ABSTRACTAgrobacterium tumefaciensgrows by addition of peptidoglycan (PG) at one pole of the bacterium. During the cell cycle, the cell needs to maintain two different developmental programs, one at the growth pole and another at the inert old pole. Proteins involved in this process are not yet well characterized. To further characterize the role of pole-organizing proteinA. tumefaciensPopZ (PopZAt), we created deletions of the five PopZAtdomains and assayed their localization. In addition, we created apopZAtdeletion strain (ΔpopZAt) that exhibited growth and cell division defects with ectopic growth poles and minicells, but the strain is unstable. To overcome the genetic instability, we created an inducible PopZAtstrain by replacing the native ribosome binding site with a riboswitch. Cultivated in a medium without the inducer theophylline, the cells look like ΔpopZAtcells, with a branching and minicell phenotype. Adding theophylline restores the wild-type (WT) cell shape. Localization experiments in the depleted strain showed that the domain enriched in proline, aspartate, and glutamate likely functions in growth pole targeting. Helical domains H3 and H4 together also mediate polar localization, but only in the presence of the WT protein, suggesting that the H3 and H4 domains multimerize with WT PopZAt, to stabilize growth pole accumulation of PopZAt.IMPORTANCEAgrobacterium tumefaciensis a rod-shaped bacterium that grows by addition of PG at only one pole. The factors involved in maintaining cell asymmetry during the cell cycle with an inert old pole and a growing new pole are not well understood. Here we investigate the role of PopZAt, a homologue ofCaulobacter crescentusPopZ (PopZCc), a protein essential in many aspects of pole identity inC. crescentus. We report that the loss of PopZAtleads to the appearance of branching cells, minicells, and overall growth defects. As many plant and animal pathogens also employ polar growth, understanding this process inA. tumefaciensmay lead to the development of new strategies to prevent the proliferation of these pathogens. In addition, studies ofA. tumefacienswill provide new insights into the evolution of the genetic networks that regulate bacterial polar growth and cell division.