Agrobacterium tumefaciens increases cytokinin production in plastids by modifying the biosynthetic pathway in the host plant

Abstract

Agrobacterium tumefaciens infects plants and induces the formation of tumors called ''crown galls'' by integrating the transferred-DNA (T-DNA) region of the Ti-plasmid into the plant nuclear genome. Tumors are formed because the T-DNA encodes enzymes that modify the synthesis of two plant growth hormones, auxin and cytokinin (CK). Here, we show that a CK biosynthesis enzyme, Tmr, which is encoded by the Agrobacterium T-DNA region, is targeted to and functions in plastids of infected plant cells, despite having no typical plastid-targeting sequence. Evidence is provided that Tmr is an adenosine phosphate-isopentenyltransferase (IPT) that creates a new CK biosynthesis bypass by using 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBDP) as a substrate. Unlike in the conventional CK biosynthesis pathway in plants, transzeatin-type CKs are produced directly without the requirement for P450 monooxygenase-mediated hydroxylation. Consistent with the plastid localization of Tmr, HMBDP is an intermediate in the methylerythritol phosphate pathway, a plastid-localized biosynthesis route for universal isoprenoid precursors. These results demonstrate that A. tumefaciens modifies CK biosynthesis by sending a key enzyme into plastids of the host plant to promote tumorigenesis.crown gall ͉ isopentenyltransferase ͉ methylerythritol phosphate pathway C ytokinins (CKs) are a group of plant hormones essential for cell division and differentiation in plants (1). Most natural CKs, including isopentenyladenine (iP) and trans-zeatin (tZ) (Fig. 1), are derivatives of N 6 -prenylated adenine (1). The prenyl side chain of CKs can be derived from the methylerythritol phosphate (MEP) pathway or the mevalonate (MVA) pathway, both of which supply common C 5 units for isoprenoid biosynthesis (2, 3). The MEP pathway widely occurs in the bacterial kingdom and the plastids of plants, whereas the MVA pathway is commonly found in the cytosol of eukaryotes (2, 3). Thus, plants have the two different isoprenoid pathways in separate subcellular compartments. Recent work has shown that the majority of the prenyl side chain of iP and tZ is derived from the MEP pathway in Arabidopsis seedlings (4).To initiate CK biosynthesis, an isoprenoid precursor is transferred to AMP, ADP or ATP by adenosine phosphateisopentenyltransferase (IPT, EC 2.5.1.27) ( Fig. 1) (5-7). At least two routes have been proposed for the formation of tZ, an active CK, in plants. In the conventional iP riboside 5Ј-monophosphate (iPRMP)-dependent pathway, dimethylallyl diphosphate (DMAPP) is used as the side chain precursor for iPRMP, which is then converted to tZ riboside 5Ј-monophosphate (tZRMP) by P450 monooxygenases (8) (CYP735A1 and CYP735A2 in Arabidopsis; Fig. 1). The other proposed pathway is an iPRMPindependent bypass, in which an unidentified hydroxylated derivative of DMAPP is directly transferred to the adenine moiety (9). A candidate for this putative substrate is 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBDP) (10), which has recently been shown to occur as an int...

The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells

“…Several published studies support the hypothesis that IPP, GPP, and GGPP are transported between the cytosolic and plastidial compartments, with a preference for transport from the plastid to the cytosol (Hemmerlin et al, 2003;Skorupinska-Tudek et al, 2008). This observation corroborates in vivo observations (Kasahara et al, 2002(Kasahara et al, , 2004Laule et al, 2003;Sakakibara et al, 2005) with inhibitor-treated Arabidopsis seedlings, cell cultures of Madagascar periwinkle (Catharanthus roseus) (Schuhr et al, 2003), and BY-2 cells (Hemmerlin et al, 2003). However, in the two latter studies, exogenous MVA was, to some extent, incorporated into plastids and used to produce plastidial isoprenoids.…”

“…Merret et al, 2007) As predicted (Hemmerlin et al, 2006), DX restored plasma membrane localization of His 6 -GFP-BD-CVIL in the presence of OC (Figure 9). The ability of DX to bypass the DXS reaction in the presence of OC confirmed the prediction that OC inhibits DXS (see also Sakakibara et al, 2005). Not surprisingly, DX did not complement the inhibition of MEP synthase by Fos.…”

“…To support the hypothesis that plastidial isoprenoid synthesis is required for the geranylgeranylation of GFP-BD-CVIL, cells were treated with oxoclomazone (OC; also referred to as ketoclomazone; Zeidler et al, 2000;Sakakibara et al, 2005), a derivative of the herbicide clomazone (also referred to as FMC 57020 or dimethazone). The latter was recognized as blocking the MEP pathway at a step before the formation of phytoene (Sandmann and Bö ger, 1989;Lange et al, 2001).…”

Roles of Arabidopsis ATP/ADP isopentenyltransferases and tRNA isopentenyltransferases in cytokinin biosynthesis

“…The Arabidopsis genome encodes seven genes belonging to a plant-specific ATP͞ADP IPT clade (AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7, and AtIPT8) (20,23,24). Overexpression of AtIPT4 or AtIPT8 confers cytokinin-independent shoot formation on calli (23,25), and overexpression of AtIPT1, 3,4,5,7, or 8 causes increased iP-type cytokinin levels in planta (25,26). These results suggest that isopentenylation of ATP and ADP makes a major contribution to the control of cytokinin biosynthesis.…”

“…Another pathway of tZ production, the iPR monophosphate-independent pathway, most likely involves transfer of the trans-hydroxylated isopentenyl moiety from 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate to adenosine phosphates, by the action of AtIPTs (28). However, this pathway has become less favored since the finding that AtIPT1 does not efficiently catalyze transfer of the trans-hydroxylated isopentenyl moiety from the likely precursor, 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate, to adenosine phosphates (26).…”