The sum and diversity of all microorganisms colonizing a plant is referred to as the plant microbiota. When grown in the same soil and environment, the composition of the microbiota varies between different plant species and genotypes, indicative of co-diversification of the microbiota with its plant hosts^2,3. Members of the microbiota form commensal relationships with and provide beneficial services to the plant host⁸, including mobilization of nutrients from soil for uptake by roots^9,10, indirect pathogen protection^11-13, and abiotic stress tolerance^3,14.

Beneficial activities are often strain-specific and depend on the presence or absence of only a few gene products. To determine the composition of the plant microbiota, microbial marker genes characterized by alternating hypervariable and conserved regions are commonly sequenced and natural nucleotide polymorphisms are employed to define different microbial taxa.

However, microbial community profiling based on marker gene sequences is insufficient for resolving community structures to a level that distinguishes near-isogenic strains with differential activities. Further, as in natural environments plants are colonized by a multitude of different microorganisms simultaneously, the establishment of the microbiota is not only dependent on microbe–host, but also microbe–microbe interactions. Thus, while attempting to identify genetic determinants that render a bacterial strain a robust member of the root microbiota using strain-specific genetic variations and mutant libraries, we started to approach the problem of how the identified genetic determinants could later be characterized in complex microbial communities, such as those found in natural environments. Thereby, we can estimate the contribution of a particular gene encoded by a member of the microbiota not only to the colonization efficiency in general but also to the prevalence of this strain in competition with other microbes on roots.

We developed the Modular Bacterial Tag (MoBacTag) toolkit with the basic idea of labelling near-isogenic strains of interest by chromosomally integrating an DNA barcode as an artificial marker gene sequence that is co-amplified during community profiling. We decided to include four different antibiotic resistance cassettes, as strains of the Arabidopsis culture collection (At-SPHERE²³) naturally encode diverse antibiotic resistances and we wished to design a toolkit that would be applicable for taxonomically diverse bacteria. As root zones differ not only in their physical properties, but also in their exudates and immune responses²⁴, we further decided to include transcription units for fluorescent proteins that allow correlation of abundances of near-isogenic strains in community profiling with strain-specific colonization patterns. To achieve these goals, we opted for GoldenGate-based modular cloning that enabled us to combine different antibiotic cassettes and fluorescent markers and allows high flexibility for easy extensions or replacement of specific expression cassettes, antibiotic resistance markers or primer binding sites.

When the first MoBacTag vectors were assembled, corresponding author Paul Schulze-Lefert, co-first author Julien Thouin and I sat down and thought about how to test the tool in a community profiling experiment. We discussed two options for a proof-of-principle experiment using either a flagellum mutant, as motility is required for efficient root colonization²⁵, or mutants of the plant growth-promoting rhizobacterium (PGPR) Pseudomonas capeferrum (P. capeferrum) WCS358, WCS358::pqqF and WCS358::cyoB, recently identified by the group of Roeland Berendsen, that are impaired in immunosuppression and root colonization²⁶. PqqF and cyoB commonly function in the production of gluconic acid and its derivative 2-keto gluconic acid, which suppress plant immunity by lowering extracellular pH locally²⁶. Suppression of host immune responses by pathogenic microbes invariably leads to increased pathogen proliferation²⁷ raising the question of whether other microbiota members also benefit indirectly from compromised host defenses. Thus, we decided to use the immunosuppressive P. capeferrum mutants for our proof-of-principle experiment to potentially gain new insights into how immunosuppression changes a bacterial community on A. thaliana roots.

We were able to label P. capeferrum wild type and the cyoB and pqqF mutants and recapitulated the colonization defects of both mutants using the DNA barcode-specific reads during community profiling, thus demonstrating the functionality of our MoBacTag toolkit. Moreover, we observed a reduction of total bacterial load on A. thaliana roots specifically upon co-colonization with the pqqF mutant revealing distinct activities of pqqF and cyoB in a microbial community context. As the pqqF mutant is impaired in the biosynthesis of PQQ, a co-factor of several dehydrogenases including the glucose dehydrogenase involved in gluconic acid production, we propose that the overall reduced microbial load on roots in the presence of the pqqF mutant results from depletion of extracellular PQQ, which might serve as a common good during root microbiota establishment and is shared between microbiota members. Thus, by using the MoBacTags we were not only able to recapitulate published data for our proof-of-principle experiment but also unexpectedly observed a phenotype specific to the pqqF immunosuppressive mutant, which would have been difficult to detect using conventional colonization estimates of near-isogenic strains.

We will deposit the MoBacTag toolkit with AddGene to allow colleagues to label their favorite members of the plant microbiota.

Thanks to Neysan Donnelly and Paul Schulze-Lefert for feedback on this article.

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