Post on 20-Feb-2023
Abstract Resistance to an yellow strain of Cucumber
mosaic virus [CMV(Y)] in Arabidopsis thaliana eco-
type C24 is conferred by the CC-NBS-LRR type R
gene, RCY1. RCY1-conferred resistance is accompa-
nied by a hypersensitive response (HR), which is
characterized by the development of necrotic local le-
sion (NLL) at the site of infection that restricts viral
spread. To further characterize the role of RCY1 in
NLL formation we have identified six recessive
CMV(Y)-susceptible rcy1 mutants, four of which con-
tain single amino acid substitutions in RCY1: rcy1-2
contains a D to N substitution in the CC domain, rcy1-
3 and rcy1-4 contain R to K and E to K substitutions,
respectively, in the LRR domain, and rcy1-6 contains a
W to C substitution in the NBS domain. The rcy1-5 and
rcy1-7 contain nonsense mutations in the LRR and
NBS domains, respectively. Although the virus sys-
temically spread in all six rcy1 mutants, HR-associated
cell death was differentially induced in these mutants.
In comparison to the wild type C24 plant, HR was not
observed in the CMV(Y)-inoculated leaves of the rcy1-
3, rcy1-5, rcy1-6 and rcy1-7 mutants. In contrast,
delayed NLL development was observed in the virus
inoculated leaves of the rcy1-2 and rcy1-4 mutants. In
addition, necrosis accompanied by elevated accumu-
lation of PR gene transcript also appeared in the non-
inoculated leaves of the rcy1-2 and rcy1-4 mutants.
Trans-complementation was observed between the
rcy1-2 and rcy1-4 alleles; in F1 plants derived from a
cross between rcy1-2 and rcy1–4, HR associated cell
death was accelerated and systemic spread of the virus
was partially suppressed than in the homozygous rcy1-2
and rcy1-4 plants. Our results suggest that the CC, NBS
and LRR domains of RCY1 are required for restriction
of virus spread but differentially impact the induction
of HR-like cell death. Furthermore, these results also
predict inter-molecular interaction involving RCY1 in
Arabidopsis resistance to CMV(Y).
Keywords Resistance gene Æ Gene-for-gene
interaction Æ Viral resistance
Abbreviations
CC Coiled-coil
CMV(Y) An yellow strain of Cucumber mosaic virus
EMS Ethyl methanesulfonate
NBS Nucleotide-binding site
NLL Necrotic local lesion
LRR Leucine-rich repeats
RCY1 RESISTANCE TO CMV(Y) 1
TIR Toll-interleukin-1 receptor
Introduction
In many cases of plant-pathogen interaction initial rec-
ognition of the pathogen occurs via the products of a
K.-T. Sekine Æ T. Ishihara Æ S. Hase Æ H. Takahashi (&)Department of Life Science, Graduate School ofAgricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Miyagi, Sendai 981-8555, Japane-mail: takahash@bios.tohoku.ac.jp
T. KusanoGraduate School of Life Sciences, Tohoku University, 2-1-1Katahira, Aoba-ku, Miyagi , Sendai 980-8577, Japan
J. ShahDivision of Biology and the Molecular, Cellular andDevelopmental Biology Program, , Kansas State University,303 Ackert Hall, Manhattan, KS 66506-4901, U.S.A
Plant Mol Biol (2006) 62:669–682
DOI 10.1007/s11103-006-9048-4
123
Single amino acid alterations in Arabidopsis thaliana RCY1compromise resistance to Cucumber mosaic virus,but differentially suppress hypersensitive response-like cell death
Ken-Taro Sekine Æ Takeaki Ishihara ÆShu Hase Æ Tomonobu Kusano Æ Jyoti Shah ÆHideki Takahashi
Received: 28 April 2006 / Accepted: 6 July 2006 / Published online: 29 August 2006� Springer Science+Business Media B.V. 2006
specific plant resistance (R) gene and a corresponding
pathogen avirulence gene. The ensuing resistance
mechanism has been termed gene-for-gene resistance
(Hammond-Kosack and Jones 1997; Dangl and Jones
2001; Hammond-Kosack and Parker 2003). In many
cases R-gene conferred resistance is accompanied by a
hypersensitive response (HR), which is characterized by
necrotic local lesions (NLL) at the site of pathogen
inoculation and the elevated expression level of the
pathogenesis-related (PR) genes. In the past decade,
several R genes that confer gene-for-gene resistance
against virus, bacteria, oomycete and fungi have been
isolated from a variety of plants species (Hammond-
Kosack and Parker 2003). A large number of R proteins
contain a conserved nucleotide binding site (NBS) and
leucine-rich repeats (LRR) (Hammond-Kosack and
Jones 1997; Dangl and Jones 2001). These NBS-LRR
proteins are further separated into two subclasses
depending on whether they have a coiled-coil (CC) do-
main or a toll-interleukin-1 receptor (TIR)-like region at
their amino-terminal (Ellis et al. 2000).
The LRR region, which is the most variable region
among closely related R proteins and is subject to
selection for diversification, is believed to modulate
recognition to the pathogen and/or interaction with
upstream activator(s) of defense responses (McDowell
et al. 1998; Noel et al. 1999; Hwang et al. 2000; Dodds
et al. 2001; Meyers et al. 2003; Tanabe et al. 2004). The
analysis of 11 alleles at the L locus in flax, which
control different resistance specificities to flax rust,
indicated that the LRR region of the L protein is an
important determinant of gene-for-gene specificity.
However, variable regions outside the LRR region also
influence expression and specificity of rust resistance
between other L alleles (Ellis et al. 1999; Luck et al.
2000). Likewise, the LRR region of the Arabidopsis
RPS2 and RPS5 proteins, which mediate resistance to
the bacterial pathogen Pseudomonas syringae
expressing the avrRpt2 and avrPphB avirulence genes,
respectively, have been postulated to contribute to
recognition of pathogen and the activation of down-
stream signal transduction (Warren et al. 1998;
Banerjee et al. 2001).
The NBS region, which is located between the amino
terminal CC/TIR and the LRR region, is believed to be
responsible for ATP hydrolysis and subsequent activa-
tion of downstream signaling (Tameling et al. 2002). The
NBS region contains three motifs found in kinases and
other motifs that are conserved in ATPases (Traut
1994). These motifs are also found in the NBS region of
the Caenorhabditis elegans, CED-4 protein and the hu-
man APAF-1 regulators of apoptosis (van der Biezen
and Jones 1998; Aravind et al. 1999). Mutational analysis
of RPS2 revealed that the NBS domain was critical for
RPS2 function (Tao et al. 2000; Axtell et al. 2001).
Furthermore, large-scale analysis of Arabidopsis RPM1,
which mediates resistance to P. syringae expressing av-
rRpm1 and avrB, indicated that the NBS plays a key role
in RPM1 stability and the RPM1-dependent activation
of HR (Tornero et al. 2002). In addition, the NBS do-
main mediates oligomerization resulting in the activa-
tion of N-terminal signaling domains in certain animal
proteins (Inohara et al. 2005).
To date, six genes conferring gene-for-gene resis-
tance to virus have been isolated from different plant
species. The N gene in Nicotiana glutinosa, which en-
codes a TIR-NBS-LRR type protein, confers resistance
to Tobacco mosaic virus (TMV) (Whitham et al. 1994).
Rx in Solanum tuberosum, HRT and RCY1 in Ara-
bidopsis thaliana, and Sw-5 and Tm-22 in Lycopersicon
esculentum, which belong to the CC-NBS-LRR class R
gene, confer resistance to Potato virus X (PVX), Tur-
nip crinkle virus (TCV), Cucumber mosaic virus
(CMV), Tomato spotted wilt virus (TSWV) and To-
mato mosaic virus (ToMV), respectively (Bendahmane
et al. 1999; Brommonschenkel et al. 2000; Cooley et al.
2000; Takahashi et al. 2002; Lanfermeijer et al. 2003).
In general, resistance conferred by these R genes is
accompanied by the development of NLLs at the pri-
mary infection site, which restrict systemic spread of
the virus (Matthews 2002). Rx-mediated resistance to
PVX is unusual in that it is usually not accompanied by
NLLs in either potato or in Nicotiana benthamiana
carrying the Rx transgene (Bendahmane et al. 1999).
However, when Agrobacterium tumefaciens carrying a
wild type Rx cDNA was infiltrated into transgenic
N. tabacum plants that expressed the PVX coat protein
(CP), which is the avirulence factor perceived by Rx,
cell death was induced at the infiltrated region (Ben-
dahmane et al. 2002). Furthermore, NLLs developed in
transgenic potato and N. benthamiana plants that
overexpressed the CP (Bendahmane et al. 1999).
Moreover, one amino acid substitution in NBS and
LRR domains of Rx that resulted in a gain-of-Rx
function phenotype also caused the activation of the
cell death even in the absence of CP, indicating that
inhibitory domains in the NBS and LRR domains
control Rx activity (Bendahmane et al. 2002). Moffett
et al. (2002) demonstrated that physical interaction can
occur in vivo between the NBS-LRR domain and the
amino-terminal CC and between the LRR and the CC-
NBS domains. They proposed that CP causes the
relaxation of intramolecular interactions within Rx
protein thereby triggering downstream resistance
mechanisms. Extensive deletion and single amino acid
substitution analysis of N has been done to understand
670 Plant Mol Biol (2006) 62:669–682
123
the function of the different domains of N in plant
defense against TMV (Dinesh-Kumar et al. 2000).
These studies have indicated that the TIR, NBS and
LRR domains of N play an indispensable role in the
induction of resistance to TMV. Furthermore, in
comparison to the wild type N protein, many single
amino acid substitutions in the TIR, NBS and LRR
domains of N resulted in the delayed onset of HR and
the inability to restrict virus spread to the primary
infection site (Dinesh-Kumar et al. 2000).
CMV is one of the best-characterized tripartite posi-
tive-sense single-stranded RNA viruses (Palukaitis and
Garcıa-Arenal 2003). A. thaliana ecotype C24 is resis-
tant to an yellow strain of CMV [CMV(Y)] but suscep-
tible to an Indonesian strain of CMV [CMV(B2)],
whereas A. thaliana ecotype Columbia (Col) is system-
ically infected by both strains (Takahashi et al. 2001).
CMV(Y) resistance in C24 is conferred by a single
dominant gene RCY1, which is located on chromosome
5. RCY1 is allelic to RPP8 and HRT (Takahashi et al.
2002) that confer race specific resistance against Pero-
nospora parasitica and TCV, respectively (Cooley et al.
2000; McDowell et al. 1998). Resistance to CMV(Y) is
accompanied by the development of NLLs and elevated
expression of the pathogenesis-related, PR1a gene, at
the primary infection site in C24 leaves (Ishihara et al.
2004; Takahashi et al. 2004a). However, nothing is
known about the molecular requirements of RCY1 in
conferring resistance to CMV(Y).
In this paper, we isolated six independent rcy1 mu-
tants which are fully susceptible to CMV(Y). Single
amino acid substitutions in the CC, NBS or LRR do-
mains of RCY1 protein were identified in four rcy1
mutants, and nonsense mutations were identified in the
other two mutants. Comparative analysis of systemic
movement of virus, cell death response, PR gene
expression between the six rcy1 mutants indicated that
these mutations in RCY1 equally compromise the
restriction of virus systemic spread but differentially
suppress HR-like cell death in CMV(Y)-infected
A. thaliana ecotype C24.
Materials and methods
Virus preparation and plant growth condition
An yellow strain of Cucumber mosaic virus [CMV(Y)]
(Tomaru and Hidaka 1960) was propagated on tobacco
(N. tabacum cv. Xanthi nc) and the virus particles were
purified as previously described (Takahashi and Eha-
ra1993). Arabidopsis thaliana ecotypes C24 and
Columbia (Col) and the rcy1 mutant plants were grown
at 25�C under continuous illumination (8,000 lux) in a
mixture of vermiculite and perlite (1:1 mixture) and
irrigated with nutrient medium every 3 days.
Virus inoculation and detection
Fully expanded leaves of 3-week-old A. thaliana were
inoculated with 100 lg/ml of CMV(Y) as previously
described (Takahashi et al. 1994). To quantify the
systemic spread of virus in CMV-infected plants, the
coat protein of CMV was detected immunologically by
a modified tissue-printing method (Takahashi et al.
2002; Sekine et al. 2004). To quantify the amount of
virus multiplication, the amount of viral coat protein
was measured by the ELISA method as described
previously (Sekine et al. 2004).
Mutagenesis and selection of CMV(Y)-susceptible
mutants
Ten thousands seeds from wild-type C24 plants were
mutagenized with 0.3% ethyl methanesulfonate (EMS)
as previously described (Redei and Koncz 1992). To
select CMV(Y)-susceptible C24 mutants, M2 seeds
generated from self-pollination of the EMS-treated M1
C24 seeds were germinated and two fully expanded
leaves of 3-week-old M2 plant were inoculated with
100 lg/ml of CMV(Y). Seven days after inoculation,
the presence of viral coat protein in the non-inoculated
upper leaves of CMV(Y)-infected plants was detected
by the tissue-printing method. Plants in which viral
coat protein was detected in their upper non-inocu-
lated leaves were considered as potential CMV(Y)-
susceptible mutants. The CMV(Y) susceptibility of
these rcy1 mutants was confirmed in M3 progeny
plants. The phenotype of each mutant was further
characterized at M4 and M5 generations.
Cloning and sequence analysis of RCY1 gene
Three individual genomic clones of RCY1 gene from each
rcy1 mutant and wild-type C24 plant were cloned and
sequenced. DNA fragments containing 5¢ and 3¢ half parts
of RCY1, which overlapped by about 500 bp in the region
containing a BsmI site, were amplified by long and
accurate PCR (LA-PCR). The template genomic DNA
for LA-PCR was isolated from fully expanded C24 leaves
by CTAB method (Murray and Thompson 1980). One
hundred ng of the genomic DNA was added to 50 ll of
reaction mixture containing 1x KOD-plus buffer pro-
vided by the manufacturer, 1 mM MgSO4, 0.2 mM each
of dATP, dCTP, dGTP and dTTP and 1 units of KOD-
Plant Mol Biol (2006) 62:669–682 671
123
plus-DNA polymerase (TOYOBO, Osaka, Japan) with
0.4 lM each of the pairs of primers: rpp8A (5¢-ATT-
GTTCTCGTACTATTCGTTAGTCGTTAC-3¢) and
rpp8B (5¢-ATCGTGGTTAGATGACAACAGTCTC-
AATTC-3¢) for 3¢ DNA fragment of RCY1 or the pairs of
primers: rpp8C (5¢-GTGTCCCAATATATGAAAGG-
CTTTACCACT-3¢) and rpp8D (5¢-CAATTTTGATT-
CCC-TGCTTGCATCATCAA-3¢) for 5¢ DNA fragment
of RCY1. The PCR reaction ran on three steps with the
program: 94�C for 2 min, followed by 30 cycles of 94�C
for 15 sec.; 55�C for 30 sec.; 68�C for 1 min. Two ampli-
fied RCY1 DNA fragments; rpp8A-rpp8B and rpp8C-
rpp8D, which have about 500 bp regions overlapping
each other, were purified as described previously (Ta-
kahashi and Ehara 1993), and then cloned into EcoRV
site of pBluescript SKII+ vector (Stratagene, La Jolla,
CA). The resulting plasmids containing rpp8A-rpp8B
and rpp8C-rpp8D fragments were designated as pRCY1/
AB and pRCY1/CD, respectively. Three independent
clones of each plasmid were sequenced by the Sanger
method using an automated DNA sequencer (ABI model
373A).
Plant transformation
Plasmid pBS+SK/RCY1 containing wild-type RCY1
was generated by cloning pRCY1/AB-derived BsmI-
SalI DNA fragment containing rpp8A-rpp8B DNA
fragment into the BsmI-SalI site of pRCY1/CD. To
construct a binary vector for plant transformation, DNA
fragment containing entire wild-type RCY1 gene was
excised from pBS+SK/RCY1 by partial digestion with
XbaI, and subcloned into the binary vector pGA482 (An
1986) and designated as pGA482/RCY1. One lg of
pGA482/RCY1 was introduced into Agrobacterium
tumefaciens LBA4404. The rcy1-3 mutant plants were
transformed with LBA4404 containing pGA482/RCY1
by vacuum infiltration (Bechtold 1998). Transgenic
plants were screened on 0.5 · MS medium (Murashige
and Skoog 1962) with 0.8% agar and 50 lg/ml kana-
mycin. Transformation of RCY1 into rcy1-3 mutant was
confirmed by detecting pGA482-derived selectable
marker NPTII in the transformants at second generation
(T2 plants) by PCR using a pair of primers: NPTII-F (5¢-ATGATTGAACAAGATGGATTG-3¢) and NPTII-R
(5¢-TCAGAAGAACTCGTCAAGAAG-3¢).
Analysis of PR-1a gene expression
Expression of PR-1a in CMV(Y)-inoculated leaves was
analyzed by northern hybridization as described pre-
viously (Takahashi et al. 2004a).
Results
Isolation of rcy1 mutants
M2 plants derived from ethyl methanesulfonate
(EMS)-mutagenized Arabidopsis ecotype C24, which
contains the wild type RCY1 gene, were inoculated
with CMV(Y). Viral growth and spread through the
plant was monitored at daily intervals between 3 –
7 days post inoculation (dpi) with an antibody raised
against the coat protein of CMV(Y). Six mutants, rcy1-
2, rcy1-3, rcy1-4, rcy1-5, rcy1-6 and rcy1-7 were iden-
tified from a screen of 30,000 M2 plants. Property of
these six mutants was summarized in Table 1. In con-
trast to the CMV(Y) resistant wild type ecotype C24
plant in which CMV(Y) coat protein accumulation was
restricted to the virus inoculated leaves, the coat pro-
tein accumulated in the virus-inoculated and uninocu-
lated leaves of the rcy1 mutants, suggesting systemic
spread and growth of the virus (Figs. 1, 2A, 2B). At
7 dpi, the coat protein accumulation in the uninocu-
lated leaves of the rcy1-2, rcy1-3, rcy1-4, rcy1-5, rcy1-6
and rcy1-7 mutants was comparable to that in the
CMV(Y) susceptible Arabidopsis ecotype Columbia
(Col), which lacks RCY1 (Fig. 1). Backcrosses of the
six CMV(Y) susceptible rcy1 mutants with the
CMV(Y) resistant wild type C24 resulted in F1 prog-
eny plants that were all resistant to CMV(Y) (Table 2),
suggesting that the CMV(Y) susceptible phenotype of
all six rcy1 mutants were due to recessive mutations.
Furthermore, F2 progeny of F1 hybrids derived from
crosses between C24 and the rcy1-2, rcy1-3, rcy1-4 and
rcy1-5 mutants segregated CMV(Y) susceptible and
CMV(Y) resistant plants in a 1:3 ratio (Table 2), con-
firming the recessive nature of the CMV(Y) suscepti-
ble phenotype conferred by rcy1-2, rcy1-3, rcy1-4 and
rcy1-5. In addition, these results indicate monogenic
inheritance of the CMV(Y) susceptible trait in these
mutants.
Characterization of CMV(Y)-infected rcy1 mutant
lines
RCY1-conferred resistance to CMV(Y) infection in
the Arabidopsis ecotype C24 is associated with the
development of HR-like necrotic local lesions (NLL)
at the site of virus inoculation. Virus is restricted to
these sites. In contrast, in the CMV(Y) susceptible
ecotype Col, which lacks the RCY1 locus, NLL does
not develop and the virus spreads and multiplies
throughout the plant, resulting in chlorosis and stun-
ting of the plant. To determine if the loss of CMV(Y)
resistance in the rcy1 mutants is paralleled by their
672 Plant Mol Biol (2006) 62:669–682
123
inability to effectively activate NLL, we compared the
timing of NLL development with the extent of viral
distribution and disease symptoms in the virus inoc-
ulated and non-inoculated leaves of each rcy1 mutant
and the wild type C24 plant. As shown in Fig. 3A, by
2 dpi the CMV(Y)-inoculated leaves of the wild type
C24 plant developed NLLs, which were accompanied
by the elevated accumulation of the pathogenesis-re-
lated PR1a gene transcript (Fig. 4). Virus was local-
ized to these NLLs (Fig. 3B). By 4 dpi, the virus
inoculated leaves of C24 had died, presumably as a
result of the HR (Fig. 3C, D). In contrast, NLLs did
not develop (Fig. 3A) and PR1a transcript did not
accumulate at elevated level (Fig. 4) in the virus-
inoculated leaves of rcy1-3, rcy1-5, rcy1-6 and rcy1-7
mutants. NLL development was delayed in rcy1-2 and
rcy1-4; a few NLL-like spots were visible by 4 dpi in
these two mutants (Fig. 3C). These late developing
NLLs in rcy1-2 and rcy1-4 were accompanied by
elevated accumulation of the PR1a transcript in the
virus inoculated leaves (Fig. 4). By 7 dpi the virus
inoculated rcy1-2, rcy1-3, rcy1-4, rcy1-5, rcy1-6 and
rcy1-7 mutant plants were stunted (Fig. 5A), the fully
expanded non-inoculated leaves exhibited chlorosis,
and the virus had systemically spread to these non-
inoculated leaves (Fig. 5B). In addition, by 10 dpi
Table 1 Summary of plants, their genotypes and their response to CMV(Y) in the presented experiments
Plants and their genotypesa Response to CMV(Y)
susceptible/ resistanceb development of lesionsc
wild C24 (RCY1/RCY1) R NLLswild Col (rpp8/rpp8) S no lesion
rcy1-2(rcy1-2/rcy1-2) S delayed NLLsrcy1-3(rcy1-3/rcy1-3) S no lesionrcy1-4(rcy1-4/rcy1-4) S delayed NLLsrcy1-5(rcy1-5/rcy1-5) S no lesionrcy1-6(rcy1-6/rcy1-6) S no lesionrcy1-7(rcy1-7/rcy1-7) S no lesion
F1(RCY1/rcy1-2) R NLLsF1(RCY1/rcy1-3) R NLLsF1(RCY1/rcy1-4) R NLLsF1(RCY1/rcy1-5) R NLLsF1(RCY1/rcy1-6) R NLLsF1(RCY1/rcy1-7) R NLLs
F1(rpp8/rcy1-2) S no lesionF1(rpp8/rcy1-3) S no lesionF1(rpp8/rcy1-4) S no lesionF1(rpp8/rcy1-5) S no lesionF1(rpp8/rcy1-6) S no lesionF1(rpp8/rcy1-7) S no lesion
F1(rcy1-2/rcy1-3) S no lesionF1(rcy1-2/rcy1-4) Sd NLLse
F1(rcy1-2/rcy1-5) S no lesionF1(rcy1-3/rcy1-4) S no lesionF1(rcy1-3/rcy1-5) S no lesionF1(rcy1-4/rcy1-5) S no lesion
a Two-week-old Arabidopsis thaliana plants were inoculated with 100lg/ml of CMV(Y). Wild ecotype plants (C24 and Col), rcy1mutants (rcy1-2, rcy1-3, rcy1-4, rcy1-5, rcy1-6 and rcy1-7), F1 plants resulted from the crosses between wild C24 and rcy1 mutants, F1plants resulted from the crosses between wild Col and rcy1 mutants, and F1 plants resulted from the crosses between each rcy1 mutantswere divided by dashed linesb The plant in which virus was systemically spread to the non-inoculated leaves, was shown as susceptible (S) response to CMV(Y), andthe plant in which virus spread was restricted within the inocuolated leaves, was shown as resistant (R) responsec The formation of necrotic local lesions (NLLs) in the virus-inoculated leaves was observed at 2 days after inoculation. However,delayed NLLs was developed at 4 days after inoculation. Plant in which NLLs were not developed, was shown as ‘‘no lesion’’d Systemic spread of virus in F1 (rcy1-2/rcy1-4) plants was partially suppressed than in the homozygous rcy1-2 and rcy1-4 plants, asshown in Fig. 2e The deveolpment of NLLs was obsvered in most virus-inoculated leaves of F1 (rcy1-2/rcy1-4) plants at 2 days after inoculation, but insome F1 plants, it was slightly delayed as shown in Table 3 and Fig. 8
Plant Mol Biol (2006) 62:669–682 673
123
necrotic symptoms developed on the unexpanded
non-inoculated leaves of the rcy1-2 and rcy1-4
mutants (Fig. 5C). As a control, we also monitored
NLL development and viral spread in the wild type
C24 and the rcy1 mutant plants inoculated with the
virulent CMV(B2) strain. NLL did not develop and
CMV(B2) systemically spread throughout the C24
and rcy1 mutant plants (data not shown).
Fig. 1 Immunologicaldetection of CMV(Y) coatprotein in leaves ofArabidopsis thaliana ecotypesC24 and Col and the rcy1mutants. Two fully expandedleaves of 3-weeks-old wildecotypes C24 and Col and M3plants of six independent rcy1mutant lines were inoculatedwith CMV(Y). At 3, 4, 5, 6and 7 days post inoculation(dpi), the distribution of virusin the whole plants wereanalyzed by the tissue-printing method using anantibody against the coatprotein of CMV(Y)
674 Plant Mol Biol (2006) 62:669–682
123
A40
5
(A)
days post inoculation
0
0.1
0.2
0.3
0.4
0.5
0.7
0.6
0
0.1
0.2
0.3
0.4
0.5
0.7
0.6
0
0.1
0.2
0.3
0.4
0.5
0.7
0.6
43
wild C24rcy1-2rcy1-3
rcy1-4rcy1-5
A40
5
(B)
days post inoculation
43
wild C24rcy1-2rcy1-4
rcy1-6rcy1-7
A40
5
(C)
days post inoculation
43
wild C24rcy1-2
rcy1-4
F1(rcy1-2/rcy1-4)F1(rcy1-4/rcy1-2)
Fig. 2 Accumulation ofCMV(Y) coat protein in virusnon-inoculated upper leaves.The amount of CMV(Y) coatprotein at 3 and 4 dpi withCMV(Y) in non-inoculatedupper leaves of the rcy1–2~rcy1–7 mutants, wild C24,F1(rcy1-2 x rcy1-4) andF1(rcy1-4 x rcy1-2) plants wasmeasured by ELISA. Datawere shown as thecombinations of wild C24,rcy1-2 and rcy1-4 plants plusrcy1-3 and rcy1-5 plants (A),wild C24, rcy1-2 and rcy1-4plants plus rcy1-6 and rcy1-7plants (B), and wild C24,rcy1-2 and rcy1-4 plants plusF1(rcy1-2 x rcy1-4) andF1(rcy1-4 x rcy1-2) plants (C).Five plants were analyzed pertreatment. The error barsindicate SD
Table 2 Genetic analysis of response to CMV(Y) in progenies crossed between the rcy1 mutants
Crossesa Generation Response to CMV(Y)b Susceptible:Resistant v2 v20.01
Number of susceptible plants Number of resistant plants Observed ratio Expected ratio
wild C24 · rcy1-2 F1 0 5 0:5 – – –F2 28 97 1:3.5 1:3 0.450 6.635
wild Col · rcy1-2 F1 5 0 5:0 – – –F2 123 0 123:0 – – –
wild C24 · rcy1-3 F1 0 5 0:5 – – –F2 32 92 1:2.9 1:3 0.043 6.635
wild Col · rcy1-3 F1 5 0 5:0 – – –F2 125 0 125:0 – – –
wild C24 · rcy1-4 F1 0 5 0:5 – – –F2 25 95 1:3.8 1:3 1.111 6.635
wild Col · rcy1-4 F1 5 0 5:0 – – –F2 120 0 120:0 – – –
wild C24 · rcy1-5 F1 0 5 0:5 – – –F2 33 90 1:2.7 1:3 0.220 6.635
wild Col · rcy1-5 F1 5 0 5:0 – – –F2 122 0 122:0 – – –
wild C24 · rcy1-6 F1 0 5 0:5 – – –wild Col · rcy1-6 F1 5 0 5:0 – – –
wild C24 · rcy1-7 F1 0 5 0:5 – – –wild Col · rcy1-7 F1 5 0 5:0 – – –
a Ecotypes C24 or Col was crossed with six indepenedent CMV(Y)-susceptible C24 muatnt lines: rcy1-2, rcy1-3, rcy1-4, rcy1-5, rcy1-6and rcy1-7. Each crosses were divided by dashed linesb Two-week-old Arabidopsis thaliana plants were inoculated with 100 lg/ml of CMV(Y) . The distribution of the coat protein of virusin CMV(Y)-inoculated plants was detected at 7 days after inoculation by the tissue printing method. The number of plants in whichCMV(Y) spread to the non-inoculated leaves (Susceptible) or was restricted within the inoculated leaves (Resistant) were counted
Plant Mol Biol (2006) 62:669–682 675
123
Cloning and sequence analysis of the rcy1 mutant
alleles
Mapping of the loci associated with CMV(Y) suscep-
tibility in the rcy1 mutants suggested that these were
located in the vicinity of the RCY1 locus on chromo-
some 5, which confers gene-for-gene resistance to
CMV(Y) (data not shown). We have previously shown
that a single copy of the RCY1 allele is sufficient to
confer resistance to CMV(Y) (Takahashi et al. 2002).
To determine if the mutations in rcy1 mutants were at
the RCY1 locus, each rcy1 mutant was crossed with the
CMV(Y) susceptible ecotype Col, which lacks RCY1. If
the CMV(Y)-susceptible phenotype in an rcy1 mutant
line was caused by a mutation at the RCY1 locus, then
all F1 plants and F2 progeny plants derived from this
cross should be susceptible to CMV(Y). In contrast, if
the mutants were not allelic to the RCY1 gene, but at a
different locus and epistatic to it, although only if
homozygous recessive, then the F1 plants from a cross
between Col plants and the various homozygous rcy1
mutants would all be resistant, because the RCY1 gene
from the parental rcy1 mutants can dominantly cause
resistance response. Furthermore, since the CMV(Y)
susceptible phenotype conferred by the rcy1 mutations
is recessive and the rcy1 alleles map in the vicinity of
RCY1, CMV(Y) susceptibility and resistance should
segregate in a near 1:1 ratio in the F2 progeny derived
Fig. 3 Necrotic symptomsand virus distribution inCMV(Y)-inoculated leaves ofthe rcy1 mutant lines.Symptoms on virus-inoculated leaves of M4plants of the rcy1-2~rcy1-7mutants and wild C24 wereobserved at 2 dpi (A) and4 dpi (C) with CMV(Y).Distribution of virus in theinoculated leaves of them wasalso analyzed at 2 dpi (B) and4 dpi (D) by the tissue-printing method. Totalprotein of CMV(Y)-inoculated wild C24 leaf couldnot be transferred into thefilter paper for the tissue-printing at 4 dpi because theinoculated leaf wascompletely died and dried
PR1a
rRNA
M V M V M V M V M V M V M V M V
wildCol
wildC24
rcy1-2
rcy1-3
rcy1-4
rcy1-5
rcy1-6
rcy1-7
Fig. 4 Expression of PR-1a in CMV(Y)-inoculated leaves of thercy1 mutant lines. PR-1a expression in virus-inoculated (V) andmock-inoculated (M) leaves of M4 plants of the rcy1-2~rcy1-7
mutants and wild ecotypes C24 and Col was analyzed bynorthern hybridization. EtBr-stained rRNA was also shown asan internal control
676 Plant Mol Biol (2006) 62:669–682
123
from these F1 hybrids. As shown in Table 2, all F1 and
F2 plants derived from crosses between Columbia and
the six rcy1 mutant plants were susceptible to CMV(Y),
suggesting that the CMV(Y)-susceptible phenotype of
the rcy1-2, rcy1-3, rcy1-4, rcy1-5, rcy1-6 and rcy1-7
mutants is caused by mutations in RCY1.
Approximately 7.5 kb genomic DNA fragments
covering the RCY1 gene were cloned from the rcy1
mutants and sequenced. The corresponding amino acid
sequence for each rcy1 allele is shown in Fig. 6. In the
rcy1-2 mutant, a G to A mutation at nucleotide position
139 of RCY1 resulted in an Aspartic acid being substi-
tuted by Asparagine at amino acid position 47 in the CC
domain. In the rcy1-3 and rcy1-4 mutants, G to A
mutations at nucleotide positions 1,649 and 2,017 of
RCY1 resulted in Arginine being substituted by Lysine
at amino acid position 550, and Glutamic acid being
substituted by Lysine at amino acid position 673,
respectively, in the putative b-strand/b-turn region of
the LRR domain. In rcy1-6, a G to A mutation at
nucleotide position 651 of RCY1 resulted in the substi-
tution of Tryptophan by Cysteine at amino acid position
217 in the NBS domain. In the rcy1-5 and rcy1-7 mutants,
G to A mutations at nucleotide positions 1,689 and 1,446
of RCY1 resulted in nonsense mutations at amino acid
positions 563 in the LRR and 482 in the NBS domains,
respectively. The recessive nature of these six mutations
suggests that they result in loss of RCY1 function. To
confirm that a loss-of-function mutation in RCY1 can
confer susceptibility to CMV(Y), the rcy1-3 mutant line
were transformed with the wild RCY1 gene to generate
stable transformants, because we considered that the
complementation experiment by wild RCY1 to one
mutant line out of six independent rcy1 mutants is en-
ough to confirm if CMV(Y)-susceptible mutant pheno-
type was resulted from loss of RCY1 function. Ten T2
progeny, which were derived from a RCY1-transformed
rcy1-3 plant, were screened for resistance to CMV(Y).
All of the segregants that contained the RCY1 transgene
were resistant to CMV(Y). Similar to the wild type
ecotype C24 plant, resistance in the seven RCY1 trans-
gene-bearing segregants was accompanied by NLL for-
mation in the virus-inoculated leaves (Fig. 7B). In
contrast, in the three segregants that lacked the RCY1
transgene, CMV(Y) systemically spread throughout the
plant, similar to the spread of CMV(Y) in the rcy1-3
mutant plant (Fig. 7A, C). The near 3:1 segregation ratio
for CMV(Y) resistant:susceptible plants in this experi-
ment suggests that the RCY1 transgene was inserted at a
single locus in the corresponding T1 transgenic plant.
Genetic interaction among the rcy1 alleles
To study the interaction between the rcy1 alleles (rcy1-
2 and rcy1-4) that result in delayed NLL development
in response to CMV(Y) inoculation and the rcy1 alleles
(rcy1-3 and rcy1-5) that abolish NLL development, we
studied the response to CMV(Y) in F1 hybrids result-
ing from crosses between these rcy1 mutants. Similar to
the rcy1-3 and rcy1-5 mutants, in F1 hybrids of the
Fig.5 Systemic symptoms and virus distribution in CMV(Y)-infected rcy1 mutant lines. At 7 dpi with CMV(Y), chlorosis andstunting symptoms developed in M4 plants of the rcy1-2~rcy1-7mutant lines but did not in wild C24 (A). Virus distribution in M4plants of the rcy1-2~rcy1-7 mutants was detected by the tissue-
printing method at 7 dpi (B). Non-inoculated upper leaves ofCMV(Y)-infected rcy1-2~rcy1-7 mutants and wild C24 at 10 dpiwere photographed (C). Necrotic symptoms appeared onlyunexpanded upper leaves of CMV(Y)-infected rcy1-2 and rcy1-4 mutants at 10 dpi with CMV(Y)
Plant Mol Biol (2006) 62:669–682 677
123
genotypes rcy1-2/rcy1-3, rcy1-2/rcy1-5, rcy1-4/rcy1-3
and rcy1-4/rcy1-5, NLLs did not develop in the
CMV(Y) inoculated leaves (Table 3). In contrast,
NLLs appeared by 4 dpi in the virus inoculated leaves
of the rcy1-2 and rcy1-4 mutants (Table 3). These re-
sults suggest that the NLL non-forming rcy1-3 and
rcy1-5 alleles are dominant over the rcy1-2 and rcy1-4
alleles. In contrast, in the F1 hybrids of the genotype
rcy1-2/rcy1-4, derived from a cross between the two
NLL forming rcy1-2 and rcy1-4 mutants, NLL-like
lesions were observed in the CMV(Y)-inoculated
leaves as early as 2 dpi and were very clear by 3 dpi
(Table 3 and Fig. 8A, C), comparable to the develop-
ment of NLLs in the CMV(Y) resistant wild type C24
plant. This is in contrast to the rcy1-2 and rcy1-4 single
mutants in which NLL-like lesions had not developed
by 2 dpi (Table 3 and Fig. 8A). Although the virus was
not restricted to the CMV(Y)-inoculated leaves in
these rcy1-2/rcy1-4 F1 hybrids, viral coat protein
accumulated to lower level in the non-inoculated
leaves of this F1 hybrid than in the rcy1-2 and rcy1-4
single mutant plants (Fig. 2C, 8B, 8D), suggesting that
systemic spread of virus was reduced in the rcy1-2/rcy1-
4 hybrid than in the corresponding single mutant
plants.
Discussion
RCY1, which is present in the CMV(Y) resistant
Arabidopsis ecotype C24, encodes a CC-NBS-LRR
(A)
(B)
(C)
(D)
(E)
(F)
(G)
Fig. 6 Amino acid sequence of mutated RCY1 protein encodedin RCY1 locus of six CMV(Y)-susceptible rcy1 mutant lines. (A)to (G) are based on putative functional motifs of RPP8(McDowell et al. 1998). Domains (B) and (D) contain coiled-coil (CC) structures. Domains (C), (D), and (E) contain NBSmotifs and conserved hydrophobic domain which are shown inboldface. Domain (F) contains 14 imperfect LRRs defined by theconserved residues shown in boldface. Amino acid positions thatwere altered in the rcy1-2, rcy1-3, rcy1-4, rcy1-5, rcy1-6 and rcy1-7 mutants are underlined. Each substituted amino acid wasshown in round brackets. Asterisk indicates the stop codoncaused by nonsense mutation
Fig. 7 Response of RCY1-transformed rcy1-3 mutant toCMV(Y). RCY1-carrying plants [RCY1(+)T2] and RCY1-lack-ing plants [RCY1(-)T2] that segregated from a T2 plant derivedfrom a RCY1-transformed rcy1-3 plant, wild C24 and the rcy1-3mutant plants were inoculated with CMV(Y). At 7 dpi, the coatprotein of virus in them was immunologically detected by thetissue-printing method (A). Necrotic local lesion (NLL) forma-tion in CMV(Y)-inoculated leaves of RCY1(+)T2, RCY1(-)T2,wild C24 and rcy1-3 mutant was observed at 2 dpi (B). TenCMV(Y)-inoculated T2 plants were segregated to CMV(Y)-resistant and susceptible plants (C). CMV(Y) systemically-infected T2 plants were indicated by the arrows
678 Plant Mol Biol (2006) 62:669–682
123
class R protein that confers gene-for-gene resistance
against CMV(Y). In comparison to C24 in which virus
is restricted to the CMV(Y) inoculated leaves,
CMV(Y) systemically spread and multiplied in the
virus non-inoculated leaves of the rcy1 mutant plants,
rcy1-2, rcy1-3, rcy1-4, rcy1-5, rcy1-6 and rcy1-7. The
accumulation of viral coat protein in the systemic tis-
sues of these CMV(Y)-inoculated rcy1 mutants was
comparable to that in the naturally CMV(Y) suscep-
tible ecotype Col. In four of these rcy1 mutants, rcy1-2,
rcy1-3, rcy1-4 and rcy1-6, missense mutations were
identified in regions encoding either the CC, NBS or
LRR domains of RCY1, indicating that these three
domains were indispensable for the activation of de-
fense mechanisms that restrict virus to the primary
infection site in the CMV(Y)-inoculated C24. How-
ever, local and systemic necrosis differentially devel-
oped in the virus-inoculated rcy1 mutants. In
comparison to the wild type C24 plant, in which HR
associated NLL were observed 2 days post inoculation
with CMV(Y), NLL-like lesions were not observed in
the virus inoculated leaves of the rcy1-3, rcy1-5, rcy1-6
and rcy1-7 mutants. In addition, unlike the virus-inoc-
ulated leaves of C24, PR1a expression was not induced
in the virus inoculated leaves of the rcy1-3, rcy1-5,
rcy1-6 and rcy1-7 mutants. In contrast, in the virus-
inoculated leaves of the rcy1-2 and rcy1-4 mutants
NLL-like lesions developed, however, only 4 days after
inoculation with virus. These lesions were accompa-
nied by the accumulation of elevated levels of tran-
script for the PR1a gene suggesting that these late
developing lesions had some hallmarks of a HR-like
response. Unlike the wild type plant and the rcy1-3,
rcy1-5, rcy1-6, rcy1-7 mutants, lesions also appeared on
the virus non-inoculated leaves of the rcy1-2 and rcy1-4
mutants, 7 days after CMV(Y) inoculation. However,
as mentioned above viral coat protein accumulated to
high levels in these lesioned tissues of rcy1-2 and rcy1-
4, comparable to levels found in the systemic leaves of
virus inoculated rcy1-3, rcy1-5, rcy1-6, rcy1-7 and Col
plants. This differential induction of necrosis among
the rcy1 mutant lines, all of which were equally sus-
ceptible to CMV(Y), suggests that some amino acid
residues in the RCY1 protein may be independently
required for regulating defenses that contribute to
restriction of virus spread and mechanisms leading to
the induction of HR-associated responses to CMV(Y).
Similarly, in tobacco carrying the N gene, which
confers gene-for-gene resistance to Tobacco mosaic
virus (TMV), it has been demonstrated that single
amino acid substitutions in the TIR, NBS and LRR
domains of N result in the delayed appearance of HR
compared with the wild type plant. Furthermore, these
late appearing lesions failed to restrict virus spread to
the primary inoculation site (Dinesh-Kumar et al.
2000). However, unlike our results with the rcy1
mutant plants, in which we did not find a strong cor-
relation between the timing and strength of HR
development in the virus-inoculated leaf and spread of
the virus to the non-inoculated leaves, the timing and
extent of HR development in the TMV-inoculated
leaves of these mutant N-bearing plants, correlated
with the extent of systemic spread of virus; systemic
spread was higher in mutants in which HR
Table 3 Symptom development in CMV(Y)-inoculated leaves of F1 progenies crossed between rcy1 mutants
Plants and their genotypesa Total number of inoculated plants Number of plants formed NLLson CMV(Y)-inoculated leavesb
2 days 4 days
wild C24 5 5 5wild Col 5 0 0
rcy1-2(rcy1-2/rcy1-2) 5 0 5rcy1-3(rcy1-3/rcy1-3) 5 0 0rcy1-4(rcy1-4/rcy1-4) 5 0 5rcy1-5(rcy1-5/rcy1-5) 5 0 0
F1(rcy1-2/rcy1-3) 5 0 0F1(rcy1-2/rcy1-4) 5 3 5F1(rcy1-2/rcy1-5) 5 0 0F1(rcy1-3/rcy1-4) 5 0 0F1(rcy1-3/rcy1-5) 5 0 0F1(rcy1-4/rcy1-5) 5 0 0
a Two-week-old Arabidopsis thaliana plants were inoculated with 100 lg/ml of CMV(Y). F1 plants resulted from the crosses betweeneach rcy1 mutants: rcy1-2, rcy1-3, rcy1-4, rcy1-5, rcy1-6 and rcy1-7, their parent mutants and wild ecotypes C24 and Col were divided bydashed linesb Number of plants developing NLLs in their CMV(Y)-inoculated leaves were counted at 2 and 4 dpi
Plant Mol Biol (2006) 62:669–682 679
123
development was slower. Hence, although restriction
of virus spread tightly correlated with the induction of
HR in TMV-infected tobacco, this does not appear to
be the case for CMV(Y)-infected C24.
Amino acid substitutions in the CC, NBS and LRR
domains of RCY1 protein may disrupt intra-molecular
interactions between these domains of RCY1. Alter-
natively, these mutations could disrupt the interaction
between RCY1 and other proteins. Inter-molecular
interactions involving NBS-LRR type R proteins have
been observed previously. For example, Moffett et al.
(2002) showed that the CC, NBS and LRR domains of
the potato Rx protein, which confers gene-for-gene
resistance to Potato virus X (PVX), physically interact
and negatively regulate defense signaling pathways
that are associated with resistance against PVX. In
contrast, trans interaction with the RIN4 protein has
been proposed to negatively regulate activation of
RPM1 and RPS2-dependent defense pathways in
Arabidopsis (Mackey et al. 2003; Axtell et al. 2003;
Belkhadir et al. 2004). Likewise, associations with the
HSP90 protein have been proposed to stabilize the
Arabidopsis RPM1 and RPS2 proteins, the tobacco N
protein and the potato Rx proteins, thereby allowing
them to interact with activation-competent defense
signal complexes (reviewed by Schulze-Lefert 2004).
The ATP-binding cassette (ABC) regions in the
NBS domains of plant R proteins and the eukaryotic
cell-death effector Apaf-1 are closely related to the
oligomerization module found in the AAA+ family of
ATPases (Inohara et al. 2005). Cryo-electron micros-
copy revealed that oligomerized Apaf-1 forms a seven-
spoke wheel-like structure (Inohara et al. 2005), which
may be important for its function. Very recently, elic-
itor-mediated oligomerization of tobacco N protein for
TMV resistance was demonstrated (Mestre and Baul-
combe 2006). Similarly, the NBS domain of RCY1 may
mediate oligomerization, which could activate signal-
ing functions of other domains in RCY1 resulting in
the activation of downstream mechanisms conferring
resistance to CMV(Y) in C24. Indeed, in comparison
to the rcy1-2 and rcy1-4 single mutants, the timely
activation of RCY1-conferred HR was restored and the
ability to restrict systemic spread of virus was partially
restored in the CMV(Y) inoculated rcy1-2/rcy1-4 hy-
brid plants. The rcy1-2 and rcy1-4 mutants are pre-
dicted to cause single amino acid substitutions in the
CC and LRR domains of RCY1, respectively. Physical
interaction between the Rcy1-2 and Rcy1-4 mutant
proteins could presumably result in trans-complemen-
tation of RCY1 activity resulting in the restoration of
HR and the ability to restrict systemic spread of virus
in the rcy1-2/rcy1-4 hybrid. Further studies on the
Fig. 8 Response to CMV(Y) in F1 progenies resulted from across between the rcy1-2 and rcy1-4 mutants. Symptoms on virus-inoculated leaves of F1 hybrid plants (rcy1-2/rcy1-4) resultingfrom a cross between the rcy1-2 and rcy1-4 mutants, their parentrcy1-2 (rcy1-2/rcy1-2) and rcy1-4 (rcy1-4/rcy1-4) mutants, andwild C24 were observed at 2 dpi (A) and 3 dpi (C) withCMV(Y). Distribution of virus in the inoculated leaves of themwas also analyzed at 2 dpi (B) and 3 dpi (D) by the tissue-printing method. Total protein of CMV(Y)-inoculated wild C24leaf could not be transferred into the filter paper for the tissue-printing at 3 dpi, because the inoculated leaf was completely diedand dried
680 Plant Mol Biol (2006) 62:669–682
123
interaction between RCY1 polypeptides and the
interaction of RCY1 with other proteins will provide
further insights into the functioning of RCY1 protein
leading to activation of HR and restriction of systemic
spread of CMV(Y) in Arabidopsis.
All of the CMV(Y)-susceptible mutants that we
identified contained mutations in RCY1. Although we
did not identify any RCY1 independent loci in our
genetic screen, CMV(Y) resistance most likely in-
volves a combination of signaling mechanisms, each of
which may partly contribute to resistance against this
virus. Our screens may have been too stringent to
identify mutations in these other genes. Indeed, we had
previously shown that SA and ethylene signaling have
a minor role in mediating RCY1-conferred resistance
to CMV(Y); resistance was partially compromised by
mutations affecting SA synthesis and ethylene signal-
ing (Takahashi et al. 2002, 2004b). Alternatively, our
inability to identify RCY1-independent loci could be
due to the lethality conferred by mutations in these
genes. Since SA, ethylene and JA signaling individually
have only a minor role in RCY1-conferred resistance to
CMV(Y), we had previously suggested that other
mechanisms may be involved in this gene-for-gene
resistance against CMV(Y) (Takahashi et al. 2002,
2004b). Improvements in the screening method for
CMV(Y)-susceptible mutants, for example, isolation of
mutants in which the restriction of virus spread is
partially compromised, may allow for isolation of
mutants in these genes involved in signaling mecha-
nisms that mediate RCY1-conferred resistance to
CMV(Y).
Acknowledgements This work was supported in part by aGrant-in-Aid for Scientific Research on Priority Areas (Molec-ular Mechanisms of Plant–Pathogenic Microbe Interaction –Toward Production of Disease Resistant Plants) and for ScientificResearch (B) (16380031) from Ministry of Education, Culture,Sports and Arts, Japan.
References
An G (1986) Development of plant promoter expression vectorsand their use for analysis of differential activity of nopalinesynthase promoter in transformed tobacco cell. Plant Phys-iol 81:86–91
Aravind L, Dixit VM, Koonin EV (1999) The domains of death:evolution of the apoptosis machinery. Trends Biochem. Sci24:47–53
Axtell MJ, McNellis TW, Mudgett MB, Hsu CS, Staskawicz BJ(2001) Mutational analysis of the Arabidopsis RPS2 diseaseresistance gene and the corresponding Pseudomonas syrin-gae avrRpt2 avirulence gene. Mol. Plant-Microbe Interact14:181–188
Axtell MJ, Staskawicz BJ (2003) Initiation of RPS2-specifieddisease resistance in Arabidopsis is coupled to AvrRpt2-directed elimination of RIN4. Cell 112:369–377
Banerjee D, Zhang XC, Bent AF (2001) The leucine-rich repeatdomain can determine effective interaction between RPS2and other host factors in Arabidopsis RPS2-mediated dis-ease resistance. Genetics 158:439–450
Bechtold N (1998) In planta Agrobacterium-mediated trans-formation of adult Arabidopsis thaliana plants by vacuuminfiltration. In: Martinez-Zapater J, Salinas J. (eds)Methods in Molecular Biology. Humana Press, TotowaNJ, pp. 259–266
Belkhadir Y, Subramaniam R, Dangl JL (2004) Plant diseaseresistance protein signaling: NBS-LRR proteins and theirpartners. Curr. Opin. Plant Biol 7:391–399
Bendahmane A, Kanyuka K, Baulcombe DC (1999) The Rxgene from potato controls separate virus resistance and celldeath responses. Plant Cell 11:781–791
Bendahmane A, Farnham G, Moffett P, Baulcombe DC (2002)Constitutive gain-of-function mutants in a nucleotide bind-ing site-leucine rich repeat protein encoded at the Rx locusof potato. Plant J 32:195–204
Brommonschenkel SH, Frary A, Frary A, Tanksley SD(2000) The broad-spectrum tospovirus resistance geneSw-5 of tomato is a homolog of the root-knot nematoderesistance gene Mi. Mol. Plant-Microbe Interact 13:1130–1138
Cooley MB, Pathirana S, Wu H-J, Kachroo P, Klessig DF (2000)Members of the Arabidopsis HRT/RPP8 family of resis-tance genes confer resistance to both viral and oomycetepathogens. Plant Cell 12:663–676
Dangl JL, Jones JDG (2001) Plant pathogens and integrateddefence responses to infection. Nature 411:826–833
Dinesh-Kumar SP, Tham W-H, Baker BJ (2000) Structure-function analysis of the tobacco mosaic virus resistance geneN. Proc. Natl. Acad. Sci. USA 97:14789–14794
Dodds PN, Lawrence GJ, Ellis JG (2001) Six amino acid changesconfined to the leucine-rich repeat b-strand/b-turn motifdetermine the difference between the P and P2 rust resis-tance specificities in flax. Plant Cell 13:163–178
Ellis JG, Lawrence GJ, Luck JE, Dodds PN (1999) Identificationof regions in alleles of the flax rust resistance gene L thatdetermine differences in gene-for-gene specificity. Plant Cell11:495–506
Ellis JG, Dodds PN, Pryor T (2000) Structure, function, andevolution of plant disease resistance genes. Curr. Opin.Plant Biol 3:278–284
Hammond-Kosack KE, Jones JDG (1997) Plant disease resistancegenes. Annu. Rev. Plant Physiol. Plant Mol. Biol 48:575–607
Hammond-Kosack KE, Parker JE (2003) Deciphering plant-pathogen communication: fresh perspectives for molecularresistance breeding. Curr. Opin. Biotech 14:177–193
Hwang C-F, Bhakta AV, Truesdell GM, Pudlo WM, WilliamsonVM (2000) Evidence for a role of the N terminus and leu-cine-rich repeat region of the Mi gene product in regulationof localized cell death. Plant Cell 12:1319–1329
Inohara N, Chamaillard M, McDonald C, Nunez G (2005) NOD-LRR proteins: role in host-microbial interactions andinflammatory disease. Annu. Rev. Biochem 74:355–383
Ishihara T, Sakurai N, Sekine K-T, Hase S, Ikegami M, ShibataD, Takahashi H (2004) Comparative analysis of expressedsequence tags in resistant and susceptible ecotypes of Ara-bidopsis thaliana infected with cucumber mosaic virus. PlantCell Physiol 45:470–480
Plant Mol Biol (2006) 62:669–682 681
123
Lanfermeijer FC, Dijkhuis J, Sturre MJG, de Haan P, Hille J(2003) Cloning and characterization of the durable tomatomosaic virus resistance gene Tm-22 from Lycopersicon es-culentum. Plant Mol. Biol 52:1037–1049
Luck JE, Lawrence GJ, Dodds PN, Shepherd KW, Ellis JG(2000) Regions outside of the leucine-rich repeats of flaxrust resistance proteins play a role in specificity determina-tion. Plant Cell 12:1367–1377
Mackey D, Belkhadir Y, Alonso JM, Ecker JR, Dangl JL (2003)Arabidopsis RIN4 is a target of the type III virulenceeffector AvrRpt2 and modulates RPS2-mediated resistance.Cell 112:379–389
Matthews REF. 2002. Plant Virology, 4th edn, Academic Press,New York.
McDowell JM, Dhandaydham M, Long TA, Aarts MGM, GoffS, Holub EB, Dangl JL (1998) Intragenic recombination anddiversifying selection contribute to the evolution of downymildew resistance at the RPP8 locus of Arabidopsis. PlantCell 10:1861–1874
Mestre P, Baulcombe DC (2006) Elicitor-mediated oligomeri-zation of the tobacco N disease resistance protein. Plant Cell18:491–501
Meyers BC, Kozik A, Griego A, Kuang H, Michelmore RW(2003) Genome-wide analysis of NBS-LRR-encoding genesin Arabidopsis. Plant Cell 15:809–834
Moffett P, Farnham G, Peart J, Baulcombe DC (2002) Interac-tion between domains of a plant NBS-LRR protein in dis-ease resistance-related cell death. EMBO J 21:4511–4519
Murashige T, Skoog F (1962) A revised medium for rapid growthand bioassays with tobacco tissue cultures. Physiol. Plan-trum 15:473–497
Murray MG, Thompson WF (1980) Rapid isolation ofhigh molecular weight plant DNA. Nucl. Acids Res 8:4321–4325
Noel L, Moores TL, van der Biezen EA, Parniske M, DanielsMJ, Parker JE, Jones JDG (1999) Pronounced intraspecifichaplotype divergence at the RPP5 complex disease resis-tance locus of Arabidopsis. Plant Cell 11:2099–2111
Palukaitis P, Garcıa-Arenal F (2003) Cucumoviruses. Adv. VirusRes 62:241–323
Redei GP, Koncz C (1992) Classical Mutagenesis. In: Koncz C,Chua N-H, Shell J (eds) Methods in Arabidopsis Research.World Scientific, Singapore, pp 16–82
Schulze-Lefert P (2004) Plant immunity: the origami of receptoractivation. Curr. Biol 14:22–24
Sekine K-T, Nandi A, Ishihara T, Hase S, Ikegami M, Shah J,Takahashi H (2004) Enhanced resistance to Cucumbermosaic virus in the Arabidopsis thaliana ssi2 mutant ismediated via an SA-independent mechanism. Mol. Plant-Microbe Interact 17:623–632
Takahashi H, Ehara Y (1993) Severe chlorotic spot symptoms incucumber mosaic virus strain Y-infected tobaccos are in-duced by a combination of the virus coat protein gene andtwo host recessive genes. Mol. Plant-Microbe Interact6:182–189
Takahashi H, Goto N, Ehara Y (1994) Hypersensitive responsein cucumber mosaic virus-inoculated Arabidopsis thaliana.Plant J 6:369–377
Takahashi H, Kanayama Y, Zheng MS, Kusano T, Hase S,Ikegami M, Shah J (2004a) Antagonistic interactions be-tween the SA and JA signaling pathways in Arabidopsismodulate expression of defense genes and gene-for-generesistance to Cucumber mosaic virus. Plant Cell Physiol45:803–809
Takahashi H, Miller J, Nozaki Y, Sukamto, Takeda M, Shah J,Hase S, Ikegami M, Ehara Y, Dinesh-Kumar SP (2002)RCY1, an Arabidopsis thaliana RPP8/HRT family resistancegene, conferring resistance to cucumber mosaic virus re-quires salicylic acid, ethylene and a novel signal transductionmechanism. Plant J 32:655–667
Takahashi H, Sekine K-T, Ishihara T, Hase S, Ikegami M, Shah J(2004b) Signal transduction pathways governing resistance toCucumber mosaic virus. In: Tsuyumu S, Leach JE, Shiraishi T,Wolpert T (eds) Genomics and Genetic Analysis of PlantParasitism and Defense. APS Press, Saint Paul pp.185–194
Takahashi H, Suzuki M, Natsuaki K, Shigyo T, Hino K, TeraokaT, Hosokawa D, Ehara Y (2001) Mapping the virus and hostgenes involved in the resistance response in Cucumbermosaic virus-infected Arabidopsis thaliana. Plant CellPhysiol 42:340–347
Tanabe T, Chamaillard M, Ogura Y, Zhu L Qiu S, Masumoto J,Ghosh P, Moran A, Predergast MM, Tromp G, Williams CJ,Inohara N, Nunez G (2004) Regulatory regions and criticalresidues of NOD2 involved in muramyl dipeptide recogni-tion. EMBO J 23:1587–1597
Tao Y, Yuan F, Leister RT, Ausubel FM, Katagiri F (2000)Mutational analysis of the Arabidopsis nucleotide bindingsite-leucine-rich repeat resistance gene RPS2. Plant Cell12:2541–2554
Tameling WIL, Elzinga SDJ, Darmin PS, Vossen JH, TakkenFLW, Haring MA, Cornelissen BJC (2002) The tomato Rgene products I-2 and Mi-1 are functional ATP bindingproteins with ATPase activity. Plant Cell 14:2929–2939
Tomaru K, Hidaka J (1960) Strains of cucumber mosaic virusisolated from tobacco plants. III. A yellow strain. Bull.Hatano Tobacco Exp. Station 46:143–149
Tornero P, Chao RA, Luthin WN, Goff SA, Dangl JL (2002)Large-scale structure-function analysis of the ArabidopsisRPM1 disease resistance protein. Plant Cell 14:435–450
Traut TW (1994) The functions and consensus motifs of ninetypes of peptide segments that form different types ofnucleotide-binding sites. Eur. J. Biochem 222:9–19
Van der Biezen EA, Jones JDG (1998) The NB-ARC domain: anovel signaling motif shared by plant resistance gene prod-ucts and regulators of cell death in animals. Curr. Biol8:R226–227
Warren RF, Henk A, Mowery P, Holub E, Innes RW (1998) Amutation within the leucine-rich repeat domain of theArabidopsis disease resistance gene RPS5 partially sup-presses multiple bacterial and downy mildew resistancegenes. Plant Cell 10:1439–1452
Whitham S, Dinesh-kumar SP, Choi D, Hehl R, Corr C, Baker B(1994) The product of the tobacco mosaic virus resistancegene N: Similarity to toll and the interleukin-1 receptor. Cell78:1101–1115
682 Plant Mol Biol (2006) 62:669–682
123