Journal of Plant Physiology 168 (2011) 1142–1145

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Journal of Plant Physiology

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Short communication

Identification of an amino acid residue required for differential recognition of a

viral movement protein by the Tomato mosaic virus resistance gene Tm-22
Michie Kobayashi a,1,2 , Ayako Yamamoto-Katou a,1 , Shinpei Katou a,1,3 , Katsuyuki Hirai a,b ,
Tetsuo Meshi a , Yuko Ohashi a , Ichiro Mitsuhara a,∗
a
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan
b
Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The Tm-2 gene of tomato and its allelic gene, Tm-22 , confer resistance to Tomato mosaic virus (ToMV)
Received 12 August 2010 and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class
Received in revised form 8 January 2011 of plant resistance (R) genes. Despite exhibiting only four amino acid differences between the products
Accepted 9 January 2011
of Tm-2 and Tm-22 , Tm-22 confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7.
An Agrobacterium-mediated transient expression system was used to study the mechanism of differen-
Keywords:
tial recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7
Movement protein
by Tm-2 and Tm-22 . Although resistance induced by Tm-2 and Tm-22 is not usually accompanied by
Tm-2
Tm-22
hypersensitive response (HR), Tm-2 and Tm-22 induced HR-like cell death by co-expression with MP of
Tomato mosaic virus a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-22 but not Tm-2 induced cell
death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR
of Tm-22 is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue
in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-22 are involved in HR cell death.
© 2011 Elsevier GmbH. All rights reserved.

Introduction ond is resistance without HR. A well-known example is the potato

Disease resistance regulated by plant resistance (R) genes is
one of the most efficient plant defense responses against pathogen
attack (Dangl and Jones, 2001). R gene products are thought to
directly or indirectly recognize products of the corresponding
pathogen avirulence (Avr) genes. Resistance responses mediated
by R genes can be phenotypically divided into two main types. The
first is resistance accompanying rapid cell death at the initial site of
invasion, known as a hypersensitive response (HR), usually result-
ing in the formation of necrotic lesions. This HR-accompanying
resistance occurs in many plant–pathogen interactions. The sec-
Abbreviations: Avr, avirulence; CC–NB-ARC–LRR, coiled-coil/nucleotide
binding-ARC/leucine-rich repeat; HR, hypersensitive response; MP, movement
protein; PVX, Potato virus X; R gene, resistance gene; ToMV, Tomato mosaic virus.
∗ Corresponding author at: Plant-Microbe Interactions Research Unit, National
Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602,
Japan. Tel.: +81 029 838 7440; fax: +81 029 838 7044.
E-mail address: mituhara@affrc.go.jp (I. Mitsuhara).
1
These authors contributed equally to this work.
2
Current address: National Institute of Floricultural Science, Tsukuba, Ibaraki
305-8519, Japan.
3
Current address: International Young Researchers Empowerment Center, Shin-
shu University, Minamiminowa 8304, Nagano 399-4598, Japan.
Rx gene, which confers extreme resistance to Potato virus X (PVX)
with little or no induction of visible necrotic lesions. Although this
type of resistance is usually not associated with HR, HR or an HR-
like response occurs in certain specific conditions. For example, an
ectopic expression of PVX coat protein, the viral elicitor against Rx,
results in the induction of Rx-dependent cell death (Bendahmane
et al., 1999).
Tm-2 and its allelic gene Tm-22 have been identified from tomato
as resistance genes for Tomato mosaic virus (ToMV) (Lanfermeijer
et al., 2003, 2005). They encode coiled-coil (CC)/nucleotide bind-
ing (NB)-ARC/leucine-rich repeat (LRR) proteins that belong to
the most common type of plant R proteins. Tm-2 and Tm-22 con-
fer extreme resistance with little or no induction of HR to ToMV
in a homozygous state but induce moderate resistance accom-
panying HR-like cell death in a heterozygous state or even in a
homozygous state at high temperatures (Hall, 1980), suggesting
that Tm-2 and Tm-22 are involved in the induction of HR cell
death.
Although only seven nucleotide differences, which lead to four
amino acid exchanges, occur in the open reading frames between
Tm-2 and Tm-22 , these R genes are known to differentially respond
to different strains of ToMV. For example, Tm-2 induces resis-
tance to the mutant strain ToMV-N3 but not to the mutant strain
ToMV-B7, whereas Tm-22 induces resistance to ToMV-B7 but not

0176-1617/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2011.01.002
 M. Kobayashi et al. / Journal of Plant Physiology 168 (2011) 1142–1145
to ToMV-N3 (Meshi et al., 1989; Weber et al., 1993). Comparison
of these two mutants and the wild-type (WT) strain revealed that
there are two amino acid changes in the region of the viral move-
ment proteins (MPs) in each strain; Cys-68 to Phe and Glu-133 to
Lys in ToMV-B7, and Ser-238 to Arg and Lys-244 to Glu in ToMV-
N3 (Meshi et al., 1989; Weber et al., 1993). Because the MPs of
ToMV are thought to function as the avirulence product for Tm-2
and Tm-22 , these results suggested that the amino acid differences
between the products of Tm-2 and Tm-22 are responsible for their
differential recognition of N3-MP and B7-MP. However, no direct
demonstration of the importance of the amino acid differences has
been reported. Here we show that Tyr-767 in LRR is important for
Tm-22 -specific B7-MP recognition.
Materials and methods
Plant materials
Four- to five-week-old Nicotiana benthamiana plants grown in
a growth chamber maintained at 25 ◦ C with 16 h of light (4000 lx)
were used for each analysis.
Vector construction
The MP sequences of ToMV (strains WT and B7; Meshi et al.,
1989) fused to the coding sequence for the single FLAG-tag were
amplified by PCR. The resulting products were inserted into the XbaI
and SacI sites of a modified version (pEl2-MCS) of the enhanced
CaMV 35S promoter-driven binary vector pBE2113 (Ohtsubo et al.,
1999).
The cDNA fragments of the tm-2, Tm-2 and Tm-22 genes were
obtained from the tomato lines GCR26, GCR236, and GCR267,
respectively, by PCR with gene-specific primers (PrRuG084 and
PrRuG86; Lanfermeijer et al., 2003). Each cDNA fragment was
fused to the coding sequence for the triple hemagglutinin
(HA)-tag and inserted into the BamHI and SacI sites of pEl2-
MCS.
A series of amino acid mutants of Tm-22 were generated using
a site-directed mutagenesis kit (Takara).
Agroinfiltration and ToMV inoculation
A mixture (OD600 = 1.0) of Agrobacterium tumefaciens strain
C58C1 cells containing an adequate R gene and an adequate MP
(1:1) suspended with a buffer (10 mM MES–NaOH, pH 5.6, 10 mM
MgCl2 , and 150 ␮M acetosyringone) was infiltrated into fourth
to sixth true leaves of N. benthamiana. For Fig. 2D, leaves were
inoculated with 10 ␮g/mL of ToMV at 24 h after agroinfiltration.
Infectious ToMV RNA was transcribed in vitro with T7 RNA poly-
merase and a linearized ToMV full-length cDNA clone. Virions were
purified from ToMV-inoculated leaves of Nicotiana tabacum L. cv.
Samsun.
Trypanblue staining
Leaves were boiled in a trypanblue solution (10 mL lac-
tic acid, 10 mL glycerol, 10 g phenol, 10 mL distilled water,
40 mL ethanol and 10 mg trypanblue) for 10 min, washed with
destaining solution (2.5 g/mL of chloral hydrate), and pho-
tographed.
Protein extraction and protein gel blot analyses
Leaves were ground using a mortar and pestle and suspended in
three volumes of extraction buffer [50 mM Tris–HCl, pH7.5, 5 mM
EDTA, 0.1% Nonidet P-40, 5 mM dithiothreitol, protease inhibitor
1143
cocktail (Roche)]. After centrifugation at 12,000 × g for 20 min at
4 ◦ C, supernatants were used for each analysis.
For protein gel blot analyses, 15 ␮g of total protein were
separated by SDS-PAGE and transferred to polyvinylidene diflu-
oride membranes (Millipore Corp.). The membrane was probed
with anti-FLAG M2 (Sigma) or anti-HA 12CA5 (Roche) antibod-
ies at a final concentration of 1 ␮g/mL and then with an alkaline
phosphatase-labeled anti-mouse IgG at a dilution of 1:2000. For
detection of ToMV-CP, we used an antiserum against the CP of TMV-
OM, which shares 84% sequence identity with ToMV-CP, generated
in rabbit. The antigen–antibody complexes were visualized with
5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium.
Results
Tm-2 and Tm-22 induce MP-dependent cell death in Nicotiana
benthamiana
Crossing between plants possessing Tm-2 or Tm-22 and trans-
genic plants expressing ToMV MP genes lead to severe growth
arrest and non-viable seeds (Lanfermeijer et al., 2004, 2005; Weber
et al., 2004). Although these results suggested that Tm-2 and Tm-22
can induce cell death through the recognition of viral MPs, there
is no direct experimental evidence confirming such a hypothe-
sis. To examine whether the induction of HR cell death by Tm-2
and Tm-22 occurs through the recognition of viral MPs, we first
generated stable transgenic tobacco plants overexpressing Tm-2 or
Tm-22 . However, these transgenic plants exhibited little cell death
in response to infection with WT ToMV (data not shown), a strain
that causes resistance for Tm-2 and Tm-22 (Fig. 1B). We there-
fore used a transient expression system with Agrobacterium and N.
benthamiana. Infiltration of Agrobacterium carrying either Tm-2 or
Tm-22 into N. benthamiana leaves caused HR-like cell death within
2 d only in the presence of WT-MP (Fig. 1C). In contrast, infiltra-
tion of tm-2, a susceptible allelic gene of Tm-2 (Lanfermeijer et al.,
2003), did not cause cell death. Thus, we used this Agrobacterium-
mediated cell death-inducing system to study the mechanism of
recognition of ToMV MPs by Tm-2 and Tm-22 .
Tm-2 and Tm-22 confer resistance to ToMV-N3 and -B7, respec-
tively (Fig. 1B). To investigate the specificity of recognition of viral
MPs by Tm-2 and Tm-22 , Tm-2 or Tm-22 were co-expressed with the
MP of B7 (B7-MP) or that of N3 (N3-MP), and the induction of cell
death was examined by using trypanblue, which stains dead cells.
Co-expression of B7-MP with Tm-22 , but not Tm-2, induced cell
death (Fig. 1D). Because the incompatible combination of N3-MP
and Tm-2 did not always induce visible necrosis in our experimen-
tal system, although the cause of these variations remains unclear,
we used B7-MP as a cell death inducer. WT-MP induced cell death
with both of these R genes. Protein gel blot analysis confirmed a
substantial accumulation of the products of Tm-2 and Tm-22 and
the viral MPs, excluding the possibility that the lack of cell death is
caused by a lower protein accumulation of either the B7-MP or R
protein (Fig. 1E).
The tyrosine residue at 767 in the LRR domain of Tm-22
determines the specificity of recognition of B7-MP.
Four amino acid differences exist between Tm-2 and Tm-22 ; two
are located in the NB-ARC and other two are located in LRR (Fig. 1A).
To assess whether the four amino acid differences are responsi-
ble for differential recognition of B7-MP, we performed amino acid
exchanges between Tm-2 and Tm-22 . B7-MP-dependent cell death
was induced in the WT Tm-22 and the mutants I257F, M286I, S769T,
I275F/M286I, I257F/S769T, M286I/S769T, and I257F/M286I/S769T
(Fig. 2A and B). No cell death was induced in the mutants Y767N,
I257F/Y767N, M286I/Y767N, Y767N/S769T, I257F/M286I/Y767N,
1144 M. Kobayashi et al. / Journal of Plant Physiology 168 (2011) 1142–1145

I257F/Y767N/S769T, and M286I/Y767N/S769T (Fig. 2A and B). Thus,

all mutants containing the amino acid exchange of Tyr-767 in Tm-
22 to Asn (Y767N) did affect the induction of cell death. All the
WT and the mutants induced cell death in the presence of WT-MP
and no cell death with vector control (supplementary Fig. 1). The
proteins of the mutated genes were accumulated in the infiltrated
leaves, excluding the possibility that the lack of HR is caused by
a lower protein accumulation of either the viral MP or R protein
(Fig. 2C).
The result shown in Fig. 2B suggests the importance of the Tyr
residue at 767 in Tm-22 in induction of B7-MP-dependent cell
death. To confirm whether the amino acid residue is also required
for resistance to ToMV-B7, Tm-22 and Tm-22 (Y767N) were tran-
siently expressed in N. benthamiana leaves, and the leaves were
inoculated with ToMV-WT or -B7 and assayed for resistance by
examining accumulation of the viral coat protein (CP). Consistent
with induction of cell death, accumulation of WT-CP was sup-
pressed in Tm-22 and Tm-22 (Y767N) compared with vector control.
In contrast, accumulation of B7-CP was suppressed in Tm-22 but not
in Tm-22 (Y767N) (Fig. 2D), suggesting that Tyr-767 contributes to
resistance to ToMV-B7.

2
Fig. 1. (A) A schematic representation of the Tm-2 and Tm-2 proteins illustrating
the positions of amino acid differences between these proteins. Numbers repre-
sent the positions of amino acid residues. (B) Phenotypic relationship between
ToMV resistance genes and ToMV strains. R and S indicate resistant and suscep-
tible, respectively. (C) Leaves stained with trypanblue 2 d after agroinfiltration of
tm-2, Tm-2 or Tm-22 with WT-MP or vector control. Circles indicate the infiltrated
zone. (D) Leaves stained with trypanblue 2 d after agroinfiltration of Tm-2 or Tm-22
with WT- or B7-MP. Circles indicate the infiltrated zone. (E) Protein gel blot analysis
of R proteins and MPs 24 h after agroinfiltration using anti-HA (␣-HA) and anti-FLAG
(␣-FLAG) antibodies. Coomassie Brilliant Blue (CBB) staining shows equal loading of
the samples.
Discussion
We have shown that Tm-2 and Tm-22 induce cell death by co-
expression with the MP of WT ToMV. Usually, resistance induced
by Tm-2 and Tm-22 is not accompanied by HR. However, the cur-
rent study indicated that Tm-2 and Tm-22 can induce resistance
responses accompanying cell death. Phenomena similar to the find-
ing in the current study have been found for some virus–host
interactions, such as the potato–PVX interaction (Bendahmane
et al., 1999, 2000). HR cell death does not always reflect the full
potential of the resistance gene (Bendahmane et al., 1999). How-

Fig. 2. (A) A series of amino acid mutants of Tm-22 . + and − indicate that induction and no induction of cell death occurred, respectively. (B) Leaves stained with trypanblue
2 d after agroinfiltration of a mixture of R gene and MP. Circles indicate the infiltrated zone. (C) Protein gel blot analysis of R proteins and MPs 24 h after agroinfiltration using
anti-HA (␣-HA) and anti-FLAG (␣-FLAG) antibodies. CBB staining shows equal loading of the samples. (D) Accumulation of viral CPs 3 d after inoculation with ToMV-WT or
-B7 was detected using anti-TMV antiserum (␣-CP). Protein gel blot analysis of R proteins using anti-HA antibody (␣-HA). CBB staining shows equal loading of the samples.
For (B) to (D), numbers correspond to amino acid mutants in (A).
 M. Kobayashi et al. / Journal of Plant Physiology 168 (2011) 1142–1145
ever, in our experimental system, induction specificity of HR cell
death correlated with resistance specificity to ToMV strains.
Tm-22 but not Tm-2 induced cell death by co-expression with
B7-MP. Several reports have indicated that recognition specificity
between highly similar NB-LRR proteins maps to the LRR domain
(Ellis et al., 2007; Rairdan and Moffett, 2006; Shen et al., 2003; Zhou
et al., 2006). The LRR domain of R proteins is thought to determine
the specific recognition of Avr proteins. Tyr-767 is located in the
12th repeat of 15 LRRs in Tm-22 . A similar result showing that
a single amino acid mutation in the C-terminal region of LRR of
Rx protein affects specific recognition of Avr factor has also been
reported (Farnham and Baulcombe, 2006). The LRR domain of Pi-
ta, a rice NB-LRR protein that confers resistance to blast fungus,
has been shown to bind specifically to its cognate Avr protein in
yeast and in vitro (Jia et al., 2000). It will be interesting to examine
whether Tm-22 interacts directly with B7-MP.
Acknowledgements
We thank the Leaf Tobacco Research Center, Japan Tobacco
Inc., for N. benthamiana seeds and S. Seo for critical reading
of the manuscript. This work was supported by the Program
for the Promotion of Basic Research Activities for Innovative
Biosciences.
1145
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jplph.2011.01.002.
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