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

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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

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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

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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

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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

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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.

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