Agrobacterium tumefaciens tumor morphology root plastid localization and preferential usage of hydroxylated prenyl donor is important for efficient gall formation.

Upon Agrobacterium tumefaciens infection of a host plant, Tumor morphology root (Tmr) a bacterial adenosine phosphate-isopentenyltransferase (IPT), creates a metabolic bypass in the plastid for direct synthesis of trans-zeatin (tZ) with 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate as the prenyl donor. To understand the biological importance of Tmr function for gall formation, we compared Tmr and Trans-zeatin secretion (Tzs) another agrobacterial IPT that functions within the bacterial cell. Although there is no significant difference in their substrate specificities in vitro, ectopic overexpression of Tzs in Arabidopsis (Arabidopsis thaliana) resulted in the accumulation of comparable amounts of tZ- and N⁶-(Δ²-isopentenyl)adenine (iP)-type cytokinins, whereas overexpression of Tmr resulted exclusively in the accumulation of tZ-type cytokinins. Ectopic expression of Tzs in plant cells yields only small amounts of the polypeptide in plastid-enriched fractions. Obligatory localization of Tzs into Arabidopsis plastid stroma by translational fusions with ferredoxin transit peptide (TP-Tzs) increased the accumulation of both tZ- and iP-type cytokinins. Replacement of tmr on the Ti plasmid with tzs, TP-tzs, or an Arabidopsis plastidic IPT induced the formation of smaller galls than wild-type A. tumefaciens, and they were accompanied by the accumulation of iP-type cytokinins. Tmr is thus specialized for plastid localization and preferential usage of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate in vivo and is important for efficient gall formation.