FEMS Microbiology Letters 179 (1999) 141^146
GFP-aided confocal laser scanning microscopy can monitor
Agrobacterium tumefaciens cell morphology and gene expression
associated with infection
Luoping Li a , Yanjun Li b , Tit Meng Lim a , Shen Q. Pan
a
a;b;
*
Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore
b
Department of Plant Pathology, China Agricultural University, Beijing 100094, China
Received 26 May 1999; received in revised form 28 July 1999; accepted 30 July 1999
Abstract
We tagged Agrobacterium tumefaciens cells with a mini-Tn5 transposon containing a promoterless gene encoding a green
fluorescent protein (GFP). Some of the GFP-tagged individual bacterial cells exhibited strong green fluorescence, which
reflected the expression levels of the GFP-tagged genes. Those cells could be readily detected with a confocal laser scanning
microscope (CLSM). We observed that the fluorescence and morphology of A. tumefaciens cells grown in plant tissues
resembled those grown in a minimal medium of low pH, which is required for expression of the virulence genes responsible for
tumorigenesis. This suggests that GFP-aided CLSM can be used to determine which growth medium is more representative of
the nutritional conditions that a pathogen encounters in plant tissues. We also observed that the fluorescence and morphology
of A. tumefaciens cells changed dramatically during the course of infection. Our data suggested that A. tumefaciens cells were
probably better fed upon successful colonization. We believe that GFP-aided CLSM can help study the fate of A. tumefaciens
cells inside plant tissues by monitoring cell morphology and gene expression associated with the infection process in
situ. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Agrobacterium; Green £uorescent protein; Confocal microscope ; In situ; Infection
1. Introduction
Cloning and characterization of virulence genes,
avirulence genes and resistance genes have generated
a great deal of both genetic and molecular information that can help us understand the molecular
* Corresponding author. Tel.: +65 874-2977;
Fax: +65 779-5671; E-mail: [email protected]
events in plant-pathogen interactions [1^3]. However, some of the questions pertaining to plant^
pathogen interactions have not been investigated intensively at the molecular level. For instance, how do
pathogens bene¢t from a successful infection? How
do pathogens live inside plant tissues? What kinds of
growth conditions do pathogens encounter inside
plant tissues? Do pathogens go through physiological changes during the course of infection? In short,
what happens to pathogens inside plant tissues has
not been closely examined, partly due to inadequate
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 4 0 3 - 6
FEMSLE 8987 9-9-99
Cyaan Magenta Geel Zwart
142
L. Li et al. / FEMS Microbiology Letters 179 (1999) 141^146
technology to monitor the fate of pathogens inside
the host plants in situ during the course of infection.
In order to study what happens to pathogenic bacteria during the course of infection, we tagged Agrobacterium tumefaciens with a mini-Tn5 transposon
containing a promoterless gene encoding a green £uorescent protein (GFP) variant, which produces
bright green £uorescence under blue or UV light.
GFP can serve as a reporter for studying biological
processes in vivo and in real time, as the £uorescence
is an intrinsic property of the protein and does not
require any cofactors, substrates, or additional gene
products [4^6]. A. tumefaciens is a soil-borne plant
pathogen that causes crown gall tumors on many
plant species by transferring a speci¢c segment (TDNA) of its tumor-inducing (Ti) plasmid into plant
cells, where the T-DNA becomes integrated into the
plant genome. The T-DNA contains the oncogenes
that cause overproduction of plant hormones and
hence tumors [7^9]. We used a confocal laser scanning microscope (CLSM) to monitor the GFPtagged bacterial cells in situ during the infection
process.
2. Materials and methods
2.1. Transposon tagging
Escherichia coli S17.1(Vpir) was transformed with
pAG408 by electroporation according to Dower et
al. [10]. The plasmid pAG408 carried a mini-Tn5
transposon containing the promoterless gfp gene
[11]. Transposition of the mini-Tn5 transposon into
A. tumefaciens C58 cells was carried out by mixing
the freshly grown cell suspension of S17.1(Vpir)(pAG408) (donor) with that of C58 (recipient) on
agar plates of MG/L [12]. The cell density was
5U107 cells ml31 for both the donor and recipient,
but the ratio of the cell suspension volume for the
donor to the recipient was 1:4. After the mixture was
spotted on the plates, they were dried for 1 h at
37³C. They were then incubated at 28³C for 24 h.
The bacterial cells were plated onto agar plates of a
minimal medium AB [12] containing 100 Wg ml31
kanamycin. The plates were incubated at 28³C for
2 days. The colonies that appeared on those AB
plates were the mini-Tn5 transposon mutants, since
FEMSLE 8987 9-9-99
the kanamycin resistance is encoded by the mini-Tn5
transposon harbored by a plasmid that can replicate
in E. coli S17.1(Vpir) (which cannot grow on the AB
medium) but not in A. tumefaciens (which is sensitive
to kanamycin).
2.2. Inoculation
The leaves of Kalanchoe daigremontiana plants
were wounded by a pin brush (28 pins, 1 cm in
diameter). Then 5 Wl of bacterial cell suspension
(grown on MG/L plates for 2 days and suspended
in MG/L at 5U108 cells ml31 ) was inoculated onto
each area of the wounds (28 wounds of 0.5 mm in
diameter) in£icted by a single pin brush puncturing.
The plants were incubated in a growth room at
26³C.
2.3. Confocal microscopy
The GFP-tagged bacterial cells were grown on
agar plates of MG/L, LB, AB, IB [12], or MS [13]
containing 100 Wg ml31 kanamycin, or on fresh Kalanchoe tissue sections which were sterile and placed
on MS medium. The bacterial cells were examined
under a CLSM (Bio-Rad MRC-500) under the 488
nm blue light laser. To observe the bacterial cells
inside plant tissues, we made free-hand sections of
the tumors at the wound sites and examined them
under the confocal microscope.
3. Results and discussion
We tagged A. tumefaciens C58 cells with the miniTn5 transposon. About 4% of transconjugant colonies exhibited bright green £uorescence under UV
light. Eight of 69 £uorescent colonies were chosen
to inoculate the leaves of Kalanchoe plants. All of
them caused tumors on the plants just like the
wild-type A. tumefaciens. The individual A. tumefaciens cells could be readily detected by CLSM. The
£uorescence facilitated easy detection of bacterial
cells inside plant tissues. In addition, the £uorescence
re£ected the expression levels of the GFP-tagged
genes, as the A. tumefaciens promoters of the GFPtagged genes controlled the GFP expression (data
not shown). One of the eight £uorescent colonies,
Cyaan Magenta Geel Zwart
L. Li et al. / FEMS Microbiology Letters 179 (1999) 141^146
143
Fig. 1. Comparison of A. tumefaciens cells grown on di¡erent growth media and plant tissue sections. CG-19 cells were grown on MG/L
(A), LB (B), AB (C), IB (D) and MS (E) media until single colonies appeared. Meanwhile, the bacterial cells were inoculated onto K. daigremontiana stem sections (F), which were placed on MS media. They were incubated at room temperature for 2 days. The bacterial cells
were examined by a confocal microscope. The scale bar represents 10 Wm.
FEMSLE 8987 9-9-99
Cyaan Magenta Geel Zwart
144
L. Li et al. / FEMS Microbiology Letters 179 (1999) 141^146
Fig. 2. A. tumefaciens cells inside plant tissues. CG-19 cells were inoculated into leaves of Kalanchoe plants, which were wounded with a
pin brush. Free-hand sections of inoculated tissues were examined under a confocal microscope at 3 (A), 5 (B), 8 (C), and 10 (D) days
after inoculation. The scale bar represents 10 Wm.
named CG-19, exhibited the strongest £uorescence
inside the plant tissues. CG-19 could cause crown
gall tumors on plants and grow on the media as
well as the parent strain. CG-19 was used subsequently to monitor the morphological changes and
£uorescence levels of A. tumefaciens cells during the
course of infection.
First, we wanted to determine which growth medium was the most representative of the growth conditions that the A. tumefaciens cells encounter in
plant tissues. We streaked CG-19 cells on agar plates
of MG/L, LB, AB, IB [12] and MS [13] containing
100 Wg ml31 kanamycin. When the single colonies
appeared on the plates, the bacterial cells were observed under the confocal microscope. As shown in
Fig. 1, the morphology and £uorescence of CG-19
FEMSLE 8987 9-9-99
cells grown in MG/L (Fig. 1A), LB (Fig. 1B) and AB
(Fig. 1C) were similar. The growth rates of CG-19
on MG/L, LB and AB plates were also similar, since
similar sizes of bacterial colonies appeared 2 days
after incubation on those plates. In contrast, the
CG-19 cells grown on IB medium (Fig. 1D) were
more £uorescent than those on MG/L, LB and AB
media (Fig. 1A^C) (it took 3 days for the bacterial
colonies to appear on the IB plates). The cells grown
on MS medium (Fig. 1E) were longer than those on
MG/L, LB, AB and IB media (Fig. 1A^D). The
growth rate of CG-19 on MS medium was the slowest among the media tested here, since it took about
10 days for the bacterial colonies to appear on the
MS plates.
We inoculated CG-19 cells directly onto fresh Ka-
Cyaan Magenta Geel Zwart
L. Li et al. / FEMS Microbiology Letters 179 (1999) 141^146
lanchoe tissue sections. The bacteria could grow to
some extent on those tissue sections. We found that
the cells grown on the stem tissue (Fig. 1F) resembled those grown on IB medium (Fig. 1D). The
cells grown on the leaf tissue (data not shown) also
looked similar to those grown in IB medium (Fig.
1D). This suggested that among the media tested, IB
(a minimal medium of pH 5.5) was the most representative of the bacterial growth conditions in plant
tissues. This is consistent with the fact that the virulence (vir) genes that are directly responsible for
processing, transfer and possibly integration of TDNA are induced in IB medium but not in MG/L,
LB and AB media [14,15]. Therefore, we believe that
GFP-aided CLSM can be utilized to determine
which growth media are most representative of certain host conditions that a pathogen encounters.
Subsequently, we wanted to study what happened
to the A. tumefaciens cells inside plant tissues during
infection. The bacterial cells inoculated into the
wounds of Kalanchoe plants were examined under
the confocal microscope at di¡erent time intervals
after inoculation. As shown in Fig. 2, the morphology and £uorescence of the bacterial cells changed
dramatically during the course of infection. Three
days after inoculation into the plants, the bacterial
cells became more £uorescent (Fig. 2A) as compared
to the cells grown on MG/L (Fig. 1A). Five days
after inoculation, however, the £uorescence became
almost invisible (Fig. 2B). Eight days after inoculation, the £uorescence appeared again (Fig. 2C). Ten
days after inoculation, the £uorescence became extremely strong (Fig. 2D). This suggested that the
expression levels of the GFP-tagged gene changed
dramatically during the course of infection. This
gene expression pattern should be speci¢c for the
infection process, since the £uorescence of another
transposon mutant (CG-9) in which the GFP was
fused onto the A. tumefaciens 16S rRNA gene was
constant inside plant tissues during infection (data
not shown). Therefore, we believe that GFP-tagging
can help monitor the in situ expression levels of the
genes relevant to the infection process. This would
be useful to further study the fate of the bacteria in
the host tissues.
In addition, the bacterial multiplication became
very apparent 10 days after inoculation (Fig. 2D).
At that time, the tumors appeared. The bacterial
FEMSLE 8987 9-9-99
145
cells also became more spherical than those grown
on IB medium (Fig. 1D) or inoculated onto plant
tissue sections (Fig. 1F). We hypothesize that the
bacterial cells were better fed upon successful colonization. Several lines of evidence support this hypothesis. (1) The T-DNA of A. tumefaciens contains
the genes that are involved in the biosynthesis of
opines, a group of novel compounds, which can be
utilized by A. tumefaciens via the opine catabolic
genes encoded on the Ti plasmid. The tumor cells
have been shown to produce opines [7]. (2) It has
been previously shown that the shape of a cell is
related to the cell growth rate. When the cells are
dividing rapidly, they tend to be smaller; when the
cells are dividing slowly, they become excessively
large [16]. (3) Our own data also support this notion
(Fig. 1). The A. tumefaciens cells grew faster and
were more spherical on MG/L, LB and AB media
than on IB and MS media. The bacterial cells grew
slower on MS than on IB medium; and they were
larger and longer on MS than on IB medium.
GFP has been used to facilitate detection of fungal
infection [17] and viral movement [18,19] as well as
symbiotic Rhizobium [20] inside plant tissues. Here
we report that GFP-aided confocal microscopy enabled us to determine that a minimal medium of pH
5.5 was the most representative of the plant's environment. This is supported by previous observations
that the A. tumefaciens vir genes are induced on this
medium [14,15]. We also observed that the morphology and £uorescence of the A. tumefaciens cells
tagged with a promoterless GFP gene changed during the course of infection. This suggests that the
expression levels of the bacterial genes relevant to
infection can be monitored in situ after these genes
are tagged with a promoterless GFP gene. In conclusion, GFP-aided CLSM can monitor the morphological changes and gene expression associated with
the infection process.
Acknowledgements
We thank C.A. Guzman for providing the plasmid
pAG408 and H.M. Soo and L.W. Tan for technical
assistance. This work was supported in part by the
National University of Singapore Academic Research Grants, RP970323 and RP3972375, and the
Cyaan Magenta Geel Zwart
146
L. Li et al. / FEMS Microbiology Letters 179 (1999) 141^146
National Natural Science Foundation of China Research Grants, 39670414 and 39625018, to S.Q.P.
[12]
References
[13]
[1] Baker, B., Zambryski, P., Staskawicz, B. and Dinesh-Kumar,
S.P. (1997) Signalling in plant-microbe interactions. Science
276, 726^773.
[2] Staskawicz, B.J., Ausubel, F.M., Baker, B.J., Ellis, J.G. and
Jones, J.D.G. (1995) Molecular genetics of plant disease resistance. Science 268, 661^667.
[3] Keen, N.T. (1990) Gene-for-gene complementarity in plantpathogen interactions. Annu. Rev. Genet. 24, 447^463.
[4] Chal¢e, M., Tu, Y., Euskirchen, G., Ward, W.W. and
Prasher, D.C. (1994) Green £uorescent protein as a marker
for gene expression. Science 263, 802^805.
[5] Wang, S. and Hazelrigg, T. (1994) Implications for bcd
mRNA localization from spatial distribution of exu protein
in Drosophila oogenesis. Nature 369, 400^403.
[6] Yeh, E., Gustafson, K. and Boulianne, G.L. (1995) Green
£uorescent protein as a vital marker and reporter gene expression in Drosophila. Proc. Natl. Acad. Sci. USA 92, 7036^7040.
[7] Kado, C.I. (1991) Molecular mechanisms of crown gall tumorigenesis. Crit. Rev. Plant Sci. 10, 1^32.
[8] Sheng, J. and Citovsky, V. (1996) Agrobacterium-plant cell
DNA transport: have virulence proteins, will travel. Plant
Cell 8, 1699^1710.
[9] Zupan, J. and Zambryski, P. (1997) The Agrobacterium DNA
transfer complex. Crit. Rev. Plant Sci. 16, 279^295.
[10] Dower, W.J., Miller, J.F. and Ragsdale, C.W. (1988) High
e¤ciency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16, 6127^6145.
[11] Suarez, A., Guttler, A., Stratz, M., Staendner, L.H., Timmis,
FEMSLE 8987 9-9-99
[14]
[15]
[16]
[17]
[18]
[19]
[20]
K.N. and Guzman, C.A. (1997) Green £uorescent proteinbased reporter systems for genetic analysis of bacteria including monocopy applications. Gene 196, 69^74.
Cangelosi, G.A., Best, E.A., Martinetti, G. and Nester, E.W.
(1991) Genetic analysis of Agrobacterium. Methods Enzymol.
204, 384^397.
Murashige, T. and Skoog, F. (1962) A revised medium for
rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15, 473^497.
Rogowsky, P.M., Close, T.J., Chimera, J.A., Shaw, J.J. and
Kado, C.I. (1987) Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58. J. Bacteriol. 169, 5101^
5112.
Winans, S.C. (1990) Transcriptional induction of an Agrobacterium regulatory gene at tandem promoters by plant-released
phenolic compounds, phosphate starvation and acidic growth
media. J. Bacteriol. 172, 2433^2438.
Kleinsmith, L.J. and Kish, V.M. (1995) Cell cycles and cell
division. In: Principles of Cell and Molecular Biology, 2nd
edn., p. 519, Harper Collins College Publishers, New York.
Spellig, T., Bottin, A. and Kahmnn, R. (1996) Green £uorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis. Mol. Gen. Genet. 252, 503^509.
Padgett, H.S., Epel, B.L., Kahn, T.W., Heinlein, M., Watanabe, Y. and Beachy, R.N. (1996) Distribution of tobacco
virus movement protein in infected cells and implications for
cell-to-cell spread of infection. Plant J. 10, 1079^1088.
Verver, J., Wellink, J., Van Lent, J., Gopinath, K. and Van
Kammen, A. (1998) Studies on the movement of cowpea mosaic virus using the jelly¢sh green £uorescent protein. Virology
242, 22^27.
Gage, D.J., Bobo, T. and Long, S.R. (1996) Use of green
£uorescent protein to visualize the early events of symbiosis
between Rhizobiumm meliloti and alfalfa (Medicago sativa).
J. Bacteriol. 178, 7159^7166.
Cyaan Magenta Geel Zwart