Mathioudakis et al., 1, MPMI

Multifaceted Capsid Proteins: Multiple Interactions suggest Multiple Roles for Pepino mosaic virus Capsid Protein

Matthaios M. Mathioudakis,1,2 Luis Rodríguez-Moreno,3 Raquel Navarro Sempere,3 Miguel A. Aranda,3 and Ioannis

Livieratos1

1Μediterranean Agronomic Institute of Chania, Department of Sustainable Agriculture, Alsylio Agrokepio, Chania 73100,

Greece;

2Plant Pathology Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, P.O.B. 269, Thessaloniki 54124,

Greece;

3Departamento de Biología del Estrés y Patología Vegetal, Centro de Edafología y Biología Aplicada del Segura (CEBAS)-

CSIC, PO Box 164, 30100 Espinardo, Murcia, Spain

Corresponding author: I. Livieratos; E-mail: livieratos@maich.gr

Pepino mosaic virus (PepMV; family Alphaflexiviridae, genus Potexvirus) is a mechanically-transmitted tomato

pathogen that over the last decade has evolved from emerging to endemic worldwide. Here, two heat shock cognate

(Hsc70) isoforms were identified as part of the coat protein (CP)/Hsc70 complex in vivo following full length PepMV

and CP agroinoculation. PepMV accumulation was severely reduced in Hsp70 VIGS-silenced and in quercetin-treated

Nicotiana benthamiana plants. Similarly, in vitro-transcribed as well as virion RNA input levels were reduced in

quercetin-treated protoplasts, suggesting an essential role for Hsp70 in PepMV replication. As for PVX, the PepMV

CP and Triple Gene Block protein 1 (TGBp1) self-associate and interact each other in vitro, but unlike in the

prototype, both PepMV proteins represent suppressors of transgene-induced RNA silencing with different modes of

action: CP is a more efficient suppressor of RNA silencing, sequesters the silencing signal by preventing its

spreading to neighbouring cells and its systemic movement. Here, we provide evidence for additional roles of the

PepMV CP and host-encoded Hsp70 in viral infection, the first as a truly multifunctional protein, able to specifically

bind to a host chaperone and to counter-attack an RNA-based defence mechanism, and the latter as an essential

factor for PepMV infection.

Pepino mosaic virus (PepMV) is a highly infectious potexvirus, which over the last twenty years has become an

endemic pathogen in tomato crops worldwide (Hanssen and Thomma, 2010). Each of the four PepMV genotypes (i.e. original

Peruvian [LP], European [EU], American [US1] and Chilean [CH2]) described to date can cause significant economical losses,

inducing mild or severe yellowing or necrotic symptoms in tomato (Hanssen et al., 2010, Hasiow-Jaroszewska et al., 2013).

The PepMV CH2 strain, which is the most widespread, has overtaken PepMV EU in Europe and the US (Gómez et al., 2009;

Ling et al., 2013). PepMV possesses a 6.4 kb single-stranded RNA genome, encoding the RNA-dependent RNA polymerase

Page 1 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 2, MPMI

(RdRp; 164 kDa), three triple gene block (TGB) proteins of 26 (TGBp1), 14 (TGBp2) and 9 (TGBp3) kDa, and the coat protein

(CP) (Aguilar et al., 2002). PepMV cDNA- and agro-infectious clones from both the EU and CH2 genotypes are available

(Hasiów-Jaroszewska et al., 2009; Duff-Farrier et al., 2011; Sempere et al., 2011) to facilitate studies on symptom induction,

resistance mechanisms, virus replication, translation and movement. In addition, an in vitro transcriptionally-active RdRp

system has been set-up, which in combination with cDNA infectious clones can assist the dissection of essential requirements

for PepMV positive- and negative-strand synthesis (Osman et al., 2012). A transcriptome analysis of tomato seedlings

inoculated with a mild or an aggressive PepMV isolate showed a differential expression of transcripts suggesting plant/virus

interactions at multiple levels (Hanssen et al., 2011). For PepMV TGBp1 and CP, specific host-virus protein interactions have

been reported, respectively with the tomato catalase 1 (CAT1) and heat shock cognate protein 70 (Hsc70) (Mathioudakis et

al., 2012, 2013). The former interaction significantly elevates the H2O2 scavenging efficiency of CAT1 to assure high levels of

virus accumulation (Mathioudakis et al., 2013). In the latter interaction, for which no biological role has yet been suggested,

Hsc70 is up-regulated and comes into contact with PepMV virions (Mathioudakis et al., 2012). The multiple roles of cytosolic

Hsp70 chaperones in various plant cellular processes have been thoroughly reviewed by Mayer and Bukau (2005). A role for

Hsp70 was firstly described almost 20 years ago in terms of responsiveness to virus infection (Aranda et al., 1996) and later in

relation to virus replication and movement and by interference with antiviral host responses (Whitham et al., 2003; Aparicio et

al., 2005; Chen et al., 2008; Hafren et al., 2010; Nagy et al., 2011; Verchot, 2012). For other potexviruses, Hsp70 is induced

upon Potato virus X (PVX) infection (Chen et al., 2008) and in the case of Bamboo mosaic virus (BaMV), a host Hsp90 has

been reported to bind specifically the viral 3’-UTR and RdRp, thus promoting viral replication (Huang et al., 2012).

Apart from their structural functions, viral CPs may be involved in virus transmission, translation, replication,

movement, modulation and in the suppression of host responses (Ivanov and Makinen, 2012). PVX CP forms complexes with

viral RNA and TGBp1 that traffic towards plant plasmodesmata to facilitate virus movement (Lough et al., 2006). PepMV CP,

the elicitor of Rx resistance (Bendahmane et al., 1995; Candresse et al., 2010), was also found to affect the nature and

severity of the induced symptoms irrespective of the viral isolate (Hasiow-Jaroszewska et al., 2013) and to be required for

virus movement (Sempere et al., 2011) as shown for other potexiviruses in conjunction with homologous and/or heterologous

interactions with TGBp1 (Verchot-Lubicz et al., 2010).

RNA silencing regulates gene expression in most eukaryotes, acting both at a transcriptional and post-transcriptional

level, whereas in plants and invertebrates, the same pathway also functions directly in anti-viral defence by targeting virus

RNA (Pumplin and Voinnet, 2013). In RNA silencing, small interfering RNAs (siRNAs; 21-25 nt long) produced by the Dicer-

like enzymes (DCL; RNase type III group) are loaded by Argonaute proteins (AGO) onto an RNA-induced silencing complex

(RISC) for cleavage and/or translational repression of the target RNA transcripts in a sequence-specific manner (Hamilton and

Baulcombe, 1999; Voinnet, 2002). To counter-attack, plant viruses express suppressors of RNA silencing. For non TGBp-

encoding viruses, the CPs may perform this function (Voinnet, 2005; Levy et al., 2008), whereas for several potexviruses

TGBp1 is the only suppressor identified to date (Voinnet et al., 2000; Senshu et al., 2009). In PepMV-infected plants, over-

expression of DCL2/4 has been reported (Hanssen et al., 2011) and no PepMV-encoded suppressor has been identified yet.

Page 2 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 3, MPMI

The essential contribution of conserved Hsp chaperones for viral infection is becoming increasingly apparent, while

reports describing additional non-structural functions of the viral CPs gradually increase (Nagy et al., 2011; Ivanov and

Makinen, 2012; Nagy and Pogany, 2012; Verchot, 2012). Here, the formation of the PepMV CP/heat shock chaperone

complex was verified in vivo, its latter component was shown to have an essential role in PepMV infection and moreover,

PepMV CP self-associates and interacts with TGBp1, both acting as suppressors of RNA silencing with diverse modes of

function. This is the first report of a potexvirus-encoded CP acting as a suppressor of RNA silencing.

RESULTS

Specific CP-Hsp70 interaction in plants following virus infection and transient expression.

In order to verify in planta the interaction between Hsc70 protein and PepMV CP (Mathioudakis et al., 2012), a

commercial One-Strep-Tag (OST) was genetically fused to the CP of PepMV-Sp13 infectious clone (Sempere et al., 2011).

The infectivity and stability of the resulting recombinant clone PepMV-Sp13-OST-CP was evaluated at different time points (8,

12, 15 days post-inoculation) both in tomato and Nicotiana benthamiana plants. In all cases, plants inoculated with PepMV-

Sp13-OST-CP showed symptoms undistinguishable to those induced by the wild type virus (data not shown) and the

presence of the recombinant RNA was RT-PCR verified using total RNA extracts (Fig. 1A). Following the stability validation,

agroinfectious vectors corresponding to PepMV-Sp13-OST-CP (pBPepXL6-OST-CP) and PepMV-Sp13 (pBPepXL6) were

used to inoculate tomato and N. benthamiana plants in order to identify host proteins interacting with OST-CP by affinity

chromatography. Crude protein extracts obtained 8 dpi from pBPepXL6-OST-CP or pBPepXL6 agroinoculated plants were

passed through specific Strep-Tactin columns and the eluted extracts were silver stained following SDS-PAGE separation.

A specific band approximately 85-90 KDa in size was differentially observed in pBPepXL6-OST-CP-treated tomato (Figure 1B)

and N. benthamiana (data not shown) extracts. Mass spectrometry of the gel-extracted protein band as well as the eluted

fraction yielded a set of 10 peptides covering approximately 20-25% of the Hsc70 amino acid sequence. The length of the

peptides varied between 13 (KNALENYSYNMRN) and 25 (KSINPDEAVAYGAAVQAAILSGEGNEKV) amino acids. Each of

the 10 peptides was blasted against the three characterized Hsc70 isoforms of Solanum lycopersicum (Lin et al., 1991; Sun et

al., 1996) and all existing Hsc70 sequences from NCBI database (XM_004250911, XM_004250910, XM_004249526,

XM_004246354, XM_004235839, XP_004235887). Two peptides (KEIAEAFLGSTVKN and RTTPSYVGFTDTERL) were

specific for Hsc70 isoform 1 (GenBank X54029) and KNQVAMNPTNTVFDAKR peptide for isoform 3 (GenBank L41253). All

seven remaining peptides showed 100% identity with at least two of the three isoforms, preventing unequivocal determination

of whether more isoforms were also present in the complex. An unspecific protein of approximately 55 kDa was also analyzed

by LC-MS/MS, resulting in several peptides belonging to the ubiquitous Ribulose 1, 5-Bisphosphate carboxylase protein.

An additional affinity chromatography experiment was carried out in N. benthamiana plants following transient

expression of PepMV CP alone. The OST-CP fusion was cloned into a binary gateway expression vector (pGWB2-OST-CP)

and co-agro-infiltrated with the Tomato bushy stunt virus (TBSV) silencing suppressor p19 into 2-weeks old N. benthamiana

Page 3 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 4, MPMI

leaves. Five dpi, agroinfiltrated leaves were collected and crude protein extracts were passed through Strep-Tactin columns.

Following gel silver staining, a band of ca. 70 kDa was confirmed by LC-MS/MS to correspond to the Hsp70 (data not shown);

whereas no additional proteins were detected.

Knock down of Hsp70 inhibits PepMV accumulation in N. benthamiana.

To dissect the effect of Hsp70 on PepMV infection, Hsp70 mRNAs were silenced using a virus-induced gene

silencing (VIGS) Tobacco rattle virus (TRV)-based agroinfiltration assay (Hayward et al., 2011). A 407 nt fragment from the 5'

end of N. benthamiana Hsp70 gene (GenBank GU575116; Hafren et al., 2010) was amplified and cloned into pTRV2 to

generate the recombinant plasmid pTRV2-Nb-Hsp. This construct was co-agroinfiltrated with the pTRV1 plasmid into N.

benthamiana leaves, resulting in Hsp-silenced (TRV-Hsp) plants exhibiting a specific severe phenotype of curling leaves,

dwarfing and leaf necrosis (15 dpi) as previously reported (Fig. 2A; Wang et al., 2009; Hafren et al., 2010). Co-agroinfiltration

of pTRV1 with pTRV2-PDS (positive control of VIGS assay) or pTRV2 (negative control) generated TRV-PDS and TRV-

control (TRV-C) plants, were also performed. The phenotype in the TRV-PDS plants was fully developed 6 dpi (data not

shown), when a set of upper leaves were mechanically inoculated with PepMV followed by plant tissue collection from the

inoculated (local) and next set of upper leaves (systemic) at 10 dpi to determine mRNA or protein levels for plant Hsp70 and

viral CP and RdRp. A semi-quantitative PCR assay confirmed high levels of silencing for the Hsp70 gene. The Hsp70 mRNA

levels in systemic -Hsp leaves were 19-fold lower and barely detectable after 30 cycles and gel overexposure when compared

to TRV-C plants (Fig. 2B). The analysis of PepMV CP gene RNA levels indicated a 15-fold reduction with detectable product

observed only after 32 PCR cycles (Fig. 2C). In the case of PepMV RdRp gRNA, the levels in TRV-Hsp plants compared to

negative control plants reached detectable levels only when 2.5 times the initial template was used followed by overexposure

(Fig. 2C; PepMV RdRp II panels). The previous data was further supported by immunoblot analysis of the same samples. The

Hsp70 expression levels of the TRV-Hsp plants were reduced by 18% in locally-inoculated leaves, whereas systemically, the

reduction of Hsp70 expression reached 75% of those in TRV-C plants (Fig. 2D). In agreement, PepMV CP levels were also

reduced by 28% locally (Hsp70 silencing levels relatively lower), and could not be detected in systemic leaves reaching a 94%

reduction compared with TRV-C plants (Fig. 2D). These data represent the outcome of three experiments using two plants per

treatment, and remarkably low levels of variability were observed among plants, within groups and between experiments. No

differences were observed in PepMV accumulation levels between TRV-C and wild-type (no TRV agroinfiltration) plants (data

not shown), confirming that a PepMV/TRV mixed infection did not affect the accumulation of the former.

Inhibition of Hsp70 expression by quercetin reduces PepMV accumulation in N. benthamiana plants and protoplasts.

A pharmacological approach to investigate PepMV accumulation levels in the absence of the host gene was applied,

using quercetin, a flavonoid inhibitor which is known to down-regulate Hsp70 mRNA expression (Hosokawa et al., 1990;

Manwell and Heikkila) and has been used in various studies with plant viruses (Wang et al., 2009, Hafren et al., 2010). N.

benthamiana leaves were infiltrated with DMSO in the presence or absence of quercetin before PepMV mechanical

Page 4 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 5, MPMI

inoculation. Plants infiltrated with DMSO/quercetin exhibited a few necrotic lesions 4 dpi (Fig. 3A) in contrast to negative

control plants, which remained completely unaffected. At 4 dpi, plant tissue was collected from PepMV-inoculated leaves and

the Hsp70 and CP protein levels were compared by immunoblot analysis. In quercetin-treated plants, Hsp70 protein levels

were significantly reduced compared to DMSO-treated plants to 50-73% of the levels in the controls, and a corresponding

decrease in PepMV CP accumulation was observed (65-92% reduction; Fig. 3B).

Both the VIGS and the pharmacological inhibition of Hsp70 expression in whole N. benthamiana plants strongly

indicated an essential role of this host factor in PepMV infection. To further investigate the role of Hsp70 in PepMV replication

or movement, we examined the effect of quercetin on PepMV RNA accumulation at a single cell level. Quercetin dissolved in

DMSO was added to isolated protoplasts prior and subsequently to PEG-mediated transfection with either two different in vitro

T7-transcribed viral mRNA or virion RNA. DMSO treatment in the absence of quercetin (D henceforth) was used as a negative

control, whereas protoplasts transfected with PepMV RNAs (referred from now on as pPepXL6) without DMSO or quercetin

served as an additional negative control [referred as (-)] of the transfection method. Total RNA extracted from PepMV-infected

N. benthamiana plants, referred as +, was used as a control of the northern blot and hybridization assay. The results clearly

showed that the effect of quercetin (Q; Fig. 3C) reduced PepMV gRNA accumulation by approximately 83-95% for both RNA

inputs. Western blot analysis of protoplast protein extracts also demonstrated the reduction of the host protein expression

levels (Fig. 3D) in the quercetin-treated samples, in agreement with the previous results. Treatment with DMSO alone had no

affect on PepMV replication for either of the two RNA inputs (Fig. 3C). These data were the outcome of three experiments with

two replicates for each RNA transfection suggesting an essential role for the Hsp70 at a particular stage of PepMV replication,

rather than virus movement.

PepMV TGBp1 and CP suppress RNA silencing in a diverse manner.

To identify potential PepMV-encoded suppressors of RNA silencing TGBp1, TGBp2, TGBp3 and CP were expressed

using the Agrobacterium-mediated transient expression system (Voinnet et al., 2000) in wild type (WT) or GFP-transgenic (line

16c) N. benthamiana plants. The first two sets of experiments tested whether each of the four PepMV-encoded proteins was

able to suppress initiation of the transgene-induced sense-post transciptional gene silencing (S-PTGS). Leaves of N.

benthamiana WT plants were co-agroinfiltrated with the pBIN/35S-mGFP4 and each of the four PepMV pGreen constructs

(pGR-TGBp1, pGR-TGBp2, pGR-TGBp3, pGR-CP) and used to test the effect of the candidate suppressor on S-PTGS

induced by overexpression of an infiltrated GFP-transgene (Fig. 4A). At 10 dpi, GFP silencing was apparent as the absence of

fluorescence in those patches infiltrated with the pGreen empty vector [pGR(-)] and the TGBp2-3 treatments, but the areas in

which PepMV TGBp1 or CP were expressed, fluorescence was maintained (Fig. 4A) for 15 and 17 dpi, respectively (data not

shown). S-PTGS suppression by CP was more efficient as assessed under UV light macroscopically (GFP fluorescence

intensity) and western blot analysis (Fig. 4B). To confirm the ability of the candidate protein to suppress the S-PTGS of a

stably expressed transgene, a second type of experiment was conducted using N. benthamiana 16c transgenic plants using

the same transient expression system (Fig. 4C). Over a period of 25 days, GFP fluorescence peaked at 3 dpi and thereafter

Page 5 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 6, MPMI

decreased, having completely disappeared by 7 dpi for the negative control, TGBp2 and TGBp3 treatments, as a result of the

induction of S-PTGS (Fig. 4C). In tissues where TGBp1 and CP were co-expressed with the 35S-mGFP4, fluorescence was

maintained for 12 and 16 dpi for the TGBp1 and CP respectively, with the latter exhibiting the highest levels of GFP intensity

(Fig. 4C). Western blot analysis at 8 dpi (Fig. 4D), in full agreement with the phenotypic analysis of the agroinfiltrated patches,

confirmed the ability of CP and TGBp1 to act as local suppressors of S-PTGS.

In a third type of experiment, the ability of TGBp1 and CP suppressors to effectively act downstream RDR

polymerase activity, through an inverted repeat transgene-induced RNA silencing pathway, was investigated. N. benthamiana

WT leaves were co-agroinfiltrated with a mixture of 35S-mGFP4 (expressing sense GFP), 35S-hpGFP (hairpin construct

expressing dsRNA GFP) in addition to TGBp1, -2, -3 or CP. The phenotypic analysis of fluorescence over a period of 25 days

was as described above. Green fluorescence peaked 3 dpi and was completely silenced at 6 dpi when maximal suppression

or no fluorescence was observed (Fig. 4E). No inhibition of silencing was observed for TGBp2 or TGBp3 in patches where

they were infiltrated together with 35S-mGFP4 plus dsRNA GFP. However TGBp1 or CP acted as local suppressors of the

inverted repeat PTGS (IR-PTGS) pathway, as their presence resulted in the maintenance of fluorescence for 11 and 15 dpi,

respectively (Fig. 4E). These results are at variance with those reported for other potexviruses (Senshu et al, 2009). As

described previously in S-PTGS experimentation, the CP-maintained fluorescence was stronger than that of TGBp1 with

higher GFP accumulation levels as verified by western blot analysis (Fig. 4F). To our knowledge, this is the first report of CP-

mediated suppression of RNA silencing amongst members of the genus Potexvirus.

The ability of PepMV CP and TGBp1 to reverse the RNA silencing mechanism and restore the expression of a

transgene in already silenced tissue was also tested. Leaves of transgenic N. benthamiana 6.4 plants with established

silencing of GFP (Tournier et al., 2006) were individually agroinfiltrated with an empty pGreen vector, or with the same vector

expressing the Tobacco etch virus (TEV) HcPro (positive control), PepMV TGBp1 or CP. GFP silencing in N. benthamiana 6.4

plants was found not to be 100% complete, as GFP was detected at very low levels in the negative controls. The restoration of

GFP expression, as assessed by the UV light examination of infiltrated patches was not observed up to 15 dpi both for TGBp1

and CP as compared to positive control HcPro (7 dpi; Fig. 5A) and was confirmed by western blot analysis (Fig. 5B).

The ability of PepMV CP and TGBp1 to interfere with the cell-to-cell or systemic spread of the RNA silencing signal

was examined. GFP expression was monitored in the neighbouring cells of infiltrated patches and in systemic leaves. To

examine the first, N. benthamiana 16c plants were co-agroinfiltrated with 35S-mGFP4 plus either pGreen empty vector or

pGreen expressing either Tomato chlorosis virus (ToCV) p22, TGBp1 or CP. The formation of red-fluorescent borders around

the infiltrated patches corresponding to the exit of the DCL4-generated siRNA 21nt silencing signal (Himber et al., 2003;

Dunoyer et al., 2005), was observed by 8 dpi in the case of pGreen empty vector. This cell-to-cell spread of the silencing

signal and its rapid systemic induction, was also observed for ToCV p22 (Fig. 5C; Canizares et al., 2008). A border was also

seen for TGBp1 at 10 dpi, indicating that TGBp1 could not sequester 21nt siRNAs and therefore prevent the movement of the

silencing signal towards the neighbouring cells (Fig. 5C). On the contrary and similarly to the Cymbidium ring spot virus

(CymRSV) p19 suppressor (Fig. 5D; Silhavy et al., 2002; Himber et al., 2003), short-distance silencing spread, as monitored

Page 6 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 7, MPMI

by the formation of the red ring front did not take place in patches where 35S-mGFP4 and CP infiltration took place (Fig. 5C-

D) demonstrating the interference of CP with the cell-to-cell spread of the silencing signal. To examine the role of TGBp1 and

CP in the systemic spread of the RNA silencing, GFP expression was monitored over a period of 40 dpi in the upper non-

infiltrated leaves. At 11 dpi, systemic silencing was observed in N. benthamiana 16c plants co-agroinfiltrated with 35S-mGFP4

and pGreen empty vector similarly to TGBp1 (12 dpi; Fig. 5E). At 30 dpi, the systemic silencing reached the upper leaves and

the flowers in both cases (Fig. 5E) and the faint fluorescence in the petals was completely lost at 35 dpi (data not shown). By

this time, either the starting point of systemic silencing at 10-12 dpi or at 30 dpi, when the silencing was complete in whole

plants, the upper leaves and flowers of infiltrated plants with 35S-mGFP4 and CP maintained the green fluorescence (as in

non-infiltrated N. benthamiana 16c plants) as showed also for the CymRSV p19 (Fig. 5E). Taken all together, these results

demonstrate that TGBp1 and CP cannot reverse established silencing, and that while TGBp1 can efficiently suppress local

RNA silencing it is not able to prevent the short- or long-distance spread of the silencing signal, while CP is able to function in

both manners.

PepMV CP and TGBp1 self-associate and interact with each other.

To assess potential homologous and heterologous interactions in planta and their subcellular localization between

the two identified PepMV viral suppressors, a bimolecular fluorescent complementation (BIFC) assay was applied by agro-

infiltration in N. benthamiana plants (Walter et al., 2004). PepMV TGBp1 and CP were fused with the C'-terminal fragment of a

yellow fluorescent protein (YFP; pCYFP-TGBp1, pCfYFP-CP) and used in all combinations with the pNYFP-TGBp1 and

pNYFP-CP constructs (Mathioudakis et al., 2012, 2013). The expression of TGBp1 and CP in infiltrated tissues was confirmed

using α-HA and α-cmyc antibodies in western blot assays (data not shown). Co-expression of pCYFP-TGBP1 and pNYFP-CP

induced the reconstitution of fluorescence demonstrating their in planta interaction, localized throughout the cytoplasm and the

nucleus (Fig. 6A). YFP fluorescence also was the outcome of pairwise co-expression of each protein fused either with the C'-

or N'-terminal YFP fragments displaying their self-interactions (Fig. 6B). The self-interactions were localized in the cytoplasm

and nucleus, evidencing no movement of each protein during their interaction with each other. No fluorescence signal was

observed in all pairwise TGBp1 and CP combinations with the empty N'- and C'-YFP vectors (Fig. 6C).

DISCUSSION

Highly conserved Hsp70/Hsc70 chaperones function in plant cells and assist protein (re)folding, import and

translocation across membranes, complex assembly and receptor signaling (Mayer and Bukau, 2005). During infection by

positive-stranded plant RNA viruses numerous essential roles have been attributed to Hsp70/Hsc70 as a result of their

specific binding to viral proteins (including CPs) or/and RNAs (Whitham et al., 2006; Nagy et al., 2011; Ivanov and Makinen,

2012; Nagy and Pogany, 2012). One important example of their involvement in viral infections is their utilization by

closteroviruses to facilitate virion assembly and intercellular movement (Peremyslov et al., 1999; Satyanarayana et al., 2000).

Page 7 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 8, MPMI

Non-specific Hsp70 induction has also been reported as a general response to positive-stranded RNA plant virus infections

(Aparicio et al., 2005).

Previously, screening a tomato cDNA library using PepMV-encoded CP in the yeast two-hybrid system was

complemented by BiFC, full-length gene-to-gene yeast-two hybrid assays, immuno-gold labeling electron microscopy and

time-course analyses of mRNA and proteins to suggest that elevated levels of Hsc70.3 accumulate and form a complex with

CP or/and virions in PepMV-infected plant cells (Mathioudakis et al., 2012). Here, we confirmed the formation of this complex

in planta and attempted to identify the exact Hsp70 isoform(s) participating in the complex. Agroinoculation of an infectious,

OST-tagged full length PepMV RNA in tomato plants showed that at least the Hsc70.3 and Hsc70.1 isoforms participate in the

complex. The actual elution of an Hsp70 protein following PepMV CP transient expression in N. benthamiana indicates that

complex formation is independent of the presence of any other PepMV-encoded protein, whereas the host contributes no

detectable, additional proteins without entirely excluding the possibility that other factors such as co-chaperones might also

participate. Similarly, a specific CP-Hsp70.3 interaction has been described for Potato virus A (PVA) in N. benthamiana, where

Hsp70.3 together with its co-chaperone CPIP prevents particle assembly in favour of viral replication and translation (Hafren et

al., 2010).

In the follow-up reverse genetics experiments, a conserved region from the 5’-end of the N. benthamiana Hsp70

gene was selected to VIGS-silence its homologues. In N. benthamiana plants, Hsp70 levels were effectively (18% local; 75%

systemic) reduced with a concomitant strong inhibition in PepMV RNA and CP accumulation (28% local; 94% systemic),

whereas an additional gentler (pharmacological) treatment with quercetin reduced the Hsp70 mRNA levels (by 50-73%)

followed by a corresponding negative effect on the PepMV accumulation (65-92% reduction). The strong suggestion of an

essential role of Hsp70 for efficient PepMV accumulation at the whole plant level, prompted the examination of its role in N.

benthamiana protoplasts, an approach that would exclude any potential involvement in virus movement. Here, the addition of

quercetin, dramatically reduced PepMV RNA levels both in the case of in vitro-transcribed viral and virion RNA. Overall, these

data support an essential role for Hsp70 in PepMV replication, while they do not exclude an additional role for Hsp70

isoform(s) during temporally and spatially diverse stages of virus infection and cannot unequivocally explain the role of

CP/Hsp70 complex. Bearing in mind that PepMV infectious clones in which the CP was substituted by GFP, retained their

replication competence in protoplasts, and that on the other hand for several CP-deficient potexvirus clones including PepMV,

the amount of positive-sense RNA accumulation was reduced, an enhancing rather than essential role of the complex in

PepMV and other potexviruses replication seems plausible (Chapman et al., 1992; Forster et al., 1992; Lough et al., 2000;

Lee et al., 2011; Sempere et al., 2011).

In the paradigm of TBSV, host Hsp70 facilitates the insertion of viral replication proteins into intracellular membranes,

and promotes folding and stability of the replication complex (RC) (Wang et al., 2009; Nagy and Pogany, 2012). The

Arabidopsis Hsc70-3 also comprises one component of the membrane-bound RC within endoplasmic reticulum (ER)-derived

vesicles, where interaction with the Turnip yellow mosaic potyvirus RdRp and a poly(A) binding protein takes place (Dufresne

et al., 2008). Similarly for Tomato mosaic virus, affinity-purified replicase, as one part of the membrane-bound RC, is

Page 8 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 9, MPMI

associated with the Hsp70, eEF1A, TOM1 and TOM2A proteins (Nishikiori et al., 2006). In yeast, Brome mosaic virus

replicase associates with Hsp70s to enhance viral RNA accumulation (Tomita et al., 2003). Beyond aiding assembly of viral

RCs, Hsp70s may contribute to virion assembly and cell-to-cell spread as in the case of poty- and pomoviruses (Haupt et al.,

2005; PYV; Hofius et al., 2007). Specific interactions of potexviruses with their host-encoded proteins including chaperones

have also been reported (Verchot-Lubicz et al., 2007; Verchot, 2012) and recently an Hsp90/RdRp/RNA 3’-end complex was

suggested to enhance BaMV replication during the initiation of negative-strand RNA synthesis (Huang et al., 2012). The J-

domain proteins are the most common co-factors of Hsp70 homologues, which identify and recruit substrates to Hsp70

through direct interactions. Experiments in silenced and overexpressing NbDnaJ plants suggested that the latter is a negative

regulator of PVX replication and movement through its interaction with CP and SL1 RNA (Cho et al., 2012).

PVX has been for decades the main source of information on the pathogenesis, cell biology and replication of

potexviruses. It is known that potexvirus CPs are essential for genome encapsidation, translational activation, cell-to-cell

movement and in the case of PVX and PepMV elicitors of Rx resistance (Bendahmane et al., 1995; Verchot-Lubicz, 2005).

Here, similarly to PVX and other potexviruses (Samuels et al., 2007; Leshchiner et al., 2008; Lu et al., 2009; Wu et al., 2011),

PepMV CP and TGBp1 were found to self-associate and interact with each other possibly to support virus movement, since

CP is indispensable for PepMV cell-to-cell and long-distance movement (Sempere et al., 2011). Increasing evidence

supporting diversification of different potexviruses is primarily highlighted by the replication of PVX and BaMV, in the

endoplasmic reticulum and chloroplasts of infected plants, respectively (Doronin and Hemenway, 1996; Lin et al., 2007).

Potexviruses movement has been proposed to depend on a number of different functions, one being TGBp1 RNA silencing

suppressor activity (Bayne et al., 2005) and its localization (Lim et al., 2010a,b), the former apparently exhibiting remarkable

variability between different potexviruses (Senshu et al., 2009). In addition to the well documented siRNA sequestration and

AGO inactivation by suppressors active against S- and IR-PTGS, Plantago asiatica mosaic potexvirus TGBp1 (exclusively

involved in S-PTGS) targets SGS3/RDR6-mediated dsRNA synthesis and therefore extends further the reported divergence of

potexvirus TGBp1 range of action (Okano et al., 2014).

PepMV-encoded TGBp1 was recently proposed to interact with tomato CAT1 in order to regulate plant homeostasis

and oxidative stress-induced plant defence to maintain high levels of the virus during infection (Mathioudakis et al., 2013) and

the data we present here show that TGBp1 also functions to counter-attack RNA silencing. For PepMV, a variation in the

sense of a “dual and complementary strategy” is shown to be implemented blocking local and systemic host antiviral RNA

silencing. Both TGBp1 and CP are able to mediate local (intracellular) suppression of two different silencing mechanisms, the

initiation of S- as well as IR-RNA silencing, with CP in both cases being the most effective acting also intercellularly and

systemically. To our knowledge, amongst all TGB-encoding plant viruses, only Potato virus M encodes an additional to and

distinct from TGBp1 suppressor of RNA silencing (CRP, Senshu et al., 2011). Overall, PepMV CP emerges as a truly multi-

functional protein. Here, its specific complex formation with a PepMV replication-essential host chaperone suggests additional

role(s) in PepMV infection, which may relate to its role as an RNA silencing suppressor. The recent report of chaperone-

mediated assembly of the RISC (Iwasaki et al., 2010) should be borne in mind. The multiple PepMV CP interactions identified

Page 9 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 10, MPMI

to date, not only suggest additional roles for this multifaceted protein, but also support the existence of significant variation

among members of the genus Potexvirus.

MATERIALS AND METHODS

Plant materials and growth conditions.

Three lines of N. benthamiana plants were used for the experiments of this work, the wild type (WT), the 16c line

which constitutively express GFP transgene (Hamilton et al., 2002), a 6.4 transgenic line in which the GFP gene silencing has

been already established (Tournier et. al., 2006). N. benthamiana seeds were sown on Murashige and Skoog medium to

germinate for 10 days at 25oC and 16:8 h light/dark. N. benthamiana and tomato seedlings were grown under standard

greenhouse conditions in growth chambers.

Construction of full length PepMV- and CP-tagged mutant plasmids.

Mutant PepMV-Sp13_OST-CP was constructed using standard overlapping PCR and molecular cloning protocols

based on the pT7PepXL6 construct (Sempere et al., 2011). For the construction of pBPepXL6-OST-CP, three overlapping

DNA fragments (PCR1, 2, 3) were separately amplified. For PCR 1, a DNA fragment containing the TGB genes, the first 36 nt

of the CP, which included a modified AGG starting triplet codon of the PepMV CP gene, was amplified from pT7PepXL6agg

(Sempere et al., 2011) using primers Pep-303 and CE-921

(GGATGACTCCATGCGCTCATGGTGGCACTTGAAGTGGCAGCAAC); primer CE-921 also including a Kozac sequence

(underlined), a new start codon (bold) and part of the OST sequence (italics). For PCR 2, a DNA fragment containing part of

the OST, the CP and the 3’UTR was amplified from pBPepPDS2a (Sempere et al., 2011) with primers CE-43 and CE-920

(CAGTTCGAAAAATCCGGAATGCCTGACACtACtCCaGTgG), which included silent mutations (lower case) to avoid the

instability of the duplicated CP sub-genomic promoter. For PCR 3, complete sequence of OST and the flanking sequence

were amplified with the CE-918 (reverse complement of CE-921) and CE-919 (reverse complement of CE-920) primers. The

PCR 2 and PCR 3 fragments were mixed and amplified in a fourth PCR step to produce a partial overlapped DNA fragment

using primers CE-918 and CE-43. Finally, PCR 1 fragment was mixed and amplified with the PCR 2-3 fragment using Pep-

303 and CE-43 primers to produce the complete overlapping fragment containing the TGB genes, the OST-CP and the 3’ UTR

of PepMV. The resulting PCR fragment was either inserted into pT7PepXL6 using the XmnI-XhoI sites to produce

pTPepXL6_OST-CP, followed by sub-cloning into the BamHI-XmaI sites of pBPepXL6 to obtain pBPepXL6-OST-CP.

For transient expression experiments, the recombinant OST-CP sequence was PCR-amplified using CE-1295

(GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGCCACCATGAGCGATGGAGTCATCC) and CE-1296

(GGGGACCACTTTG TACAAGAAAGCTGGGTCTTAAAGTTCAGGGGGTGCGTCTATCGCG) primers and cloned into the

pGWB2 gateway binary vector (Nakagawa et al., 2007). A PCR fragment containing the att sites was recombined into the

Page 10 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 11, MPMI

pDONR221 vector and subsequently, the pDONR221-OST-CP vector was LR recombined into the pGWB2 destination vector

resulting in the pGWB2-OST-CP DNA construct.

In planta pull-down assays and mass spectrometry.

Upper, non-inoculated leaves from tomato and N. benthamiana plants agro-inoculated with pBPepXL6-OST-CP,

pBPepXL6, pGWB2-OST-CP/pBP19 or pBP19 recombinant constructs were collected and crushed in liquid nitrogen before

addition of an extraction buffer (25 mM Tris-HCl at pH: 7.5, 10% Glycerol, 1 mM EDTA, 150 mM NaCl, 10 mM DTT, 2%

PVPP, 0.1% Tween-20 and Roche protease inhibitor cocktail [ROCHE]). Following centrifugation at 14,000 rpm for 10 min, the

supernatant was gently incubated with 15 µg/ml of avidin for 1 hour at 4oC. Extracts were passed through a Strep-Tactin

MacroPrep column according the manufacturer’s instructions and the recombinant OST-CP fusion protein was eluted in six

aliquots (E1-E6) of 100 µl in volume. Samples from E2, E3 and E4 were loaded in 10% SDS-PAGE gels to separate the

recombinant from the interacting proteins and silver stained bands were excised and destained before downstream HPLC-

MS/MS analysis.

The separation and analysis of the tryptic digests of the samples were performed with a HPLC/MS system consisting

of an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a µ-well plate auto-sampler and

a capillary pump, connected to an Agilent Ion Trap XCT Plus Mass Spectrometer (Agilent Technologies, Santa Clara, CA,

USA) using an electrospray (ESI) interface. Data processing was performed using the Data-Analysis program for LC/MSD

Trap Version 3.3 (Bruker Daltonik, GmbH, Germany) and Spectrum Mill MS Proteomics Workbench (Rev A.03.02.060B,

Agilent Technologies, Santa Clara, CA, USA). The MS/MS search against the appropriate NCBI database was performed with

the following criteria: identity search mode; tryptic digestion with 2 maximum missed cleavages; carbamidomethylated

cysteines; peptide charge +1, +2, +3; mono-isotopic masses; peptide precursor mass tolerance 2.5 Da; product ion mass

tolerance 0.7 amu; ESI ion trap instrument; minimum matched peak intensity 50%; STY phosphorylation, oxidized methionine,

and N-terminal glutamine conversion to pyroglutamic acid as variable modifications.

Silencing Hsp70 by VIGS and quercetin.

To study the biological role of the PepMV CP/Hsp70 interaction in viral infection, the Hsp70 homologue genes were

silenced using the previously described pTRV1 and pTRV2 vectors (Liu et al., 2002; kindly provided by Prof. S. Dinesh-

Kumar, University of California, Davis, USA). For this purpose, a 407 bp in size fragment (93% amino acid identity with the

tomato Hsc70.3) from the 5΄ end of N. benthamiana Hsp70 gene (GeneBank GU575116) was PCR-amplified from total RNA

extracts and with the primers listed in Table 1 (Supplementary Data). The DNA amplicons were cloned in the pTRV2 vector

via BamHI-XhoI, to generate the plasmids pTRV2-Nb-Hsp. The pTRV2-PDS construct was used as a silencing control (kindly

provided by S.P. Dinesh-Kumar). All of the constructs were introduced into the Agrobacterium GV2260 cells and infiltrated into

young 3rd-4th leaves of N. benthamiana as described previously (Hayward et al., 2011).

Page 11 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 12, MPMI

The sampling and testing methodology performed in previous work about the biological role of the catalase-TGBp1

interaction (Mathioudakis et al., 2013) was also applied herein. Briefly, the systemic leaves six days after the TRV agro-

infiltration were mechanically inoculated by PepMV and total RNAs and protein extracts were obtained from the inoculated and

systemically PepMV-infected leaves, 4 days after PepMV inoculation, for the estimation of the protein and mRNA levels of

Hsp70, CP and RdRp by western blotting and semi-quantitative PCR using the primers listed in Table 1 (Supplementary

Data).

In parallel with the Hsp70 VIGS-silencing, a second assay was applied with the use of quercetin, an Hsp70 protein

inhibitor. N. benthamiana plants were firstly infiltrated with 1 mM quercetin in DMSO and after one hour, the plants were

mechanically inoculated with PepMV inoculum, as previously described (Wang et al., 2009; Hafren et al., 2010). DMSO-

Infiltrated plants were inoculated with PepMV and they consisted the negative controls. The Hsp70 and virus accumulation

levels were analysed on local leaves by immunoblot analysis 4 days after quercetin application.

PepMV virion and in vitro-transcribed RNA preparation for protoplasts transfection.

Preparation of PepMV purified virions was previously described (Mathioudakis et al., 2012). The infectious PepMV

cDNA full-length clone pT7PepXL6 (Sempere et al., 2011) was linearized by KpnI and in vitro-transcribed in the presence of

cap analogue using the mMessage mMachine kit (Ambion), according to the manufacturer's instructions. N. benthamiana

mesophyll protoplasts isolation was carried out according to standard protocols and approximately 106 protoplasts were

polyethylene glycol (PEG)-mediated transfected either using 10 or 5 µg of the in vitro-transcribed or virion RNA respectively as

previously reported by Navas-Castillo et al. (1997). Protoplasts transfected with no viral RNA represented the mock control.

The quercetin inhibitor, dissolved in DMSO, was added to the protoplasts at a concentration of 200 µM 30 min prior as well as

after PEG-transfection with PepMV RNAs, whereas protoplasts treated with the same concentration of DMSO were used as

control. Protoplasts were incubated at 25oC under continuous light and harvested 24 h post transfection for RNA analysis.

Screening for RNA-silencing suppressors and their functional studies

The full length coding sequences of PepMV TGBp1, TGBp2, TGBp3 and CP ORFs were PCR-amplified using the

pLMPepMV15 construct (Aguilar et al., 2002) as template and specific primers listed in Table 1 (Supplementary Data). PCR-

amplified DNA products were firstly cloned into the pGEM-T easy vector and subsequently subcloned into the binary vector

pGreen300 (35S-BI/GST) using the BamHI-XhoI sites. The four pGreen constructs pGR-TGBp1, pGR-TGBp2, pGR-TGBp3

and pGR-CP were transformed into A. tumefaciens cells strain AGL-1 by electroporation. The AGL-1 cells contained the

plasmid combination pSoup, providing replication functions in trans for 35S/BI-GST (Hellens et al., 2000).

The commonly used A. tumefaciens-mediated transient gene expression of the candidate suppressor and the

reporter gene was used to study the PTGS phenomenon (Llave et al., 2000; Voinnet et al., 2000; Johansen and Carrington,

2001). This assay involves the co-infiltration of N. benthamiana leaves with a mixture from individual agrobacterium cells

suspension of the GFP reporter gene together with putative suppressor candidates and showing the preservation of the GFP

Page 12 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 13, MPMI

expression according to the suppressor activity of the candidate protein. Three individual experiments sets were carried out,

each one in 3 replicates, as described previously (Kataya et al., 2009). Briefly, N. benthamiana WT and transgenic 16c leaves

were co-agroinfiltrated with the pariwise combination of 35S-mGFP4 construct (Haseloff et al., 1997; expressing the GFP) and

each of the pGR-plasmids indicated above to study the S-PTGS mechanism, whereas N. benthamiana WT leaves co-

agroinfiltrated with the pairwise combination of each PepMV protein together with 35S-mGFP4 and a hp-GFP construct

(Koscianska et al., 2005; producing dsRNA-GFP molecules) were used for the hp-PTGS study. The pBIN/35S-mGFP4 and

pFGC5941/35S-hpGFP plasmids were kindly provided by Dr J. Haseloff (Cambridge University, UK) and Dr. K. Kalantidis

(University of Crete, Geece), respectively. As positive control, the ToCV p22 (pGR-p22) suppressor (Cañizares et al. 2008)

was used, wheres the empty pGreen300 vector served as negative control. Daily observations were undertaken for GFP

fluorescence by long-wavelength UV light (Sylvania H44GS 100W, USA), whereas images were taken by a PENTAX digital

camera with an UV filter.

To study the involvement of the candidate suppressors in cell-to-cell and long-distance spread of the RNA silencing

signal, N. benthamiana 16c plants were co-agroinfiltrated with the 35S-mGFP4 plus either the pGR(-), pGR-p22 ToCV, pGR-

p19 of CymRSV, pGR-CP or pGR-TGBp1. The monitoring of the GFP expression was observed under the UV light in a period

of 9 to 35 dpi.

Some RNA silencing suppressors like TEV HcPro have the ability to reverse the silencing phenomena and restore

the GFP expression in already silenced tissue of the tested transgene. In the present study, we individually agroinfiltrated

sGFP-expressing transgenic N. benthamiana 6.4 plants leaves (Tournier et al., 2006) with pGR-HCPro (kindly provided by J.

J. Lopez-Moya, CSIC, Barcelona, Spain) or pGR-CP or pGR-p26 or the pGreen empty vector and the GFP expression was

monitored for 7 days.

BiFC assay.

To study in planta homologous and heterologous interactions of PepMV CP and TGp1, the binary vectors pSPYNE-

35S and pSPYCE-35S, allowing the expression of the proteins of interest as fusion to the N’- (NYFP) or C’-terminal half

(CYFP) of the YFP together with a c-myc (pSPYNE-35S) or HA (pSPYCE-35S) affinity tag (Walter et al., 2004), were used. N.

benthamiana WT fully expanded leaves were co-agroinfiltrated with the following pairwise combinations: pNYFP-TGBp1 and

pCYFP-TGBp1, pNYFP-CP and pCYFP-CP, pNYFP-CP and pCYFP-TGBp1 constructs, together with the TBSV p19 RNA-

silencing suppressor, as described previously (Mathioudakis et al., 2012). To generate the pCYFP-CP and pCYFP-TGBp1

constructs, the full length coding sequences of CP and TGBp1 genes were subcloned from pG-YFP-CP (A/S) and pG-YFP-

p26 (S/S) constructs (Mathioudakis et al., 2012; 2013), in frame, into the binary vector pSPYCE-35S via AscI-SalI and SpeI-

SalI sites, respectively. The bZIP63 transcription factor was used as positive control (Walter et al., 2004), whereas

combinations of pSPYNE-35S (pNYFP) and pSPYCE-35S (pCYFP) plasmids, with the viral-constructs, were used as negative

controls. Images were taken from the epidermal cell layers 3-4 dpi as described previously (Mathioudakis et al., 2012).

Page 13 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 14, MPMI

Protein and RNA analysis and semi-quantitative RT-PCR.

For PepMV mechanical inoculations, identical volumes of virus inoculum were used, whereas in control plants the

plant sap of a healthy plant was used. Total proteins were extracted from 0.1g either from PepMV infected or agroifiltrated

plant tissue as described previously (Mathioudakis et al., 2012). Proteins from protoplasts were isolated using the Trizol

reagent (Invitrogen) using the manufacturer's instructions. Immunoblot analysis was used to study the GFP accumulation in

the suppressor experiments and the Hsp70 and PepMV CP accumulation levels in VIGS assays, using anti-GFP (Abcam),

anti-Hsp70 (Stressgen) and anti-PepMV CP (Neogen) antibodies. Other specific proteins were detected by Western blotting

using anti-HA rat polyclonal (Roche; pSPYCE-35S constructs) and anti-c-myc mouse monoclonal antibodies (Roche;

pSPYNE-35S constructs), respectively. All the primary antibodies were conjugated to alkaline phosphatase (AP) and the

products were visualized using the NBT-BCIP substrates (Promega).

Total RNA was extracted from leaf tissue or protoplasts using the Trizol reagent according to the manufacturer's

instructions. Northern blot analysis using a digoxigenin-labeled PepMV CP probe was performed as described previously

(Sempere et al., 2011). Chemiluminescent detection was carried out using the DIG-labelling kit (Roche). RNA extraction from

PepMV virus particles was performed as previously described (AbouHaidar et al., 1998). RNA concentrations were estimated

by a NanoDrop spectrophotometer.

A semi-quantitative RT-PCR was applied in VIGS experiments on the systemic leaves using specific primer pairs

(Table 1; Supplementary Data) amplifying a partial fragment of the Hsp70, CP and RdRp genes, as previously described

(Mathioudakis et al., 2012). The PCR DNA and protein bands were visualized using the Gel DocTM XR imager system and

their intensities were quantified as absorbance units using Quantity One analysis software (BioRad Laboratories).

ACKNOWLEDGEMENTS

Matthaios Mathioudakis is a recipient of an Onassis Foundation PhD scholarship. Work in Aranda´s lab was supported by

grant AGL2012-37390 from Ministerio de Economía y Competitividad (Spain). We would like to thank Dr. Carolyn Owen for

critically reading the manuscript and Dr. Tomas Canto (CSIC) for technical assistance with the BiFC assays.

Page 14 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 15, MPMI

LITERATURE CITED

AbouHaidar, M. G., Xu, H. M., and Hefferon, K. L. 1998. Potexvirus isolation and RNA extraction. Plant Virol. Protoc. 81: 131-

143.

Aguilar, J. M., Hernandez-Gallardo, M. D., Cenis, J. L., Lacasa, A., and Aranda, M. A. 2002. Complete sequence of the

Pepino mosaic virus RNA genome. Arch. Virol. 147: 2009-2015.

Aparicio, F., Thomas, C. L., Lederer, C., Niu, Y., Wang, D. W., and Maule, A. J. 2005. Virus induction of heat shock protein 70

reflects a general response to protein accumulation in the plant cytosol. Plant Physiol. 138: 529-536.

Aranda, M. A., Escaler, M., Wang, D., and Maule, A. J. 1996. Induction of HSP70 and polyubiquitin expression associated with

plant virus replication. Proc. Natl. Acad. Sci. U.S.A. 93: 15289-15293.

Bayne, E. H., Rakitina, D. V., Morozov, S. Y., and Baulcombe, D. C. 2005. Cell-to-cell movement of Potato potexvirus X is

dependent on suppression of RNA silencing. Plant J. 44: 471-482.

Bendahmane, A., Kohm, B. A., Dedi, C., and Baulcombe, D. C. 1995. The coat protein of Potato virus X is a strain-specific

elicitor of Rx1-mediated virus resistance in potato. Plant J. 8: 933-941.

Candresse, T., Marais, A., Faure, C., Dubrana, M. P., Gombert, J., and Bendahmane, A. 2010. Multiple coat protein mutations

abolish recognition of Pepino mosaic potexvirus (PepMV) by the potato Rx resistance gene in transgenic tomatoes. Mol.

Plant-Microbe. Interact. 23: 376-383.

Canizàres, M. C., Navas-Castillo, J., and Moriones, E. 2008. Multiple suppressors of RNA silencing encoded by both genomic

RNAs of the Crinivirus, Tomato chlorosis virus. Virology 379: 168-174.

Chapman, S., Hills, G., Watts, J., and Baulcombe, D. 1992. Mutational analysis of the coat protein gene of Potato virus X:

effects on virion morphology and viral pathogenicity. Virology 191: 223-230.

Chen, Z. R., Zhou, T., Wu, X. H., Hong, Y., Fan, Z. F., and Li, H. F. 2008. Influence of cytoplasmic heat shock protein 70 on

viral infection of Nicotiana benthamiana. Mol. Plant Pathol. 9: 809-817.

Cho S. Y., Cho W. K., Sohn S. H., and Kim K.H. 2012. Interaction of the host protein NbDnaJ with Potato virus X minus-strand

stem-loop 1 RNA and capsid protein affects viral replication and movement. Biochem. Biophys. Res. Commun. 417: 451–

456.

Doronin, S. V., and Hemenway, C. 1996. Synthesis of Potato virus X RNAs by membrane-containing extracts. J Virol 70:

4795-4799.

Duff-Farrier, C., Boonham, N., and Foster, G. R. 2011. The generation of Pepino mosaic virus infectious clones; investigating

the link between genotype and phenotype. Phytopathology 101: S46.

Dufresne, P. J., Thivierge, K., Cotton, S., Beauchemin, C., Ide, C., Ubalijoro, E., Laliberté, J. F., and Fortin, M. G. 2008. Heat

shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus induced membrane

vesicles. Virology 374: 217-227.

Page 15 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 16, MPMI

Dunoyer, P., Himber, C., and Voinnet, O. 2005. Dicer-Like 4 is required for RNA interference and produces the 21-nucleotide

small interfering RNA component of the plant cell-to-cell silencing signal. Nat. Genet. 37: 1356-1360.

Forster, R. L. S., Beck, D. L., Guilford, P. J., Voot, D. M., van Dolleweerd, C. J., and Andersen, M. T. 1992. The coat protein of

White clover mosaic potexvirus has a role in facilitating cell-to-cell transport in plants. Virology 191: 480-484.

Gómez, P., Sempere, R. N., Elena, S. F., and Aranda, M. A. 2009. Mixed infections of Pepino mosaic virus strains modulate

the evolutionary dynamics of this emergent virus. J. Virol. 83: 12378-12387.

Hafrén, A., Hofius, D., Rönnholm, G., Sonnewald U., and Mäkinen K. 2010. HSP70 and its co-chaperone CPIP promote

potyvirus infection in Nicotiana benthamiana by regulating viral coat protein functions. Plant Cell 22: 523-535.

Hamilton, A. J., Baulcombe, D. C. 1999. A species of small antisense RNA in posttranscriptional gene silencing in plants.

Science 286: 950-952.

Hamilton, A., Voinnet, O., Chappell, L., and Baulcombe, D. 2002. Two classes of short interfering RNA in RNA silencing.

EMBO J. 21: 4671-4679.

Hanssen I. M, and Lapidot, M., and Thomma, B. P. H. J. 2010. Emerging viral diseases of tomato crops. Mol. Plant Microbe

Interact. 23: 539-548.

Hanssen, I. M., and Thomma, B. P. H. J. 2010. Pepino mosaic virus: A successful pathogen that rapidly evolved from

emerging to endemic in tomato crops. Mol. Plant Pathol. 11: 179-189.

Hanssen, I. M., Peter van Esse, H., Ballester, A. R., Hogewoning, S. W., Parra, N. O., Paeleman, A., Lievens, B., Bovy, A. G.,

and Thomma, B. P. H. J. 2011. Differential tomato transcriptomic response induced by Pepino mosaic virus isolates with

differential aggressiveness. Plant Physiology 156: 301-318.

Haseloff, J., Siemering, K. R., Prasher, D. C., and Hodge, S. 1997. Removal of a cryptic intron and subcellular localization of

green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. U.S.A. 94:

2122-2127.

Hasiów-Jaroszewska, B., Borodynko, N., and Pospieszny, H. 2009. Infectious RNA transcripts derived from cloned cDNA of a

Pepino mosaic virus isolate. Arch. Virol. 154: 853-856.

Hasiów-Jaroszewska, B., Paeleman, A., Ortega-Parra, N., Borodynko, N., Minicka, J., Czerwoniec, A., Thomma, B. P. H. J.,

and Hanssen, I. N. 2013. Ratio of mutated versus wild-type coat protein sequences in Pepino mosaic virus determines

the nature and severity of yellowing symptoms on tomato plants. Mol. Plant Pathol. 14: 923-933.

Haupt, S., Cowan, G. H., Ziegler, A., Roberts, A. G., Oparka, K. J., Torrance, L. 2005. Two plant-viral movement proteins

traffic in the endocytic recycling pathway. Plant Cell 17: 164-181.

Hayward, A., Padmanabhan, M., and Dinesh-Kumar, S. P. 2011. Virus-induced gene silencing in Nicotiana benthamiana and

other plant species. Methods Mol. Biol. 678: 55-63.

Hellens, R. P., Edwards, E. A., Leyland, N. R., Bean, S., Mullineaux, P. M. 2000. pGreen: a versatile and flexible binary Ti

vector for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 42 819-832.

Page 16 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 17, MPMI

Himber, C., Dunoyer, P., Moissiard, G., Ritzenthaler, C., and Voinnet, O. 2003. Transitivity dependent and -independent cell-

to-cell movement of RNA silencing. EMBO J. 22, 4523-4533.

Hofius, D., Maier, A. T., Dietrich, C., Jungkunz, I., Bornke, F., Maiss, E., and Sonnewald, U. 2007. Capsid protein-mediated

recruitment of host DnaJ like proteins is required for Potato virus Y infection in tobacco plants. J. Virol. 81: 11870-11880.

Hosokawa, N., Hirayoshi, K., Nakai, A., Hosokawa, Y., Marui, N., Yoshida, M., Sakai, T., Nishino, H., Aoike, A., and Kawai, K.

1990. Flavonoids inhibit the expression of heat shock proteins. Cell Struct. Funct. 15: 393-401.

Huang, Y. W., Hu, C. C., Liou, M. R., Chang, B. Y., Tsai, C. H., Meng, M., Lin, N. S., and Hsu, Y. H. 2012. Hsp90 interacts

specifically with viral RNA and diffentially regulates replication initiation of Bamboo mosaic virus and associated satellite

RNA. PLoS Pathog. 8: e1002726.

Ivanov K. I, and Mäkinen, K. 2012. Coat proteins, host factors and plant viral replication. Curr. Opin. Virol. 6: 712-718.

Iwasaki, S., Kobayashi, M., Yoda, M., Sakaguchi, Y., Katsuma, S., Suzuki, T., and Tomari, Y. 2010. Hsc70/Hsp90 chaperone

machinery mediates ATP-dependent RISC loading of small RNA duplexes. Mol. Cell 39: 292-299.

Johansen, L. K., and Carrington, J. C. 2001. Silencing on the spot. Induction and suppression of RNA silencing in the

Agrobacterium-mediated transient expression system. Plant Physiol. 126: 930-938.

Kataya, A. M. R., Mohamed N. S.,Kalantidis, K., and Livieratos, I. C. 2009. Cucurbit yellow stunting disorder virus p25 is a

suppressor of post-transcriptional gene silencing. Virus Res., 145: 48-53.

Koscianska, E., Kalantidis, K., Wypijewski, K., Sadowski, J., and Tabler, M. 2005. Analysis of RNA silencing in agroinfiltrated

leaves of Nicotiana benthamiana and Nicotiana tabacum. Plant Mol. Biol. 59: 647-661.

Lee, C. C., Ho, Y. N., Hu, R. H., Yen, Y. T., Wang, Z. C., Lee, Y. C., Hsu, Y. H., and Meng, M. 2011. The interaction between

Bamboo mosaic virus replication protein and coat protein is critical for virus movement in plant hosts. J. Virol. 85: 12022-

12031.

Levy, A., Dafny-Yelin, M., and Tzfira, T. 2008. Attacking the defenders: plant viruses fight back. Trends Microbiol. 16: 194-197.

Leshchiner, A. D., Minina, E. A., Rakitina, D. V., Vishnichenko, V. K., Solovyev, A. G., Morozov, S. Y., and Kalinina, N. O.

2008. Oligomerization of the Potato virus X 25-kD movement protein. Biochemistry 73: 50-55.

Lim, H. S., Vaira, A. M., Reinsel, M. D., Bae, H., Bailey, B. A., Domier L. L., and Hammond, J. 2010a. Pathogenicity of

Alternanthera mosaic virus is affected by determinants in RNA-dependent RNA polymerase and by reduced efficacy of

silencing suppression in a movement-competent TGB1. J. Gen. Virol. 91: 277-287.

Lim, H. S., Vaira, A. M., Domier, L. L., Lee, S. C., Kim, H. G.,and Hammond, J. 2010b. Efficiency of VIGS and gene

expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression.

Virology 402: 149-163.

Lin, T.Y., Duck, N.B., Winter, J., Folk, W.R. (1991). Sequences of two hsc 70 cDNAs from Lycopersicon esculentum. Plant

Molecular Biology 16, 475-478.

Lin, J. W., Ding, M. P., Hsu, Y. H., and Tsai, C. H. 2007. Chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is

required for efficient accumulation of Bamboo mosaic virus. Nucleic Acids Res. 35: 424-432.

Page 17 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 18, MPMI

Ling, K. S., Li, R., and Bledsoe, M. 2013. Pepino mosaic virus genotype shift in North America and development of a loop-

mediated isothermal amplification for rapid genotype identification. Virology J. 10: 117.

Liu, Y., Schiff, M., Marathe, R., and Dinesh-Kumar, S. P. 2002. Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required

for N-mediated resistance to Tobacco mosaic virus. Plant J. 30: 415-429.

Llave, C., Kasschau, K.D., Carrington, J.C. (2000). Virus-encoded suppressor of posttranscriptional gene silencing targets a

maintenance step in the silencing pathway. Proc. Natl. Acad. Sci. U.S.A. 97: 13401-13406.

Lough, T. J., Netzler, N. E., Emerson, S. J., Sutherland, P., Carr, F., Beck, D. L., Lucas, W. J., and Forster, R. L. 2000. Cell-to-

cell movement of Potexviruses: Evidence for a ribonucleoprotein complex involving the coat protein and first triple gene

block protein. Mol. Plant-Microbe Interact. 13: 962-974.

Lough, T. J., Lee, R. H., Emerson, S. J., Forster, R. L., and Lucas, W. J. 2006. Functional analysis of the 5′ untranslated

region of Potexvirus RNA reveals a role in viral replication and cell-to-cell movement. Virology 351: 455-465.

Lu, H. C., Chen, C. E., Tsai, M. H., Wang, H. I., Su, H. J., and Yeh, H. H. 2009. Cymbidium mosaic potexvirus isolate-

dependent host movement systems reveal two movement control determinants and the coat protein is the dominant.

Virology 388: 147-159.

Manwell, L. A., and Heikkila, J. J. 2007. Examination of KNK437- and quercetin-mediated inhibition of heat shock-induced

heat shock protein gene expression in Xenopus laevis cultured cells. Comp. Biochem. Physiol. A 148521-148530.

Mathioudakis, M. M, Veiga, R., Ghita, M., Tsikou, D., Medina, V., Canto, T., Makris, A. M., and Livieratos, I. C. 2012. Pepino

mosaic virus capsid protein interacts with a tomato heat shock protein cognate 70 in yeast, in planta and in vivo. Virus

Res. 163: 28-39.

Mathioudakis, M. M., Veiga, R., Canto, T., Medina, V., Mossialos, D., Makris, A. M., and Livieratos, I. 2013. Pepino mosaic

virus triple gene block protein 1 (TGBp1) interacts with and increases tomato catalase 1 activity to enhance virus

accumulation. Mol. Plant Pathol. 14: 589-601.

Mayer, M. P., and Bukau, B. 2005. Hsp70 chaperones: cellular functions and molecular mechanism. Cell. Mol. Life Sci. 62:

670-684.

Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., Toyooka, K., Matsuoka, K., Jinbo, T., and Kimura,

T. 2007. Development of series of Gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for

plant transformation. Journal Biosc. & Bioengineering 104: 31-41.

Nagy, P. D., and Pogany, J. 2012. The dependence of viral RNA replication on co-opted host factors. Nat. Rev. Microbiol. 10:

137-149.

Nagy, P. D., Wang, R. Y., Pogany, J., Hafrén, A., and Mäkinen, K. 2011. Emerging picture of host chaperone and cyclophilin

roles in RNA virus replication. Virology 411: 374-382.

Navas-Castillo, J., Albiach-Martí, M. R., Gowda, S., Hilf, M. E., Garnsey, S. M., and Dawson, W. O. 1997. Kinetics of

accumulation of Citrus tristeza virus RNAs. Virology 228: 92–97.

Page 18 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 19, MPMI

Nishikiori, M., Dohi, K., Mori, M., Meshi, T., Naito, S., and Ishikawa., M. 2006. Membrane-bound Tomato mosaic virus

replication proteins participate in RNA synthesis and are associated with host proteins in a pattern distinct from those that

are not membrane bound. J. Virol. 80: 8459-8468.

Okano, Y., Senshu, H., Hashimoto, M., Neriya, Y., Netsu, O., Minato, M., Yoshida, T., Maejima, K., Oshim, K., Komatsu, K.,

Yamaji, Y., and Namba, S. 2014. In planta recognition of a double-stranded RNA sunthesis protein complex by a

potexviral RNA silencing suppressor. Plant Cell, in press http://dx.doi.org/10.1105/tpc.113.120535.

Osman, T., Olsthoorn, R. C. I., and Livieratos, I. C. 2012. In vitro template-dependent synthesis of Pepino mosaic virus

positive- and negative-strand rna by its rna-dependent rna polymerase. Virus Res. 167: 267-272.

Peremyslov, V. V., Hagiwara, Y., and Dolja V. V. 1999. HSP70 homolog functions in cell-to-cell movement of a plant virus.

Proc. Natl. Acad. Sci. U.S.A. 96: 14771-14776.

Pumplin N., and Voinnet O. 2013. RNA silencing suppression by plant pathogens: defence, counter-defence and counter-

counter-defence. Nat. Rev. Microbiol. 11: 745-760.

Samuels, T. D., Ju, H. J., Ye, C. M., Motes, C. M., Blancaflor, E. B., and Verchot-Lubicz, J. 2007. Subcellular targeting and

interactions among the Potato virus X TGB proteins. Virology 367: 375-389.

Satyanarayana, T., Gowda, S., Mawassi, M., Albiach-Marti, M. R., Ayllon, M. A., Robertson, C., Garnsey, S. M., and Dawson,

W. O. 2000. Closterovirus encoded HSP70 homolog and p61 in addition to both coat proteins function in efficient virion

assembly. Virology 278: 253-265.

Sempere, R. N., Gómez, P., Truniger, V., and Aranda, M. A. 2011. Development of expression vectors based on Pepino

mosaic virus. Plant Methods 7: 6.

Senshu, H., Ozeki, J., Komatsu, K., Hashimoto, M., Hatada, K., Aoyama, M., Kagiwada, S., Yamaji, Y., and Namba, S. 2009.

Variability in the level of RNA silencing suppression caused by triple gene block protein 1 (TGBp1) from various

potexviruses during infection. J. Gen.Virol. 90: 1014-1024.

Senshu, H., Yamaji, Y., Minato, N., Shiraishi, T., Maejima, K., Hashimoto M., Miura, C., Neriya, Y., and Namba S. 2011. A

dual strategy for the suppression of host antiviral silencing: two distinct suppressors for viral replication and viral

movement encoded by Potato virus M. J. Virol. 85: 10269-10278.

Silhavy, D., Molnar, A., Lucioli, A., Szittya, G., Hornyik, C., Tavazza, M., and Burgyan, J. 2002. A viral protein suppresses

RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J. 21: 3070-3080.

Sun, S.W., Chung, M.C., Lin, T.Y. (1996). The structure and expression of an hsc70 gene from Lycopersicon esculentum.

Gene 170, 237-241.

Tomita, Y., Mizuno, T., Diez, J., Naito, S., Ahlquist, P., and Ishikawa, M. 2003. Mutation of host DnaJ homolog inhibits Brome

mosaic virus negative-strand RNA synthesis. J. Virol. 77: 2990-2997.

Tournier, B., Tabler, M., and Kalantidis, K. 2006. Phloem flow strongly influences the systemic spread of silencing in GFP

Nicotiana benthamiana plants. Plant J. 47: 383-394.

Page 19 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 20, MPMI

Verchot, J. 2012. Cellular chaperones and folding enzymes are vital contributors to membrane bound replicaiton and

movement complexes during RNA virus infection. Front. Plant Sci. 3: 275.

Verchot-Lubicz, J., Torrance, L., Solovyev A. G., Morozov, S. Y., Jackson, A. O., and Gilmer, D. 2010. Varied movement

strategies employed by triple gene block-encoding viruses. Mol. Plant-Microbe Interact. 23: 1231-1247.

Verchot-Lubicz, J., Ye, C. M., and Bamunusinghe, D. 2007. Molecular biology of potexviruses: recent advances. J. Gen. Virol.

88: 1643-1655.

Voinnet, O. 2002. RNA silencing: small RNAs as ubiquitous regulators of gene expression. Curr. Opin. Plant Biol. 5: 444-451.

Voinnet, O. 2005. Induction and suppression of RNA silencing: insights from viral infections. Nat. Rev. Gen. 6: 206-220.

Voinnet, O., Lederer, C., and Baulcombe, D. C. 2000. A viral movement protein prevents spread of the gene silencing signal in

Nicotiana benthamiana. Cell 10: 157-167.

Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., Blazevic, D., Grefen, C., Schumacher, K.,

Oecking, C., Harter, K., and Kudla, J. 2004. Visualization of protein interactions in living plant cells using bimolecular

fluorescence complementation. Plant J. 40: 428-438.

Wang, R. Y., Stork, J., and Nagy, P. D. 2009. A key role for heat shock protein 70 in the localization and insertion of

Tombusvirus replication proteins to intracellular membranes. J. Virol. 83: 3276-3287.

Whitham, S. A., Quan, S., Chang, H. S., Cooper, B., Estes, B., Zhu, T., Wang, X., and Hou, Y. M. 2003. Diverse RNA viruses

elicit the expression of common sets of genes in susceptible Arabidopsis thaliana plants. Plant J. 33: 271-283.

Whitham, S. A., Yang, C., and Goodin, M. M. 2006. Global impact: elucidating plant responses to viral infection. Mol. Plant-

Microbe Interact. 19: 1207-1215.

Wu, C. H., Lee, S. C., and Wang, C. W. 2011. Viral protein targeting to the cortical endoplasmic reticulum is required for cell-

cell spreading in plants. J. Cell Biol. 193: 521-535.

Page 20 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 21, MPMI

Figures Legends

Fig. 1. Detection of the OST-CP recombinant protein in tomato plants. A, RT-PCR from total-RNA isolated following PepMV-

SP13 (WT) and PepMV-SP13_OST-CP inoculation. B, Silver-stained acrylamide gel showing the eluted fractions (E2-E4)

following agro-inoculation of tomato plants with pBPepXL6-OST-CP and pBPepXL6 12 days post-inoculation. Broad range

protein molecular weight marker (Promega) (M) was included in the gel, and the sizes of its proteins are shown in the right

side of the panel.

Fig. 2. Knock-down of Hsp70 plants by virus-induced gene silencing (VIGS) Tobacco rattle virus (TRV)-based assay reduces

the Pepino mosaic virus (PepMV) accumulation in Nicotiana benthamiana plants. A, (Top panel) Phenotypes of N.

benthamiana plants 5 days (left) after Hsp70 silencing by TRV-VIGS system. The photographs were taken one to two days

before PepMV inoculation of the upper leaves. At 17 days after TRV infection the Hsp70-silenced plants died (right). (Bottom

panel) Phenotypes of N. benthamiana-TRV control plants (TRV-C; left) and the Hsp70-silenced (TRV-Hsp; right) plants after

10 days of TRV infection and 4 days of PepMV inoculation. The photographs were taken after the collection of plant tissue for

mRNA and protein analysis. (1), (2) and (3) indicate the TRV-agroinfiltrated, PepMV-mechanically inoculated (local) and

PepMV-systemically infected leaves, respectively. B, Quantification of Hsp70 mRNA levels by semi-quantitative

polymerase chain reaction (PCR) in PepMV inoculated N. benthamiana TRV-C and TRV-Hsp plants, as indicated. The

amplification cycles are indicated above the panels. C, Quantification of the coat protein (CP) and RNA-dependent RNA

polymerase (RdRp) subgenomic and genomic mRNA levels by semi-quantitative PCR in PepMV inoculated N. benthamiana

TRV-C and TRV-Hsp plants, as indicated. In PepMV RdRp II panel 2.5-fold times more template of the initial template

amount (PepMV RdRp I) was used. The amplification cycles are indicated above the panels. D, Western blotting in

systemic leaves of N. benthamiana TRV-C and TRV-Hsp plants, 4 days after PepMV infection, using α-Hsp70 (top panel) and

α-PepMV CP (middle panel) antibodies. The small unit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein

(LC; bottom panel) stained with Coomassie Brilliant Blue served as protein loading control.

Fig. 3. Inhibition of Hsp70 mRNA expression by quercetin treatment reduces the Pepino mosaic virus (PepMV) accumulation

in Nicotiana benthamiana plants and protoplasts. A, Phenotypes of either DMSO- or quercetin-treated N. benthamiana plants

4 days after PepMV inoculation. The arrows indicate the DMSO- or quercetin- treated leaves. B, Western blotting of PepMV-

inoculated leaves treated either with DMSO (lane 1) or quercetin (lanes 2-4, corresponding to different plants) 4 days after

inoculation, using α-Hsp70 (top panel) and α-PepMV CP (second panel) antibodies. The third panel is an overexposure of the

second one and the asterisk indicates the faint CP band. C, Northern blotting of total RNA samples prepared from N.

benthamiana protoplasts treated either with DMSO or quercetin (labeled as D and Q, respectively) 24 h post transfection using

either two different in vitro transcribed PepMV RNAs (pPepXL6 clone) or RNA extracted from purified PepMV virions. (-) and -

represent total RNAs from protoplasts transfected either with PepMV RNA without any treatment or without viral RNA (mock),

Page 21 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 22, MPMI

respectively; + represents total RNA from PepMV infected N. benthamiana leaves used as control of the Northern assay. Viral

genomic RNA bands are indicated by an arrow. The ethidium-bromide stained gel (bottom panel) shows rRNA from

protoplasts samples. D, Western blotting of N. benthamiana protoplasts transfected with PepMV and treated either with

DMSO, quercetin or untreated 24 h post transfection, using α-Hsp70 antibodies. The last lane represents total protein extract

from protoplasts without viral transfection. In B and D the small unit of ribulose-1,5-bisphosphate carboxylase/oxygenase

(Rubisco) protein (LC; bottom panel) stained with Coomassie Brilliant Blue served as protein loading control.

Fig. 4. Pepino mosaic virus (PepMV) TGBp1 and CP proteins suppress RNA silencing of GFP in Nicotiana benthamiana

plants species. A, Suppression of ssRNA-induced RNA silencing (S-PTGS) of GFP in N. benthamiana wild type plants.

Leaves were infiltrated with mixtures of Agrobacterium cultures harboring the 35S-mGFP4 construct either in combination with

the pGreen empty vector [pGR(-); negative control] or with the pGreen constructs expressing the PepMV TGBp1 (pGR-

TGBp1), TGBp2 (pGR-TGBp2), TGBp3 (pGR-TGBp3), CP (pGR-CP) and the Tomato chlorosis virus (ToCV) p22 (positive

control). UV light images were taken 10 days post-infiltration (dpi). B, Western blot analysis of GFP protein levels extracted

from the infiltrated patches as indicated above each lane at 10 dpi, using α-GFP antibodies (top panel). C, Suppression of

ssRNA-induced RNA silencing (S-PTGS) of GFP in N. benthamiana 16c plants. Leaves were infiltrated with mixtures of

Agrobacterium cultures harboring the 35S-mGFP4 construct either in combination with the pGR(-) or with pGR-TGBp1, pGR-

TGBp2, pGR-TGBp3, pGR-CP and the ToCV p22. UV light images were taken at 8 dpi. D, Western blot analysis of GFP

protein levels extracted from the infiltrated patches as indicated above each lane at 8 dpi, using α-GFP antibodies (top panel).

The first line represents protein extract from a patch only infiltrated with GFP, whereas the last line represents protein extract

sample from a non-infiltrated patch. The one asterisk indicates the endogenous GFP and with two asterisks the transiently

expressed GFP. E, Suppression of dsRNA-induced RNA silencing (IR-PTGS) of GFP in N. benthamiana wild type plants.

Leaves were infiltrated with mixtures of Agrobacterium cultures harboring the 35S-mGFP4 and 35S-hpGFP constructs either

in combination with the pGR(-) or with pGR-TGBp1, pGR-TGBp2, pGR-TGBp3, pGR-CP and the ToCV p22. UV light images

were taken 6 dpi. F, Western blot analysis of GFP protein levels extracted from the infiltrated patches as indicated above each

lane at 8 dpi, using α-GFP antibodies (top panel). The first line represents protein extract from a patch only infiltrated with

GFP, whereas the last line represents protein extract sample from a non-infiltrated patch. In B, D and F the small unit of

ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein (LC; bottom panel) stained with Coomassie Brilliant Blue

served as protein loading control.

Fig. 5. Pepino mosaic virus (PepMV) TGBp1 and CP suppressors are functionally divergent. A, Effect of PepMV TGBP1 and

CP in restoration of GFP expression in already silenced tissue (reversal silencing assay). Leaves of Nicotiana benthamiana

6.4 transgenic line (established GFP silencing) were infiltrated individually with Agrobacterium cultures of the pGreen empty

vector [pGR(-); negative control] or the pGreen constructs expressing the PepMV TGBp1 (pGR-TGBp1), TGBp2 (pGR-

TGBp2), TGBp3 (pGR-TGBp3), CP (pGR-CP) and the Tobacco etch virus Hc-Pro (positive control). UV light images were

Page 22 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Mathioudakis et al., 23, MPMI

taken 7 days post-infiltration (dpi). B, Western blot analysis of GFP protein levels extracted from infiltrated patches with Hc-

Pro (1), pGR-TGBp1 (2), pGR-CP (3) and pGR(-) (4) at 10 dpi, using α-GFP antibodies (top panel). The small unit of ribulose-

1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein (LC; bottom panel) stained with Coomassie Brilliant Blue served

as protein loading control. C-D, Interference of PepMV TGBp1 and CP with the cell-to-cell movement of the silencing signal of

GFP. Leaves from N. benthamiana 16c plants were infiltrated mixtures of Agrobacterium cultures harboring the 35S-mGFP4

construct either in combination with the pGR(-) or with pGR-TGBp1, pGR-CP, Tomato chlorosis virus (ToCV) p22 and

Cymbidium ringspot virus (CymRSV) p19. Arrows indicate a red-fluorescent ring around the infiltrated patch corresponding to

the exit of the silencing signal. UV light images were taken 10 dpi. E, Interference of PepMV TGBp1 and CP with the systemic

(long-distance) spread of the silencing signal of GFP. Leaves from N. benthamiana 16c plants were infiltrated as described in

C-D. UV light images of the plants were taken at 12 and 30 dpi.

Fig. 6. In planta subcellular localization of Pepino mosaic virus (PepMV) viral-viral interactions by bimolecular fluorescent

complementation (BiFC) in Nicotiana benthamiana cells. Reconstitution of YFP fluorescence was visualized by confocal laser

scanning microscopy after co-expression of the fusion proteins. A, Co-infiltration of pCYFP-TGBp1 and pNYFP-CP revealed

their interaction in cytoplasm and nucleus, but not the nucleolus using YFP and DAPI filter. B, Co-infiltration of pCYFP-CP with

pNYFP-CP (upper panel) and pCYFP-TGBp1 with pNYFP-TGBp1 (lower panel) revealed their self-interactions in cytoplasm

and nucleus. In A-B, the third column (right) represents the overlay image from generated from YFP and DAPI filters. C, Co-

infiltrations of pCYFP-TGBp1 with pNYFP, pCYFP and pNYFP-TGBp1, pCYFP-CP with pNYFP and pCYFP and pNYFP-CP

(negative controls), respectively; c, cytoplasm; n, nucleus; Bars: denote 75 mm.

Page 23 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Fig. 1

8 dpi 12 dpi 15 dpi WT WT A)

B)

100

75

50

25

35

150

CP

HSP70

RUBISCO

M E2 E3 E4 E2 E3 E4

pBPepXL6-OST-CP pBPepXL6

Page 24 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Fig. 2

(3)

(2)

(3) (2)

(3)

TRV-Hsp TRV-C (1)

(1)

A) B)

EF-1a

Hsp

TRV-C TRV-Hsp

20 25 30 20 25 30 cycles

C)

24 28 32 24 28 32 cycles

EF-1a

PepMV CP

PepMV RdRp I

PepMV RdRp II

α-Hsp70

α-PepMV CP

LC

TRV-Hsp TRV-C D)

TRV-C TRV-Hsp (1)

(3) (2)

TRV-Hsp

5dpi TRV-Hsp

17dpi

Page 25 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Fig. 3

α- Hsp70

α-PepMV CP

1 2 3 4

LC

Overexposure *

Quercetin DMSO

Quercetin + PepMV DMSO + PepMV Quercetin + PepMV DMSO +PepMV

A) B)

+ 1 2 3 4 5 6 7 8 9 10 11 12 - + 14 15 16 17 18 19 -

In vitro RNA transcripts Virion RNA

(-) D Q (-) D Q (-) D Q

rRNA

PepMV gRNA

C)

D)

(-) D Q

α- Hsp70

LC

pPepXL6 + pPepXL6 +

PepMV +

Page 26 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Fig. 4

- + + -

TGBp2 TGBp3 p25 CP

pGR(-) ToCV p22

TGBp2 TGBp3

ToCV p22 pGR(-)

TGBp1 CP

pGR(-) ToCV p22

TGBp2 TGBp3

ToCV p22 pGR(-)

TGBp1 CP

35S-mGFP4 +

- α-GFP

- LC

A) B)

E) F)

- α-GFP

- LC

35S-mGFP4 + 35S-hpGFP +

pGR(-) ToCV p22

TGBp2 TGBp3

ToCV p22 pGR(-)

TGBp1 CP

C) D) 35S-mGFP4 +

** * - α-GFP

- LC

Page 27 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Fig. 5

HcPro pGR(-)

- α-GFP

- LC

A) B)

pGR(-)

ToCV p22

TGBp1

CP

pGR(-) CymRSV p19

CP

1 2 3 4

TGBp1 CP

C) D)

E)

pGR(-) p19 TGBp1 CP

pGR(-) p19 TGBp1 CP

12 dpi

30 dpi

Page 28 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Fig. 6

CYFP-TGBp1

NYFP-TGBp1

n

C

YFP DAPI Overlay A)

B)

C) CYFP-TGBp1

NYFP

CYFP-CP

NYFP

NYFP-TGBp1

CYFP

NYFP-CP

CYFP

C

C

n

n

C

C n

n

CYFP-TGBp1

NYFP-CP

CYFP-CP

NYFP-CP

Page 29 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.

Tables

Table 1. List of oligonucleotide primers used in this study.

Primers Primer sequence (5΄- 3΄)a Restriction sites Application assay

Hsp70-VIGS-F GGATCCGATTTACTGATACTGAGCGTC BamHI VIGS Hsp70-VIGS-R CTCGAGTGTAGGCTCATTGATAATACG XhoI VIGS

PepMV-CP-PTGS-F GGATCCATGCCTGACACAACACCTGTTG BamHI RNA silencing

PepMV-CP-PTGS-R CTCGAGTTAAAGTTCAGGGGGTGCGTCT XhoI RNA silencing PepMV-TGBp1-PTGS-F GGATCCATGTCCTCATACTTATCTTCA BamHI RNA silencing

PepMV-TGBp1-PTGS-R CTCGAGCTAGGAGTTAGACCTGGCATTT XhoI RNA silencing

PepMV-TGBp2-PTGS-F GGATCCATGCCAGGTCTAACTCCTAGAGC BamHI RNA silencing

PepMV-TGBp2-PTGS-R CTCGAGTCAATGATGGCCTGACAATGGTTG XhoI RNA silencing

PepMV-TGBp3-PTGS-F GGATCCATGTCCTCATACTTATCTTCATT BamHI RNA silencing

PepMV-TGBp3-PTGS-R CTCGAGCTAATTTTCAAATTTAGGAAAAC XhoI RNA silencing

sqPepMV-CP-F GGCAGCCAAATGAAAAAGAA n.i. Semi-quantitative PCR

sqPepMV-CP-R TTAGCTCCTCCCATGTGTCC n.i. Semi-quantitative PCR sqPepMV-RdRp-F CAAGCCAAAGATGAΑΑ n.i. Semi-quantitative PCR sqPepMV-RdRp-R TCAATGTGTTGCTTTTGGA n.i. Semi-quantitative PCR sqEF-1a-Fb GATTGGTGGTATTGGAACTGTC n.i. Semi-quantitative PCR sqEF-1a-Rb AGCTTCGTGGTGCATCTC n.i. Semi-quantitative PCR

a Restriction enzymes used in primers are underlined

b Primers used in Mathioudakis et al., 2012 n.i.; not included

Page 30 of 30M

olec

ular

Pla

nt-M

icro

be I

nter

actio

ns "

Firs

t Loo

k" p

aper

• h

ttp://

dx.d

oi.o

rg/1

0.10

94/M

PMI-

07-1

4-01

95-R

• p

oste

d 08

/27/

2014

T

his

pape

r ha

s be

en p

eer

revi

ewed

and

acc

epte

d fo

r pu

blic

atio

n bu

t has

not

yet

bee

n co

pyed

ited

or p

roof

read

. The

fin

al p

ublis

hed

vers

ion

may

dif

fer.