Transcriptional profiling of rhizobacteria during plant-root colonization using CRAGE-RB-PI-seq

Characterizing microbial gene expression during plant root colonization is key to better understanding plant-microbe interactions. However, such studies have been technically challenging because microbial RNA is present at low levels compared to the abundant plant RNA in collected samples. The goal of this study is to develop a synthetic biology approach for transcriptomic analysis of root-colonizing bacteria. To achieve this, a randomly barcoded promoter-library insertion sequencing (RB-PI-seq) method was developed using Pseudomonas simiae WCS417 as a model organism. Barcoded promoter libraries were integrated into the chromosome of P. simiae WCS417 using chassis-independent recombinase-assisted genome engineering (CRAGE). Both in vitro and in planta assays were performed using a pooled cell library. Promoter activities were quantified by targeted RNA-seq and DNA-seq, with RNA-derived barcode counts normalized to DNA-derived counts for each promoter.