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. In chapter 1, we established a collection of maize root bacteria and in an extensive highthroughput 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. Overall gram-positive bacteria were more MBOA-tolerant, indicating that the cell wall properties are one important component defining MBOA tolerance. Tolerance to MBOA correlated positively with the benzoxazinoiddependent colonisation of maize roots by the bacteria. This study revealed that benzoxazinoids selectively act as antibiotics on members of the maize root microbiome and that their capacity to tolerate benzoxazinoids enhanced root colonisation. We propose that tolerance to secreted antimicrobial compounds presents an important mechanism for structuring the microbial community on plant roots. In chapter 2, 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. In chapter 3, we designed two synthetic communities (SynComs) differing in their ability to metabolise benzoxazinoids to investigate how benzoxazinoid tolerance and metabolism affect a microbial community. We found that bacteria cooperate to tolerate and metabolise benzoxazinoids. The BX-metabolising SynCom had higher MBOA tolerance than the non- Summary 2 metabolising SynCom. Further, MBOA structured these SynComs differently, by inhibiting the susceptible strains. The BX-metabolising SynCom bacteria cooperate to metabolise MBOA to form N-(2-hydroxy-4-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 MBOAdegrading Microbacterium with a Pseudomonas that acetylates the unstable intermediate thereby redirecting the metabolism to HMPAA. 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 BXproducing 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.