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.

Full text

Page 1

www.landesbioscience.com Plant Signaling & Behavior 1145
Plant Signaling & Behavior 4:12, 1145-1147; December 2009; © 2009 Landes Bioscience
articLe aDDenDum
articLe aDDenDum
Key words: Agrobacterium rhizogenes,
auxin-autotrophic cell, auxin biosyn-
thesis, hairy root, plant cell division, Ri
plasmid, T-DNA, aux, rol, tobacco BY-2
cells
Submitted: 08/25/09
Accepted: 08/25/09
Previously published online:
www.landesbioscience.com/journals/psb/
article/9904
*Correspondence to:
Yoshihiro Mano; Email: y-mano@wing.ncc.u-tokai.ac.jp
A
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
autotrophy.
uxin-autonomous growth in vitro
may be related to the integration
Hairy Roots Induced by
Agrobacterium rhizogenes
Infection
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
pRiA4, respectively.4
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
Mano1,2,*
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

Page 2

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
autotrophy.
(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
Plant Hormones
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
root formation.14,19
Characterization of Tobacco BY-2
Cells Transformed by
Agrobacterium rhizogenes
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)
(LSD medium).
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.

Page 3

www.landesbioscience.com Plant Signaling & Behavior 1147
23. Lemcke K, Schmülling T. A putative rolB gene homo-
logue of the Agrobacterium rhizogenes TR-DNA has
different morphogenetic activity in tobacco than rolB.
Plant Mol Biol 1998; 36:803-8.
24. Mano Y, Nabeshima N, Matsui C, Ohkawa H.
Production of tropane alkaloids by hairy root cultures
of Scopolia japonica. Agric Biol Chem 1986; 50:2715-
22.
25. Mano Y, Ohkawa H, Yamada Y. Production of
tropane alkaloids by hairy root cultures of Duboisia
leichhardtii transformed by Agrobacterium rhizogenes.
Plant Sci 1989; 59:191-201.
26. Mano Y, Matsuhashi M. A novel life cycle arising
from leaf segments in plants regenerated from horse-
radish hairy roots. Plant Cell Rep 1995; 14:370-4.
27. Ambros PF, Matzke AJ, Matzke MA. Localization
of Agrobacterium rhizogenes T-DNA in plant chro-
mosomes by in situ hybridization. EMBO J 1986;
5:2073-7.
28. Jouanin L, Guerche D, Pamboukdjian N, Tourneur
C, Casse-Delbart F, Tourneur J. Structure of T-DNA
in plants regenerated from roots transformed by
Agrobacterium rhizogenes strain A4. Mol Gen Genet
1987; 206:387-92.
29. Alpizar E, Dechamp E, Lapeyre-Montes F,
Guilhaumon C, Bertrand B, Jourdan C, et al.
Agrobacterium rhizogenes-transformed roots of coffee
(Coffea arabica): conditions for long-term prolifera-
tion, and morphological and molecular characteriza-
tion. Ann Bot 2008; 101:929-40.
30. Boulanger F, Berkaloff A, Richaud F. Identification of
hairy root loci in the T-regions of Agrobacterium rhizo-
genes Ri plasmids. Plant Mol Biol 1986; 6:271-9.
31. Nemoto K, Hara M, Goto S, Kasai K, Seki H, Suzuki
M, et al. The aux1 gene of the Ri plasmid is sufficient
to confer auxin autotrophy in tobacco BY-2 cells. J
Plant Physiol 2009; 166:729-38.
32. Linsmaier EM, Skoog F. Organic growth factor
requirements of tobacco tissue cultures. Physiol Plant
1965; 18:100-27.
33. Onouchi H, Yokoi K, Machida C, Matsuzaki H,
Oshima Y, Matsuoka K, et al. Operation of an
efficient site-specific recombination system of
Zygosaccharomyces rouxii in tobacco cells. Nucl Acids
Res 1991; 19:6373-8.
13. Spena A, Schmulling T, Koncz C, Schell J.
Independent and synergistic activity of the rol A, B
and C loci in stimulating abnormal growth in plants.
EMBO J 1987; 6:3891-9.
14. White FF, Taylor BH, Huffman GA, Gordon MP,
Nester EW. Molecular and genetic analysis of the
transferred DNA regions of the root-inducing plasmid
of Agrobacterium rhizogenes. J Bacteriol 1985; 164:33-
44.
15. Cardarelli M, Mariotti D, Pomponi M, Spanò L,
Capone I, Costantino P. Agrobacterium rhizogenes
T-DNA genes capable of inducing hairy root pheno-
type. Mol Gen Genet 1987; 209:475-80.
16. Vilaine F, Charbonnier C, Casse-Delbart F. Further
insight concerning the TL region of the Ri plasmid
of Agrobacterium rhizogenes strain A4: transfer of a
1.9 kb fragment is sufficient to induce transformed
roots on tobacco leaf fragments. Mol Gen Genet 1987;
210:111-5.
17. De Paolis A, Mauro ML, Pomponi M, Cardarelli M,
Spano L, Costantino P. Localization of agropine-
synthesizing functions in the TR region of the root-
inducing plasmid of Agrobacterium rhizogenes 1855.
Plasmid 1985; 13:1-7.
18. Jouanin L. Restriction map of an agropine-type Ri
plasmid and its homologies with Ti plasmids. Plasmid
1984; 12:91-102.
19. Offringa IA, Melchers LS, Regensburg-Tuink AJG,
Costantino P, Schilperoort RA, Hooykaas PJJ.
Complementation of Agrobacterium tumefaciens
tumor-inducing aux mutants by genes from the
TR-region of the Ri plasmid of Agrobacterium rhizo-
genes. Proc Natl Acad Sci USA 1986; 83:6935-9.
20. Sinkar VP, White FF, Furner IJ, Abrahamsen M,
Pythoud F, Gordon MP. Reversion of aberrant plants
transformed with Agrobacterium rhizogenes is associ-
ated with the transcriptional inactivation of the
TL-DNA genes. Plant Physiol 1988; 86:584-90.
21. Levesque H, Delepelaire P, Rouzé P, Slightom J,
Tepfer D. Common evolutionary origin of the central
portions of the Ri TL-DNA of Agrobacterium rhizo-
genes and the Ti T-DNAs of Agrobacterium tumefa-
ciens. Plant Mol Biol 1988; 11:731-44.
22. Gaudin V, Camilleri C, Jouanin L. Multiple regions
of a divergent promoter control the expression of the
Agrobacterium rhizogenes aux1 and aux2 plant onco-
genes. Mol Gen Genet 1993; 239:225-34.
References
1. Chilton MD, Drummond MH, Merio DJ, Sciaky D,
Montoya AL, Gordon MP, et al. Stable incorporation
of plasmid DNA into higher plant cells: the molecular
basis of crown gall tumorigenesis. Cell 1977; 11:263-
71.
2. Moore L, Warren G, Strobel G. Involvement of a
plasmid in the hairy root disease of plants caused by
Agrobacterium rhizogenes. Plasmid 1979; 2:617-26.
3. Chilton MD, Tepfer DA, Petit A, David C, Casse-
Delbart F, Tempe J. Agrobacterium rhizogenes inserts
T-DNA into the genome of the host plant root cells.
Nature 1982; 295:432-4.
4. White FF, Nester EW. Relationship of plasmids
responsible for hairy root and crown gall tumorigenic-
ity. J Bacteriol 1980; 144:710-20.
5. Yadav NS, Vanderleyden J, Bennett DR, Barnes WM,
Chilton M-D. Short direct repeats flank the T-DNA
on a nopaline Ti plasmid. Proc Natl Acad Sci USA
1982; 79:6322-6.
6. Wang K, Herrera-Estrella L, Montagu MV, Zambryski
P. Right 25 bp terminus sequence of the nopaline
T-DNA is essential for and determines direction
of DNA transfer from Agrobacterium to the plant
genome. Cell 1984; 38:455-62.
7. Barker RF, Idler KB, Thompson DU, Kemp JD.
Nucleotide sequence of the T-DNA region from
Agrobacterium tumefaciens octopine Ti plasmid pTi
15955. Plant Mol Biol 1983; 2:335-50.
8. Huffman GA, White FF, Gordon MP, Nester GW.
Hairy root inducing plasmid: physical map and
homology to tumor inducing plasmid. J Bacteriol
1984; 157:269-76.
9. Zambryski P, Tempé J, Schell J. Transfer and func-
tion of T-DNA genes from Agrobacterium Ti and Ri
plasmids in plants. Cell 1989; 56:193-201.
10. Vilaine F, Casse-Delbart F. Independent induction of
transformed roots by the TL and TR regions of the
Ri plasmid of agropine type Agrobacterium rhizogenes.
Mol Gen Genet 1987; 206:17-23.
11. Slightom JL, Durand-Tardif M, Jouanin L, Tepfer
D. Nucleotide sequence analysis of TL-DNA of
Agrobacterium rhizogenes agropine type plasmid:
Identification of open-reading frames. J Biol Chem
1986; 261:108-21.
12. Spano L, Mariotti D, Cardarelli M, Branca C,
Costantino P. Morphogenesis and auxin sensitivity of
transgenic tobacco with different complements of Ri
T-DNA. Plant Physiol 1988; 87:479-83.
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.