CRAGE-RB-PI-seq enables transcriptional profiling of rhizobacteria during plant-root colonization

Plant roots release a wide array of metabolites into the rhizosphere, shaping microbial communities and their functions. While metagenomics has expanded our understanding of these communities, little is known about the physiology of their members in host environments. Transcriptome analysis via RNA sequencing is a common approach to learning more, but its use has been challenging because plant RNA masks bacterial transcripts. To overcome this, we developed randomly-barcoded promoter-library insertion sequencing (RB-PI-seq) and combined it with chassis-independent recombinase assisted genome engineering (CRAGE), using Pseudomonas simiae WCS417 as a model rhizobacterium. This method enables targeted amplification of barcoded transcripts, bypassing plant RNA interference and allowing measurement of thousands of promoter activities during root colonization. Our analysis revealed time-resolved dynamics of promoter activities, highlighting early transcriptional reprogramming as a key determinant of successful colonization. Additionally, we discovered that transcriptional activation of xanthine dehydrogenase and a lysozyme inhibitor are crucial for evading plant immune system defenses. This framework is scalable to other bacterial species and provides new opportunities for understanding rhizobacterial gene regulation in native environments.