Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes.
ABSTRACT Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to silence a meristem gene, proliferating cell nuclear antigen and disease resistant N gene in tobacco and RCY1 gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad range of plant species.
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ABSTRACT: Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification.Frontiers in Microbiology 11/2014; 5:595. · 3.94 Impact Factor
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ABSTRACT: The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process. Citation: Kaido M, Abe K, Mine A, Hyodo K, Taniguchi T, et al. (2014) GAPDH-A Recruits a Plant Virus Movement Protein to Cortical Virus Replication Complexes to Facilitate Viral Cell-to-Cell Movement. PLoS Pathog 10(11): e1004505. doi:10.1371/journal.ppat.1004505 Copyright: ß 2014 Kaido et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported in part by a Grant-in-Aid (22580047) for Scientific Research C and a Grant-in-Aid (25450058) for Scientific Research C from the Japan Society for the Promotion of Science (http://www.jsps.go.jp/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.PLoS Pathogens 11/2014; · 8.06 Impact Factor
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ABSTRACT: We investigated the protective effects of a viral vector based on an Apple latent spherical virus (ALSV) harboring a segment of the Bean yellow mosaic virus (BYMV) genome against mosaic diseases in pea, broad bean, and eustoma plants caused by BYMV infection. In pea plants pre-inoculated with the ALSV vaccine and challenge inoculated with BYMV expressing green fluorescence protein, BYMV multiplication occurred in inoculated leaves, but was markedly inhibited in the upper leaves. No mosaic symptoms due to BYMV infection were observed in the challenged plants pre-inoculated with the ALSV vaccine. Simultaneous inoculation with the ALSV vaccine and BYMV also prevented mosaic symptoms in broad bean and eustoma plants, and BYMV accumulation was strongly inhibited in the upper leaves of plants treated with the ALSV vaccine. Pea and eustoma plants were pre-inoculated with BYMV followed by inoculation with the ALSV vaccine to investigate the curative effects of the ALSV vaccine. In both plant species, recovery from mosaic symptoms was observed in upper leaves and BYMV accumulation was inhibited in leaves developing post-ALSV vaccination. These results show that ALSV vaccination not only prevents mosaic diseases in pea, broad bean, and eustoma, but that it is also effective in curing these diseases.Viruses 11/2014; 6(11):4242-57. · 3.28 Impact Factor
Apple latent spherical virus vectors for reliable and effective virus-induced gene
silencing among a broad range of plants including tobacco, tomato,
Arabidopsis thaliana, cucurbits, and legumes
Aki Igarashia, Kousuke Yamagataa, Tomokazu Sugaia, Yukari Takahashia, Emiko Sugawaraa,
Akihiro Tamuraa, Hajime Yaegashib, Noriko Yamagishia, Tsubasa Takahashic, Masamichi Isogaia,
Hideki Takahashid, Nobuyuki Yoshikawaa,b,c,⁎
aPlant Pathology Laboratory, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan
bThe United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8550, Japan
cThe 21st Century Center of Excellence Program, Iwate University, Morioka 020-8550, Japan
dDepartment of Life Science, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
a b s t r a c ta r t i c l ei n f o
Received 17 November 2008
Returned to author for revision
20 December 2008
Accepted 10 January 2009
Available online 25 February 2009
Apple latent spherical virus vector
Virus-induced gene silencing
Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of
endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit
of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout
phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and
economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes
persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to
silence a meristem gene, proliferating cell nuclear antigen and disease resistant N gene in tobacco and RCY1
gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for
long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad
range of plant species.
© 2009 Elsevier Inc. All rights reserved.
Virus infection induces an RNA-mediated defense mechanism that
targets viral RNAs in a nucleotide sequence-specific manner in plants,
commonly referred to as RNA silencing (Waterhouse et al., 2001;
Vance and Vaucheret 2001; Voinnet 2005). When the virus carries
sequences of plant genes, virus infection triggers virus-induced gene
silencing (VIGS) that results in the degradation of endogenous mRNA
homologous to the plant genes through a homology-dependent RNA
degradation mechanism (Lu et al., 2003a,b; Waterhouse and
Helliwell, 2002). VIGS has been shown to have great potential as a
reverse-genetics tool for studies of gene functions in plants, and it has
several advantages compared with other functional genomics
approaches (Burch-Smith et al., 2004). For example, VIGS can knock
down the expression of a gene without the need to generate
transgenic plants, and it can identify a loss-of-function phenotype
for a specific gene within a single generation. In addition, VIGS can
overcome the problem of redundancy by using a target sequence from
the highly conserved region of a gene family. Thus, VIGS offers an easy
way to determine the functions of the genes in a short time, and it can
also be applied to high throughput functional genomics in plants
(Benedito et al. 2004; Burch-Smith et al., 2004; Lu et al., 2003a,b;
Godge et al., 2008).
So far, several virus vectors designed for VIGS in plants have been
developed (Godge et al., 2008), including vectors of Tobacco mosaic
virus (TMV) (Kumagai et al.,1995), Potato virus X (PVX) (Chapman et
al.,1992; Ruiz et al.,1998), Tomato golden mosaic virus (TGMV) (Peele
et al., 2001), and Tomato yellow leaf curl China virus satellite DNA (Tao
and Zhou, 2004). These VIGS vectors have successfully silenced
endogenous genes like phytoene desaturase (PDS) in Nicotiana
benthamiana plants. Tobacco rattle virus (TRV)-derived vectors can
be used for VIGS in Solanum species including tomato, potato, N.
benthamiana, and Arabidopsis thaliana (Brigneti et al., 2004; Burch-
Smith et al., 2006; Cai et al., 2006; Fu et al., 2005; Liu et al., 2002a;
Ratcliff et al., 2001). The modified TRV-vectors are compatible with
the GATEWAY system allowing restriction- and ligation-free cloning
and large-scale functional genomics screening (Liu et al., 2002a).
Cabbage leaf curl virus (CbLCV) for VIGS in A. thaliana (Turnage et al.,
Virology 386 (2009) 407–416
⁎ Corresponding author. Plant Pathology Laboratory, Faculty of Agriculture, Iwate
University, Morioka 020-8550, Japan. Fax: +81 196 23 6150.
E-mail address: firstname.lastname@example.org (N. Yoshikawa).
0042-6822/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/yviro
2002), Turnip yellow mosaic virus (TYMV) in A. thaliana (Pflieger et al.,
2008), satellite TMV (STMV) in N. tabacum (Gossele et al., 2002),
Barley stripe mosaic virus (BSMV) in barley (Holzberg et al., 2002;
Lacomme et al., 2003), Pea early browning virus (PEBV), Bean pod
mottlevirusandCucumbermosaicvirus in legumespecies(Constantin
et al., 2004; Nagamatsu et al., 2007; Zhang and Ghabrial, 2006),
African cassava mosaic virus (ACMV) in cassava (Fofana et al., 2004),
and Brome mosaic virus in monocotyledonous plants (Ding et al.,
2006) were reported as vectors for other hosts.
One of the limitations of VIGS technology is that the most reliable
and effective VIGS vectors have a limited host range and are functional
only in some plant species, especially in the model plant N.
benthamiana (Goodin et al., 2008). Moreover, some of the viruses
used in VIGS induce symptoms that confuse the phenotype caused by
silencing of the target gene. So, the development of reliable VIGS
vectors for additional plant species will be very useful for the
development of plant genomics (Burch-Smith et al., 2004).
Apple latent spherical virus (ALSV), classified into a newly
established genus Cheravirus (Le Gall et al., 2007), has isometric
virus particles c. 25 nm in diameter, and it contains two ssRNA species
(RNA 1 and RNA 2) and three capsid proteins (Vp25, Vp20 and Vp24)
(Li et al.,2000). ALSV is composedof two components, MandB, which
are thought to contain two molecules of RNA 2 and a single molecule
of RNA 1, respectively (Li et al., 2000). RNA 1 (6813nt excluding the 3′
poly (A) tail) has a single open reading frame (ORF) encoding a
polypeptide of 243K, which contains the consensus motifs of the
protease cofactor, the NTP-binding helicase, the cysteine protease and
the RNA polymerase from the N-terminus (Li et al., 2000). RNA 2
(3385ntexcludingthe3′poly(A) tail)alsohasa single ORFencodinga
polypeptide of 119K/108K containing a 53K/42K movement protein
(MP) on the N-terminal side and three capsid proteins in the C-
terminal region (Li et al., 2000). Recently, we constructed infectious
cDNA clones of ALSV-RNAs and modified them into viral vectors for
the expression of foreign genes in plants (Li et al., 2004). ALSV vectors
expressing fluorescence proteins have been used to trace the cell-to-
cell movement of ALSV in infected plant tissues and for analyzing the
distribution of identical virus populations labeled with different
fluorescence proteins in co-infected plants (Yoshikawa et al., 2006;
Takahashi et al., 2007). An ALSV vector expressing green fluorescent
protein(GFP-ALSV)wasalsoused foranalysisof VIGS ofa transgene in
tobacco plants expressing GFP (Yaegashi et al., 2007).
Here, we describe how ALSV vectors effectively induce reliable
VIGS of endogenous genes among a broad range of plants, i.e., Ni-
cotiana species (N. tabacum, N. occidentalis, N. glutinosa, and N.
benthamiana), Solanum lycopersicum, A. thaliana, cucurbit species
(Cucumis sativus, C. melo, Cucurbita pepo, Citrullus lanatus, Luffa
cylindrical, and Lagenaria siceraria), and legume species (Glycine
max, Pisum sativum, Vigna angularis, and V. unguiculata). As ALSV does
not induce any obvious symptoms in most host plants, ALSV vectors
can be used for functional genomics in the host plants. This is also the
first report on virus vectors for VIGS in cucurbit species.
Symptomless infection of ALSV vectors in most host plant species
We previously reported that ALSV infected Chenopodium quinoa,
Tetragonia expansa, C. amaranticolor, and Beta vulgaris, the latter two
symptomlessly (Li et al., 2000). Further examination of the ALSV host
range showed that the virus could systemically infect the following
herbaceous plants: Gomphrena globosa and Celosia cristata in the
family Amaranthaceae; A. thaliana in Brassicaceae; C. sativus, C. melo,
C. pepo, C. lanatus, L. cylindrical, and L. siceraria in Cucurbitaceae; G.
max, P. sativum, and V. angularis, V. unguiculata in Leguminaceae; Li-
naria maroccana in Scrophulariaceae; and N. benthamiana, N. occiden-
talis, N. glutinosa, N. tabacum, S. lycopersicum, and Petunia hybrida in
Solanaceae. Among these plants, soybean, cowpea, and cucurbit plants
initially developed chlorotic spots in a few upper leaves, followed by
the development of symptomless leaves. Other plants showed no
obvious symptoms on both inoculated and upper leaves.
Silencing of endogenous genes in Nicotiana species and tomato with
To test whether ALSV vectors could act as effective inducers for the
silencing of endogenous genes in Nicotiana species, we first targeted
the tobacco sulfur (SU) gene that encodes a magnesium chelatase
subunit required for chlorophyll production. The SU gene fragment
(321 nt) was inserted into an ALSV-RNA2 vector (Fig. 1), and the
resulting virus (tSU-ALSV) was inoculated into the plants (6 to 8 true
leaf stage) of four Nicotiana species (N. benthamiana, N. tabacum, N.
glutinosa, and N. occidentalis). The inoculated plants started todevelop
yellow leaf symptom along the veins on upper uninoculated leaves
from 7 to 10 days post inoculation (dpi) dependingon the species, and
then newly developed leaves, petioles and stems of infected plants
showed a highly uniform yellowing which is a knockout phenotype
typical of Su inhibition, indicating that the SU gene had been silenced
by tSU-ALSV (Fig. 2A, Table 1). The SU silencing persisted for more
than three months in four Nicotiana species, and the recovery of the
silencing phenotype was never observed during the experiments. A
semi-quantitative PCR analysis indicated that the levels of SU mRNA
were reduced in the silenced leaves (Fig. 3A). Mechanical inoculation
of Nicotiana plants with tSU-ALSV resulted in 100% infection in
separate experiments, and all plants developed the same knockout
phenotype. The plants inoculated with a wild-type (wt) ALSV did not
show any viral symptoms nor a change of leaf color (data not shown).
Inoculationof tSU-ALSV intotomato plantsalso resulted in a highly
uniform yellowing phenotype typical of Su inhibition (Fig. 2B), and
Fig.1. Schematic representation of the infectious cDNAclones of ALSV-RNA1 (pEALSR1) and ALSV-RNA2 vector (pEALSR2L5R5) with artificial processing sites by duplicating the Q/G
cleavage site between 42KP and VP25. A target gene can be inserted between 42KP and VP25 using Xho I, Sma I and Bam HI restriction sites. P35S, Cauliflower mosaic virus 35S
promoter; Tnos, nopaline synthase terminator; PRO-co, protease cofactor; HEL, NTP-binding helicase; C-PRO, cysteine protease, POL, RNA polymerase; 42KP, 42K movement protein;
Vp25, Vp20, and Vp24, capsid proteins.
A. Igarashi et al. / Virology 386 (2009) 407–416
the SU silencing phenotype was maintained for more than one month
after the start of silencing.
We next targeted a phytoene desaturase (PDS) gene essential for
the production of carotenoid pigments. All four Nicotiana species
inoculated with tPDS201-ALSV containing a 201 bp fragment of
tobacco PDS gene developed a white photo-bleached phenotype
typical of PDS inhibition from 8 to 14 dpi depending on which Nicoti-
ana species it was. Then, a uniform white photo-bleached phenotype
appeared on newly developed leaves, and the phenotype was
maintained for more than two months after the start of silencing
(Fig. 2C, Table 1). A semi-quantitative PCR analysis indicated that PDS
mRNA was strikingly reduced in the silenced white leaves (Fig. 3A).
To investigate whether ALSV vectors can be used to silence the
genes expressed in meristematic tissues, we tested the proliferating
cell nuclear antigen (PCNA) gene used to evaluate the silencing of a
meristem-expressed gene (Peele et al., 2001; Tao and Zhou, 2004).
When ALSV containing a fragment (291nt) of PCNA gene (PCNA-
ALSV) was inoculated to tobacco, chlorosis, malformation, and severe
dwarfing appeared on newly developed leaves (Fig. 2D), indicating
that the PCNA gene in tobacco plants was effectively silenced.
Fig. 2. Virus-induced gene silencing in plants infected with ALSV vectors. (A) Silencing of SU gene in tobacco cv Xanthi nc (30 dpi), N. benthamiana (14 dpi), N. occidentalis (23 dpi),
and N. glutinosa (24 dpi) infected with tSU-ALSV. (B) Tomato (cv. Oogata fukuju) infected with wtALSV (left) and tSU-ALSV (right) 31 dpi. (C) Silencing of PDS gene in tobacco
infected with tPDS-ALSV 75 dpi. (D) Leaf malformation of tobacco infected with PCNA-ALSV 30 dpi. (E) A. thaliana Col plants infected with wtALSV (left, three plants), atPDS-ALSV
(center), and CH42-ALSV (right) 30 dpi. (F) C. sativus cv. Tsubasa infected with wtALSV (left), cuSU-ALSV (center), and cuPDS-ALSV (right) 27 dpi. (G) C. melo cv. Earis Knight
infected with wtALSV (left), cuSU-ALSV (center), and cuPDS-ALSV (right) 31 dpi. (H) G. max cv. Suzukari infected with soyPDS-ALSV 50 dpi. (I) P. sativum cv. Denkou infected with
soyPDS-ALSV 15 dpi. (J) V. angilaris cv. Benidainagon infected with soyPDS-ALSV 51 dpi.
A. Igarashi et al. / Virology 386 (2009) 407–416
Malformation of leaves and severe dwarfing of the plants were
observed similarly in all four Nicotiana species tested (Table 1).
Silencing of CH42 and PDS genes in A. thaliana with ALSV vectors
In order to determine whether ALSV vectors are functional as
effective silencing inducers of endogenous genes in A. thaliana, the
fragments (201 bp) of a Chlorata42 (CH42) gene encoding aprotopor-
phyrin–IX Mg-chelatase and a PDS gene from A. thalianawere inserted
into ALSV-RNA2 vectors, and the resulting viruses were inoculated
into A. thaliana plants at the 8 true-leaves stage. All plants inoculated
with ALSV containing a CH42 gene fragment (CH42-ALSV) started to
develop yellow phenotype along the veins on uninoculated upper
leaves 10 dpi, and then newly developed leaves, petioles and stems
showed a highly uniform knockout phenotype of SU (Fig. 2E). The
silencing was maintained for more than 1 month. Infection with ALSV
containing A. thaliana PDS gene (AtPDS-ALSV) also resulted in the
white photo-bleached silencing phenotype 10 dpi, and the silencing
was maintained for more than 1 month (Fig. 2E). A semi-quantitative
PCR analysis indicated that the levels of both CH42- and PDS-mRNAs
in the silenced leaves were strikingly reduced (Fig. 3B). On the other
hand, plants infected with wtALSV did not show any obvious
symptoms nor a change of leaf color (Fig. 2E).
Silencing of PDS and SU genes in cucurbit species with ALSV vectors
The PDS and SU gene fragments (300 bp) amplified from mRNAs of
cucumber leaves were inserted into ALSV-RNA2 vectors. The resulting
viruses (cuPDS-ALSV and cuSU-ALSV) were inoculated to cotyledons
of cucumber (C. sativus), melon (C. melo), zucchini (C. pepo),
watermelon (C. lanatus), sponge gourd (L. cylindrical), and bottle
gourd(L. siceraria). Allspecies inoculated withcuPDS-ALSV developed
white leaves that have a highly uniform PDS knockout phenotype,
indicating that the PDS gene had been silenced by cuPDS-ALSV (Figs.
2F, G, Table 1). Similarly, the plants infected with cuSU-ALSV
developed yellow leaves typical of Su inhibition, indicating that the
SU gene had been silenced by cuSU-ALSV (Figs. 2F, G, Table 1). The
growth of silenced plants was severely suppressed due to the loss of
chlorophyll. As ALSV infection was restricted to the inoculated leaves
of pumpkin plants (Cucurbita moschata), no silencing phenotypes
appeared on either inoculated or uninoculated upper leaves of plants
inoculated with cuPDS-ALSV or cuSU-ALSV. A semi-quantitative PCR
analysis indicated that the levels of a SU-mRNA and a PDS-mRNA in
the silenced leaves were strikingly reduced in the silenced leaves of
melon and sponge gourd plants (Figs. 3C, D).
Silencing of PDS gene in legume species with ALSV vectors
The 300-bp fragment of a PDS gene from soybean plants was
inserted into ALSV-RNA2 vectors, and the resulting viruses (soyPDS-
ALSV)were inoculated to primaryleavesof soybean, pea,Adzuki bean,
and cowpea plants. Inoculated soybean, pea and Adzuki bean plants
initiated the development of white spots on the third trifoliate true
leaf 10 to 14 dpi, and then showed highly uniform white photo-
bleached phenotype in the fourth or fifth true leaves of the plants,
indicating that the PDS gene had been silenced (Figs. 2H, I, J). The PDS
silencing on these plants persisted for a month, though the growth of
plants was severely suppressed due to loss of chlorophyll. In cowpea
plants inoculated with soyPDS-ALSV, infected leaves showed yellow
color instead of bleached white phenotype (data not shown).
Silencing of R genes in N. tabacum and A. thaliana with ALSV vectors
The tobacco N gene confers resistance to TMV and encodes a
protein belonging to a Toll-interleukin-1 receptor – nucleotide
binding site – leucine-rich repeat (TIR-NSB-LRR) class (Whitham
et al., 1994). We constructed ALSV vectors containing fragments of N
gene from N. tabacum cv. Xanthi nc and tested whether the vectors
can be used to silence the N gene in N. tabacum. The wtALSV and ALSV
containing fragments of N gene (TIRN-ALSV and TIRF-ALSV) were
mechanically inoculated onto three leaves (the first to third true
leaves of four-leaf stage) of tobacco (cv. Xanthi nc). The SU-ALSV was
also inoculated to tobacco to monitor the progress of VIGS in each leaf
(Fig. 4A-a). After 30 days, the upper leaves (the tenth and eleventh
leaves) of tobacco plants infected with wtALSV (wtALSV-tobacco),
TIRN-ALSV (TIRN-ALSV-tobacco), and TIRF-ALSV (TIRF-ALSV-
tobacco) were then inoculated with purified Tomato mosaic virus
(ToMV) (100 ng/ml). In wtALSV-tobacco plants inoculated with
ToMV, local necrotic lesions appeared on the inoculated leaves at 2 dpi
(Fig. 4A-b). ToMV had localized near local lesions and never moved to
upper uninoculated leaves. On the other hand, in TIRN-ALSV- and
TIRF-ALSV-tobacco inoculated with ToMV, necrotic lesions started to
develop at 3 dpi, and the lesion numbers were about half of that in
wtALSV-tobacco (Table 2 and Fig. 4A-c,d). These plants then
developed necrosis on the stem and petioles of newly developed
upper leaves and stunted severely (Fig. 4A-e,f), indicating that ToMV
moved to upper uninoculated leaves from the inoculated leaves.
Enzyme linked immunosorbent assay of ToMV-inoculated leaves
showed that ToMV was distributed on green tissues among necrotic
lesions in TIRN-ALSV- or TIRF-ALSV-tobacco plants, in contrast to
ToMV which was detected only on tissues containing local lesions in
We next examined whether ALSV vectors can be used for the
silencing of the R gene in A. thaliana. The RCY1 gene found in A.
thaliana ecotype C24 is an R gene to a yellow strain of Cucumber
mosaic virus (CMV-Y) and is characterized by the development of a
necrotic local lesion at the site of infection that restricts viral spread
(Takahashi et al., 1994, 2002). ALSV vectors containing 300-bp
fragments of RCY1 gene (RCYN-ALSV and RCYCEN-ALSV) were
constructed and tested whether the vectors can be used to silence
RCY1 gene in transgenic A. thaliana ecotype Columbia (Col-RCY1)
(Takahashi et al., 2002). RCYN-ALSV and RCYCEN-ALSV were
mechanically inoculated onto two true leaves (three-true leaf stages)
of Col-RCY1 plants. After 14 days, the upper leaves (the fifth and sixth
Plant species in which VIGS of trans- and endogenous genes was effectively induced by
Plant speciesSymptoms by virus infectiona
CH42, PDS, RCY1
SU, PDS, PCNA
SU, PDS, PCNA
GFP, SU, PDS, PCNA
GFP, SU, PDS, PCNA, N
a−, no symptoms. ±, appearance of faint chlorotic spots on a few leaves followed by
development of normal symptomless leaves.
bCH42, Chlorata 42 gene encoding aprotoporphyrin–IX Mg-chelatase; PDS, phytoene
desaturase; RCY1, a resistant gene to an yellow strain of Cucumber mosaic virus in A.
thaliana; SU, sulfur gene that encodes a magnesium chelatase subunit; PCNA,
proliferating cell nuclear antigen; GFP, green fluorescence protein; N, a resistant gene
to Tobacco mosaic virus in N. glutinosa. Silencing of GFP gene was reported by Yaegashi
et al. (2007).
A. Igarashi et al. / Virology 386 (2009) 407–416
true leaves) of Col-RCY1 plants infected with RCYN-ALSV, RCYCEN-
ALSV, or wtALSV were then inoculated with CMV-Y and analyzed
regarding the systemic movement of CMV-Y in inoculated plants. In
most Col-RCY1 plants pre-inoculated with wt ALSV, CMV induced
necrotic local lesion in inoculated leaves and did not move to upper
uninoculated leaves (Table 3 and Fig. 4B). In contrast, CMV-Y
systemically infected Col-RCY1 plants and the infected plants showed
severe stunting (Table 3 and Fig. 4B). Immunoblot and tissue blot
analyses confirmed that CMV-Y was systemically distributed through-
out the plants previously inoculated with RCYN-ALSV or RCYCEN-
ALSV (Fig. 4C). Thus, ALSV vectors containing fragments of RCY1 gene
successfully silenced RCY1 gene, and the plants showed a loss of
resistance to CMV-Y.
Influence of insert length on efficiency and stability of VIGS
To investigate the influence of insert length on the efficiency and
the stability of VIGS, ALSV vectors carrying different lengths of a PDS
gene (Fig. 5A) were inoculated to the second to fourth true leaves of
tobacco plants (5-true leaf stage) and were investigated regarding
their efficiencies and the stability of VIGS. The plants infected with
PDS108′-, PDS102-, PDS201-, PDS300-, or PDS408-ALSV vectors
showed a white photo-bleached phenotype typical of PDS inhibition
on the sixth to ninth true leaves. Interestingly, tobacco plants
inoculated with PDS108-ALSV always developed yellow green leaves
in contrast to white color leaves induced by other vectors (Fig. 5B).
Tobacco plants inoculated with PDS156-ALSV also developed cream
color leaves (Fig. 5B). Measurements of chlorophyll contents clearly
showed the difference among white, yellow green, and cream color
phenotypes (Fig. 5C). These results indicated that the length and/or
positions of inserted sequences affected the efficiencies of VIGS.
Another striking difference among vectors carrying different lengths
of a PDS gene was found on the stability of VIGS. Plants inoculated
with PDS408-ALSV and PDS300-ALSV initially developed white
colored leaves followed by the development of leaves showing mosaic
phenotypes consisting of white and green islands after 30 to 40 dpi.
RT-PCR analysis indicated that ALSV vectors in green islands had lost
PDS sequences, in contrast to viruses in the white area which
maintained their sequences. Plants infected with PDS108-ALSV,
PDS102-ALSV, PDS108′-ALSV, PDS156-ALSV, and PDS201-ALSV
showed highly uniform knockout phenotypes of PDS for more than
two months (Fig. 2), indicating that the ALSV vector carrying ~200-bp
PDS sequences is suitable for long term silencing.
We previously reported that the infection of ALSV expressing GFP
(GFP-ALSV) induced VIGS of a transgene (GFP gene) in transgenic
tobacco plants, and the VIGS persisted for more than three months
(Yaegashi et al., 2007). Transgenes are known to often be more
susceptible to VIGS than endogenous plant genes are. In the present
study, we investigated whether ALSV vectors could act as effective
inducers for the silencing of endogenous genes in plants. Our results
showed that ALSV vectors carrying plant endogenous gene sequences
effectively induced VIGS of the genes in plants including A. thaliana,
four Nicotiana species (N. tabacum, N. occidentalis, N. glutinosa, N.
benthamiana), tomato, four legume species (G. max, P. sativum, V.
angularis, and V.unguiculata), and six cucurbit species (C. sativus, C.
melo, C. pepo, C. lanatus, L. cylindrical, and L. siceraria) (Table 1). To the
bestof ourknowledge, this is the first reportonvirus vectorsuseful for
VIGS in cucurbit species (Godge et al., 2008).
several advantages for VIGV inplants. Firstly, ALSV is a latent virus that
infectsmosthost plantswithoutshowinganysymptoms.Thisis oneof
the requirements for reliable and effective plant virus vectors used for
VIGS to assess gene functions (Burch-Smith et al. 2004; Godge et al.,
2008). Second, ALSVinducesa highly uniformknockout phenotype on
the entire area of most leaves in infected plants as shown in Fig. 2. The
Fig. 3. RT-PCR analysis of SU, PDS or CH42 mRNA levels in silenced (ALSV vectors-infected) and non-silenced (wtALSV-infected) leaves of (A) tobacco, (B) A. thaliana, (C) C. melo, and
(D) L. cylindrical cv. Oonagahechima. Ubiquitin gene in tobacco and actin genes in A. thaliana, C. melo, and L. cylindrical were used as internal controls. Lane NC represents the controls
in which the reverse transcriptase-free RT reaction mix was used as a template in the reaction (30 cycles).
A. Igarashi et al. / Virology 386 (2009) 407–416
ALSV vectors also have high stability and the VIGS persisted
throughout plant growth in infected plants, though the stability of
VIGS depends on the kind and the length of inserted genes. Thirdly, an
ALSV vector can also be used to successfully silence a meristem gene
(PCNA) in Nicotiana species, similar to a TRV vector that is able to
penetrate growing points (Ratcliff et al., 2001). It is reported that PVX
may not be effective in assessing the function of genes involved in
shoot, leaf, flower, and fruit development (Burch-Smith et al., 2004).
Fourthly, ALSV vectors can facilitate efficient VIGS among a broad
range of plants including model plants, A. thaliana and Nicotiana
species including tobacco, and economically important crops such as
tomato, cucurbits, and legumes. Most plant virus vectors for VIGS are
Fig. 4. Silencing of N gene in tobacco (A) and RCY1 gene in A. thaliana (B and C). (A) (a) Tobacco plants infected with Su-ALSV (left) and TIRF-ALSV (right) 30 dpi. (b–d) Appearance
of necrotic lesions on ToMV-inoculated leaves of plants pre-infected with wtALSV (b), TIRN-ALSV (c), and TIRF-ALSV (d). (e) Necrosis on the stem and petiole of upper leaves of
tobacco plants pre-infected with TIRF-ALSV 24 days after ToMV inoculation. (f) Severe stunting of tobacco plants pre-infected with TIRF-ALSV (lower three plants) compared with
normal development of plants pre-infected wtALSV (upper three plants) after 24 days after ToMV inoculation. (B) Symptoms of CMV-Y-inoculated A. thaliana (Col-RCY1) plants pre-
infected with wtALSV (left), RCYN-ALSV (center), and RCYCEN-ALSV (right). (C) Tissue blotanalysisof CMV-Y-inoculated Col-RCY1 plants pre-infected with wtALSV and RCYN-ALSV.
Whole CMV-Y-inoculated plants pre-infected with wtALSV or RCYN-ALSV were placed and crushed between two layers of filter paper, and one paper was probed with an antiserum
against ALSV (anti-ALSV) and the other with an antiserum against CMV (anti-CMV).
Number of local lesions induced by ToMV in leaves of tobacco (cv. Xanthi nc) pre-
inoculated with wtALSV, TIRN-ALSV, and TIRF-ALSV
Virus vectorsNumber of inoculated leaves Number of local lesionsa±S.D.
aAveragenumberoflocallesions±standarddeviationper 1cm2of inoculatedleaves.
Systemic infection of CMV-Y in A. thaliana Col-RCY1 pre-inoculated with ALSV vectors
containing RCY1 gene
Virus vectors Number of plants systemically infected
with CMV-Y/inoculated plants
aTwo plants without showing any symptoms were found to be systemically infected
with CMV-Y by immunoblot analysis, though the signals were so weak compared with
those of plants pre-inoculated with RCYN-ALSV and RCYCEN-ALSV.
A. Igarashi et al. / Virology 386 (2009) 407–416
functionalonly in someplantspecies,especiallyin N. benthamiana. For
proven recalcitrant to the use of most VIGS vectors besides a satellite
virus-induced silencing system (SVISS) by the satellite tobacco mosaic
virus (Burch-Smith et al., 2004; Godge et al., 2008; Gossele et al.,
ALSV vectors effectively induced VIGS of several genes (GFP, SU, PDS,
and PCNA) in tobacco and other Nicotiana species. In addition, we
could successfully silence the N gene in tobacco cv. Xanthi nc, and
ToMV moved into upper uninoculated leaves in silenced tobacco
plants. On the tobacco N gene, it was shown that Rar1, EDS1 and NPR1/
NIM1 like genes are required for N-mediated resistance in transgenic
N. benthamiana containing the tobacco N gene using TRV-vectors
(Liu et al., 2002b). We believe that ALSV vectors can be used for
functional analysis of several genes associated with the N-mediated
signaling pathway in original tobacco plants.
It is worth noting that tobacco plants infected with PDS108-ALSV
developed yellow green leaves that are clearly distinguished from
white leaves induced by PDS102-ALSV and PDS108′-ALSV (Fig. 5B).
This indicates that a nucleotide sequence and/or a position on a target
gene inserted into an ALSV-RNA2 vector affects the phenotype
induced by VIGS, even if the length of the inserted sequence was
equivalent. This is inconsistent with the results showing that in BSMV
vectors caring fragments of PDS ranging from 128 to 584 nucleotides,
the insert length has been reported to influence stability but not
efficiency of VIGS (Bruun-Rasmussen et al., 2007). When ALSV vectors
were used for VIGS of a specific gene, the length and position of a
target sequence should be carefully selected.
Fig. 5. (A) Schematic representation of the positions and length of PDS genes inserted into ALSV vectors. The number above the shaded box is a nucleotide number of the tobacco PDS
gene (Genebank accession no. AJ571699). (B) Photo-bleached phenotypes of PDS inhibition on tobacco cv Xanthi nc infected with tPDS-ALSV vectors containing different lengths of
PDS genes shown in (A). (C) Relative chlorophyll contents (SPAD values) in non-silenced leaves infected with wtALSV (wt) and in silenced leaves infected with tPDS-ALSV vectors
containing different lengths of PDS genes shown in (A).
A. Igarashi et al. / Virology 386 (2009) 407–416
One of the disadvantages of an ALSV vector comes from its gene
expression strategy of virus genome. As the proteins encoded by ALSV
genome were expressed by polyprotein synthesis followed by
proteolytic processing, it is necessary to ligate target sequences in
frame to the cloning sites of the ALSV vector. This makes it difficult to
apply an ALSV vector for high throughput functional genomics as
reported byother vectors(Burch-Smith et al.,2004; Lu et al.,2003a,b).
There have also been reports on effective and convenient methods for
inoculation of the VIGS vectors into plants (Godge et al., 2008). These
are based on methods using Agrobatererium tumefaciens, e.g.,
agroinfiltration and “Agrodrench” (Fu et al., 2005; Ryu et al., 2004).
At present, it is necessary to prepare ALSV-infected leaves as inocula
for inducing VIGS in plants, because direct inoculation of infectious
ALSV-cDNA clones was less efficient. Studies on convenient methods
for delivery of ALSV vectors into plants are under way.
Nevertheless, we think that ALSV vectors are a valuable tool to
determine the functions of interested genes in plants because of their
effective VIGS inducers among a broad range of plant species. In
particular, ALSVvectors areuseful for functionalgenomicsof cucurbits
and legumes in which systems to generate transgenic plants are not
Materials and methods
Construction of ALSV vectors
The sequences of tobacco SU, PDS, and PCNA genes were amplified
(5′-gagggattagaatcccagtt-3′ corresponding to nt positions 31–50) and
SU351(−) (5′-ctggatctgaattgaacgga-3′ complementary to nt positions
332–351) for SU gene (Genebank accession no. AJ571699), tPDS2(+)
(5′-gcactcaactttataaaccc-3′ corresponding to nt positions 2–21) and
tPDS409(−) (5′-cttcagttttctgtcaaacc-3′ complementary to nt posi-
tions 390–409) for PDS gene (AJ616724), and PCNA61(+) (5′-
cctaaccctaatttccccag-3′ corresponding to nt positions 61–80) and
PCNA480(−) (5′-tcactgtcaatgtccatccag-3′ complementary to nt posi-
tions 461–480) (AF305075), respectively. The amplified DNAs were
cloned into pGEMR-T Easy vectors, and all cloned sequences were
confirmed by automated dye-terminator sequencing using an ABI 310
sequencer. To construct a plasmid for tSU-ALSV, a DNA fragment was
amplified by using a cloned SU-DNA as a template and a primer pair
tSU33Xho(+) and tSU350Bam(−) (Table 4). The DNA product was
double-digested with Xho I and Bam HI and ligated to
pEALSR2L5R5GFP restricted with the same enzymes (Fig.1) (Yaegashi
et al., 2007).
To construct ALSV vectors containing different sizes of PDS gene,
DNA fragments were amplified using primer pairs, tPDS2Xho(+) and
tPDS109Bam(−), tPDS157Bam(−), tPDS202Bam(−), tPDS301Bam
(−), or tPDS409Bam(−) (Table 4). The DNA products were inserted
to a RNA2 vector as described above, and the resulting viruses were
designated tPDS108-ALSV, tPDS156-ALSV, tPDS201-ALSV, tPDS300-
ALSV, and tPDS408-ALSV, respectively (Fig. 5). PDS102-ALSV and
PDS108′-ALSV were also constructed using primer pairs, tPDS110Xho
An ALSV vector carrying tobacco PCNA gene (PCNA-ALSV) was
constructed using a primer pair tPCNA113Xho(+) and tPCNA403Bam
(−) as described above.
The sequences of cucumber SU and PDS genes were amplified from
mRNA samples from cucumber leaves using primer pairs, cuSU1(+)
(5′-ACAGCATTGGAAGAGAATTGG-3′ corresponding to nt positions
1–21) and cuSU507(−) (5′-CCCACGCCCAGTATCTTTAA-3′ comple-
mentary to nt positions 488–507) for a SU gene (DQ641092), and
cuPDS31(+) (TTTGGGGCTTATCCCAATGT corresponding to nt posi-
tions 31–50) and cuPDS900(−) (TTCCAACATGGACTGGTTTG comple-
mentary to nt positions 881–900) for a PDS gene (EF159942),
respectively. The amplified DNAs were cloned into pGEMR-T Easy
vectors for sequencing. To construct plasmids for cuSU-ALSV and
cuPDS-ALSV, DNA fragments were amplified using cloned DNAs as
templates and primer pairs cuSU1Xho(+) and cuPDS330Bam(−)
(Table 4) for SU gene and cuPDS31Xho(+) and cuSU300Bam(−)
(Table 4) for PDS gene, respectively. The DNA product was double-
digested with Xho I and Bam HI and ligated to pEALSR2L5R5GFP
restricted with the same enzymes.
The sequence of soybean PDS gene was amplified from soybean
leaves using a primer pair soyPDS1(+) (5′-GAATTCCTTCTACG-
TACTGC-3′ corresponding to nt positions 1–20) and soyPDS1020(−)
(5′-ACCTCATCAGCTACCCGTTC-3′) designed from soybean PDS gene
(AF305075). The amplified DNAs were cloned and sequenced as
described above. To construct an ALSV vector containing soybean PDS
(soyPDS-ALSV), a DNA fragment was then amplified by using cloned
soyPDS-DNA as a template and a primer pair, soyPDS494Xho(+) and
soyPDS793Bam(−) (Table 4). The DNA product was double-digested
with Xho I and Bam HI and ligated to pEALSR2L5R5GFP restricted with
the same enzymes.
The partial sequence of tobacco N gene was amplified from mRNA
samples from N. glutinosa leaves using a primer pair, Ngene-TIR(+)
(5′-ATGGCATCTTCTTCTTCTTC-3′ corresponding to nt positions 52–71)
nt positions 411–530) designed from N gene (U15605). The amplified
vectors containing N gene (TIRN-ALSV and TIRF-ALSV), DNA fragments
were amplified using cloned DNAs as templates and primer pairs
NTIR52Xho(+) and NTIR252Bam(−) (Table 4) for TIRN-ALSV, and
NTIR52Xho(+) and NTIR528Sma(−) (Table 4) for TIRF-ALSV. The DNA
products were ligated to an ALSV-RNA2 vector as described above.
ALSV vectors containing RCY1 gene (RCYN-ALSV and RCYCEN-
ALSV) were constructed as follows: RCY1-DNA fragments were
amplified using RCY1 full-length cDNA clone #1 as a template and
primer pairs RCY1-1Xho(+) and RCY1-300Bam(−) (Table 4) for
RCYN-ALSV and RCY1-1300Xho(+) and RCY1-1599Bam(−) (Table 4)
for RCYCEN-ALSV. The DNA products were ligated to an ALSV-RNA2
vector as described above.
List of primers used to construct ALSV vectors for VIGS
+: sense primer, −; antisense primer.
Restriction sites are underlined.
A. Igarashi et al. / Virology 386 (2009) 407–416
The constructed vectors were purified from large-scale cultures of
Escherichia coli JM109 using a QIAGEN plasmid Maxi kit (QIAGEN,
Japan) and then mechanically inoculated to C. quinoa plants (Li et al.,
2004). After two to three weeks, leaves with symptoms were
homogenized in 3 volumes of extraction buffer (0.1 M Tris–HCl,
pH7.8, 0.1 M NaCl, 5 mM MgCl2) and reinoculated to C. quinoa plants.
Plant materials and growing conditions
The following plants were used for VIGS of endogenous genes by
ALSV vectors: A. thaliana ecotype Columbia (Col), N. tabacum cv.
Xanthi nc, N. occidentalis, N. glutinosa, N. benthamiana, S. lycopersicon
cvs. Kouju and Oogata fukuju, G. max cv. Suzukari, P. sativum cv.
Denkou, V. angularis cv. Benidainagon, V. unguiculata cv. Akadanesan-
jakuoonaga, C. sativus cv.Tubasa, C. melo cv. Earis Knight, C. pepo cv
Diner, C. lanatus cv. Zuisyo, L. cylindrical cv. Oonagahechima, and
L. siceraria cv. Oonagayuugao.
N.tabacum, N. glutinosa, and N. benthamiana plants inoculated with
ALSV vectors were grown in a growth chamber under conditions at
25 °C and daylength of 16 h. Inoculated A. thaliana and N. occidentalis
plants were grown in a chamber under conditions at 22 °C and
daylength of 8 h. Other plants were grown in a greenhouse at natural
conditions at a minimum temperature of 20 °C with supplementary
light (daylength of 16 h) in the winter season.
RNA extraction and semi-quantitative RT-PCR
Silenced and non-silenced leaves (0.1 g) were homogenized with
500 μl of 0.1 M Tris–HCl,10 mM EDTA, pH8.0, 0.1 M LiCl,1% SDS, 500 μl
of a phenol/chloroform (1:1). After centrifugation, the supernatants
were re-extracted with chloroform and then mixed with an equal
volume of 4 M LiCl. The pellets were collected by centrifugation,
washed with 70% ethanol, and dissolved in 50 μl of TE buffer (0.1 M
Tris,10 mM EDTA, pH8.0). The nucleic acid samples were then treated
with RNase free DNase I (TAKARA), and the resulting RNAs were
finally dissolved in distilled water at a concentration of 1 μg/μl.
First strand cDNA was synthesized using 2 μg of RNA, oligo(dT)
primer, and Rever Tra Ace reverse transcriptase (TOYOBO). Semi-
PCR amplifications were performed for 15, 18, 21, 27, and 30 cycles.
Tobacco ubiquitin gene (Genebank accession no. NTU66267) in Nicoti-
ana species, A. thaliana actin gene (U39449) in A. thaliana, cucumber
actingene(AB010922) incucurbitspecies, andpeaactingene(X67666)
in legume species were used as internal controls.
Measurements of chlorophyll contents in PDS-silenced leaves
Relative chlorophyll contents in silenced leaves infected with ALSV
vectors containing PDS genes were determined using SPAD-502
(MINOLTA Co. Ltd). Measurements were made at fifteen spots (5
spots per leaf) on three leaves per plant.
VIGS of R genes in tobacco and A. thaliana
The N gene tobacco (N. tabacum cv. Xanthi nc) and transgenic A.
thaliana Col expressing a RCY1 gene (Col-RCY1) (Takahashi et al.,
2002) were also used for VIGS of R genes against ToMV and CMV-Y,
TIRN-ALSV, TIRF-ALSV, or wt ALSV was inoculated to the first to
third true leaves of tobacco plants (four-leaf stage). After 30 days,
upper leaves (the ninth to eleventh true leaves) of infected tobacco
plants were inoculated with ToMV (100 μg ml−1), and the plants were
grown in a growth chamber for 1 month.
In the experiment of VIGS in Col-RCY1, inocula of ALSV vectors
(RCYN-ALSV, RCYCEN-ALSV, and wtALSV) were prepared as follows:
Infected C. quinoa leaves (10 g) were homogenized with 30 ml of an
extraction buffer, clarified by bentonite, precipitated by polyethy-
lene glycol, and finally dissolved in 1 ml of a extraction buffer (Li et
al., 2000). ALSV vectors were inoculated to the first and second true
leaf of Col-RCY1 (three-leaf stage). After 14 days, upper leaves (the
fifth to seventh true leaf) of infected Col-RCY1 (eight-leaf stage)
were inoculated with CMV-Y (100 μg ml−1), and the plants were
grown in a growth chamber in controlled conditions as described
Enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay was conducted as described
previously (Takahashi et al., 2007).
Tissue blot analysis
Direct tissue immunoblotting analysis was conducted as described
We thank Prof. N. Kawai, Iwate University for his helpful advice.
This work was supported in part by Grant-in-Aids for KAKENHI (no.
19380026, 18380196, 20380025) and the 21st Century of Excellence
Program from the Ministry of Education, Culture, Sports, Science and
Technology of Japan.
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