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Plant roots are colonized by microbial communities which can promote growth, provide nutrients and protection against pathogens for the host. Plant specialized metabolites exuded by roots, like benzoxazinoids (BX) produced by maize, can structure root-associated microbial communities. However, the underlying mechanisms are poorly understood. The present thesis aimed to uncover the contribution of i) bacterial tolerance to benzoxazinoids, ii) bacterial metabolisation of benzoxazinoids and iii) the interactions of both for structuring root bacterial communities. First, we established a collection of maize root bacteria and in an extensive high-throughput in vitro screening we uncovered that benzoxazinoids inhibit bacterial growth in a strain-dependent and compound-dependent manner. Among the benzoxazinoids tested, 6-methoxybenzoxazolin-2(3H)-one (MBOA), which is the dominant metabolite structuring maize rhizosphere microbiomes, is the most selective compound. Tolerance to MBOA correlated positively with the benzoxazinoid-dependent colonisation of maize roots by the bacteria. We propose that tolerance to secreted antimicrobial compounds presents an important mechanism for structuring the microbial community on plant roots. Second, we identified maize root bacteria that metabolise the abundant benzoxazinoid in the rhizosphere, MBOA to 2-amino-7-methoxyphenoxazin-3-one (AMPO). The characteristic red colour of AMPO enabled us to develop a simple plate assay to screen the maize root bacteria strain collection for their ability to metabolise MBOA to AMPO. Few bacterial lineages including Sphingobium and Microbacterium convert MBOA to AMPO. AMPO-forming bacteria were enriched on roots of BX-producing but not of BX-deficient plants. We utilized the phenotypic diversity within the genus of Microbacteria to identify an N-acyl homoserine lactonase (BxdA) as the key enzyme for converting MBOA to AMPO. This study demonstrated the specific recruitment of adapted bacteria on BX-producing roots that can metabolise the host secondary metabolites. Third, we studied benzoxazinoid tolerance and metabolisation in a community context. We found that bacteria cooperate to tolerate and metabolise benzoxazinoids in synthetic microbial communities. Benzoxazinoid metabolisation conferred MBOA tolerance to the SynComs. Cooperation lead to the formation of an alternative MBOA conversion product, N-(2-hydroxy-methoxyphenyl)acetamide (HMPAA), a dominant metabolite that is not formed by single strains. We discovered that HMPAA is formed by the combined activity of an MBOA-degrading Microbacterium with a Pseudomonas. This study demonstrated that bacteria on maize roots cooperate to metabolise benzoxazinoids and as a community, they benefitted from an enhanced tolerance to these compounds. This research reveals microbiological and biochemical mechanisms of how plant specialized metabolites contribute to shape the root microbiota. The ability of maize root bacteria to tolerate and metabolise benzoxazinoids are important traits defining their abundance on BX-producing maize roots. We demonstrate that strains when combined in synthetic communities, they divide labour and cooperate to metabolise and tolerate benzoxazinoids. The deepened understanding of how plant specialized metabolites sculpt community composition provides a tool to selectively steer microbiome structure. Further work is required to understand how bacterial BX-mediated mechanisms affect microbiomes to harness the functions of microbial communities for sustainable agriculture.

The western corn rootworm (WCR) decimates maize crops worldwide. One potential way to control this pest is treatment with entomopathogenic nematodes (EPNs) that harbor bacterial symbionts that are pathogenic to insects. However, WCR larvae sequester benzoxazinoid secondary metabolites that are produced by maize and use them to increase their resistance to the nematodes and their symbionts. Here we report that experimental evolution and selection for bacterial symbionts that are resistant to benzoxazinoids improve the ability of a nematode–symbiont pair to kill WCR larvae. We isolated five Photorhabdus symbionts from different nematodes and increased their benzoxazinoid resistance through experimental evolution. Benzoxazinoid resistance evolved through multiple mechanisms, including a mutation in the aquaporin-like channel gene aqpZ. We reintroduced benzoxazinoid-resistant Photorhabdus strains into their original EPN hosts and identified one nematode–symbiont pair that was able to kill benzoxazinoid-sequestering WCR larvae more efficiently. Our results suggest that modification of bacterial symbionts might provide a generalizable strategy to improve biocontrol of agricultural pests.