The Plant Journal (2002) 32, 195–204
Constitutive gain-of-function mutants in a nucleotide
binding site–leucine rich repeat protein encoded at the Rx
locus of potato
Abdelhafid Bendahmaney Garry Farnham, Peter Moffett and David C. Baulcombe
The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK
Received 25 April 2002; revised 13 June 2002; accepted 14 June 2002.
For correspondence (fax þ44 1603 250024; e-mail david.baulcombe@sainsbury-laboratory.ac.uk).
y
Present address: INRA-URGV, 2 Rue Gaston Crémieux CP 5708, 91057 Evry Cedex, France.
Summary
Rx in potato encodes a protein with a nucleotide binding site (NBS) and leucine-rich repeats (LRR) that
confers resistance against Potato virus X. The NBS and LRR domains in Rx are present in many disease
resistance proteins in plants and in regulators of apoptosis in animals. To investigate structure-function
relationships of NBS-LRR proteins we exploited the potential of Rx to mediate a cell death response. With
wild-type Rx cell death is elicited only in the presence of the viral coat protein. However, following random
mutagenesis of Rx, we identified mutants in which cell death is activated in the absence of viral coat
protein. Out of 2500 Rx clones tested there were seven constitutive gain-of-function mutants carrying eight
independent mutations. The mutations encoded changes in the LRR or in conserved RNBS-D and MHD
motifs of the NBS. Based on these findings we propose that there are inhibitory domains in the NBS and
LRR. The constitutive gain-of-function phenotypes would be due to deletion or modification of these
inhibitory domains. However activation of Rx is not simply release of negative regulation by the LRR and
adjacent sequence because deleted forms of Rx that lack constitutive gain of function mutations are not
active unless the protein is overexpressed.
Keywords: potato virus X; coat protein; hypersensitive response; cell death.
Introduction
Many disease resistance (R) genes in plants encode proteins with nucleotide binding site (NBS) and leucine rich
repeat (LRR) domains (Ellis et al., 2000; Hammond-Kosack
and Jones, 1997). Genetic evidence indicates that these
NBS-LRR proteins are receptors that interact, directly or
indirectly, with elicitors produced by the pathogen. Following recognition there is activation of downstream signalling
pathways leading to disease resistance and a hypersensitive response (HR) that is manifested as cell death (Shirasu
and Schulze-Lefert, 2000).
In common with other receptors it is generally considered
that NBS-LRR proteins have a modular structure with separate recognition and signalling domains. The LRR is a
candidate recognition domain because it is the most variable region in closely related NBS-LRR proteins and is
under selection to diverge (Meyers et al., 1998; Noel et al.,
1999). Functional analysis of recombinant R proteins also
indicates that recognition specificity resides in the LRR (Ellis
ß 2002 Blackwell Publishing Ltd
et al., 1999). However, there is indirect evidence that the
LRR can contribute to signalling as well as recognition.
(Banerjee et al., 2001; Warren et al., 1998)
The amino terminal region in NBS-LRR R proteins is
thought to be the major signalling domain. In most NBSLRR proteins this region includes either a Toll-interleukin
receptor (TIR)-like domain or a putative coiled-coil (CC)
structure (Ellis et al., 2000). In Arabidopsis, TIR-NBS-LRR
proteins apparently signal through the EDS1 lipase-like
protein, whereas CC-NBS-LRR proteins require pathways
involving NDR1 (Aarts et al., 1998). RPS7 and RPP8 are NBSLRR proteins but do not have absolute dependency on
EDS1 or NDR1 and it is thought that they signal through
a novel, as yet uncharacterized, pathway (McDowell et al.,
2000). In some NBS-LRR proteins the amino terminal
domain may be involved in recognition as well as signalling. For example, from the novel resistance specificity of
recombinant L6, L2 and LH flax rust R genes, it seems likely
195
196 Abdelhafid Bendahmane et al.
that recognition can be influenced by a TIR domain (Ellis
et al., 1999; Luck et al., 2000).
The NBS is positioned between the putative signalling
domain and the LRR. Its precise function is not known but
there are clues from sequence motifs that are also present
in the NBS of CED-4 (C. elegans) and APAF-1 (human)
regulators of apoptosis (Aravind et al., 1999; van der Biezen
and Jones, 1998). The common sequences include the P
loop (kinase 1), kinase 2 and kinase 3a and other motifs
found in ATPase proteins from both prokaryotes and eukaryotes (Aravind et al., 1999). Mutation analysis indicates that
at least some of these motifs are required for the function of
R proteins (Axtell et al., 2001; Dinesh-Kumar et al., 2000; Tao
et al., 2000; Tornero et al., 2002). By analogy to APAF-1 and
similar proteins, it seems likely that ATPase activity in NBSLRR proteins mediates conformational changes necessary
for downstream signalling and disease resistance (Hu et al.,
1999).
Indirect evidence from the nematode resistance gene Mi
indicates how the activity of NBS-LRR proteins could be
controlled (Hwang et al., 2000). Recombinant Mi proteins
containing elements of a non-functional Mi homologue
activate an HR in the absence of elicitor. To explain this
result it was proposed that there are intra-molecular interactions between domains of Mi. In the wild-type protein in
the absence of elicitor these interactions would prevent
signalling of disease resistance and cell death. Modification of the interaction, either following elicitor recognition
or by mismatch of interacting residues in the recombinant
proteins, would allow the signalling domains of Mi to
activate the response pathways. The lesion mimic phenotype of a natural recombinant of the Rp1 resistance gene in
maize (Sun et al., 2001) is also consistent with this interpretation.
Here we investigate structure-function relationships in
the CC-NBS-LRR protein encoded by Rx. Rx confers
resistance against Potato virus X (PVX) in potato (Cockerham, 1970) and the elicitor is the viral coat protein (CP)
(Bendahmane et al., 1995). Rx-mediated resistance is unusual in that it is not normally associated with an HR either in
potato or as a transgene in Nicotiana species (Bendahmane
et al., 1999). However transgenic expression of the CP elicits
an Rx-dependent HR in potato and in Nicotiana species
(Bendahmane et al., 1999). Our approach to the analysis of
Rx involved screening for Rx mutants that mediate an HR in
the absence of the elicitor CP. We also tested the overexpression of wild type and deleted forms of Rx for an
elicitor-independent HR. Our results imply that conserved
motifs in the NBS and LRR regions of Rx regulate a signalling domain in the amino terminal region. As deletion or
mutation of these motifs leads to gain of Rx function it is
likely that they have an inhibitory role in the wild-type
protein. Presumably this inhibitory function is abrogated
in the presence of the coat protein elicitor.
Results
Random mutagenesis of Rx
To address the possible role of the NBS and other domains
in Rx we screened for Rx mutants in which an HR was
activated in the absence of the elicitor PVX CP. The mutants
were generated by PCR of Rx cDNA under conditions
leading to misincorporation of nucleotides. The PCR
products were then inserted between the promoter and
transcriptional terminator of Rx (Figure 1a, pR-AT) and
the constructs were assembled in the T-DNA region of an
Agrobacterium binary plasmid vector. A library of Rx
mutants was generated and individual clones were assayed
for HR induction by infiltration of liquid Agrobacterium
cultures into leaves of Nicotiana tabacum. All experiments
were repeated at least three times with several replicates in
each experiment and the data, as presented here, were
consistent and reproducible in all instances.
Agrobacterium cultures carrying a wild-type Rx cDNA
construct (pR-Rx; Figure 1a) did not induce an HR when
infiltrated into non-transformed N. tabacum leaves. However, when this construct was infiltrated into transgenic N.
tabacum producing the CP of PVX (Spillane et al., 1997),
there was necrosis after 48 h and death of the infiltrated
region by 72 h (Figure 1b). Presumably the transiently
expressed Rx had recognized the elicitor CP and activated
signalling leading to cell death.
Out of 2500 mutant Rx clones tested there were seven that
induced an HR in the leaves of non-transformed N. tabacum.
Figure 1(b) illustrates the HR phenotype of one of these constitutive gain-of-function mutants (AT25). Four of the
mutants (AT39, AT193, AT25, and AT7) induced an HR within
48 h of Agrobacterium infiltration. The others (AT32, AT72
and AT28) induced an HR that was delayed until 48–72 h post
infiltration. These gain-of-function mutantsall showedan HR
in the presence of the coat protein (Figure 1b).
Constitutive gain-of-function mutants are modified in the
NBS and LRR domains
DNA sequence analysis revealed that each of the constitutive gain-of-function forms of Rx carries between three and
11 amino acid substitutions relative to the wild type. To
identify the amino acid substitutions implicated in the
activation of Rx we constructed a series of recombinant
molecules incorporating elements of the wild type and
constitutive gain-of-function mutants. For each of the
gain-of-function mutants we identified recombinant molecules with a single amino acid mutation that induced an
elicitor-independent HR on non-transformed N. tabacum
using the Agrobacterium infiltration assay.
In each of the pR-AT39, pR-AT193, pR-AT25, pR-AT32,
pR-AT72 and pR-AT28 clones a single mutation was
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 195–204
Gain of Rx function by mutation and overexpression
197
Figure 1. Induction of HR by a constitutive gain-of-function mutant of Rx.
(a) Schematic representation of T-DNA constructs for expression of Rx
cDNA. The cDNA inserts of wild-type or mutant forms of Rx were inserted
between Rx promoter (pR) and transcriptional terminator (ter). The black
box indicates the cDNA or either wild-type (wt) Rx cDNA or the cDNA of
mutant (AT) forms of Rx. LB and RB indicate the left and right border of the TDNA.
(b) Expression of wild type Rx and the constitutive gain-of-function mutant
pR-AT25 in N. tabacum. The Rx constructs were the wild type Rx (pR-Rx)
or the constitutive gain-of-function mutant (pR-AT25). Agrobacterium cultures carrying pR-Rx or pR-AT25 was infiltrated into leaves of eithernon-transformed (NT) or transgenic N. tabacum (CP) expressing the PVX
CP from the 35S promoter. The leaves were photographed 4 days after
infiltration.
responsible for the HR (Figure 2a). In each instance the
timing of the HR was the same with the single amino acid
mutant and the corresponding progenitor clone. The pRAT7 clone carried two mutations that were independently
responsible for activation of an HR (Figure 2a). The pR-AT7derived recombinants with single amino acid mutations
caused an HR that was slower than with the progenitor
pR-AT7. Presumably the faster HR of the progenitor pR-AT7
was due to an additive effect of the two mutations.
Three out of these eight mutations (F393I, D399V, E400K)
are in an eight amino acid interval close to the RNBS-D
(CFLY) motif that is conserved in R proteins (HammondKosack and Jones, 1997; Meyers et al., 1999). Two other
mutations led to the same amino acid change (D460V) in the
conserved MHD motif (Hammond-Kosack and Jones, 1997)
(Figure 2a,b) but are derived independently because, in the
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 195–204
Figure 2. The sequence of the Rx constitutive gain-of-function mutants.
(a) Sequence changes responsible for constitutive gain-of-function phenotypes. The diagram shows the changes at the nucleotide and protein level
that are responsible for the constitutive gain-of-function phenotype.
(b) The predicted protein sequence of Rx. The sequence is divided into three
domains corresponding to the amino terminal CC domain, the NBS domain
and the LRR. The NBS domain includes the P loop, kinase2 and kinase 3a
motifs of the NBS (Aravind et al., 1999) and the RNBS-C, GLPL, RNBS-D and
MHD motifs that are conserved in NBS-LRR proteins (Meyers et al., 1999; van
der Biezen and Jones, 1998). The residues modified in the constitutive gainof-function mutants are shown with a black background and the mutant
residues are in bold above the main sequence. The LRR and C terminus
region includes the LRR and the amide rich and acidic tail regions at the
carboxy terminus of Rx (Bendahmane et al., 1999). The LRR consensus
motifs are shown in blue and are aligned. The conserved VLDL motif in LRR3
is underlined.
198 Abdelhafid Bendahmane et al.
pR-AT39 and pR-AT193 progenitors, they were associated
with different mutations that did not affect the Rx response
(AB, data not shown). The remaining three constitutive
gain-of-function mutations were in LRR2 (H519R), LRR3
(D543E) and LRR11 (H738R) (Figure 2a,b). The LRR3 mutation is in a motif, VLDL, that is present in LRR3 of many NBSLRR R proteins (Axtell et al., 2001; Banerjee et al., 2001) (G.
Farnham, unpublished data).
The Rx gain-of-function phenotype is dependent on a
wild-type P loop motif and on SGT1
In principle the constitutive gain-of-function mutants could
elicit an HR because the encoded proteins are toxic in the
Agrobacterium-infiltrated leaves. Alternatively these mutant
proteins could have activated the downstream pathway
that is involved in the Rx-mediated response to the PVX
CP. To test these possibilities we introduced alanine substitutions (G175a þ K176A) in the P loop (K1) motif of the
NBS (Aravind et al., 1999) (Figure 2) and assayed the HR
phenotype with the Agrobacterium infiltration assay in nontransgenic and CP transgenic plants. We predicted that an
HR related to Rx-mediated resistance would depend on integrity of the conserved P loop, whereas an HR due to toxic
effects of protein over-expression in the Agrobacterium infiltration assay would be unaffected by disruption of the NBS.
As predicted the P loop Rx mutants failed to show an HR
in the infiltrated region of the CP transgenic plants (Figure
3a) indicating that activation of downstream signalling
requires integrity of the NBS. Similarly, when the P loop
mutation was introduced into Rx gain-of-function mutants,
there was no HR in non-transformed N. tabacum leaves.
Identical results were obtained with HA epitope tagged
versions of Rx and the constitutive gain-of-function
mutants. Thus, Rx(HA) was fully functional in an Agrobacterium infiltration HR assay in the CP transgenic N. tabacum
and was readily detected by Western blot analysis with HA
antibody in non-transgenic plants (Figure 3b). The P loop
mutants of Rx(HA), D543E(HA) and D460V(HA) were also
non-functional in the HR assay although the encoded proteins were detected by Western analysis (Figure 3b). From
these results we can rule out that the lack of an HR with the P
loop mutants was due to protein destabilization. A more
likely explanation is that the P loop has the same function in
wild type and gain-of-function mutants of Rx. This interpretation is reinforced by our finding (AB, data not shown)
that mutations in residues D244A þ D245A (kinase 2) or
G330A þ P332A (GLPL) abrogated the ability of both wildtype and constitutive gain-of-function mutants to produce
an HR in the infiltration assay.
Further indication that the gain-of-function phenotype is
relevant to Rx mediated resistance was based on an HR
assay in N. benthamiana following virus induced gene
silencing (VIGS) of SGT1 loci. SGT1 proteins are required
for many types of disease resistance responses (Austin
et al., 2002; Azevedo et al., 2002) including the HR associated with Rx (Figure 3c)(Peart et al., 2002). However
expression of SGT1 loci is not required for cell death
induced by ethanol, sodium azide or sodium chloride (Peart
et al., 2002). Based on these findings we predicted that the
constitutive gain-of-function HR would only be compromised by silencing of SGT1 if it were a bona fide resistance
response. A toxic response would not be compromised by
SGT1 silencing. The HR tests in SGT1 silenced plants were
carried out with D460V and H519R þ D543E þ H738R in
which three LRR gain-of-function mutations were combined in one construct. The SGT1 silencing was induced
by infection of N. benthamiana with a tobacco rattle virus
vector (Ratcliff et al., 2001) construct carrying an SGT1
cDNA insert.
The results shown in Figure 3(c) show that SGT1 expression is required for an HR induced by the mutant Rx
proteins. This requirement is not because SGT1 silencing
causes reduced levels of Rx (P. Moffett, unpublished data).
It is likely therefore that the constitutive gain-of-function
phenotype represents Rx-mediated resistance rather than a
toxic response. The SGT1 requirement for other gain-offunction mutants could not be tested because, for reasons
that we do not understand, they did not produce an elicitorindependent HR in N. benthamiana unless they accumulate
at very high levels due to co-expression in the presence of a
suppressor of gene silencing (A. Bendahmane, G. Farnham
and P. Moffett unpublished observations). In our hands
virus-induced silencing is much more effective in N.
benthamiana than in N. tabacum (Ana Montserrat Martin
Hernandez, unpublished observations).
Overexpression of Rx
As a second approach to structure-function analysis of Rx
we used the Agrobacterium infiltration assay to overexpress full length and deleted Rx constructs in the absence
of elicitor (Figure 4a). We predicted that overexpression of
the full length Rx in N. tabacum would activate resistance in
the absence of elicitor, as with other NBS-LRR proteins
(Oldroyd and Staskawicz, 1998; Tao et al., 2000). We also
predicted that deletion of domains required for downstream signalling would prevent the overexpression phenotype. However, deletions in inhibitory domains would
either have no effect or would enhance the overexpression
phenotype. The overexpression constructs all included a
carboxy terminal HA tag so that the level of the Rx proteins
could be monitored by immuno-blotting with HA antibody.
As predicted, expression of full length Rx(HA) under the
strong constitutive 35S RNA promoter of CaMV (Figure 4a)
induced an HR that developed by 72 h post infiltration in the
absence of PVX coat protein. The same protein, either with
or without the HA tag did not induce an HR when expressed
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 195–204
Gain of Rx function by mutation and overexpression
199
Figure 3. Constitutive gain-of-function phenotypes require P loop-dependent signalling and SGT1.
(a) Rx constructs were infiltrated into N. tabacum that was either non-transgenic or that expressed the PVX CP. Production of an HR was monitored over a period
of 4 days. The Rx constructs were either in a wild-type background or with constitutive gain-of-function mutations E440K, D460V or H519R þ D543E þ H738R. The
P loop motif in these constructs was either wild type or had GK residues at position 175 and 176 modified to AA (K1). HR indicates an HR within 48 h of infiltration
and ‘-’indicates no HR.
(b) Western blot analysis of HA tagged versions of Rx and constitutive gain-of-function mutants with P loop (K1) mutations. The constructs were either the wildtype Rx with an HA tag (RxHA), K1 mutant Rx with an HA tag (RxHAK1) and RxHA constructs with double constitutive gain-of-function and K1 mutations
((D543E)K1 and (D460V)K1). The constructs were transiently expressed using Agrobacterium infiltration (O.D.600 ¼ 0.2). Samples were taken at 3 days post
infiltration, fractionated by SDS-PAGE and the HA tagged proteins were detected with anti-HA antibody. The HR or ‘-’symbols indicate whether the proteins were
able to mediate an HR within 48 h either in the presence or absence of CP.
(c) N. benthamiana plants were infected with TRV:00 or TRV:SGT to silence SGT1 expression. After 21d Agrobacterium infiltration was used to introduce Rx
constructs into the upper infected leaves of the plants either individually or in combination with constructs carrying a 35S CP construct. The PVX constructs were
either wild type Rx in the presence (1) or absence (2) of CP, D460V (3) or H519R þ D543E þ H738R (4). The images show that Rx mediated an HR in TRV:00-infected
plants either as a wild-type Rx in the presence of the CP construct or as a constitutive gain-of-function mutant (right leaf) but that SGT1 silencing compromised
the resistance-associated HR (left leaf).
from the genomic Rx promoter (Figure 1b). The level of
Rx(HA) was higher from the 35S construct than with the Rx
promoter (Figure 4b) and we conclude that the HR is caused
by overexpression of Rx.
Overexpression of three deleted versions of Rx(HA)
(382D, 318D and 293D) resulted in an HR that developed
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 195–204
more rapidly (< 48 h) than with the full length protein (72 h).
The 282D protein induced a very slow HR (> 96 h) and the
1D138 amino terminal deletant was inactive. These deleted
proteins were HA tagged but we could not detect the 382D,
318D and 293D proteins in N. tabacum because rapid HR
occurred before they had accumulated to detectable levels.
200 Abdelhafid Bendahmane et al.
Figure 4. Over expression of Rx deletion mutants.
(a) A scale diagram of Rx showing the CC and
LRR regions, as in Figure 2. In the NBS domain
the locations are shown of the P loop(K1), kinase
2 kinase 3a and other motifs that are conserved
in NBS-LRR proteins including APAF-1 and
CED4. The lines indicate the extent of deletions
in HA tagged constructs relative to these conserved motifs. The figures in the right hand side
column indicate the timing of an HR with these
constructs when transiently expressed from a
35S promoter in the absence of CP (NR ¼ no
response).
(b) Western analysis of RxHA expressed in N.
tabacum in an Agrobacterium transient assay
(O.D.600 ¼ 0.2) either from the Rx promoter
(pB1)or from the 35S promoter (35S). The Rx
constructs encoded either the wild-type protein
of the P loop (K1) mutants. Protein extracts were
prepared 40 h post-infiltration, prior to the onset
of HR, separated by SDS-PAGE and detected by
anti-HA immuno-blotting. An asterisk denotes
that in these samples, five-fold less protein
extract was loaded.
(c) Western analysis of RxHA and deletion constructs expressed in an Agrobacterium transient
assay (O.D. 600 ¼ 0.5)from the 35S promoter of
CaMV in N. benthamiana. Protein extracts from
duplicate experiments were prepared 48 h postinfiltration, separated by SDS-PAGE and detected by anti-HA immuno-blotting.
However in N. benthamiana none of these proteins induced
an HR and they could be detected at 48 h post infiltration by
western analysis (Figure 4c). All of the deleted forms of Rx
were substantially less abundant than full length Rx.
Based on the assumption that accumulation in N.
benthamiana is indicative of levels in N. tabacum these
results show that HR-inducing activity is enhanced if the
carboxy terminal part of the protein is removed. Thus 382D,
318D and 293D elicited a rapid HR in N. tabacum, despite
their accumulation at a lower level than the full length
protein. This interpretation is consistent with the proposal,
based on the gain-of-function mutant phenotype described
above, that there are negative regulatory functions in Rx
between residues 382 and the carboxy terminus.
The removal of sequence on the amino terminal side of
residue 293 either reduced (282D) or abolished (1D138) HRinducing activity, even though these proteins were at least
as abundant as those with a rapid HR phenotype (Figure 4c).
The 282D construct differs from the 293D construct that
elicited a more rapid HR in that it lacks a complete kinase 3
motif and the completely inactive 1D138 is deleted in the CC
domain. It is likely therefore that the CC domain and the
kinase 3 motif of the NBS are essential for downstream
signaling capability of Rx.
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 195–204
Gain of Rx function by mutation and overexpression
Discussion
Regulatory domains in Rx and other NBS-LRR proteins
In this paper we report two findings related to the function
of Rx and, presumably, other NBS-LRR proteins. First we
describe constitutive gain-of-function mutations in the central and carboxy terminal regions of the Rx protein and,
second, we show that an HR induced by overexpression of
Rx can be accelerated by deletion of conserved sequence
motifs in the NBS. We conclude that these Rx phenotypes
are related to signalling rather than recognition because
they involve an elicitor-independent HR.
In one scenario a constitutive gain-of-function mutation
would have fortuitously generated specific structures of Rx
that normally exist only in the presence of elicitor. Alternatively, the gain-of-function mutations could have an
indirect effect by, for example, disruption of inhibitory
domains in Rx that prevent signalling in the absence of
elicitor. Formally, from the data presented here, neither
interpretation can be ruled out. However it is unlikely that
random mutagenesis would repeatedly generate new specific structures involved in downstream signalling. A more
likely scenario is that the mutation has a disruptive effect
and that the constitutive gain-of-function mutations affect
inhibitory domains in Rx. A similar interpretation, invoking
inactivation of inhibitory domains, was used to explain a
constitutive gain-of-function phenotype in a mutant Pto
protein (Rathjen et al., 1999).
Other NBS-LRR proteins that, like Rx, may be subject to
negative regulation include Nod1 in humans. This protein
exhibits enhanced signalling activity when over-expressed
with a deletion in the LRR (Inohara et al., 1999) and it was
concluded that the LRR prevents signalling by amino terminal domains. The analysis of the nematode resistance
protein Mi is also consistent with negative regulatory
domains in NBS-LRR proteins. Recombinants of Mi and
an inactive homologue induced an elicitor-independent HR
(Hwang et al., 2000) like the constitutive gain of function Rx
mutants described here. To explain this phenotype, it was
proposed that there are intramolecular interactions in Mi
(Hwang et al., 2000). In the recombinants or, presumably in
the presence of the Mi elicitor, these inhibitory interactions
would be disrupted so that signalling domains would
become exposed and available for interaction with components of the downstream pathway.
201
unpublished data) including RPS5 in Arabidopsis. RPS5
encodes an NBS-LRR protein and confers resistance
against Pseudomonas syringae. However a VLDL motif
mutant allele, rps5-1, has a complex phenotype involving
loss of RPS5-mediated resistance and suppression of resistance conferred by other NBS-LRR proteins (Warren et al.,
1998).
To reconcile the interference phenotype of rps5-1 and the
constitutive gain-of-function of D543E we propose that
these mutations both disrupt a negative regulatory domain
in the respective NBS-LRR proteins. Complete inactivation
of this domain, as in D543E, would release the negative
regulation so that the Rx protein could interact with downstream components of the signalling pathway and activate
an HR in the absence of elicitor. Partial inactivation of this
domain could allow the NBS-LRR protein to interact with
downstream signalling components but, as may be the
case with rps5-1, without activation of signalling. In that
scenario, if the downstream signalling components are
required by other NBS-LRR proteins, a partial loss-of-function would interfere with the respective signalling pathways.
Other NBS-LRR motifs, including RNBS-D (Meyers et al.,
1999) and MHD (van der Biezen and Jones, 1998) are
implicated in Rx inhibitory domains by constitutive gainof-function mutant phenotypes of D399V, E400K and D460V
(Figure 2). The MHD motif had not been targeted previously
in mutational analysis of NBS-LRR proteins (Axtell et al.,
2001; Dinesh-Kumar et al., 2000; Tao et al., 2000; Tornero
et al., 2002) and so the data presented here provide the first
direct analysis of its function.
Other than Rx, the most extensively mutagenised NBSLRR protein is RPM1 (Tornero et al., 2002). A total of 53
missense loss-of-function alleles were identified at this
locus of which only one is at or adjacent to the conserved
motifs that were modified in the Rx gain-of-function alleles.
The one exception (rpm1-12) affected the RNBS-D motif
that was modified in the AT72 and AT25 alleles of Rx (Figure
2). However the mutated amino acid in rpm1-12 is not
conserved in Rx and therefore is not directly comparable
with our constitutive gain-of-function alleles. The other
RPM1 mutants, like the gain-of-function mutants of Rx,
were predominantly either within or on the carboxy terminal side of the NBS. However, it is not possible to draw
additional conclusions by cross referencing between the
two sets of data because the gain- or loss-of-function
screens would identify different functional domains.
Conserved sequence motifs in NBS-LRR proteins
Most of the constitutive gain-of-function mutations in Rx
are in or close to sequence motifs that are common to many
NBS-LRR proteins. For example the D543E (Figure 2) mutation is in the VLDL motif that is present in the third LRR of
many NBS-LRR proteins (Axtell et al., 2001) (G. Farnham,
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 195–204
Amino terminal domains
Our constitutive gain-of-function mutants are not directly
informative about signalling domains in Rx. However, since
the HR would require signalling, the phenotypes of the Rx
deletion mutants (1D138, 382D, 318D and 293D; Figure 4) are
202 Abdelhafid Bendahmane et al.
indirectly informative. They indicate, consistent with previous suggestions (Aarts et al., 1998), that signalling
involves the amino terminal domain in combination with
the P loop, kinase 2 and kinase 3a motifs. It is striking that
none of the constitutive gain-of-function mutations was
within this region of the Rx protein. Perhaps, as suggested
above, random mutagenesis leads primarily to disruption
of functional domains rather than the generation of specific
structures that are required for interaction with components of the signalling pathway.
A gain of function analysis of the nematode resistance
gene Mi led to the conclusion that the amino terminal
domain was a regulator of signalling mediated by the
LRR. This interpretation differs from that made here about
Rx and it may be that activation of Mi- and Rx-mediated
resistance involves different mechanisms. However, if as
seems likely, the NBS-LRR domains have multiple functions
in recognition, activation and response signalling these two
sets of data are not necessarily incompatible. The artificial
Mi recombinants would have been informative about some
but not all of these functions. Similarly the Rx point mutants
would have provided information about a different set of
functions. For more complete understanding of recognition
and response in disease resistance, it will be necessary to
generate high resolution secondary and tertiary structures
of Rx and other NBS-LRR proteins and their elicitors.
Experimental procedures
Rx constructs
pB1 is a modified pBIN19 plasmid (Bevan, 1984) that carries a
transcription cassette comprising 3 kb of the Rx promoter and a
1.5-kb Rx terminator separated by an XbaI and a SacI cloning sites
(Bendahmane et al., 2000). All Rx derivative mutants were cloned
between the XbaI and the SacI cloning sites. All constructs were
sequenced to confirm the absence of PCR-derived mutations.
To construct pR-Rx, Rx cDNA was PCR amplified and the PCR
product was cloned into the XbaI and SacI sites of pB1 to create
pR-Rx.
The 35S promoter constructs were all in the pBIN61 binary
vector. pBIN61 is a modified pBIN19 binary vector that carries a
transcription cassette comprising the CaMV 35S promoter and
terminator. To construct the pBIN61 binary vector, the transcription cassette containing the CaMV 35S promoter and terminator
was released by digestion with KpnI and XhoI from the plasmid
pJIT61 (kindly provided by P. Mullineaux, JIC, Norwich, UK). The
transcription cassette was then ligated to the pBIN19 plasmid
vector digested with KpnI and SalI to create pBIN61. To construct
35S-Rx, Rx cDNA was PCR amplified and cloned into the XbaI and
SmaI sites of pBIN61.
RxHA was constructed via PCR, using a primer that introduced a
BamHI site at the extreme carboxyl terminus of the Rx open
reading frame followed by the HA epitope tag. Similarly the
truncated constructs were generated by PCR from the Rx cDNA
with 30 primers introducing a BamHI site. The PCR products were
cloned into the Xba1/BamH1 sites of PBin61-RxHA and subsequently tranferred to pB1.
Full details of all constructs are available on request and biomaterials are available through our website (http://www.sainsbury-laboratory.ac.uk).
PCR mutagenesis
Random mutagenesis of the Rx gene was performed under conditions similar to those previously described (Shafikhani et al.,
1997). The PCR was carried out using the primers RxP1 and Rxac4,
which flank the Rx ORF. The PCR reaction contained (100 ml final
volume) 10 mM Tris (pH 8.3), 50 mM KCl, 0.05% Nonidet P-40, 7 mM
MgCl2, 0.15 mM MnCl2, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,
1 mM dTTP, 0.3 mM of both primers, 50 ng of template and 5 U Taq
DNA polymerase (Gibco-BRL). PCR was performed for 35 cycles: 15 s
at 948C, 15 s at 558C, and 2 min at 728C. The PCR products were
digested with XbaI and SacI, gel purified and cloned in the binary
vector pB1 in E. coli. Plasmid DNA was purified from 10000 colonies
and electroporated into A. tumefaciens strain C58C1 carrying the
virulence helper plasmid pCH32 (Hamilton et al., 1996).
Site-directed mutagenesis
Oligonucleotide-directed mutagenesis was used to introduce specific mutations into the Rx cDNA or into the mutants E400K, D460V
and D543E þ H738R. The site directed mutations were confirmed
by sequence analysis. The mutations of the P loop were at Rx
codon 175 from GGG(G) to GCG(A) and at codon 176 from AAA(K)
to GCA(A). The mutations of the kinase Za motif were at Rx codon
244 from GAT(D) to GCT(A) and at codon 245 from GAC(D) to
GCC(A). The mutations of the GLPL motif were at codons 330 from
GGA(G) to GCA(A) and 332 from CCT(P) to GCT(A).
Agrobacterium-mediated transient expression
Agrobacterium-mediated transient expression was performed
under conditions similar to those described previously (Bendahmane et al., 1999). The binary Ti-plasmid vector constructs were
transformed into A. tumefaciens strain C58C1 carrying the virulence helper plasmid pCH32 (Hamilton et al., 1996). The transformants were inoculated into 5 ml L-broth medium supplemented
with 50 mg ml-1 kanamycin and 5 mg ml-1 tetracycline and grown at
288C overnight. Cells were precipitated and resuspended to the OD
of 0.5 in solution containing 10 mM MgCl2, 10 mM MES pH 5.6 and
150 mM acetosyringone. The cells were left at room temperature on
the bench for 2 h before infiltration into N. tabacum leaves. The
infiltrations were either into-non-transformed N. tabacum or into
transgenic N. tabacum expressing the PVX CP (Spillane et al.,
1997). The transient expression was more efficient in the first true
leaf of young N. tabacum seedlings.
DNA sequencing and analysis
The sequencing reactions were performed using a dye terminator
cycle sequencing reaction kit (Perkin-Elmer, Cambridge, UK).
Sequence reactions were resolved on ABI377 automated sequencer (Applied Biosystems ABI, La Jolla, CA, USA). Sequence contigs
were assembled using UNIX versions of the Staden programs
package (Staden, 1996).
Immunodetection
To prepare protein samples, two leaf discs (approximately 35 mg)
were ground in 150 ml of 8 M urea, followed by addition of 75 ml of
ß Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 195–204
Gain of Rx function by mutation and overexpression
3X SDS loading buffer and boiling for 10 min Fifty ml of protein
extract was separated by 10% SDS-PAGE followed by transfer to
Immun-blot PVDF membrane (Bio-Rad, Hemel Hempstead, UK).
Membranes were incubated successively in 5% skim milk powder
in TBST (25 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.1% Tween-20),
0.1 ng ml1 of 3F10 anti-HA antibody (Roche, Lewes, UK) and
finally with goat anti rat HRP conjugate (Sigma, Poole, UK). Proteins were visualised using the chemiluminesence kit ECL Plus
(Pharmacia, Little Chalfont, UK).
Virus induced gene silencing (VIGS)
VIGS of the N. benthamiana homologue of SGT1 using a tobacco
rattle virus vector (Ratcliff et al., 2001) has been described elsewhere (Peart et al., 2002). The procedures involved inoculation of
14-day-old N. benthamiana plants with TRV vectors either without
an insert (TRV:00) or carrying an N. benthamiana SGT1 insert
(TRV:SGT). The ability of these infected plants to support an Rxmediated HR was then tested after a further 14–21 days in the
leaves that were systemically infected with TRV.
Acknowledgements
We thank Erik Van der Biezen for comments on the manuscript.
The work was supported by the Gatsby Charitable Foundation and
the Biotechnology and Biological Sciences Research Council. P.
Moffett is supported by an EMBO long-term fellowship. Recombinant viruses and transgenic plants were contained in greenhouses
under DEFRA license PHL 161/4080.
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