Glutamate facilitates root colonization by plant growth-promoting rhizobacteria Bacillus subtilis in tomato seedlings

The influence of microbially and plant-synthesized compounds on colonization of plant growth-promoting rhizobacteria (PGPR) on various regions of the plant root is underexplored. Here, we examine the influence of surfactin and glutamate on the level and specificity of colonization of Bacillus subtilis along different regions of tomato root by adding exogenous compounds and testing mutated bacterial strains. First, B. subtilis PY79 was modified to express a full-length phosphopantetheinyl transferase (sfp) native to other surfactin-producing B. subtilis strains, and surfactin biosynthesis was observed under static incubation at 25°C in plant culturing media. Microscopy was performed using PY79 sfp^- (surfactin null), PY79 sfp⁺ (surfactin overproducing), and the wild isolate B. subtilis UD1022 to map the colonization of each strain along the entire root of young tomato plants incubated at 25°C. In addition, experiments involving supplementation of surfactin or glutamate were also performed to evaluate the root colonization of sfp⁺ and sfp^- strains. Root mapping of a tomato seedling shows that B. subtilis prefers to colonize near the mature region of the root and that colonization patterns vary based on exogenous metabolite concentration. Inclusion of glutamate in the media or through transient priming of the plant prior to bacterial inoculation strongly promoted root colonization by B. subtilis strains (both sfp⁺ and sfp^-). In addition, the data shows that the lab strains were less efficient in colonization compared to the wild-type B. subtilis strain. Interestingly, in the presence of glutamate, microbes lost their preference for colonization at the mature region, instead colonizing along the entire root. Overall, our work reveals a preference for colonization of these B. subtilis strains to the mature region of tomato in the absence of glutamate supplementation and demonstrates a smaller than anticipated role of biosynthesized or supplemented surfactin on root colonization, at least in a hydroponic culturing format.IMPORTANCEPlants are associated with large communities of microbes across the rhizosphere. However, comparatively little is known about the drivers underpinning the diversity of microbes, especially regarding how they are recruited by plants. Growing evidence indicates that the rhizospheric microbiome supports plant growth in response to biotic and abiotic stresses. Of late, the usage of a synthetic community of plant growth-promoting rhizobacteria (PGPR), especially Bacillus subtilis, has been recognized for its role as a potential biofertilizer and bio-fungicide agent. The role of PGPR-derived metabolites has been debated as a driver for enhanced root colonization. However, the knowledge pertaining to where and how PGPR colonize on the root surface is currently unknown. Therefore, it is prudent to elucidate the role of bacterially derived compounds and other carbon sources in the rhizosphere that drive root colonization.