- Wiley Online Library

The Plant Journal (2009) 57, 905–917
doi: 10.1111/j.1365-313X.2008.03737.x
RKP, a RING finger E3 ligase induced by BSCTV C4 protein,
affects geminivirus infection by regulation of the plant
cell cycle
Jianbin Lai1,2, Hao Chen1,2, Kunling Teng2, Qingzhen Zhao2,3, Zhonghui Zhang1,2, Yin Li1, Liming Liang1, Ran Xia2,
Yaorong Wu2, Huishan Guo4 and Qi Xie1,2,*
1
State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen (Zhongshan) University, 135 West Xin-Gang Road,
Guangzhou 510275, China,
2
State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental
Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China,
3
School of Life Science, Liaocheng University, Liaocheng 252059, China, and
4
State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Microbiology, Chinese Academy
of Sciences, Datun Road, Beijing 100101, China
Received 2 September 2008; revised 22 October 2008; accepted 28 October 2008; published online 4 December 2008.
*
For correspondence (fax +86 10 64889351; e-mail [email protected]).
Summary
The C4 protein from Curtovirus is known as a major symptom determinant, but the mode of action of the C4
protein remains unclear. To understand the mechanism of involvement of C4 protein in virus–plant
interactions, we introduced the C4 gene from Beet severe curly top virus (BSCTV) into Arabidopsis under a
conditional expression promoter; the resulting overexpression of BSCTV C4 led to abnormal host cell division.
RKP, a RING finger protein, which is a homolog of the human cell cycle regulator KPC1, was discovered to be
induced by BSCTV C4 protein. Mutation of RKP reduced the susceptibility to BSCTV in Arabidopsis and
impaired BSCTV replication in plant cells. Callus formation is impaired in rkp mutants, indicating a role of RKP
in the plant cell cycle. RKP was demonstrated to be a functional ubiquitin E3 ligase and is able to interact with
cell-cycle inhibitor ICK/KRP proteins in vitro. Accumulation of the protein ICK2/KRP2 was found increased in
the rkp mutant. The above results strengthen the possibility that RKP might regulate the degradation of ICK/
KRP proteins. In addition, the protein level of ICK2/KRP2 was decreased upon BSCTV infection. Overexpression
of ICK1/KRP1 in Arabidopsis could reduce the susceptibility to BSCTV. In conclusion, we found that RKP is
induced by BSCTV C4 and may affect BSCTV infection by regulating the host cell cycle.
Keywords: RING finger E3 ligase, cell cycle, geminivirus, RKP, ICK/KRP proteins.
Introduction
Geminiviruses are a group of single-stranded DNA viruses
that infect a large range of plants and cause considerable
agricultural losses. Their small genomes are amplified
through a rolling-circle mechanism in plant cell nuclei
(Gutierrez, 1999; Hanley-Bowdoin et al., 2000; Lazarowitz,
1992). The limited coding capacity of geminiviruses means
that their replication is heavily dependent on host factors.
However, many infected cells are differentiated cells that
have exited the cell cycle and cannot support DNA replication. Therefore, geminiviruses must cause the host cells
to re-enter the cell cycle to create an environment suitable
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd
for replication (Morra and Petty, 2000; Nagar et al., 1995,
2002).
There is evidence to support the proposition that amplification of geminiviruses is coupled with DNA replication of
host cells. For instance, dsDNA replicative forms are much
more abundant in S-phase nuclei of cultured cells (Accotto
et al., 1993). Proliferating cell nuclear antigen (PCNA), an
accessory protein of DNA polymerase d, is induced in
differentiated cells due to the presence of the Rep protein
of Tomato golden mosaic virus (TGMV) (Nagar et al., 1995).
Some viral replication-associated proteins (Begomovirus
905
906 Jianbin Lai et al.
Rep or Mastrevirus RepA) may interfere with the host cell
cycle by interacting with RBR, an activator of G/S-phase
transition in plant cells (Ach et al., 1997; Grafi et al., 1996;
Kong et al., 2000; Liu et al., 1999; Xie et al., 1995). The
physical interaction between Tomato yellow leaf curl Sardinia virus (TYLCSV) REn/Rep and tomato PCNA may also
contribute to the cell-cycle switch (Castillo et al., 2003).
Recently, microarray analysis of the Arabidopsis transcriptome in response to Cabbage leaf curl virus (CaLCuV)
infection has suggested that geminiviruses modulate the
plant cell cycle by differential impacts on the CYCD/RBR/E2F
regulatory network and promotion of progression into the
endocycle (Ascencio-Ibanez et al., 2008).
Infection by many monopartite geminiviruses, such as
Beet curly top virus (BCTV), leads to a vein-swelling phenotype associated with abnormal cell division in host plants
(Stanley et al., 1986). BCTV C4 protein has been proposed as
a major determinant of pathogenesis that contributes to the
symptom of the host in BCTV infection (Stanley and Latham,
1992). In addition, expression of BCTV C4 protein in transgenic Nicotiana benthamiana results in ectopic cell division
(Latham et al., 1997; Piroux et al., 2007). Previous studies
also uncovered differential roles of C4 protein homologs
encoded by some bipartite geminiviruses. For instance, the
AC4 protein of TGMV may be a virus movement factor
(Pooma and Petty, 1996), and the AC4 protein of African
cassava mosaic virus (ACMV) may be a gene silencing
suppressor (Chellappan et al., 2005; Vanitharani et al., 2004).
In eukaryotes, the mechanisms of cell-cycle regulation are
conserved. Cell-cycle progression in plants is controlled by
cyclin-dependent kinase (CDK)/cyclin complexes (De Veylder
et al., 2007; Dewitte and Murray, 2003). The activities of CDK/
cyclin complexes may be negatively regulated by CDK
inhibitors (CKIs) (Sherr and Roberts, 1999; Verkest et al.,
2005b). There are seven proteins in Arabidopsis related to
the mammalian protein CKI p27Kip1, known as Kip-related
proteins (KRPs) or interactors/inhibitors of Cdc2 kinases
(ICKs) (De Veylder et al., 2001; Jakoby et al., 2006). Overexpression of ICK/KRPs in Arabidopsis blocks plant growth
through reduction of cell numbers (De Veylder et al., 2001;
Zhou et al., 2003). In mammalian cells, the degradation of
CKI p27Kip1 is mediated by two pathways, the nuclear SCF
ubiquitin ligase Skp2 pathway, and the cytoplasmic ubiquitin ligase KPC (Kip1 ubiquitination-promoting complex)
pathway that includes KPC1 and KPC2 (Kamura et al., 2004;
Kotoshiba et al., 2005). Recently, a similar redundant control
pathway has been found in plants (Ren et al., 2008). However, the degradation mechanism of ICK/KRPs might be
more complicated, for instance ICK4/KRP6 is degraded by
RING-H2 group F1a (RHF1a) and RHF2a, two RING-type E3
ligases, during Arabidopsis gametogenesis (Liu et al., 2008).
This study focuses on C4 protein encoded by Beet severe
curly top virus (BSCTV) (formerly named the BCTV CFH
strain) (Fauquet et al., 2008; Park et al., 2002, 2004), a species
of Curtovirus. In this report, we describe a geminivirus
BSCTV C4-inducible Arabidopsis protein, similar to human
KPC1, named AtKPC1. During the course of this study, this
gene was reported by Ren et al. (2008) in their study of
turnover the Arabidopsis cell-cycle inhibitor KRP1, and
named RKP (related to KPC1). To avoid confusion, the name
RKP is used throughout this paper. We found that mutation
of RKP in Arabidopsis reduces susceptibility to BSCTV
infection and impairs BSCTV replication in plant cells. Callus
division is impaired in rkp mutants. In addition, RKP acts as a
functional ubiquitin E3 ligase, and ICK/KRPs may be its
targets. The protein level of ICK2/KRP2 was decreased in
BSCTV infection. Overexpression of ICK1/KRP1 in Arabidopsis could reduce susceptibility to BSCTV. Thus, RKP,
which is induced by C4 protein, could affect BSCTV infection
by regulating the plant cell cycle.
Results
Expression of BSCTV C4 in Arabidopsis induces abnormal
cell division
The C4 protein from BCTV, a close species of BSCTV, is
known to be a symptom determinant in BCTV infection, but
the molecular function and the role of C4 in virus–plant
interactions remains unclear. To reveal the molecular function of the C4 protein in plant cells, we cloned the C4 gene
from BSCTV and expressed it in Arabidopsis. To achieve
this, the 264 bp coding sequence (CDS) of the C4 gene,
encoding a small protein containing 87 amino acids (Figure 1a), was introduced using the flower-dip transformation
method into a transgenic vector under the control of the
CaMV 35S promoter to produce transgenic plants constitutively overexpressing C4. However, no transgenic plants
were obtained in several individual transformations.
Instead, we used green callus-like tissues that were found
on the selection plates under light growth conditions
(Figure 1b). When viewed under a microscope, the callus cell
type was similar to that of callus obtained by the normal
tissue culture method. This indicates that overexpression of
BSCTV C4 may induce abnormal cell division/differentiation,
and that normal transgenic lines could not be generated. To
resolve this problem, we cloned the C4 gene into the pER8
vector, in which expression of C4 protein is under the control
of the lexA-VP16-ER (XVE)-inducible promoter, whose
expression is induced by treatment of estrodiol (Zuo et al.,
2000). We obtained transgenic plants in which expression of
C4 was not detected under standard growth conditions but
was detected on medium including the inducer estrodiol
(Figure 1c). Interestingly, growth of pER8-C4 plants was
normal on Murashige and Skoog (MS) plates but was
blocked when C4 was induced. Curled cotyledons and short
roots were observed when the seeds of pER8-C4 plants
were germinated on MS plates including 2 lM estrodiol
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 905–917
RING finger E3 ligase RKP affects geminivirus replication 907
(b)
(c)
(e)
(d)
Figure 1. BSCTV C4 induces abnormal cell division in Arabidopsis.
(a) BSCTV genome; the C4 gene is indicated in red.
(b) Callus-like plants generated by constitutive expression of BSCTV C4.
(c) C4 expression under estrodiol treatment in pER8-C4 plants. RNA from
seedlings treated with 2 lm estrodiol for various times was hybridized with
32
P-labeled C4 probe. 28S rRNA is shown as a loading control.
(d) The growth of pER8-C4 plants was blocked when C4 expression was
induced. Growth of the pER8-C4 plant was normal on MS medium (left) but
was blocked when C4 was induced by 2 lM estrodiol (ES) (right).
(e) Calli generated from pER8-C4 plants after long-term estrodiol induction.
After ES induction for approximately 40 days, the blocked plants would
develop into calli without addition of exogenous hormones.
In the microarray data, the expression of one gene, which
is identical to the putative gene At2g22010 in the Arabidopsis database, attracted our attention as its expression was
approximately eight times greater than that in the control.
BLAST analysis demonstrated that this gene might be the
homolog of KPC1 in humans. Thus we named it AtKPC1. The
amino acid sequence identity between KPC1 and AtKPC1 is
27% overall, but there are two highly conserved domains,
including a SPRY domain, which acts as a protein interaction
region, in the N-terminus, and a RING finger domain in the
C-terminus. During our research, this gene was named RKP
(related to KPC1) by Ren et al. (2008). In their study, the
protein level of the cell-cycle negative regulator KRP1 was
found to increase in the rkp mutant. To avoid confusion, we
have adopted the gene name RKP here also.
To verify the array data, we performed semi-quantitative
RT-PCR to check the expression of RKP. The expression of
RKP was indeed increased in pER8-C4 plants treated with
estrodiol for 16 h, under which conditions the C4 gene was
expressed (Figure 2a). This result was further confirmed by a
transient assay in which the promoter of the RKP was fused
(a)
(b)
–
+
RKP
ACT1
1.0
(Figure 1d). More surprisingly, after several weeks under
estrodiol induction, the blocked seedlings turned into calli
structures similar to those that constitutively express C4
(Figure 1e). These data indicate that C4 could induce
abnormal cell division in transgenic plants.
C4
5.0
GUS activity
pmoles × mg–1 × min–1
(a)
400
300
200
100
0
CK
(c)
CK
0
4
C4
BSCTV
8
12
0
4
8
12
DPI
RKP
RKP expression is induced by C4 protein and BSCTV
infection
To dissect the molecular function of C4 protein in plants, we
examined the effect of the C4 protein on expression of host
genes. We performed genome-wide expression studies
using ATH1 Arabidopsis whole-genome microarray chips.
mRNA was isolated from 2-week-old seedlings of pER8-C4
and pER8-GFP (comprising the GFP gene cloned into the
same vector) treated with estrodiol for 16 h, and subjected to
microarray analysis. Compared with the pER8-GFP control,
expression levels of various genes were increased or
decreased in pER8-C4 plants. Among them, the expression
of a few genes related to cell division was found to be
altered. Several interesting genes were chosen for study,
and this paper focuses on the function of one gene changed
in microarray.
ACT1
1.0
1.2
1.2
1.2
1.0
1.8
2.0
1.4
Figure 2. RKP expression is induced by C4 protein and BSCTV infection.
(a) RT-PCR analysis was used to confirm RKP expression results from the
microarray data. RNA was extracted from pER8-GFP ()) and pER8-C4 (+)
seedlings treated with 2 lM estrodiol for 16 h. ACTIN1 was used as an internal
control. The numbers below the gel indicate the relative expression ratios.
(b) Effects of RKP expression by C4 in Arabidopsis mesophyll protoplasts.
A plasmid carrying the GUS reporter gene under the control of the RKP
promoter was transfected into mesophyll protoplasts together with the
pCambia1300-221 (CK) or pCambia1300-221-C4 (C4) expression vectors. GUS
activity was measured after 16 h. Error bars represent SE (triplicate measurements).
(c) RT-PCR analysis of RKP expression in plants infected with BSCTV. RNA
was extracted from inoculated leaves infected with pCambia1300-BSCTV
carrying 1.8 copies of the BSCTV genome or pCambia1300 (CK) at various
time points. ACTIN1 was used as an internal control. The numbers below the
gel indicate the relative expression ratios.
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 905–917
908 Jianbin Lai et al.
to the GUS reporter gene. This construct was co-transfected
with either pCambia1300221-C4 or the control vector into
Arabidopsis mesophyll protoplasts. Compared to the vector
control, GUS activity in protein extracts from protoplasts
was obviously enhanced when C4 was co-expressed (Figure 2b). Together, these results indicated that RKP expression is induced by C4 protein. To examine whether RKP
expression is also induced during the infection of BSCTV
carrying the C4 gene, RNA extracted from local leaves
(rosette leaves) of Arabidopsis inoculated by BSCTV or the
vector control was used for semi-quantitative RT-PCR analysis. Compared to the vector control, the RNA level of RKP
increased in leaves infected by BSCTV (Figure 2c). Accordingly, BSCTV could induce RKP expression in Arabidopsis,
and RKP may play a role in BSCTV infection.
Mutation of RKP reduces the susceptibility to BSCTV
in Arabidopsis
BSCTV C4 expression, as well as BSCTV infection, induced
expression of the endogenous gene RKP. To show whether
RKP plays a role in BSCTV infection, the reverse genetics
approach was used. Three independent T-DNA insertion
lines, rkp-1 (SAIL_3_E03), rkp-2 (WiscDsLox466C1) and rkp-3
(SALK_121005), which showed loss of function of RKP, were
obtained from the ABRC seed stock center. The T-DNA insertion positions are indicated in Figure 3(a) and homozygous
mutants were verified by PCR using RKP gene-specific and
T-DNA border primers (Figure S1). All alleles were confirmed
by RT-PCR. Full-length RKP was not detected in any of the
three mutants (Figure 3b). Because the RING finger domain is
located at the end of the RKP C-terminus, the ligase activity of
the protein should be affected in all the mutants.
Under standard growth conditions, we did not observe
any apparent morphological phenotype in rkp mutants. To
investigate whether loss of function of RKP affects BSCTV
infection, the rkp mutant and control plants were agroinoculated with BSCTV (Grimsley et al., 1986). Conventional
agro-inoculation of Arabidopsis by BSCTV requires manual
infiltration through wounds on individual plants (Lee et al.,
1994; Park et al., 2002). In our study, the procedure was
improved to be more efficient (see Experimental procedures). The number of plants with symptoms was counted
every day post-inoculation. In independent repeated experiments, all three rkp alleles displayed delayed symptom
appearance and a reduced ratio of symptomatic plants
compared to control plants (Figure 3c). Total DNA from the
whole plants (overground tissues of the inoculated plants) at
various time points post-inoculation was extracted and
subjected to a DNA gel-blot using the whole BSCTV genome
DNA as the probe. As a result, the accumulation of viral DNA
in rkp mutants was approximately 40–50% of that in control
plants 15 days post infection in repeated experiments
(Figure 3d). Together, these results indicate that mutation
of RKP results in reduced susceptibility to BSCTV infection in
Arabidopsis.
BSCTV DNA replication is decreased in rkp mutants
Virus resistance in plants has at least two possible reasons:
impairment of virus DNA replication leading to reduction of
virus accumulation, or restriction of virus movement from
cell to cell to reduce the spread of virus. To discover the
reason for reduced susceptibility in rkp mutants, several
experiments were performed. First, we examined viral DNA
accumulation in the inoculated rosette leaves of symptomatic plants after BSCTV infection. In this analysis, because
leaves that expanded after infection were excluded, BSCTV
DNA accumulation must primarily be a result of viral DNA
replication. DNA gel results indicated that the level of viral
DNA in the inoculated rosette leaves of rkp mutants was
lower (approximately 60% of samples from leaves 16 days
post infection in repeated experiments) than that in control
plants (Figure 3e). This indicates that virus replication is
affected in rkp mutants.
To confirm this result and to exclude an effect of shortdistance movement of the virus in the inoculated rosette
leaves, a transient replication assay was performed in the
Arabidopsis mesophyll protoplasts. In this assay, protoplasts
are free of cell walls and there is no viral movement among
the cells. The mesophyll protoplasts from the rkp mutant and
control plants were transfected by the BSCTV construct. Total
DNA was extracted from the protoplasts at various times after
transfection, and was subjected to DNA gel blots to examine
the newly replicated viral DNA. The data showed that virus
replication was reduced to approximately 40% in protoplasts
of the rkp-1 mutant (Figure 3f). These two results prove that
replication of BSCTV is impaired in the absence of RKP.
Callus formation is impaired in rkp mutants
Several reasons led us to propose that RKP is involved in
plant cell-cycle control. First, replication of viral DNA is
known to be associated with the host plant cell cycle. Second, RKP is the homolog of the cell-cycle regulator KPC1 that
negatively regulates p27Kip1 in humans. Third, in this study,
we have shown that the replication of BSCTV is impaired in
rkp mutants. Thus, we would like to examine whether loss of
function of RKP affects the plant cell cycle. Because no
apparent developmental phenotypes were observed in rkp
mutants, a callus formation assay was used to examine the
cell-cycle process (Kim et al., 2006; Riou-Khamlichi et al.,
1999). Hypocotyls of rkp mutants and wild-type plants were
cut into segments and were cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and
6-benzylaminopurine (6-BA) (Riou-Khamlichi et al., 1999).
Calli were actively induced on both wild-type and mutant
segments. However, callus growth was reduced on the rkp
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 905–917
RING finger E3 ligase RKP affects geminivirus replication 909
(a)
(b)
P3
P1 + P2
P3 + P4
P4
p745
LBb1
ACT1
(d)
W
T
(c)
100
rk
p2
rkp-3
rk
p1
LB1
rkp-2
WT
rkp
-1
rkp
-2
rkp
-3
P2
rkp-1
rk
p3
P1
Symptomatic plants (%)
9 12 15 9 12 15 9 12 15 9 12 15
80
DPI
OC
LIN
WT
60
rkp-1
40
SC
SS
rkp-2
20
rkp-3
0
0.
2
0.
4
1.
0
0.
1
0.
3
0.
4
0.
1
0.
3
0.
4
0.
1
0.
3
0.
5
T
DPI
rkp
0 4 8 12 16 0 4 8 12 16
-1
(f)
W
0 4 8 12 16
rkp
W
T
-1
(e)
DPI
-3
9 10 11 12 13 14 15
8
rkp
7
0 2 4
0 2 4
DPI
OC
LIN
OC
LIN
SC
SC
SS
mutant segments compared to that on the control segment
(Figure 4a). Similar results were obtained when root
segments were used for callus induction (Figure 4b). These
results demonstrate that cell division is impaired in rkp
mutants under hormone treatments.
Interestingly, when the hypocotyls and roots of transgenic
plants carrying an RKP promoter–GUS construct were cultured on callus formation mediums, the GUS activity increased significantly (Figure 4c,d). This suggests that expression
of RKP is induced in dividing cells, and supports the hypothesis that RKP might be involved in plant cell-cycle regulation.
0.4
0.0
0.3
0.0
0.4
1.0
0.3
0.7
0.6
0.6
0.8
1.0
0.0
0.1
0.4
0.6
0.6
0.0
0.1
SS
0.0
0.2
Figure 3. Mutation of RKP reduces susceptibility
to BSCTV in Arabidopsis.
(a) Genomic structure showing the positions of
three T-DNA insertions in RKP. Closed boxes
represent exons, and lines between closed boxes
represent introns. P1/P2 and P3/P4 indicate the
two primer pairs used for RT-PCR. LB1, p745 and
LBb1 indicate primers specific to the T-DNA left
borders of the three mutants.
(b) RT-PCR analysis of the RKP transcripts in
wild-type and T-DNA insertion mutant seedlings.
The primer pairs used are shown in (a). ACTIN1
was used as an internal control.
(c) Agro-inoculation of BSCTV on wild-type and
rkp mutant plants. Plants were infected by
BSCTV carried by Agrobacterium tumefaciens
EHA105 at an absorbance at 600 nm of 0.02. The
values shown are the percentage of plants that
display systemic disease symptoms at various
days after inoculation (DPI). Data are from four
independent experiments (35 plants per line in
each experiment).
(d) Relative levels of BSCTV DNA accumulation in
whole plants. After BSCTV infection, DNA from a
mixture of overground tissues of inoculated wildtype or rkp mutant plants at various time points
was used for DNA gel blotting. Ethidium bromidestained genomic DNA served as the loading
control. OC, open circular double-stranded DNA;
LIN, linear double-stranded DNA; SC, supercoiled
double-stranded DNA; SS, single-stranded DNA.
(e) Relative levels of BSCTV DNA accumulation in
inoculated leaves of symptomic plants. Only the
total DNA from inoculated rosette leaves of the
plants that displayed symptoms was used for
DNA gel blotting.
(f) Relative levels of BSCTV DNA accumulation in
Arabidopsis mesophyll protoplasts. Total DNA
was extracted from protoplasts at various time
points after BSCTV transfection and subjected to
DNA gel blot.
The experiments for which results are shown in
(d)–(f) were repeated twice with similar results
(data not shown). The values below the gels
indicate the relative total viral DNA accumulation
level.
RING domain including conserved Cys and His residues.
Therefore, we assume that RKP has ubiquitin E3 ligase
activity. To confirm our hypothesis, RKP fused with the GST
tag was expressed in Escherichia coli, and was purified using
GST affinity beads from the soluble fraction. In the presence
of wheat (Triticum aestivum) E1 and human E2 (UBCh5b),
ubiquitination activity was observed in the presence of purified GST–RKP (Figure 5a). This assay showed that RKP is a
functional ubiquitin E3 ligase that may regulate degradation
of targeted proteins by the 26S proteasome pathway.
RKP interacts with ICK/KRPs
RKP is a functional E3 ligase
Previous research has shown that RING finger-containing
proteins play roles as ubiquitin E3 ligases (Xie et al., 2002).
The C-terminus of RKP contains a conserved C3HC4-type
Human KPC1 is known to act as a ubiquitin ligase to regulate
the degradation of p27Kip1 at G1 phase, therefore we analyzed the similarity of protein sequences between p27Kip1
and the Arabidopsis ICK/KRP family. Seven ICK/KRP proteins
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 905–917
910 Jianbin Lai et al.
RKP, the protein levels of ICK/KRP proteins may be supposed to be decreased during BSCTV infection. We used
ICK2/KRP2 to prove this hypothesis. 35S-myc-ICK2/KRP2
and 35S-GFP plasmids were co-transfected with either
pCambia-BSCTV or the pCambia vector control into
Arabidopsis mesophyll protoplasts. Total proteins were
extracted from the protoplasts 1-day post-transfection and
applied to protein gel blots. The level of myc-ICK2/KRP2
proteins was decreased during BSCTV infection compared
to the vector control (Figure 5d), whereas the level of
myc-ICK2/KRP2 mRNA was not affected by BSCTV in this
assay. Therefore, the protein level of ICK2/KRP2 is affected
by BSCTV infection.
An interesting question is whether the changes in ICK/
KRP levels in plants affect the susceptibility to BSCTV. It
has been reported that overexpression of ICK/KRP proteins
in plants results in smaller size and serrated leaves by
inhibition of cell-cycle progression (De Veylder et al., 2001;
Zhou et al., 2002). To verify the effect of expression of ICK/
KRP gene on BSCTV infection, the ICK1/KRP1 gene
under the control of the 35S promoter was introduced
into Arabidopsis, and several transgenic lines with
various levels of leaf size reduction were obtained. To
avoid experimental error, a transgenic line with
serrated leaves but a similar leaf size to wild-type control
was used for virus infection (Figure S2a,b). In independent
repeated experiments, the 35S-ICK1/KRP1 plants displayed
delayed symptom appearance and a reduced proportion
of symptomatic plants compared to wild-type plants
(Figure 5e). This result indicates that overexpression of
ICK1/KRP1 in Arabidopsis could reduce susceptibility to
BSCTV.
contained a conserved domain at their C-termini. Interestingly, this domain is similar to the p27Kip1 N-terminal
domain that interacts with human KPC1. This suggests that
RKP may interact with ICK/KRP proteins. An in vitro pulldown assay was performed to test our hypothesis. All
members of the ICK/KRP family except ICK3/KRP5 could be
produced as fusion proteins with GST tags in E. coli, and
were purified using affinity beads. RKP labeled with 35S on
its methionine residues was generated by in vitro transcription and translation using wheatgerm extracts. After
binding and washing, we found that RKP was able to interact
with all six ICK/KRP proteins in vitro but not with the GST
protein itself as a control (Figure 5b).
Because it has been reported recently that degradation of
KRP1 is mediated by RKP (Ren et al., 2008), we examined
whether the degradation of other ICK/KRPs is also regulated
by RKP. ICK2/KRP2, a cell-cycle inhibitor in G1/S phase and a
regulator of leaf development (De Veylder et al., 2001;
Verkest et al., 2005a), was chosen for a detailed study. There
was no obvious difference in the RNA level of myc-ICK2/
KRP2 between the rkp-1 and control plants. However, the
protein level of myc-ICK2/KRP2 was higher in the mutant
than in the wild-type (Figure 5c). This result is similar to that
obtained for ICK1/KRP1 in the rkp mutant (Ren et al., 2008).
Therefore, the degradation of at least two ICK/KRP family
members is regulated by RKP.
Inter-play between BSCTV infection and the ICK/KRP
protein level
Because ICK/KRP proteins may be the targets of RKP in
certain situations and BSCTV can induce the expression of
(a)
(b)
WT
rkp-1
WT
rkp-1
rkp-2
rkp-3
rkp-2
rkp-3
2,4-D + KT
2,4-D + 6BA
(c)
(d)
CK
2,4-D + 6BA
CK
Figure 4. Callus formation is impaired in rkp
mutants.
(a) Hypocotyl callus formation in wild-type and
rkp mutants. The hypocotyls of 2-week-old plants
were cut down and transferred onto medium
containing 100 ng l)1 2,4-D and 100 ng l)1 6-BA.
Photographs were taken 10 days later. Scale
bar = 1 cm.
(b) Root callus formation in wild-type and rkp
mutants. The roots of 8-day-old plants were cut
off and transferred onto medium containing
with 100 ng l)1 2,4-D and 300 ng l)1 kinetin.
Photographs were taken 12 days later. Scale
bar = 1 cm.
(c) RKP promoter–GUS expression pattern during hypocotyl callus formation. Hypocotyls of the
transgenic plants were cut and moved onto MS
medium or MS medium containing 100 ng l)1
2,4-D and 100 ng l)1 6-BA for 4 days and stained
to detect GUS activity. Scale bar = 1 cm.
(d) RKP promoter–GUS expression pattern during root callus formation. Roots of the transgenic
plants were cut and moved onto MS medium or
MS medium containing 100 ng l)1 2,4-D and
300 ng l)1 kinetin for 6 days and stained to detect
GUS activity. Scale bar = 1 cm.
2,4-D + KT
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 905–917
RING finger E3 ligase RKP affects geminivirus replication 911
Discussion
Much work has been performed to analyze the interaction
between geminiviruses and host plants, but the mechanism
remains unclear. Our results suggest that an Arabidopsis
RING-type E3 ligase RKP, which is induced by the C4 protein
of BSCTV, affects geminivirus replication by regulation of
the cell cycle.
BSCTV C4 protein enhances host cell division
It has been suggested that geminiviral DNA replication is
coupled to host cell-cycle transition due to the requirement
for cellular factors (Gutierrez, 2000a). In our study, the calluslike tissues produced by overexpression of C4 in Arabidopsis
suggest that abnormal host cell division is induced by the
BSCTV C4 protein.
Previous research has shown that infection by most
geminiviruses (bipartite Begomoviruses and Mastreviruses)
does not lead to abnormal cell division and an increase
in cell numbers. However, infection by curtoviruses,
such as BCTV, is associated with morphological changes
(for instance, vein swelling and leaf curling) due to
interference with the host cell cycle (Gutierrez, 2000b). It
is therefore interesting to determine why cell division is
induced and whether it is necessary for virus amplification. Mutation analysis of BCTV genes suggests that the
molecular basis for infection symptom is dependent on
the viral protein BCTV C4 (Stanley and Latham, 1992).
Abnormal cell division resulting from expression of
BCTV C4 in Nicotiana benthamiana confirms that this
gene alone can initiate host cell division (Latham et al.,
1997). This evidence supports our hypothesis that cell
division is enhanced in transgenic Arabidopsis expressing
BSCTV C4.
Conversely, C4 protein from Tomato yellow leaf curl virus
(TYLCV) was reported to be involved in virus movement,
because the C4 mutant retained the ability to replicate in
tomato protoplasts and was able to infect Nicotiana benthamiana plants systemically with a lower virus DNA level
(Jupin et al., 1994; Rojas et al., 2001). It is interesting to
determine whether the role of C4 in cell division and virus
movement are compatible.
The molecular mechanism of action of BSCTV C4 is
unclear, but previous studies have provided some insight on
how C4 interferes with the host cell cycle. First, C4 might
interact physically with some cell-cycle regulators in a
manner similar to Rep/RepA proteins, which interact with
RBR, but no cell-cycle factor has been reported. Second, C4
may affect the metabolism or distribution of hormones. For
example, the brassinosteroid signaling pathway may be
regulated by BSCTV C4 (Piroux et al., 2007). Third, C4 may
regulate the expression of host genes directly or indirectly to
control the cell cycle. For instance, the expression of RKP is
upregulated by BSCTV C4. C4 may control transcription or
act as a gene silencing suppressor to regulate the RNA level
of some host genes (Vanitharani et al., 2004).
RKP acts as a cell-cycle regulator in Arabidopsis
Because C4 induces abnormal cell division, we suspect that
expression of several important cell-cycle regulators must
be altered in the presence of C4. After analysis of microarray
data, RKP was chosen as a candidate due to its similarity to
human KPC1, which acts as a regulator of p27kip1 during the
cell cycle (Kamura et al., 2004). Therefore, we speculate that
RKP might be involved in regulation of the plant cell cycle in
Arabidopsis. There are no apparent morphological phenotypes for the rkp mutants, although there is only one copy for
RKP in the Arabidopsis genome. This finding may be interpreted in two ways. First, there may be redundant pathways.
In human cells, the cyclin-dependent kinase inhibitor p27Kip1
is degraded at the G0–G1 transition of the cell cycle by two
ubiquitin/proteasome pathways, independently mediated
by the nuclear ubiquitin ligase SCF (Skp2) and the cytoplasmic ubiquitin ligase KPC complex (Kamura et al., 2004).
Because these two pathways appear to be redundant, the
Skp2 homolog in Arabidopsis may work as a regulator by
itself to retain cell-cycle balance even in the absence of RKP.
The other reason may be that RKP plays a role in regulating
the cell cycle under special conditions. RKP expression is
low under normal conditions, but is induced dramatically by
the BSCTV C4 protein. The callus formation experiment
supports the hypothesis that cell division is impaired in rkp
mutants under hormone treatments. At the same time, the
GUS assay suggests that RKP expression is highly induced
in dividing cells.
Human KPC1 is a functional ubiquitin ligase that regulates the degradation of p27kip1 directly (Kamura et al.,
2004; Kotoshiba et al., 2005). Our data demonstrate that
RKP also has ubiquitin ligase activity in vitro. The ICK/KRP
protein family, known cell-cycle inhibitors, share a conserved domain with p27kip1. Because this domain could
interact with cell cycle-dependent kinase as well as KPC1 in
human cells, ICK/KRPs may be the targets of RKP. Recently,
it has been reported that the degradation of ICK1/KRP1 is
regulated by Skp2 and RKP in Arabidopsis (Ren et al.,
2008). Our data suggest that ICK/KRPs may be the targets of
RKP in vitro and in vivo. In plants, the members of the ICK/
KRP family may play different roles at different cell-cycle
checkpoints and in different tissues (De Veylder et al., 2001;
Menges and Murray, 2002; Verkest et al., 2005b). Therefore,
the fundamental function of RKP in plant cells needs to be
studied in detail. Some other E3 ligases have been reported
to regulate the stability of ICK/KRPs with the manner of
member specificity (Liu et al., 2008), thus the abundance
and specificity of this complexity needs to be further
addressed.
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 905–917
912 Jianbin Lai et al.
RKP may affect BSCTV replication by cell-cycle regulation
When plants are infected by geminiviruses, the cell cycle
may be altered in terminally differentiated cells. For
example, PCNA, an accessory factor for DNA polymerase,
was induced by TGMV in differentiated cells (Nagar et al.,
1995). In our experiment, virus DNA replication was reduced in rkp mutants. Therefore, RKP might be involved in
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 905–917
RING finger E3 ligase RKP affects geminivirus replication 913
a cell-cycle switch affected by BSCTV infection in
differentiated cells. RKP upregulation would trigger the
degradation of ICK/KRPs and enhance the activity of cell
cycle-dependent kinases to accelerate G1/S cell-cycle transitions, which can provide a suitable environment for virus
replication. Interestingly, many components of the cell
cycle, including cyclin-dependent kinase inhibitors, are the
targets of viruses in mammalian cells. For instance, degradation of p27Kip1 is triggered by the Kaposi’s sarcoma
virus cyclin–cdk6 complex (Ellis et al., 1999; Mann et al.,
1999), and recruitment of SCF Skp2 activity to cyclin A
complexes by Epstein–Barr virus protein EBNA3C results in
ubiquitination and SCF Skp2-dependent degradation of
p27 (Knight et al., 2005). In our experiment, the protein
level of ICK2/KRP2 was also found to decrease in BSCTV
infection. These results suggest that cyclin-dependent kinase inhibitors are critical factors in the cell cycle, and are
always the targets of viruses in both plants and animals.
If the cell cycle is blocked at G1 phase, virus infection may
be interrupted. There is no evidence for this hypothesis in
plants, but animal virus research can help us to understand
virus resistance mechanisms in rkp mutants. In animal cells,
cell-cycle interference could affect virus infection. The
replication of herpes simplex virus type 1 is inhibited in
some temperature-sensitive cell-cycle mutant cells (Yanagi
et al., 1978). Regulation of the activity of cyclin-dependent
kinases, a key factor in cell-cycle progression, is an effective
strategy for virus inhibition. For instance, roscovitine, a
cyclin-dependent kinase inhibitor, prevents replication of
varicella-zoster virus (Taylor et al., 2004). Human cytomegalovirus replication is inhibited by the expression of a CDK2
dominant negative mutant (Bresnahan et al., 1997). Modification of cyclin-dependent kinase inhibitor (CKI) p21 expression altered HIV-1 infection by regulation of the cell cycle
(Zhang et al., 2007a). This evidence from mammalian cell
research indicates that cell-cycle arrest inhibits virus replication. In rkp mutants, the C4 protein could not enhance
expression of the RKP gene to enhance degradation of ICK/
KRPs, even after BSCTV infection, so the activity of CDKs
remains inhibited by ICK/KRPs. Delay in the G1/S cell-cycle
transition may disturb the BSCTV replication environment,
and thus could be a new strategy to reduce susceptibility to
viruses in plants (Figure 5f). This model may be valid
because expression of ICK1/KRP1 led to the reduction of
BSCTV infectivity. There may be further cell-cycle genes
related to virus replication and infection. Because the RKP
and ICK/KRP genes are important in regulating G1/S transition, plants that show impaired cell-cycle G1/S transition
may show reduced susceptibility. However, with respect to
virus replication, the influence of cell division and endoreplication may be different and complicated. Thus more
evidence is required to establish a precise model for the
interaction between the cell cycle and virus infection. In rkp
mutants, infection by BSCTV was not abolished but only
impaired, which might be due to the redundant pathways
for ICK/KRP degradation. If the SCF Skp2 pathway is also
destroyed, susceptibility to the virus may decrease more
drastically.
C4 protein could induce cell division but appeared not to
be related to geminivirus replication (Jupin et al., 1994). The
host cell cycle may be affected cooperatively by C4, Rep or
other viral proteins. When C4 is mutated, other proteins may
regulate the host cell cycle to establish a suitable environment for DNA replication. It is an interesting question
whether the expression of RKP is regulated by other
geminvirus proteins. The mechanism of RKP expression
regulation requires further investigation. Thus, further functional dissection of RKP is necessary for a complete understanding of the interaction between the cell cycle and virus
infection in plants.
Experimental procedures
Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia was used for this study.
Seeds were surface-sterilized with 10% bleach for 20 min and rinsed
three times with sterile water. Sterile seeds were suspended in
0.15% agarose and plated on MS medium plus 1.5% sucrose. Plates
Figure 5. E3 ubiquitin ligase activity of RKP, and interaction between RKP and ICK/KRPs.
(a) E3 ubiquitin ligase activity of RKP. The GST–RKP fusion protein was assayed for E3 activity in the presence of E1 (from wheat), E2 (UBCh5b), and 6x His-tagged
ubiquitin (Ub). The numbers on the left indicate the molecular masses of marker proteins in kilodaltons. GST itself was used as a negative control. Nickel–
horseradish peroxidase was used to detect His-tagged ubiquitin.
(b) In vitro pull-down assay between RKP and ICK/KRPs. RKP was translated in vitro and labeled with 35S. ICK/KRPs fused with GST were expressed in E. coli and
purified. After binding, RKP protein was detected by radioactivity (top panel). The bottom panel shows the GST and GST–ICK/KRPs used for the assay. Asterisks
indicate the degraded forms of some GST–ICK/KRP proteins.
(c) Protein level of myc-ICK2/KRP2 in wild-type and rkp-1 protoplasts. 35S-myc-ICK2/KRP2 and 35S-GFP were transiently expressed in protoplasts of wild-type and
rkp-1. Antibodies to myc and GFP were used for protein gel blotting. RNA from the protoplasts was used for RT-PCR to check the mRNA levels of myc-ICK2/KRP2 and
GFP.
(d) Protein level of myc-ICK2/KRP2 in BSCTV infection. The plasmids 35S-myc-ICK2/KRP2 and 35S-GFP were co-transfected with either BSCTV or vector control (CK).
Antibodies to myc and GFP were used for protein gel blotting. RNA from the protoplasts was used for RT-PCR to check the mRNA levels of myc-ICK2/KRP2 and GFP.
(e) Agro-inoculation of BSCTV on wild-type and 35S-ICK1/KRP1 transgenic plants. Plants were infected by BSCTV carried by Agrobacterium tumefaciens EHA105 at
an absorbance at 600 nm of 0.02. The values shown are the percentage of plants that display systemic disease symptoms at various days after inoculation. Data are
from two independent experiments (30 plants per line in each experiment).
(f) Proposed model for the role of RKP in the cell cycle and BSCTV replication.
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 905–917
914 Jianbin Lai et al.
were stratified in darkness for 2–4 days at 4C and then transferred
to a tissue culture room at 22C under a 16 h light/8 h dark photoperiod. After 2–3 weeks, seedlings were potted in soil and placed in
a growth chamber at 22C and 70% relative humidity under a 16 h
light/8 h dark photoperiod.
GGCTTGTCATTTG-3¢/5¢-GACAAGAACCGAATTGCGTG-3¢, and P3/
P4). ACTIN1 expression levels were monitored using forward
and reverse primers (F, 5¢-CATCAGGAAGGACTTGTACGG-3¢; R,
5¢-GATGGACCTGACTCGTCATAC-3¢ to serve as an internal control.
GUS bioassays
Construction of the BSCTV plasmid
An infectious clone of BSCTV (previously named the BCTV CFH strain)
(Stenger, 1994; Stenger et al., 1990), pCFH (ATCC number PVMC-6),
was obtained from the American Type Culture Collection (ATCC,
Manassas, VA, USA). An EcoRI–BamHI fragment (0.8 copy of genome) and an EcoRI–EcoRI fragment (full genome) were introduced
from pCFH to binary vector pCambia1300 to generate pCambiaBSCTV, carrying 1.8 copies of the BSCTV genome as a tandem repeat.
Transformation vectors and construction of transgenic
plants
To produce 35S-C4 plants, a 264 bp fragment containing BSCTV C4
cDNA was cloned into the vector pCambia1300-221 in which transgene expression is under the control of the CaMV 35S promoter.
Inducible C4 plants were generated by cloning C4 cDNA into the
vector pER8, in which transgene expression is under the control of
the XVE-inducible promoter. For the RKP promoter/GUS fusion
construct, a 5¢ flanking sequence (2 kb promoter region just upstream of the ATG start codon of RKP) was amplified from genomic
DNA by PCR and verified by sequencing. The PCR fragment was
cloned into the PstI–XbaI site of binary vector pCambia1300-221 to
obtain a transcriptional fusion of the RKP promoter and the GUS
coding sequence. For the 35S-ICK1/KRP1 construct, the full-length
coding sequence (CDS) of ICK1/KRP1 was cloned into the vector
pCanG, modified from pCambia, in which transgene expression is
under the control of the CaMV 35S promoter. Transformation of
Arabidopsis was performed by the vacuum infiltration method
(Bechtold and Pelletier, 1998), using Agrobacterium tumefaciens
strain EHA105.
Plants carrying the RKP promoter fused with the GUS gene were
treated under various conditions and used for histochemical
detection of GUS expression. Materials were stained at 37C overnight in 1 mg ml)1 5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid
(X-Gluc), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 0.03% Triton X-100 and 0.1 M sodium phosphate buffer, pH
7.0. To test the induction of GUS expression on callus formation
medium, hypocotyls of 2-week-old transgenic seedlings were
transferred onto medium containing 100 ng l)1 2,4-D and 100 ng l)1
6-BA for 4 days; roots of 8-day-old transgenic seedling were transferred onto medium containing 100 ng l)1 2,4-D and 300 ng l)1
kinetin for 6 days. For the protoplast GUS activity assay, the
proteins were extracted and detected using a GUS reporter gene
activity detection kit (MGT-M0877; FLUOstar OPTIMA, BMG
Labtech, http://www.bmglabtech.com/), according to the manufacturer’s instructions.
BSCTV agro-inoculation
Rosette leaves of 4-week-old plants were agro-inoculated with
BSCTV (Briddon et al., 1989; Grimsley et al., 1986). A suspension of
Agrobacterium tumefaciens strain EHA105 (Hood et al., 1993) carrying pCambia1300-BSCTV (carrying 1.8 copies of the BSCTV genome) at a dose of 0.02 (absorbance at 600 nm of 0.02) and mixed
with 1% carborundum (320 grit, C192-500, Fisher Scientific, http://
www.fishersci.com) was sprayed onto the leaves using an airbrush
(SIL.AIR, http://www.silentaire.com) with an air pressure of 75 psi
(1 psi = 6.89 kPa). The inoculated plants were covered for one night
and grown in another greenhouse at a higher temperature (26C).
Symptoms appeared approximately 10 days after inoculation.
Verification of RKP T-DNA insertion mutants
DNA gel blot
Seeds of the T-DNA insertion lines rkp-1 (SAIL_3_E03), rkp-2 (WiscDsLox466C1) and rkp-3 (SALK_121005) were obtained from the
Arabidopsis Biological Resource Center (Ohio State University,
Columbus, OH, USA). Homozygous mutants were identified by PCR
from genomic DNA using T-DNA left border primers (LB1, 5¢-GCC
TTTTCAGAAATGGATAAATAGCCTTGCTTCC-3¢; p745, 5¢-AACGTCC
GCAATGTGTTATTAAGTTGTC-3¢; LBb1, 5¢-GCGTGGACCGCTTGCT
GCAACT-3¢) and RKP-specific primers (P3, 5¢-CAAGTAATCAGT
CTGACCCTG-3¢; P4, 5¢-TCATGTGCTTCTTTTGTGACC-3¢).
For the DNA gel blot, total DNA was extracted using CTAB buffer.
Genomic DNA was stained using ethidium bromide as a loading
control. After depurination and neutralization, total DNA was
transferred to Hybond N+ nylon membranes (Amersham Pharmacia
Biotech, http://www5.amershambiosciences.com/) by upward capillary transfer in 0.4 M NaOH solution. The membranes were
hybridized at 65C using the whole genome of BSCTV, labeled
with [a-32P]dCTP, as a probe. Signal intensity was measured using
IMAGEJ (National Institutes of Health, http://www.rsbweb.nih.gov/ij/).
Semi-quantitative RT-PCR amplification
Protoplast transformation
To examine the expression of RKP by RT-PCR, DNase I-treated total
RNA was denatured and subjected to reverse transcription using
SuperScript II (200 units per reaction; Invitrogen, http://www.
invitrogen.com/) at 37C for 1 h followed by heat inactivation of the
reverse transcriptase at 70C for 15 min. To determine the changes
in RKP expression in C4 transgenic plants and BSCTV infection, PCR
amplification was performed using RKP forward (RT Fw, 5¢-TT
CGTAGTTACACACTTCAAC-3¢) and reverse (RT Rev, 5¢-TCATGTGCT
TCTTTTGTGACC-3¢) primers. To check RKP expression in rkp
mutant plants, two pairs of primers were used (P1/P2, 5¢-TGG CGCT
Mesophyll protoplasts were isolated from rosette leaves of
4-week-old Arabidopsis in the soil and transfected with plasmid
DNA based on a previously described protocol (Yoo et al., 2007),
except that the leaves were sterilized in 70% ethanol for approximately 1 min; the entire operation was performed under sterile
conditions. Transfected cells were kept in the dark at room
temperature. For the BSCTV replication assay, approximately
3 · 105 cells were collected at 0, 2 and 4 days after transfection for
DNA extraction. Total genomic DNA was extracted from the cells
according to a previously described protocol (Fontes et al., 1994).
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2009), 57, 905–917
RING finger E3 ligase RKP affects geminivirus replication 915
Newly replicated viral DNA was identified by DNA gel blot. For the
GUS activity assay, pCambia1300-221–promoterRKP::GUS was
transformed together with pCambia1300 or pCambia1300-C4, and
the protoplasts were collected 16 h later. For detection of the mycICK2/KRP2 protein level in the rkp mutant, 35S-myc-ICK2/KRP2
was prepared by inserting the PCR-amplified coding region of
ICK2/KRP2 fused with MYC in the N-terminus of ICK2/KRP2 to the
pBA002 vector under the control of the 35S promoter. The plasmid was transformed into protoplasts of wild-type and rkp-1
plants; the pGFP2 plasmid, in which the GFP gene was under the
control of the 35S promoter, was transfected alongside for an
internal control. For detection of myc-ICK2/KRP2 protein level
during BSCTV infection, 35S-myc-ICK2/KRP2 and pGFP2 plasmids
were co-transfected with either pCambia-BSCTV or vector control
into wild-type protoplasts.
KRP6
(At3G19150),
ICK5/KRP7
(At1G49620),
ICK6/KRP3
(At5G48820), ICK7/KRP4 (At2G32710) and ACTIN1 (At2G37620).
Acknowledgments
We would like to thank Dr Nam-Hai Chua from the Laboratory of
Plant Molecular Biology, Rockefeller University for kindly providing
us with the pER8 vector, the Arabidopsis Biological Resource Center
at Ohio State University for providing the T-DNA insertion lines, and
Mr Sanyuan Tang for technical assistance. This research was supported by grants CNSF30325030/30530400 from the Chinese Natural
Science Foundation (CNSF). Q.X. is supported by grants KSCX2YW-N-010 and CXTD-S2005-2 from the Chinese Academy of Science. H.G. is supported by CNSF grant 30525004.
E3 ubiquitin ligase activity assay
Supporting Information
The entire RKP ORF was cloned into the PGEX-6P-1 vector and expressed in Escherichia coli. Fusion proteins were prepared according to the manufacturer’s instructions (GE Healthcare, http://www.
gehealthcare.com). The in vitro E3 ligase assays were performed as
described previously (Zhang et al., 2007b).
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Diagnostic PCR of the T-DNA inserted at three positions
in RKP.
Figure S2. The developmental phenotype and molecular character
of the 35S-ICK1/KRP1 transgenic line for BSCTV inoculation.
Please note: Wiley-Blackwell are not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
In vitro pull-down assay
RKP labeled with 35S on its methionine residues was generated by
in vitro transcription and translation using a T7/T3-coupled TnT kit
(Promega, http://www.promega.com/). ORFs of ICK/KRP proteins
were cloned into the PGEX-6P-1 vector and expressed in Escherichia coli. RKP (4 ll) labeled with 35S on its methionine residues
was mixed with 1 lg GST proteins in 1 ml GST-binding buffer
(GBB: 50 mM Tris at pH 8.0, 120 mM NaCl, 1 mM DTT, 0.5% NP-40,
1 mM PMSF [Amesco, http://www.amescodavao.com]), and incubated at room temperature for 60 min. The glutathione–Sepharose beads (GE Healthcare) were then rinsed five times in GBB
containing 0.5 M NaCl and twice with GBB. Bound proteins were
released by boiling in SDS sample buffer at 90C for 4 min and
subjected to SDS–PAGE. After running the gel, protein was fixed
to the gel using isopropanol 25%/acetic 10% for 30 min and
washing for 5 min with distilled water, after which the gel was
soaked with 1 M Na-salicylate for 45 min, washed again with
water, and vacuum-dried at 80C. Autoradiography exposure was
performed at )70C.
Protein gel blot analysis
Protein extracts were prepared by grinding material in homogenization buffer (Yoo et al., 2007). Protein gel blotting was performed
according to standard procedures using primary anti-c-myc antibody (9E10; Santa Cruz Biotechnology, http://www.scbt.com) and
anti-GFP antibodies (JL-8; Clontech, http://www.clontech.com/),
followed by secondary goat anti-mouse antibody conjugated
to horseradish peroxidase, and visualized using chemiluminescence as instructed by the manufacturer (ECL; Amersham
Pharmacia).
Accession numbers
The Arabidopsis Genome Initiative locus identifiers for the major
genes mentioned in this paper are given in parentheses as follows:
BSCTV C4 (GeneID 1489875), RKP (At2G22010), ICK1/KRP1
(At2G23430), ICK2/KRP2 (At3G50630), ICK3/KRP5 (At3G24810), ICK4/
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