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Molecular Microbioiogy (1993) 10(3), 597-605
Restoration of attachment, virulence and noduiation of
Agrobacterium tumefaciens chvB mutants by rhicadhesin
Saskia Swart,* Gerrit Smit, Ben J.J. Lugtenberg and
Jan W. Kijne
institute of Moiecular Plant Sciences, Leiden University,
Nonnensteeg 3. 2311 VJ Leiden, The Netheriands.
Summary
In contrast to wild-type Agrobacterium tumefaciens
strains, p-1,2-glucan-deficjent chvB mutants were
found to be unable to attach to pea root hair tips. The
mutants appeared to produce rhicadhesin, the protein that mediates the first step in attachment of Rhizobiaceae cells to plant root hairs, but the protein
was inactive. Both attachment to root hairs and virulence of the ChvB mutants could be restored by treatment of the plants with active rhicadhesin, whereas
treatment of plants with |3-1,2-glucan had no effect on
attachment or virulence. Moreover, nodulation ability
of a chvB mutant carrying a Sym plasmid could be
restored by pretreatment of the host plant with rhicadhesjn. Apparently the attachment-minus and avirulence phenotype of chvB mutants is caused by tack
of active rhicadhesin, rather than directly being
caused by a deficiency in p-1,2-glucan synthesis. The
results strongly suggest that rhicadhesin is essential
for attachment and virulence of A. tumefaciens cells.
They also indicate that the mechanisms of binding of
Agrobacterium and Rhizobium bacteria to plant target cells are similar, despite differences between
these target cells.
Introduction
Attachment of Rhizobium and Agrobacterium bacteria,
both members of the Rhizobiaceae family, to host plant
cells is an early step in the establishment of the plantbacterium associations leading to the formation of nitrogen-fixing root nodules and plant tumours, respectively.
Attachment of Agrobacterium and Rhizobium cells
appears to be a two-step process in which binding to the
plant target cell surface is followed by accumulation of the
bacteria on the plant cell (Matthysse, 1983; Kljne e( a/.,
Received 7 June, 1993; revised and accepted 26 July, 1993. *For correspondence. Tel. (71) 275059; Fax (71) 275088.
1988; Smit et ai, 1989b; 1992), The majority of genes
that are responsible for tumour induction by Agrobacterium are located on the Ti (tumour-inducing) plasmid
(Van Larebeke etai., 1974; Zaenen etai., 1974), whereas
most nodulation genes of Rhizobium are localized on the
Sym (symbiosis) plasmid (Johnston etai., 1978; Nuti et
ai., 1979; Hirsch etai., 1980), Introduction of a Ti piasmid
into Rhizobium can yield tumorigenic Rhizobium strains,
whereas Agrobacterium harbouring a Sym plasmid can
nodulate certain leguminous plants (Hooykaas et ai.,
1977; 1981; 1985). In contrast, genes involved in the
attachment of Agrobacterium to cells of host plants are
localized on the bacterial chromosome (Douglas et ai.,
1982; Smit etai., 1987). Similarly, attachment of Rhizobium to the root hairs of host plants is chromosomally
encoded (Smitefa/., 1986; Kijne ef a/., 1988).
Cell-to-cell attachment is considered to be a prerequisite for plant tumour formation. Plant molecules involved
in the attachment process are poorly characterized.
Recently a vitronectin-like protein of plant origin was proposed to play a role in attachment of Agrobacterium
(Wagner and Matthysse, 1992). Several bacterial chromosomal loci are reported to be involved in the ability of
Agrobacterium tumefaciens to attach to plant cells (Douglas etai., 1982; Cangelosi etai., 1987; Matthysse. 1987;
Thomashow etai., 1987). The corresponding mutants are
all avirulent. Two of these loci. chvB and exoC. are
required for the production of p-1,2-glucan (Puvanesarajah etai., 1985; Cangelosi etai., 1987). cftvSmutants do
not produce cyclic p-1.2-glucan, since the chvB gene
encodes a 235 kDa cytoplasmic membrane protein, that
is required for p-1,2-glucan synthesis (Zorreguieta et al.,
1988). A third mutant, chvA, is deficient in the excretion of
cyclic p-1,2-glucan from the cell (Cangelosi et ai., 1989;
Inon de lannino and Ugalde, 1989; O'Connel and Handelsman. 1989), However, the mutant still exports some
p-1,2-glucan (O'Connel and Handelsman, 1989) and has
been reported to have a leaky phenotype (Van Veen et
ai., 1987), Since p-1.2-glucan mutants studied so far were
found to be attachment deficient, ^-1,2-glucan was
hypothesized to be necessary for attachment. However, it
should be noted that these mutants show a pleiotropic
phenotype (Douglas et ai.. 1985; Puvanesarajah et ai,
1985; Cangelosi etai., 1987; Thomashow etai, 1987).
Results from our laboratory showed that binding of Rhizobiaceae to the surface of plant root hairs is mediated by
598
S. Swart, G. Smit, B. J. J. Lugtenberg and J. W. Kijne
Table 1. Attachment of Agrobacterium and Rhizobium cells to pea root
haJrtips^.
I attachment in class''
Stfain
characteristics
t
2
3
A. tumetaciens
LBA1010
1251
A1011
A1020
A1045
ME117
Wild type
pTi-. pCr-cured
ChvB
ChvB
chvB
ChvB
12
9
73
75
90
63
33
25
24
24
9
15
55
66
3
1
1
2
7
23
70
R. leguminosarum bv. viciae
24B
Wild type
a. Bacteria were incubated with the roots as described previously (Smit et
ai., 1986).
b. Class 1, no attached bacteria; class 2, tew bacteria, directly attached to
the root hair; class 3, many attached bacteria, forming two or more layers
of bacteria or a cap-like aggregate at the tip ot the root hair (cap formation).
rhicadhesin, a calcium-binding protein located on the bacterial cell surface (Smit et al., 1989a,b). Since chvB
mutants are unable to attach to plant cells, and since it
was unknown whether these bacteria produce rhicadhesin, we studied the attachment characteristics of these
mutants to pea root hair tips in relation to production of
rhicadhesin. Study of chvB mutants was preferred over
chvA mutants because of the leaky phenotype of chvA
mutants. The results show that the attachment-minus and
avirulence phenotypes of chvB mutants result from a lack
of active rhicadhesin and not from a lack of p-1.2-glucan.
Furthermore, evidence is presented that in the case of
nodulation by Sym plasmid-carrying Agrobacterium cells
attachment is a prerequisite for infection, being dependent on the presence of active rhicadhesin.
plasmid-cured strain, and tested for the presence of rhicadhesin, that is, for its ability to inhibit attachment of A.
tumefaciens and R. leguminosarum cells to pea root
hairs. As shown for A. tumefaciens in Table 2, cell surface
components isolated from chvB mutants did not possess
rhicadhesin activity, whereas cell surface preparations
isolated from A. tumefaciens wild-type and plasmid-cured
cells did. The same results were obtained with inhibition
of attachment of R leguminosarum 248. This result indicates that no active rhicadhesin is present at the cell surface of chvS mutants. For one c/ii/S mutant strain, Al O i l ,
the culture medium as well as a sonicated cell extract
were tested for adhesin activity, since it could not be
excluded that these mutants still produce rhicadhesin, but
are somehow affected in transiocation or anchoring of the
adhesin. No adhesin activity was found in either fraction
(Table 2} indicating that strain A1011 is affected in the
production of active rhicadhesin.
In order to check whether the chvB mutant produces
rhicadhesin in an inactive form, ME117 bacteria were
sheared and the rhicadhesin purification method was followed. By SDS-PAGE, a protein band of about 14kDa
was observed for rhicadhesin purified from strain ME117
(Fig. 1, lane A) at the same position as expected for wildtype rhicadhesin (Fig. 1, lane B) (Smit etaL, 1991). Since
several concentrations of rhicadhesin isolated from chvB
mutants were tested for activity and no inhibition of
attachment of wild-type cells was observed (data not
shown), it was concluded that ME117 produces rhicadhesin in an inactive form.
Table 2. Influence of cell surface preparations, cell extract and culture
supernatant from various A. tumetaciens stfains on attachment of A.
fumeCac/ens wild-type strain LBA1010 to pea root hairtips^.
7o attachment in class"
Results
Attachment ability of A. tumefaciens chvB mutants
The chvB mutants grown in TYC and harvested at various
growth stages were found to be unable to attach to pea
root hair tips, in contrast to the wild-type strain LBA1010,
the Ti plasmid-cured strain LBA1251, and Rhizobium
leguminosarum 248 (Table 1). This result is consistent
with earlier reports on attachment to tobacco and Zinnia
cells (Douglas et al., 1982: 1985). The chvB mutants
appeared to be affected in the first step of attachment,
since the majority of the root hairs were devoid of adhering bacteria.
chvB mutants do not synthesize active rhicadhesin
Cell surface preparations were isolated from A. tumefaciens chvB mutants, the wild-type strain and the Ti
Addition to standard
assay
2
3
9
31
60
Cell surface preparation from
LBA1010
1251 (pTi-. pCr-cured)
A1011 (ChvB)
M020 {chvB\
A1045 (c/1 vB)
ME117(c/iv6)
56
43
12
19
10
5
25
34
28
28
26
32
19
23
60
53
64
63
Culture supernatant from
A1011{chvBi
11
32
57
14
28
58
None
1
Cell extract from
AlOti {ChvBi
a. Additions corresponding to 4 ml bacterial culture were incubated with
the pea roots. A. tumetaciens LBA1010 cells were harvested from TY
medium, suspended in phosphate bufter and incubated with the roots for
2h.
b. See Table 1, footnote b.
Role of rhicadhesin in virulence and nodulation
599
reported by O'Connel and Handelsman (1989) concerning attachment of chvB mutants to Zinnia cells.
Restoration of virulence of A. tumefaciens chvB mutants
by wild-type rhicadhesin
A
B
Fig. 1. SDS-PAGE of purified rhicadhesin isolated from +1.8 x lO'^ cells
of A. tumefaciens chvB mutant strain ME117 (A) and of A. tumefaciens
wild-type strain LBA1010 (B).
Restoration of attachment ability of chvB mutants by wildtype rhicadhesin
R. leguminosarum 248 and A. tumefaciens LBA1010
cells, grown under Ca^*-limiting conditions, are unable to
attach to pea root hair tips (Smit etai., 1987; 1991). However, after pre-incubation of pea roots with purified rhicadhesin such bacteria appeared to attach to pea root hairs.
This physiological restoration of attachment ability is most
likely to be caused by binding of the bacteria to rootbound rhicadhesin (Smit ef al., 1991). The results presented in Table 3 show that attachment ability of chvB
mutant ME117 could be restored after pretreatment of
roots with rhicadhesin, isolated from wild-type A. tumefaciens, followed by incubation with mutant cells grown in
TY medium, that is, under Ca^*-limiting conditions. Also
rhicadhesin isolated from R. leguminosarum bv. viciae
was able to restore attachment (data not shown). However, wild-type levels of attachment, such as that found
with restoration of attachment of wild-type Rhizobium and
Agrobacterium grown under Ca^*-limiting conditions,
were not reached. This indicates that binding of rhicadhesin to the cell surface of the chvB mutants is indeed
possible, but is less efficient than to wild-type strains of
Agrobacterium and Rhizobium. Restoration of attachment
ability was also found when chioramphenicol (50)igmr')
was present duhng the attachment assay (data not
shown). No restoration of attachment ability was found
after protease treatment of rhicadhesin (Tabie 3). Cells of
strain ME117 grown in TYC medium did not adhere to the
rhicadhesin-pretreated roots (Table 3). Pretreatment of
roots with p-1,2-glucan did not result in restoration of
attachment ability of ME117 cells (Table 3). The presence
of |3-1,2-glucan during the incubation with bacteria had no
effect on the attachment of ME117 cells; however, this
treatment did not inhibit attachment of wild-type strains
(data not shown). This result is In agreement with results
Virulence of chvB mutants on Kaianchoe daigremontiana
and Kaianchoe tubiflora plants was tested after treatment
of plants with purified rhicadhesin, isolated from A. tumefaciens or R. leguminosarum 248, The chvB mutants,
A1011 and ME117, grown in TY or TYC, appeared to be
avirulent in the absence of rhicadhesin (less than 1% of
the wound sites developed a small tumour) (Fig.2B).
Since attachment ability of chvB mutants could be optimally restored by rhicadhesin after growth of the cells
under Ca^*-limiting conditions, bacteria grown under such
conditions were used for testing the possible restoration
of virulence. Treatment of K. daigremontiana leaf wound
sites with rhicadhesin isolated from A. tumefaciens
LBA1010, prior to inoculation with the chvB mutants,
resulted in tumour formation on an average of 50% of the
wound sites (Fig.2E). No tumours were observed when
rhicadhesin was added in the absence of bacteria (data
not shown). Moreover, no tumours were formed after
treatment with rhicadhesin pretreated with protease
(Fig. 2C). Addition of p-1,2-glucan to the wound sites prior
to inoculation with ME117 did not result in tumour formation (Fig.2D), This result is in agreement with earlier
reports (O'Connel and Handelsman. 1989). Addition of
Table 3. Influence ot pretreatment ol pea roots with purified rhicadhesin
and [i-1,2-glucan isolated from wild-type A. tumefaciens on attachment of
cells of ,4. tumefaciens chvB n\u\an\ ME117topea root hair lips".
%• attachment in class''
Bacterial growth
medium"
Root
pretreatment
TY
TYC
TY
TYC
TY
None
None
Rhicadhesin
Rhicadhesin
Protease-treated
rhicadhesin"
p-1,2-glucan
p-i ,2-glucan
0-1,2-glucan
+rhicadhesin
TY
TYC
TY
1
2
3
73
74
14
75
23
23
48
25
4
3
38
0
70
69
65
25
23
27
5
8
8
11
50
39
a. Roots were incubated with rhicadhesin (lOngml ') and/or [i-1,2-glucan
(0.5tigmr'). The roots were washed in phosphate buffer betore incubation with the bacteria. Agrobacterium cells were harvested, suspended in
phosphate bufter and incubated with the roots for 2 h. See the Experimental procedures section for details.
b. TY medium contatns (per litre); tryptone (Difco), 5.0g and yeast extract
(Oifco), 3 0 g . TYC medium is TY medium supplemented with 7mM
CaClj.
c. See Table 1, footnote b.
d- Rhicadhesin (100 ng ml"^) was treated with proteinase K (1 mg m r ' ) for
60 mm at 37'-C prior to incubation with the roots.
600
S. Swan. G. Smit, B. J. J. Lugtenberg and J. W. Kijne
B
leguminosarum bv. phaseoli strain RCC3622 forms nodules on siratro (Fig.3A). Also A. tumefaciens strain
LBA2728, grown in TY or TYC medium, forms nodules on
siratro (Fig. 3B). However, c^vSstrain LBA2753, grown in
either TY or TYC medium, does not form nodules on siratro
(Fig. 3C). Pretreatment of the roots with rhicadhesin followed by inoculation with LBA2753 cells, grown in TY
medium, resulted in the formation of several nodules per
plant {Fig. 3D). Incubation of fresh plants, not pretreated
with rhicadhesin, with plant medium of the nodulated siratro plants did not result in nodulation. This indicates that;
{i) no nodulating wild-type bacteria were present in the
medium of the nodulated plants, and {ii) LBA2753 did not
permanently regain nodulation ability. Apparently, pretreatment of roots with rhicadhesin is required for nodulation by LBA2753. Similar results were obtained when
bean was used as a test plant.
Discussion
cf)vB mutants of A. tumefaciens were found to be unable
Fig. 2. Restoration of virulence of A. tumefaciens chvB mutant strain
ME117on K. daipremonftana by treatmeni of the wound sites with rhicadhesin, isoiated from A tumefaciens wild type. Before mocuiaton with A
tumefaciensWM-t^pe strain LBAIOIO (A) or chvSmutant strain ME117
(B-F). wound sites were incubated for 30 mm with either buffer (A, B), rtiicadhesin treated with proteinase K(C), lOiag |3-1,2-glucan (D), lOngrtiicadhesin(E)of botti(J-1,2-glucanandrhicadhesin(F). Seethe Experimental procedures sec\\or\ lor 6e\a\\s.
both rhicadhesin and [i-1,2-glucan prior to inoculation
gave on average a greater number of, and larger tumours
than treatment with rhicadhesin oniy (Fig.2F). Similar
results were obtained when K. tubiflora was used as a
test plant (data not shown). The use of rhicadhesin purified from R. leguminosarum strain 248 ied to identical
results {data not shown).
Restoration ofnodulation of A. tumefaciens chvB
mutants
Van Veen etai (1987) showed that, unlike wiid-type A.
tumefaciens (Hooykaas et al., 1985). chvB mutants harbouring a Sym plasmid of R. leguminosarum bv. phaseoli
are not capable of nodulating bean. To investigate if this
phenomenon was because of the lack of active rhicadhesin, nodulation of siratro (Macroptiiium atropurpureum)
by LBA2753 ceils grown in TY was initially tested with and
without pretreatment of the roots with purified rhicadhesin
isolated from strain LBAIOIO. Siratro was chosen
because of its ease as a test system. Wild-type R.
Fig. 3. Nodulation of siratro after inoculation with (A) R. leguminosarum
bv. phaseoii strain RCC3622, (B) A., tumefaciens sUam LBA2728. (C) A.
tumefaciens chvB strain LBA2753, (D) A tumefaciens chvB strain
LBA2753 after trealment of the rools with rhicadhesin, isolated from A.
fumefeciens strain LBAIOIO. See the Experimental procedures section
for details
Role of rhicadhesin in virulence and nodulation
to attach to pea root hairs. According to the model of Smit
etal. {1987; 1989b), attachment appeared to be affected
in the first step, that is, direct attachment of the bacteria to
the surface of the root hair tip. For Rhizobium, this step is
non-host-plant-specific and mediated by rhicadhesin.
SDS-PAGE of a rhicadhesin preparation of the chvB
mutant ME117 yielded a protein band of about 14kDa
running at the same position as wild-type rhicadhesin.
However, rhicadhesin preparations isolated from chvB
mutants were found to be devoid of adhesin activity.
Attachment ability of chvB mutants could be restored by
addition of active wild-type rhicadhesin. Presumably chvB
mutants produce an inactive form of rhicadhesin. It was
concluded that lack of active rhicadhesin is directly
responsible for the attachment deficiency of chvB
mutants and that other components that are not produced
by these mutants do not seem to be directly involved in
attachment. Since we were able to isolate rhicadhesin
from the cell surface, lack of translocation to the cell surface is not responsible for the attachment deficiency of
cfivB mutants. The fact that more than one chvB mutant
strain has been used and none displays rhicadhesin
activity (Table 2) renders the possibility of a {point) mutation in the gene encoding rhicadhesin unlikely. The defect
may be caused by improper processing of the rhicadhesin
protein or to impaired Ca^* binding. The role of p-1,2-glucan in activation of rhicadhesin will be discussed later.
Restoration of attachment ability of chvB mutants by rhicadhesin did not result in wild-type attachment levels,
leaving the possibility for requirement of a second efficiency-increasing factor. It is not very likely that this factor
is extracellular p-1,2-glucan since addition of p-1,2-glucan
did not result in enhancement of attachment, neither in
absence or presence of rhicadhesin. Taken together
these results are consistent with the hypothesis that, like
in the case of Rhizobium, the first step in the attachment
of A. tumefaciens is mediated by rhicadhesin. Douglas et
al. (1982) reported that chvB mutants are not affected in
the synthesis of cellulose fibrils. Our results are consistent with this obsetvation, since accumulation of mutant
cells on the plant cell surface (class 3 attachment), a process mediated by cellulose fibrils (Matthysse, 1983; Smit
et al., 1987), is observed after treatment of the roots with
active rhicadhesin.
Binding of rhicadhesin to Rhizobium and Agrobacterium cells is mediated by Ca^"" ions. Only Rhizobiaceae
cells grown under Ca^'^-limiting conditions or grown with
calcium and treated with EDTA, were found to be able to
bind to rhicadhesin-treated pea roots. Under such growth
conditions rhicadhesin bound to bacteria is released into
the medium, which probably enables binding of the cells
to root-bound adhesin (Smit etai. 1989b; 1991). Accordingly, in order to achieve binding of rhicadhesin to the cell
surface of the chvB mutants, the level of Ca^^ ions on the
601
cell surface of the mutants had to be tow. Furthermore,
several phenotypic changes in the cell envelope of chvB
mutants could be prevented by growth under calcium-limiting conditions {S. Swart et al., submitted), yielding a
more wild-type-like surface for the mutant cells, which
may be necessary for anchoring on root-bound rhicadhesin.
Virulence of chvB mutants on K. daigremontiana and K.
tubifiora plants was restored by treatment of the wound
sites with rhicadhesin prior to inoculation with chvB
mutants. On average, the number of tumours was lower
in comparison with that induced by the wild-type strain,
although the size of the tumours was similar. This result
corresponds with the suboptimal restoration of attachment ability found after pretreatment of roots or bacteria
with rhicadhesin. Treatment of wound sites with j3-1,2-glucan prior to inoculation did not result in tumour formation
by ChvB mutants, strongly suggesting that restored
tumour formation is preceded by restored attachment.
Taken together, it can be concluded that lack of active rhicadhesin on the cell surface of the chvB mutants causes
the attachment-minus and virulence-minus phenotype of
the chvB mutants, rather than lack of p-1,2-glucan, as
proposed by Puvanesarajah etal. (1985). However, it is
possible that endogenous p-1,2-glucan is necessary for
production of active rhicadhesin. The active rhicadhesin
used was isolated from a [5-1,2-glucan-producing wildtype strain. Under hypo-osmotic conditions p-1,2-glucan
may be required for proper membrane assembling, which
may be necessary for correct anchoring of rhicadhesin.
Beta-1,2-glucan may also be required for stability of rhicadhesin, such as cyclodextrins, that are used for protein
stabilization {Szejtti, 1991). This could explain why a
greater number, and larger tumours were found in the
case where both rhicadhesin and p-1,2-glucan were
added in the restoration of virulence. A. tumefaciens chvA
mutants still export some of the wild-type amount of p-1,2glucan {O'Connel and Handelsman, 1989). This may
explain why chvA mutants have a leaky phenotype with
regard to attachment and rhicadhesin activity (S. Swart,
unpublished results) and virulence and nodulation {Van
Veen etal., 1987). Dylan etai (1986) reported homology
between the chvA and chvB loci of A. tumefaciens and
so-called ndvA and ndvB (for nodulation development)
loci in Rhizobium meliloti. In reports on R. meliloti ndvB
mutants it is proposed that p-1,2-glucan is not absolutely
required for nodule development but might play a role in
enhancement of nodulation (Dylan etai, 1990).
Wild-type A. tumefaciens cells harbouring a Sym plasmid of H. leguminosarum bv. phaseoli are able to nodulate siratro and bean whereas c/ivS mutants harbouring a
Sym plasmid cannot {Van Veen e( al., 1987; this study).
We demonstrated that, only after treatment of the roots of
siratro or bean plants with rhicadhesin, strain LBA2753
602
S. Swart, G. Smit, B. J. J. Lugtenberg and J. W. Kijne
Table 4. Sacterial strains.
Strain
Genotype or phenotype
Reference/Source
LBA1010
A. tumefaciens C58
chromosome, pTiB6
A. tumefaciens C58
chromosome, pTi- and
pCr-cured
A. tumefaciens C58
chromosome chvB::Jn5
A. tumefaciens C58
chromosome chv&.:Tn5
A. tumefaciens C58
chromosome chvB:.Tn5
A. tumefaciens C58
chromosome
chvS::Tn3::HoHo1
A. (umefec/ensLBAIOlO
pSym9 ot RCC3622
A. lumefaciens M E n 7 ,
pSym9 of RCC3622
R. ieguminosarum
bv. phaseoli (wild-type)
R. ieguminosarum
bv. viciae (wild-type)
Koekman era/. (1982)
LBA1251
A1011
A1020
A1045
ME117
LBA2728
LBA2753
RCC3622
248
Rosenberg and Huguet
(1984)
Douglas e( af. (1982)
Douglas ef a/. (1982)
Douglas efa/. (1982)
Douglas sfaA (1985)
Van Veen e/a/.(1987)
Van Veen efa/. (1987)
Hooykaas efa/. (1985)
Josey efa/. (1979)
encoded (Douglas etai., 1982; Matthysse, 1987; Smit ef
al., 1986; 1987). (iii) Bacteria from both genera produce
rhicadhesin, and, moreover, rhicadhesin, purified from
one genus, can inhibit attachment of cells of the other
genus to legume root hair tips (Smit e/a/., 1989b). (tv) For
both genera attachment is a two-step process. The second step, mediated by cellulose fibrils, is not essential for
the establishment of a successful association (Matthysse,
1983; Smit etaL, 1987). The first step, mediated by rhicadhesin, is necessary for an interaction between plant
and bacterium (Smit ef aL, 1989b; this paper), (v) The
attachment ability to root hairs as well as virulence of
ChvB mutants can be restored by addition of rhicadhesin
isolated from either Rhizobium or Agrobacterium (this
paper), (vi) The virulence of chvB mutants on Kaianchoe
as well as nodulation of bean and siratro by cArvS mutants
harbouring a Sym plasmid of R. leguminosarum bv.
phaseoli can be restored by pretreatment of the roots with
rhicadhesin (this paper).
Experimental procedures
was able to nodulate these roots, Heterologous complementation of A. tumefaciens chvB mutants with R. meliloti
ndvB genes and vice versa demonstrated the functional
interchangeability of the chv and ndv loci (Dylan ef al.,
1986). Also ndvB mutants of R. meliloti were found to be
affected in infection (Geremia et ai, 1987). However
ndvB mutants form pseudonodules whereas chvB
mutants harbouring a Sym plasmid do not form nodules at
all (Van Veen ef al., 1987). Furthermore, ndvB mutants
form abortive infection threads and show attachment,
although diminished (Dylan et aL, 1990). Indeed, we
found rhicadhesin activity in cell surface preparations of a
ndvB mutant (S. Swart, unpublished results). Apparently,
a mutation in the gene encoding the intermediate protein
in the p-1,2-glucan synthesis, chvB in the case of A. tumefaciens or ndvB in the case of R. meliloti, does not result
in the same phenotype for these Rhizobiaceae. Rhicadhesin activity might be regulated in a different manner in
A. tumefaciens and R. meliloti.
Comparision of binding properties of Rhizobium and
Agrobacterium
The following evidence supports the hypothesis that Rhizobium and Agrobacterium bind to the surface of plant
celis through a similar mechanism, (i) The chromosome of
bacteria from each genus contains the necessary information for establishing an association with host plants of
the other genus, provided that the proper plasmid (Sym or
Ti) is present (Hooykaas etai., 1977; 1981; 1985; Kondorosi etaL, 1982; Hirsch et ai., 1985). (ii) The binding
properties of cells of both genera are chromosomally
Bacterial strains and culture conditions
Bacterial strains of A. tumefaciens and R. leguminosarum used
are listed in Table 4. Compositions of the media A+ and LC
(Smit etai., 1987) have been described previously. TY medium
contains (per litre); tryptone (Difco), 5.0g and yeast extract
(Difco), 3.0 g. TYC medium is TY medium supplemented with 7
mM CaClg. Rhizobium and Agrobacterium species were maintained on solid A+ and solid LC medium, respectively. For
attachment assays, bacteria were cultivated on a rotary shaker
(180 r.p.m.) at 28^C in 100 ml Erienmeyer flasks, containing
50 ml TYC medium. For purification ot rhicadhesin, bacteria
were grown in either 500 ml or 21 Erienmeyer flasks containing
250 ml or 1.25 I TYC medium, respectively.
Plants and plant culture conditions
Seeds of pea {Pisum sativum cv. finale; Cebeco) were surface
sterilized and cultivated as described previously (Smit et al.,
1986). Seeds of siratro {Macroptilium atropurpureum, a generous gift from Dr B. G. Rolfe, Australian National University,
Canberra, Australia) were surface sterilized as described for
pea seeds and germinated on solid Jensen agar (Vincent,
1970) at 28'^C for about 20 h. Subsequently, plants were grown
in liquid Jensen medium at 28''C in a growth chamber. The light
intensity at table surface was 20000 iux during a 16h lightperiod. K. daigremontiana and K. tubiflora plants were cultivated as described by Ooms efa/- (1980). Bean seeds (Phaseolus vuigaris cv. prelude) were surface sterilized and
germinated between wetted Whatman filter paper at 22°C.
After germination for 4d the seedlings were grown in glass
tubes on solid Jensen medium slopes at 22°C in a growth
chamber with light intensity of 20 000 lux during a 16h lightperiod.
Role of rhicadhesin in virulence and nodulation
Attachment assay
The attachment assay used has been described previously by
Smit etal. (1986). Briefly, roots were immersed in a suspension
of bacteria, washed 10 times and attachment was quantified by
randomly screening at least 100 developing root hairs with a
phase-contrast microscope. Attachment was classified in three
classes: class 1, no attached bacteria; class 2, few bacteria,
directly attached to the root hair; class 3, many attached bacteria, forming two or more layers of bacteria or a cap-like aggregate at the tip of the root hair (cap formation). The percentage
of root hairs of each class was calculated. The variability of the
test is about 10% and depends largely on the condition of the
roots.
Purification of rhicadhesin
Procedures for the purification of rhizobial rtiicadhesin (Smit ef
ai, 1989b), isolation of cell surface preparations from A. tumefaciens strains (Smit etai, 1989a), and testing the presence of
rhicadhesin in culture supernatants of cells (Smit et al., 1991)
have been described previously. Presence of rhicadhesin
inside bacteria was examined after sonication of the cells for 5x
30s in a sonifier cell disrupter (Branson Sonic Power Comp.).
After sonication of the cells, the suspension was clarified by
ultracentrifugation (100 000 x gfor 2 h) and tested for adhesin
activity.
SDS-PAGE and protein staining
SDS-PAGE was performed as descibed by Schagger and Von
Jagow (1987). Proteins were silver-stained according to Oakley etai (1980), The molecular masses of the marker proteins
were; bovine serum albumin, 66 kDa; egg albumin, 45 kDa;
carbonic anhydrase, 29 kDa; p-lactoglobu!in, 18 kDa; and
lysozyme, 14 kDa.
Isolation ofp-1,2-glucan
Stationary cells of A. tumefaciens LBA1010, grown in TYC.
were harvested by centrifugation at 10000 x pfor lOmin. Pellets were extracted with 1 % trichloroacetic acid (TCA) for
30 min at room temperature. TCA extracts were neutralized,
concentrated, chromatographed on Sephadex G-50 (Pharmacia), and tested according to the method of Miller e/a/. (1986).
Test for rhicadhesin activity
Activity of rhicadhesin was determined by measuring the ability
to inhibit attachment of LBAIOIO cells to pea root hair tips. To
test whether a fraction contained active rhicadhesin, two lateral
pea roots were incubated together with the fraction in 1.0 ml of
25 mM potassium phosphate buffer, pH 7-5, for 60 min at room
temperature on a rotary table (2 r.p.m.). Controls were incubated for 60 min in buffer only. After this treatment the roots
were washed five times in phosphate buffer and incubated with
bacteria in an attachment assay as described above (see also
Smit era/., 1989b).
Restoration of attachment ability of A. tumefaciens chvB
mutants was tested by incubating rhicadhesin-treated roots
603
with bacteria grown in TY as described in the attachment
assay.
Virulence assays
In the greenhouse K- daigremontiana and K. tubifiora plants
were wounded on leafs and stems, respectively. A solution of
rhicadhesin (2-10ng per wound site) and/or p-1,2-glucan
(10 ng per wound site) in 25 mM phosphate buffer, pH7,5, was
added in a volume of 5-25^1 per wound site. Controls were
incubated with phosphate buffer only. Ten to twenty microlitres
of A. tumefaciens chvB mutant cells, grown in TY medium, harvested and suspended in phosphate buffer to an Agjo value of
1.0, were added to each wound site 30 min after incubation
with rhicadhesin or buffer. As a control wild-type A. tumefaciens was added to wound sites. Tumour formation was quantified three weeks after inoculation.
Nodulation assay
After germination, sirato seedlings were transferred to Jensen
medium (5-6 seedlings per glass tube containing 25 ml
medium) and grown overnight at 28°C in the dark. Subsequently, an appropriate number of roots were treated with
0.1 fig ml"' rhicadhesin in Jensen medium for 30 min. Twentyfive microlitres of A. tumefaciens LBA2753 cells, grown in TY
medium, harvested and resuspended in Jensen medium to an
A620 value of 0.1, were added to each tube. Strains LBA2728
and RCC3622 were used as controls. Nodulation was quantified 3 weeks after inoculation.
After germination, bean seedlings were grown on Jensen
medium for 3d in the dark before inoculation. One millilitre
25 mM phosphate buffer, pH7.5, or 1.0 ml rhicadhesin
(0.5|igmr') in phosphate buffer was added to the roots and
after 1 h the roots were inoculated with 3.0 ml of A. tumefaciens
strain LBA2753 cells, grown in TY medium, harvested and
resuspended in Jensen medium to an Agao value of 0.05.
Strains LBA2728 and RCC3622 were used as controls. Nodulation was quantified 5 weeks after inoculation.
Acknowledgements
This work was supported by the Foundation for Fundamental
Biological Research (BION), which is subsidized by the Netherlands Organization of Scientific Research (NWO). The authors
thank Trudy Logman, Teun Tak and Bram Wetselaar for supply
of test plants, and Ron van Veen for helpful discussions.
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