<title>Abstract</title>The Casparian Strip (CS) constitutes a physical diffusion barrier to water and nutrients in plant roots, and is formed by the polar deposition of lignin polymer in the endodermis. This precise pattern of lignin deposition is thought to be mediated by the scaffolding activity of membrane-bound Casparian Strip domain proteins (CASPs). However, we show that endodermis-specific receptor-like kinase 1 (ERK1) and ROP Binding Kinase1 (RBK1) are also involved in this intricate process, with the former playing an essential role both in the localization of CASP1 and in lignin deposition. We further characterised ERK1 and determined its subcellular localisation in the cytoplasm and nucleus of the endodermis, as well as provide evidence for its involvement in a signalling pathway together with the circadian clock regulator, Time for Coffee (TIC). We also show that disruption to CS organisation and increased suberisation in the endodermis due to loss of function of either <italic>ERK1</italic> or <italic>TIC</italic> collectively leads to an altered root microbiome composition. Thus, our work reveals additional players in the complex cascade of signalling events operating in the root endodermis to establish both the CS diffusion barrier and the microbial composition of the rhizosphere.