While beneficial plant-microbe interactions are common in nature, direct evidence for the evolution of bacterial mutualism is scarce. Here we use experimental evolution to causally show that initially plant-antagonistic Pseudomonas protegens bacteria evolve into mutualists in the rhizosphere of Arabidopsis thaliana within six plant growth cycles (6 months). This evolutionary transition is accompanied with increased mutualist fitness via two mechanisms: (i) improved competitiveness for root exudates and (ii) enhanced tolerance to the plant-secreted antimicrobial scopoletin whose production is regulated by transcription factor MYB72. Crucially, these mutualistic adaptations are coupled with reduced phytotoxicity, enhanced transcription of MYB72 in roots, and a positive effect on plant growth. Genetically, mutualism is associated with diverse mutations in the GacS/GacA two-component regulator system, which confers high fitness benefits only in the presence of plants. Together, our results show that rhizosphere bacteria can rapidly evolve along the parasitism-mutualism continuum at an agriculturally relevant evolutionary timescale.

• Arguably, symbiotic arbuscular mycorrhizal (AM) fungi have the broadest host range of all fungi, being able to intracellularly colonize root cells in the vast majority of all land plants. This raises the question how AM fungi effectively deal with the immune systems of such a widely diverse range of plants. •Here, we studied the role of a nuclear-localization signal containing effector from Rhizophagus irregularis, called Nuclear Localized Effector1 (RiNLE1), that is highly and specifically expressed in arbuscules. •We show that RiNLE1 is able to translocate to the host nucleus where it interacts with the plant core nucleosome protein Histone 2B (H2B). RiNLE1 is able to impair the mono-ubiquitination of H2B, which results in the suppression of defense-related gene expression and enhanced colonization levels. • This study highlights a novel mechanism by which AM fungi can effectively control plant epigenetic modifications through direct interaction with a core nucleosome component. Homologs of RiNLE1 are found in a range of fungi that establish intimate interactions with plants, suggesting that this type of effector may be more widely recruited to manipulate host defense responses.

Beneficial root-associated microorganisms carry out many functions that are essential for plant performance. Establishment of a bacterium on plant roots, however, requires overcoming many challenges.

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.

SummaryEven though beneficial plant-microbe interactions are commonly observed in nature, direct evidence for the evolution of bacterial mutualism in the rhizosphere remains elusive. Here we use experimental evolution to causally show that initially plant-antagonistic Pseudomonas protegens bacterium evolves into mutualists in the rhizosphere of Arabidopsis thaliana within six plant growth cycles (6 months). This evolutionary transition was accompanied with increased mutualist fitness via two mechanisms: i) improved competitiveness for root exudates and ii) enhanced capacity for activating the root-specific transcription factor gene MYB72, which triggers the production of plant-secreted scopoletin antimicrobial for which the mutualists evolved relatively higher tolerance to. Genetically, mutualism was predominantly associated with different mutations in the GacS/GacA two-component regulator system, which conferred high fitness benefits only in the presence of plants. Together, our results show that bacteria can rapidly evolve along the parasitism-mutualism continuum in the plant rhizosphere at an agriculturally relevant evolutionary timescale.