Arch Virol (2006) 151: 2349–2363DOI 10.1007/s00705-006-0819-7

Post-transcriptional gene silencing in controllingviruses of the Tomato yellow leaf curl

virus complex

M. K. Abhary1, G. H. Anfoka1, M. K. Nakhla2, and D. P. Maxwell2

1Department of Biotechnology, Faculty of Agricultural Technology, Al-Balqa’AppliedUniversity, Al-Salt, Jordan

2Department of Plant Pathology, University of Wisconsin-Madison,Madison, WI, U.S.A.

Received December 29, 2005; accepted June 9, 2006Published online July 27, 2006 c© Springer-Verlag 2006

Summary. Tomato yellow leaf curl disease (TYLCD) is caused by a group ofgeminiviruses that belong to the Tomato yellow leaf curl virus (TYLCV) complexand are transmitted by the whitefly (Bemisia tabaci Genn.). The disease causesgreat yield losses in many countries throughout the Mediterranean region andthe Middle East. In this study, the efficacy of post-transcriptional gene silencing(PTGS) to control the disease caused by TYLCV complex was investigated. Non-coding conserved regions from the genome of TYLCV, Tomato yellow leaf curlvirus – mild, tomato yellow leaf curl Sardinia virus, tomato yellow leaf curlMalaga virus, and tomato yellow leaf curl Sardinia virus – Spain [2] were selectedand used to design a construct that can trigger broad resistance against differentviruses that cause tomato yellow leaf curl disease. The silencing construct wascloned into an Agrobacterium-binary vector in sense and antisense orientationand used in transient assay to infiltrate tomato and Nicotiana benthamiana plants.A high level of resistance was obtained when plants were agro-infiltrated withan infectious clone of the Egyptian isolate of TYLCV (TYLCV-[EG]) or chal-lenge inoculated with TYLCV, TYLCV-Mld, and TYLCSV-ES[2] using whitefly-mediated transmission 16–20 days post infiltration with the silencing construct.Results of the polymerase chain reaction showed that the resistance was effectiveagainst all three viruses. Furthermore, dot blot hybridization and PCR failed todetect viral DNA in symptomless, silenced plants. A positive correlation betweenresistance and the accumulation of TYLCV-specific siRNAs was observed insilenced plants. Together, these data provide compelling evidence that PTGS canbe used to engineer geminivirus-resistant plants.

2350 M. K. Abhary et al.

Introduction

Tomato yellow leaf curl disease (TYLCD) is caused by a complex of plantviruses that belong to two species of the genus Begomovirus in the familyGeminiviridae [8]. The disease causes severe damage to tomato (Lycopersiconesculentum Mill.) crops in many tropical and subtropical regions worldwide [15].Members of three species of the TYLCV complex are known to cause TYLCDin the Mediterranean basin: tomato yellow leaf curl virus (TYLCV, formerlyTYLCV-Israel), tomato yellow leaf curl Sardinia virus (TYLCSV, formerlyTYLCV-Sardinia) and tomato yellow leaf curl Malaga virus (TYLCMalV) [1, 17,23, 41]. In addition to the Mediterranean basin, these viruses cause serious lossesin tomato crops in the tropical Africa, Southeast Asia, North America, SouthAmerica, and the Caribbean region [15, 31] and are transmitted by the whitefly(Bemisia tabaci Genn.) (Homoptera: Aleyrodidae) in a circulative manner [13].Most begomoviruses possess a bipartite genome, composed of two approximately2.6-kb DNA components. TYLCV is unusual in that it has a monopartite genome,composed of a single genomic DNA. The genome consists of six open readingframes (ORFs) encoding proteins approximately 10 kDa or greater [3, 34]. ORFsV1 and V2 (encoding the coat protein [CP]), are in the viral sense and overlap,whereas the other four ORFs, C1 (encoding the replication-associated protein[Rep]), C2, C3, and C4, are in the complementary sense and also overlap. Anapproximately 300-nt intergenic region (IR) contains the origin of replication,which includes a potential stem-loop structure that is involved in the rolling-circlereplication of viral ssDNA [21, 24].

In Jordan, TYLCV was first reported in 1978 as the causal agent of TYLCD[27]. Since then, TYLCV has spread to all main vegetable-producing regions andbecome the limiting factor for tomato production during the summer and fall,causing up to 100% yield loss. In addition to TYLCV, tomato yellow leaf curlvirus – Mild (TYLCV-Mld) and tomato yellow leaf curl Sardinia virus – Spain [2](TYLCSV-ES[2]) were recently reported in Jordan [2].

Farmers in Jordan use traditional control measure such as chemical control ofthe whitefly vector, use of yellow traps and fine screens, and intercropping withcucurbits to reduce the damage caused by TYLCV complex viruses, however,these measures have failed to make tomato production profitable under severeinfection.

RNA silencing is a host defence mechanism targeted against invasive ormobile RNA elements, such as viruses or transposable retro-elements, leadingto sequence-specific RNA degradation [6, 12, 28, 39]. It is highly conservedin plants and animals and known as post-transcriptional gene silencing (PTGS)in plants. PTGS was discovered in plants, and a closely related phenomenon,RNA interference, is known to occur in a wide range of organisms includingCaenorhabditis elegans and Neurospora crassa [14, 18, 32]. This strategy is ableto adapt to all sorts of viruses, because its specificity is dictated by the sequenceof the viral genome itself. Another feature of PTGS is that a signal that triggerssilencing is not restricted to individual plant cells, but can spread from the site ofinfection, generating a response in more distant tissues [29].

Post-transcriptional gene silencing of TYLCV complex viruses 2351

Silencing can be triggered in plants by replicating viruses, double-strandedRNA molecules, and foreign genes (transgenes) that allow the production of highlevels of normal or aberrant messenger RNAs [29, 42, 43], which become dsRNAby host-encoded RNA-dependent RNA polymerase activity. These dsRNAs arecleaved by Dicer-like enzymes into short interfering RNAs (siRNAs) of between21 and 26 nt in length, which then promote RNA degradation by forming a multi-component RNA-induced silencing complex that destroys cognate mRNA [16, 43].

A PTGS-based strategy to control gemeniviruses was demonstrated whentobacco and tomato plants were transfected with dsRNA derived from the AC1gene ofAfrican cassava mosaic virus and transgenes developed from the intergenicregion (IR) and the Rep gene of TYLCV plants were highly resistant to cottonleaf curl virus and TYLCV, respectively [4, 45].

Geminiviruses, in spite of being ssDNA viruses with no dsRNA phase in theirreplication cycle, have been shown to be involved in the gene silencing pathway.They are able to trigger the host PTGS system with the production of specific virus-derived siRNAs and have evolved an active mechanism to counteract silencing[11, 25]. The uniqueness of geminiviruses is that they encode at least two typesof PTGS suppressor proteins such as AC2 and AC4, indicating that these virusesevolved differently with regard to interaction with the host.

The objective of this work was to explore the use of non-coding conservedregions of the TYLCV genome to develop a construct that can trigger an efficientresistance in tomato and N. benthamiana against members of two species in theTYLCV complex.

Materials and methods

Plant materials and insect cultures

Three week-old seedlings of tomato, local line (981), which is highly susceptible to TYLCV,and thirty day-old N. benthamiana plants were planted in plastic pots, kept in an insect-proofgreenhouse and used in all experiments of this study. The whiteflies (B. tabaci Genn.) weremaintained on cotton (Gossypium hirsutum L.) plants in insect-proof cages held at 26 ◦C.

Construction of TYLCV infectious clone

Tomato leaves showing severe TYLCD symptoms were collected from the Ismailia gover-norate in Egypt and used to develop an infectious clone of TYLCV. Total DNA was extractedfrom these leaves as previously described [40]. The primer pairs PTYV1v1164/PTYC1C2196and PTYC1v2182/ PTYC3c1320 (Table 1) were used to amplify two fragments, A and B,respectively. These fragments were digested with SphI/PstI and PstI/NcoI, and ligated topGEM-5zf+ to get pTYEG14. The monomer and dimer were obtained when the pTYEG14was digested with SphI and cloned into pGEM5zf+. A head-to-tail dimer of the monomericinsert from pTYEG14 was cloned into pGEM-5zf, creating pTYEG7. The dimer was re-moved from this plasmid and cloned into pSL1180 plasmid using ScaI and PstI restrictionenzymes. Then digested with ScaI and BamHI and cloned in the binary vector pBin302to give the infectious TYLCV-[EG] clone, which has been sequenced and deposited in theGenBank (AY594174). Agrobacterium tumefaciens strain LBA 4404 was transformed withthe recombinant plasmid, and the infectivity was tested by agro-infiltration of tomato andN. benthamiana plants using the syringe-spot method [22].

2352 M. K. Abhary et al.: Post-transcriptional gene silencing of TYLCV complex viruses

Construction of the silencing trigger construct

The silencing trigger construct (STC) was designed based on non-coding conserved re-gions of different viruses belonging to the TYLCV complex. These viruses were TYLCV(X15656),TYLCV-Mld (X76319),TYLCSV-ES[2] (L27708), tomato yellow leaf curl Malagavirus (TYLCMalV) (AF271234), and tomato yellow leaf curl virus-Sardinia TYLCSV-(Sar)(X61153) [17]. The computer software DNAMANTM (Lynnon BioSoft, Quebec, Canada) wasused for alignment analysis. Three noncoding fragments of the virus genome denoted C1C2,C2C3, andV1V2 (Fig. 1A), were selected and amplified using specific oligonucleotide primers(Table 1) that had BamHI/AscI or XbaI/SwaI restriction sites. The polymerase chain reaction(PCR) consisted of 3 µl (10–20 ng) DNA, 2.5 µl of 10× PCR buffer, 2.5 µl Mg2+ (25 mM),2.5 µl of dNTPs (1 µM), 1 µl of each primer (10 µM), and 5 U of Taq polymerase (Promega,WI, USA) in a total volume of 25 µl. PCR cycle parameters were as follows: denaturation at94 ◦C for 3 min followed by 30 cycles of 94 ◦C for 1 min, annealing at 55 ◦C for 45 sec, exten-sion at 72 ◦C for 1 min, one cycle at 94 ◦C for 1 min, 55 ◦C for 1 min, and 72 ◦C for 5 min. Theamplified fragments (C1C2, C2C3 and V1V2) were cloned separately into the pCRII vectorusing the TA cloning kit (Invitrogen, CA, USA) according to the manufacturer’s instructions.After that, fragments C1C2 and C2C3 were removed from their vectors by restriction digestion

Fig. 1. Maps of TYLCV and the silencing trigger construct. A Genome organization of theEgyptian isolate of tomato yellow leaf curl virus (TYLCV-[EG]). Open reading frames (ORFs)are shown as black arrows; V denotes ORFs on the virion strand, and C denotes ORFs onthe complementary strand. IR indicates the intergenic region. Boxes indicate the position ofthe non-coding regions of the virus genome selected to design the silencing trigger constructand the primer pair used to amplify each fragment. B A diagram demonstrating the binaryvector (pFGC5941) engineered with the silencing trigger construct. LB left border of T-DNA;RB right border of T-DNA; p35S CaMV35S promoter; V1V2, C1C2 and C2C3 fragments ofTYLCV genome used to develop the silencing construct and arranged in sense and antisenseorientations; CHSA chalcone synthase A gene; BAR gene encoding phosphinothricin acetyl

transferase

Tabl

e1.

Des

crip

tion

ofol

igon

ucle

otid

epr

imer

sus

edin

this

stud

y

Prim

erSe

quen

ce5′

→3′

Ntp

ositi

onTa

rget

Ref

eren

ce

STC

TY

C2C

3FA

TAT

CT

CG

AG

TTA

AG

AA

AC

GA

CC

AG

TC

TG

AG

GC

1235

–125

7T

his

stud

yT

YC

2C3R

TTA

AA

AG

CT

T AT

GG

AT

TC

AC

GC

AC

AG

GG

GA

AC

1482

–146

3C

2C3

TY

C1C

2FA

TATA

AG

CT

TT

TAC

GC

CT

TAT

TG

GT

TT

CT

TC

TT

GG

C15

41–1

566

TY

C1C

2RA

TAT

GG

AT

CC

AT

TTA

AA

T AT

GC

AA

CC

TT

CG

TC

AC

CC

TA16

32–1

614

C1C

2

TY

V1V

2FT

CA

TT

CTA

GA

GG

CG

CG

CC

CG

AA

GG

TT

CG

CC

GA

AG

GC

TG

350–

369

TY

V1V

2RA

TT

TC

TC

GA

GA

TAT

CA

TC

CC

GC

TG

CT

CA

TAA

G55

7–53

6V

1V2

STC

vT

CA

TT

CTA

GA

GG

CG

CG

CC

CG

AA

GG

TT

STC

cA

TAT

GG

AT

CC

AT

TTA

AA

TAT

GC

AA

CC

STC

TY

LC

VPT

YIR

v21

TT

GA

AA

TG

AA

TC

GG

TG

TC

CC

21–4

1N

akhl

aet

al.(

1993

)C

ompl

exPT

YIR

c287

TT

GC

AA

GA

CA

AA

AA

AC

TT

GG

GA

CC

287–

263

IR

TY

v233

7A

CG

TAG

GT

CT

TG

AC

AT

CT

GT

TG

AG

CT

C23

37–2

364

IRof

TY

c138

AA

GT

GG

GT

CC

CA

CA

TAT

TG

CA

AG

AC

138–

125

TY

LC

VT

Ym

V26

64A

TT

GA

CC

AA

CA

TT

TT

TAC

AC

TTA

TC

CC

2664

–269

1IR

ofA

nfok

aet

al.[

2]T

Yc1

38A

AG

TG

GG

TC

CC

AC

ATA

TT

GC

AA

GA

C13

8–12

5T

YL

CV

-[M

ld]

Alm

V25

16T

TT

TAT

TT

GT

TG

GT

GT

TT

GTA

GT

TG

AA

G25

16–2

544

IRof

Alm

C11

5A

TAT

TG

AT

GG

TT

TT

TC

AA

AA

CT

TAG

AA

G11

5–86

TY

LC

SV-E

S[2]

TY

LC

V-

PTY

V1v

1164

GTA

CG

AG

AA

CC

ATA

CT

GA

AA

AC

GC

CT

967–

992

Thi

sst

udy

[EG

]PT

YC

1C21

96A

AA

TC

TG

CA

GA

TG

AA

CTA

GA

AG

AG

TG

GG

2024

–205

1Fr

agm

entA

PTY

C1v

2182

TAG

GC

CA

TG

GC

CG

CG

CA

GC

GG

AA

TAC

AC

G19

86–2

010

PTY

C3c

1320

GG

TT

CT

GC

AG

CA

GA

GC

AG

TT

GA

TC

AT

GTA

TT

G11

47–1

168

Frag

men

tB

2354 M. K. Abhary et al.

using BamHI-HindIII and HindIII-XhoI and religated together into the pBluescript KS+TM

vector (Stratagen, WA, USA). Recombinant pBluescript KS+TM was digested again withHindII-XhoI to excise the C1C2–C2C3 fragment. This fragment (C1C2–C2C3) was thenligated back to the pCRII vector that harbored the V1V2 fragment in the BamHI-XhoI site.The full-length STC (C1C2, C2C3 and V1V2) in the pCRII vector was digested with AscI andSwaI to give the sense orientation and with BamHI andXbaI to give the antisense orientation,leaving the chalcone synthase intron in the middle. Finally, the STC in sense and antisenseorientations was ligated into the pFGC5941 binary vector (Fig. 1B) and used to infiltratetomato and N. benthamiana plants as previously described [22].

Transient delivery of the silencing trigger construct

Recombinant A. tumefaciens cells harboring the binary vector pFGC5941 that contain the STCwere grown overnight at 28 ◦C in 50-ml flasks containing LB medium and 5 µg/ml kanamycin.When the OD600 reached 0.6–1.0, cells were harvested by centrifugation at 5000 rpm for5 min. Bacterial cells were resuspended in MS medium containing vitamins to a final OD600 of0.3–0.5. Using a 5-ml syringe, the bacterial solution was injected into leaves of N. benthamianaor the fully expanded second leaf of tomato plants [22]. Plants infiltrated with A. tumefaciensharboring an empty pFGC5941 vector were used as control. Six days after infiltration, theexpression of the STC in infiltrated leaves was confirmed by PCR using the STCv/STCcprimer pair (Table 1).

Challenge inoculation

Challenge inoculation of silenced and control tomato and N. benthamiana plants was doneby whitefly-mediated transmission and by agro-infiltration.

Whitefly-mediated transmission

The virus isolate that was used throughout this study for whitefly-mediated transmissionconsisted of a mixture of two TYLCV stains (TYLCV, TYLCV-Mld) and TYLCSV-ES[2] thatoriginated from a tomato field in the Jordan Valley and was maintained in tomato by whitefly-mediated transmission. In summary, whitefly transmission was done as follows: adult non-viruliferous whiteflies were given a 48-h acquisition access period on tomato plants infectedwith TYLCV complex. After that, whiteflies were provided with a 48-h inoculation accessperiod on STC-infiltrated (silenced) and control plants, at a density of 50–100 whitefliesper plant. Following inoculation, plants were sprayed with Imidacloprid (Confidor, Bayer,Leverkusen, Germany), kept in an insect-proof greenhouse, and monitored for developmentof symptoms.

Agro-infiltration

Leaves of tomato and N. benthamiana previously infiltrated with STC were agro-infiltratedwith the TYLCV-[EG] infectious clone as follows: the bacterial culture was grown overnight at28 ◦C and then diluted to OD600 0.3–0.5. Plant leaves were infiltrated using the A. tumefaciens-mediated transient expression system [22].

Resistance assay

The severity of TYLCD symptoms on silenced and control plants was assessed at 14, 21,and 28 days post inoculation and graded according to the scale previously described [19]: 0(no visible symptoms, inoculated plants show same growth and development as uninoculatedplants), 1 (very slight yellowing of leaflet margins on apical leaf), 2 (some yellowing andminor curling of leaflet ends), 3 (a wide range of leaf yellowing, curling, and cupping, with

Post-transcriptional gene silencing of TYLCV complex viruses 2355

some reduction in size, yet plants continue to develop), and 4 (very severe plant stunting andyellowing and pronounced cupping and curling; plants cease to grow).

Minimum time needed to trigger the resistance against TYLCV complex

A time-course analysis of STC-expressing tissue was done to examine the initiation of theRNA silencing and, subsequently, the resistance against TYLCV complex. Tomato plantswere infiltrated with the STC as previously mentioned and then divided into two groups. Thefirst group was challenge inoculated with the infectious TYLCV-[EG] clone, while the secondgroup was challenged by viruliferous whiteflies. Leaf tissues were collected from control andsilenced tomato plants 5, 10, 15 and 20 days post inoculation and used for virus and siRNAdetection.

Detection of TYLCV complex

Non-radioactive dot blot hybridization and PCR were used to detect TYLCV, TYLCV-Mld,and TYLCSV-ES[2] in silenced and control plants. Total DNA was extracted from leaf tissuesusing a modification of the Dellaporta heat extraction method [38]. For dot blot hybridization,a general DNA probe was developed from the IR of TYLCV as previously described [2] andused to detect TYLCV complex using the Alk Phos Direct Hybridization kit (AmershamPharmacia, Piscataway, NJ, USA) according to the manufacturer’s instructions. In addition,PCR was applied to detect the TYLCV complex in silenced and control plants using specificprimer pairs (Table 1) [2].

Extraction of TYLCV-specific siRNA

The siRNAs were extracted from control and silenced tomato plants as follows: Leaf tissueswere collected from the uppermost fully expanded leaf of tomato plants challenge inoculatedwith viruliferous whiteflies 4, 8, 12, 16 and 20 days post STC infiltration. Total RNA wasextracted from 1g leaf tissue as previously described [20]. Low-molecular-weight RNA wasprecipitated by suspending total RNA in 1 M NaCl2 with 20% PEG 8000 and centrifuged at15,000 rpm for 20 min. The supernatant was transferred to a new tube and siRNAs wereprecipitated with two volumes of 95% ethanol. The resulting pellet was resuspended in20 µl ddH2O. siRNAs were subjected to polyacrylamide gel electrophoresis using an 8%polyacrylamide gel, and bands were visualized after staining the gel with eithium bromide.After that, siRNAs were transferred into a nylon membrane and fixed using UV crosslinker(Fluo-Link FLX, Vilber Lourmat, Eberhardzell, Germany). To identify the siRNA, two probeswere developed from the C1C2 and IR regions of the TYLCV genome and used in dot blothybridization.

Results

Evaluation of the resistance triggered against TYLCV-[EG]

The silencing construct developed in this study was able to trigger a high levelof resistance in tomato and N. benthamiana plants against the most commonviruses of the TYLCV complex. As shown in Table 2, all silenced tomato andN. benthamiana plants failed to develop TYLCD symptoms when they werechallenge inoculated with TYLCV-[EG] 16 days post STC-infiltration, whereas,control plants showed typical disease symptoms three weeks post inoculation.The disease severity in control plants increased with time, and at the end ofthe experiments (28 days post inoculation) all control N. benthamiana plants

2356 M. K. Abhary et al.

Table 2. Efficiency of the resistance triggered against TYLCV-[EG]

Days post TYLCV inoculation

14 21 28

Plant/treatment Control STC Control STC Control STC

L. esculentum 0 ± 0.0 0 ± 0.0 1 ± 0.90 0 ± 0.0 2 ± 0.88 0 ± 0.0N. benthamiana 0 ± 0.0 0 ± 0.0 2 ± 1.0 0 ± 0.0 4 ± 0.0 0 ± 0.0

Severity of tomato yellow leaf curl disease caused by TYLCV-[EG] in silenced and controlplants. Plants were challenge inoculated with infectious TYLCV-[EG] clone 16 days postSTC-infiltration. Data present the mean ± standard deviation. The experiment was repeatedthree times and each treatment consisted of 5 replicates

developed severe symptoms, while, control tomato plants showed yellowing andmild leaf curling.

To determine if the resistance induced by the STC affected the cell-to-cellmovement of TYLCV particles, which subsequently leads to a delay in symptomdevelopment, a few silenced (symptomless) and control (mild symptoms) tomatoplants were kept under greenhouse conditions and observed for symptom develop-ment. Sixty days after challenge inoculation, none of the silenced plants showeddiseases symptoms, whereas, severe TYLCV symptoms were observed in controlplants.

Time period between construct infiltration and challenge inoculation

The time interval between STC infiltration and the induction of resistance againstthe TYLCV complex was investigated. The data presented in Fig. 2A show that allcontrol and silenced tomato and N. benthamiana plants developed typical TYLCDsymptoms when they were challenged by viruliferous whiteflies 4 days postSTC infiltration. However, a significant decrease in the number of silencedN. benthamiana and tomato plants that showed TYLCD symptoms was observedwhen challenge inoculation was done 8 days post STC-infiltration. The high-est level of resistance in N. benthamiana could be achieved when plants werechallenged 12 days post STC-infiltration. At this time, the average number ofsymptomatic silenced tomato plants did not exceed 1. This number deceased whenthe time interval between STC-infiltration and challenge inoculation increased to20 days. However, most of the control tomato plants showed disease symptoms.

The same time interval (8 days) was also necessary to trigger the resis-tance in silenced tomato and N. benthamiana plants when they were challengedwith the infectious TYLCV-[EG] clone. As demonstrated in Fig. 2B, all silencedN. benthamiana plants that were challenged 12 days post infiltration were resistant,whereas 4.7% of control plants developed TYLCD symptoms. On the otherhand, 2.3% of silenced tomato plants showed typical disease symptoms whenthey were challenged 12 days post inoculation. When the time interval between

Post-transcriptional gene silencing of TYLCV complex viruses 2357

Fig. 2. Time period between infiltration of tomato (dashed line) and N. benthamiana (rigidline) plants with the silencing construct and the induction of the resistance triggered againstthe TYLCV complex. Plants were either infiltrated with an empty vector (� − � � − �) orwith a vector harboring the silencing construct (� − � • − •) and challenge inoculated withviruliferous whiteflies carrying TYLCV, TYLCV-Mld, and TYLCSVES[2] (A) or with theinfectious TYLCV-[EG] clone (B). Disease development was recorded until the end of theexperiment (28 days post inoculation). Data represent the mean of three experiments. Each

treatment consisted of 5 replicates. Bars represent standard deviation

STC infiltration and challenge inoculation increased to 16 days, the number ofsymptomatic tomato plants did not exceed 1. The highest level of resistance wasobserved when tomato plants were challenged 20 days post STC infiltration.

Effect of gene silencing on the accumulation of TYLCV

To investigate the effect of the resistance on the accumulation of viral DNA, leaftissues from all treatments shown in Fig. 2 were collected at five-day intervalsand used for dot blot hybridization. The intensity of the signals on the membraneswas compared with the intensity of different concentrations of TYLCV amplicon

2358 M. K. Abhary et al.

(250, 125, 62.5, 31 and 15 ng/ml). Hybridization results confirmed the data ob-tained from the disease progress curve (Fig. 2). When plants were challenged 4 or8 days post STC-infiltration, viral DNA could be detected in control as well as insilenced plants 10 days post inoculation. The concentration of virus in these plantsranged between 15 and 30 ng/µl. This is in accordance with the data presented inFig. 2A, where more than 3.3% of plants challenged 8 days post STC infiltrationdeveloped disease symptoms. On the other hand, when the time interval betweenSTC-infiltration and challenge inoculation increased to 16 or 20 days, no viralDNA could be detected in silenced plants, while detectable levels of viral DNAcould be observed in control plants 5–10 days after challenge inoculation (datanot shown).

PCR is a very sensitive technique and can detect low concentrations of thetarget DNA. Therefore, all symptomless plants that were challenge inoculated 16and 20 days post STC-infiltration were subjected to PCR using specific primerpairs. The expected sizes of TYLCV (634 bp), TYLCSV-ES[2] (433 bp), andTYLCV-Mld (316 bp) could only be amplified from control plants challenged withthe TYLCV complex, whereas no bands could be detected when DNA extractedfrom control, unchallenged, or silenced tomato plants was used as template in thePCR reaction (Fig. 3).

Accumulation of siRNA

To investigate the correlation between the resistance and the abundance ofTYLCV-specific siRNAs, RNA fractions were extracted from tomato plants shown inFig. 2B, 20 days post inoculation and enriched for siRNAs. siRNAs could onlybe detected in silenced plants, suggesting that siRNA might have originated from

Fig. 3. Detection of viruses of the tomato yellow leaf curl virus complex by polymerase chainreaction in tomato plants. Plants were agro-infiltrated with either an empty vector (control)or with a vector harboring the silencing trigger construct (silenced) and 20 days later werechallenge inoculated with TYLCV complex (TYLCV, TYLCSV-ES[2] and TYLCV-Mld)using viruliferous whiteflies. Total DNA preparations were obtained 30 days post challengeinoculation from tomato plant used as inoculum source (1–3); control plants challenged withTYLCV complex (4, 7 and 10); silenced plants (5, 8 and 11); control, unchallenged plants(6, 9 and 12). PCR was performed using TYLCV (1 and 4–6), TYLCSV-ES[2]-(2 and 7–9)

and TYLCV-Mld-(3 and 10–12) specific primers

Post-transcriptional gene silencing of TYLCV complex viruses 2359

the TYLCV genome. To test this hypothesis, siRNA were hybridized with twoprobes corresponding to the C1C2 and IR regions of the TYLCV genome. Datafrom dot blot hybridization showed that siRNAs hybridized with the C1C2 probebut not with the IR probe (not shown). A hybridization signal of high intensitywas observed when siRNA was extracted from plants challenged inoculated 12,16, and 20 days post STC infiltration.

Discussion

TYLCV is among the most important limiting factors that affect tomato productionin many Mediterranean countries. Recently, several laboratories worldwide havetried to introduce resistance to TYLCV by transforming plants with viral-derivedsequences [5, 7, 9, 34–36]. Nevertheless, only partial resistance has been obtained,suggesting that TYLCV possesses some special features that make it able toovercome pathogen-derived resistance. In the light of previous studies that showedthe ability of TYLCV to form new recombinants with other members of its speciesin mixed infected hosts [30, 33], the aim of this work was to apply the technology ofgene silencing to trigger resistance against different viruses that cause TYLCD. Toachieve this goal, the conserved regions of five viruses belonging to the TYLCVcomplex that represent the most sensitive domains of the virus, including thereplicase active site [10], the transcription enhancer, promoter, the start codon ofthe coat protein, and the silencing suppressor C2 [26], were selected to develop asilencing construct. Sequence alignment showed high similarities in (V1V2, C1C2and C2C3) regions between TYLCV, TYLCV-Mld, TYLCMalV, TYLCSV-ES[2]and TYLCSV. Previous studies demonstrated that small fragments of the TYLCVRep gene alone can induce a homologous silencing effect that can be broken byother virus species [25]. Similarly, it has been reported that different genes or frag-ments of the TYLCV genome can induce a high level of resistance, depending onthe construct design [45]. Effective silencing construct designs are reviewed in [44].

Although agro-inoculation is a common technique used in different studies toinfect plants, we included whitefly-mediated inoculation to challenge silencedplants to examine the response of these plants when they were subjected tohigh pressure from TYLCV. The data presented in this study demonstrate thatthe developed silencing construct was very efficient in protecting tomato andN. benthamiana plants against the TYLCV complex. It is also interesting tonote that the level of resistance in N. benthamiana challenged by viruliferouswhiteflies or agro-infiltration was almost the same. None of the silenced plantsshowed disease symptoms when they were challenged 12 days post infiltrationand no viral DNA could be detected either by hybridization or by PCR. On theother hand, the technique used for challenge inoculation affected the level of theresistance triggered in tomato plants. For example, in tomato plants challengeinoculated 12 days post STC infiltration, the number of silenced, symptomless,plants was at least two times more in plants challenged by viruliferous whitefliesthan in plants challenged using the infectious TYLCV-[EG] clone. A possibleexplanation for this is that whitefly-mediated transmission was less efficient indelivering the virus to the vascular tissues than agro-infiltration. This might be true,

2360 M. K. Abhary et al.

since in ago-infiltration the whole lower surface of tomato leaves were infiltratedwith the bacterial culture harboring the virus genome, and therefore the numberof introduced viral particles using this technique might be much higher thanwhen introduced by whiteflies. In addition, in whitefly-mediated transmission,three viruses were introduced at the same time into tomato cells. These virusesmight compete with each other for the cell’s resources, and this was not the casewhen plants were challenged with agro-infiltration. Further investigations on therole of mixed infection on the level of resistance triggered against the TYLCVcomplex have to be done in which silenced and control plants are challengedwith TYLCV, TYLCV-Mld, and TYLCV-ES[2] separately. In contrast to tomato,12 days between STC infiltration and challenge inoculation were enough totrigger a high level of resistance in N. benthamiana. The ease of infiltration ofN. benthamiana leaves with STC, compared to tomato leaves, might contribute tothe high level of resistance obtained. In addition, 12 days might be long enoughfor the silencing signals to move from STC-infiltrated N. benthamiana cells toupper plant tissues but not enough to reach all upper parts of tomato plants. Thisis in agreement with the fact that the highest level of resistance in tomato plantswas obtained when plants were challenged 20 days post STC infiltration. This isin accordance with previous studies that showed a time period is required for thesilencing signals to move to different plant parts [37].

Although the Agrobacterium-mediated transient expression assay is a rapidand useful method to analyze genes and gene products in plants [22], the successof this technique to deliver the transgene of interest to all infiltrated tissue dependson many factors among which the texture of the tissues to be infiltrated and thepressure used to inject the bacterial suspension are the most important. Therefore,it was not expected that infiltrated tomato and N. benthamiana cells would receivethe same amount of STC which, subsequently, would affect the level of resistanceobtained in these plants. This might explain the data shown in Fig. 2, wherefew silenced tomato plants developed TYLCD symptoms even though they werechallenged 20 days post STC infiltration.

Since the aim of this study was to develop a construct that can provide ahigh level of resistance against different viruses of TYLCV complex, it wasimportant to test the stability of the resistance triggered by this construct whensilenced plants were challenged by more than one virus known to cause TYLCD.To accomplish this, plants were challenged with TYLCV, TYLCV–Mld, andTYLCSV-ES[2] by whitefly transmission. The absence of detectable viral DNAin symptomless silenced plants by either nucleic acid hybridization or PCR makesthe resistance demonstrated in this study superior to that of commercially availableresistances derived from wild Lycopersicon spp. This suggests that the resistancemay be immunity.A growing body scientific reports have demonstrated that plantstransformed with constructs that produce siRNAs are capable of inducing a highlevel of resistance against DNA and RNA viruses [4, 5].

Several attempts have been made to engineer tomato plants resistant to TYLCVvia a gene silencing strategy. In some cases the resistance has been overcome whensilenced plants were challenged with other strains of the virus that can silence the

Post-transcriptional gene silencing of TYLCV complex viruses 2361

homologous transgene. For example, it has been showed that transgenic expressionof a truncated form of TYLCSV replication-associated protein (Rep210) confersresistance to the homologous virus but not to a Spanish isolate (TYLCSV-ES1)[25]. Contrary to these results, data presented in this work showed that STC wasable to trigger a high level of resistance in tomato and N. benthamiana plantsagainst three viruses belonging to two species of TYLCV complex. To shedmore light on the stability of the resistance developed in this study, transgenictomato plants expressing the STC should be evaluated under field conditionswhere high virus pressure is expected during the spring and autumn seasons inthe Jordan Valley. Furthermore, field experiments will provide more informationon the effect of different PTGS suppressors on the resistance triggered by thedeveloped silencing construct, since tomato is a natural host for many viruses, likecucumber mosaic virus, tobacco mosaic virus and potato virus Y, which possesthis feature.

Since the STC reported in this work was developed based on the conservedregions of different viruses of TYLCV complex, it will be interesting to knowif it will also be effective against members of other species of TYLCV likeTYLCMalV, tomato yellow leaf curl Thailand virus, and tomato yellow leaf curlChina virus.

Acknowledgment

This research was supported in part by the Middle East Research and Cooperation (MERC)project M21-037.

References1. Accotto GP, Navas-Castillo J, Noris E, Moriones E, Louro D (2000) Typing of tomato

yellow leaf curl viruses in Europe. Eur J Plant Pathol 106: 179–1862. Anfoka GH, Abhary M, Nakhla MK (2005) Molecular identification of species of the

Tomato yellow leaf curl virus complex in Jordan. J Plant Pathol 87: 61–663. Antignus EY, Cohen S (1994) Complete nucleotide sequence of an infectious clone of a

mild isolate of tomato yellow leaf curl virus (TYLCV). Phytopathology 84: 707–7124. Asad S, Haris WA, Bashir A, Zafar Y, Malik KA, Malik NN, Lichtenstein CP (2003)

Transgenic tobacco expressing geminiviral RNAs are resistant to the serious viralpathogen causing cotton leaf curl disease. Arch Virol 148: 2341–2352

5. Baulcombe DC (1996) Mechanisms of pathogen-derived resistance to viruses intransgenic plants. Plant Cell 8: 1833–1844

6. Baulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing. CurrOpin Plant Biol 2: 109–113

7. Bendahmane M, Gronenborn B (1997) Engineering resistance against tomato yellow leafcurl virus (TYLCV) using antisense RNA. Plant Mol Biol 33: 351–357

8. Briddon RW, Markham PG (1995) Family Geminiviridae. In: Murphy FA, Fauquet CM,Bishop DHL, Ghabrial SA, Jarvis AW, Martelli GP, Mayo MA, Summers MD (eds) VirusTaxonomy: Classification and Nomenclature of Viruses. Sixth Report of the InternationalCommittee on Taxonomy of Viruses. Springer, Wien, New York, pp 158–165

9. Brunetti A, Tavazza R, Noris E, Lucioli A, Accotto GP, Tavazza M (2001) Transgenicallyexpressed T-Rep of tomato yellow leaf curl Sardinia virus acts as a trans-dominant-negative mutant, inhibiting viral transcription and replication. J Virol 75: 10573–10581

2362 M. K. Abhary et al.

10. Campos-Olivas R, Louis JM, Clerot D, Gronenborn B, Gronenborn AM (2002) Thestructure of a replication initiator unites diverse aspects of nucleic acid metabolism.PNAS 99: 10310–10315

11. Chellappan P, Vanitharani R, Fauquet CM (2004) siRNA accumulation correlates withhost recovery in DNA virus infected hosts and gene silencing targets specific viralsequences. J Virol 78: 7465–7477

12. Chicas A, Macino G (2001) Characteristics of post-transcriptional gene silencing.EMBO Rep 2: 992–996

13. Cohen S, Antignus Y (1994) Tomato yellow leaf curl virus, a whitefly-borne geminivirusof tomatoes. Adv Dis Vector Res 10: 259–288

14. Cogoni C, Macino G (1999) Gene silencing in Neurospora crassa requires a proteinhomologous to RNA-dependent RNA polymerase. Nature 399: 166–169

15. Czosnek H, Laterrot H (1997) A worldwide survey of tomato yellow leaf curl viruses.Arch Virol 142: 1391–1406

16. Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and22-nucleotide RNAs. Genes Dev 15: 188–200

17. Fauquet CM, Bisaro DM, Briddon RW, Brown JK, Harrison BD, Rybicki EP, Stenger DC,Stanley J (2003) Revision of taxonomic criteria for species demarcation in the familyGeminiviridae, and an updated list of begomovirus species. Arch Virol 148: 405–421

18. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent andspecific genetic interference by double stranded RNA in Caenorhabditis elegans. Nature391: 806–811

19. Friedmann M, Lapidot M, Cohen S, Pilowsky M (1998) A novel source of resistance totomato yellow leaf curl virus exhibiting a symptomless reaction to viral infection. J AmSoc Hortic Sci 123: 1004–1007

20. Gauthier ER, Madison SD, Michel RN (1997) Rapid RNA isolation without the use ofcommercial kits: application to small tissue samples. Pflugers Arch – Eur J Physiol 433:644–668

21. Hanley-Bowdoin L, Settlage SB, Orozco BM, Nagar S, Robertson D (1999)Geminiviruses: models for plant DNA replication, transcription, and cell cycleregulation. Crit Rev Plant Sci 18: 71–106

22. Johansen LK, Carrington JC (2001) Silencing on the spot. Induction and suppressionof RNA silencing in the Agrobacterium-mediated transient expression system. PlantPhysiology 126: 930–938

23. Kheyr-Pour A, Bendahmane M, Matzeit V, Accotto GP, Crespi S, Gronenborn B (1991)Tomato yellow leaf curl virus from Sardinia is a whitefly-transmitted monopartitegeminivirus. Nucleic Acids Res 19: 6763–6769

24. Laufs J, Traut W, Heyraud F, Matzeit V, Rogers SG, Schell J, Gronenborn B (1995) Invitro cleavage and joining at the viral origin of replication by the replication initiatorprotein of tomato yellow leaf curl virus. Proc Natl Acad Sci USA 92: 3879–3883

25. LucioliA, Noris E, BrunettiA, Tavazza R, RuzzaV, CastilloAG, Bejarano ER,Accotto GP,Tavazza M (2003) Tomato yellow leaf curl Sardinia virus rep-derived resistance tohomologous and heterologous geminiviruses occurs by different mechanisms and isovercome if virus-mediated transgene silencing is activated. J Virol 77: 6785–6798

26. LukeA, Randles J, Rezaian M (2004) Host responses to transient expression of individualgenes encoded by tomato leaf curl virus. MPMI 17(1): 27–33

27. Makkouk KM (1978) A study on tomato viruses in the Jordan Valley with specialemphasis on tomato yellow leaf curl. Plant Dis Rep 62: 259–262

28. Matzke MA, Matzke AJM, Pruss G, Vance VB (2001) RNA-based silencing strategiesin plants. Curr Opin Gene Devel 11: 221–227

29. Meins F Jr (2000) RNA degradation and models for post-transcriptional gene silencing.Plant Mol Biol 43: 261–273

Post-transcriptional gene silencing of TYLCV complex viruses 2363

30. Monci F, Sanchez-Campos S, Navas-Castillo J, Moriones E (2002) A naturalrecombination between the geminiviruses Tomato yellow leaf curl Sardinia virus andTomato yellow leaf curl virus exhibits a novel pathogenic phenotype and is becomingprevalent in Spanish populations. Virology 303: 317–326

31. Moriones E, Navas-Castillo J (2000) Tomato yellow leaf curl virus, an emerging viruscomplex causing epidemics worldwide. Virus Res 71: 123–134

32. Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthasegene into petunia results in reversible co-suppression of homologous genes in trans.Plant Cell 2: 279–289

33. Navas-Castillo J, Sanchez-Campos S, Noris E, Louro D, Accotto GP, Moriones E (2000)Natural recombination between Tomato yellow leaf curl virus-Is and Tomato leaf curlvirus. J Gen Virol 81: 2797–2801

34. Noris E, Hidalgo E, Accotto GP, Moriones E (1994) High similarity among the tomatoyellow leaf curl virus from the west Mediterranean basin: The nucleotide sequence ofan infectious clone from Spain. Arch Virol 135: 165–170

35. Noris E, Accotto GP, Tavazza R, Brunetti A, Crespi S, Tavazza M (1996) Resistance totomato yellow leaf curl geminivirus in Nicotiana benthamiana plants transformed witha truncated viral C1 gene. Virology 224: 130–138

36. Noris E, Lucioli A, Tavazza R, Caciagli P, Accotto GP, Tavazza M (2004) Tomatoyellow leaf curl Sardinia virus can overcome transgene-mediated RNA silencing of twoessential viral genes. J Gen Virol 85: 1745–1749

37. Palauqui JC, Elmayan, Pollien JM, Vaucheret H (1997) Systemic acquired silencing:transgene-specific post-transcriptional silencing is transmitted by grafting from silencedstocks to non-silenced scions. EMBO J 16: 4738–4745

38. Potter JL, Nakhla MK, Mejıa L, Maxwell DP (2003) PCR and DNA hybridizationmethods for specific detection of bean-infecting begomoviruses in the Americas andCaribbean. Plant Dis 87: 1205–1212

39. Prins M, Goldbach R (1996) RNA-mediated virus resistance in transgenic plants. ArchVirol 141: 2259–2276

40. Rojas MR, Gilbertson RL, Russell DR, Maxwell DP (1993) Use of degenerate primersin the polymerase chain reaction to detect whitefly-transmitted geminiviruses. Plant Dis77: 340–347

41. Sanchez-Campos S, Dıaz JA, Monci F, Bejarano ER, Reina J, Navas-Castillo J,Aranda MA, Moriones E (2002) High genetic stability of the begomovirus Tomatoyellow leaf curl Sardinia virus in southern Spain over an 8-year period. Phytopathology92: 842–849

42. Voinnet O (2001) RNA silencing as a plant immune system against viruses. TrendsGenet 17: 449–459

43. Waterhouse PM, Wang MB, Lough T (2001) Gene silencing as an adaptive defenceagainst viruses. Nature 411: 834–842

44. Wesley SV, Helliwell CA, Smith NA, Wang M, Rouse DT, Liu Q, Gooding PS, Singh SP,Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001)Construct design for efficient, effect and high throughput gene silencing in plants. Plant J27: 581–590

45. Yang Y, Sherwood TA, Patte CP, Hiebert E, Polston JE (2004) Use of Tomato yellowleaf curl virus (TYLCV) Rep gene sequences to engineer TYLCV resistance in tomato.Phytopathology 94: 490–496

Author’s address: Ghandi H. Anfoka, Department of Biotechnology, Faculty of Agri-cultural Technology, Al-Balqa’ Applied University, Al-Salt 19117, Jordan; e-mail: anfoka@wanadoo.jo