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A simplifed method o constructing inectious cloneso Begomovirus
employing limited restriction enzymedigestion o products o rolling
circle amplifcation.
AbstractMost infectious clones of geminiviruses consist of
(partial)tandem repeats of viral genomes in the vectors, which
usuallyinvolve tedious, multi-step assemblies of genomic
fragmentsin the construction process. A simplied procedure
wasdevised to circumvent these problems, which employs
limitedrestriction digestion of multimeric viral genomes produced
byrolling circle amplication (RA), followed by direct cloninginto
appropriate vectors. !he e"ciency of the procedure, andinfectivity
of the dimeric constructs it produced, were
demonstrated using three di#erent geminiviruses, namelyageratum
yellow vein virus, tomato leaf curl virus, and s$uashleaf curl
virus.
%eminiviruses are characteri&ed by single-stranded,
circular'A (ssc'A) genomes, which are encapsidated in
geminatevirions approimately *+ nm nm (/tanley et al., 01).2hite3y
transmitted geminiviruses with mono- or bi-partitessc'Agenomes
comprise the genus Begomovirus of the
family %eminiviridae. Although ecised unit-length 'A orcloned 'A
containing single copies of certain geminivirusesis infectious
(e.g., /tanley and !ownsend, *4+56 7onilla-Ramire& et al.,
*4486 9nseld et al., 0:6 ;apidot et al., 08),earlier studies have
shown that most infectious clones re$uire(partially) tandem-repeat
constructs of geminivirus genomesto increase their infectivity
(7oulton and 'avies, *4++6hatani et al.,*44*6 'onson et al.,
*4++6
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8/9/2019 A Simplified Method of Constructing Infectious Clones
of Begomovirus Employing Limited Restriction Enzyme Digest
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have to be monitored to select clones that contain
tandemlyrepeated viral genomes.
Recently, rolling circle amplication (RA) technology hasbeen
developed for linear or geometric amplication of target'A for
research and diagnostic purposes (@ire and >u, *4416;iu et al.,
*4456 ;i&ardi et al., *44+) with sensitivitiesapproaching those
of polymerase chain reactions (R), andmultimeric infectious clones
of geminiviruses have beengenerated using the RA techni$ue
(Bnoue-agata et al.,0:6 Caible et al., 056Dnierim and Maiss, 08).
Cowever,the procedures used in these studies still re$uire etra
stepsfor fragment assembly following etensive digestions of theRA
products with di#erent restriction en&ymes. Bn this study,
a simplied procedure was devised, which limited thedigestion of
RA reaction products with restriction en&ymes togenerate
tandemly repeated concatamers of %eminivirusgenomes for the
construction of infectious clones. !hefeasibility of this methodwas
demonstrated using threedi#erent begomoviruses as templates.
A an-!ou isolate of Ageratum yellow vein Taiwan virus(AEF!F-G!H,
%en7an= accession no.
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8/9/2019 A Simplified Method of Constructing Infectious Clones
of Begomovirus Employing Limited Restriction Enzyme Digest
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and combined with 1 Il of reaction bu#er plus .0 Il of
en&ymemi (containing hi04 'A polymerase and random heamersin 1K
glycerol). !he reaction mitures were then incubatedfor *+ h at P,
followed by inactivation of the en&yme at 51P for * min.
Agarose gel analysis revealed mitures of RAreaction products longer
than *0 =bp (@ig. 0A, lane 0),indicating that the circular 'A
templates in the samples hadbeen etensively amplied.
@ig. *. /chematic representation of the wor=3ow andconstruction
of multimeric clones of begomoviruses by limiteddigestion of
rolling circle amplication (RA) products.Ageratum yellow vein
!aiwan virus an-!ou isolate (AEF!F-G!H) was used as a
representative for circular templates in
the following diagrams. (A) Bllustration of the wor=3ow.
(*)Annealing of random heamers (short arrows) to single-stranded
circular 'A templates6 (0)
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of Begomovirus Employing Limited Restriction Enzyme Digest
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@ig. 0. onstruction of multimeric infectious clones of AEF!F-G!H
with limitedrestriction en&yme digestion. (A) Agarose gel
analysis ofrestriction en&yme digestion products. !he polymeric
'Asgenerated by rolling circle amplication were digested
withrestriction en&yme 7amCB, using 0 units for * h at 8 P
(lane) or .0 unit for *1 min at 01 P (lane :). !he
digestionproducts were then analy&ed by electrophoresis through
*Kagarose gel and staining with ethidium bromide. ;ane *,
'Asi&e mar=er6 lane 0, undigested RA products as the
negativecontrol. (7) /outhern blot analysis of the products of
limiteddigestion. !o verify identities, the partially digested (.0
unitof 7amCB for *1 min at 01 P) RA products (lane S*1 minT)was
/outhern-transferred to a nitrocellulose membrane
following electrophoresis through *K agarose gel
andhybridi&ed with 'igoigenin-labeled probes specic to
AEF!Fgenomic 'A(2u et al., 08).
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of Begomovirus Employing Limited Restriction Enzyme Digest
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genomes were identied by the probes (lane S*1 minT). !heproduct
approimately *.: =bp long (indicated by the SVT sign)also
hybridi&ed with the probes, suggesting that the 'Aproducts were
incomplete copies of the AEF!F genome insteaof satellite 'As, 'A-*
or 'A I, associated with AEFF(/aunders and /tanley, *4446 /aunders
et al., 0), becausesatellite 'As do not share signicant similarity
with theAEF!F genome. Cowever, the possibility that these
'Asrepresent a defective 'A of AEF!F could not be ruled out.
@ollowing purication by phenol etraction and
ethanolprecipitation, the partially digested RA products
wereinserted into the 7amCB site of the binary vector p7in*4(7evan,
*4+:) (@ig. *7). !o eamine whether the presence of a
constitutive promoter could increase the infectivity of
the%eminivirus constructs, the 1/ promoter region ofauli3ower
mosaic virus (aMF 1/ promoter) was ampliedby R with the primer pair
1/Cind@, 1I-
!%AA%!!%A!%!%-I and 1/CindR, 1I%!AA%!!A%%!!AAA!%-I using pass
plasmid('ing et al., *441) as the template, digested with
restrictionen&yme CindBBB, and inserted into the CindBBB site
of p7in*4vector immediately upstream of the %eminivirus 'A
insertion site, resulting in a new vector, namely p7in*4-
1/(@ig. *7).
!o test whether this simplied procedure is applicable to
otherviruses with circular genomes, dimeric constructs of two
otherbegomoviruses, tomato leaf curl virus (!;F, a
mono-partitebegomovirus) and s$uash leaf curl virus (/$;F, with
bi-partite genome designated as'AAand'A7), isolatedrespectively
from !ainan and Eunlin ounty in !aiwan, weregenerated following the
same procedure. !he selected clonesshownin @ig. *7 and !able * were
identied on the basis ofrestriction digestions and 'A
se$uencing.
!he integrity and infectivity of the begomovirus clones
wereassayed by agroinfection (%rimsley et al., *4+5, *4+86
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benthamiana plants were inoculated with
Agrobacteriumtumefaciensstrain 1+* harboring either p7in*4-1/
vectoralone or dimeric constructs of the begomoviruses
describedabove. As shown in !able *, the dimeric constructs
showedinfection rates ranging from 50.1 to *K. !he orientations
ofthe inserts relative to aMF 1/ promoter did not a#ect
theinfectivity the infection rate was *K in the case ofp7inAE:.0,
the same as that in the case of the maUority ofconstructs with the
opposite orientation. Cowever, whetherthese clones share similar 'A
accumulation levels orreplication e"ciencies is un=nown, as the
$uantities ofreplication intermediates corresponding to these
clones werenot determined. !he release of circular viral 'A
duringreplication from the input dimer template, which might
result
from the nic=ing and re-ligating activities of viral Rep
protein(Ceyraud-itsch=e et al., *4416 ;aufs et al., *441) or
therecombination events between the homologous se$uences(/tenger et
al., *44*6 Rigden et al., *445), followed bysubse$uent 'A synthesis
in plant nuclei may havecontributed mainly to the intracellular
viral growth irrespectiveof the orientations of the clones.
!able * Bnfectivity of various begomovirus constructs in N.
benthamiana plants inoculated by agroinfection.
!o verify the presence of AEF!F-G!H in the a#ected leaves ofthe
inoculated N. benthamiana, 2estern blot analysis wasperformed to
detect the viral coat protein () which furtherconrmed the
infectivity of these dimeric constructs (@ig. ).
!he results indicated that the dimeric clones constructedusing
limited restriction en&yme digestion of the RA productsstill
remained highly infectious.
@ig. . onrmation of infectivity by2estern blot
analysis.!otalproteins e$uivalent to 0.1 mg of fresh-leaf weight
from eachsample were separated using a *0.1K polyacrylamide
gelcontaining *K sodium dodecyl sulphate (;aemmli, *48),transferred
to a membrane, and probed with antiserumspecic to the coat protein
() of AEF!F, followed by color
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of Begomovirus Employing Limited Restriction Enzyme Digest
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development using nitroblue tetra&olium (7!) and
1-bromo-:-chloro--indodyl phosphate (7B) as substrates (/ambroo=and
Russell, 0*). !he identities of the samples are indicatedon top of
each lane. ;ane AEF!F-, E. coli epressed ofAEF!F (*1 ng) as a
positive control. !he position of AEF!F-is indicated on the
right.
Bn conclusion, the simplied procedure reduces the timere$uired
for the construction of infectious clones ofgeminiviruses. !he need
to select single-cut restrictionen&ymes and to verify the
orientation of the inserts iseliminated. 7y appropriately modifying
the conditions ofrestriction digestion, the procedure can be
applied to studiesof viruses with circular genomes.
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Un mtodo simplifcado de la construccin de clonesinecciosos de
Begomovirus empleando limitadadigestin con enzimas de restriccin de
los productosde amplifcacin por crculo rodante.
Abstracto
;a mayorWa de los clones infecciosos de geminivirus consistenen
repeticiones en tXndem (parcial) de los genomas virales en
los vectores, $ue generalmente implican tediosas, conUuntosde
varios pasos de fragmentos genYmicos en el proceso
deconstrucciYn.9n procedimiento simplicado se concibiY paraevitar
estos problemas, $ue emplea la digestiYn de restricciYnlimitado de
genomas virales multimZricas producidos poramplicaciYn por cWrculo
rodante (RA), seguido por clonaciYndirecta en vectores
apropiados.;a ecacia del procedimiento,y la infectividad de los
constructos dimZricos $ue produUo, sedemostraron utili&ando
tres diferentes geminivirus, virus de lavena amarilla ageratum a
saber, el virus de enrollamiento delas hoUas de tomate, y virus del
enrollamiento de la hoUas$uash.
;os geminivirus se caracteri&an por (ssc'A) genomas de
unasola hebra, A' circular, $ue estXn encapsulados en
virionesgeminadas aproimadamente *+ nm nm(/tanley etal., 01).Mosca
7lanca transmite geminivirus conssc'Agenomes mono o bi-partita
comprender elgZnero Begomovirusde la familia %eminiviridae.Aun$ue
el
A' unidad de longitud etirpado o A' clonado $uecontiene copias
individuales de ciertos geminivirus esinfecciosa (por
eUemplo,/tanley y !ownsend, *4+56. 7onilla-RamWre& et al.,
*4486 9nseld et al., 0:6 ;apidot etal, 08),
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aumentar su infectividad(7oulton y 'avies, *4++6. hatani
etal,*44*6'onson et al., *4++6iong y ?hou, 056 >ionget al.,
08).or otraparte, las orientaciones de los insertos tienen $ue
sermonitoreados para seleccionar clones $ue contienen repetidasen
tXndem genomas virales.
Recientemente, la tecnologWa de amplicaciYn por cWrculo
rodante (RA) ha sido desarrollado para la amplicaciYn linealo
geomZtrica de A' diana para nes de investigaciYn y
dediagnYstico(fuego y >u, *4416;iu et al., *4456;i&ardi
etal, *44+)con sensibilidades se aproiman a las de reaccionesen
cadena de la polimerasa (R), y clones infecciososmultimZricas de
geminivirus se han generado utili&ando latZcnica de
RA(Bnoue-agata et al, 0:6.. Caible et al.,056Dnierim y Maiss,
08)./in embargo, losprocedimientos utili&ados en estos estudios
todavWa re$uieren
pasos adicionales para el montaUe fragmento siguientesetensas
digestiones de los productos de RA con diferentesen&imas de
restricciYn.
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describen%rimsley et al.(*4+8).7revemente, la hoUa sencilla(,* g
de peso fresco) se moliY a un polvo no en nitrYgenolW$uido, se
descongela en tampYn de etracciYn de A'
I; (1 mM !ris-Cl, pC +,, 0 mM
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de A' de diferentes longitudes de unidad de genoma
diana(indicados por \*>\, \0>\, y \>\).;os signos
\tallo-bucle\indican el origen de replicaciYn de geminivirus.(7)
'iagramasde los constructos infecciosas para agroinfecciYn.
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\viriYn A'\) se utili&Y como control positivo para indicar
laposiciYn relativa de la unidad de longitud del genoma viral.
ara generar dWmeros en tXndem de AEF!F A' genYmicopara la
clonaciYn directa, los productos de las reacciones RA(*Ig) se
puricaron mediante etracciYn con fenol y sesometieron a digestiYn
ya sea limitada, usando ,0 unidadde la en&ima de restricciYn
7amCB a 01 P durante *1 min o ala etensa la digestiYn usando 0
unidades a 8 P durante * hcomo control.;as digestiones se
terminaron mediante laadiciYn de formamida y
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p7in*4(7evan, *4+:) (@ig. *7).ara eaminar si la presenciade un
promotor constitutivo podrWa aumentar la infectividaddel
geminivirus construcciones, la regiYn del promotor 1/ delvirus del
mosaico de la coli3or (aMF 1/) se amplicY porR con el par de
cebadores 1/Cind@, 1I-
!%AA%!!%A!%!%-I y 1/CindR, 1I%!AA%!!A%%!!AAA!%-I utili&ando
el plXsmidopass('ing et al.,*441)como plantilla, se digiriY con
laen&ima de restricciYn CindBBB, y se insertY en el sitio
CindBBB delvector p7in*4 inmediatamente aguas arriba del sitio
deinserciYn de A' de geminivirus, resultando en un nuevovector, a
saber p7in*4 - 1/(@ig. *7).
ara comprobar si este procedimiento simplicado es
aplicable a otros virus con genomas circulares,
construccionesdimZricas de otros dos begomovirus, hoUa de tomate
curl virus(!;F, un begomovirus mono-partita) y la hoUa de
calaba&acurl virus (/$;F, con genoma bipartito
designadoas'AAand'A7 ), aislado, respectivamente, de !ainan y
elcondado de Eunlin en !aiwXn, se generaron siguiendo elmismo
procedimiento.;os clones seleccionadosshownin@ig.*7 y enla !abla
*se identicaron sobre la basede las digestiones de restricciYn y
secuenciaciYn de A'.
;a integridad y la infectividad de los clones de begomovirusse
ensayaron mediante agroinfecciYn(%rimsley et al, *4+5,*4+86.
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de replicaciYn correspondientes a estos clones.;a liberaciYnde
A' viral circular durante la replicaciYn de la entrada dedWmero
plantilla, $ue podrWan resultar de las actividadesnic=ing y
re-ligadura de la proteWna Rep viral(Ceyraud-itsch=e et al, *4416..
;aufs et al, *441)o los eventos derecombinaciYn entre las
secuencias homYlogas(/tengeret al,*44*6.. Rigden et al,
*445),seguido por la sWntesis de A'posterior en los n]cleos de las
plantas pueden habercontribuido principalmente al crecimiento viral
intracelular,independientemente de las orientaciones de los
clones.
!abla *;a infectividad de diferentes begomovirus construyeen
N.benthamianaplantas inoculadas por agroinfecciYn.
ara vericar la presencia de AEF!F- G!H en las hoUasafectadas del
N.inoculadobenthamiana,se reali&Y anXlisis detransferencia
2estern para detectar la proteWna de la cubiertaviral (), $ue
conrmY ademXs la infectividad de estasconstrucciones dimZricas(@ig.
).;os resultados indicaron $uelos clones dimZricos construidos
usando limitada digestiYncon en&imas de restricciYn de los
productos de RA todavWapermanecieron altamente infecciosa.
@ig.. onrmaciYn de las proteWnas analysis.!otal infectividadblot
by2estern e$uivalente a 0,1 mg de peso fresco de hoUasde cada
muestra fueron separados usando un gel depoliacrilamida al *0,1K
$ue contenWa *K de dodecil sulfato desodio (;aemmli, *48), se
transrieron a una membrana, y sesondeY con antisuero especWco para
la proteWna de cubierta() de AEF!F, seguido por el desarrollo de
color utili&andonitroa&ul de tetra&olio (7!) y fosfato
de 1-bromo-:-cloro--indodyl (7B) como sustratos (/ambroo= y
Russell, 0*). ;asidentidades de las muestras se indican en la parte
superior de
cada carril.arril AEF!F-, E.coliepresa de AEF!F (*1ng) como
control positivo.;a posiciYn de AEF!F- se indica ala derecha.
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restricciYn de un solo corte y para vericar la orientaciYn delos
insertos se elimina.or apropiadamente la modicaciYn delas
condiciones de digestiYn de restricciYn, el procedimientose puede
aplicar a los estudios de los virus con genomascirculares.
