Laird, J., McInally, C., Carr, C., Doddiah, S., Yates, G., Chrysanthou, E., Khattab, A., Love, AJ, Geri

14 Pages·2013·1.67 MB·English

Laird, J., McInally, C., Carr, C., Doddiah, S., Yates, G., Chrysanthou, E.,
Khattab, A., Love, A.J., Geri, C., Sadanandom, A., Smith, B.O., Kobayashi,
K., and Milner, J.J. (2013) Identification of the domains of cauliflower
mosaic virus protein P6 responsible for suppression of RNA-silencing and
salicylic acid-signalling. Journal of General Virology, 49 (12). pp. 2777-
2789. ISSN 0022-1317

Copyright © 2013 SGM.

http://eprints.gla.ac.uk/86415/

Deposited on: 19 Nov 2013

Enlighten – Research publications by members of the University of Glasgow
http://eprints.gla.ac.uk
JournalofGeneralVirology(2013),94,2777–2789 DOI10.1099/vir.0.057729-0
Identification of the domains of cauliflower mosaic
virus protein P6 responsible for suppression of
RNA silencing and salicylic acid signalling
Janet Laird,1 Carol McInally,1 Craig Carr,1 Sowjanya Doddiah,1
Gary Yates,1 Elina Chrysanthou,1 Ahmed Khattab,1 Andrew J. Love,13
Chiara Geri,1,2 Ari Sadanandom,3 Brian O. Smith,4 Kappei Kobayashi5
and Joel J. Milner1
Correspondence 1PlantScience Research Theme,School of LifeSciences andInstitute ofMolecular Cellular and
JoelJ.Milner Systems Biology, College ofMedical, Veterinary & Life Sciences,University of Glasgow,
[email protected] Glasgow G128QQ, UK
2Istituto diBiologiaeBiotechnologia Agraria, Consiglio NazionaleDelle Richerche, Pisa,Italy
3School of Biological andBiomedical Sciences, DurhamUniversity, DurhamDH13LE, UK
4InstituteofMolecularCellularandSystemsBiology,CollegeofMedical,Veterinary&LifeSciences,
University of Glasgow,Glasgow G128QQ, UK
5PlantMolecular Biology andVirology, Faculty ofAgriculture, Ehime University, Ehime 790-8566,
Japan
Cauliflowermosaicvirus(CaMV)encodesa520aapolypeptide,P6,whichparticipatesinseveral
essentialactivities inthevirus life cycle includingsuppressing RNAsilencing andsalicylic acid-
responsivedefencesignalling.WeinfectedArabidopsiswithCaMVmutantscontainingshortin-
framedeletions within the P6ORF. A deletion inthe distalend ofdomainD-I (the N-terminal
112aa) of P6did not affect virusreplication butcompromised symptom development and
curtailedtheabilitytorestoreGFPfluorescenceinaGFP-silencedtransgenicArabidopsisline.A
deletion intheminimum transactivator domainwasdefective invirusreplicationbutretained the
capacity tosuppress RNA silencinglocally. Symptom expression inCaMV-infected plants is
apparently linked tothe ability tosuppress RNAsilencing. When transiently co-expressed with
tomatobushystuntvirusP19,anelicitorofprogrammedcelldeathinNicotianatabacum,WTP6
suppressed the hypersensitive response,butthree mutants,two withdeletions withinthe distal
endofdomainD-IandoneinvolvingtheN-terminalnuclearexportsignal(NES),wereunabletodo
so.Deleting the N-terminal 20aaalsoabolished thesuppression ofpathogen-associated
molecular pattern-dependent PR1aexpression following agroinfiltration. However,thetwo other
deletions indomain D-Iretained this activity, evidence thatthemechanisms underlying these
functionsarenotidentical.TheD-IdomainofP6whenexpressedalonefailedtosuppresseither
celldeathorPR1aexpressionandistherefore necessarybutnotsufficient forallthreedefence
Received 30July2013 suppressionactivities.Consequently,concernsaboutthebiosafetyofgeneticallymodifiedcrops
Accepted 19September2013 carryingtruncated ORFVI sequences appearunfounded.
INTRODUCTION contains six known genera of which the most extensively
studied member is cauliflower mosaic virus (CaMV), the
Members of the family Caulimoviridae of pararetroviruses
type member of the genus Caulimovirus. CaMV has a
infect plants and replicate by reverse transcription of a
genome of ~8 kb comprising six major ORFs (I–VI). Five
circular dsDNA genome (Haas et al., 2002). The family
of the six major virus proteins are translated sequentially
from a single polycistronic RNA, the 35S RNA (Ryabova
3Presentaddress:Cell&MolecularSciences,JamesHuttonResearch et al., 2002, 2004). This unusual translational strategy
Institute,Invergowrie,DundeeDD25DA,UK. is found in members of only two genera of viruses,
Caulimovirus and the closely related Soymovirus (Ryabova
Twosupplementaryfiguresandonetableareavailablewiththeonline
versionofthispaper. et al., 2002, 2006).
057729G2013SGM PrintedinGreatBritain 2777
J.Lairdandothers
P6, a 62 kDa polypeptide encoded by CaMV ORFVI, was (a)
initially identified as the major component of cytoplasmic
a
inclusion bodies, which constitute the sites of virus
b
assembly (Haas et al., 2002). P6, which is translated from RH bip
its own monocistronic mRNA, plays an essential role in c
cc Path miniTAV RB-a RB-b
different aspects of virus replication. It functions in 30 112 242 310 400 520
infected cells to facilitate translation of the downstream
ORFs on the 35S RNA (Bonneville et al., 1989; Zijlstra &
1a 1b
Hohn, 1992) via interaction with components of the
D-I D-II D-III D-IV
translational machinery (Bureau et al., 2004; Leh et al.,
2000;Parketal.,2001;Ryabovaetal.,2004),amechanism
(b)
known as translation transactivation (TAV). P6 prevents
ribosome detachment at the stop codon, enabling poly- TAVD2(Δ40–76)
peptide synthesis to reinitiate at the next start codon
(Ryabova et al., 2004). TAVD3(Δ80–110)
At least four more roles for P6 have been identified. P6 TAVD6(Δ166–201)
interacts with at least two of the other CaMV proteins
involved in aphid transmission, P2 and P3 (Lutz et al., P6: Δ3–20
2012). It forms cytoplasmic inclusion bodies of various
P6: T1–112
sizes; the smaller ones associate with microtubules and the
endoplasmic reticulum and move dynamically along actin P6: T1–200
filaments(Harriesetal.,2009).Thismovementisprobably
essential for intracellular virus trafficking and involves the P6: T111–520
interaction of P6 with the CaMV movement protein P1
P6: T183–520
(Hapiak et al., 2008) and CHUP1, which mediates
association between chloroplasts and the cytoskeleton. P6: T1–112: Δ3–20
These findings suggest that P6 subverts the mechanism
responsible for chloroplast movement for intracellular
Fig.1.MapoftheP6domainsandmutantsusedinthisstudy.(a)
trafficking of CaMV (Angel et al., 2013).
SchematicrepresentationofP6domains:aminoacidnumbersat
P6 is the major genetic determinant of virus pathogenicity theboundariesofknowndomainsareindicated.Openboxesshow
(Baughman et al., 1988; Kobayashi & Hohn, 2004; Schoelz the coiled-coil (cc) a-helix, pathogenicity/host-range/avirulence
etal.,1986;Stratford&Covey,1989)andexpressionfroma (Path),minimumtransactivator(miniTAV),RNaseH(RH)andRNA
transgeneresultsinasymptom-likephenotype(Baughman binding(RB-aandRB-b).Thebipartitenuclearlocalizationsignals
et al., 1988; Cecchini et al., 1997; Zijlstra et al., 1996). P6 (NLS; bip) and three non-conventional NLS (a, b and c) are
exhibitsvirus-encodedsuppressorof RNA silencing (VSR) indicated by diamonds above and cross-hatching. The self-
association domains D-I to D-IV and subdomains 1a and 1b are
activity (Haas et al., 2008; Love et al., 2007), probably
indicatedbysolidlines.DatafromHaasetal.(2005),Haasetal.
through its interaction with the dsRNA-binding protein
(2008), Kobayashi & Hohn (2004) and Hapiak et al. (2008). (b)
DRB4 (Haas et al., 2008), a component of the Dicer4
Deletions in P6 coding sequences in CaMV-TAV mutants and in
complex.
thecorrespondingP6expressionconstructs.Filledboxesindicate
Finally, ectopic expression of P6 in Arabidopsis and Nico- sequencefromCaMVCM1841,shadedboxesindicatesequence
tiana benthamiana profoundly affects signalling mediated derived from CaMV Cabb B-JI and open boxes indicate internal
by salicylic acid (SA), jasmonic acid, ethylene and auxin deletions.
(Geri et al., 2004; Love et al., 2012; Smith, 2007). The
ability of P6 to manipulate multiple components of the
host defence suggests its central role as a pathogenicity
predicted nuclear localization signals (NLSs) (Cerritelli
determinant during virus infection, and the pleiotropic
et al., 1998; De Tapia et al., 1993; Haas et al., 2008;
phenotypesthatresultfromtransgene-mediatedexpression
Kobayashi & Hohn, 2003; Ryabova et al., 2004). De Tapia
inplantaderivefromitsactivityasapathogenicityeffector.
et al. (1993) identified aa 111–242 (domain D-II) as
How does a single protein achieve such a diverse range of containing the minimum functional domain (miniTAV
activities? Outwith closely related members of the family domain) able to facilitate TAV. The miniTAV domain
Caulimoviridae, P6 has no obvious homologues and its overlaps an RNase H domain (Cerritelli et al., 1998) and
three-dimensional structure is unknown. Li & Leisner contains the interaction motif for RL18; those for RL24,
(2002)definedfourdomainsbasedonself-association,and eIF4G and eIF2B are located within domain D-III
sequenceanalysishasrevealedseveralstructuralmotifsand (Ryabova et al., 2004). Domains D-II and D-IV are
functional domains (Fig. 1a). These include RNA binding, involved in the interaction with CHUP1 (and presumably
RNase H, a short N-terminal helical domain and several therefore intracellular trafficking (Angel et al., 2013). P6 is
2778 JournalofGeneralVirology94
DomainanalysisofCaMVproteinP6
a nucleocytoplasmic shuttle protein, and both nuclear 2a). Plants inoculated with CaMV-TAVD2 and CaMV-
localization and export functions are essential for infectiv- TAVD6 did not develop any symptoms (Fig. 2a). With
ity. Mutation of a nuclear export signal (NES) at the CaMV-TAVD3 inoculation, plants were usually asympto-
N terminus abolishes infectivity and mutation of the matic,althoughby28daysp.i.theoccasionalleafexhibited
predicted NLS within the C-terminal domains abolishes subtleveinclearing.Wemeasuredvirustitresat28daysp.i.
both VSR activity and infectivity (Haas et al., 2008; usingELISA(Fig.2c).Col-0plantsinoculatedwithCaMV-
Kobayashi & Hohn, 2004). CW containedhightitres ofvirus,but CaMV-TAVD2 and
CaMV-TAVD6werenotdetectablebyELISA.Surprisingly,
AlthoughTAVactivityand virus-trafficking functionshave
despitethelackofsymptoms,titresofCaMV-TAVD3were
been mapped, the domain(s) responsible for VSR activity
consistently verysimilartotitresof CaMV-CW.Thus,this
and SA signalling suppression remain(s) to be identified.
deletion did not appear to significantly reduce virus
Domain D-I plays a major role in pathogenicity and host
accumulation, at least under the conditions of our ex-
range and acts as an avirulence domain in Arabidopsis and
periment,butprofoundlyaffectedsymptomdevelopment.
Solanaceous hosts (Agama et al., 2002; Baughman et al.,
1988; Palanichelvam et al., 2000; Palanichelvam & Schoelz, We next tested whether we could complement the
2002;Schoelzetal.,1986;Stratford&Covey,1989).D-Ihas mutations in ORFVI by providing P6 from the transgenic
beendividedintosubdomain1a,comprisingtheN-terminal Arabidopsis line A7, which expresses P6 at levels similar to
30 aa containing the NES (Haas et al., 2008; Haas et al., those in infected plants (Cecchini et al., 1997) (Fig. 2b).
2005),andsubdomain1b(aa31–110)containingavirulence Plantsstartedtoexhibitsubtlesymptomsofleafdistortion
andpathogenicityfunctions.Mutantswithdeletionswithin ataround14daysp.i.,andby28daysp.i.,stuntingandleaf
1b retain replication competence but exhibit delayed virus distortion were visible on all A7 plants inoculated with
spreadinturnip(Kobayashi&Hohn,2004). CaMV-CW. Plants inoculated with CaMV-TAVD6 also all
WecarriedoutinfectionstudiesusingCaMVmutantswith developed symptoms similar to CaMV-CW and at around
deletionsinP6andfoundthatatleastonemutationwithin the same time, but those inoculated with CaMV-TAVD2
subdomain 1b abolished both VSR activity and symptom and CaMV-TAVD3 did not. Titres of CaMV-CW were
development without significantly reducing systemic virus approximately 30% of those in Col-0 plants, consistent
titre. We transiently expressed WT and mutant P6 in with our previous reports of reduced titres in P6
NicotianabenthamianaandNicotianatabacumandassayed transgenics (Love et al., 2007, 2012). All three mutants
the ability to suppress expression of an SA-responsive also accumulated to significant titres (Fig. 2c). These
marker gene, PR1a, and cell death in response to a gene- results suggested that functional P6 provided from a
for-gene elicitor. Deletions in subdomains 1a and 1b transgenecanactintranstofacilitatethereplicationofthe
abolished VSR activity and also abolished the suppression mutants. ORFVI sequences provided from a transgene can
ofthecell-deathresponseseenwithWTP6.However,only undersomecircumstancesrecombinewithdefectiveCaMV
the deletion in subdomain 1a eliminated suppression of genomeswheninfectionproceedsoveranextendedperiod
PR1a expression. Domain D-I evidently plays an essential (Kira´ly et al., 1998). Although we cannot absolutely rule
roleinseveralpathogenicityeffectoractivities.Suppression out the possibility of recombination in our complementa-
ofRNAsilencingandcelldeathmaybefunctionallylinked, tionexperiments,webelievethatitisunlikelybecausevirus
but suppression of SA-responsive gene expression must titres for the WT and mutants were similar at 28 days p.i.
involve an at least partially independent mechanism. and, in the case of CaMV-TAVD6, symptoms started to
appear at a similar relatively early stage in infection. The
absence of symptoms in A7 plants infected with CaMV-
RESULTS TAVD2andCaMV-TAVD3suggestedthat,evenwhenWT
P6 is provided from atransgene, symptom development is
InfectivityofWTandP6deletionmutantsofCaMV blocked in the presence of virus-encoded P6 containing
in Arabidopsis deletions in subdomain 1b. We did not observe this with
CaMV-TAVD6 in which the deletion affects the miniTAV
CaMV mutants with in-frame deletions in subdomain 1b
domain.
of P6 are replication competent in turnip but show delay-
ed long-distance spread (Kobayashi & Hohn, 2004). We
inoculated WT Arabidopsis (ecotype Col-0) with WT virus VSR activity of CaMV deletion mutants
(CaMV-CW) and three mutants (Fig. 1b). CaMV-TAVD2
The transgenic Arabidopsis line GxA contains a 35S–GFP
and CaMV-TAVD3 carry deletions in subdomain 1b,
transgene whose expression is silenced by a second
whilst CaMV-TAVD6 has a deletion in the miniTAV
transgene, a potato virus X amplicon containing part of
domain and cannot replicate in turnip.
the GFP-coding sequence (Dalmay et al., 2000; Schwach
Agroinoculation with CaMV-CW was remarkably effi- et al., 2005). CaMV infection of GxA suppresses silencing
cient,withsymptomsappearingat~13dayspost-infection of the GFP transgene, restoring strong fluorescence to
(p.i.) and essentially 100% of plants developing obvious infected tissue (Love et al., 2007). We used this assay to
stunting, leaf distortion and mosaics by 28 days p.i. (Fig. comparetheVSRactivitiesofWTandmutantvirus.Virus
http://vir.sgmjournals.org 2779
J.Lairdandothers
(a)
(i) Uninoculated (ii) CaMV-CW (iii) CaMV-TAVD2 (iv) CaMV-TAVD3 (v) CaMV-TAVD6
(b)
(i) Uninoculated (ii) CaMV-CW (iii) CaMV-TAVD2 (iv) CaMV-TAVD3 (v) CaMV-TAVD6
(c)
1.2
1
U 0.8
A Col 0
e
s titr 0.6 A7
u GxA
Vir 0.4
0.2
0
CW TAVD2 TAVD3 TAVD6
Virus genotype
Fig.2.InfectivityofCaMV(WTandmutants)onWTandP6transgenicArabidopsis.(a)SymptomsonCol-0at28daysp.i.:(i)
uninfected,(ii)CaMV-CW,(iii–v)CaMVTAVmutantsasindicated.Bar,2cm.(b)SymptomsonP6transgenicplants(lineA7)
at 28 days p.i.: (i) uninfected, (ii) CaMV-CW, (iii–v) CaMV TAV mutants as indicated. Bar, 2cm (note difference in scale
betweenaandb).(c)Virustitresat28daysp.i.inCol-0,A7andGxAplantsdeterminedbyELISA.Barsshowsmeantitres
(±SD)ofthreetissuesampleseachcomprisingthreepooledplants.Titresinarbitraryunits(AU)arenormalizedtothemeanof
Col-0plantsinfectedwithCaMV-CW.
levels in GxA were similar to those in Col-0, with CaMV- contained no detectable titres of virus. With CaMV-
CW and CaMV-TAVD3 accumulating to high titres but TAVD2,we did not observe any fluorescent cells, but with
CaMV-TAVD2andCaMV-TAVD6undetectablebyELISA CaMV-TAVD6 we consistently observed fluorescence in
(Fig. 2c). As with Col-0, only CaMV-CW induced groups of cells within every leaf we examined between 8
symptoms in GxA. and11daysp.i.(Fig.3b),albeitatlowerintensitythanwith
CaMV-CW; by 14 days p.i. fluorescence had become
We examined the upper leaves for GFP fluorescence using
undetectable. Kobayashi & Hohn (2003) showed using
confocal microscopy (Fig. 3a). Uninoculated controls
sensitive PCR that CaMV-TAVD6 is unable to replicate in
showed no detectable fluorescence, even at high gain, but
single cells. However, agroinoculation could provide
tissuefromCaMV-CW-infectedplantsconsistentlyshowed
transient P6 expression through direct transcription of
strong fluorescence. We did not observe any fluorescent
ORFVI (from its own 19S promoter) of the replication-
cells in systemic leaves of GxA inoculated with any of the
incompetent CaMV-TAVD6 genome (Kobayashi & Hohn,
threemutants,despitethehightitresofCaMV-TAVD3.To
2003,2004).Thisresultdemonstratesthatdeletingaa166–
test for local silencing suppression (around the sites of
201 does not abolish VSR activity.
inoculation), we examined inoculated leaves (Fig. 3b).
With CaMV-CW, by 8–11 days p.i. we consistently
Mutations in P6 affect the ability to suppress SA-
observed groups of cells showing strong GFP fluorescence.
responsive cell death
Inoculatedleaves recoveredfrom themicroscope slide and
assayed by ELISA all contained moderate to high titres of Expression of P6 from a transgene in Arabidopsis reduces
virus (with some leaf-to-leaf variation). Titres of CaMV- and delays cell death following treatment with SA or
TAVD3 in inoculated leaves were similar to those of inoculation withan avirulent pathogen (Loveet al., 2012).
CaMV-CW but we did not observe any GFP fluorescence. To identify the domain(s) responsible for this activity, we
LeavesinoculatedwithCaMV-TAVD2andCaMV-TAVD6 exploited the ability of tomato bushy stunt virus (TBSV)
2780 JournalofGeneralVirology94
DomainanalysisofCaMVproteinP6
(a)
(i) Uninoculated (ii) CaMV-CW (iii) CaMV-TAVD2 (iv) CaMV-TAVD3 (v) CaMV-TAVD6
(b)
(i) Uninoculated (ii) CaMV-CW (iii) CaMV-TAVD2 (iv) CaMV-TAVD3 (v) CaMV-TAVD6
Fig.3.GFPfluorescenceinleavesofGxAplantsinoculatedwithCaMVWTandmutants.(a)Confocalmicroscopeimagesof
representative upperleaves ofplantsat28daysp.i.: (i)uninoculated, (ii)CaMV-CW, (iii–v)CaMV TAVmutants.Thepanels
showlow-magnificationimagesofGFPfluorescence.Allpanelsweretakenatthesamemicroscopegainsettings.(b)Confocal
microscopeimagesofrepresentativeinoculatedleavesofplantsat28daysp.i.:(i)uninfected,(ii)CaMV-CW,(iii–v)CaMVTAV
mutants.Notethattheimagesin(b)aretakenatahighermagnificationthanthosein(a).Allpanelsweretakenwiththesame
microscopegainsettings.Bars,100mm.
P19 to elicit a gene-for-gene hypersensitive response (HR) suppresstheHRelicitedbyP19(Fig.4h,i).Neitherdidthe
in N. tabacum in an SA-dependent manner (Angel & corresponding C-terminally truncated variants (P6:T111–
Schoelz, 2013; Sansregret et al., 2013). Agrobacterium- 520 and P6:T183–520) (Fig. 4j, k). The ability to suppress
mediated expression of P19 in N. tabacum gave a strong cell death thus broadly paralleled the ability to suppress
HRthatwascompleteby36 hbutcouldbeextendedto3– RNA silencing, with deletions in both D-I subdomains,
5 days by reducing the Agrobacterium titre to one-quarter but not in the miniTAV domain, affecting this activity.
the usual level. At the higher titre of P19, co-infiltration However,theD-Idomainexpressedalonewasnotsufficient
with a hypervirulent strain of Agrobacterium containing to suppress cell death; therefore other regions of P6 must
pGWB-P6BJIW or pGWB-P6CW delayed the onset of HR alsoberequiredforthisactivity.
by approximately 24 h. At the reduced titre of P19, co-
infiltrationwitheitherWTP6constructsubstantiallyhalted
Mutations in P6 affect the ability to suppress
theprogress of thisHR compared with co-infiltration with
expression of an SA-responsive marker gene
emptyvector(EV)(Fig.4a,b,f).NeitherWTnormutantP6
elicitedHRintheabsenceofP19. Agroinfiltration of N. benthamiana elicits pathogen-
associated molecular pattern (PAMP)-responsive expres-
We cloned the P6 coding sequences from the three CaMV
sion of PR1a, a reliable marker of SA-responsive gene
mutants into Ti expression vectors and tested their ability
tosuppressHR(Fig.4).IncontrasttoP6:CW(wild-typeP6 expression (Volko et al., 1998); this response is strongly
from CaMV CM1841), neither of the two mutants with suppressedbytransientexpressionofP6(Loveetal.,2012).
deletions in subdomain 1b, P6:D2 and P6:D3, was able to P6:CW gave the expected reduction in PR1a transcripts to
suppressthedevelopmentofHR,butP6:D6,withadeletion ~30% that with EV (Fig. 5a). We anticipated that P6
in the miniTAV domain, suppressed cell death with an mutants with deletions in subdomain 1b might also fail to
efficiencysimilar toWT(Fig. 4c–e).TheN-terminalsubdo- suppress PR1a expression. However, P6:D2 and P6:D3 as
main1acontainstheNES,andmutationsthatabolishnuclear well as P6:D6 all reduced PR1a transcripts to a broadly
export also abolish VSR activity (Haas et al., 2008). We similar level to that of P6:CW (Fig. 5a). All three mutants
thereforedeleted18ofthe20 aaattheNterminustoproduce evidently retained the ability to suppress SA-responsive
what we predicted would be a functionally equivalent gene expression. In contrast, infiltration with P6:D3–20
construct,P6:D3–20(Fig.1b).Whentransientlyco-expressed resulted in levels of PR1a expression similar to EV (Fig.
withP19,P6:D3–20wasunabletosuppressHR(Fig.4g). 5b). Therefore, sequences required for suppression of SA-
responsive gene expression are present in subdomain 1a
To testwhether the D-I domain was able to suppressP19-
but apparently not 1b.
induced HR in the absence of the C-terminal domains, we
produced a further series of expression constructs (for We next investigated whether expressing the N-terminal
details,seeFig.1b).Truncatedpolypeptidescomprisingthe domain alone was sufficient to suppress PR1a expression.
N-terminal 112or200 aa(P6:T1–112,P6:T1–200)didnot P6:T1–112 and P6:T1–200 not only failed to reduce PR1a
http://vir.sgmjournals.org 2781
J.Lairdandothers
(a) EV (b) P6:CW (c) P6:D2 (d) P6:D3
(e) P6:D6 (f) P6:BJIW (g) P6:Δ3–20 (h) P6:T1–112
(i) P6:T1–200 (j) P6:T111–520 (k) P6:T183–520
Fig.4.SuppressionofTBSVP19-dependentcelldeathbyco-infiltrationwithWTandmutantvariantsofP6.Photographsof
leaf patches 4days after co-agroinfiltration with a construct expressing P19 plus EV control (a) or WT or mutant P6 as
indicated(b–k).Allimagesareshownatsimilarmagnification.Bar,1.0cm.
transcript levels but also produced a consistent increase of distribution.With P6:D6-GFP,weobservedveryfewsmall
more than twofold over and above EV controls (Fig. 5b). inclusion bodies, although the large ones were still
The D-I domain is therefore not sufficient for suppression abundant (Fig. 6a, v). Domain D-II, which contains the
of SA-dependent gene expression but may play some role deletion in P6:D6 (aa 166–201), has been identified as
in this activity because expression on its own promoted interacting with CHUP1 in connection with intracellular
elevated expression of PR1a. virus trafficking (Angel et al., 2013). The region may be
required for cytoskeletal association and for the formation
of small inclusion bodies.
Intracellular localization of mutant P6
The deletion in P6:D3–20–GFP included three residues
Totestwhetherlossofdefencesuppressionactivityinsome
identifiedasessentialfornuclearexport(Haasetal.,2008),
mutants might be attributable to mislocalization and to
so we anticipated that it would show enhanced co-
confirm appropriate expression of P6, we analysed its
localization with DAPI. Unexpectedly, the nuclear local-
intracellular localization after transiently expressing the
ization was similar to that of WT (Fig. 6b, ii). Haas et al.
mutant forms of P6 as C-terminal GFP fusions in N.
(2005) expressed the N-terminal 110 aa alone and
benthamiana (Fig. 6). GFP-tagged P6BJIW (wild-type P6
compared localization with the same polypeptide with
from CaMV Cabb B-JI) suppressed PR1a expression with
the N-terminal a-helix deleted. We produced equivalent
an efficiency similar to a myc-tagged construct (see
constructs (although with a C-terminal GFP tag), P6:T1–
Methods), indicating that this activity was unaffected by
112–GFP and P6:T1–112:D3–20–GFP. Whereas P6:T1–
the GFP tag (data not shown). Epidermal cells expressing
112–GFP showed no nuclear localization whatsoever,
WT P6 from the two isolates CM1841 and Cabb B-JI
P6:T1–112:D3–20–GFP co-localized strongly with DAPI
showed identical patterns of intracellular fluorescence,
(Fig. 6b, iii, iv). P6:T111–520–GFP, which lacks the entire
reminiscentofthosereportedbyHarriesetal.(2009)(Fig.
D-I domain (including the NES), also showed enhanced
6a, i and ii). Cells contained cytoplasmic inclusion bodies
nuclear localization (Fig. 6, v). Our results are therefore
that were highly variable in size. Large numbers of small
consistent with deletion of the N-terminal 20 aa affecting
inclusion bodies were present, some of which appeared to
nuclear export. Haas et al. (2005) fused GFP to the N
be associated with cytoplasmic strands. Large inclusion
terminus;ouruseofaC-terminaltagmightaccountforthe
bodies were often clustered around the nucleus, but we
differences for full-length P6.
observed only weak GFP fluorescence co-localizing with
DAPI(Fig.6b,i),consistentwiththefindingsofHaasetal.
(2005) who reported rapid nuclear export of P6. Sequence conservation of domain D-I across
members of the Caulimoviridae
Localization of P6:D2-GFP and P6:D3-GFP was indistin-
guishable from that of the WT (Fig. 6a, ii–iv). These We used the programs Jalview (Waterhouse et al., 2009)
deletions did not cause obvious changes in intracellular and JPred3 (Cole et al., 2008) to align the sequences of P6
2782 JournalofGeneralVirology94
DomainanalysisofCaMVproteinP6
(a) The N-terminal sequences of the soymoviruses are shorter
1.2 than those of the caulimoviruses and are rather diverse
V (Fig.S1).Possibly,membersofthegenusSoymoviruslacka
E 1.0
o functional D-I domain.
e t
ativ 0.8
el
s r 0.6 DISCUSSION
pt
nscri 0.4 WehaveshownthattheN-terminaldomainofP6contains
a sequencesessentialforitsactivitiesasasuppressorofRNA
1a tr 0.2 silencing and of SA-dependent defence responses. These
R
P appear to be distinct from its TAV and virus-trafficking
0.0 functions.Deletingthedistalendofsubdomain1b(aa 80–
U EV CW D2 D3 D6 110) abolished VSR activity within the context of an
infectiousvirusclone.Thesamedeletionalsoabolishedthe
(b) 3.5 ability to suppress one aspect of SA-dependent signalling,
cell death triggered by the elicitor TBSV P19, but not
V 3.0 another, PAMP-driven PR1a expression. The mechanisms
E
o underlying these three activities may therefore overlap but
ve t 2.5 are clearly not identical. However, the N-terminal sub-
elati 2.0 domain1a,whichincludestheNES,isessentialforallthree.
s r
pt CaMV-TAVD6, with a deletion within the miniTAV
cri 1.5 domain, consistently produced a transient silencing sup-
s
n
a tra 1.0 prerteasisniosnVSiRnaicntoivciutyla.tAedsClaeaMveVs-,TeAvVidDen6ciesctohmatpltehtieslymunuatabnlet
1
PR 0.5 to replicate in protoplasts (Kobayashi & Hohn, 2003), we
assume that P6 mRNA is transcribed directly from CaMV-
0.0
U EV BJIW Δ3–20 T1–112T1–200 TAVD6 genomes introduced by agroinoculation. Although
we were unable to detect CaMV-TAVD2 accumulation in
inoculated leaves using ELISA, we might have expected
Fig. 5. Quantification of PR1a expression in N. benthamiana similar limited P6 expression by direct transcription of the
leaves following transient expression of WT and mutant P6 by T-DNA following agroinoculation. If so, the failure of
agroinfiltration. (a) PR-1a transcripts, determined by quantitative CaMV-TAVD2 to stimulate similar transient GFP expres-
PCR, in N. benthamiana leaves harvested 48h after agroinfiltra- sionininoculatedleavessuggeststhatittoomaybedeficient
tion. Samples were uninfiltrated leaves (U), and leaves infiltrated inVSRactivity.
withAgrobacteriumcarryingthefollowingvectors:pGWB17(EV),
P6:CW(CW),P6:D2(D2),P6:D3(D3)andP6:D6(D6).(b)PR- Suppression of RNA silencing by VSR is a major
1a transcripts, determined as above. Samples were uninfiltrated contributor to symptom induction (Burgya´n & Havelda,
leaves (U), and leaves infiltrated with Agrobacterium carrying the 2011). Despite titres similar to WT virus, CaMV-TAVD3
followingvectorspGWB17(EV),P6BJIW(BJIW),P6:D3–20(D3– was essentially asymptomatic on Col-0 plants, suggesting
20),P6:T1–112(T1–112)andP6:T1–200(T1–200).Barsshow that the symptoms of CaMV infection (at least in
means±SD (in arbitrary units) of three independent biological Arabidopsis) are probably linked to the VSR activity of
samples each comprising three pooled infiltrated leaf sections. P6. Complementation by transgene-derived P6 allowed all
ValueswerenormalizedtovaluesforEV. three mutants to replicate, but, whereas CaMV-CW and
CaMV-TAVD6causedobviousstuntingandleafdistortion
in A7, CaMV-TAVD3 and CaMV-TAVD2 were both
from CaMV with ten other members of the genus Cauli-
asymptomatic. As A7 produces high levels of WT P6
movirus and four members of the genus Soymoviruses. P6
(Cecchinietal.,1997),theabsenceofsymptomsinCaMV-
from all ten caulimoviruses showed significant homology
TAVD2- and CaMV-TAVD3-infected plants must be a
with CaMV over almost the entire sequence, but none of
dominant-negative effect, presumably linked to the loss of
the soymoviruses showed significant similarity to the
VSR activity, further evidence that CaMV-TAVD2 is also
caulimovirus D-I domain. (Figs 7 and S1, available in
defective in this respect.
JGV Online). Within domain D-I, homology varied
between different members of the genus Caulimovirus ArolefordomainD-Iindefence suppression isconsistent
(Fig. 7), but there was notable sequence conservation with its identification as the major genetic determinant of
between aa 67 and 88, in particular the GK(D/E)X(S/ virus pathogenicity, host range and avirulence (Kobayashi
T)NPLXXXXLXK motif (aa 74–88) conserved in 10 of 11 & Hohn, 2003; Palanichelvam & Schoelz, 2002; Schoelz &
sequences. Interestingly, this motif extends across the Shepherd, 1988; Stratford & Covey, 1989). Both subdo-
junction between the TAVD2 and TAVD3 deletions. mainsareinvolved.Subdomain1aclearlyplaysanessential
http://vir.sgmjournals.org 2783
J.Lairdandothers
(a)
(i)
P6:BJIW
(ii) P6:CW (iii) P6:TAVD2 (iv) P6:TAVD3 (v) P6:TAVD6
(b)
(i) P6:BJIW
DAPI GFP Merge
(ii) P6:Δ3-20
DAPI GFP Merge
(iii) P6:T1-112
DAPI GFP Merge
(iv)
P6:T1-112:Δ3-20
DAPI GFP Merge
(v) P6:T111-520
DAPI GFP Merge
Fig. 6. Intracellular localization of WT and mutant P6 tagged with GFP. Confocal microscope images of tissue from N.
benthamiana leaves 3days after agroinfiltration with WT and mutant variants of P6 fused at the C terminus to GFP. GFP
fluorescenceisgreenandDAPIfluorescence(nuclearstaining)isblue.(a)IntracellulardistributionofWTandTAVDmutantP6:
(i)P6BJIWinasingleepidermalcell.Representativelargeinclusionbodiesareindicatedbyyellowarrowsandsmallinclusion
bodiesbypinkarrows.(ii)P6CW.Yellowandpinkarrowsareasin(i),whilstbluearrowsindicatenuclei(DAPIstaining).(iii–v)
P6:D2, P6:D3 and P6:D6. Bars, 100mm. (b) High-magnification images showing nuclear localization of WT and truncated
formsofP6:(i)P6BJIW,(ii)P6:D3–20,(iii)P6:T1–112,(iv)P6:T1–112:D3–20and(v)P6:T111–520.Panelsfromlefttoright:
DAPI,GFP,merge.Nuclearfluorescenceisindicatedbyarrows.Bars,20mm.
2784 JournalofGeneralVirology94
DomainanalysisofCaMVproteinP6
role in pathogenicity as deleting it eliminated both the Theeffectsofmutationsinsubdomain1bonVSRactivityand
suppression of cell death and PAMP-responsive gene thesuppressionoftheHRelicitedbyP19implythatitmust
expression. Correct localization of P6 may be required play a key role in these functions. The motif GK(D/E)X(S/
for these activities. Deletions within subdomain 1b T)NPLXXXXLXK, which spans the ends of the TAVD2 and
abolished the ability to suppress cell death in our assay TAVD3 deletions, is very highly conserved across 10/11
but not the ability to suppress PAMP-triggered expression members of the genus Caulimovirus. Such a degree of
of PR1a. The apparent discrepancy between the effects of sequence homology provides additional support for the
deletions in subdomain 1b on these two different SA- importanceofthisregionofP6tomembersofthisgenus.
dependentresponsesmaybeexplainedbytherecentreport
The pleiotropic phenotype(s) of P6-transgenic Arabidopsis
thatextremeresistancetoTBSVinN.tabacumiselicitedby
(Geriet al., 2004; Love et al., 2012; Smith, 2007) would be
a complex of P19 plus small interfering RNAs (siRNAs)
most elegantly accounted for by a common underlying
(Sansregret et al., 2013). Interaction with DRB4, a
mechanism, perhaps all involving RNA silencing, rather
component of the Dicer4 complex (Haas et al., 2008),
than by diverse direct interactions with multiple signalling
providesaprobablemechanism fortheVSRactivityofP6.
intermediates. However, because deletion mutants of
As Dicer4 is involved in generating siRNAs, both defence
subdomain 1b suppressed PAMP-responsive PR1a expres-
suppression activities of P6 (on RNA silencing and SA
sion with a similar efficiencyto WT P6, VSR activitymust
signalling) could potentially play a role in inhibiting the
not be essential for this activity.
HR elicited by P19.
Transgene-mediated expression of VSRs elicits pleiotropic
The truncated proteins P6:1–112 and P6:1–200 elicited
effects on jasmonic acid and other phytohormone
elevated levels of PR1a transcripts compared with EV
controls. The C-terminal region of P6, which is absent responses (Endres et al., 2010; Lewsey et al., 2010;
from these constructs, contains four predicted NLSs (Fig. Lozano-Dura´n et al., 2011; Yang et al., 2008), and CaMV
1a).NuclearlocalizationisrequiredforVSRactivity(Haas infection is accompanied by profound changes in
et al., 2008), and our results are consistent with it also microRNA (miRNA) and trans-acting siRNA (tasiRNA)
being essential for cell death and suppression of SA- populations(Blevinsetal.,2006;Moissiard&Voinnet,2006;
dependent gene expression. We did not observe obvious Shivaprasad et al., 2008). The 59 leader sequence of the
differences in nuclear localization between WT and CaMV 35S RNA is a target for all four Arabidopsis Dicer
truncated proteins (Fig. 6). However, because the N- complexes, producing siRNAs that appear to target host
terminalNESpromotesveryefficientre-exportofP6from transcripts(Blevinsetal.,2006;Moissiard&Voinnet,2006;
the nucleus, even WT P6, which is actively imported into Shivaprasadetal.,2008),evidenceofacomplexinteraction
the nucleus (Haas et al., 2005), gave only weak GFP mediated at least partially by RNA silencing. miRNAs and
fluorescence within nuclei. tasiRNAs regulate signalling pathways involving auxin
CaMV IQ3HM11 1 63
HLV IQ5J1S0 1 64
LLDAV IB2CXY6 1 88
CERV IP05401 1 47
DaMV-HollIA9QKR8 1 94
EVCV IB2D1N1 1 58
MMV IQ8JTA1 27 120
DaMV IA9UD04 1 96
FMV IQ67458 1 96
CmYLCV IQ7TBL3 1 116
SVBV IQ88443 1 87
Consensus
MEE–+L+A–LRL+LQQ–E––EKIL+LELK++++KI–––––––––––SLYEES+SSQ+++VSP+REK–QTE+SPLQTADGK++–+NPLKPDAL+KSITE+T–+S+L+D+S–KLVG+TK–S
CaMV IQ3HM1164 140
HLV IQ5J1S0 65 140
LLDAV IB2CXY6 89 169
CERV IP05401 48 134
DaMV-HollIA9QKR8 95 144
EVCV IB2D1N1 59 134
MMV IQ8JTA1121 176
DaMV IA9UD04 97 150
FMV IQ67458 97 154
CmYLCV IQ7TBL3117 184
SVBV IQ88443 88 146
Consensus
SD–––++SSPVQT+SGKDSSNPLMADSLPKS++–VQT–SRLVK––––––––––IPEDFSLRPN––––––––––––––––D–––––QGIPIP–KSE–HSSVA–PSY+E++IQ P+SG+KY
Fig. 7. Alignment of sequences of the N-terminal amino acids of P6 from caulimoviruses. The sequences from CaMV,
horseradish latent virus (HLV), lamium leaf distortion associated virus (LLDAV), carnation etched ring virus (CERV), dahlia
mosaic virus-Holland (DaMV-Holl), eupatorium vein clearing virus (EVCV), mirabilis mosaic virus (MMV), dahlia mosaic virus
(DaMV),figwortmosaicvirus(FMV),cestrumyellowleafcurlingvirus(CmYLCV)andstrawberryveinbandingvirus(SVBV)that
precedetheRNaseHdomain(aa140inCaMV)arealigned,withtheconsensussequenceshownbelowinlogoform.Residues
arecolouredaccordingtotheCLUSTAL_XcolouringschemeandtheUniprotaccessionnumbersindicated.
http://vir.sgmjournals.org 2785