The Plant Journal 62 Up-regulation of LSB1 GDU3 affects ... Journal 2010-Chen … · Up-regulation of LSB1/GDU3 affects geminivirus infection by activating the salicylic acid pathway

Up-regulation of LSB1/GDU3 affects geminivirus infectionby activating the salicylic acid pathway

Hao Chen1,2, Zhonghui Zhang1,2, Kunling Teng2, Jianbin Lai1,2, Yiyue Zhang1,2, Yiliang Huang1, Yin Li1, Liming Liang1,

Yiqin Wang2, Chengcai Chu2, Huishan Guo3 and Qi Xie1,2,*

1Stake Key Laboratory for Biocontrol, 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, and3State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Microbiology,

Chinese Academy of Sciences, Beijing 100080, China

Received 14 October 2009; revised 20 November 2009; accepted 16 December 2009; published online 11 February 2010.*For correspondence (fax +86 10 64889351; e-mail [email protected]).

SUMMARY

Geminiviruses include a large number of single-stranded DNA viruses that are emerging as useful tools to

dissect many fundamental processes in plant hosts. However, there have been no reports yet regarding the

genetic dissection of the geminivirus–plant interaction. Here, a high-throughput approach was developed to

screen Arabidopsis activation-tagged mutants which are resistant to geminivirus Beet severe curly top virus

(BSCTV) infection. A mutant, lsb1 (less susceptible to BSCTV 1), was identified, in which BSCTV replication

was impaired and BSCTV infectivity was reduced. We found that the three genes closest to the T-DNA were

up-regulated in lsb1, and the phenotypes of lsb1 could only be recapitulated by the overexpression of GDU3

(GLUTAMINE DUMPER 3), a gene implicated in amino acid transport. We further demonstrated that activation

of LSB1/GDU3 increased the expression of components in the salicylic acid (SA) pathway, which is known to

counter geminivirus infection, including the upstream regulator ACD6. These data indicate that up-regulation

of LSB1/GDU3 affects BSCTV infection by activating the SA pathway. This study thus provides a new approach

to study of the geminivirus–host interaction.

Keywords: functional genomics, geminivirus, plant–microbe interaction.

INTRODUCTION

Geminiviruses are composed of numerous plant single-

stranded DNA viruses that have a large host range, including

many economically important crops. Their virion particles

are geminate and 18–30 nm in size. Geminivirus genomes,

which are composed of one or two circular single-stranded

DNA (ssDNA) molecules of 2.5–3.0 kb in length, are repli-

cated through double-stranded DNA (dsDNA) intermediates

within the nuclei of host cells by a rolling circle mechanism

(Fauquet et al., 2008; Gutierrez, 1999; Hanley-Bowdoin et al.,

2000).

The extremely limited coding capacity of the geminivirus

genomes makes these viruses dependent upon host factors

to complete their life cycle. In fact, geminiviruses have

become an extremely useful molecular tool for studying

many fundamental processes in plants, e.g. transcriptional

regulation, DNA replication, cell cycle control, and macro-

molecular trafficking (Gutierrez, 1999). To date, two

strategies have been applied to isolate host factors involved

in the geminivirus–host interaction. The first is screening for

host proteins that interact with the geminivirus-encoded

proteins using two hybrid systems (Bagewadi et al., 2004;

Castillo et al., 2004; Fontes et al., 2004; Hao et al., 2003; Kong

and Hanley-Bowdoin, 2002; Luque et al., 2002; McGarry

et al., 2003; Selth et al., 2005; Xie et al., 1996). The second

strategy is to use microarrays to uncover host factors whose

expression is modified by geminivirus infection (Ascencio-

Ibanez et al., 2008; Lai et al., 2009; Trinks et al., 2005).

However, there have been no reports yet regarding the

genetic dissection of the geminivirus–plant interaction.

Geminiviruses fall into four genera on the bases of host

range, insect vector, and genome organization (Fauquet

et al., 2008). There are five species in the Curtovirus genus,

Beet curly top virus (BCTV), Beet mild curly top virus

(BMCTV), Beet severe curly top virus (BSCTV), Horseradish

12 ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd

The Plant Journal (2010) 62, 12–23 doi: 10.1111/j.1365-313X.2009.04120.x

curly top virus (HrCTV), and Spinach curly top virus (SpCTV)

(Fauquet et al., 2008). BCTV (formerly known as BCTV-

Logan/California strain), BSCTV (formerly known as BCTV-

CFH strain), and SpCTV have been shown to be able to infect

the model plant Arabidopsis thaliana (Lee et al., 1994; Park

et al., 2002; Baliji et al., 2007). Previous research identified

two ecotypes of Arabidopsis (Ms-0 and Pr-0) that are

resistant to BCTV and another ecotype (Cen-0) that is

tolerant to hypervirulent BSCTV (Lee et al., 1994; Park et al.,

2002). Interestingly, such phenotypes were both determined

by a single locus. However, the corresponding genes have

not yet been cloned.

Salicylic acid (SA), a key signaling molecule for plant

disease resistance, is synthesized by plants to induce

accumulation of pathogenesis-related (PR) proteins and

establish both local and systemic acquired resistance

(SAR) against a diverse range of pathogens. At least three

types of SA regulators have been described (Lu et al., 2009).

The type I regulators include enzymes involved in SA

biosynthesis, e.g. SA INDUCTION-DEFICIENT 2 (SID2),

which converts chorismate to isochorismate for SA biosyn-

thesis (Wildermuth et al., 2001). The type II regulators, e.g.

ACCELERATED CELL DEATH 6 (ACD6), ENHANCED DISEASE

SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4

(PAD4), and SID1/EDS5 (Falk et al., 1999; Jirage et al., 1999;

Nawrath et al., 2002; Lu et al., 2003), affect accumulation of

SA, but may not be biosynthetic enzymes. The type III

regulators transduce signals downstream of SA, e.g. NON-

EXPRESSOR OF PR GENES 1 (NPR1) (Cao et al., 1997). The

type II SA regulator ACD6 is a plasma membrane protein that

has 34 homologs in Arabidopsis (Lu et al., 2003). A gain-of-

function mutation of ACD6 (acd6-1) results in increased

expression of ACD6-1, EDS1, PAD4, and NPR1, confers

resistance to Pseudomonas syringae and Hyaloperonospora

parasitica (Song et al., 2004), and induces accumulation of

high levels of SA (Rate et al., 1999). The acd6-1 gain-of-

function phenotype can be partially suppressed by pad4,

eds5, npr1, and sid2 (Lu et al., 2009). Thus, ACD6 is proposed

to function upstream of SA synthesis and other regulatory

genes that modulate SA synthesis, including PAD4, EDS5,

and possibly EDS1.

The SA pathway is typically activated by RNA viruses

(Whitham et al., 2006), but equivalent information has not

been available until recently for geminiviruses. Ascencio-

Ibanez et al. performed a global analysis of the Arabidop-

sis transcriptome upon infection with the geminivirus

Cabbage leaf curl virus (CaLCuV) and revealed that CaLCuV

also triggers a pathogen response via the SA pathway

(Ascencio-Ibanez et al., 2008). Furthermore, they showed

that Arabidopsis cpr1 (constitutive expresser of PR genes)

plants, in which SA-mediated SAR is constitutively acti-

vated marked with an elevated endogenous level of SA

and increased PR gene expression (Bowling et al., 1994),

were less susceptible to CaLCuV infection, indicating that

constitutive activation of the SA pathway impairs infection

by geminivirus.

In the last decade, activation tagging has been used in

Arabidopsis to generate not only conventional loss-of-

function mutants but also gain-of-function mutants by

insertion of T-DNA containing enhancers or promoters in

the Arabidopsis genome. This produces an unbiased

up-regulation of the genes flanking the T-DNA (Weigel et al.,

2000). Screens in Arabidopsis activation tagged mutants

have been shown to be an effective approach for identifying

genes with redundant functions, which are abundant in

eukaryotic genomes, and genes that are required for funda-

mental processes in plants, since loss-of-function mutation

of these genes leads to early embryonic or gametophytic

lethality (Weigel et al., 2000; Borevitz et al., 2000; Ito and

Meyerowitz, 2000; Pilot et al., 2004).

To further elucidate the mechanism of the geminivirus–

plant interaction, taking advantage of the Arabidopsis

genetic resources and the finding that the geminivirus

BSCTV can infect Arabidopsis, a high-throughput approach

was developed to screen mutants resistant to BSCTV

infection in Arabidopsis activation tagged mutants. One

mutant, lsb1 (less susceptible to BSCTV 1), was identified in

which BSCTV DNA replication was impaired and BSCTV

infectivity was reduced. This phenotype was caused by the

elevated expression of LSB1/GDU3 (GLUTAMINE DUMPER 3),

a gene implicated in amino acid transport. We further found

that activation of LSB1/GDU3 increased the expression of

several components of the SA pathway, including the

upstream regulator ACD6. Thus, this study indicates that

up-regulation of LSB1/GDU3 affects geminivirus infection by

activating the salicylic acid pathway.

RESULTS

Screen for mutants resistant to BSCTV infection

To further dissect the geminivirus–plant interaction, we

established a strategy to screen Arabidopsis activation tag-

ged mutants which are resistant to BSCTV. This pool of

mutants was chosen based on the consideration that if

up-regulation of any gene(s) can be shown in our studies

to reduce susceptibility to BSCTV in Arabidopsis, a similar

effect might be easier to recapitulate in other susceptible

plant hosts, especially economically important crops, to

increase resistance against geminiviruses.

In the laboratory, Arabidopsis can be successfully inocu-

lated with BSCTV by agroinoculation (Lee et al., 1994; Park

et al., 2004, 2002). However, conventional agroinoculation of

Arabidopsis with BSCTV on is performed by infiltrating

individual plants manually at the wounds produced by

needle puncture, which is inconvenient for mass inocula-

tion. We successfully improved the convenience of inocula-

tion by applying the airbrush technique (Whitham et al.,

1999) while retaining a desirable infection efficiency (the

LSB1/GDU3, geminivirus and the SA pathway 13

ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 12–23

details are described in Experimental Procedures). Typical

BSCTV symptoms, including severely curled and deformed

inflorescences and leaves, stunted growth, and accumula-

tion of anthocyanins (Figure 1b), started to appear in

infected plants around 8 days post-inoculation, and over

97% of the inoculated wild-type plants became symptomatic

3 weeks post-inoculation (Figure 1c). Because this approach

had a desirable inoculation efficiency and reproducibility it

was applied in the subsequent mutant screen.

About 10 000 lines from the pSKI015 activation tagged

mutant collection (Weigel et al., 2000), obtained from The

Arabidopsis Information Resource (TAIR), were inoculated

in the first screen (Figure S1a in Supporting Information). In

total, 169 plants survived (Figure S1b). Since previous work

has shown that geminiviruses are not transmitted to seeds

(Peele et al., 2001; Sudarshana et al., 1998), to further

confirm the resistance of these candidate lines and eliminate

false positives seeds from the surviving plants were col-

lected individually (Figure S1c) and a second inoculation

was performed on their progeny (Figure S1d). Multiple lines

with less susceptibility to BSCTV were obtained from this

screen. Among these lines, lsb1 (less susceptible to BSCTV 1)

displayed a reduced ratio of symptomatic plants upon

BSCTV inoculation. A study of lsb1, especially its attenuation

of BSCTV infection, is presented in this paper.

BSCTV infectivity is reduced in lsb1

We found that the ratio of symptomatic plants in lsb1 was

reduced upon BSCTV inoculation (Figure 2a). Only about

63% of lsb1 plants showed systemic symptoms upon BSCTV

inoculation, compared with nearly 100% of the control

plants (Figure 2a); however, there was no obvious difference

regarding symptom severity in lsb1 compared with control

plants. To analyze whether there was accumulation of virus

in the asymptomatic lsb1 plants, total DNA from the

aboveground tissues of these plants was extracted and

subjected to a DNA gel blot with the whole BSCTV genome

as the probe. As shown for a representative experiment in

Figure 2(b), there was no detectable viral DNA in the

majority of these plants, except for a few plants that con-

tained a low level of viral DNA compared with that in

symptomatic plants. This result further confirms that the

majority of these asymptomatic plants are not infected

systemically by BSCTV.

To exclude the possibility that the reduction of symptom-

atic lsb1 plants upon BSCTV inoculation was due to uneven

spraying or defects in Agrobacterium tumefaciens-mediated

T-DNA transfer, a transient expression assay was performed

utilizing the binary T-DNA vector pCambia1305.1 (CAMBIA,

http://www.cambia.org/), which contains the b-glucuroni-

dase gusA gene harboring an intron. The gusA gene is under

the control of the cauliflower mosaic virus (CaMV) 35S

promoter, and the intron in the gusA gene permits expres-

sion of this gene only in plant cells and not in the host

BSCTV (1.0mer)HygRB35S

E

LB

B BE

BSCTV (0.8mer)

35SpolyA1 kb

2

3 4

1

0

20

40

60

80

100

0.02 0.2 2O.D600 of EHA105 (pCambiaBSCTV)

Sym

pto

mat

ic p

lan

ts (

%)

(a)

(b)

(c)

Figure 1. Modification of conventional agroinoculation.

(a) Schematic representation of the T-DNA region of the pCambiaBSCTV

construct containing the linearized, partial tandem-repeated and double-

stranded Beet severe curly top virus (BSCTV) genome. LB, left border;

RB, right border; 35S, 35S promoter; Hyg, hygromycin resistance; E, EcoRI;

B, BamHI.

(b) Symptoms of BSCTV infection in Arabidopsis (Columbia ecotype). 1,

Stunted growth of infected plants. Left, healthy wild-type plants; right, plants

infected with BSCTV. 2, Severely curled and deformed inflorescences of

infected plants. 3, Detailed representation of the deformed inflorescences.

Left, healthy wild-type inflorescence; right, inflorescence infected with

BSCTV. 4, Accumulation of anthocyanins in the infected plants 6 weeks

post-inoculation.

(c) Infectivity of BSCTV in Arabidopsis (Columbia ecotype) wild-type plants

with different doses of modified agroinoculation. Values shown are percent-

ages of plants that displayed systemic disease symptoms 3 weeks post-

inoculation with BSCTV at different doses, which represent typical data from

at least three replicates with 40 plants in each experiment. EHA105 (pCambia-

BSCTV), the Agrobacterium tumefaciens strain EHA105 containing pCambia-

BSCTV.

14 Hao Chen et al.


bacteria. Arabidopsis plants were inoculated with the sus-

pension of EHA105 containing this plasmid in the same

manner as BSCTV inoculation. The expression of GUS with a

punctate pattern on the leaves could be observed 3 days

post-inoculation. Similar densities of blue GUS spots on the

rosette leaves of both the lsb1 and wild-type plants were

observed, as shown in Figure 2(c) and (d) for a representa-

tive experiment. This observation demonstrates that the

reduction of symptomatic lsb1 plants upon BSCTV inocula-

tion does not result from a deficiency in inoculation.

The observation that there were some asymptomatic lsb1

plants containing a low level of viral DNA suggests that

replication of BSCTV in lsb1 may be impaired but not

inhibited. To investigate whether BSCTV DNA replication in

lsb1 was affected, a transient replication assay was con-

ducted in Arabidopsis mesophyll protoplasts, which are

devoid of a cell wall so there is no viral movement among

the cells. The mesophyll protoplasts from both the lsb1 and

wild-type plants were transfected with pCambia-BSCTV

DNA. Then, total DNA from the protoplasts was isolated at

various time points post-transfection and subjected to DNA

gel blot probed with the whole BSCTV genome to examine

the newly replicated viral DNA. As shown in Figure 2(e) for a

representative experiment, newly replicated BSCTV DNA

was detected 2 days post-transfection in both lsb1 and wild-

type protoplasts, but the amount in the lsb1 protoplasts was

much lower than that in the wild-type protoplasts. This result

reveals that replication of BSCTV DNA is impaired in lsb1.

Collectively, these results suggest that reduced BSCTV

infectivity in lsb1 may be attributed to attenuated viral DNA

replication. This observation is consistent with a previous

study that found that BSCTV DNA accumulates in the tissues

of a tolerant Arabidopsis ecotype Cen-0, but at a much lower

level than that in a highly susceptible ecotype SKKU (Park

et al., 2002). These observations suggest that there might be

a threshold level of viral DNA accumulation required for

systemic infection of geminiviruses. In addition, we did not

observe any obvious morphological phenotypes in lsb1,

except that lsb1 plants were slightly smaller than wild-type

plants (Figure S2a).

The expression levels of the three genes closest to the

T-DNA are up-regulated

To uncover the genetic changes responsible for the pheno-

types of lsb1, the T-DNA insertion site in lsb1 was deter-

mined by plasmid rescue (Weigel et al., 2000). Genomic

0

20

40

60

80

100

1 3 5 7 9 11 13 15 17 19 21Days post-inoculation

Sym

pto

mat

ic p

lan

ts (

%)

lsb1

WT

Asymptomatic lsb1 plants

WT

lsb1

Newlyreplicated

DNA

*

0 2 4 0 2 4 dpt

WT lsb1

Newlyreplicated

DNA

0

5

10

15

20

25

30

WT lsb1Sp

ot

nu

mb

er o

f G

US

(cm

–2)

Plantgenomic

DNA

Plantgenomic

DNA

(a)

(b)

(d) (e)

(c)

Figure 2. Beet severe curly top virus (BSCTV)

infectivity is reduced in lsb1.

(a) Agroinoculation of BSCTV in wild-type (WT)

and lsb1 plants. Values shown are percentages of

plants (n = 40, three replicates � SD) that dis-

played systemic disease symptoms at different

days post-inoculation (OD600: 2.0).

(b) A DNA gel blot analysis of BSCTV DNA

accumulation from either symptomatic (lane* 1)

or asymptomatic lsb1 plants 3 weeks post-inoc-

ulation. Blots were probed with specific probes

for the whole BSCTV genome. Loading control:

genomic DNA from inoculated plant.

(c) Leaves from WT and lsb1 plants were stained

to examine GUS gene expression 3 days post-

inoculation with EHA105 harboring pCam-

bia1305.1. Bar = 2 mm.

(d) Statistics of the GUS gene expression on the

leaves from either WT or lsb1 plants (per cm2).

Values are the means (n = 10) � SD of typical

data replicated at least three times.

(e) Replication of BSCTV in mesophyll proto-

plasts from either WT or lsb1 plants.

Newly replicated viral double stranded DNA at 0,

2, and 4 days post-transfection was detected by

DNA gel blot. dpt, days post-transfection. Load-

ing control: genomic DNA from protoplasts.



DNA recovered by HindIII or SpeI both identified the same

site on chromosome V (18250–18323 in the BAC MRI1). A

series of PCR amplifications with appropriate primers

located either in the T-DNA or the flanking genomic DNA

also confirmed this result (Figure S3).

According to annotations in TAIR, the T-DNA insertion

does not interrupt any apparent open reading frames

(ORFs). As lsb1 is an activation tagged mutant, it is

possible that expression levels of the genes close to the

T-DNA are elevated. Previous studies of pSKI015 activation

tagged mutants have shown that in the majority of cases

up-regulated genes were found immediately adjacent to

the inserted CaMV35S enhancers, at distances ranging

from 380 to 3.6 kb (Pilot et al., 2004; Weigel et al., 2000).

Consistent with this observation, RNA gel blot analysis

showed that the expression levels of the genes in the 6-kb

region flanking the CaMV35S enhancers, which are close

to the T-DNA right border, including AT5G57690 (encoding

a putative diacylglycerol kinase, DGK) and AT5G57700

(encoding a putative BNR/Asp-box repeat family protein),

were evidently up-regulated in lsb1 (Figure 3a,b). Interest-

ingly, the expression of another gene, AT5G57685 (also

called GDU3), about 4 kb away from the left border of the

T-DNA, was also up-regulated (Figure 3a,b). To further

verify this pattern and investigate whether there were

more up-regulated genes close to the T-DNA insertion site,

a cDNA microarray analysis was conducted to examine the

global expression profile in lsb1. The microarray analysis

confirmed the result of the RNA gel blot, except that the

expression of AT5G57685 could not be detected since

there was no corresponding probe set in Affymetrix chip

ATH1-121501, and we did not discover obviously enhanced

expression for other genes that flanked the T-DNA (data

not shown). Together, these results establish that expres-

sion levels of the three genes closest to the T-DNA were

elevated. We then speculated that the phenotypes of lsb1

might be caused by up-regulation of one or more of the

three candidate genes.

Elevated expression of LSB1/GDU3 leads to the phenotypes

of lsb1

To determine which gene(s) is responsible for the pheno-

types of lsb1, transgenic plants overexpressing each of the

three genes were generated by fusing the coding sequences

(CDS) or genomic DNA downstream of the CaMV 35S pro-

moter. There are two splicing variants of AT5G57700, and

thus both cDNAs were cloned and overexpressed sepa-

rately. Overexpression of these genes in the transgenic

plants was verified by RNA gel blot (Figure 4a,c,e), and

BSCTV was agroinoculated on six independent F2 lines for

each expression cassette. As shown in Figure 4(b) and (d),

the ratios of the symptomatic 35S-AT5G57690 and 35S-

AT5G57700 plants (including both variants) were not

reduced compared with the ratio of the symptomatic control

plants. In contrast, the ratio of the symptomatic 35S-

AT5G57685 plants was noticeably reduced compared with

that of the symptomatic control plants (Figure 4g). Further

analysis showed that accumulation of BSCTV DNA was also

reduced in the 35S-AT5G57685 protoplasts (Figure 4f). In

addition, the 35S-AT5G57685 plants were smaller than the

control plants, consistent with the phenotypes of lsb1 (Fig-

ure S2b). Together, these results demonstrate that the ele-

vated expression of AT5G57685 leads to the phenotypes

of lsb1. Based on these observations, AT5G57685 was

renamed LSB1/GDU3.

Enhanced expression of LSB1/GDU3 increases the

expression of genes involved in the SA pathway and the

response to geminivirus

To further investigate the mechanism underlying the atten-

uated BSCTV infection caused by up-regulation of LSB1/

GDU3, we searched for genes whose expression was regu-

lated by LSB1/GDU3. During the course of this study,

another group found that infection with the geminivirus

CaLCuV triggered a pathogen response in Arabidopsis,

probably via the SA pathway, as revealed by global analysis

LB H BastarpUC19 S 4X35SE RB

1 kb

HS

AT5G57690(DGK)

AT5G57700(BNR)

AT5G57685(GDU3)

WT lsb1

AT5G57690(DGK)

AT5G57700(BNR)

AT5G57685(GDU3)

rRNA

(a)

(b)

Figure 3. Schematic representation of the T-DNA insertion and the three

up-regulated genes close to the T-DNA in lsb1.

(a) Genomic context of the T-DNA insertion in lsb1. Bastar, Basta resistance;

LB, left border; pUC19, pUC19 plasmid; RB, right border; 4X35SE, four copies

of the 35S enhancer; H, HindIII; S, SpeI.

(b) RNA gel blot analysis of the accumulation of AT5G57685, AT5G57690, and

AT5G57700 in wild-type (WT) and lsb1 plants. The 28s rRNA is shown as a

loading control.

16 Hao Chen et al.


of Arabidopsis gene expression (Ascencio-Ibanez et al.,

2008). Furthermore, they showed that the Arabidopsis cpr1

plants, in which SA-mediated SAR is constitutively activated

marked with elevated endogenous level of SA and increased

expression of the PR genes (Bowling et al., 1994), did not

display typical symptoms at 25 days after inoculation with

0

20

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60

80

100

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lan

ts (

%)

0

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100

Sym

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%)

AT5G57690(DGK)

rRNA

Vector 35S-DGK

Vector Variant1 Variant2

AT5G57700(BNR)

rRNA

Vector Variant1 Variant2

Line no.

Line no.

Line no.

Vector 35S-DGK

Line no.

35S-BNR

35S-BNR

1.1

2.2

1.1

2.2

3.1

4.1

5.3

6.1

1.1

2.1

3.5

4.2

5.1

6.1

1.1

2.2

1.1

2.2

3.1

4.1

5.3

6.1

1.1

2.1

3.5

4.2

5.1

6.1

1.1

2.2

2.1

4.1

5.3

7.1

8.2

9.1

1.1

2.2

2.1

4.1

5.3

7.1

8.2

9.1

(a) (b)

(c)

(d)

AT5G57685(GDU3)

rRNA

Line no.

0 2 4 0 2 4 DPTNewly

replicatedDNA

Vector 35S-GDU3 (8.1)

Vector 35S-GDU3

1.1

2.2

2.2

3.3

4.1

5.2

7.1

8.1

Plantgenomic

DNA

(e)

(f)

0

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ts (

%)

Vector 35S-GDU3

Line no. 1.1

2.2

2.2

3.3

4.1

5.2

7.1

8.1

(g)

Figure 4. Overexpression of LSB1/GDU3 recapitulates the phenotype of lsb1.

(a) RNA gel blot analysis of AT5G57690 (DGK) expression in two vector control lines and six independent 35S-AT5G57690 (DGK) lines. The 28s rRNA is shown as a

loading control.

(b) Agroinoculation of Beet severe curly top virus (BSCTV) in the transgenic plants identified in (a). Values shown are percentages of plants (n = 40, three

replicates � SD) that displayed systemic disease symptoms at 4 weeks post-inoculation (OD600: 2.0).

(c) RNA gel blot analysis of AT5G57700 (BNR) expression in two vector control lines and 12 independent 35S-AT5G57700 (BNR) lines (including six lines of variant 1

and six lines of variant 2). The 28s rRNA is shown as a loading control.

(d) Agroinoculation of BSCTV in the transgenic plants identified in (c). Values shown are percentages of plants (n = 40, three replicates � SD) that displayed

systemic disease symptoms at 4 weeks post-inoculation (OD600: 2.0).

(e) RNA gel blot analysis of AT5G57685 (GDU3) expression in two vector control lines and six independent 35S-AT5G57685 (GDU3) lines. The 28s rRNA is shown as a

loading control.

(f) Replication of BSCTV in vector and 35S-AT5G57685 (LSB1/GDU3) protoplasts. Newly replicated viral double stranded DNA at 0, 2, and 4 days post-transfection

was detected by DNA gel blot. dpt, days post-transfection. Loading control: genomic DNA from protoplasts.

(g) Agroinoculation of BSCTV in the transgenic plants identified in (e). Values shown are percentages of plants (n = 40, three replicates � SD) that displayed

systemic disease symptoms at 4 weeks post-inoculation (OD600: 2.0).



CaLCuV (Ascencio-Ibanez et al., 2008). This observation

suggests that constitutive activation of the SA pathway may

compromise geminivirus infection.

Coincidentally, the microarray analysis showed that four

genes involved in the SA pathway, including ACD6,

an upstream regulator of SA synthesis and signaling

in Arabidopsis (Dong, 2004; Lu et al., 2003, 2005), PCC1

(PATHOGEN AND CIRCADIAN CONTROLLED 1,

AT3G22231), a SA-induced and NPR1-dependent response

gene (Sauerbrunn and Schlaich, 2004), WAK1 (CELL WALL-

ASSOCIATED KINASE 1, AT1G21250), another SA-induced

and NPR1-dependent response gene (He et al., 1998), and

PR5 (pathogenesis-related protein 5, AT1G75040), another

downstream marker of the SA pathway (Hu and Reddy,

1997), were up-regulated in lsb1, and this result was

confirmed by RNA gel blots (Figure 5a). Since the T-DNA

containing 35S enhancers in lsb1 is located in chromo-

some V, up-regulation of these genes in lsb1 might not be

the direct effect of the insertion, but the effect of enhanced

expression of the three genes flanking the T-DNA, includ-

ing LSB1/GDU3. To determine whether enhanced expres-

sion of these genes is caused by up-regulation of LSB1/

GDU3, we examined expression of these genes in the 35S-

LSB1/GDU3 plants. As shown in Figure 5(a), expression of

these genes was also elevated in the 35S-LSB1/GDU3

plants. This result indicates that activation of LSB1/GDU3

can further up-regulate the expression of genes involved in

the SA pathway.

After examining the published microarray data in Arabid-

opsis upon CaLCuV infection, we noticed that the expression

of ACD6, WAK1, and PR5 was increased during CaLCuV

infection, while expression of PCC1 was not. As for LSB1/

GDU3, there is no information regarding its gene expression

upon various treatments, due to the absence of a corre-

sponding probe set. CaLCuV and BSCTV belong to different

geminivirus genera (Fauquet et al., 2008). To explore

whether these genes also respond to BSCTV infection, their

expression levels upon inoculation with BSCTV were exam-

ined. Total RNA from the aboveground tissues of both

control and inoculated plants sampled at various time points

post-inoculation was extracted and subjected to a RNA gel

blot. As shown in Figure 5(b) for a representative experi-

ment, the expression levels of these genes, including LSB1/

GDU3 itself, were affected by BSCTV infection. Specifically,

the expression levels of the majority of these genes were

up-regulated at 7 days post-BSCTV inoculation, and this

pattern was maintained until 14 days post-inoculation. In

contrast, PCC1 expression was suppressed during the same

time course, which suggests that the expression of this gene

might be also regulated by other pathway(s) simultaneously

during BSCTV infection. Taken together, these results

establish that activation of LSB1/GDU3 can further

up-regulate the expression of genes involved in the gemini-

virus–host interaction.

lsb1

35S-LSB1/GDU3

2.2 8.1VectorWT

LSB1/GDU3

ACD6

PCC1

WAK1

PR5

Actin

7 14

Vector BSCTV

7 14 dpi

LSB1/GDU3

ACD6

PCC1

WAK1

PR5

Actin

Ratio of symptomatic plants (%)

WT 97.5 ± 2.5

cpr1 0 ± 0

cpr5 0 ± 0

acd6-1 0 ± 0

(a)

(b)

(c)

Figure 5. Enhanced expression of LSB1/GDU3 increased expression of genes

involved in the salicylic acid (SA) pathway and response to geminivirus.

(a) RNA gel blot analysis of LSB1/GDU3, ACD6, PCC1, WAK1, PR5, and ACTIN1

expression levels in the lsb1 and 35S-LSB1/GDU3 plants. Expression of

ACTIN1 was used as an internal control. This experiment was repeated twice

with similar results.

(b) RNA gel blot analysis of LSB1/GDU3, ACD6, PCC1, WAK1, PR5, and ACTIN1

expression levels during Beet severe curly top virus (BSCTV) infection.

Expression of ACTIN1 was used as an internal control. This experiment was

repeated twice with similar results.

(c) cpr1, cpr5, and acd6-1 display strong resistance to BSCTV. Agroinoculation

of BSCTV in wild-type (WT), cpr1, cpr5, and acd6-1 plants. Values shown are

percentages of plants (n = 40, three replicates � SD) that displayed systemic

disease symptoms at 3 weeks post-inoculation (OD600: 2.0).

18 Hao Chen et al.


To examine whether constitutive activation of the SA

pathway could also compromise BSCTV infection, we inoc-

ulated with BSCTV cpr1, cpr5 (Bowling et al., 1997) and

acd6-1 (Rate et al., 1999) plants in which the SA pathway was

constitutively activated. We found that none of these

mutants showed typical BSCTV symptoms 3 weeks post-

inoculation, while the majority of the inoculated wild-type

plants were highly symptomatic by this time (Figure 5c).

Only around 10% inoculated cpr5 plants, but not cpr1 and

acd6-1 plants, eventually developed mild symptoms (data

not shown). This result indicates that constitutive activation

of the SA pathway can compromise geminivirus infection.

Enhanced expression of GDU1 displays phenotypes similar

to LSB1/GDU3

LSB1/GDU3 is one of six members of a plant-specific family

of genes. Overexpression of each member of this family

leads to the same phenotypes, including increases in the

free amino acid levels in the leaves and obvious glutamine

secretion from the hydathodes of their leaves (Pratelli et al.,

2008; Pilot et al., 2004; Pratelli and Pilot, 2006); however, the

latter phenotype was not observed when lsb1 and gdu1-6D,

one recapitulation line of GDU1 (Pilot et al., 2004; Pratelli

and Pilot, 2006), were grown in our greenhouse, suggesting

that this phenotype may be dependent on the environment.

To date, the molecular and biochemical identity of the

members of this family remain unknown. To explore whe-

ther other members of the GDU gene family are also

involved in the geminivirus–host interaction, we inoculated

BSCTV on gdu1-6D. Surprisingly, gdu1-6D displayed full

resistance to BSCTV and none of the inoculated gdu1-6D

plants showed typical BSCTV symptoms post-inoculation

(Figure 6a). We further examined the expression of the SA-

related genes whose expression was up-regulated in lsb1

and found that the expression levels of all of these genes

were also increased in gdu1-6D (Figure 6b). Together, these

results indicate that enhanced expression of GDU1 displays

phenotypes similar to LSB1/GDU3.

DISCUSSION

The geminivirus–plant interaction still remains poorly

understood. In a screening for mutants resistant against

BSCTV infection in Arabidopsis activation tagged mutants,

we identified a mutant named lsb1. The study of lsb1

revealed that the elevated expression of LSB1/GDU3, a gene

implicated in amino acid metabolism and transport, was

able to impair replication of BSCTV DNA and confer reduced

susceptibility to BSCTV. We further demonstrated that acti-

vation of LSB1/GDU3 increased the expression levels of

components of the SA pathway that are known to counter

geminivirus infection, including the upstream regulator

ACD6. Thus, this study indicates that up-regulation of LSB1/

GDU3 affects geminivirus infection by activating the salicylic

acid pathway.

A new strategy to dissect the geminivirus–host interaction:

forward genetics

In the last decade, the main strategy used to dissect the

geminivirus–plant interaction was to determine host factors

that interact with viral proteins through a two-hybrid screen

and then examine the impact of these protein–protein

interactions by reverse genetics. This strategy is direct and

effective, because many host factors involved in the

geminivirus–plant interaction have been identified through

this strategy (Hao et al., 2003; Kong and Hanley-Bowdoin,

2002; McGarry et al., 2003; Xie et al., 1996). However, this

approach cannot uncover underlying processes in the virus–

host interaction besides protein–protein interaction. In

addition, as these protein–protein interactions are identified

outside the context of real viral infection, these observations

might even be somewhat biased.

Another strategy used to dissect the geminivirus–plant

interaction is mining genes whose expression is affected by

geminivirus infection through microarray technology and

then testing the involvement of these genes in the gemini-

virus–plant interaction by reverse genetics. This strategy is

also feasible and can provide a complete picture of the

geminivirus–plant interaction (Ascencio-Ibanez et al., 2008;

Lai et al., 2009; Trinks et al., 2005). Nevertheless, it seems

Ratio of symptomatic plants (%)

WT 97 ± 3

gdu1-6D 0 ± 0

gdu1-6DWT

GDU1

ACD6

PCC1

WAK1

PR5

Actin

(a)

(b)

Figure 6. Up-regulation of GDU1 displays phenotypes similar to LSB1/GDU3.

(a) Agroinoculation of Beet severe curly top virus (BSCTV) in wild-type (WT)

and gdu1-6D plants. Values shown are percentages of plants (n = 40, three

replicates � SD) that displayed systemic disease symptoms at 4 weeks post-

inoculation (OD600: 2.0).

(b) RNA gel blot analysis of GDU1, ACD6, PCC1, WAK1, PR5, and ACTIN1

expression levels in the gdu1-6D plants. Expression of ACTIN1 was used as an

internal control. This experiment was repeated twice with similar results.



difficult to locate the key nodes of the transcriptional

network which are essential, or at least important, for the

virus–host interaction due to the large set of genes uncov-

ered by microarray analysis. For example, although it has

been shown that geminivirus CaLCuV triggers a pathogen

response via the SA pathway, Arabidopsis mutant lines of

key regulators of this pathway (PAD4, MPK4, SID2, NPR1,

and WRKY70) displayed no obvious differences with respect

to the timing or severity of symptoms upon CaLCuV

infection compared with wild-type plants (Ascencio-Ibanez

et al., 2008).

Based on this context, we applied a new strategy,

forward genetics, to study the geminivirus–plant interac-

tion. The unbiased screen provides an opportunity to

isolate host mutations that affect any aspect of the virus–

host interaction, not just the level of interaction between

viral and host proteins. In addition, it would be easier to

reach the key nodes of the geminivirus–plant interaction

network through a phenotype-orientated screen. Using this

strategy, we successfully isolated and characterized one

mutant, in which geminivirus BSCTV DNA replication was

impaired and infectivity was reduced, and established the

involvement of LSB1/GDU3 during geminivirus–plant inter-

action. This indicates that the new strategy is feasible and

can be further applied to research into the geminivirus–

plant interaction.

Activation of LSB1/GDU3 or GDU1 may counter BSCTV

infection by activating the SA pathway

LSB1/GDU3 was originally identified as a member of a plant-

specific gene family that could be implicated in amino acid

transport (Pratelli et al., 2008; Pilot et al., 2004). However,

the molecular and biochemical identities of the members of

this family are still unclear. In this study, LSB1/GDU3 was

discovered to be involved in the geminivirus–host interac-

tion through a genetic screen for mutants resistant to the

geminivirus BSCTV; its overexpression attenuates replica-

tion of BSCTV DNA and renders Arabidopsis plants less

susceptible to BSCTV. This finding highlights the use of

geminiviruses as a tool to decipher the functions of plant

genes. Furthermore, we found that the elevated expression

of LSB1/GDU3 dramatically up-regulated the expression of

several genes involved in the SA pathway, including ACD6,

PCC1, WAK1, and PR5, and all of these genes, including

LSB1/GDU3 itself, responded to BSCTV infection. Combined

with the observation that cpr1, cpr5, and acd6-1, the three

mutants in which the SA pathway is constitutively activated,

can compromise geminivirus infection, these results sug-

gest that up-regulation of LSB1/GDU3 may activate the SA

pathway to impair geminivirus infection. In addition, we

found that enhanced expression of GDU1, a homolog of

LSB1/GDU3, displays phenotypes similar to LSB1/GDU3 in

both BSCTV resistance and up-regulation of SA pathway

components. This suggests that the members of this gene

family, in addition to LSB1/GDU3, might play similar roles in

the geminivirus–host interaction.

A previous study has established that SA treatment

interferes with tobacco mosaic virus (TMV, an RNA virus)

replication in TMV-susceptible tobacco tissues (Chivasa

et al., 1997). However, there has been no evidence that

activation of the SA pathway can also impair replication of

plant DNA viruses. In this study, our observations imply that

elevated expression of LSB1/GDU3 may activate the SA

pathway to attenuate replication of geminivirus DNA and

thus suggest that the same mechanism may also affect

replication of DNA viruses in plant cells.

Our study is based on the activation tagged mutant of

LSB1/GDU3, in which elevated expression of LSB1/GDU3

up-regulated SA-related genes and led to lower susceptibil-

ity to BSCTV. However, the knockdown alleles of LSB1/GDU3

exhibit no apparent phenotypes regarding the expression

levels of these SA-related genes and the susceptibility to

BSCTV (data not shown). This finding may be due to

functional redundancy, since there are five homologs of

LSB1/GDU3 in the Arabidopsis genome which share two

highly conserved domains (Pilot et al., 2004). In fact, the

original identification of the six members of the GDU family

and the 35 members of the ACD6 family were both attributed

to the isolation and characterization of gain-of-function

mutants, while the phenotypes of their loss-of-function

mutants have not been described or are not significant

(Pilot et al., 2004; Lu et al., 2003). This possibility actually

highlights the utility of activation tagged mutants in our

screen. In addition, previous studies have established that

individual knockout mutations, even in the key components

of the SA pathway, do not have a dramatic impact on

infection with RNA viruses and geminiviruses in compatible

hosts, which is in contrast to the enhanced disease suscep-

tibility that these mutations confer to some bacterial and

fungal pathogens (Huang et al., 2005; Ascencio-Ibanez et al.,

2008). Therefore, the observation that the single mutation of

LSB1/GDU3 did not lead to enhanced susceptibility to

BSCTV does not surprise us.

EXPERIMENTAL PROCEDURES

Plant growth, agroinoculation, and transformation

Seeds of A. thaliana plants were sown on Murashige and Skoog(MS) plates containing 1% agar and 1.5% sucrose, pH 5.7, stratifiedat 4�C for 2 days, and grown for about 2 weeks (16-h light, 22�C)before they were transferred to the soil to grow in a greenhouse(24�C,16-h light). The activation tagged mutants (ecotype Columbia,Col-7) for mass inoculation with BSCTV were obtained from theArabidopsis Biological Resource Center.

Rosette leaves of 4-week-old plants were agroinoculated withBSCTV (Briddon et al., 1989; Grimsley et al., 1986). Briefly, thesuspension of the A. tumefaciens strain EHA105 (Hood et al., 1993)containing pCambia-BSCTV (described below) with an opticaldensity at 600 nm (OD600) of 2.0, mixed with 1% carborundum(320 grit, C192-500; Fisher Scientific, http://www.thermofisher.com/),

20 Hao Chen et al.


was sprayed onto the leaves using the airbrush technique(Whitham et al., 1999) at an export pressure of 75–80 p.s.i. duringspraying. The inoculated plants were covered for one night forrecovery.

To generate transgenic plants, Arabidopsis (ecotype Col-7) wastransformed via floral dip (Clough and Bent, 1998) with A. tumefac-iens strain EHA105 harboring the appropriate plasmids. Transgenicplants were screened on MS plates containing kanamycin (50 lgml)1). The T3 homozygous lines were used for the inoculation assay.

Plasmid construction

An infectious clone of BSCTV (Stenger, 1994; Stenger et al., 1990),named pCFH (ATCC number: PVMC-6), was obtained from theAmerican Type Culture Collection (ATCC, http://www.lgcstandards-atcc.org/). To construct a partial tandem repeat of the BSCTV gen-ome on the binary vector pCambia1300 (CAMBIA), an EcoRI–BamHIfragment of pCFH (2.4 kb) was first cloned into pCambia1300digested with the same enzymes to generate pCambiaBSCTV0.8.The complete genome of BSCTV, excised with EcoRI from pCFH,was then cloned into pCambiaBSCTV0.8 linearized with EcoRI togenerate pCambiaBSCTV, which harbors 1.8 copies of the BSCTVgenome as a tandem repeat.

For the transgenic construct of LSB1/GDU3, a DNA fragmentcontaining the 447-bp coding sequence was obtained by RT-PCRwith the following primers: overGDU3F, 5¢-ATCGGATCCATGGA-AGGAAGACAATATTAC-3¢ and overGDU3R, 5¢-CTGACTAGTTCA-ATGTGTCTCACCGTTAC-3¢. This fragment was then cloned intothe binary vector pCanGHA as a transcriptional fusion with theCaMV 35S promoter. For the transgenic construct of the locusAT5G57690, a DNA fragment harboring the 2407-bp genomic DNAsequence from ATG to TGA was amplified by PCR using thefollowing primers: kinasegenomicF, 5¢-CATTCTAGAATGGAATC-ACCGTCGATTG-3¢ and kinasegenomicR, 5¢-GCTGAATTCTCAA-TCTCCTTTGACGACC-3¢. This fragment was then cloned intoanother binary vector named VIP96, which is transcribed underthe control of the 35S promoter. For the transgenic constructs oflocus AT5G57700, which has two splicing variants (1059 and1044 bp), these variants were obtained by RT-PCR with the follow-ing primers: overBNRLongF for the longer variant, 5¢-ATA-GTCGACATGAAAAGTTCTCAGATGTCAG-3¢; overBNRshortF forthe shorter variant, 5¢-ATAGTCGACATGTCAGAAACAGAATTTAAAG-3¢; and overBNRR for both variants, 5¢-TCGACTAGTTTAATT-AATGTTTGGTTCGTAC-3¢. The two variants were further cloned intothe binary vector pCanGHA.

GUS staining

Whole rosette leaves were first washed in 0.1 M sodium phosphatebuffer (pH 7.0) before vacuum infiltration for 10 min in stainingbuffer containing 1 mg ml)1 5-bromo-4-chloro-3-indolyl-b-D-glucu-ronic acid (X-Gluc), 5 mM potassium ferricyanide, 5 mM potassiumferrocyanide, 10% methanol, and 0.1 M sodium phosphate buffer(pH 7.0). The tissue was then stained at 37�C overnight.

Protoplast assay

Mesophyll protoplasts were isolated from 4-week-old Arabidopsis(Col-7) rosette leaves in the soil and transfected with pCam-biaBSCTV DNA based on the protocol described previously (Yooet al., 2007). Improvements of the original protocol include sterili-zation of the leaves in 70% ethanol for about 1 min before beingsubjected to enzyme digestion, and conduction of the whole oper-ation in sterile conditions. Transfected cells were kept in the dark atroom temperature. Approximately 3 · 105 cells were removed at 0,2, and 4 days post-transfection for DNA extraction. Total genomic

DNA was extracted from the cells according to a previouslydescribed protocol (Fontes et al., 1994). Newly replicated viral DNAwas identified by DNA gel blot with 32P-labeled BSCTV genome asthe probe.

Plasmid rescue

The method has been described previously (Weigel et al., 2000).Briefly, genomic DNA from lsb1 was extracted using the E.Z.N.A. SPPlant DNA Kit (Omega Bio-Tek, http://www.omegabiotek.com/),digested with HindIII or SpeI, ligated using T4 ligase (Fermentas,http://www.fermentas.com/), and transformed into XL1-blue MRF(Stratagene, http://www.stratagene.com/) competent cells. Geno-mic DNA was sequenced using the primers 5¢-TCTAGAT-CCGAAACTATCA-3¢ for the plasmid rescued with HindIII and5¢-TTGCGACAACATGTCGAGG-3¢ for the plasmid rescued withSpeI.

Nucleic acid gel bots

For the DNA gel blot, total DNA was extracted with cetyl trimethylammonium bromide (CTAB) buffer [2· CTAB, 100 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)-Cl, pH 8.0, 1.4 M NaCl,20 mM EDTA, pH 8.0, 2% CTAB, and 0.2% b-mercaptoethanol addedprior to use]. After depurination and neutralization, total DNA wastransferred to Hybond N+ nylon membranes (Amersham PharmaciaBiotech, http://www.gelifesciences.com) by upward capillarytransfer in 0.4 M NaOH solution. For the RNA gel blot, total RNA wasextracted by LiCl precipitation (Verwoerd et al., 1989). Appropriateamounts of total RNA, 30 lg for the detection of endogenous genesor 5 lg for the detection of overexpressed genes, were loaded andtransferred to Hybond N+ nylon membranes by upward capillarytransfer in 10· SSC solution.

Either DNA or RNA blots were hybridized at 65�C with DNAprobes labeled with [a)32P]dCTP using a Ready-Primed labeling kit(Amersham International). The primers used to probe for LSB1/GDU3 were probeGDU3F, 5¢-GAAGACAATATTACCCTCC-3¢, andprobeGDU3R, 5¢-CATCATGACCTAGATATGG-3¢; the primers usedto probe for AT5G57690 were probekinaseF, 5¢-ATGGAATCACC-GTCGATTG-3¢, and probekinaseR, 5¢-GAGGATCCACAATTTCACC-3¢; the primers used to probe for AT5G57700 were probeBNRF,5¢-CTTGAGACATTCACATTTCC-3¢, and probeBNRR, 5¢-GCTTGA-CTCCATCAATACC-3¢; the primers used to probe for ACD6 wereACD6UP, 5¢-CCTTCTATTCGAGCAAAACTC-3¢, and ACD6DOWN,5¢-TTTGCAGCCGAATGAATTGG-3¢; the primers used to probe forWAK1 were WAK1UP, 5¢-GGTGGCTATTTTCTTCTCCC-3¢, andWAK1DOWN, 5¢-TGCTCGCATGTCTGATTTCC-3¢; the primers usedto probe for PCC1 were Forward, 5¢-ACTGTCGACATGAATCAA-TCCGCGCAAAA-3¢, and reverse, 5¢-AGCACTAGTTTACTCTGATGT-ACAGAGG-3¢; and the primers used to probe for PR5 were Forward,5¢-GCCACAGACTTCACTCTAAG-3¢, and Reverse, 5¢-TAAACCTCTCA-CAGGCACTC-3¢. Signal intensities were measured using a Phos-phorImager (Bio-Rad, http://www.bio-rad.com).

ACKNOWLEDGEMENTS

We would like to thank the Arabidopsis Biological Resource Centerat Ohio State University for providing the activation tagged mutantsand Mr Sanyuan Tang for technical assistance. We are grateful toDr Guillaume Pilot (Carnegie Institution, USA), Dr Xinnian Dong(Duke University, USA), Dr Jianmin Zhou (National Institute ofBiological Sciences, China), and Dr Jean T. Greenberg (University ofChicago, USA) for providing the seeds of gdu1-6D, cpr1, cpr5, andacd6-1, respectively. This research was supported by grantsCNSF30325030/30530400 from the Chinese Natural Science Foun-dation (CNSF). QX is supported by grants KSCX2-YW-N-010 and



CXTD-S2005-2 from the Chinese Academy of Science. HG is sup-ported by CNSF grant 30525004.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Screening for mutants resistant to Beet severe curly topvirus (BSCTV) infection.Figure S2. lsb1 and 35S-LSB1/GDU3 were both smaller than therespective control plants.Figure S3. Confirmation of the T-DNA insertion site in lsb1 by PCR.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

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The Plant Journal 62 Up-regulation of LSB1 GDU3 affects ... Journal 2010-Chen … · Up-regulation of LSB1/GDU3 affects geminivirus infection by activating the salicylic acid pathway