Dissection of Genes in Plant Immunity Suppressing Bacteria that Impact Fitness and Plant Colonization

Abstract

Plant root exudates shape the structure and function of the rhizosphere microbiome by providing chemical cues and substrates that influence microbial survival, colonization, and interaction with the host. These exudates can select for beneficial microbes that support plant growth or suppress immunity, making them critical in mediating plant-microbe interactions. This study focuses on two Arabidopsis thaliana (Arabidopsis) associated, immunity-suppressive bacterial strains, Dyella japonica MF79 (MF79) and Brevundimonas sp. MF374 (MF374), which use distinct genetic mechanisms to suppress root immune responses. To evaluate how the root exudate composition influences bacterial fitness, transposon mutant libraries of MF374 and MF79 were grown in exudates derived from Arabidopsis Col-0, fls2 mutant, and cyp79b2/b3 mutant lines. Genome-wide fitness profiling using random barcode transposon-site sequencing (RB-TnSeq) and Random Forest classification revealed minimal fitness changes in response to flg22 treatment, but strong genotype-specific fitness differences driven by root exudate composition. In MF374, candidate genes involved in osmotic regulation, cell wall synthesis, oxidative stress response, and translation were identified as major contributors to fitness in root exudate environments. Targeted gene deletions confirmed reduced fitness in immune-active or metabolite-rich exudates; however, root colonization assays demonstrated that these fitness effects did not always translate to differences in colonization capacity. This framework enabled the identification and functional assessment of bacterial genes important for exudate-mediated fitness and adaptation. The findings offer insight into how variation in root exudate composition influences the assembly, persistence, and adaptation of immunity-suppressive bacteria within the root microbiome and have broad implications for understanding plant health and developing strategies for microbiome-based agricultural applications.