Communication In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder Replicates in Cucumber Protoplasts

Carolyn A. Owen 1, Romy Moukarzel 1, Xiao Huang 2, Mona A. Kassem 3,4, Eleonora Eliasco 2, Miguel A. Aranda 3, Robert H. A. Coutts 5 and Ioannis C. Livieratos 1,*

1 Department of Sustainable Agriculture, Mediterranean Agronomic Institute of Chania, Alsylio Agrokepio, Chania GR-73100, Greece; [email protected] (C.A.O.); [email protected] (R.M.) 2 Sir Alexander Fleming Building, Department of Biological Sciences, Imperial College, London SW7 2AZ, UK; [email protected] (X.H.); [email protected] (E.E.) 3 Departamento de Biología del Estrés y Patología Vegetal, Centro de Edafología y Biología Aplicada del Segura (CEBAS)-CSIC, P.O. Box 164, 30100 Espinardo, Murcia, Spain; [email protected] (M.A.K.); [email protected] (M.A.A.) 4 Centro de Investigacion en Quimica Aplicada, CIQA-CONACYT, Saltillo 25294, Mexico 5 Department of Biological and Environmental Sciences, School of Life and Medical Sciences, University of Hertfordshire, College Lane, Hatfield, Hertfordshire AL10 9AB, UK; [email protected] * Correspondence: [email protected]; Tel.: +30-28210-35050; Fax: +30-28210-35001

Academic Editor: Thomas Hohn Received: 22 March 2016; Accepted: 6 June 2016; Published: 14 June 2016

Abstract: Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8∆) comprising the 51- and 31-UTRs plus the 31-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8∆ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants.

Keywords: criniviruses; whitefly-transmitted viruses; Cucurbit yellow stunting disorder virus; infectious clones; protoplasts

Cucurbit yellow stunting disorder virus (CYSDV; genus Crinivirus) is among the most important cucurbit pathogens worldwide, causing diseases with serious economic impact [1,2]. CYSDV is efficiently transmitted by two of the most invasive species of the Bemisia tabaci complex, the Mediterranean (MED, previously referred to as biotype Q) and the Middle East-Asia Minor 1 (MEAM1, biotype B). As both MED and MEAM1 are now distributed throughout the sub-tropical and temperate regions where cucurbit cultivation chiefly occurs, their ubiquity has been a major factor in the emergence and spread of a number of important viral pathogens including CYSDV [3]. Because criniviruses cannot be mechanically transmitted, full-length infectious cDNA clones are essential for the experimental infection of model plants in the absence of whiteflies and for the creation and

Viruses 2016, 8, 170; doi:10.3390/v8060170 www.mdpi.com/journal/viruses Viruses 2016, 8, x 2 of 7 Viruses 2016, 8, 170 2 of 7 creation and propagation of recombinant viruses for reverse genetics studies to examine the propagationfunction(s) of of viral recombinant sequences virusesand RNA for structures. reverse genetics studies to examine the function(s) of viral sequencesThe large and size RNA and structures. complexity of crinivirus , and the propensity of their cDNA clones to rearrangeThe large or size to cause and complexity toxicity in ofbacteria crinivirus has genomes,limited the and production the propensity of infectious of their clones cDNA to clones only threeto rearrange criniviruses. or to causeIn the toxicity first instance in bacteria infectious has limited transcripts the production of the genus of infectious type species clones toLettuce only infectiousthree criniviruses. yellows virus In the (LIYV), first instance with expression infectious of transcripts the viral sequences of the genus driven type speciesby bacteriophageLettuce infectious RNA polymeraseyellows virus promoters,(LIYV), with were expression used to of transfect the viral toba sequencescco protoplasts driven by [4]. bacteriophage These clones, RNA in conjunction polymerase promoters,with later versions were used permitting to transfect Agrobacterium tobacco protoplasts-mediated transformation [4]. These clones, of plants in conjunction with LIYV with cDNAs later versionsunder the permitting control of theAgrobacterium CaMV 35S-mediated promoter [5], transformation generated important of plants insights with LIYV into cDNAs LIYV biology under [6]. the Itcontrol was shown of the CaMV that RNA1 35S promoter is competent [5], generated to replicate important alone insights [4], and into to LIYVdrive biology the trans [6].-replication It was shown of LIYVthat RNA1 RNA2 is in competent an asynchronous to replicate manner alone [7]. [4], The and RNA1-encoded to drive the trans p34-replication was shown of LIYVto be essential RNA2 in for an RNA2asynchronous replication manner [7], and [7]. Theto bind RNA1-encoded single strand p34ed wasRNA shown [8]. The to beRNA2-encoded essential for RNA2 heat replicationshock [7], 70and homologue to bind single (Hsp70h), stranded p59, RNA CP, [8 ].and The CPm RNA2-encoded were shown heat to be shock virion protein structural 70 homologue (Hsp70h),[9], with p59,CPm CP, being and CPmthe sole were determinant shown to be virionof whitefly structural transmission proteins [9 ],[9–11]. with CPm Lettuce being chlorosis the sole virus determinant (LCV) infectiousof whitefly clones transmission [12,13] [ 9have–11]. Lettucealso revealed chlorosis further virus (LCV) aspects infectious of crinivirus clones [12 biology,,13] have including also revealed the synchronousfurther aspects replication of crinivirus of LCV biology, RNA1 including and RNA2 the synchronous molecules, and replication the existence of LCV of RNA1 defective and RNA2RNAs arisingmolecules, from and the the former. existence Shifts of in defective vector populati arisingon dynamics from the have former. greatly Shifts reduced in vector the agricultural population threatdynamics from have LIYV, greatly and reduced LCV has the agriculturallittle economic threat impact from LIYV, on agricultural and LCV has production. little economic Therefore impact researchon agricultural is now production. focused on Thereforethe more researcheconomically is now important focused oncriniviruses the more economically[2]. Recently, importantinfectious clonescriniviruses were [reported2]. Recently, for Tomato infectious chlorosis clones virus were (ToCV), reported a forcrinivirusTomato responsible chlorosis virus for(ToCV), severe adamage crinivirus to tomatoresponsible production for severe that damage also infects to tomato other import productionant crop that species also infects including other sweet important pepper crop and speciespotato [14].including Here, sweetthe first pepper CYSDV and infectious potato [14 clones]. Here, able the to first replicate CYSDV in cucumber infectious protoplasts clones able are to replicate described. in cucumberThroughout, protoplasts detection are described. of the genomic (g) RNA species and co-terminal subgenomic (sg) RNAs derivedThroughout, from RNA1 detection and RNA2 of the was genomic conducted (g) RNA by hybridisation species and co-terminalof northern subgenomicblots. The dig-labelled (sg) RNAs riboprobesderived from used RNA1 were and transcribed RNA2 was from conducted cloned cDNAs by hybridisation encoding ofp22 northern and p26, blots. the most The dig-labelled3′-proximal genesriboprobes of CYSDV used were RNA1 transcribed and RNA2, from respectively. cloned cDNAs Figure encoding 1A shows p22 and the p26, hybridisation the most 31-proximal patterns obtainedgenes of CYSDVfrom RNA RNA1 extracted and RNA2, from respectively.CYSDV-infected Figure cucumber1A shows leaves the hybridisation from the field, patterns using obtained p22 (left panel),from RNA and extracted p26 (right from panel), CYSDV-infected negative ( cucumber−) sense riboprobes. leaves from Each the field, probe using hybridised p22 (left panel),to the correspondingand p26 (right panel),gRNA, negativeplus a number (´) sense of smaller riboprobes. species. Each The probe smallest hybridised and most to the prominent corresponding RNA speciesgRNA, plusdetected a number in the ofRNA1 smaller hybridisation species. The was smallest determined and most to be prominent the p22 sgRNA RNA species on the detectedbasis of its in sizethe RNA1(ca. 900 hybridisation bp). was determined to be the p22 sgRNA on the basis of its size (ca. 900 bp).

Figure 1. CYSDV RNA1 replicates independently of RNA2 in cucumbercucumber protoplasts.protoplasts. Positive and negative strand strand accumulation accumulation occurs occurs with with similar similar kinetics, kinetics, but butwith with quantitative quantitative asymmetry. asymmetry. (A) (NorthernA) Northern blots blots of total of total RNA RNA extracted extracted from from fiel fieldd samples samples of of naturally naturally CYSDV CYSDV infected infected cucumber plants showing gRNA and positive co-terminalco-terminal RNARNA speciesspecies hybridisinghybridising withwith aa ((´−)) sense p22 probe (left panel, RNA1), and aa ((´−)) sense sense p26 p26 probe (right panel, RNA2).RNA2). The The position position of of the sgRNA for CYSDV p22 is indicated; (B) Northern blot of total RNA extractedextracted 36 h and 72 h p.i. from protoplasts transfected inin conjunction conjunction with with CYSDV CYSDV RNA1, RNA1, hybridised hybridised with awith (´) sense a (− p22) sense probe. p22 UC probe. = uninfected UC = uninfectedcontrol; (C )control; Time course (C) Time of RNA1 course transfected of RNA1 protoplasts transfected hybridised protoplasts with hybridised (´) and (+)with sense (−) and probes. (+) senseDuplicate probes. northern Duplicate blots northern were hybridised blots were with hybrid dig-labelledised with riboprobes dig-labelled corresponding riboprobes tocorresponding the (´) sense (topto the panel) (−) sense and (top (+) sense panel) (middle and (+) panel) sense of(middle the CYSDV panel) p22 of the gene. CYSDV The lower p22 gene. panel The shows lower the panel EtBr showsstained the loading EtBr controls.stained loading The positions controls. of The the gRNApositions bands of the and gRNA the p22 bands sgRNA and are the indicated. p22 sgRNA are indicated.

Viruses 2016, 8, 170 3 of 7

A full length cDNA clone of CYSDV gRNA1 (9127 bp) was constructed by amplifying a series of seven overlapping cDNA fragments from CYSDV dsRNA using Pfu polymerase (Promega, Madison, WI, USA), with each pair of adjacent fragments sharing a unique restriction site occurring in the sequence. The first PCR primer introduced a NotI site to facilitate cloning and a T7 polymerase binding site immediately upstream of the viral 51-UTR. A G/C substitution was made at the final nucleotide of the 31-UTR to introduce a terminal XhoI site to allow generation of a linear transcription template. The restricted PCR fragments were serially cloned as double ligation pairs into the plasmid vector pBluescript, to produce pBS_T7CYSDVRNA1. The insert was sequenced, and the purified linearised construct was used as a template to produce capped mRNA transcripts using the mMessage mMachine system (Thermo Fisher Scientific, Waltham, MA, USA). CYSDV RNA1 transcripts were tested for replication competence in Nicotiana benthamiana mesophyll protoplasts, using a polyethyleneglycol (PEG)-mediated transfection protocol, as described by Mathioudakis et al. [15]. However, in repeated experiments, in which 10 µg CYSDV RNA1 capped transcripts were introduced per million protoplasts, no replication was detected. While N. benthamiana is not a natural host for CYSDV, the RNA1 of the criniviruses LIYV, ToCV, and the phylogenetically close relative of CYSDV, LCV, all have been demonstrated to replicate in protoplasts of this species [4,13,14]. Similarly, for the Citrus tristeza virus (CTV), replication of virion RNA in N. benthamiana protoplasts was more pronounced than in those isolated from its natural woody host Citrus sinensis [16]. The CYSDV RNA1 construct was then tested in cucumber (Cucumis sativus c.v. Ravenna) first-leaf mesophyll protoplasts, isolated by the same procedure as for the N. benthamiana protoplasts but with the centrifugation force increased to 190ˆ g to compensate for the smaller cell size. The cucumber protoplasts were less robust than those of N. benthamiana and were difficult to maintain in culture beyond 72 h, but following transfection RNA1 replication was detected at 36 h post inoculation (p.i.), and was further increased at 72 h (Figure1B). In time-course experiments, the input mRNA was detectable on inoculation but had reduced to undetectable levels after 12 h (Supplementary Figure S1), while bands corresponding to de novo synthesized positive strand gRNA and the p22 sgRNA were detected 24 h p.i. The amounts of both species increased further at 48 h and remained high at 72 h p.i. (Figure1C upper panel). No alteration in the relative intensity of the two species was observed throughout the time course, and the hybridisation pattern was the same as that seen from infected plant tissue. The intensity of the two bands was approximately equal; as their respective sizes are 9.1 kb and 0.9 kb, transcription of the p22 sgRNA is proportionally greater. On longer exposures, faint bands corresponding to RNA species of ca. 1600 bp, and 1400 bp were visible; these, on the basis of their sizes were determined to be the 31-co-terminal sgRNAs for CYSDV p5 and p25, respectively. The prominent p22 sgRNA band was also detected consistently in RNA isolated from different CYSDV-infected melon and cucumber samples, indicating the absence of temporal regulation of p22 sgRNA transcription in natural infections, as well as in transfected protoplasts. Analogous results have also been reported for the p34 sgRNA of LIYV [4,7] and the p23 sgRNA of LCV [13], which are also transcribed from the most 31-proximal coding sequence of each respective crinivirus RNA1. On a duplicate blot hybridised with a p22 positive sense (+) probe, a single band corresponding to the negative sense RNA1 was detected that first appeared at 24 h, increased in intensity at 48 h, and remained high at 72 h p.i. (Figure1C middle panel). The CYSDV gRNA1 ( ´) strand hybridisation signal was weaker than that seen with the equivalently-labelled p22 (´) probe, requiring longer exposures to reveal the bands, indicating that the replication of CYSDV RNA1 is asymmetric, with accumulation of CYSDV gRNA1 (+) strand being greater than that of the (´) strand (Figure1C). Crinivirus genomes exhibit a generally conserved architecture, while each has unique features [1,6]. Interspecies variation is seen in RNA 1 downstream of RdRp, where 1–3 ORFs occur, for which the predicted amino acid homology among the proteins encoded by similarly-sized and equivalently-positioned genes is low. For three criniviruses, proteins encoded by genes in this region of RNA1 have been reported to be suppressors of RNA silencing: Sweet potato chlorotic stunt virus Viruses 2016, 8, x 4 of 7

Viruses(SPCSV)2016, p228, 170 [17], ToCV p22 [18] and CYSDV p25 [19]. In SPCSV the p34 gene encodes an RNase4 of 7 type III protein bearing no resemblance to any other viral protein, that has been shown to enhance the RNA silencing suppression activity of p22 [17], and to mediate synergism between SPCSV and (SPCSV) p22 [17], ToCV p22 [18] and CYSDV p25 [19]. In SPCSV the p34 gene encodes an RNase type Sweet potato feathery mottle virus (SPFMV) [20]. ToCV p22 has been recently shown to preferentially III protein bearing no resemblance to any other viral protein, that has been shown to enhance the RNA bind dsRNA species and to protect them from degradation [21]. In CYSDV the p22 gene exhibits no silencing suppression activity of p22 [17], and to mediate synergism between SPCSV and Sweet potato homology with any characterised protein and has yet to have any function ascribed to it. The early feathery mottle virus (SPFMV) [20]. ToCV p22 has been recently shown to preferentially bind dsRNA and sustained transcription of the p22 sgRNA in transfected protoplasts, and its maintained species and to protect them from degradation [21]. In CYSDV the p22 gene exhibits no homology with presence in natural CYSDV infections, suggests its importance for the initiation and promotion of any characterised protein and has yet to have any function ascribed to it. The early and sustained infection and underlines the necessity of further investigations to characterise its function(s) and transcription of the p22 sgRNA in transfected protoplasts, and its maintained presence in natural intracellular localisation. CYSDV infections, suggests its importance for the initiation and promotion of infection and underlines To construct a full-length cDNA clone for CYSDV gRNA2 (7976 bp), two overlapping cDNA the necessity of further investigations to characterise its function(s) and intracellular localisation. fragments were amplified from total RNA extracted from CYSDV-infected plants using Pfu To construct a full-length cDNA clone for CYSDV gRNA2 (7976 bp), two overlapping cDNA polymerase (Promega). Overlapping PCR was used to produce full-length cDNAs, which were fragments were amplified from total RNA extracted from CYSDV-infected plants using Pfu polymerase cloned into pGEM-T-Easy vector (Promega). A blunt-ended (Q5; NEB, Ipswitch, MA, USA) PCR (Promega). Overlapping PCR was used to produce full-length cDNAs, which were cloned into product amplified from one RNA2 clone (pGEMT_CM8), using primers corresponding to the pGEM-T-Easy vector (Promega). A blunt-ended (Q5; NEB, Ipswitch, MA, USA) PCR product amplified natural ends of the CYSDV RNA2 sequence that introduced a T3 RNA polymerase site 5′ of the viral from one RNA2 clone (pGEMT_CM8), using primers corresponding to the natural ends of the CYSDV sequence, was used as the template to produce capped mRNA transcripts. On transfection of 10 μg RNA2 sequence that introduced a T3 RNA polymerase site 51 of the viral sequence, was used as the CYSDV RNA2 mRNA alone into protoplasts no replication was detected. However, upon template to produce capped mRNA transcripts. On transfection of 10 µg CYSDV RNA2 mRNA alone co-transfection with equimolar RNA1 mRNA (10 μg RNA1, 8.8 μg RNA2/106 protoplasts), into protoplasts no replication was detected. However, upon co-transfection with equimolar RNA1 replication of RNA2 was observed. The full-length positive-strand CYSDV RNA2 was detectable in mRNA (10 µg RNA1, 8.8 µg RNA2/106 protoplasts), replication of RNA2 was observed. The full-length total protoplast RNA extracts hybridised with the CYSDV p26 (−) riboprobe at 24 h, significantly positive-strand CYSDV RNA2 was detectable in total protoplast RNA extracts hybridised with the increased in intensity at 48 h and remained elevated at 72 h p.i. (Figure 2). A number of less intense CYSDV p26 (´) riboprobe at 24 h, significantly increased in intensity at 48 h and remained elevated at bands corresponding to smaller RNA species could be observed at the limit of detection. These 72 h p.i. (Figure2). A number of less intense bands corresponding to smaller RNA species could be presumably correspond to 3′ co-terminal sgRNAs, as at least six such species have been predicted to observed at the limit of detection. These presumably correspond to 31 co-terminal sgRNAs, as at least arise from CYSDV RNA2 [22]. The ability of CYSDV RNA1, and not RNA2, to replicate alone in six such species have been predicted to arise from CYSDV RNA2 [22]. The ability of CYSDV RNA1, protoplasts indicates that as for LIYV, LCV and ToCV [7,13,14] the latter RNA does not encode any and not RNA2, to replicate alone in protoplasts indicates that as for LIYV, LCV and ToCV [7,13,14] proteins required for virus replication. Attempts to RT-PCR amplify de novo defective 5′- and the latter RNA does not encode any proteins required for virus replication. Attempts to RT-PCR 3′-co-terminal CYSDV RNA2 species analogous with those described by Rubio and co-workers amplify de novo defective 51- and 31-co-terminal CYSDV RNA2 species analogous with those described [23,24] from transfected protoplasts were unsuccessful. by Rubio and co-workers [23,24] from transfected protoplasts were unsuccessful.

Figure 2. Replication of CYSDV RNA2 occurs in the presence of RNA1, with similar kinetics and a small Figure 2. Replication of CYSDV RNA2 occurs in the presence of RNA1, with similar kinetics and a temporal delay. A time-course northern blot of total RNA extracted from protoplasts co-transfected small temporal delay. A time-course northern blot of total RNA extracted from protoplasts with equimolar amounts of CYSDV RNA1 and RNA2, hybridised with a dig-labelled riboprobe co-transfected with equimolar amounts of CYSDV RNA1 and RNA2, hybridised with a dig-labelled corresponding to the (´) sense CYSDV p26 gene. The position of the RNA2 gRNA is indicated. riboprobe corresponding to the (−) sense CYSDV p26 gene. The position of the RNA2 gRNA is The apparent shift in the gRNA band at 72 h p.i. is an electrophoresis artefact. indicated. The apparent shift in the gRNA band at 72 h p.i. is an electrophoresis artefact.

TheThe relative relative expression expression patterns patterns of of CYSDV CYSDV RNA1 RNA1 and and RNA2, RNA2, recall recall the the virtually virtually synchronous synchronous replicationreplication observedobserved for for LCV LCV RNA1 RNA1 and and RNA2 RNA2 in to inbacco tobacco protoplasts protoplasts transfected transfected with either with virions either virions[25], or [ 25infectious], or infectious RNA transcripts RNA transcripts [13]. These [13]. re Thesesults resultscontrast contrast with those with seen those for seen LIYV, for LIYV,where wheretransfection transfection of virion of virion RNA RNAinto tobacco into tobacco protoplasts protoplasts resulted resulted in 24 in h 24delays h delays in the in theinitiation initiation and and attainment of maximal replication of RNA2, relative to RNA1 [7]. This indicates that for CYSDV,

Viruses 2016, 8, x 5 of 7

Virusesattainment2016, 8, of 170 maximal replication of RNA2, relative to RNA1 [7]. This indicates that for CYSDV,5 of as 7 for LCV, RNA2 transcription is effected by RNA1, with a minimal lag period in its appearance. asFolding-analysis for LCV, RNA2 of transcription the 3′-UTR regions is effected of criniviruses by RNA1, with predicts a minimal the presence lag period of characteristic in its appearance. stem Folding-analysisloop structures ofand the terminal 31-UTR regionspseudoknots of criniviruses that have predicts been shown the presence in other of characteristic viral sequences stem to loop be structuresimportant andfor the terminal initiation pseudoknots of (−) strand that RNA have beensynthesis shown [26]. in otherComparison viral sequences of the 3′-UTR to be importantregions of forRNA1 the and initiation RNA2 of from (´) strandCYSDV, RNA LIYV, synthesis and SPCSV [26]. revealed Comparison conserved of the predicted 31-UTR regions secondary of RNA1 structure and RNA2in all cases, from CYSDV,except for LIYV, LIYV and RNA2 SPCSV which revealed lacked conserved one of the predicted four hairpin secondary loops structureand the pseudoknot in all cases, exceptstructure for [27]. LIYV This RNA2 variation which lackedmight account one of the for four the hairpinsimilar loopsreplication and the kinetics pseudoknot of LCV structure and CYSDV [27]. ThisRNA1 variation and RNA2, might and account the delay for the in RNA2 similar synthesis replication for kinetics LIYV. of LCV and CYSDV RNA1 and RNA2, and theAn delay artificial in RNA2 defective synthesis CYSDV for LIYV. RNA2 cDNA clone was created by BglII restriction and re-ligationAn artificial of pGEMT_CM8 defective CYSDV to excise RNA2 nucleotides cDNA clone 1116-6325 was created of RNA2. by Bgl FollowingII restriction PCR and amplification re-ligation of(LA pGEMT_CM8 Taq; Clontech, to excisePala Alto, nucleotides CA, USA) 1116-6325 of the of 2.8 RNA2. kbp modified Following sequence PCR amplification using primers (LA Taq;that Clontech,introduced Pala a T3 Alto, polymerase CA, USA) binding of the site 2.8 5′ kbpof the modified viral sequence sequence and usingan Nru primersI site to thatallow introduced restriction aat T3 its polymerasenatural end, bindingthe product site was 51 of cloned the viral as pGEMT_T3CM8 sequence and anΔ. NruThisI construct site to allow served restriction as a template at its naturalfor the end,production the product of a wascapped cloned mRNA as pGEMT_T3CM8 transcript, RNA2∆. ThisΔ, shown construct in schematic served as arepresentation template for the in productionFigure 3A, which of a capped comprises mRNA 95% transcript, of the 5′-UTR, RNA2 the∆, showndistal half in schematicof the CPm representation gene, the p26 ingene Figure and3 theA, which3′-UTR comprises of RNA2. 95%When of transfected the 51-UTR, into the cucu distalmber half protoplasts of the CPm with gene, equimolar the p26 gene RNA1 and (10 the μg 3RNA1,1-UTR of3.1 RNA2. μg RNA2 WhenΔ/10 transfected6 protoplasts), into RNA2 cucumberΔ accumulated protoplasts to with high equimolar levels with RNA1 similar (10 kineticsµg RNA1, to CYSDV 3.1 µg RNA2RNA2∆ (Figure/106 protoplasts), 3B). No replication RNA2∆ accumulated was seen in tothe high absence levels of with RNA1. similar Thus, kinetics the last to CYSDV62 nucleotides RNA2 (Figure(1116–1177)3B). No of replication the CYSDV was RNA2 seen in 5 the′-UTR, absence the ofsix RNA1. RNA2-encoded Thus, the last pr 62oteins nucleotides that were (1116–1177) deleted of(ORFs5-10: the CYSDV p5, RNA2 Hsp70h, 51-UTR, p6, thep59, six p9, RNA2-encoded CP), and the proteins partially that deleted were deleted CPm, (ORFs5-10:are all dispensable p5, Hsp70h, for p6,CYSDV p59, p9,RNA2 CP), replication. and the partially Previously, deleted mutation CPm, st areudies all dispensableconducted on for LIYV CYSDV had RNA2demonstrated replication. that Previously,the individual mutation elimination studies of conducted the five largest on LIYV RNA2-encoded had demonstrated proteins that (Hsp70h, the individual p59, CP, elimination CPm, and of thep26) five via largest the introduction RNA2-encoded of stop proteins codons (Hsp70h, to abort translation p59, CP, CPm, did andnot p26)affect via the the accumulation introduction of of either stop codonsRNA1 toor abortRNA2, translation indicating did that not none affect is the required accumulation for LIYV of either RNA2 RNA1 replication or RNA2, [7]. indicatingMuch shorter that noneexposure is required times forwere LIYV required RNA2 to replication reveal the [7]. RNA2 MuchΔ shorter band exposurein comparison times werewith requiredthat from to RNA2, reveal theindicating RNA2∆ higherband inlevels comparison of replication with thatof the from form RNA2,er. Similarly, indicating it has higher previously levels ofbeen replication shown for of thethe former.(unipartite Similarly, ) it has closteroviruses previously been Beet shown yellows for virus the (unipartite(BYV) and genome)CTV, that closteroviruses cDNA clones inBeet which yellows all virusnon-replicase(BYV) and genes CTV, were that cDNA deleted clones generated in which RNA all non-replicasethat replicated genes to higher were deleted levels than generated full-length RNA thatconstructs replicated in N. to benthamiana higher levels protoplasts than full-length [28,29]. constructs in N. benthamiana protoplasts [28,29].

FigureFigure 3.3. AnAnartificial artificial defective defective RNA2 RNA2 transcript transcript replicates replicates in in cucumber cucumber protoplasts protoplasts in thein the presence presence of RNA1.of RNA1. (A) ( SchematicA) Schematic showing showing the relationship the relationsh betweenip between CYSDV CYSDV RNA2 RNA2 and RNA2 and∆ RNA2, a deletionΔ, a deletion mutant createdmutant bycreated removing by removing nucleotides nucleotides 1116–6325 1116–6325 of CYSDV of CYSDV RNA2; ( BRNA2;) a time-course (B) a time-course northern northern blot of total blot RNAof total extracted RNA extracted from the from protoplasts the protoplasts co-transfected co-transfected with equimolar with equimolar amounts amounts of CYSDV of CYSDV RNA1 RNA1 and RNA2and RNA2∆, hybridisedΔ, hybridised with with a dig-labelled a dig-labelled riboprobe riboprobe of the of (the´) sense(−) sense CYSDV CYSDV p26 p26 gene. gene. The The position position of RNA2of RNA2∆ isΔ indicated. is indicated.

AA numbernumber ofof featuresfeatures ofof thethe T3CM8T3CM8∆Δ cloneclone makemake itit aa usefuluseful tooltool toto definedefine thethe 331-′- andand 551-UTR′-UTR sequencessequences requiredrequired forfor thethe replicationreplication ofof CYSDVCYSDV RNA2:RNA2: itsits smallsmall sizesize allowsallows readyready amplificationamplification andand easyeasy purification,purification, andand makesmakes itit anan idealideal templatetemplate forfor thethe rapidrapid generationgeneration ofof mutantsmutants usingusing high-fidelityhigh-fidelity PCR-drivenPCR-driven mutationmutation strategies.strategies. The replication of its RNARNA transcripttranscript toto highhigh levelslevels

Viruses 2016, 8, 170 6 of 7 within 36 h in protoplasts will facilitate rapid simultaneous evaluation of panels of mutant defective RNAs without recourse to whole plant studies. Introduction of a reporter gene such as that encoding green fluorescent protein immediately downstream of the 51-UTR could obviate the need for northern hybridization by allowing the photometric quantitation of the replication of RNA2 mutants and dissection of translation-essential domains within the exceptionally long 51-UTR which, at 1177 bp, is considerably longer than that of any other known plant virus [6,22]. CYSDV is a prominent crinivirus exerting a major negative impact on cucurbit production in several areas of the world, a phenomenon exacerbated by the lack of effective whitefly control measures. It is anticipated that containment of CYSDV and other whitefly-associated plant viruses will be most easily achieved via the development of resistant plant varieties or mechanisms to block virus transmission [1]. Central to these aims are the creation of genetic tools to allow dissection of plant-virus [14], virus-vector [10,30] and vector-plant [31] interactions at a molecular level. The construction of CYSDV cDNA infectious clones represents a first step towards this goal.

Acknowledgments: This work was supported by a joint research project (09SYN-22-617) funded by the European Union, the Greek State (Ministry of Development-General Secretariat of Research and Technology, GSRT) and the private sector (E.P.A.N.II). The authors would like to thank Dr. Anastasia Tsagkarakou for kindly providing information on the B. tabaci complex. Author Contributions: Carolyn A. Owen, Xiao Huang, Eleonora Eliasco, Romy Moukarzel, and Mona A. Kassem conducted the experiments; Robert H.A. Coutts, Miguel A. Aranda, Carolyn A. Owen, and Ioannis C. Livieratos designed the experimental work; Carolyn A. Owen, Miguel A. Aranda, Robert H.A. Coutts and Ioannis C. Livieratos wrote the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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