Bacterial-fungal interactions are endemic phenomena in the rhizosphere, where bacterial partners may utilize the fungal mycelium for reaching to new niches. Previously in our lab, an Enterobacter sp. strain, named Enterobacter sp. En-Cren, was isolated from the hyphae of the soil pathogen fungus Rhizoctonia solani AG2-2IIIB (Rs-cren), the causal agent of turfgrass brown patch disease. Endohyphal En-cren can be released from damaged hyphae as a free-living bacterium and rapidly migrate along the outside of hyphae. While most identified bacterial hitchhikers of the fungal highway are flagellum-dependent, En-cren does not require flagellum to move along the fungal hyphae of Rs-cren. To identify the determinant factors for the hyphal motility, we created an En-cren mutant library using transposon Tn5. Mutants were screened and identified for loss of ability to migrate along fungal hyphae. Complementation and functional gene characterization are undergoing. This study will contribute to the knowledge of bacterial-fungal interaction and bacteria distribution in the mycorrhizosphere.