27 combinations (100 samples) of four P. syringae pv. tomato DC3000 and nine Arabidopsis thaliana genotypes. Bacterial suspension was syringe infiltrated to plant leaves at OD600=0.5. At 6 h after infection, the infected leaves were harvested and bacterial cells were isolated, followed by RNA extraction. Ribosomal RNA of bacteria and plants were removed with a commercial kit to enrich bacterial mRNA. Then, cDNA libraries were generated and RNA-Seq was performed. Bacteria grown in liquid media (Minimal medium and/or King’s B medium) were also analyzed. For most of the samples, three independent replicates were taken.