Experimental evolution-driven identification of Arabidopsis rhizosphere competence genes in Pseudomonas protegens

Beneficial plant root-associated microorganisms carry out a range of functions that are essential for plant performance. Establishment of a bacterium on plant roots, however, requires overcoming several challenges, including the ability to outcompete neighboring microorganisms and suppression of plant immunity. Forward and reverse genetics approaches have led to the identification of diverse mechanisms that are used by beneficial microorganisms to overcome these challenges such as the production of iron-chelating compounds, biofilm formation, or downregulation of plant immunity. However, how such mechanisms have developed from an evolutionary perspective is much less understood. In an attempt to study bacterial adaptation in the rhizosphere, we employed an experimental evolution approach to track the physiological and genetic dynamics of root-dwelling Pseudomonas protegens CHA0 in the Arabidopsis thaliana rhizosphere under axenic conditions. This simplified binary one plant, and one bacterium system allows for the amplification of key adaptive mechanisms for bacterial rhizosphere colonization. We found that mutations in global regulators, as well as in genes for siderophore production, cell surface decoration, attachment, and motility accumulated in parallel in our evolutionary experiment, underlining several different strategies of bacterial adaptation to the rhizosphere. In total we identified 35 mutations, including single-nucleotide polymorphisms, smaller indels and larger deletions, distributed over 28 genes in total. Altogether these results underscore the strength of experimental evolution to identify key genes and pathways for bacterial rhizosphere colonization, as well as highlighting a methodology for the development of elite beneficial microorganisms with enhanced root-colonizing capacities that can support sustainable agriculture in the future.