Construction of a synthetic infectious cDNA clone of Grapevine Algerian latent virus

15 Pages·2014·4.14 MB·English

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RESEARCH Open Access
Construction of a synthetic infectious cDNA clone
of Grapevine Algerian latent virus (GALV-Nf) and
its biological activity in Nicotiana benthamiana
and grapevine plants
Arianna Lovato1, Franco Faoro2,3, Giorgio Gambino3, Dario Maffi2, Marcella Bracale4, Annalisa Polverari1*
and Luca Santi5
Abstract
Background: Grapevine Algerian latent virus (GALV) is a tombusvirus first isolated in1989 from an Algerian
grapevine (Vitis spp.) plant and more recently from water samples and commercial nipplefruit and statice plants. No
further reportsof natural GALV infections in grapevine have been publishedin the last two decades, and artificial
inoculations of grapevine plants have not been reported.We developed and tested a synthetic GALV construct for
theinoculationof Nicotiana benthamiana plants and different grapevine genotypesto investigate the ability of this
virus to infect and spreadsystemicallyin different hosts.
Methods: We carried out a phylogenetic analysis of allknownGALV sequences and anepidemiological survey of
grapevine samplesto detect the virus. A GALV-Nf clone under thecontrol ofthe T7promoter was chemically
synthesized based on thefull-lengthsequence of thenipplefruitisolate GALV-Nf, theonly available sequenceatthe
time theprojectwas conceived, and the infectious transcripts were testedin N. benthamiana plants. A GALV-Nf-
based binary vector was then developed for theagroinoculation of N. benthamiana and grapevine plants. Infections
were confirmed by serological and molecular analysis and the resulting ultrastructural changes were investigated in
both species.
Results: Sequence analysis showed thatthe GALVcoat protein is highly conserved among diverse isolates. The first
epidemiological survey ofcDNAs collected from 152grapevine plants with virus-like symptomsdid not reveal the
presence of GALVin any of thesamples. The agroinoculation of N. benthamiana and grapevineplants with the
GALV-Nf binary vector promoted efficient infections,as revealed by serologicaland molecular analysis. The GALV-Nf
infection ofgrapevine plants was characterizedin more detail by inoculating different cultivars, revealing distinct
patterns of symptom development. Ultrastructural changes induced by GALV-Nf inN. benthamianawere similar to
thoseinduced by tombusvirusesinotherhosts, but the cytopathological alterations in grapevine plantswere less
severe.
Conclusions: This is the firstreport describing thedevelopmentof a synthetic GALV-Nf cDNA clone,itsartificial
transmission to grapevine plants and the resulting symptomsand cytopathological alterations.
Keywords: Plant viruses, Synthetic biology, Grapevine, GALV, Cytopathology
*Correspondence:[email protected]
1DepartmentofBiotechnology,UniversityofVerona,StradaleGrazie15,
37134Verona,Italy
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Background basedontheavailablegenomesequenceoftheGALViso-
Grapevine Algerian latent virus (GALV) was first iso- late from nipplefruit (GALV-Nf [6]). These vectors were
lated in Italy in 1989 from an Algerian vine infected by used to inoculate N. benthamiana and grapevine plants,
Grapevine fanleaf virus (GFLV) [1]. GALV was consid- which allowed us to investigate the ability of synthetic
ered a latent virus because the infected plant showed GALV-Nf constructs to replicate, spread systemically and
only GFLV-related symptoms, and there have been no assembleintonormalvirusparticles.Wecharacterizedthe
further reports describing the detection of GALV in resulting infections in detail, describing the development
grapevine plants in the last 25 years. GALV has subse- ofsymptomsandthecytopathologicalfeaturesofinfection
quently been isolated from waterways in western Sicily inbothspecies.
[2], from ditches and streams in agricultural areas of
Germany [3], from water samples and different wild
plants [4], and also from rivers and Gypsophila panicu- Results
lata [5]. GALV has also been isolated from Dutch sam- VariabilityofGALVisolates
ples of commercially-grown statice plants and the We collected all available published GALV sequences by
groundwater of a statice production glasshouse [5], in searchingthe NCBI Nucleotide Database, which yielded a
Japan from commercially-cultivated nipplefruit [6] and single complete genome sequence derived from a nipple-
Limonium sinuatum [7], and in Korea from Limonium fruit isolate (GALV-Nf) plus seven further GALV coat
sinense [8]. The potential economic consequences of this protein mRNA sequences. A further complete genome
virus in the grapevine industry are unknown because it sequencederivedfromagrapevineisolate[1]wasalsode-
has been detected only once in a mixed infection [1] but posited recently, which hereafter we describe as GALV-
it caused severe stunting, chlorotic spots and mosaic Vv.2todistinguishitfromtheGALV-Nfsequence.
symptoms in nipplefruit and statice plants [6,7]. Several The nucleotide and deduced amino acid sequences of
experimental hosts have been identified in plant families the CP genes were aligned using ClustalW2 and a Per-
such as Solanaceae, Chenopodiaceae and Amarantha- cent Identity Matrix was produced in order to evaluate
ceae[1-3,5-7]. sequence similarities. The CP sequences from different
GALV was assigned to the genus Tombusvirus in the isolates showed amino acid sequence identities ranging
family Tombusviridae based on its biological and physi- from 91.49 to 99.73% whereas the nucleotide sequence
cochemical properties [1,3]. The type member of this identities ranged from 84.03 to 99.73% (Table 1). The
family is Tomato bushy stunt virus (TBSV), which has CP sequence lengths were also variable, comprising 376
been studied in detail to characterize the molecular biol- amino acids in most cases but increasing to 378 and 381
ogy ofthetombusviruses [9].TBSVhasbeenusedtode- residues for the Shunter River and GALV-Vv.1 isolates,
velop expression vectors [10,11] and gene silencing respectively (Figure 1). Based on these available CP
approaches [12,13]. The GALV genome is a positive amino acid sequences, a phylogenetic tree was con-
single-stranded RNA ~4730 nucleotides in length, com- structed, showing the sequences clustered in three dis-
prising a 5′ untranslated region (UTR), at least five open tinct groups with high bootstrap values (Figure 2). The
reading frames (ORFs) [6,14] and a 3′ UTR. The p33 Water Doss, Lim 3 and GALV-Vv isolates clustered in
and p92 ORFs encode the replicase proteins which gen- group I. The Lim 4, Gyp 2, Limo-08 and GALV-Nf iso-
erate two major subgenomic RNAs, one for the p40 coat lates clustered in group II. Finally, the Shunter River iso-
protein (CP) and the other containing two nested ORFs latewasthesolerepresentativeofgroup III.
for the production of the p24 movement protein (MP) For completeness, the genomic sequences of the
and the p19 multifunctional protein, which also func- GALV-Vv [GenBank: KJ534082.1] and GALV-Nf [Gen-
tions as a silencing suppressor [15]. GALV is assembled Bank: AY830918.1] isolates were compared, revealing an
into 30-nm icosahedral particles which accumulate in overallidentity of93.46%.
the cytoplasm. Infection results in the formation of spe-
cific membrane-associated vesicles at the periphery of
peroxisomes in Gomphrena globosa [1]. Furthermore, GALVepidemiologicalsurveyinacollectionofgrapevine
peripheral vesiculation of mitochondria and chloroplasts samples
[1] and dark staining rod-like structures in the cyto- We analyzed 152 cDNAs from grapevine accessions of
plasm and in the stroma of mitochondria and chloro- diverse provenance for the presence of GALV, including
plasts[3]were observedinChenopodiumquinoa. grapevine cultivars from commercial vineyards in the
Therehave been no reports of Agrobacterium-mediated northern, central and southern regions of Italy and
GALV infection and attempts to inoculate grapevine from germplasm collections of different grapevine ge-
plantswithnaturalisolatesofthevirushavethusfarfailed notypes from other countries such asAlgeria, Germany,
[1,6]. We therefore constructed synthetic GALV vectors Portugal, Greece, Cyprus, Armenia, Slovakia, Romania
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Table1PercentidentitymatrixofaminoacidandnucleotidecoatproteinsequencesfromdifferentGALVisolates
Isolates GALV-Vv.1 GALV-Vv.2 Waterdoss S.River Gyp2 Lim3 Lim4 GALV-Nf Limo-08
GALV-Vv.1 ― 99.73 99.20 93.35 96.28 99.94 93.88 95.48 95.74
GALV-Vv.2 99.91 ― 99.47 93.62 96.28 98.2 93.88 95.74 96.01
WaterDoss 99.13 99.73 ― 93.09 95.74 99.73 93.35 95.21 95.48
S.River 85.08 85.5 85.29 ― 94.41 92.82 91.49 93.88 94.15
Gyp2 91.71 92.57 92.1 85.75 ― 92.28 95.31 98.14 98.67
Lim3 99.73 99.73 99.66 85.37 92.18 ― 93.09 94.95 95.21
Lim4 90.66 90.98 90.42 84.03 95.31 90.58 ― 94.68 95.21
GALV-Nf 92.04 92.13 92.04 85.85 96.46 91.87 93.81 ― 99.47
Limo-08 91.87 91.95 91.87 85.85 96.20 91.69 94.08 98.14 ―
Deducedaminoacid(abovehorizontalbars)andnucleotide(belowhorizontalbars)sequencesfromdifferentisolates(fromAlgeria:GALV-Vv.1,GALV-Vv.2;from
Germany:WaterDoss,SchunterRiver(S.River),Gyp2;fromNetherlands:Lim3,Lim4andfromJapan:GALV-Nf,Limo-08)werealignedandapercentidentity
matrixwascreated.Thelowestandthehighestsequenceidentitiesbetweendifferentisolatesareshowninbold.
and France (Additional file 1: Table S1). These samples We screened the samples for the presence of GALV
comprised different American and Asiatic species com- by RT-PCR, using a primer set designed to amplify a
merciallycultivatedorusedforbreedingorrootstockpro- conserved portion of the GALV-Nf and GALV-Vv CP
duction as well as a number of V. vinifera cultivars and coding regions as well as the same CP fragment of most
accessions. oftheremainingGALVisolates,exceptShunterRiverand
Figure1AminoacidsequencealignmentofpredictedcoatproteinsderivedfromdifferentGALVisolates.Predictedproteinsequences
ofdifferentisolates(GALV-Vv.1,GALV-Vv.2,WaterDoss,SchunterRiver(S.River),Gyp2,Lim3,Lim4,GALV-Nf,Limo-08)werealigned.Theprotruding
coatproteindomainsareshowninbold.
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Figure2PhylogenetictreeofpredictedcoatproteinsderivedfromdifferentGALVisolates.Predictedproteinsequencesofdifferent
isolateswerealignedandthephylogeneticthreewasconstructedusingtheneighbor-joiningmethod(fromAlgeria:GALV-Vv.1,GALV-Vv.2;from
Germany(DE):WaterDoss,SchunterRiver,Gyp2;fromNetherlands(NL):Lim3,Lim4andfromJapan:GALV-Nf,Limo-08).
Lim 4. No GALV infection was detected in any of the the transcriptional control of the T7 promoter plus a
samples. SrfI site for vector linearization, which is required to
produce the correct 3′ viral end by in vitro transcription
Constructionoffull-lengthGALV-NfcDNAclones [16,17]. We added a multiple cloning site (MCS) down-
We developed and tested three different infectious stream of the p24 coding region at the unique BstBI re-
GALVclones (Figure 3A, B, C). First, two GALV-Nf par- striction site to create vector T7-MCS-GALV-Nf, which
tial sequences were synthesized and manipulated to pro- contains two new unique restriction sites (BglII and
duce the final T7-GALV-Nf vector (Figure 3A). This XhoI) thus allowing the further functionalization of this
clone contains the full-length GALV-Nf sequence under vector in future studies. Three different stop codons,
Figure3SchematicrepresentationoftheinfectiousGALV-NfcDNAclones.GALV-NfsequencesunderthecontrolofT7promoter(T7-GALV-Nf
andT7-MCS.GALV-Nf)werelinearizedwithSrfIandusedfortheinvitroproductionofinfectioustranscripts(detailsofT7transcriptionsiteandSrfI
cleavagesiteareshowninpanelA).ApolylinkerwasinsertedbyBstBIcleavageintheT7-GALV-NfconstructobtainingtheT7-MCS.GALV-Nfvector(the
BglIIandXhoIsitesofthepolylinkerareshowninboldandunderlinedinthesequencereportedinpanelB).Theviralsequence,placedunderthe
controloftheCaMV35Spromoter(35S)andthenosterminator(NOS),wasintroduced(usingSacI/AscIandAsiSI/XbaIsites)betweenthepK7WG2left
andrightborders(RBandLB)producingthepK7WG2-MCS.HRz.GALV-NfbinaryvectorforA.tumefaciens-mediatedinfection.Todesignafunctional5′
viralend,thejunctionbetweenthe35Sandtheviralsequencewasobtainedbyligatingblunt-endfragmentsproducedbydigestingthe35Ssequence
withStuIandtheviralsequencewithDraI(detailsshowninpanelC).ThesequenceoftheHepatitisdeltavirusantigenomicribozyme(HRz)was
introducedtoallowtheproductionofacorrect3′viralendfollowingribozymeautocleavage(panelC).T7:T7promoter;p33andp92:RNA-dependent
RNApolymerases;p40:coatprotein(CP);p24:movementprotein(MP);p19:silencingsuppressor.
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one for each reading frame, were designed upstream the chlorotic spots appeared on the inoculated leaves 4 days
MCS to prevent the formation of aberrant viral proteins post-inoculation (dpi) (Figure 4A) followed by the devel-
(Figure 3B). Finally, the pK7WG2-MCS.HRz.GALV-Nf opment of systemic veinlet chlorosis at 7 dpi, starting
binary vector was produced (using the SacI/AscI and from the proximal part of the leaf (Figure 4B). GALV-Nf
AsiSI/XbaI restriction sites) placing the GALV-Nf se- infection was confirmed in systemically infected leaves by
quence under the control of the CaMV 35S promoter DAS-ELISA(datanotshown).However,only~30%ofthe
and inserting the HRz ribozyme and nos terminator se- plantswereinfectedineachexperiment.
quences immediately downstream of the viral 3′ UTR We developed the GALV-Nf binary vector described
(Figure 3C). This vector can be delivered to plants by abovetoimprovetheefficiencyofinfection,anddelivered
agroinfiltration and allows the production of GALV-Nf the vector to N. benthamiana plants by leaf agroinfiltra-
RNAs almost equivalent to the viral genome. In particu- tion. Upper non-infiltrated leaves displayed light mottling
lar, by using the StuI-DraI ligation strategy described with some necrotic spots, and apical leaf necrosis at 12
under Materials and Methods, a functional viral 5′ UTR dpi (Figure 4C). The efficiency of infection in agroinfil-
sequence corresponding to the +1 transcriptional start trated plants was ~90% in almost all experiments. Sys-
site can be produced, lacking only two nucleotides from temic spreading was confirmed in all symptomatic plants
the original GALV-Nf genome sequence. The HRz ribo- by RT-PCR analysis (Figure 4D) and by tissue-print im-
zyme can produce the precise viral 3′ terminus by self- munoassays in the same plants (Figure 5A, B). GALV-Nf
cleavage[11]. particleswerepurifiedfromsystemicallyinfectedleavesof
N. benthamiana plants rubbed with the T7-GALV-Nf
InfectivityofGALV-NfclonesinN.benthamianaplants transcripts (Figure 5C) or following agroinfiltration with
The infectivity of GALV-Nf transcripts derived from the binary vector (Figure 5D). Immunosorbent electron
the T7-GALV-Nf construct was investigated by rub- microscopy(ISEM)revealedinbothcasesthepresenceof
inoculating approximately 20N. benthamiana plants correctly-assembled 32-nm particles with the expected
with different lots of infectious transcripts. Coalescing icosahedralmorphology.
Figure4LocalandsystemicGALV-NfsymptomsonN.benthamianaplantsandGALVdetectionbyRT-PCR.(A)Localand(B)systemic
GALV-Nfsymptoms7daysaftertheinoculationwithT7-GALV-Nfviraltranscriptsand(C)systemicsymptomsobservedonN.benthamianaplants
12daysafteragroinfiltrationwiththebinaryvector.(D)RT-PCRdetectionofGALVinfectiononsystemicleavesofagroinfiltratedplants(D1,2,3)
andahealthyplant(D4).w:waternegativecontrol.
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Figure5SerologicalGALV-Nfdetectioninsystemically-infectedN.benthamianaleavesandanalysisofviralparticlesbyelectron
microscopy.Tissue-printimmunoassayusingaGALV-specificantibodyonsystemically-infectedleavesof(A)agroinfiltratedand(B)healthyplants.
Purifiedviralparticlesfrom(C)T7-GALV-Nfinfectedor(D)agroinfiltratedN.benthamianaplantsdetectedbyimmunosorbentelectronmicroscopy.
CytopathologyofAgrobacterium-mediatedGALV-Nf InfectivityofGALV-Nfingrapevineplants
infectioninN.benthamianaplants Plantlets representing different grapevine genotypes
Insystemically-infectedleaves,icosahedralvirusparticles were inoculated with the binary vector by agroinfiltra-
were present in almost all tissues, in the cytoplasm and tion. It is important to assess symptom development in
vacuoles but not in other organelles (Figure 6). In some completely virus-free plants, so we investigated the out-
heavily-infected cells, large virus crystals occupied most comes of infection in virus-free plants regenerated from
ofthe celllumen(Figure6A,C)togetherwithaggregates somatic embryos representing the cultivars Brachetto,
of virions in membrane-bound enclaves (Figure 6D). Syrah and Nebbiolo, to provide solid evidence that
The other major cytopathic features of infected cells in- GALV-Nf can produce symptoms following artificial in-
cluded the presence of multivesicular bodies formed by fection. Different symptoms were observed in emerging
stacked vesicles containing a fibrillar network (Figure 6B) leaves 5 weeks after infiltration (Figure 7A, B, C) and
andalteredchloroplastsshowingthylakoiddisorganization these symptoms were absent in the corresponding
and vesiculation (Figure 6E). Thylakoid disorganization healthy plants (Figure 7D, E, F). Regenerated Brachetto
was observed in almost all chloroplasts of the parenchy- plants occasionally developed small chlorotic or necrotic
malmesophyllcells.Insomeofthesechloroplasts,vesicles spots along the veins (Figure 7A) whereas Syrah plants
similar to those forming the multivesicular bodies were showed mild vein clearing or mottling (Figure 7B). No
also visible, either singly or in small groups, and stacked other developmental abnormalities were observed in ei-
on the thylakoid membranes (Figure 6F). The analysis of ther cultivar. In contrast, Nebbiolo plants showed mal-
multiple sections through the multivesicular bodies did formations of the whole leaf lamina, chlorotic patches
not reveal the origin of these structures. In cells from and dark green blistering on the leaves (Figure 7C) to-
leaves at the late stage of infection, the chloroplast struc- gether with weak and stunted shoot growth (Figure 7G).
ture was completely disrupted (Figure 6G) and electron- These symptoms were not observed in healthy controls
densetubularstructureswerevisible inthestroma.These (Figure 7H).
structures were also present in some mitochondria Additional grapevine genotypes were tested for symp-
(Figure6H),althoughthemorphologyoftheperoxisomes tom development, i.e. V. vinifera cv. Sultana and cv.
and mitochondria did not show significant changes in Corvina derived from certified mother-plants, and V.
mostoftheinfectedcells. riparia cv. Gloire de Montpellier. In each case, plant
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Figure6GALV-Nfcytopathologyinsystemically-infectedN.benthamianaleaves.(A)Epidermalcellfilledwithvirusparticles(V)and
showingsomemultivesicularbodies(arrows),oneofthemenlargedin(B).Virionsaredistributedthroughoutthecytoplasm,oraggregatedinalarge
crystal(C),orenclosedinmembrane-boundenclaves(D).Aplastid,enlargedin(E),showsthylakoiddisorganizationandvesiculation.Similarchloroplast
alterationsarevisiblealsoinparenchymamesophyllcells(F)and,insomeinstances,vesiclessimilartothoseformingthemultivesicularbodiesare
observed(arrows).Incellsatalatestageofinfection,thechloroplaststructureappearscompletelydisrupted(G)andelectron-densetubularstructures
(T)arevisibleinthestroma.Thesestructuresaresometimespresentalsoinmitochondria(H).N,nucleus;P,peroxisome;L,lipidglobule.Blackbars=
200nm;whitebars=400nm.
growth was unaffected, shoots developed normally and observedirregularchloroticveinbandingofthemajorleaf
theplantsreachedthesamesizeandgrowinghabitsasun- veinsinV.ripariaplants(Figure7I),aslightupwardcurl-
infected controls. Mild systemic symptoms were visible ing of leaf margins in Sultana plants (Figure 7J) and light
only on emerging leaves about 5 weeks after infiltration mottling in Corvina leaves (Figure 7K). These symptoms
(Figure 7I, J, K), and these were absent in GALV-free were consistent across all experiments, with an infection
plants of the same genotypes (Figure 7L, M, N). We efficiency of ~90%, suggesting that artificial GALV-Nf
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infection can lead to the development of different symp-
tomsindifferentgenotypes.
The systemic infection of grapevine plants by GALV-
Nf was confirmed by RT-PCR analysis of apical leaves
representing the six infected genotypes (Figure 8A) and
by tissue-print immunoassay (Figure 8B, C) confirming
that the virus can replicate and spread systemically in all
the cultivars wetested.
CytopathologyofAgrobacterium-mediatedGALV-Nf
infectioningrapevine
Ultrastructural analysis of systemically-infected leaves
(mainly in the Syrah plants showing vein clearing and
mottling) confirmed that the most severely affected cells
were those adjacent to small veins, particularly in the
spongy mesophyll (Figure 9A). These cells showed differ-
ent stages of plasmolysis and membrane rupture, and the
chloroplasts appeared swollen compared to matched, un-
infected control leaves (Figure 9B, E). At the ultrastruc-
tural level, the altered chloroplasts showed evidence of
thylakoid disorganization, although without vesiculation
(Figure 9C), and numerous virus particles were spread
throughout the surrounding cytoplasm (Figure 9D). The
mitochondria were occasionally swollen, and almost de-
voidofcristaeandstroma(Figure9C).Cellsaroundsmall
veins sometimes showed incipient necrosis (Figure 9F),
and contained dense cytoplasm in which it was still pos-
sible to distinguish virus-like particles (Figure 9G). The
mitochondria of infected parenchyma cells in small veins
were vacuolated and devoid of cristae (Figure 9H) and
small vesicles weresometimes present in dilated cisternae
derivedfromtheendoplasmicreticulum(Figure9I).How-
ever, multivesicular bodies like those observed in N.
benthamiana werenotobserved intheinfected grapevine
cells. All the ultrastructural alterations described above
were also present in the symptomatic leaves of Nebbiolo
plants.Theonlyultrastructuralfeatureassociatedwiththe
peculiar symptoms observed in Nebbiolo plants was the
significantreduction in chloroplastnumber in cells repre-
sentingthelight-greenareasoftheleaf(datanotshown).
Figure7SystemicGALV-Nfsymptomsingrapevineplants.
Discussion
GALV-Nfsymptomsinduced5weeksafterinfiltrationwiththe
GALVisdetectedoccasionallyintheenvironment
GALV-Nf-basedbinaryvectorindifferentcultivarsregeneratedfrom
somaticembryos:V.vinifera(A)Brachetto,(B)Syrah,(C)Nebbiolo GALV is a tombusvirus that can cause severe symptoms
comparedtocorrespondinghealthyplants(D,E,F).Severestunting in certain plants, including nipplefruit and statice [6,7],
andleafalterationsobservedonaNebbioloplant(G)5weeksafter butithasbeenisolatedonlysporadicallyoverthetwo de-
infiltrationwiththeGALV-Nf-basedbinaryvectorcomparedtoa
cades since the discovery of the original grapevine isolate
correspondinghealthyplant(H).GALV-Nfsymptomsinduced
in 1986 [1]. It is most often found in environmental wa-
5weeksafterinfiltrationwiththeGALV-Nf-basedbinaryvectoron
differentgrapevinegenotypes:(I)V.ripariacv.GloiredeMontpellier; ters,afeaturecommontootherwater-borneplantviruses
V.vinifera(J)Sultana,(K)Corvina,comparedtocorresponding thatcanpersistoutsidethehostforalongtime[18].Nat-
GALV-freeplants(L,M,N). ural GALV infections occur with a low incidence in most
countries and the virus is not considered a threatening
pathogen by the European and Mediterranean Plant Pro-
tection Organization (EPPO). Accordingly, our extensive
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Figure8DetectionofGALV-Nfinsystemically-infectedgrapevineplants.(A)RT-PCRanalysisofpK7WG2-MCS.HRz.GALV-Nfinfectedsystemic
leavesof(1)V.riparia,orV.viniferacv.(2)Sultana,(3)Corvina,(4)Nebbiolo,(5)Syrah,(6)Brachetto,andoncorrespondingnon-inoculatedplants
(7,8,9,10,11,12).w:waternegativecontrol.Tissue-printimmunoassayonsystemically-infectedCorvinaleaf(B)andonaGALV-freeleaf(C).
survey of 152 grapevine plants from different locations AfunctionalGALVgenomecanbecreatedbysynthetic
showed no evidence of naturally-occurring GALV infec- biology
tion. GALV was also not detected in deep sequencing ex- Viruses have been used to demonstrate the potential of
periments carried out on leaves, petioles and phloem synthetic biology because they have small genomes that
scrapes collected from grapevines showing symptoms of can be synthesized de novo with great precision. The
viraldisease[19-22]. principle was demonstrated by synthesizing an artificial
replicon based on Hepatitis C virus [24] and this was
GALVcoatproteinsequenceshavediversifiedintothree followed by the complete fabrication of poliovirus [25]
majorclusters and bacteriophage ϕX174 [26]. Plant viruses are particu-
The paucity of natural GALVsamples means that few se- larly suitable for this approach because the majority pos-
quences have been deposited in public databases. When sess small ssRNA(+) genomes that can be manipulated as
our experiments began there were only seven GALVcoat cDNAandtranscribedinvitroorinplanta,andsynthetic
protein sequences from different isolates in the NCBI Nu- biology is therefore the ideal solution if no natural tem-
cleotideDatabase[5-7,23],allidentifiedbasedonbiological plate is available, as is the case for GALV. Thus far, only
features and serological reactions against an antibody pro- Tobacco mosaic virus (TMV) has been synthesized de
duced against theoriginal 1986isolate [1]. Therewas only novo,although the initialsyntheticgenome was not infec-
onefull-lengthGALVgenomesequencefromanipplefruit tiousduetothepresenceoferrorsintheoriginalsequence
isolate [6] although the genome sequence of the original deposited in 1982 [27]. The authors aligned the original
1986 GALV isolate [1] was deposited during the course of sequence with more recent strains and identified the
our work [GenBank: KJ534082.1]. The sequences were changes required to synthesize an infectious clone [28].
highly conserved across isolates as anticipated, but as pre- Chimeric sequences were also tested to determine the re-
viouslyreportedtherewereminordifferencesinthelength gions of the viral genome responsible for pathogenicity
oftheCPopenreadingframe[5].Theseminordifferences and host range [28]. Improvements in DNA sequencing
do not prevent the original antiserum cross-reacting with and synthesis now make such errors increasingly unlikely
all known isolates [1-6]. We found that the CP sequences and reduce the need for the detailed genetic analysis of
clusteredintothreemajorgroupsbutthesedidnotcorrel- synthetic genomes [28]. We thereforeused synthetic biol-
ate with the geographic origin, with the exception of the ogy to produce infectious clones based on the GALV-Nf
Japanese isolates which tended to show particularly strong genome sequence and tested their ability to infect N.
conservation. benthamianaandgrapevineplants.
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Figure9GALV-Nfcytopathologyofsystemically-infectedgrapevineleaves.(A)Virusinfectedcellsareeasilyidentifiedevenunderalight
microscopeclosetosmallveins(X)andshowdifferentstagesofplasmolysisandmembranerupture.Chloroplastsofspongymesophyll(arrow)
appearswollen,incomparisonwiththosevisibleinanuninfectedcontrolleaf(B).Attheultrastructurallevel(C),swollenchloroplasts(Ch)show
thylakoiddisorganization,andnumerousvirusparticles(V)arevisibleinthesurroundingcytoplasm(D);mitochondria(M)arealsoswollen,
apparentlywithoutstromaandalmostdevoidofcristae.Anuninfectedcontrolcellisvisiblein(E)forcomparison.Cellsaroundsmallveins
sometimesshowincipientnecrosis(F)withdensecytoplasminwhichvirus-likeparticlesarestilldistinguishable(G).Infectedparenchymacellsof
smallveins(H)showmitochondriawithvacuolizationandlossofcristaeandsmallvesiclesaresometimespresentinapparentlydilatedER
cisternae(I).N,nucleus;P,peroxisome;Ps,plasmodesmata.Blackbars=400nm;whitebars=100nm,ifnototherwisestated.