This poster was presented at the New Phytologist Editor-in-Chief Symposium: "Microbes as hidden or prominent players in plant life" held in Tartu, Estonia, between 8–10 July 2025 at the Oecologicum, University of Tartu. The bacterial genus Sphingomonas is a common colonizer of major crop leaves and roots. It is non-pathogenic and often provides growth promoting benefits to plants, making it a promising candidate for agricultural inoculants. To enable targeted applications, we aim to uncover the genetic basis of Sphingomonas’ beneficial interactions with plants. To pinpoint essential genes for plant colonization, we are performing large-scale mutant screens on the Sphingomonas genome. We have generated a pool of ~300,000 mutants for the model beneficial strain Sphingomonas melonis FR1. In our mutagenesis process, we use an innovative method known as RB-TnSeq, which allows for identifying mutants in a population simply by amplicon sequencing of their unique barcodes. We are applying these mutant populations to in-vitro and in planta conditions to identify differentially represented mutants across conditions. Those mutants missing in-planta may represent genes critical for plant colonization. In parallel, we are using a CRISPR RNA-guided integrase-based reverse genetics system to disrupt specific genes that we believe may play an essential role in plant interactions or bacterial colonization, and also to introduce genetic barcodes for easily quantifying mutants and wild-type strains by amplicon sequencing. The expected outcome will be genes required for Sphingomonas' success on plants.