research article use of posttranscription gene silencing in...

Research ArticleUse of Posttranscription Gene Silencing inSquash to Induce Resistance against the Egyptian Isolate ofthe Squash Leaf Curl Virus

Omnia Taha1 Inas Farouk1 Abdelhadi Abdallah2 and Naglaa A Abdallah2

1Agricultural Genetic Engineering Research Institute ARC Cairo 12613 Egypt2Department of Genetics Faculty of Agriculture Cairo University Cairo 12619 Egypt

Correspondence should be addressed to Naglaa A Abdallah naglaaabdallahagrcuedueg

Received 10 November 2015 Revised 12 January 2016 Accepted 3 February 2016

Academic Editor Anita Nag

Copyright copy 2016 Omnia Taha et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Squash leaf curl virus (SqLCV) is a bipartite begomovirus affecting squash plants It is transmitted by whitefly Bemisia tabacibiotype B causing severe leaf curling vein banding and molting ending by stunting In this study full-length genomic clone ofSqLCVEgyptian isolated and posttranscriptional gene silencing (PTGS) has been induced to develop virus resistanceTheNoubariaSqLCV has more than 95 homology with Jordon Israel Lebanon Palestine and Cairo isolates Two genes fragment from SqLCVintroduced in sense and antisense orientations using pFGC5049 vector to be expressed as hairpin RNA The first fragment was348 bp from replication associated protein gene (Rep) The second fragment was 879 bp representing the full sequence of themovement protein gene (BC1) Using real-time PCR a silencing record of 97 has been recorded to RepTrAP construct as aresult it has prevented the appearance of viral symptoms in most tested plants up to two months after infection while constructcontaining the BC1 gene scored a reduction in the accumulation of viral genome expression as appearing in real-time PCR results46-fold giving a silencing of 79 which had a positive effect on symptoms development in most tested plants

1 Introduction

The familyCucurbitaceae comprisesmany vegetable and fruitcrops including cucumbers squashes luffas melons andwatermelons Squash (Cucurbita pepo) is a major vegetablecrop grown all over the world from the cold region to thetropical one [1] The world production of squash reportedby FAO statistics for 2002 exceeded 177 million tons from24million ha China is the most important producer on theworld while in Africa Egypt is one of the 10 top producersranking as the eighth country from the world production ofsquash depending on last report of FAO 2012

There aremany pests that significantly affect the yield andfruit quality of squash Among these diseases viral diseasesare more important as they cause heavy economic lossesThere are more than 35 viruses that have been isolated fromthe family Cucurbitaceae [2]

Geminiviridae represents the largest family of plant DNAviruses that infect a broad range of plants causing severe

losses in the yield of economically important crops withintropical and subtropical regions Geminiviruses are single-stranded circular DNA viruses that cause severe damageto wide range of economically important crops worldwide[3] They are characterized by twinned icosahedra particlesand have emerged globally as one of the most economicallyimportant pathogens Begomovirus is the largest genus of theGeminiviridae family whose genus has two types classifieddepending on their genome into monopartite and bipartiteBipartite begomoviruses genome has either one or two circu-lar single strand DNA named components A and B each oneof them has its specific role in the virus life cycle [4] Compo-nent A contains 5 genes named replication associated protein(Rep or AC1) replication enhancer protein (REn or AC3)transcription activator protein (TrAP or AC2) AC4 and coatprotein (CP or AV1) Component B contains 2 genes namedmovement protein (MP or BC1) and nuclear shuttle protein(NSP or BV1) [5] NSP and MP genes play significant rolein virus movement either from the nucleus to the cytoplasm

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2016 Article ID 6053147 9 pageshttpdxdoiorg10115520166053147

2 International Journal of Genomics

or from cell to cell through plasmodesmata respectivelySquash leaf curl virus (SqLCV) is transmitted by its whiteflyvector Bemisia tabaci and it induces severe stunting and leafcurling in squash plants and causes severe losses in cucurbitproduction The first record of SqLCV in the Middle Eastwas in Israel in 2003 subsequently it has spread to neighborcountries such as Egypt [6] Jordan [7] Palestine [8] andLebanon [9] The spreading of the SqLCV within Egypt wasquickly as it took 5 years to spread from one governorateto another [6 10 11] Restriction enzymes of RCA productscombined with a cloning strategy showed being a veryefficient method to obtain full genome sequences This tech-nique allowed the easy detection cloning and sequencingof a mixed infection and the identification of the respectivebegomoviruses on the same sample [12ndash14]

The small interference RNA (siRNA) or posttranscrip-tional gene silencing (PTGS) occurs naturally in plants andacts as an antiviral defense mechanism The siRNA has beenwidely used as amethod to engineer resistance against virusesin plants including nuclear-replicating geminiviruses whichhave no double-stranded RNA phase in their replicationcycleTheproduced virus specific siRNA from the breakdownof dsRNA induces the degradation of homologous mRNAsequences resulting in hindering the virus form expressingits genes thus limiting virus replication process [15]

In this study the two viral components of the EgyptianSqLCV Noubaria isolate were cloned and their sequenceswere submitted to the GenBank from the cloned viralgenome two regions one from early and one from lateexpressed genes which were used to trigger PTGS againstSqLCV using agroinfiltration as a transient experimentmethod The first region is located at the overlapped regionbetween Rep and TrAP genes The second one represents thegene BC1 which has an important role in virus spreadingfrom cell to cell causing systemic infection

2 Materials and Methods

21 Cloning Viral Genome Geminivirus-infected squashplants were collected from different cultivated open fieldsof Noubaria village at Beheira Governorate The collectedplant samples showed symptoms of Squash leaf curl virus(SqLCV) such as severe systemic stunting and leaf curlingNonviruliferous whiteflies (Bemisia tabaci) were obtainedfrom rearing whiteflies on healthy cotton plants in an isolatedinsectary for at least amonthWhiteflies were fed for amonthon infected squash plants collected from the field to acquirethe virus and used to infect healthy squash plants

Total DNA from infected plants was extracted by usingCTAB method [16] Preliminary PCR for viral detection wascarried out using the degenerate primers V324 (+) primer 51015840-GCCYATRTAYAGRAAGCC MAG-31015840 and C889 (minus) primer51015840-GGRTTDGARGCATGHGTACATG-31015840 specific for white-fly transmitted geminiviruses [17] to amplify 570 bp fragmentFor cloning the complete viral genome the amplifiedmethodwas used Total DNA extracted from infected squash plantswas subjected to 12059329 amplification essentially according tothe manufacturerrsquos instructions (Fermentas) The amplifiedproducts were denatured at 65∘C for 10min and run on

a 1 agarose gel to confirm amplification The ampliPhiproduct was digested independently with selected restrictionenzymes that is EcoRI HindIII BamHI SalI SacI PstIXbaI BglII KpnI NcoI XmaI Xho1 SmaI SphI and SwaI(Fermentas fast digest) and separated in an agarose gel Theenzymes that could cut at a single site in the genome gener-ating genome size fragment was selected for further cloningin pBluescript II SK (minus) Sequencing was carried out andwas subjected to GenBank (GenBank accession A KP795975B KP795976)

22 Primer Design and PCR Amplification To assess silenc-ing effect of the selected viral fragments potentially useful inthe control of SqLCV two fragments were selected from theviral sequences to cover partial RepTrAP and full BC1 genesTwo sets of primers were designed to amplify the selectedregions by using Vector NTI Software RepTrAP primersequences are 51015840-CCCACATAATTACTTGAGCGGCCAT-31015840 and 51015840-TTAGCAATCCTGTGCTGTGCTTTGA-31015840 whileBC1 gene primer pairs are 51015840-ATGGGTTCTCAATTAGTT-CCACCC-31015840 and 51015840-GCTTAGGGATTTTGGAGTGCT-CGGG-31015840 Restriction sites XhoI and XmaI added to theforward primers and AscI and BamHI to the reverse primersto facilitate cloning in sense and antisense forms in the binaryvector pFGC5041within the intron regionThe expected sizesof the selected regions were 348 bp and 879 bp for clonedviral genome to amplify the selected region The sizes ofthe selected fragments were 348 bp and 879 bp for RepTrAPand BC1 genes respectively PCR was carried out using FlexiGoTaq DNA polymerase (Promega USA) Cloned viralgenome was used as a template in PCR reaction with aconcentration of 100 ng 5120583L of 5x Flexi GoTaq buffer 2120583Lof 25mMMgCl

2 05 120583L of 10mMdNTPs 05 120583L 10 pmol of

each primer 025120583L GoTaq enzyme and ddH2O to reach a

total volume of 25120583L the amplification was carried out in aBioRadT100USAThe reactionwas performedwith an initialcycle at 94∘C for 4min followed by 30 cycles of denaturationstep at 94∘C for 1min annealing at 50∘C for 30 sec and anextension cycle at 72∘C for 1min followed by a final cycle at72∘C for 7min

23 Cloning of Silencing Fragments The amplified fragmentswere cloned initially into pGEM-Teasy Vector System sub-sequently RePTrAP and BC1 fragments were digested usingXhoI andXmaI andAscI andBamHI for cloning in pFGC5041silencing binary vector (supplied by Richared JorgensenUniversity of Arizona USA) Each fragment was cloned intothe expression vector pFGC5041 in which the transgeneis cloned in both orientations separated within the ChsAintron and under the control of the 35S promoter and theOSC terminatorThe cloning of amplified fragments has beencarried out in two steps by using the inner restriction siteson the primers Double digestion with FastDigest enzymesXmaI and BamHI was used for the sense orientation into theplant vector pFGC5041 the outer restriction enzymes pairXhoI and AscI were used to clone the antisense orientationAll enzymes used for cloning were FastDigest restrictionenzymes supplied fromThermo Scientific

International Journal of Genomics 3

After confirming the presence of the gene fragments bysequencing pFGC-RepTrAP and pFGC-BC1 were trans-formed to electrocompetent Agrobacterium LB4404 by elec-troporation [18] Electroporation was carried out using Bio-Rad Gene Pulser set at 25 120583F 200Ω and 25 kV Transformedbacteria were detected by colony PCR using specific primersfor each construct

24 Transient Expression of Silencing Fragments in SquashPlants Squash plants (Cucurbita pepo cv Eskandrani) weregrown in the biocontainment greenhouse in the AgricultureGenetic Engineering Research Institute (AGERI) at 28∘Cwith 16 hr light8 hr dark For each treatment twenty plantswere used Plants at the stage of two true leaves wereinfiltrated with the recombinant Agrobacterium Infiltrationmedia were prepared as follows 5mL overnight culturewas used to inoculate 50mL of LB medium containing 1 1kanamycin and 05 1 streptomycin and incubated overnightat 28∘C Upon centrifugation of the 50mL overnight cultureit has been resuspended in infiltration media containing10mMMgCl

2 10mM MES and 20120583M acetosyringone and

then was incubated at room temperature for 2 hr till itsOD600

reached 20 Agrobacterium harboring senseantisenseRepTrAp construct and senseantisense BC1 construct infil-trated employing needleless 5mL syringe using the SyringeSpotted Technique developed by Johansen and Carrington[19] After 1 week from infiltration viruliferous whitefliesfeed on infiltrated plant for 48 hr inoculation access periodThe treated plants were sprayed with imidacloprid (ConfidorBayer Leverkusen Germany) to get rid of the whitefliesPlants infiltrated with silencing constructs were assayed forpositive inoculation with specific PCR primers for each frag-ment PCR-positive plants were evaluated for their resistanceto the viral infection

25 Resistance Evaluation Observation of symptom devel-opment was done in 30 days after inoculation The accu-mulation of viral DNA in infected agrobacterium plants wasdetermined by RT-qPCR using the (leaves weight 05ndash101 g)total DNA extracted from upper leaves [20]

Primers for RT-qPCR were designed by using Inte-grated DNA Technologies website tools [21] The frag-ment of BC1 amplified by 51015840-CGGATTTGACCAGTC-TTCTCTC-31015840 and 51015840-GGGCACCGACAGTAAAGATAC-31015840 as forward and reverse primer respectively resulted infragment with length 102 bp In case of RepTrAP a frag-ment with length 105 bp was produced by those primers51015840-TATCCAAGGTGCGATGTTCC-31015840 as forward and 51015840-ATGTTCCTCTCTGGCCATTC-31015840 as reverse one Anneal-ing temperature was 60∘C for both fragments while 18srRNA was used as internal control for gene expressionSquash plants were kept for 10 days after virus infectionthen samples were collected for RT-qPCR detection andtotal RNA was extracted using SV Total RNA IsolationSystem cDNAwas synthesized by using 31015840 SMARTerRACEcDNA Amplification Kit The RT-qPCR was performed in aAB7300 Real-Time PCR System (Applied Biosystems USA)using SYBR Premix ExTaqII from TAKARA (Catalognumber RR82SW TAKARA Japan) RT-qPCR analysis was

M 87654321

Figure 1 PCR amplification on squash plant samples with degener-ate primers from Cp gene of geminivirus M Molecular Marker 1 kbFermentas lanes 1ndash6 squash plant samples lane 7 positive controland lane 8 negative control

M 1 2 3 4 5 6 7 8 9 10

26 kb

3kb2kb1kb

Figure 2 Restriction enzyme digestion of TempliPhi product ofsquash leaf curl virus M Marker 1 kb Fermentas lane 1 SacI lane 2XhoI lane 3 EcoR I lane 4BamH I lane 5NotI lane 6 SmaI lane 7XmaI lane 8 NcoI lane 9 SwaI and lane 10 undigested TempliPhiproduct

performed as described by manufacturer In real-time PCRprimers were tested using conventional PCR resulting in theamplification of fragments monitored at the expected sizewhere Rep primers gave a 105 bp fragment while BC1 gave a102 bp fragment with no primer dimer development

3 Results

31 Squash Leaf Curl Virus Isolation All plant samplesfrom different fields gave the expected band with PCR usingthe degenerate primer of CP core region indicating thatthese samples were infected with geminivirus (Figure 1) Thedigestion of amplified products resulted in 4 unique cutswith enzymes EcoRI BglII BamHI and SalI (Figure 2) whileonly Xba1 enzyme cut twice (Figure 3) Linearized fragmentswith the expected size were cloned in the correspondingsite into the E coli vector pSKminus Three constructed plasmidsfrom cloning of the linearized genome with EcoRI BamHIand SalI were sent for sequencing and the sequences wereconfirmed by similarity search using BLAST at National Cen-ter for Biotechnology Information (NCBI) Results showedthat EcoRI gave component B while SalI and BamHI gavecomponent A

Identity ratio showed high similarity results reaching99 homology to component A with other SqLCV isolatesComponent B showed less similarity than component A but


M 1 2 3 4 5 6 7

26 kb3kb2kb

13 kb

Figure 3 Restriction enzyme digestion of TempliPhi product of squash leaf curl virus M Marker 1 kb Fermentas lane 1 NotI lane 2 BglIIlane 3 PstI lane 4 HindIII lane 5 XbaI lane 6 KpnI and lane 7 undigested TempliPhi product

AC4

DNA-A DNA-B

AC1

AC3

AC2

347bp

347bp

AV1

XhoI XmaI BamHI AscI XhoI XmaI BamHI AscI

BC1

BV1

849bp

849bp

RepTrAP fragment BC1 gene

Figure 4 Illustration of silencing fragments RepTrAP and BC1 sites on full genome of squash leaf curl virus

the identity ratio is still high giving 99homologywithCairoisolate (DQ2850201) 98 homology with Palestine isolate(KC4414661) Jordon isolates (JX4445741 and JX1312821)Israel isolate (HQ1844371) and Lebanon (HM3683741) 96homology with Kaliobeya isolate (KJ5799541) and 95homology with California isolate (DQ2850171) and Arizonaisolate (DQ2850181)

32 Generation of the RNAi Construct and Squash PlantTransformation To study SqLCV resistance using siRNAstrategy on squash plants two putatively effective fragmentsfrom the whole virus genome of the SqLCVA Noubariawere chosen to form short dsRNA structure for enhancingposttranscriptional gene silencing (Figure 4) The first frag-ment was designed targeting the RepTrAP with a length of

347 bp while the second fragment was designed to containthe full BC1 gene with length of 849 bp PCR analysis wascarried out to amplify both fragments giving the expectedband size as showed in Figure 5 The insertion of the firstfragment in the antisense orientation into the pFGC5041vector was confirmed by restriction digestion using XhoI andAscI enzymes while BamHI and XmaI enzymes were usedto confirm the insertion of the second fragment in the senseorientation The full RNAi cassette expression was driven bythe e35S promoter and the OSC 31015840 terminator (Figure 6)Positive clones of transformed Agrobacterium LB4404 withconstructs pFGC5041-Rep and pFGC5041-BC1were screenedusing colony PCR technique (data not shown)

To assess the expression of silencing fragments C pepoplant leaveswere infiltrated in a transientmanner Plants were


M 1 2

1

500bp

Figure 5 Molecular isolation for squash leaf curl virus genes using PCR with primers specific to Rep fragment and BC1 gene M Marker100 bp plus Fermentas lane 1 Rep 31015840 end fragment from base 701 to 1049 bp and lane 2 full BC1 gene

LB LB

RB RB

Step 1 ligation Step 2 ligation

P-35S

P-35S

CHSA intron CHSA intron

1st orientation 1st orientation

2nd orientation

Xho1

Xma1

Xma1

Xma1

BamHI BamHI

BamHI

AscI

OCS-3998400OCS-3998400

Figure 6 Schematic representation of the cloning strategy of the fragments RepTrAP and BC1 in sense and antisense orientation into thebinary vector pFGC5941 done in two steps using the inner and outer restriction enzyme sites

then left for a week and subsequently subjected to infectionwith viruliferous whiteflies raised on infected squash plantswith SqLCV To detect the presence of viral genome ininfiltrated plants PCR was carried out after inoculation on adaily basis Primers of SqLCV coat protein gene were used inconfirming PCR as a positive control After the 7th day afterinfection noninfiltrated control plants gave positive resultsin PCR however pFGC-RepTrAP and pFGC-BC1 infiltratedplants gave negative results in PCR To monitor quantitativeviral accumulation in infiltrated squash plants real-time PCRanalyses were performed on infiltrated plants

33 Evaluation of the Resistance Imparted by the RNAi Con-structs by Transient Assay

331 Monitoring Viral Symptoms All noninfiltrated squashplants inoculated with viruliferous whiteflies carrying theSqLCV developed typical viral symptoms with severe yel-lowing leaf curling and a reduced leaflet size Such plantsceased to grow failing to flower and produce fruits while theuntreated plants remained the negative control (Figure 7(a))

One month after inoculation by the pFGC-RepTrAPvector all plants were symptomless while after two months


(a) (b)

(c) (d)

Figure 7 Symptoms evaluation after 2 months after infection (a) Health squash plant (b) infected plants with typical SqLCV symptoms (c)infiltrated plant with pFGC-RepTrAP produced plants with mild symptoms and (d) infiltrated plant with pFGC-BC1 produced plants withmild and moderate symptoms

Table 1 Symptom severity of SqLCV during 2 months on infiltrated squash plants after SqLCV-viruliferous whiteflies feed on it

Treatment Total number ofplants treated

Symptoms after 1 month Symptoms after 1 monthNo symptoms + ++ +++ No symptoms + ++ +++

pFGC-BC1 construct 5 25 35 05 05 15 35 15 05pFGC-RepTrAP construct 5 55 05 05 05 45 15 05 05pFGC5041 empty vector 5 05 25 35 05 05 05 15 45minusve control untreated 5 55 05 05 05 55 05 05 05

only 1 plant of the total 5 plants gave mild symptoms = +(Figure 7(c)) Meanwhile in the case of infiltrating the plantswith pFGC-BC1 construct one month after inoculationplants produced 2 symptomless plants and 3 plants with mildsymptoms By the end of the second month only 1 plantshowedbeing symptomless 3withmild symptoms and 1withmoderate symptoms = ++ (Figure 7(d)) (Table 1) Controlplants treated with empty pFGC5041 showed early symptomsafter two weeks and severe symptoms after one month ofinoculation (Figure 7(b))

332 Quantitative Screening of Viral Gene Expression Quan-titative analysis using RT-qPCR assaywas carried out to studythe level of the expression of the two viral genes RepTrAPand BC1 in the infiltrated squash Total RNA extracted frominfiltrated plants was used to synthesize cDNA and wassubjected to RT-qPCR In addition RT-qPCRwas performedfrom infectedC pepo plants as a positive control reaction andnontemplate DNA reaction that represent negative controlwhich had Ct values equivalent to the total number of cyclesused in the RT-qPCR experiment resulting in curves that


Table 2 Quantitative real-time PCR estimation of viral expression levels for SqLCV in plants infiltrated by pFGC-RepTrAP and pFGC-BC1

RepTrAP gene BC1 geneCt ofcontrol

Ct of controlreference

Ct oftreatment

Ct of treatmentreference

Ct ofcontrol


Ct oftreatment


Replica 1 2426 1647 2928 1628 2707 1692 2941 1664Replica 2 2455 1600 2939 1619 2794 1677 2989 167Replica 3 2407 1638 2987 1598 2745 1689 2944 1682Average 2429 1628 2951 1615 2748 1686 2958 1672ΔCt = Cts minus Ctrlowast 801 1301 1063 1286ΔΔCt = Ctc minus Cttlowast 5 223Fold of change = 2ΔΔCt 003125 0213Silencing = (1 minus ΔΔCt) times 100 97 79lowastCts = Ct values of sample Ctr = Ct value of housekeeping gene for the same sample Ctc = Ct values of control Ctt = Ct values of treatment

79

0

02

04

06

08

1

12

Relat

ive g

ene e

xpre

ssio

n

ControlBC1RepTrAP

97

Figure 8 siRNA-mediated silencing of RepTrAP and BC1 wasassessed using ΔΔCt method to determine relative gene expressionfrom qPCR data using 18s rRNA as an endogenous reference geneThe cells exhibited siRNA of RepTrAP reduced the mRNA level by97 while siRNA of BC1 reduced mRNA level by 79 comparedwithmRNA of control samples which fully expressed the squash leafcurl virus genes

did not achieve the threshold level thus indicating that thesereactions contained no detectable viral DNA

The level of virus detection was significantly lower inall infiltrated plants than in the control plants Infiltratedplants with pFGC-RepTrAP construct showed significantshifting in Ct value compared with the control Average Ctresulting fromRe pFGC-RepTrAP treatment was 2951 whilean average Ct value of control plants was 2429 In pFGC-RepTrAP samples the fold of change is calculated using theΔΔCt and it showed a 32-fold decrease thus silencing percentwas calculated to reach 97 Also pFGC-BC1 constructdisplayed a slight shifting in Ct values compared with that inthe control Treated samplesCt values averagewas 2958whilecontrol Ct values average was 2748 resulting in a decrease47-fold (Table 2) The calculated expression of BC1 gene hasdecreased by 79 giving showing a good degree of plantresistance against the respected virus but not as efficient aspFGC-RepTrAP construct (Figure 8)

4 Discussion

SqLCV has become amajor concern for cultivation of squashin the Middle East region Thus it is important to study thevast diversity of SqLCV and also the epidemic implications

Small circular DNA is preferentially amplified by RCAwhich makes the ssDNA genome of the geminivirusesideal substrate for RCA In addition during viral repli-cation through complementary strand synthesis (CSR) ofthe circular ssDNA rolling circle replication (RCR) andrecombination-dependent replication (RDR) various DNAintermediates are produced [22] In this work the rolling cir-cle amplification (RCA) technology was used for molecularcloning of the SqLCV genome because of its simplicity highsensitivity and proofreading against misincorporation ofnucleotides It can be used to amplify sequences without thenecessity of previous knowledge of it [12 23] The sequenceof the SqLCV-Noubaria isolate obtained in this work showedhigh similarities to other isolates in the region The lowsequence variation observed among SqLCV isolates in theMiddle East region was explained by Lapidot et al [24] Theypredict that uncontrolledmovement of viruliferous whitefliesamong countries causes viruses readilymoved across countryboundaries in this region preventing strong genetic differen-tiation of these viruses among neighbor countries

Studies on crops harboring virus resistance indicatedthe possibility of engineering the resistance by differenttransgene-based approaches [25] During the past decadethere has been considerable evidence for the successful usingof siRNA to protect plants against viruses in plum papayawatermelon and potato which has led to the developmentof virus-resistant transgenic crops [26ndash30] In this studythe same technology has been used by the incorporation ofsense and antisense fragments separated in pFGC5041 vectorwithin the CHSA intron to be transcribed and hybridizedwith itself to form a hairpin structure [31] However RNAsilencing degree induced in infiltrated plants could be diverseaccording to the gene used

Two regions of squash leaf curl viral genomewere selectedfor cloning the first was RepTrAP and the second was thefull length of BC1 gene The selected region RepTrAP frag-ment was chosen according to its location representing theend of theRep gene and the beginning ofTrAP in overlapping


manner which may be putatively stronger than one of themalone as it could lead to the silencing of both of them while incase of BC1 gene it was selected because of its important func-tion in systemic spreading of viral genome through the plas-modesmata as its silencing attempt is expected to limit theinfection and inhibit virus transmission from cell to cell

The two constructed plasmids pFGC-RepTrAP andpFGC-BC1 were prepared and used to infiltrate squashplants Resistance was measured either by monitoring symp-toms development or by real-time PCR In real-time PCRthree replicates from each construct were tested whereasdetection of gene expressionwasmeasured by threshold cycle(Ct) Thus higher Ct value means less amount of template inthe sample which indicates fewer titrations of virus particlesDepending on previous studies using the same virus 18srRNAwas chosen to be used as the reference gene in real-timePCR [32] To calculate relative gene expressionΔΔCtmethodwas used where target gene expressionwas normalized to ref-erence gene expression within the same sample to correct anyvariation that could affect the study [33]

Results of RepTrAP fragment in real-time PCR haveproved that there is a vital role for this fragment on the Repgene expression as it has been decreased 32-fold and a silenc-ing of 97has been achieved through this constructThis sig-nificant result could be due to the key role of the Rep proteinwhich is the most essential protein for viral replication [34]In addition transcription activator protein gene is overlappedwith Rep gene by 50 bp at its 31015840 end this overlapping hasplayed a significant role in silencing of both genes and gavemore efficient silencing percent this could be clear becausethe transcription activator protein alone is acting as a genesilencing suppressor [35] so its early silencing may help theplants to resist higher

The construct that contains the movement protein geneshowed a degree of resistance accomplishment but being lessefficient than the RepTrAP construct A decrease in the virusquantity 47-fold has been achieved thismay be due to the lateexpression of this gene in the virus machinery or it may bedue to the effect of its longer fragment size In former studiesfragments size showed efficient silencing ranged from sim110 to550 bp [36 37] while in the case ofBC1 gene used in this studyit was 879 bp so it might be a factor that affect the silencingpercentage

Symptoms developed in all treated plants in each treat-ment completed the story with the real-time PCR results bygiving ratio in plant resistance in case of pFGC-RepTrAPconstruct more than pFGC-BC1 construct

We have demonstrated that hairpin constructs expressingeither 348 bp of the RepTrAP gene or 879 bp for the BC1genes can control SqLCV However siRNA produced fromusing the RepTrAP construct proved to be more efficientin controlling the virus than BC1 construct It is clear fromseveral studies that the siRNA strategy is more efficient at the51015840 and 31015840 ends of the gene [36]

5 Conclusion

Both the RT-PCR and the symptoms screening at the green-house showed that the expression level of the Rep gene as

one of the early genes could play the most significant rolein silencing when it is employed as a posttranscriptional genesilencing and tool for controlling the squash leaf curl virusThe BC1 gene could also give a degree silencing but we provedthat it is less effective than the Rep gene Therefore the 31015840end fragment of the Rep gene for producing stable transfor-mation should be used to measure effect of construct on thetransgenic C pepo plants and reach high level of resistance toSqLCV on large populations in the field

Abbreviations

SqLCV Squash leaf curl virusDNA Deoxyribonucleic acidRNA Ribonucleic acidRep Replication associated proteinBC1 Movement proteinTrAP Transcription activator proteinsiRNA Small interference RNAPTGS Posttranscriptional gene silencing

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] A S Moffat ldquoPlant pathology geminiviruses emerge as seriouscrop threatrdquo Science vol 286 no 5446 p 1835 1999

[2] M Ozaslan T Aytekin B Bas I Halil Kilic I Didem Afacanand D S Dag ldquoVirus diseases of cucurbits in Gaziantep-Turkeyrdquo Plant Pathology Journal vol 5 no 1 pp 24ndash27 2006

[3] S Mansoor R W Briddon Y Zafar and J Stanley ldquoGemi-nivirus disease complexes an emerging threatrdquo Trends in PlantScience vol 8 no 3 pp 128ndash134 2003

[4] C Gutierrez ldquoStrategies for geminivirus DNA replicationand cell cycle interferencerdquo Physiological and Molecular PlantPathology vol 60 no 5 pp 219ndash230 2002

[5] G Sunter and D M Bisaro ldquoTransactivation of geminivirusAR1 and BR1 gene expression by the viral AL2 gene productoccurs at the level of transcriptionrdquo Plant Cell vol 4 no 10 pp1321ndash1331 1992

[6] A G Farag M A Amer H A Amin and H M MayzadldquoDetection of bipartite gemininviruses causing squash leafcurl disease in Egypt using polymerase chain reaction andnucleotide sequencerdquo Egyptian Journal of Virology vol 2 pp239ndash253 2005

[7] AAl-MusaGAnfoka SMisbehMAbhary andFHAhmadldquoDetection and molecular characterization of Squash leaf curlvirus (SLCV) in Jordanrdquo Journal of Phytopathology vol 156 no5 pp 311ndash316 2008

[8] M S Ali-Shtayeh R M Jamous E Y Husein and M YAlkhader ldquoFirst report of Squash leaf curl virus in squash(Cucurbita pepo) melon (Cucumis melo) and Cucumber(Cucumis sativa) in the northern best bank of the Palestinianauthorityrdquo Plant Disease vol 94 no 5 p 6402 2010

[9] H Sobh J Samsatly M Jawhari C Najjar A Haidar andY Abou-Jawdah ldquoFirst report of Squash leaf curl virus inCucurbits in Lebanonrdquo Plant Disease vol 96 no 8 p 1231 2012


[10] A M Idris A Abdel-Salam and J K Brown ldquoIntroduction ofthe newworld Squash leaf curl virus to Squash (Cucurbita pepo)in Egypt a potential threat to important food cropsrdquo PlantDisease vol 90 no 9 p 1262 2006

[11] K A El-Dougdoug H S Abd El-Kader I A Hamad E AAhmed and A F Abd El-Monem ldquoIdentification of squash leafcurl virus (Egyptian Isolate)rdquo Australian Journal of Basic andApplied Sciences vol 3 no 4 pp 3470ndash3478 2009

[12] A K Inoue-Nagata L C Albuquerque W B Rocha andT Nagata ldquoA simple method for cloning the complete bego-movirus genome using the bacteriophage 12059329 DNA poly-meraserdquo Journal of Virological Methods vol 116 no 2 pp 209ndash211 2004

[13] T Paprotka L S Boiteux M E N Fonseca et al ldquoGenomicdiversity of sweet potato geminiviruses in a Brazilian germ-plasm bankrdquo Virus Research vol 149 no 2 pp 224ndash233 2010

[14] T Paprotka V Metzler and H Jeske ldquoThe complete nucleotidesequence of a new bipartite begomovirus from Brazil infectingAbutilonrdquo Archives of Virology vol 155 no 5 pp 813ndash816 2010

[15] SVWesley CAHelliwell NA Smith et al ldquoConstruct designfor efficient effective and high-throughput gene silencing inplantsrdquo Plant Journal vol 27 no 6 pp 581ndash590 2001

[16] J J Doyle and J L Doyle ldquoA rapid DNA isolation procedure forsmall quantities of fresh leaf tissuerdquo Phytochemical Bulletin vol19 pp 11ndash15 1987

[17] J K Brown A M Idris I Torres-Jerez G K Banks and S DWyatt ldquoThe core region of the coat protein gene is highly usefulfor establishing the provisional identification and classificationof begomovirusesrdquoArchives of Virology vol 146 no 8 pp 1581ndash1598 2001

[18] J Sambrook T Maniatis E F Fritsch and Cold Spring HarborLaboratory Molecular Cloning A Laboratory Manual J Sam-brook E F Fritsch and T Maniatis Eds Cold Spring HarborLaboratory Press Cold Spring Harbor NY USA 2nd edition1987

[19] L K Johansen and J C Carrington ldquoSilencing on the spotInduction and suppression of RNA silencing in the Agrobac-terium-mediated transient expression systemrdquoPlant Physiologyvol 126 no 3 pp 930ndash938 2001

[20] T M Fulton J Chunwongse and S D Tanksley ldquoMicroprepprotocol for extraction of DNA from tomato and other herba-ceous plantsrdquo PlantMolecular Biology Reporter vol 13 no 3 pp207ndash209 1995

[21] Euidtdnacom ldquoIntegrated DNA TechnologiesmdashHomerdquo 2015httpseuidtdnacomsite

[22] H Jeske M Lutgemeier and W Preiss ldquoDNA forms indicaterolling circle and recombination-dependent replication of Abu-tilonmosaic virusrdquoTheEMBO Journal vol 20 no 21 pp 6158ndash6167 2001

[23] D Haible S Kober and H Jeske ldquoRolling circle amplificationrevolutionizes diagnosis and genomics of geminivirusesrdquo Jour-nal of Virological Methods vol 135 no 1 pp 9ndash16 2006

[24] M Lapidot D Gelbart A Gal-On et al ldquoFrequent migrationof introduced cucurbit-infecting begomoviruses amongMiddleEastern countriesrdquo Virology Journal vol 11 no 1 p 181 2014

[25] D B CollingeH J L JoslashrgensenO S Lund andM F LyngkjaeligrldquoEngineering pathogen resistance in crop plants current trendsand future prospectsrdquoAnnual Review of Phytopathology vol 48pp 269ndash291 2010

[26] A Callaway D Giesman-Cookmeyer E T Gillock T L Sitand S A Lommel ldquoThemultifunctional capsid proteins of plant

RNA virusesrdquo Annual Review of Phytopathology vol 39 pp419ndash460 2001

[27] F-J Jan S-Z Pang C Fagoaga and D Gonsalves ldquoTurnipmosaic potyvirus resistance in Nicotiana benthamiana derivedby post-transcriptional gene silencingrdquo Transgenic Researchvol 8 no 3 pp 203ndash213 1999

[28] K-S Ling H-Y Zhu and D Gonsalves ldquoResistance toGrapevine leafroll associated virus-2 is conferred by post-transcriptional gene silencing in transgenic Nicotiana ben-thamianardquo Transgenic Research vol 17 no 4 pp 733ndash740 2008

[29] R Scorza A Callahan L Levy V Damsteegt K Webb and MRavelonandro ldquoPost-transcriptional gene silencing in plumpoxvirus resistant transgenic European plum containing the plumpox potyvirus coat protein generdquo Transgenic Research vol 10no 3 pp 201ndash209 2001

[30] S Urcuqui-Inchima A-L Haenni and F Bernardi ldquoPotyvirusproteins a wealth of functionsrdquo Virus Research vol 74 no 1-2pp 157ndash175 2001

[31] N Agrawal P V N Dasaradhi A Mohmmed P Malhotra RK Bhatnagar and S K Mukherjee ldquoRNA interference biologymechanism and applicationsrdquoMicrobiology andMolecular Bio-logy Reviews vol 67 no 4 pp 657ndash685 2003

[32] P E Abrahamian and Y Abou-Jawdah ldquoDetection and quanti-tation of the new world Squash leaf curl virus by TaqMan real-time PCRrdquo Journal of Virological Methods vol 191 no 1 pp 76ndash81 2013

[33] J Haimes andM KelleyDemonstration of a ΔΔ C q CalculationMethod to Compute Relative Gene Expression from qPCR DataThermo Fisher Scientific Lafayette Colo USA 2010

[34] J S Elmer L Brand G Sunter W E Gardiner D M Bisaroand S G Rogers ldquoGenetic analysis of the tomato goldenmosaicvirus II The product of the AL1 coding sequence is requiredfor replicationrdquoNucleic Acids Research vol 16 no 14 pp 7043ndash7060 1988

[35] R Vanitharani P Chellappan J S Pita and C M FauquetldquoDifferential roles of AC2 and AC4 of cassava geminivirusesin mediating synergism and suppression of posttranscriptionalgene silencingrdquo Journal of Virology vol 78 no 17 pp 9487ndash9498 2004

[36] A A Rezk N A Abdallah A M A Salam M K NakhlaH M Mazyad and D P Maxwell ldquoTransgene-mediated RNAsilencing of TYLCV genes affecting the accumulation of viralDNA in plantsrdquo Arab Journal of Biotechnology vol 9 no 1 pp143ndash158 2005

[37] C-Y Lin W-S Tsai H-M Ku and F-J Jan ldquoEvaluation ofDNA fragments covering the entire genome of a monopartitebegomovirus for induction of viral resistance in transgenicplants via gene silencingrdquo Transgenic Research vol 21 no 2 pp231ndash241 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


or from cell to cell through plasmodesmata respectivelySquash leaf curl virus (SqLCV) is transmitted by its whiteflyvector Bemisia tabaci and it induces severe stunting and leafcurling in squash plants and causes severe losses in cucurbitproduction The first record of SqLCV in the Middle Eastwas in Israel in 2003 subsequently it has spread to neighborcountries such as Egypt [6] Jordan [7] Palestine [8] andLebanon [9] The spreading of the SqLCV within Egypt wasquickly as it took 5 years to spread from one governorateto another [6 10 11] Restriction enzymes of RCA productscombined with a cloning strategy showed being a veryefficient method to obtain full genome sequences This tech-nique allowed the easy detection cloning and sequencingof a mixed infection and the identification of the respectivebegomoviruses on the same sample [12ndash14]

The small interference RNA (siRNA) or posttranscrip-tional gene silencing (PTGS) occurs naturally in plants andacts as an antiviral defense mechanism The siRNA has beenwidely used as amethod to engineer resistance against virusesin plants including nuclear-replicating geminiviruses whichhave no double-stranded RNA phase in their replicationcycleTheproduced virus specific siRNA from the breakdownof dsRNA induces the degradation of homologous mRNAsequences resulting in hindering the virus form expressingits genes thus limiting virus replication process [15]

In this study the two viral components of the EgyptianSqLCV Noubaria isolate were cloned and their sequenceswere submitted to the GenBank from the cloned viralgenome two regions one from early and one from lateexpressed genes which were used to trigger PTGS againstSqLCV using agroinfiltration as a transient experimentmethod The first region is located at the overlapped regionbetween Rep and TrAP genes The second one represents thegene BC1 which has an important role in virus spreadingfrom cell to cell causing systemic infection

2 Materials and Methods

21 Cloning Viral Genome Geminivirus-infected squashplants were collected from different cultivated open fieldsof Noubaria village at Beheira Governorate The collectedplant samples showed symptoms of Squash leaf curl virus(SqLCV) such as severe systemic stunting and leaf curlingNonviruliferous whiteflies (Bemisia tabaci) were obtainedfrom rearing whiteflies on healthy cotton plants in an isolatedinsectary for at least amonthWhiteflies were fed for amonthon infected squash plants collected from the field to acquirethe virus and used to infect healthy squash plants

Total DNA from infected plants was extracted by usingCTAB method [16] Preliminary PCR for viral detection wascarried out using the degenerate primers V324 (+) primer 51015840-GCCYATRTAYAGRAAGCC MAG-31015840 and C889 (minus) primer51015840-GGRTTDGARGCATGHGTACATG-31015840 specific for white-fly transmitted geminiviruses [17] to amplify 570 bp fragmentFor cloning the complete viral genome the amplifiedmethodwas used Total DNA extracted from infected squash plantswas subjected to 12059329 amplification essentially according tothe manufacturerrsquos instructions (Fermentas) The amplifiedproducts were denatured at 65∘C for 10min and run on

a 1 agarose gel to confirm amplification The ampliPhiproduct was digested independently with selected restrictionenzymes that is EcoRI HindIII BamHI SalI SacI PstIXbaI BglII KpnI NcoI XmaI Xho1 SmaI SphI and SwaI(Fermentas fast digest) and separated in an agarose gel Theenzymes that could cut at a single site in the genome gener-ating genome size fragment was selected for further cloningin pBluescript II SK (minus) Sequencing was carried out andwas subjected to GenBank (GenBank accession A KP795975B KP795976)

22 Primer Design and PCR Amplification To assess silenc-ing effect of the selected viral fragments potentially useful inthe control of SqLCV two fragments were selected from theviral sequences to cover partial RepTrAP and full BC1 genesTwo sets of primers were designed to amplify the selectedregions by using Vector NTI Software RepTrAP primersequences are 51015840-CCCACATAATTACTTGAGCGGCCAT-31015840 and 51015840-TTAGCAATCCTGTGCTGTGCTTTGA-31015840 whileBC1 gene primer pairs are 51015840-ATGGGTTCTCAATTAGTT-CCACCC-31015840 and 51015840-GCTTAGGGATTTTGGAGTGCT-CGGG-31015840 Restriction sites XhoI and XmaI added to theforward primers and AscI and BamHI to the reverse primersto facilitate cloning in sense and antisense forms in the binaryvector pFGC5041within the intron regionThe expected sizesof the selected regions were 348 bp and 879 bp for clonedviral genome to amplify the selected region The sizes ofthe selected fragments were 348 bp and 879 bp for RepTrAPand BC1 genes respectively PCR was carried out using FlexiGoTaq DNA polymerase (Promega USA) Cloned viralgenome was used as a template in PCR reaction with aconcentration of 100 ng 5120583L of 5x Flexi GoTaq buffer 2120583Lof 25mMMgCl

2 05 120583L of 10mMdNTPs 05 120583L 10 pmol of

each primer 025120583L GoTaq enzyme and ddH2O to reach a

total volume of 25120583L the amplification was carried out in aBioRadT100USAThe reactionwas performedwith an initialcycle at 94∘C for 4min followed by 30 cycles of denaturationstep at 94∘C for 1min annealing at 50∘C for 30 sec and anextension cycle at 72∘C for 1min followed by a final cycle at72∘C for 7min

23 Cloning of Silencing Fragments The amplified fragmentswere cloned initially into pGEM-Teasy Vector System sub-sequently RePTrAP and BC1 fragments were digested usingXhoI andXmaI andAscI andBamHI for cloning in pFGC5041silencing binary vector (supplied by Richared JorgensenUniversity of Arizona USA) Each fragment was cloned intothe expression vector pFGC5041 in which the transgeneis cloned in both orientations separated within the ChsAintron and under the control of the 35S promoter and theOSC terminatorThe cloning of amplified fragments has beencarried out in two steps by using the inner restriction siteson the primers Double digestion with FastDigest enzymesXmaI and BamHI was used for the sense orientation into theplant vector pFGC5041 the outer restriction enzymes pairXhoI and AscI were used to clone the antisense orientationAll enzymes used for cloning were FastDigest restrictionenzymes supplied fromThermo Scientific









M 87654321


M 1 2 3 4 5 6 7 8 9 10

26 kb

3kb2kb1kb



3 Results




M 1 2 3 4 5 6 7

26 kb3kb2kb

13 kb


AC4

DNA-A DNA-B

AC1

AC3

AC2

347bp

347bp

AV1


BC1

BV1

849bp

849bp








M 1 2

1

500bp


LB LB

RB RB


P-35S

P-35S



2nd orientation

Xho1

Xma1

Xma1

Xma1

BamHI BamHI

BamHI

AscI

OCS-3998400OCS-3998400







(a) (b)

(c) (d)












Ct oftreatment


Ct ofcontrol


Ct oftreatment



79

0

02

04

06

08

1

12

Relat

ive g

ene e

xpre

ssio

n

ControlBC1RepTrAP

97




4 Discussion












5 Conclusion



Abbreviations




References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









M 87654321


M 1 2 3 4 5 6 7 8 9 10

26 kb

3kb2kb1kb



3 Results




M 1 2 3 4 5 6 7

26 kb3kb2kb

13 kb


AC4

DNA-A DNA-B

AC1

AC3

AC2

347bp

347bp

AV1


BC1

BV1

849bp

849bp








M 1 2

1

500bp


LB LB

RB RB


P-35S

P-35S



2nd orientation

Xho1

Xma1

Xma1

Xma1

BamHI BamHI

BamHI

AscI

OCS-3998400OCS-3998400







(a) (b)

(c) (d)












Ct oftreatment


Ct ofcontrol


Ct oftreatment



79

0

02

04

06

08

1

12

Relat

ive g

ene e

xpre

ssio

n

ControlBC1RepTrAP

97




4 Discussion












5 Conclusion



Abbreviations




References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M 1 2 3 4 5 6 7

26 kb3kb2kb

13 kb


AC4

DNA-A DNA-B

AC1

AC3

AC2

347bp

347bp

AV1


BC1

BV1

849bp

849bp








M 1 2

1

500bp


LB LB

RB RB


P-35S

P-35S



2nd orientation

Xho1

Xma1

Xma1

Xma1

BamHI BamHI

BamHI

AscI

OCS-3998400OCS-3998400







(a) (b)

(c) (d)












Ct oftreatment


Ct ofcontrol


Ct oftreatment



79

0

02

04

06

08

1

12

Relat

ive g

ene e

xpre

ssio

n

ControlBC1RepTrAP

97




4 Discussion












5 Conclusion



Abbreviations




References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


M 1 2

1

500bp


LB LB

RB RB


P-35S

P-35S



2nd orientation

Xho1

Xma1

Xma1

Xma1

BamHI BamHI

BamHI

AscI

OCS-3998400OCS-3998400







(a) (b)

(c) (d)












Ct oftreatment


Ct ofcontrol


Ct oftreatment



79

0

02

04

06

08

1

12

Relat

ive g

ene e

xpre

ssio

n

ControlBC1RepTrAP

97




4 Discussion












5 Conclusion



Abbreviations




References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)












Ct oftreatment


Ct ofcontrol


Ct oftreatment



79

0

02

04

06

08

1

12

Relat

ive g

ene e

xpre

ssio

n

ControlBC1RepTrAP

97




4 Discussion












5 Conclusion



Abbreviations




References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Ct oftreatment


Ct ofcontrol


Ct oftreatment



79

0

02

04

06

08

1

12

Relat

ive g

ene e

xpre

ssio

n

ControlBC1RepTrAP

97




4 Discussion












5 Conclusion



Abbreviations




References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








5 Conclusion



Abbreviations




References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article use of posttranscription gene silencing in...

Documents