RKP, a RING finger E3 ligase induced by BSCTV C4 protein ... J.-Lai.pdf · RKP, a RING ﬁnger E3 ligase induced by BSCTV C4 protein, affects geminivirus infection by regulation of

RKP, a RING finger E3 ligase induced by BSCTV C4 protein,affects geminivirus infection by regulation of the plantcell 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,*

1State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen (Zhongshan) University, 135 West Xin-Gang Road,

Guangzhou 510275, China,2State 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,3School of Life Science, Liaocheng University, Liaocheng 252059, China, and4State 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.*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 repli-

cation. Therefore, geminiviruses must cause the host cells

to re-enter the cell cycle to create an environment suitable

for replication (Morra and Petty, 2000; Nagar et al., 1995,

2002).

There is evidence to support the proposition that ampli-

fication 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

ª 2008 The Authors 1Journal compilation ª 2008 Blackwell Publishing Ltd

The Plant Journal (2008) doi: 10.1111/j.1365-313X.2008.03737.x

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 Sardi-

nia 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 transcrip-

tome 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 pheno-

type 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 trans-

genic 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). Overex-

pression 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 ubiqu-

itin 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). How-

ever, 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 Arabid-

opsis 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 func-

tion 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 (Fig-

ure 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 constitu-

tively 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

2 Jianbin Lai et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), doi: 10.1111/j.1365-313X.2008.03737.x

(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.

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.

In the microarray data, the expression of one gene, which

is identical to the putative gene At2g22010 in the Arabidop-

sis 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

2 4 8 16 1 0

C4 rRNA

h

ES – +

(a) (b)

(e) (c)

(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 with32P-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.

CK C4

GU

S a

ctiv

ity

pm

ole

s x

mg

–1 x

min

–1

RKP

C4– +

ACT1

DPI

RKP

ACT1

0 4 8 12 0 4 8 12

BSCTV CK

1.0

1.0 5.0

1.2 1.2 1.2 1.0 1.8 2.0 1.4

100

200

300

400

0

(a) (b)

(c)

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 measure-

ments).

(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.

RING finger E3 ligase RKP affects geminivirus replication 3


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 (Fig-

ure 2b). Together, these results indicated that RKP expres-

sion 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 anal-

ysis. Compared to the vector control, the RNA level of RKP

increased in leaves infected by BSCTV (Figure 2c). Accord-

ingly, 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 inser-

tion 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 agro-

inoculated 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 proce-

dures). The number of plants with symptoms was counted

every day post-inoculation. In independent repeated exper-

iments, 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 symptom-

atic 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 short-

distance 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. Sec-

ond, 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 sup-

plemented 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



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 cul-

tured on callus formation mediums, the GUS activity incre-

ased significantly (Figure 4c,d). This suggests that expression

of RKP is induced in dividing cells, and supports the hypoth-

esis that RKP might be involved in plant cell-cycle regulation.

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

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 puri-

fied 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

Human KPC1 is known to act as a ubiquitin ligase to regulate

the degradation of p27Kip1 at G1 phase, therefore we ana-

lyzed the similarity of protein sequences between p27Kip1

and the Arabidopsis ICK/KRP family. Seven ICK/KRP proteins

DPI

Sym

pto

mat

ic p

lan

ts (

%)

0

20

40

60

80

100

7 8 9 10 11 12 13 14 15

WT

rkp-1

rkp-3

rkp-2

OCLIN

SC SS

DPI9 12 15

WT

rkp-

1

9 12 15 9 12 15 9 12 15

rkp-

2

rkp-

3

0.2

0.4

1.0 0.1

0.3

0.4

0.1

0.3

0.4

0.1

0.3

0.5

rkp-

1

rkp-

3DPI0 4 8 12 16

WT

0 4 8 12 16 0 4 8 12 16

OCLIN

SCSS

0.0

0.2

0.6

0.8

1.0

0.0

0.1

0.4

0.6

0.6

0.0

0.1

0.3

0.7

0.6

OCLIN

SC

SS

0 2 4 0 2 4 DPI

WT

rkp-

1

0.0

0.4

1.0

0.0

0.3

0.4

WT

rkp-

1rk

p-2

rkp-

3

P1 + P2

P3 + P4

ACT1

(a)

(c)

(e) (f)

(d)

(b)P1

P2

P3

P4

LBb1LB1 p745

rkp-1 rkp-2 rkp-3

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 wild-

type or rkp mutant plants at various time points

was used for DNA gel blotting. Ethidium bromide-

stained 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.



contained a conserved domain at their C-termini. Interest-

ingly, 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 pull-

down 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 tran-

scription 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

RKP, the protein levels of ICK/KRP proteins may be sup-

posed 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.

WT

rkp-2

2,4-D + 6BA

rkp-1

rkp-3

2,4-D + KT

WT

rkp-2

rkp-1

rkp-3

CK 2,4-D + 6BA CK 2,4-D + KT

(a) (b)

(d) (c)

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 dur-

ing 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 dur-

ing 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.



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 callus-

like 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 amplifica-

tion. 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 benth-

amiana 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 pheno-

types for the rkp mutants, although there is only one copy for

RKP in the Arabidopsis genome. This finding may be inter-

preted 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 cyto-

plasmic 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 regu-

lates 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 con-

served 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.



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 re-

duced in rkp mutants. Therefore, RKP might be involved in



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 tran-

sitions, 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, deg-

radation 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 ki-

nase 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 cytomeg-

alovirus replication is inhibited by the expression of a CDK2

dominant negative mutant (Bresnahan et al., 1997). Modifi-

cation of cyclin-dependent kinase inhibitor (CKI) p21 expres-

sion 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 repli-

cation. 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 transi-

tion, 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 endo-

replication 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 environ-

ment 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 func-

tional dissection of RKP is necessary for a complete under-

standing 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 rinsedthree times with sterile water. Sterile seeds were suspended in0.15% 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.



were stratified in darkness for 2–4 days at 4�C and then transferredto a tissue culture room at 22�C under a 16 h light/8 h dark photo-period. After 2–3 weeks, seedlings were potted in soil and placed ina growth chamber at 22�C and 70% relative humidity under a 16 hlight/8 h dark photoperiod.

Construction of the BSCTV plasmid

AninfectiouscloneofBSCTV(previouslynamedtheBCTVCFHstrain)(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 gen-ome) and an EcoRI–EcoRI fragment (full genome) were introducedfrom pCFH to binary vector pCambia1300 to generate pCambia-BSCTV, 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 C4cDNA was cloned into the vector pCambia1300-221 in which trans-gene expression is under the control of the CaMV 35S promoter.Inducible C4 plants were generated by cloning C4 cDNA into thevector pER8, in which transgene expression is under the control ofthe XVE-inducible promoter. For the RKP promoter/GUS fusionconstruct, a 5¢ flanking sequence (2 kb promoter region just up-stream of the ATG start codon of RKP) was amplified from genomicDNA by PCR and verified by sequencing. The PCR fragment wascloned into the PstI–XbaI site of binary vector pCambia1300-221 toobtain a transcriptional fusion of the RKP promoter and the GUScoding sequence. For the 35S-ICK1/KRP1 construct, the full-lengthcoding sequence (CDS) of ICK1/KRP1 was cloned into the vectorpCanG, modified from pCambia, in which transgene expression isunder the control of the CaMV 35S promoter. Transformation ofArabidopsis was performed by the vacuum infiltration method(Bechtold and Pelletier, 1998), using Agrobacterium tumefaciensstrain EHA105.

Verification of RKP T-DNA insertion mutants

Seeds of the T-DNA insertion lines rkp-1 (SAIL_3_E03), rkp-2 (Wis-cDsLox466C1) and rkp-3 (SALK_121005) were obtained from theArabidopsis Biological Resource Center (Ohio State University,Columbus, OH, USA). Homozygous mutants were identified by PCRfrom genomic DNA using T-DNA left border primers (LB1, 5¢-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC-3¢; p745, 5¢-AACGTCCGCAATGTGTTATTAAGTTGTC-3¢; LBb1, 5¢-GCGTGGACCGCTTGCTGCAACT-3¢) and RKP-specific primers (P3, 5¢-CAAGTAATCAGTCTGACCCTG-3¢; P4, 5¢-TCATGTGCTTCTTTTGTGACC-3¢).

Semi-quantitative RT-PCR amplification

To examine the expression of RKP by RT-PCR, DNase I-treated totalRNA was denatured and subjected to reverse transcription usingSuperScript II (200 units per reaction; Invitrogen, http://www.invitrogen.com/) at 37�C for 1 h followed by heat inactivation of thereverse transcriptase at 70�C for 15 min. To determine the changesin RKP expression in C4 transgenic plants and BSCTV infection, PCRamplification was performed using RKP forward (RT Fw, 5¢-TTCGTAGTTACACACTTCAAC-3¢) and reverse (RT Rev, 5¢-TCATGTGCTTCTTTTGTGACC-3¢) primers. To check RKP expression in rkpmutant plants, two pairs of primers were used (P1/P2, 5¢-TGG CGCT

GGCTTGTCATTTG-3¢/5¢-GACAAGAACCGAATTGCGTG-3¢, and P3/P4). ACTIN1 expression levels were monitored using forwardand reverse primers (F, 5¢-CATCAGGAAGGACTTGTACGG-3¢; R,5¢-GATGGACCTGACTCGTCATAC-3¢ to serve as an internal control.

GUS bioassays

Plants carrying the RKP promoter fused with the GUS gene weretreated under various conditions and used for histochemicaldetection of GUS expression. Materials were stained at 37�C over-night in 1 mg ml)1 5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid(X-Gluc), 5 mM potassium ferricyanide, 5 mM potassium ferrocya-nide, 0.03% Triton X-100 and 0.1 M sodium phosphate buffer, pH7.0. To test the induction of GUS expression on callus formationmedium, hypocotyls of 2-week-old transgenic seedlings weretransferred 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 trans-ferred 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, theproteins were extracted and detected using a GUS reporter geneactivity detection kit (MGT-M0877; FLUOstar OPTIMA, BMGLabtech, http://www.bmglabtech.com/), according to the manufac-turer’s instructions.

BSCTV agro-inoculation

Rosette leaves of 4-week-old plants were agro-inoculated withBSCTV (Briddon et al., 1989; Grimsley et al., 1986). A suspension ofAgrobacterium tumefaciens strain EHA105 (Hood et al., 1993) car-rying pCambia1300-BSCTV (carrying 1.8 copies of the BSCTV gen-ome) at a dose of 0.02 (absorbance at 600 nm of 0.02) and mixedwith 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 nightand grown in another greenhouse at a higher temperature (26�C).Symptoms appeared approximately 10 days after inoculation.

DNA gel blot

For the DNA gel blot, total DNA was extracted using CTAB buffer.Genomic DNA was stained using ethidium bromide as a loadingcontrol. After depurination and neutralization, total DNA wastransferred to Hybond N+ nylon membranes (Amersham PharmaciaBiotech, http://www5.amershambiosciences.com/) by upward cap-illary transfer in 0.4 M NaOH solution. The membranes werehybridized at 65�C using the whole genome of BSCTV, labeledwith [a-32P]dCTP, as a probe. Signal intensity was measured usingIMAGEJ (National Institutes of Health, http://www.rsbweb.nih.gov/ij/).

Protoplast transformation

Mesophyll protoplasts were isolated from rosette leaves of4-week-old Arabidopsis in the soil and transfected with plasmidDNA based on a previously described protocol (Yoo et al., 2007),except that the leaves were sterilized in 70% ethanol for approx-imately 1 min; the entire operation was performed under sterileconditions. Transfected cells were kept in the dark at roomtemperature. For the BSCTV replication assay, approximately3 · 105 cells were collected at 0, 2 and 4 days after transfection forDNA extraction. Total genomic DNA was extracted from the cellsaccording to a previously described protocol (Fontes et al., 1994).



Newly replicated viral DNA was identified by DNA gel blot. For theGUS activity assay, pCambia1300-221–promoterRKP::GUS wastransformed together with pCambia1300 or pCambia1300-C4, andthe protoplasts were collected 16 h later. For detection of the myc-ICK2/KRP2 protein level in the rkp mutant, 35S-myc-ICK2/KRP2was prepared by inserting the PCR-amplified coding region ofICK2/KRP2 fused with MYC in the N-terminus of ICK2/KRP2 to thepBA002 vector under the control of the 35S promoter. The plas-mid was transformed into protoplasts of wild-type and rkp-1plants; the pGFP2 plasmid, in which the GFP gene was under thecontrol of the 35S promoter, was transfected alongside for aninternal control. For detection of myc-ICK2/KRP2 protein levelduring BSCTV infection, 35S-myc-ICK2/KRP2 and pGFP2 plasmidswere co-transfected with either pCambia-BSCTV or vector controlinto wild-type protoplasts.

E3 ubiquitin ligase activity assay

The entire RKP ORF was cloned into the PGEX-6P-1 vector and ex-pressed in Escherichia coli. Fusion proteins were prepared accord-ing to the manufacturer’s instructions (GE Healthcare, http://www.gehealthcare.com). The in vitro E3 ligase assays were performed asdescribed previously (Zhang et al., 2007b).

In vitro pull-down assay

RKP labeled with 35S on its methionine residues was generated byin vitro transcription and translation using a T7/T3-coupled TnT kit(Promega, http://www.promega.com/). ORFs of ICK/KRP proteinswere cloned into the PGEX-6P-1 vector and expressed in Escher-ichia coli. RKP (4 ll) labeled with 35S on its methionine residueswas 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 incu-bated at room temperature for 60 min. The glutathione–Sepha-rose beads (GE Healthcare) were then rinsed five times in GBBcontaining 0.5 M NaCl and twice with GBB. Bound proteins werereleased by boiling in SDS sample buffer at 90�C for 4 min andsubjected to SDS–PAGE. After running the gel, protein was fixedto the gel using isopropanol 25%/acetic 10% for 30 min andwashing for 5 min with distilled water, after which the gel wassoaked with 1 M Na-salicylate for 45 min, washed again withwater, and vacuum-dried at 80�C. Autoradiography exposure wasperformed at )70�C.

Protein gel blot analysis

Protein extracts were prepared by grinding material in homogeni-zation buffer (Yoo et al., 2007). Protein gel blotting was performedaccording to standard procedures using primary anti-c-myc anti-body (9E10; Santa Cruz Biotechnology, http://www.scbt.com) andanti-GFP antibodies (JL-8; Clontech, http://www.clontech.com/),followed by secondary goat anti-mouse antibody conjugatedto horseradish peroxidase, and visualized using chemilumines-cence as instructed by the manufacturer (ECL; AmershamPharmacia).

Accession numbers

The Arabidopsis Genome Initiative locus identifiers for the majorgenes 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/

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 ofPlant Molecular Biology, Rockefeller University for kindly providingus with the pER8 vector, the Arabidopsis Biological Resource Centerat Ohio State University for providing the T-DNA insertion lines, andMr Sanyuan Tang for technical assistance. This research was sup-ported by grants CNSF30325030/30530400 from the Chinese NaturalScience Foundation (CNSF). Q.X. is supported by grants KSCX2-YW-N-010 and CXTD-S2005-2 from the Chinese Academy of Sci-ence. H.G. is supported by CNSF grant 30525004.

Supporting Information

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Diagnostic PCR of the T-DNA inserted at three positionsin RKP.Figure S2. The developmental phenotype and molecular characterof the 35S-ICK1/KRP1 transgenic line for BSCTV inoculation.Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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