Genome-wide interrogation of genetic requirements for root colonization by CRISPRi-seq in Bacillus subtilis

Bacillus subtilis is a known plant growth promoting rhizobacterium. Yet, the genetic basis for effective B. subtilis root colonization remains unclear. Here, we applied genome-wide CRISPR interference sequencing (CRISPRi-seq) to systematically identify bacterial genes required for early Arabidopsis thaliana root colonization by the undomesticated B. subtilis strain NCIB3610. Complementary bacterial RNA-seq experiments in the same conditions further revealed a distinct transcriptional state of root-associated cells. Together, we identified 249 B. subtilis genes impacting root colonization. We reveal a critical, biofilm-independent, role of cell envelope remodeling during early root association, specifically wall teichoic acid synthesis and D-alanylation. Moreover, we observed a metabolic shift toward TCA-cycle-driven aerobic growth and utilization of fructose, induction of secondary metabolite biosynthesis, as well as active repression of sporulation, competence and prophage programs. Our work establishes CRISPRi-seq as a powerful tool for dissecting bacterial-plant interactions relevant to sustainable agriculture.