Function of the aux and rol genes of the Ri plasmid in plant cell division in vitro.
ABSTRACT Auxin-autonomous growth in vitro may be related to the integration and expression of the aux and rol genes from the root-inducing (Ri) plasmid in plant cells infected by agropine-type Agrobacterium rhizogenes. To elucidate the functions of the aux and rol genes in plant cell division, plant cell lines transformed with the aux1 and aux2 genes or with the rolABCD genes were established using tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells. The introduction of the aux1 and aux2 genes enabled the auxin-autonomous growth of BY-2 cells, but the introduction of the rolABCD genes did not affect the auxin requirement of the BY-2 cells. The results clearly show that the aux genes are necessary for auxinautotrophic cell division, and that the rolABCD genes are irrelevant in auxin autotrophy.
Article: Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis.Cell 07/1977; 11(2):263-71. · 32.40 Impact Factor
Article: Involvement of a plasmid in the hairy root disease of plants caused by Agrobacterium rhizogenes.[show abstract] [hide abstract]
ABSTRACT: Agrobacterium rhizogenes causes a proliferation of roots on plants that it infects. This is in contrast to Agrobacterium tumefaciens which causes gall or tumor formation on its hosts. A large molecular weight plasmid (1.1 × 108) in A. rhizogenes strain A4 is correlated with the infectivity of this organism. However, this plasmid apparently carries additional information not vital to the infection process. Experimental evidence supporting these conclusions is: (i) A. rhizogenes A4 loses infectivity when all or part of the plasmid is lost after treatment with ethidium bromide or after heating at 37 °C. (ii) There occurs successful conjugational transfer of the A4 plasmid in planta to a noninfectious, antibiotic-resistant A. radiobacter. Infectious transconjugants were antibiotic resistant and contain a plasmid comparable to that of A. rhizogenes A4. (iii) A. rhizogenes A4 and the transconjugants possessed identical EcoR1 restriction endonuclease patterns, whereas three ethidium bromide-treated isolates that were noninfectious but plasmid containing had lost or gained bands in the pattern. The infectious plasmid of A. rhizogenes A4 has been designated pHrA4. Some potential benefits of the A. rhizogenes plasmid to agriculture are discussed.Plasmid 11/1979; 2(4):617-26. · 1.52 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Agrobacterium rhizogenes, which induces hairy root disease of dicotyledonous plants1, is closely related to Agrobacterium tumefaciens, the causative agent of crown gall disease1–3. Virulence in both species is conferred by large plasmids4–7. Infected plant tissue synthesizes novel metabolites, opines8–11, that are not found in normal plant tissues. The pattern of opines synthesized is determined by the type of virulence plasmid in the bacterium, and in general virulence plasmids confer on the host bacterium the ability to catabolize the same opines (refs 8,12, 13 and A.P. et al., in preparation). Opine synthesis persists when the affected plant tissue is cultivated in vitro in the absence of the pathogenic bacterium9–11,14,15, which in the case of crown gall tumours is a consequence of gene transfer from the pathogen to the plant cells. A small specific part of the tumour-inducing (Ti) plasmid of A. tumefaciens, termed T-DNA (transferred DNA), is incorporated into host plant nuclear DNA16–21 and transcribed into mRNA18,22–25. A specific region of T-DNA confers the ability to synthesize the characteristic opine26,27. Synthesis of opines is thus a natural example of genetic engineering in which the agent is the Ti plasmid of,A. tumefaciens. The discovery of opines in roots induced by A. rhizogenes (ref. 11 and A.P. et al., in preparation) suggested that they too might contain T-DNA derived from the virulence plasmid of the pathogen. We report here DNA hybridization studies that confirm this hypothesis.02/1982; 295(5848):432-434.
www.landesbioscience.com Plant Signaling & Behavior 1145
Plant Signaling & Behavior 4:12, 1145-1147; December 2009; © 2009 Landes Bioscience
Key words: Agrobacterium rhizogenes,
auxin-autotrophic cell, auxin biosyn-
thesis, hairy root, plant cell division, Ri
plasmid, T-DNA, aux, rol, tobacco BY-2
Previously published online:
Yoshihiro Mano; Email: email@example.com
and expression of the aux and rol genes
from the root-inducing (Ri) plasmid in
plant cells infected by agropine-type
Agrobacterium rhizogenes. To elucidate
the functions of the aux and rol genes
in plant cell division, plant cell lines
transformed with the aux1 and aux2
genes or with the rolABCD genes were
established using tobacco (Nicotiana
tabacum) Bright Yellow-2 (BY-2) cells.
The introduction of the aux1 and aux2
genes enabled the auxin-autonomous
growth of BY-2 cells, but the intro-
duction of the rolABCD genes did not
affect the auxin requirement of the
BY-2 cells. The results clearly show that
the aux genes are necessary for auxin-
autotrophic cell division, and that the
rolABCD genes are irrelevant in auxin
uxin-autonomous growth in vitro
may be related to the integration
Hairy Roots Induced by
The plant pathogen A. rhizogenes has
a large Ri plasmid and induces hairy
root disease, causing root proliferation
from the infection site. When A. rhizo-
genes infects plants, a portion of the Ri
plasmid, the T-DNA, is transferred to
the plant cell, integrated into the plant
genome, and expressed as polyadenylated
mRNA.1-4 The T-DNA is defined by left-
border and right-border sequences, which
are cis-acting 25-bp direct repeats.5-7
The most studied A. rhizogenes strains,
which belong to the group of agropine-type
strains, have two T-DNA regions on their
Ri plasmid. These regions, designated the
TL-DNA and TR-DNA, can be indepen-
dently transferred to the nuclear genome
of infected plant cells.3,8-10 A. rhizogenes
strains 15834 and A4 are typical agropine-
type strains, and contain the essentially
identical large plasmids pRi15834 and
The Ri TL-DNA carries approximately
18 potential genes,11 of which four genes,
rol A, B, C and D, have a fundamental role
in the induction of hairy root syndrome
in tobacco,10,12-16 although nothing is
known about the functions of the proteins
encoded by these genes. The Ri TR-DNA
carries the genes responsible for opine syn-
thesis17 and the genes representing two
steps of auxin biosynthesis, referred to as
aux1 and aux2 (also referred to as tms1
and tms2, respectively).8,14,18-22 The Ri
TR-DNA also contains a rolB homolog,
but the function of the rolB (TR) gene
product is unknown.23
Hairy roots induced by A. rhizogenes
infection can grow in phytohormone-free
medium in vitro after the elimination of
bacteria.24-26 Phenotype variations among
hairy roots have been attributed to dif-
ferences in the genes from the TL-DNA
and TR-DNA regions that have been inte-
grated into the plant genome.24,25,27,28 In
one example, the TR-DNA from the Ri
plasmid bearing the aux1 and aux2 genes
was never integrated in the cells of coffee
hairy roots, even though the TL-DNA
Function of the aux and rol genes of the Ri plasmid in plant cell
division in vitro
Keiichirou Nemoto,1 Masamitsu Hara,2 Masashi Suzuki,3 Hikaru Seki,3 Atsuhiro Oka,4 Toshiya Muranaka3,5 and Yoshihiro
1Graduate School of Bioscience; and 2Department of Biological Science and Technology; Tokai University; Numazu, Shizuoka Japan; 3RIKEN Plant Science
Center; Yokohama, Kanagawa Japan; 4Institute for Chemical Research; Kyoto University; Uji, Kyoto Japan; 5Plant Biotechnology Division; Yokohama City
University; Yokohama, Kanagawa Japan
Addendum to: Nemoto K, Hara M, Goto S,
Kasai K, Seki H, Suzuki M, Oka A, Muranaka T,
Mano Y. The aux1 gene of the Ri plasmid is suffi-
cient to confer auxin autotrophy in tobacco BY-2
cells. J Plant Physiol 2009; 166:729–38; PMID:
18986729; DOI: 10.1016/j.jplph.2008.09.006
1146 Plant Signaling & Behavior Volume 4 issue 12
the rolABCD genes through selection
based on the vector-derived kanamycin
and hygromycin resistance markers in
the presence of auxin. None of the trans-
genic cell lines was able to multiply with-
out auxin. These results imply that the
rolABCD genes are irrelevant in auxin-
autotrophic cell division.
Transgenic BY-2 Cells
Transformed with the aux Genes
Multiply Without Plant Hormones
To elucidate the functions of the aux
genes in plant cell division, we established
another type of transgenic BY-2 cell line.
The aux2, aux1 and rolB (TR) genes
with their own promoter regions and the
additional region Z were isolated from
pRi15834 by PCR using PfuTurbo® DNA
Polymerase (Stratagene) with the forward
primer TR-F21 (5'-TCG ATT GTA CTG
AAT CGG ATT TTC AAG GGT-3') and
the reverse primer TR-R21 (5'-CAC CAG
GGC GAG ATA AGC TTT CAG GTC
A-3'), and inserted into the EcoRV site
of pER1A.31 This construct served as the
entry vector to transfer the aux2, aux1 and
rolB (TR) genes and the region Z into the
binary destination vector pBCR-112 (Seki
et al. unpublished data) via the Gateway
system (Invitrogen). The resulting plas-
mid pF21BZ was modified to construct
the plasmids pF21B, which contains the
aux2, aux1 and rolB (TR) genes; pF21,
which contains the aux2 and aux1 genes;
and pFZ, which contains the region Z
(Fig. 2). These plasmids were introduced
into A. tumefaciens GV3101 (pMP90), and
BY-2 cells were infected with the resulting
A. tumefaciens strains for transformation.
The transgenic cell lines grown on
kanamycin-containing LSD medium were
tested to examine their auxin-autotrophic
characteristics. Among the transgenic cell
lines, the percent of auxin-autotrophic cell
lines was 11% when inoculating GV3101
containing pF21BZ, 15% for pF21B, 13%
for pF21 and 0% for pFZ. These results
show that the aux1 and aux2 genes are
essential for plant cell division in vitro.
In conclusion, these results clearly
show that the aux genes are necessary for
auxin-autotrophic cell division, and that
the rolABCD genes are irrelevant in auxin
(TL) loci (3,244 bp) in the TL-DNA and
the aux1 and rolB (TR) loci (3,405 bp) in
the TR-DNA, respectively. In a previous
study, we showed that BYHR-7 is trans-
genic for both the TR- and TL-DNAs,
whereas BYHR-3 is only transgenic for the
TR-DNA.31 Figure 1 shows that the aux1
and rolB (TR) loci were detected in the
genomes of all of the transgenic lines, but
the rolA and rolB (TL) loci were detected
only in the genomes of transgenic lines
BYHR-1 and BYHR-7. These results show
that BYHR-1 and BYHR-7 are transgenic
for both the TR- and TL-DNAs, whereas
the other lines are possibly only transgenic
for the TR-DNA.
Transgenic BY-2 Cells
Transformed with the rolABCD
Genes Cannot Multiply Without
The rolABCD genes, along with their
own promoter region, were isolated from
pRi15834 by PCR using Phusion® High-
Fidelity DNA Polymerase (NEB) with
the forward primer TL-FA (5'-GCC ACG
CAA CTC GAG ATA TTC CCA C-3')
and the reverse primer TL-RD (5'-GCG
ACG AAT TCA AAC AAG CCC GTC
T-3'), and subcloned into the EcoRV site
of pBluescript II SK+ (Stratagene). This
construct was digested with Acc65I and
XbaI, and inserted into the binary vec-
tor pGAH33 that had been digested with
Acc65I and XbaI. The resulting plasmid,
pGABCD, was introduced into A. tume-
faciens GV3101 (pMP90). To elucidate
the functions of the rol genes in plant
cell division, we established 125 trans-
genic BY-2 cell lines transformed with
bearing the rol genes was always trans-
ferred.29 Various experiments, performed
by different researchers, have demon-
strated that the aux genes do not play a
major role in hairy root disease, whereas
the rol genes are necessary and sufficient
to induce hairy roots.13,15,30 However, there
have been contradictory reports of the
contributions of aux1 and aux2 to plant
tumorigenicity; some have suggested that
these aux genes are important for hairy
Characterization of Tobacco BY-2
Cells Transformed by
Auxin-autonomous growth may be related
to the integration and expression in trans-
formed cells of aux and rol genes from the
Ri plasmid that enable the production of
endogenous auxin and/or trigger auxin
signal transduction. To elucidate the func-
tions of the aux and rol genes in plant cell
division, we generated transformed plant
cell lines by infecting BY-2 cells with
A. rhizogenes strain 15834.31 Tobacco BY-2
cells require only auxin for growth, and
are usually cultured in Linsmaier-Skoog
(LS) medium32 containing 0.2 μg mL-1 of
2,4-dichlorophenoxyacetic acid (2,4-D)
The transgenic cell lines, established
by further culturing through seven pas-
sages in phytohormone-free medium,
were named BYHR-1, 3, 5, 7, 8 and 9. To
determine the presence or absence of the
T-DNA in the genomes of the transgenic
lines, PCR analysis was performed using
the primer sets TL-F1/TL-R1 and TR-F7/
TR-R5,31 which cover the rolA and rolB
Figure 1. Pcr analysis to detect the aux1 and rolB (tr) loci in the tr-Dna (a) and the rolA
and rolB (tL) loci in the tL-Dna (B) in the genomes of the transgenic cell lines BYHr-1 (lane 2),
BYHr-3 (lane 3), BYHr-5 (lane 4), BYHr-7 (lane 5), BYHr-8 (lane 6) and BYHr-9 (lane 7). Pcr
amplification was performed with genomic DNAs (2 μg) from BY-2 cells (lane 1) and the transgen-
ic cell lines, followed by agarose gel electrophoresis. numbers to the right of the gel correspond
to those of the Pcr fragments. m, λ Hindiii size marker.
www.landesbioscience.com Plant Signaling & Behavior 1147
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different morphogenetic activity in tobacco than rolB.
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Figure 2. Binary vector construct harboring several genes of the pri15834 tr-Dna. LB and rB indicate the left and right borders, respectively. the
arrows above and below the tr-Dna indicate the positions of representative genes that are transcribed from the left-to-right and right-to-left direc-
tions. the bar shows the location and size of the Pcr product obtained using the primer set tr-F21/tr-r21.