In planta bacterial RNA-Seq without 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. Two leaves from three four-and-one-half week-old A. thaliana plants were infiltrated with either 0.005% dimethyl sulfoxide (DMSO, mock) or 500 nM flg22 using a needleless syringe. Twenty hours post-infiltration, plants were inoculated with a bacterial suspension at an OD600 of 0.75 (10^9 CFU ml-1) of Pto DC3000. Seven hours after Pto DC3000 inoculation, leaves were collected for RNA extraction. Three independent replicates (including 3 technical replicates for flg22 treated samples) were taken.