Agrobacterium tumefaciens Tumor Morphology Root
Plastid Localization and Preferential Usage of
Hydroxylated Prenyl Donor Is Important for Efficient
Gall Formation1[C][W][OA]
Nanae Ueda, Mikiko Kojima, Katsunori Suzuki, and Hitoshi Sakakibara*
RIKEN Plant Science Center, Tsurumi, Yokohama 230–0045, Japan (N.U., M.K., H.S.); and Department
of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima,
Hiroshima 739–8526, Japan (K.S.)
Upon Agrobacterium tumefaciens infection of a host plant, Tumor morphology root (Tmr) a bacterial adenosine phosphateisopentenyltransferase (IPT), creates a metabolic bypass in the plastid for direct synthesis of trans-zeatin (tZ) with 1-hydroxy2-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.
Cytokinins are a group of phytohormones that regulate various aspects of plant growth and development, such as maintenance of stem cell systems in
shoots and roots (Kurakawa et al., 2007; Müller and
Sheen, 2008; Gordon et al., 2009; Zhao et al., 2010;
Tokunaga et al., 2012), nodule organogenesis (Murray
et al., 2007; Tirichine et al., 2007; Kouchi et al., 2010),
and root vascular development (Higuchi et al., 2004;
Hutchison et al., 2006; Mähönen et al., 2006; Ishida
et al., 2008; Kondo et al., 2011). This hormone is also
synthesized by some phytopathogenic bacteria, such
as Agrobacterium tumefaciens, Rhodococcus fascians, and
Pseudomonas savastanoi, and consequently plays a key
1
This work was supported by the Ministry of Education, Culture,
Sports, Science, and Technology in Japan (Grant-in-Aid for Scientific
Research [B] no. 21370023 to H.S.).
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Hitoshi Sakakibara ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.112.198572
1064
role for plant tumorigenesis (Morris, 1986; Goethals
et al., 2001; Pertry et al., 2009). A. tumefaciens infection
of plant cells induces the formation of “crown gall”
tumors. This neoplastic morphogenesis is induced by
the expression of genes encoded within the T region of
Ti plasmids (Van Larebeke et al., 1974). Upon infection, the T region is introduced into host plant cells by
a type IV secretion system (Alvarez-Martinez and
Christie, 2009) and integrated into the nuclear genome
(Chilton et al., 1977; Zhu et al., 2000; Zupan et al.,
2000). The T region contains genes involved in the
biosynthesis of cytokinins, auxin, and opines, and the
genes are expressed by the host plant transcription/
translation machinery (Suzuki et al., 2009). Aberrant
cell proliferation caused by the overproduction of cytokinins and auxin in infected cells results in tumor
formation (Escobar and Dandekar, 2003).
Cytokinins are adenine derivatives that carry an
isoprene-derived side chain at the N6 terminus (Mok
and Mok, 2001; Sakakibara, 2006). The first step of
cytokinin biosynthesis is catalyzed by adenosine
phosphate-isopentenyltransferase (IPT). The T regions
of Ti plasmids commonly contain an IPT gene (tmr;
Akiyoshi et al., 1984; Barry et al., 1984). Tmr catalyzes
the condensation of AMP with 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate (HMBDP) or dimethylallyl
diphosphate (DMAPP) to form trans-zeatin (tZ) riboside 59-monophosphate or N6-(Δ2-isopentenyl)adenine
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Specialization of Tmr for Efficient Gall Formation
(iP) riboside 59-monophosphate, respectively, which
are precursors of cytokinins (Akiyoshi et al., 1984;
Barry et al., 1984; Sakakibara et al., 2005; Sakakibara,
2006). Tmr is localized in the plastids of host plants
despite lacking a typical transit peptide at the N terminus.
The enzyme modifies cytokinin biosynthesis by recruiting HMBDP, an intermediate product of the
methylerythritol phosphate pathway in plastids, to
directly produce tZ riboside 59-monophosphate without CYP735A-mediated transhydroxylation (Takei
et al., 2004; Sakakibara et al., 2005). Despite the
similar affinity of Tmr for HMBDP and DMAPP in
vitro (Sakakibara et al., 2005; Sugawara et al., 2008),
crown galls and Tmr-overexpressing transgenic plants
almost exclusively contain tZ and its conjugates (tZ-type
cytokinins; Morris, 1986; Faiss et al., 1997; Sakakibara
et al., 2005), implying an unknown mechanism for
preferential HMBDP usage by Tmr in vivo. In addition
to tmr, nopaline-type Ti plasmids encode another IPT
gene (tzs) within the virulence region, which is induced
by plant phenolic compounds (John and Amasino,
1988). The Tzs and Tmr proteins are structurally similar,
with 51.3% identity at the amino acid level, whereas
higher plant IPTs have lower similarity to Tmr (approximately 20% identity). In vitro studies indicate that
both Tzs and Tmr utilize HMBDP and DMAPP with
similar affinity (Krall et al., 2002; Sugawara et al., 2008).
Tzs is localized on the bacterial cell surface and may
contribute to the interactions of A. tumefaciens with the
host plants via cytokinin production (Aly et al., 2008).
The two A. tumefaciens IPTs are involved in cytokinin
production under spatially and temporally separated
conditions. However, the functional specialization of
Tmr for the shortcut pathway and the plastid localization of tZ precursor biosynthesis and tumor formation
are not well understood. As examined so far, the ability
to transform directly HMBDP into trans-zeatin riboside
59-phosphates (tZRPs) is only found in Tmr, suggesting
that Tmr is uniquely specialized for tumorigenesis. A
comparison of the biochemical and functional features
between Tmr and Tzs should provide a clue about the
specialization. In this study, we examined the activity of
Tzs as an alternative to Tmr in terms of preferential
substrate usage and plastid localization. We also evaluated the tumor-forming ability of genetically modified
A. tumefaciens strains whose tmr was replaced with another IPT. Our results strongly suggest that Tmr plastid
localization and the preferential usage of HMBDP in
vivo are both important for efficient gall formation.
RESULTS
Tmr Uses HMBDP Preferentially in Planta, But Tzs
Does Not
Previously, we have shown that Tmr localizes in the
plastids of host plant cells and directly produces tZ
nucleotide using HMBDP and that transgenic Arabidopsis (Arabidopsis thaliana) plants that overexpress
Tmr predominantly accumulate tZ-type cytokinins
(Sakakibara et al., 2005). To examine the specialization
of Tmr in terms of prenyl donor substrate usage in
vivo, we compared postinduction concentrations of
tZRPs and N6-(Δ2-isopentenyl)adenine riboside 59phosphates (iPRPs) in Arabidopsis plants that were
transformed with either tzs or tmr (Sakakibara et al.,
2005) under the control of a dexamethasone (DEX)inducible promoter (Fig. 1; Supplemental Table S1).
Induction of Tzs expression by DEX treatment resulted
in the production of roughly equal amounts of iPRPs
and tZRPs (Fig. 1A). This result contrasts with our
observation of a clear difference in iPRP and tZRP
synthesis in Tmr-overexpressing Arabidopsis (Fig. 1B;
Sakakibara et al., 2005). The overall accumulation of
cytokinins in Tzs-overexpressing Arabidopsis plants
was substantially lower than in Tmr-overexpressing
plants (Fig. 1), even though the substrate affinities of
Tmr and Tzs for HMBDP and DMAPP are similar in
Figure 1. Cytokinin accumulation in transgenic Arabidopsis lines
overexpressing agrobacterial IPTs. Transgenic Arabidopsis seedlings
harboring pTA-Tzs (A; Tzs-ox), pTA-Tmr (B; Tmr-ox), and pTA-7001
empty vector (C; Control) were grown for 18 d on MGRL-agar plates
and then sprayed with 30 mM DEX. After 0, 4, 7, and 24 h, the whole
seedlings were harvested and cytokinin contents were measured. Only
the results of iPRPs and tZRPs are presented. Error bars represent SD of
three biological replicates. FW, Fresh weight. The complete data set is
presented in Supplemental Table S1.
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Ueda et al.
vitro (Sakakibara et al., 2005; Sugawara et al., 2008),
and the kcat/Km values of Tmr are rather lower than
those of Tzs (Supplemental Fig. S1). These results
suggest that Tmr preferentially uses HMBDP in planta
but Tzs does not and that Tmr has higher catalytic
activity than Tzs in vivo.
Plastid Import Is Largely Limited to Tmr
Translational fusions of Tzs were made with GFP at
the C terminus (Tzs-GFP) under the control of the
cauliflower mosaic virus 35S promoter and introduced
into Arabidopsis cells by particle bombardment (Fig. 2).
When the fusion gene was transiently expressed in leaf
and root cells, the GFP fluorescence of Tzs-GFP was
distributed in plastids, nuclei, and cytosol (Fig. 2, A–C)
whereas Tmr-GFP was restricted to plastids (Fig. 2, D–
F), as reported previously (Sakakibara et al., 2005). To
further obtain information about localization, chloroplasts were enriched from stable transformants of
Arabidopsis shoots in which Tzs expression was induced by DEX treatment for 24 h, and Tzs polypeptide
was detected with anti-Tzs antibody in immunoblot
assays. Very little Tzs polypeptide was recovered in the
chloroplast-enriched fraction, but plastidic Gln synthetase concentrations in the chloroplast fraction proteins,
used as a control, were comparable to total leaf proteins
(Fig. 2G). It should be noted that Tmr polypeptide had
been recovered in the chloroplast-enriched fraction using the same experimental method (Sakakibara et al.,
2005). This result indicates that the ability of Tmr to be
imported into plastids is not well conserved in Tzs.
Obligatory Plastid Localization of Tzs Does Not Change
Substrate Use Preference
To understand the relationship between Tmr plastid
localization and its preferential use of HMBDP, a
fusion protein construct was made between the plastid-target transit peptide and Tzs at the N terminus
(TP-Tzs). Because of this arrangement, it was assumed
that TP-Tzs was translocated to plastid stroma via the
Toc/Tic system (Soll and Schleiff, 2004; Inaba and
Schnell, 2008). Since A. tumefaciens infected galls are
usually induced in nonphotosynthetic organs, we used
the nonphotosynthetic-type ferredoxin III (Fd III; Hase
et al., 1991). Because the cleavage site of Fd III is between Met-55 and Ala-56 (Hase et al., 1991; Suzuki
et al., 1991), mature polypeptide from TP-Tzs in plastid
was expected to have an extra Ala residue at the N
terminus. To test whether this extra Ala residue introduced at the N terminus of Tzs affects the catalytic
activity of TP-Tzs, we expressed the recombinant
proteins with a poly-His tag in Escherichia coli, purified
the protein, and checked the enzyme activity in vitro.
The poly-His-tagged recombinant Tzs protein with Nterminal Met-Ala has catalytic activity for HMBDP and
DMAPP comparable to wild Tzs, but the activity of
TP-Tzs is not detectable (Supplemental Fig. S2).
Figure 2. Subcellular localization of Tzs in Arabidopsis cells. A to F,
Distribution of GFP fusion proteins. Translational fusion genes (TzsGFP [A–C] and Tmr-GFP [D–F]) were transiently expressed in Arabidopsis leaf epidermal cells (A, B, D, and E) or root cells (C and F) by
particle bombardment. Bars = 10 mm. B and E are superimpositions of
chloroplast autofluorescence to A and D, respectively. n, Nucleus; p,
plastid. G, Immunoblot analysis. Arabidopsis seedlings harboring pTATzs were grown for 18 d on MGRL-agar plates and then treated with
(+) or without (2) 30 mM DEX for 24 h. Total leaf proteins (Total) and
the chloroplast fraction proteins (Chl.) were prepared. Proteins
equivalent to 0.2 mg of chlorophyll were separated by SDS-PAGE.
Polypeptides were detected by immunoblotting using antibodies
against Tzs or Gln synthetase (GS). GS1 and GS2 represent cytosolic
and plastidic GS, respectively.
When the chimeric TP-Tzs-encoding gene was
translationally fused to GFP at the C terminus (TP-TzsGFP) and transiently expressed in Arabidopsis mesophyll cells, GFP fluorescence was only observed in
chloroplasts (Fig. 3A). We generated stable transgenic
Arabidopsis lines overexpressing the TP-Tzs fusion
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Specialization of Tmr for Efficient Gall Formation
under the control of the DEX-inducible promoter. After the induction of TP-Tzs expression by DEX treatment, accumulation of the Tzs polypeptide in leaf
chloroplasts and root plastids was examined by immunoblots. Although the accumulation of mature Tzs
protein in leaves was higher in TP-Tzs-ox in comparison with Tzs-ox, the polypeptides were not present in
high concentrations in the chloroplast-enriched fractions of two independent transgenic lines (TP-Tzs-ox
#1 and #2; Fig. 3B). In the roots of TP-Tzs-ox (TP-Tzsox #2), Tzs polypeptides were extracted at more elevated levels in the plastid-enriched fraction. The signal
ratio of total to plastid-enriched fractions in Tzs was
comparable to plastidic Gln synthetase (Fig. 3C, right
panels). These results suggest that TP-fused Tzs is efficiently transported into plastids in TP-Tzs-ox line
plants, especially in the nonphotosynthetic organs.
We harvested the transgenic Arabidopsis seedlings
under the same conditions to assay cytokinin (Fig. 3D;
Supplemental Table S2). Although the accumulation of
cytokinins was remarkably higher in transgenic Arabidopsis plants expressing TP-Tzs (TP-Tzs-ox) in
comparison with Tzs-ox, the major species of cytokinins were all iPRPs in two independent transgenic
lines (Fig. 3D). These results suggest that the imported
TP-Tzs is processed to form a functional enzyme, but if
any Tzs is localized in plastids, it could not preferentially use HMBDP as a prenyl donor.
Comparison of Tumor Formation Efficiency with Tmr
and Tzs
Figure 3. Forced localization of TP-Tzs into Arabidopsis plastids. A,
Distribution of TP-Tzs-GFP fusion protein in an Arabidopsis cell. TPTzs-GFP was transiently expressed in Arabidopsis leaf mesophyll cells
by particle bombardment. B and C, Immunoblot analysis. B, A transgenic Arabidopsis line harboring pTA-Tzs (Tzs-ox) and two independent transgenic Arabidopsis lines harboring pTA-TP-Tzs (TP-Tzs-ox #1
and #2) were grown for 18 d on MGRL-agar plates and then treated
with (+) or without (2) 30 mM DEX for 24 h. Total leaf proteins (Total)
and chloroplast-enriched fraction proteins (Chl.) were prepared. Proteins equivalent to 0.2 mg of chlorophyll were subjected to immunoblot analysis. The white arrowhead indicates the mobility of mature
Tzs. The less mobile polypeptide in TP-Tzs-ox #1, indicated with the
black arrowhead, is probably unprocessed precursor. C, Arabidopsis
lines Tzs-ox and TP-Tzs-ox #2 were first grown on MGRL-agar plates
for 24 d with 12 h of light/12 h of dark and then hydroponically grown
with MGRL salt for 18 d with 18 h of light/6 h of dark. After treatment
of the roots with (+) or without (2) DEX, total root proteins (Total) and
the plastid-enriched fraction proteins (Pl.) were prepared. Six micrograms of protein for Tzs-ox and 4 mg for TP-Tzs-ox was subjected to
immunoblot analysis. GS1 and GS2, Cytosolic and plastidic Gln synthetase (GS), respectively. D, Cytokinin accumulation in transgenic
To evaluate Tmr specialization for tumorigenesis,
we compared tumor formation efficiency among
A. tumefaciens strains expressing Tmr, Tzs, or TP-Tzs.
We first disrupted tzs within the vir region of pTiSAKURA in A. tumefaciens MAFF301001 rif to exclude
any effects of authentic Tzs-derived cytokinins, and we
defined this strain (301001tzs2) as the wild type for
this study (Table I). The tmr gene on the Ti plasmid
was then replaced with tzs or TP-tzs by homologous
recombination in the A. tumefaciens 301001tzs2 strain,
and the genotypes in the recombinant strains were
designated as tmr/Tzs and tmr/TP-Tzs, respectively. A
recombinant A. tumefaciens strain whose tmr gene was
replaced with AtIPT1 was also generated, and the
genotype was designated as tmr/AtIPT1. AtIPT1
localizes in plastids and exclusively uses DMAPP
as its prenyl donor substrate (Kasahara et al., 2004;
Sakakibara et al., 2005). To exclude the possibility of
Arabidopsis lines overexpressing Tzs or TP-Tzs. Transgenic Arabidopsis
seedlings of Tzs-ox #1 and #2 (independent lines) and TP-Tzs-ox #1
and #2 were grown for 18 d on MGRL-agar plates; half were subjected
to DEX treatment (+) and half were not treated as controls (2). After 24
h, the seedlings were harvested and cytokinin contents were analyzed.
Only iPRP and tZRP concentrations are presented. Error bars represent
SD of three biological replicates. FW, Fresh weight. The complete data
set is presented in Supplemental Table S2.
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Ueda et al.
Table I. A. tumefaciens strains used for tumor-forming assays
Gmr, Gentamycin resistant; Rif r, rifampicin resistant.
Strain
MAFF301001 rif
301001tzs2
tmr/Tzs
tmr/TP-Tzs
tmr/AtIPT1
Relevant Properties
Source or Reference
Rif
tzs::Gmr in MAFF301001 rif; Rif r, Gmr; used as the wild type
tmr/Tzs replacement mutant of 301001tzs2; Rifr, Gmr
tmr/TP-Tzs replacement mutant of 301001tzs2; Rifr, Gmr
tmr/AtIPT1 replacement mutant of 301001tzs2; Rifr, Gmr
Sonoda et al. (2002)
This study
This study
This study
This study
r
differences in expression efficiency, we replaced tmr
with the other gene’s reading frame without any nucleotide insertions or deletions (for details, see “Materials and Methods”). The recombinant A. tumefaciens
strains were introduced into Kalanchoe daigremontiana
leaves, and tumor formation was measured 34 d after
infection (Fig. 4). In Kalanchoe, the tumorigenesis due
to tmr/Tzs was substantially less than with the wild
type (Fig. 4A). tmr/TP-Tzs strains induced significantly more tumor formation than strains carrying
tmr/Tzs but were still much lower than the wild type.
When we replaced tzs with AtIPT1 (tmr/AtIPT1), tumorigenesis increased but still did not reach the wildtype level (Fig. 4A). Because it was difficult to obtain
enough soluble protein from tumors for immunoblot
analysis, expression of the exogenous genes was confirmed by reverse transcription (RT)-PCR. Exogenous
cytokinin biosynthesis genes were all expressed
(Supplemental Fig. S3). We also analyzed cytokinin
and auxin (indole-3-acetic acid [IAA]) concentrations
in the tumors (Fig. 4, B and C; Supplemental Table S3).
Since the tumors generated from tmr/Tzs and tmr/TPTzs strain infections were too small to analyze individually, we pooled each group. Uninfected Kalanchoe
leaves served as the control tissue. Accumulation of
IAA was higher in all tumor tissues, suggesting successful A. tumefaciens infection with expression of the
introduced auxin biosynthesis genes. tZ-type and iPtype cytokinins were the dominant forms in the wildtype A. tumefaciens and tmr/AtIPT1-infected tumors,
respectively (Fig. 4B; Supplemental Table S3). The tmr/
Tzs- and tmr/TP-Tzs-infected tumors contained much
less cytokinin than the wild type or tmr/AtIPT1 and
predominantly accumulated iP types, namely the iPRPs
(Fig. 4C). The tmr/TP-Tzs-infected tumors contained
significantly higher amounts of iPRPs than tmr/Tzsinfected tumors. These results indicate that the expression of Tmr in Kalanchoe should form the largest tumors,
that they predominantly accumulate tZ-type cytokinins,
and that the introduction of alternative IPTs does not
complement the loss of wild-type capabilities.
To further evaluate tumor formation efficiencies in a
natural host-pathogen interaction, we infected rose
(Rosa sp. ‘Princess Michiko’) stems with the same set of
A. tumefaciens strains and evaluated tumorigenesis by
measuring gall weights (Fig. 5). Although there was a
wide range in tumor weights, the results were essentially the same as in the Kalanchoe assay (Fig. 5A). Galls
induced by 301001tzs2 (the wild type) were largest,
galls induced by tmr/Tzs were the smallest, and those
induced by tmr/TP-Tzs and tmr/AtIPT1 were significantly larger than tmr/Tzs-induced tumors but smaller
than those induced by the wild type. Consistent with
gall formation efficiency, tZ-type cytokinins were
dominantly accumulated in wild-type galls but iP-type
cytokinins predominated in other galls (Fig. 5, B and C;
Supplemental Table S4). These results suggest that Tzs,
as with plant IPT, is not sufficient to replace Tmr for tZ
production and that cytokinin synthesis by Tmr is a
critical aspect of specialization during tumor formation.
DISCUSSION
In this study, we demonstrate that Tmr is specialized among IPTs for translocation into plastids, selective usage of HMBDP as its prenyl donor, and highly
efficient tZ biosynthesis during tumor formation in
A. tumefaciens infected galls. This Tmr-specific property is clearly important for larger tumor formation,
because alternative IPTs could not complement the
capabilities. Although Tzs was not efficiently translocated into plastids, it is intriguing that the fluorescence of Tzs-GFP chimeric protein was observed in
plastids as well as cytosol and nuclei (Fig. 2). Dilution
of Tzs polypeptide in the intact chloroplast-enriched
fraction denied a tight association of Tzs to the chloroplast envelope membrane and efficient translocation
of Tzs into the stroma (Fig. 2G). However, GFP fluorescence distribution implies that plastid translocation
function is partially conserved in Tzs. In fact, our experiments suggest that a trace amount of Tzs polypeptide could be translocated into stroma, because a
small amount of Tzs polypeptide was detected after
treatment with proteases (Supplemental Fig. S4).
Given that tzs occurs in the nopaline-type Ti plasmids
whereas tmr is common to all Ti plasmids so far examined, it could be hypothesized that tzs is derived
from an ancestral tmr by gene duplication. If so, plastid
translocation signals have been mostly lost in Tzs. As
for selective usage of HMBDP, even when Tzs was
imported into plastids, it could not preferentially use
the hydroxylated substrate (Fig. 3), indicating that this
property is not due to subcellular localization but is
an intrinsic property of Tmr. At present, there is no
evidence to explain the preferential usage of HMBDP
in plastids. The in vitro properties of Tmr tend to be
DMAPP-philic: the kcat/Km for DMAPP is about 10
times higher than that for HMBDP (Supplemental
Fig. S1). Therefore, there must be a mechanism to
control Tmr use of HMBDP in plastids. One possible
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Specialization of Tmr for Efficient Gall Formation
and DMAPP might be spatially biased in the plastid
stroma, because the chemical properties, such as hydrophobicity, of HMBDP and DMAPP should be
Figure 4. Effect of the replacement of tmr by other IPTs on A. tumefaciens-infected tumor formation in Kalanchoe. A, Tumor formation
assay with Kalanchoe by genetically modified A. tumefaciens. Cell
concentrations of genetically modified A. tumefaciens strains
301001tzs2 (wild type [WT]), tmr/Tzs, tmr/TP-Tzs, and tmr/AtIPT1
were adjusted to 5 3 1010 cells mL21. Data sets marked with single
and double asterisks differ significantly as assessed by Student’s t test at
P , 0.05 and P , 0.01, respectively (n = 47). B and C, Hormone
analysis of the tumors. Concentrations of cytokinins and IAA in tumors
and mock-inoculated control leaves were analyzed after 29 d by
UPLC-MS/MS. C shows the y axis-magnified graph of B. FW, Fresh
weight. The complete data set is presented in Supplemental Table S3.
[See online article for color version of this figure.]
mechanism is the formation of an enzyme complex
containing Tmr and HMBDP synthase (GcpE; Hecht
et al., 2001). Complex formation would make it possible to channel HMBDP directly from GcpE to Tmr.
Another possibility is that the distribution of HMBDP
Figure 5. Effect of the replacement of tmr with other IPTs on
A. tumefaciens infected tumor formation in rose. A, Tumor formation
assay with rose by genetically modified A. tumefaciens strains. The
galls were photographed 54 d after inoculation, excised with a razor
blade, and weighed. Data sets marked with double asterisks differ
significantly as assessed by Student’s t test at P , 0.01 (n = 24 for the
wild type [WT], n = 40 for tmr/Tzs, n = 36 for tmr/TP-Tzs, n = 22 for
tmr/AtIPT1). B and C, Hormone analysis of the tumors. Concentrations
of cytokinins in the tumors and mock-inoculated control stems were
analyzed by UPLC-MS/MS. C shows the y axis-magnified graph of B.
FW, Fresh weight. The complete data set is presented in Supplemental
Table S4. [See online article for color version of this figure.]
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Ueda et al.
different. If the spatial distribution of HMBDP and
DMAPP is not uniform within the plastid, and if Tmr
is colocalized with HMBDP, Tmr could preferentially
access HMBDP. Further analysis of the suborganellar
localization of Tmr in plastids, and searching for Tmrinteracting proteins, will give us a hint to understand
the molecular basis of efficient tZ production.
When Tzs was expressed in Arabidopsis under the
control of the same promoter system as Tmr, cytokinin
accumulation was consistently lower than with Tmr
(Fig. 1). On the other hand, forced translocation of Tzs
into plastids substantially increased accumulation in
two independent lines (Fig. 3D). A similar effect was
observed in Kalanchoe and rose tumors (Figs. 4C and
5C). These results suggest that plastid stroma is more
suitable for the catalytic reaction of agrobacterial IPT,
such as pH, substrate concentration, and/or magnesium ion concentration.
It should be noted that in the tumor formation
assays, much larger amounts of tZ-type cytokinins
were accumulated in galls expressing Tmr than iP-type
cytokinins in galls expressing Tzs or a plant IPT (Figs.
4 and 5). This result suggests that there is an advantage
to tZ production for effective tumorigenesis. Given
that tZ has generally lower affinity for cytokinin oxidase (Bilyeu et al., 2001), a cytokinin-degrading enzyme, Tmr might have adapted to produce biologically
stable cytokinin species.
Amino acid identity between Tmr and Tzs is about
51%, and the differences among the amino acid residues likely determine functional specialization in vivo.
The atomic structure of Tzs has been solved (Sugawara
et al., 2008). A comparison of Tmr and Tzs at the
atomic level could provide a clue about the structural
basis for the specialization of Tmr in the host plant cell.
MATERIALS AND METHODS
Plants and Bacteria
Arabidopsis (Arabidopsis thaliana) ecotype Columbia was used for generating transgenic plants. Arabidopsis plants were grown on MGRL salt medium (Fujiwara et al., 1992) with 1% (w/v) Suc and 1% (w/v) agar at 22°C
under fluorescent illumination at an intensity of 70 mmol m22 s21 with a
photoperiod of 16 h of light/8 h of dark unless otherwise stated. For tumor
formation assays, rose (Rosa sp. ‘Princess Michiko’) and Kalanchoe daigremontiana were grown in soil in a greenhouse at the RIKEN Plant Science
Center. Agrobacterium tumefaciens strain MAFF301001 rif (Sonoda et al., 2002),
which harbors a nopaline-type Ti plasmid (pTi-SAKURA; Suzuki et al., 2000),
was used for generating A. tumefaciens mutants and tumor formation assays.
A. tumefaciens strain GV3101 (pMP90) was used for generating transgenic
plants. Escherichia coli strain S17-1 lpir (Simon et al., 1983) was used as the
host strain for conjugation with A. tumefaciens strain MAFF301001 rif. E. coli
strain BL21 was used for the expression of recombinant proteins.
transit peptide region plus Ala (Met-1 to Ala-56) was amplified by RT-PCR
with oligo(dT)12-18 primers for RT and specific primers TP(Fd)-Afl3-59 and TP
(Fd)-Nco1-39 for subsequent amplification. The DNA amplicon was cloned
into pCR-BluntII-TOPO vector (Invitrogen). To obtain cDNA encoding MATzs, which has two additional amino acid residues, Met and Ala, at the N
terminus, the QuickChange Site-Directed Mutagenesis Kit (Stratagene) was
used with pQE-Tzs (Sugawara et al., 2008) as the template along with primers
MA-Tzs QC-59 and MA-Tzs QC-39, containing the two amino acid codons. The
resulting plasmid was named pQE-MA-Tzs. After confirmation of the nucleotide sequences, the AflIII-NcoI fragment of the transit peptide region was ligated into the NcoI site of pQE-MA-Tzs to yield pQE-TP-Tzs.
DNA containing the TP-tzs region was amplified by PCR with pQE-TP-Tzs
and primers TP-Bsa1-Sal1-59 and Tzs-Spe-39 and then cloned into pCR-BluntIITOPO. After confirmation of the nucleotide sequence, the SalI/SpeI fragment was
ligated into the XhoI/SpeI site of pTA7001 (Aoyama and Chua, 1997) to yield a
construct containing the DEX-inducible TP-tzs gene (pTA-TP-Tzs). For the construction of pTP-Tzs-GFP, which contains a chimeric gene of TP-Tzs and GFP,
DNA containing TP-tzs was amplified by PCR with pQE-TP-Tzs and primers TPBsa1-Sal1-59 and Tzs-Bsa1-Nco1-GGGS-39 and then cloned into pCR-BluntII-TOPO.
After confirmation of the nucleotide sequence, the BsaI fragment was ligated into
the SalI/NcoI site of sGFP (S65T) vector (Niwa et al., 1999) to yield 35Spro:TP-Tzs,
whose expression was controlled by the cauliflower mosaic virus 35S promoter.
Generation of Transgenic Arabidopsis Expressing Tzs
and TP-Tzs
The coding region of Tzs was ligated into pTA7001 to yield pTA-Tzs. pTATzs and pTA-TP-Tzs were introduced into Arabidopsis by the floral dip method
(Clough and Bent, 1998). T3 homozygous progeny were used for experiments.
Hormone Measurements
Extraction and determination of cytokinins and IAA from Arabidopsis
transgenic lines overexpressing IPTs or galls were performed as described
previously with an ultra-performance liquid chromatography-tandem mass
spectrometry (UPLC-MS/MS) apparatus (AQUITY UPLC System/Quattro
Ultima Pt; Waters) with an octadecylsilyl column (AQUITY UPLC BEH C18,
1.7 mm, 2.1 3 100 mm; Waters; Kojima et al., 2009).
Observation of Intracellular Distribution with GFP
Fusion Proteins
The coding region of tzs was ligated into the sGFP (S65T) vector to yield
35Spro:Tzs. The 35Spro:Tzs, 35Spro:TP-Tzs, and 35Spro:Tmr (Tmr-GFP; Sakakibara
et al., 2005) constructs were introduced into leaf epidermal cells or root cells of
2-week-old Arabidopsis seedlings by particle bombardment (Bio-Rad; PDU1000/He). Transient expression was observed by confocal laser scanning fluorescence microscopy (Olympus; Fluoview IX5) after overnight incubation.
Western-Blot Analysis
About 400 mg of Arabidopsis seedling tissues was homogenized in 2
volumes of ice-cold extraction buffer (50 mM Tris-HCl and 150 mM NaCl, pH
7.5). Intact chloroplasts were isolated from Arabidopsis seedlings as described
previously (Jackson et al., 1998). Root plastids were enriched as described
previously (Emes and England, 1986; Sakakibara et al., 1992a). The proteins
were separated by SDS-PAGE followed by western blotting. Polyclonal antibodies against maize cytosolic Gln synthetase (Sakakibara et al., 1992b) were
used for the detection of Arabidopsis Gln synthetase isoproteins.
Tzs Antibodies
Monoclonal antibodies against Tzs were prepared by Kohjin Bio.
DNA Primers
Primer sequences used in this study are given in Supplemental Table S5.
Construction of TP-Tzs Expression Plasmids
To generate chimeric TP-tzs, which has the transit peptide of maize (Zea
mays) Fd III (Hase et al., 1991) at the N terminus of tzs, cDNA encoding the
IPT Activity Assay
Recombinant Tzs, MA-Tzs, and TP-Tzs, which has a His6 tag at the C terminus, were overexpressed in E. coli strain BL21 containing pQE-Tzs, pQE-MATzs, and pQE-TP-Tzs, respectively. After purification over a nickel-nitrilotriacetic
acid agarose superflow column (Qiagen), IPT activity was measured by using a
modified nonradioisotopic assay as described previously (Sugawara et al., 2008).
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Specialization of Tmr for Efficient Gall Formation
Homologous Recombination of tmr
In order to generate A. tumefaciens mutants, the sacB-based gene-replacement
strategy was employed using pK18mobsacB vector (Schäfer et al., 1994). To disrupt tzs in the vir region of Ti plasmid pTi-SAKURA, a gentamycin-resistant gene
cassette (Gmr) was made by PCR with pUC19Gm (Yamamoto et al., 2009) as the
template and primers Gm MluI 59 and Gm MluI 39 and then cloned into pCRBluntII-TOPO. A 3.7-kb DNA fragment containing tzs and its upstream and
downstream 1.5-kb flanking regions was amplified by PCR with pTi-SAKURA
and primers Pre-Tzs and Post-Tzs and then cloned into pCR-BluntII-TOPO to yield
pTOPO-Tzs+. After confirmation of the nucleotide sequences, an MluI fragment of
Gmr was ligated into the MluI site of pTOPO-Tzs+ to yield pTOPO-Tzs+Gmr. The
BamHI/XbaI fragment was ligated into the BamHI/XbaI site of pK18mobsacB to
yield pK18mobsacB-Tzs+Gmr. The conjugation of pK18mobsacB-Tzs+Gmr to A.
tumefaciens MAFF301001 rif was performed by the method of Uraji et al. (2002).
After selection of the first homologous recombination with kanamycin and gentamycin, a second recombination was selected on Luria-agar medium containing
Suc. Disruption of the gene was confirmed by Southern-blot analysis.
To replace tmr in the T region with tzs, TP-tzs, and AtIPT1, a 3.6-kb DNA
fragment containing tmr and its upstream and downstream 1.5-kb flanking
regions was amplified by PCR with pTi-SAKURA and primers Pre-Tmr and
Post-Tmr and then cloned into pCR-BluntII-TOPO to yield pTOPO-Tmr+. For
replacement with tzs and AtIPT1, ScaI and AvrII sites were generated at the
start and stop codons of tmr, respectively, by the QuickChange Site-Directed
Mutagenesis Kit with the following primers: tmr-QC Sca1 and tmr-QC Sca1
comp for ScaI, tmr-QC Avr2 and tmr-QC Avr2 comp for AvrII. The mutagenized plasmid was named pTOPO-Tmr+*. tzs and AtIPT1 coding regions
were amplified by PCR with the following primers: Tzs Sca1 59 and Tzs Spe-39
for tzs, AtIPT1 Sca1 59 and pIPT1-2 for AtIPT1, and then cloned into pCRBluntII-TOPO. After confirmation of the nucleotide sequences, the ScaI/SpeI
fragment containing tzs or AtIPT1 was ligated into the ScaI/AvrII site of
pTOPO-Tmr+*. Then, ScaI and the AvrII-SpeI fusion site were deleted with the
QuickChange Site-Directed Mutagenesis Kit with the following primers: tmr/
tzs-QC59 and tmr/tzs-QC59comp for deletion of the ScaI site, tmr/tzs-QC39
and tmr/tzs-QC39comp for the AvrII-SpeI fusion site from tzs, tmr/ipt1-QC59
and tmr/ipt1-QC59comp for the ScaI site, and tmr/ipt1-QC39 and tmr/ipt1QC39comp for the AvrII-SpeI fusion site from AtIPT1. Deletions were confirmed by nucleotide sequencing. A SpeI/SphI fragment containing tzs or
AtIPT1 flanked with Tmr upstream and downstream regions was cloned into
the XbaI/SphI site of pK18mobsacB. To replace tmr with TP-tzs, the TP region
(168 bp) and pK18mobsacB-tmr/TZS were amplified by PCR with the following primers: TP-IF-59 and TP-IF-39 for TP, tmr/tzs+1500-IF-59 and tmr/tzs
+1500-IF-39 for pK18mobsacB-tmr/TZS. These fragments were ligated with
the In-Fusion Dry-Down PCR Cloning Kit (Clontech). Double-crossover
homologous recombinations were carried out as described above.
Tumor Formation Assays
A. tumefaciens cells were cultured overnight at 28°C in L-broth supplemented
with the appropriate antibiotics. The bacterial cells were collected by centrifugation, and the cell concentrations were adjusted to 5 3 1010 cells mL21 by diluting
with fresh L-broth medium. The leaves of Kalanchoe plants were wounded with a
toothpick. A bacterial cell suspension (2 mL, equivalent to 1 3 108 cells) was inoculated onto each wound area. Leaves at different developmental stages were
used for inoculations. The tumors were photographed 34 d after inoculation. The
tumorigenic symptoms were classified from 0 (none) to 5 (severe). The tumors
were harvested by excision with a razor blade for further analysis. Rose stems were
wounded with a hypodermic needle, and the bacterial cell suspension (2 mL) was
inoculated onto each wound area. Tumors were photographed 54 d after inoculation. The galls were harvested by excision with a razor blade for further analysis.
RT-PCR Analysis
Eight days after Kalanchoe inoculation with A. tumefaciens total RNA was
prepared from the inoculated area containing developing tumors using the
Fast Pure RNA Kit (Takara) with RNase-free DNase I. cDNA was synthesized
using SuperScript III reverse transcriptase (Invitrogen) with gene-specific
primers: tmr-RT for tmr, tzs-RT for tzs and TP-tzs, AtIPT1-RT for AtIPT1, and
KD-26S-1R for 26S rRNA. The primers used for the following PCRs were tmr
RT-PCR-F1 and tmr RT-PCR-R1 for tmr, tzs-RT-PCR-F3 and tzs-RT-PCR-R5
for tzs, TP-RT-PCR-F4 and TP-RT-PCR-R4 for TP-tzs, AtIPT1-RT-PCR-F3 and
AtIPT1-RT-PCR-R3 for AtIPT1, and KD-26S-1F and KD-26S-2R for 26S rRNA.
Nucleotide sequence data from this article can be found in the GenBank/
EMBL data libraries under the accession numbers as follows: tmr (NC_002147),
tzs (NC_002147), Fd III (M73831), AtIPT1 (AB062607), and 26S rRNA
(AF274651). Nucleotide sequences used in this study are as follows: Tmr
(NP_053424), Tzs (NP_053379), Fd III (BAA19251), and AtIPT1 (BAB59040).
The Arabidopsis Genome Initiative code for AtIPT1 is At1g68460.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Kinetic parameters of Tmr and Tzs.
Supplemental Figure S2. Effect of Met-Ala or transit peptide addition to
the N terminus of Tzs on catalytic activity.
Supplemental Figure S3. Detection of transcripts in tumors on Kalanchoe
by RT-PCR.
Supplemental Figure S4. Protease sensitivity assay with chloroplasts from
Tzs-overexpressing Arabidopsis.
Supplemental Table S1. Quantification of cytokinins in transgenic Arabidopsis lines overexpressing A. tumefaciens IPTs.
Supplemental Table S2. Quantification of cytokinins in transgenic Arabidopsis lines overexpressing Tzs or TP-Tzs.
Supplemental Table S3. Quantification of cytokinins and auxin in galls on
Kalanchoe leaves.
Supplemental Table S4. Quantification of cytokinins in galls on rose.
Supplemental Table S5. Primers used in this study.
ACKNOWLEDGMENTS
We thank Dr. Yasunori Machida (Nagoya University) for kindly providing
Kalanchoe. We also thank Drs. T. Hase and M. Nakai (Osaka University)
for providing us with antibodies against maize Fd and Arabidopsis Toc159,
respectively.
Received April 16, 2012; accepted May 11, 2012; published May 15, 2012.
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