The bacterium Pantoea stewartii ssp. stewartii causes Stewart's wilt disease in corn. Pantoea stewartii is transmitted to plants via corn flea beetles, where it first colonizes the apoplast causing water-soaked lesions, and then migrates to the xylem and forms a biofilm that blocks water transport. Bacterial quorum sensing ensures that the exopolysaccharide production necessary for biofilm formation occurs only at high cell density. A genomic-level transposon sequencing (Tn-Seq) analysis was performed to identify additional bacterial genes essential for survival inplanta and to provide insights into the plant-microbe interactions occurring during wilt disease. A mariner transposon library of approximately 40 000 mutants was constructed and used to inoculate corn seedlings through a xylem infection model. Cultures of the library grown in Luria-Bertani (LB) broth served as the invitro pre-inoculation control. Tn-Seq analysis showed that the number of transposon mutations was reduced by more than 10-fold for 486 genes inplanta compared with the library that grew in LB, suggesting that they are important for xylem survival. Interestingly, a small set of genes had a higher abundance of mutants inplanta versus invitro conditions, indicating enhanced strain fitness with loss of these genes inside the host. Inplanta competition assays retested the trends of the Tn-Seq data for several genes, including two outer membrane proteins, Lon protease and two quorum sensing-associated transcription factors, RcsA and LrhA. Virulence assays were performed to check for correlation between growth/colonization and pathogenicity. This study demonstrates the capacity of a Tn-Seq approach to advance our understanding of P.stewartii-corn interactions.