Developing a Massively Parallel Reporter Assay to Profile in Planta transcriptional Activities of Plant-Associated Bacteria

Plant roots excrete a variety of metabolites into the rhizosphere and create unique habitats for the microbial communities. Recent metagenomic studies have considerably enriched our genomic knowledge of the plant microbiome. However, much less is studied about the rhizobacterial physiology in association with host plants. One approach to understand it is to measure their transcriptional activities, yet the existing methodologies have limitations: conventional methods such as fluorescent reporters and qPCR are limited to measure a handful of promoter activities, while utilizing genome-wide transcriptomics has been challenging due to a small fraction of bacterial transcripts that are hindered by plant RNA. To this objective, synthetic biology offers an alternative approach. A massively parallel reporter assay (MPRA), which employs DNA barcode as a reporter of promoter activity, allows simultaneous measurements of thousands of promoter activities in a simple workflow. In this study, we developed MPRA in a model rhizobacterium Pseudomonas simiae WCS417, leveraging CRAGE (chassis-independent recombinase-assisted genome engineering) to construct a library of strains with barcoded promoters. We established a high-throughput measurement of in planta promoter activities of P. simiae WCS417 without taking care of plant RNA. Furthermore, we identified a set of uniquely regulated promoters in their process of Arabidopsis root colonization and verified these regulated genes substantially influence the root-colonization efficiency. The framework developed here is efficient and scalable, thereby providing an increasing opportunity to understand gene regulation and physiology of rhizobacteria in their host environments.