Mungbean yellow mosaic virus (MYMV) AC4 suppressespost-transcriptional gene silencing and an AC4 hairpin RNAgene reduces MYMV DNA accumulation in transgenic tobacco

Sukumaran Sunitha • Gnanasekaran Shanmugapriya •

Veluthambi Balamani • Karuppannan Veluthambi

Received: 1 October 2012 / Accepted: 4 February 2013 / Published online: 17 February 2013

� Springer Science+Business Media New York 2013

Abstract Mungbean yellow mosaic virus (MYMV) is a

legume-infecting geminivirus that causes yellow mosaic

disease in blackgram, mungbean, soybean, Frenchbean and

mothbean. AC4/C4, which is nested completely within the

Rep gene, is less conserved among geminiviruses. Much

less is known about its role in viral pathogenesis other than

its known role in the suppression of host-mediated gene

silencing. Transient expression of MYMV AC4 by agro-

infiltration suppressed post-transcriptional gene silencing

in Nicotiana benthamiana 16c expressing green fluores-

cence protein, at a level comparable to MYMV TrAP

expression. AC4 full-length gene and an inverted repeat of

AC4 (comprising the full-length AC4 sequence in sense and

antisense orientations with an intervening intron) which

makes a hairpin RNA (hpRNA) upon transcription were

introduced into tobacco by Agrobacterium-mediated leaf

disc transformation. Leaf discs of the transgenic plants

were agroinoculated with partial dimers of MYMV and

used to study the effect of the AC4-sense and AC4 hpRNA

genes on MYMV DNA accumulation. Leaf discs of two

transgenic plants that express the AC4-sense gene dis-

played an increase in MYMV DNA accumulation. Leaf

discs of six transgenic plants containing the AC4 hpRNA

gene accumulated small-interfering RNAs (siRNAs) spe-

cific to AC4, and upon agroinoculation with MYMV they

exhibited a severe reduction in the accumulation of

MYMV DNA. Thus, the MYMV AC4 hpRNA gene has

emerged as a good candidate to engineer resistance against

MYMV in susceptible plants.

Keywords Geminivirus resistance � Hairpin RNA �Legume-infecting begomovirus � RNA silencing �Silencing suppression � siRNA

Introduction

Geminiviruses are single-stranded DNA viruses with

characteristic twinned icosahedral capsid structures that

replicate via double-stranded DNA intermediates in infec-

ted cell nuclei [1, 2]. The genome structures of geminivi-

ruses are either monopartite or bipartite, with DNA A and

DNA B components of approximately 2.5–3.0-kb in length.

Geminiviruses are classified into four genera, Mastrevirus,

Curtovirus, Topocuvirus and Begomovirus based on their

genome organization, host-range and the type of insect

vector [3]. Mungbean yellow mosaic virus (MYMV-

[IN:Vig]) and Mungbean yellow mosaic India virus (MY-

MIV) are whitefly-transmitted bipartite begomoviruses

which infect the legume crops blackgram, mungbean,

soybean, Frenchbean and mothbean and cause an approx-

imate annual yield loss of US $300 million [4, 5]. DNA A

of MYMV encodes the replication-associated protein (Rep/

AC1), transcriptional activator protein (TrAP/AC2), repli-

cation enhancer protein (REn/AC3), pre-coat protein

(AV2) and the coat protein (CP/AV1). DNA B encodes the

nuclear shuttle protein (NSP/BV1) and the movement

protein (MP/BC1).

DNA A of bipartite begomoviruses harbour an addi-

tional open reading frame (ORF), AC4, nested completely

within the Rep gene. AC4 was first studied in DNA A of

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11262-013-0889-z) contains supplementarymaterial, which is available to authorized users.

S. Sunitha � G. Shanmugapriya � V. Balamani �K. Veluthambi (&)

Department of Plant Biotechnology, School of Biotechnology,

Madurai Kamaraj University, Madurai 625021, India

e-mail: kveluthambi@rediffmail.com

123

Virus Genes (2013) 46:496–504

DOI 10.1007/s11262-013-0889-z

Tomato golden mosaic virus (TGMV) by Elmer et al. [6].

The introduction of a premature translation termination

codon in the AC4 ORF, which did not alter the Rep ORF,

did not affect TGMV replication in Nicotiana benthami-

ana. Similar results were observed when mutated AC4

genes of TGMV, Bean golden mosaic virus (BGMV) and

Potato yellow mosaic virus (PYMV) were tested on

Nicotiana tabacum, Datura stramonium [7] and N. benth-

amiana [8] plants and in the tobacco cell suspension cul-

ture [9]. The reports indicated that either the AC4 ORF of

bipartite begomoviruses is non-functional in the tested

hosts or is functionally redundant with one or more of the

essential viral-encoded proteins. Interestingly, African

cassava mosaic virus (ACMV) Rep with its cognate AC4

and Tomato yellow leaf curl virus-China (TYLCV-C) Rep

with its cognate C4 triggered systemic necrosis in N.

benthamiana when expressed using the potato virus X

vector. However, AC4 and C4 individually neither induced

nor enhanced the hypersensitive response [10].

The C4 gene of monopartite viruses manifests several

functions. The C4 genes of Beet curly top virus (BCTV)

[11], Tomato leaf curl virus (TLCV) [12–14] and Tomato

leaf curl virus-Australia (ToLCV) [15] act as major

symptom determinants. Jupin et al. [16] found that C4 of

tomato-infecting TYLCV was involved in systemic

movement. Similarly, Beet severe curly top virus (BSCTV)

C4 facilitated systemic movement of the virus in N.

benthamiana and Arabidopsis thaliana through its non-

specific DNA binding property [17]. The symptom deter-

minant property of C4 of monopartite geminiviruses was

widely attributed to its interaction with hormone signalling

factors [18, 19] and regulation of cell cycle factors [20, 21].

Since the functional differences between AC4 of bipartite

and C4 of monopartite geminiviruses are so clear, it is

speculated that the AC4 and C4 ORFs clearly distinguish

bipartite and monopartite geminiviruses, respectively [12].

The functionally divergent AC4 and C4 of bipartite and

monopartite geminiviruses converge in functioning as a

silencing suppressor. ACMV AC4 and TYLCV-C C4

suppress host RNA silencing and trigger Rep-mediated

necrosis in N. benthamiana [10]. Vanitharani et al. [22]

identified ACMV-Cameroon (ACMV-[CM]) AC4 and Sri

Lankan cassava mosaic virus (SLCMV) AC4 as suppres-

sors of post-transcriptional gene silencing (PTGS). The

suppression of the host defence mechanism by AC4 of

ACMV-[CM] resulted in synergistic, mixed infection of

East African cassava mosaic Cameroon virus (EACMCV).

AC4 of ACMV-[CM] bound specifically to the single-

stranded microRNAs (miRNAs) and small-interfering

RNAs (siRNAs), and inhibited the cleavage of target

mRNA. The resultant up-regulation of the target mRNA

caused developmental defects in Arabidopsis thaliana [23].

AC4 of EACMCV, with an N-myristoylation motif, was

demonstrated to be a membrane-bound protein involved in

symptom determination and suppression of the systemic

spread of silencing in N. benthamiana [24]. C4 of Bhendi

yellow vein mosaic virus (BYVMV) [25] and ToLCV [26]

are examples of monopartite geminivirus C4 with silencing

suppression and symptom determination properties.

In this study, MYMV AC4 was investigated for its role

in the suppression of PTGS. Conventional pathogen-

derived resistance and RNA silencing-mediated resistance

were evaluated in N. tabacum plants expressing the

MYMV AC4-sense gene or the AC4 hairpin RNA (hpRNA)

gene. Plants expressing the AC4-sense gene did not inhibit

MYMV DNA accumulation in the leaf disc agroinoculation

assay, whereas the hairpin AC4 (hpAC4) plants that accu-

mulated high levels of the AC4 siRNAs displayed a pro-

nounced reduction in MYMV DNA accumulation. We

report for the first time that AC4 of MYMV, a legume-

infecting geminivirus, acts as a suppressor of PTGS.

Materials and methods

Viral clones and plasmids

Accession numbers of DNA A and DNA B (KA22) of

MYMV-[IN:Vig] in EMBL/Genbank are AJ132575 and

AJ132574, respectively. The binary plasmid pBI-mgfp5-

ER which harbours the mgfp5-ER gene in pBI121 was used

to trigger gfp silencing in N. benthamiana 16c [27]. The

binary plasmid pCAM-AC4-gus, which harbours the

MYMV AC4-sense gene, was constructed as follows: A

377-bp HincII/DraI AC4 ORF from MYMV DNA A (co-

ordinates 2489–2112) was cloned in the corresponding

sites of pJIC-35S [28]. The 1.1-kb AC4 cassette with the

CaMV 35S promoter and polyadenylation signal was

excised using EcoRV and cloned in the SmaI site of

pCAMBIA2301. The resulting clone was mobilized by

triparental mating into the Agrobacterium tumefaciens

strain LBA4404 (pSB1). pSB1 harbours virB, virG and

virC genes of pTiBo542 [29].

The construction of the binary plasmid pCAM-AC2-gus,

which harbours the MYMV AC2 gene, and its mobilization

into the A. tumefaciens strain EHA105 have been described

previously [30]. The binary plasmid pML-hpAC4, which

harbours the MYMV AC4 hpRNA gene, was mobilized

into the A. tumefaciens strain C58C1 (pGV2260) [31].

Agroinfiltration

N. benthamiana 16c plants which harboured the mgfp5-ER

gene [27] were grown on vermiculite and sand (1:1) mix-

ture at 25 �C with 16-h-day and 8-h-dark photoperiod in a

growth chamber. Six weeks after germination, the plants

Virus Genes (2013) 46:496–504 497

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were subjected to agroinfiltration as described by Llave

et al. [32]. Visual detection of GFP fluorescence was done

5 days-post-agroinfiltration using a hand-held long-wave

UV lamp (UVP Inc., San Gabriel, USA).

Tobacco transformation

Tobacco (N. tabacum L. cv. Wisconsin38) leaf discs were

transformed using A. tumefaciens as described by Sun-

ilkumar et al. [33]. Transgenic shoots were selected on the

shoot-induction medium (Murashige and Skoog [MS] salts

[34], B5 vitamins, 0.5 lM NAA, 4 lM BAP, 3 % [w/v]

sucrose, 0.8 % [w/v] agar, pH 5.7) containing 100 mg/l

kanamycin (for pCAM-AC4-gus-plants) or 5 mg/l phos-

phinothricin (for pML-hpAC4-plants) and were kept for

root induction on the plant establishment medium (MS

salts, 1 mg/l folic acid [w/v], 100 mg/l myoinositol [w/v],

0.4 mg/l thiamine [w/v], 0.057 lM indole-3-acetic acid,

0.14 lM kinetin, 3 % [w/v] sucrose, 0.9 % [w/v] agar, pH

5.7). The primary transformants were axenically main-

tained by subculturing shoots on the plant establishment

medium at 6-week intervals.

Southern and northern blot analysis

Total plant DNA was extracted as described by Rogers and

Bendich [35]. DNA concentration was estimated using the

Hoechst dye 33258 in the DyNA Quant 200 fluorometer

(Hoefer Scientific Instruments, San Francisco, USA). DNA

(10 lg) from control and transgenic plants was digested

with appropriate restriction enzymes and electrophoresed

in a 0.8 % agarose gel in 19 Tris–borate-EDTA (TBE)

buffer, depurinated and subjected to Southern blot analysis

with [a-32P]dCTP-labelled probes. Viral titre determination

of agroinoculated leaf discs was performed by electro-

phoresis of undigested total plant DNA (5 lg) in a 0.8 %

agarose gel in 19 TNE (40 mM Tris–acetate, pH 7.5,

20 mM sodium acetate, 2 mM EDTA) buffer [36]. Total

RNA was extracted using the Tri Reagent (Sigma-Aldrich,

St. Louis, USA) and northern blot analysis was carried out

as described by Pawlowski et al. [37].

siRNA analysis

Total RNA was extracted and the small RNAs were frac-

tionated using the RNA clean up protocol from the Midi

RNeasy kit (Qiagen GmBH, Hilden, Germany). Speed vac-

dried small RNA fraction (20 lg) was loaded onto a 15 %

polyacrylamide gel with 8 M urea and was electrophoresed

at 300 V. Small RNAs were blotted onto the positively

charged nylon membrane (Roche Diagnostics, Indianapo-

lis, USA) using the Transblot-SD semidry transfer appa-

ratus (Bio-Rad, Hercules, CA) in 19 TBE at 7 V for

45 min. The membrane was crosslinked twice in a UV

cross linker (Hoefer Scientific Instruments, San Francisco,

USA). A 0.4-kb AC4 fragment was labelled using the

MegaprimeTM DNA labelling system (Amersham Biosci-

ences Ltd., Little Chalfont, UK) and [a-32P]dCTP.

Hybridization was performed at 37 �C for 16–20 h. Post-

hybridization washes were performed as follows: The

hybridization solution was discarded and the blots were

washed four times with 29 SSC/0.2 % SDS. Each wash

was done for 20 min at 50 �C in the hybridization oven.

Tobacco leaf disc agroinoculation assay to study

MYMV replication

Leaf discs (8 mm diameter) were cut from 6-week-old,

axenically grown tobacco plants and were agroinoculated

with the A. tumefaciens strain Ach5 harbouring the partial

dimers of both DNA A and DNA B of MYMV-[IN:Vig]

[38, 39]. After 2-day co-cultivation, the leaf discs were

transferred onto tobacco shoot-induction medium supple-

mented with 250 mg/l cefotaxime. Ten days-post-inocula-

tion (dpi) total DNA was extracted from the agroinoculated

leaf discs and subjected to Southern blot analysis.

Results

Evaluation of MYMV AC4 as a silencing suppressor

Agroinfiltration of N. benthamiana 16c [27] was performed

to evaluate whether MYMV AC4 acts as a silencing sup-

pressor. RNA silencing of the gfp transgene can be trig-

gered by transient expression of the gfp gene (trigger) and

consequently, the agroinfiltrated leaf will exhibit red fluo-

rescence. In contrast, co-agroinfiltration of a silencing

suppressor gene with the gfp gene (trigger ? suppressor)

will yield green fluorescence in the infiltrated zone.

Six-week-old N. benthamiana 16c plants were agroin-

filtrated with the A. tumefaciens strain containing the P35S-

gfp binary vector (pBI-mgfp5-ER). Five days-post-infil-

tration, local gfp silencing of the infiltrated area and the rest

of the leaf was observed as red fluorescence (Fig. S1Ab).

To evaluate the effect of MYMV AC4 on silencing trig-

gered by gfp expression, the 16c plants were co-agroinfil-

trated with a 1:1 (v/v) mixture of the A. tumefaciens strains

expressing the P35S-gfp and P35S-AC4 genes. To ensure

equivalent expression levels, identical 35S promoters were

used in the two binary vectors. The co-agroinfiltrated area

displayed green fluorescence under UV light (Fig. S1Ac).

MYMV-TrAP has been reported to function as a PTGS

suppressor [40] and hence was used as a positive control

for this experiment; plants co-agroinfiltrated with P35S-

gfp ? P35S-TrAP displayed green fluorescence in the

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infiltrated zone (Fig. S1Ad). Mock-infiltrated 16c plants,

infiltrated with just the medium, exhibited green fluores-

cence (Fig. S1Aa). These findings suggest that both TrAP

and AC4 of MYMV function as PTGS suppressors.

Northern blot analysis of agroinfiltrated leaf tissue was

performed using a probe complementary to the gfp gene.

The agroinfiltrated area was harvested under UV light and

total RNA was extracted. Samples from mock (M)-infil-

trated sections unexpectedly accumulated very low levels

of gfp mRNA (Fig. 1, gfp transcript was observed only

upon longer exposure, which is not shown). Agroinfiltra-

tion of P35S-gfp was expected to trigger silencing and

degrade the gfp transcript, and although red fluorescence

was observed (Fig. S1Ab) a considerable amount of gfp

transcript was still present. This unexpected observation

suggests that the silencing trigger did not completely

degrade the gfp transcript made from the agroinfiltrated

P35S-gfp. Leaves co-agroinfiltrated with the trigger

(gfp) ? AC4 accumulated much higher levels of gfp

mRNA, similar to that observed in gfp ? TrAP co-agro-

infiltrated plants (Fig. 1). To determine the increase of gfp

transcript level in the leaves agroinfiltrated with the TrAP

construct or AC4 construct, densitometry analysis of the

autoradiogram and the ethidium bromide-stained gel

(Fig. 1) was done using the AlphaEaseTM software (Ver-

sion 5.5, Alpha Innotech Corporation, San Leandro, USA).

The data showed that 2.1- and 1.8-fold increase in gfp

transcript occurred upon co-agroinfiltration with AC4 and

TrAP constructs, respectively, in comparison to agroinfil-

tration with gfp alone.

Analysis of transgenic tobacco plants transformed

with the MYMV AC4-sense gene

The binary plasmid pCAM-AC4-gus, which harbours the

MYMV AC4-sense gene, the neomycin phosphotransfera-

seII (nptII) gene and the gus gene under the transcriptional

control of CaMV 35S promoters (Fig. 2a) was used to

transform tobacco. Six transgenic plants were established

on selection medium containing 100 mg/l kanamycin and

the T-DNA integration was studied by Southern blotting.

Genomic DNA from all six (AC4-1 to AC4-6) plants dis-

played hybridization of one to five junction hybridization

fragments when probed with the nptII coding sequence

(Fig. 2b). To check whether the integrated T-DNA contains

the complete AC4 gene, PCR analysis was done using the

AC4 gene-specific primers. A 0.4-kb fragment was

expected to amplify in plants harbouring the complete AC4

gene. Amplification of 0.4-kb fragment was observed in

plants AC4-1, AC4-2, AC4-3, AC4-5 and AC4-6 (Fig. 2c).

Plant AC4-4 did not harbour the transgene. Thus, the plants

AC4-1, AC4-2, AC4-3, AC4-5 and AC4-6 were inferred to

contain the complete AC4 gene.

Expression of the AC4 gene in the AC4-sense

transgenic plants

Northern blot analysis with the AC4 probe was carried out in

the transgenic plants AC4-1, AC4-2, AC4-5 and AC4-6 which

harbour the complete AC4 gene. The AC4 transcript accu-

mulated in plants AC4-1 and AC4-5 (Fig. 3a), whereas plants

AC4-2 and AC4-6 failed to accumulate the AC4 transcript.

The effect of the AC4-sense gene on the accumulation

of MYMV DNA

Leaf discs from a non-transformed control plant and four

transgenic plants which harbour integrated copies of the

AC4 gene were agroinoculated with the wild-type A. tum-

efaciens strain Ach5 containing the partial dimers of both

DNA A and DNA B of MYMV [38]. Viral DNA levels in

the leaf discs were determined by Southern blot analysis

using the MYMV DNA A probe at 10 days-post-infection

(dpi). In the agroinoculated leaf discs from the control

plants, a 1.8-kb band that corresponds to both single-

stranded DNA (ss) and supercoiled double-stranded DNA

(sc) [36] was intense and a 2.8-kb band that corresponds to

both open circular double-stranded DNA (oc) and linear

double-stranded DNA (lin) was weak (Fig. 3b). Densi-

tometry analysis of the autoradiogram (Fig. 3b) was done

Fig. 1 Northern blot analysis of agroinfiltrated Nicotiana benthami-ana 16c plants. Total RNA from the infiltrated areas of mock-

infiltrated leaves (M), P35S-gfp infiltrated leaves (gfp), P35S-

gfp ? P35S-AC4 infiltrated leaves (AC4 ? gfp), and P35S-

gfp ? P35S-TrAP infiltrated leaves (TrAP ? gfp) was probed with

the gfp coding sequence. The 18S rRNA portion of the ethidium

bromide-stained gel is placed at the bottom. A weak band of the gfptranscript was observed in the mock-infiltrated leaves (lane M) upon

longer exposure (not shown in the figure)

Virus Genes (2013) 46:496–504 499

123

using the AlphaEaseTM software (Version 5.5, Alpha In-

notech Corporation, San Leandro, USA). IDV at the ss/sc

position in lane C (control) was 7,452. In the AC4-sense

transgenic plants AC4-2 and AC4-6, which do not accu-

mulate the AC4 transcript, MYMV DNA levels were

comparable to that in the control plants (the IDV of plants

AC4-2 and AC4-6 were 9,744 and 8,786, respectively). In

the two AC4-sense transgenic plants AC4-1 and AC4-5,

which express the AC4 transcript, there was an increase in

MYMV DNA levels in comparison to that in the control

plants (the IDV values for AC4-1 and AC4-5 plants were

12,432 and 14,518, respectively). It is clear from these

results that expression of the AC4-sense gene in transgenic

plants promotes the accumulation of MYMV DNA.

Generation of AC4 hpRNA transgenic tobacco plants

An AC4 hpRNA cassette comprising the CaMV 35S pro-

moter, MYMV AC4 inverted repeat and ocs polyadenyla-

tion signal was cloned as a NotI fragment in the

pMLBART binary vector to make pML-hpAC4 [31]. This

final construct contains the bialaphos resistance gene (bar)

under the transcriptional control of the nopaline synthase

(nos) promoter as the plant selectable marker (Fig. 4).

Southern blot analysis of eight transgenic plants has been

previously reported [31]. Six transgenic plants (hpAC4-1 to

hpAC4-6), which display two to six junction fragments and

harbour the complete hpAC4 cassette [31], were used in

this study. siRNA levels were determined by northern blot

analysis of a non-transformed tobacco plant and six hpAC4

transgenic plants using a probe specific for AC4. Different

transgenic plants exhibited variation in the level of AC4

siRNAs; plants hpAC4-1 and hpAC4-4 accumulated mod-

erate levels, plants hpAC4-2, hpAC4-3 and hpAC4-5

accumulated high levels (Fig. 5a) and hpAC4-6 accumu-

lated a low level of the AC4 siRNAs.

The effect of the AC4 hpRNA gene on MYMV DNA

accumulation

Leaf discs of a non-transformed tobacco plant and six

hpAC4 transgenic plants were agroinoculated with the

Fig. 2 Analysis of tobacco plants transformed with the binary vector

pCAM-AC4-gus. a The T-DNA of pCAM-AC4-gus. RB T-DNA

border-right; P35S Cauliflower mosaic virus (CaMV) 35S promoter;

nptII neomycin phosphotransferaseII gene; 35S 30 CaMV 35S

polyadenylation signal; nos 30 nopaline synthase polyadenylation

signal; LB T-DNA border-left. The nptII probe used for hybridization

has been marked in a bold line. The junction fragment size ([2.5-kb)

has been marked with a dashed arrow. b The nptII probe-based

Southern blot analysis of tobacco plants transformed with the binary

vector pCAM-AC4-gus. Total DNA (10 lg) from six transformants

AC4-1 to AC4-6 (1–6) was digested with HindIII and the blot was

probed with the nptII coding sequence. Total DNA from the non-

transgenic tobacco plant digested with HindIII (C) was used as the

negative control. c PCR analysis of tobacco plants transformed with

pCAM-AC4-gus with AC4-specific primers. DNA (100 ng) from the

untransformed tobacco plant (C) and from the six transgenic plants

(AC4-1 and AC4-6) was used as the PCR template. The binary

plasmid pCAM-AC4-gus (50 pg) was used as the positive control.

Amplification of a 0.4-kb fragment was expected in transgenic

plants and the binary plasmid control (P). W water control;

M 1-kb ? marker

500 Virus Genes (2013) 46:496–504

123

A. tumefaciens strain Ach5 harbouring the partial dimers of

both DNA A and DNA B of MYMV. Total DNA was

extracted from the leaf discs and Southern blot analysis

was performed with the MYMV DNA A probe. Agroin-

oculated leaf discs from the control tobacco plant accu-

mulated viral DNA (1.8-kb band) that corresponds to both

ssDNA and scDNA (Fig. 5b). Leaf discs of hpAC4-plants,

which accumulate a high level of the AC4 siRNAs (plants

hpAC4-2, hpAC4 -3 and hpAC4-5) and moderate level of

siRNAs (plants hpAC4-1 and hpAC4-4), completely

blocked MYMV DNA accumulation. The plant hpAC4-6,

which displays a much lower level of the AC4 siRNAs, had

a reduced level of MYMV DNA accumulation (Fig. 5b).

The leaf disc agroinoculation experiment was repeated

thrice and similar results were obtained.

Discussion

AC4/C4 ORFs of geminiviruses are completely embedded

within AC1/C1, just in a different ORF. AC4 of bipartite

geminiviruses [10] and C4 of monopartite geminiviruses

[11–13, 15] are pathogenicity determinants. The pathoge-

nicity property of geminiviral proteins is associated with

their ability to suppress host-mediated RNA silencing [30,

41–43].

MYMV-TrAP was shown to suppress silencing by

transactivating the host suppressor genes [40]. The ability

of ACMV-[CM] and EACMCV to encode more than one

potential silencing suppressor with varying efficiencies

raised the question whether MYMV AC4 also displayed

silencing suppressor activity. AC4 of MYMV suppressed

gfp silencing in transient silencing assays in N. benthami-

ana 16c (Fig. S1Ac). Analysis of the gfp transcript accu-

mulation revealed that the efficiency of silencing

suppression by AC4 is comparable to that of TrAP of

MYMV (Fig. 1). This is in contrast to results with that of

ACMV-[CM] and SLCMV, in which AC4 acts as a strong

suppressor and TrAP acts as a weak suppressor [22].

Interestingly, in EACMCV and ICMV, AC4 acted as a

weak suppressor and TrAP acted as a strong suppressor

[22]. The observation that MYMV encodes two suppres-

sors (TrAP and AC4) with similar efficiencies is intriguing

and more detailed analysis is required to understand the

significance of such a strategy evolved by MYMV.

The silencing suppression of AC4 of ACMV-[CM] was

mediated by its specific binding to miRNAs and siRNAs

and thus leading to inhibition of target mRNA cleavage

[23]. While 34 of the total 99 amino acids of MYMV AC4

were predicted by the BindN database [44] to possess an

RNA binding property, 38 of the 140 amino acids of

ACMV-[CM] AC4 have a potential RNA binding capa-

bility (data not shown). It speculates that the silencing

suppression of MYMV AC4 is similar to that of ACMV-

[CM] AC4, although experimental evidence is needed to

confirm the RNA binding property of MYMV AC4.

Although the generation of transgenic callus [45] and

plants [46] of blackgram with marker and reporter genes

has been reported, Agrobacterium-mediated blackgram

transformation has not yet become a routine method.

Alternatively, a viral replication assay using tobacco leaf

discs has proven to be a useful method to evaluate how

Fig. 3 Expression analysis of the AC4-sense transgenic tobacco

plants and leaf disc agroinoculation assay to study the effect of AC4on MYMV DNA accumulation. a Northern blot analysis of pCAM-

AC4-gus-transformed plants. Total RNA from the non-transgenic

plant (C) and four transgenic plants AC4-1, -2, -5 and -6 (1, 2, 5 and

6) was probed with the AC4 coding sequence. The 18S rRNA portion

of the ethidium bromide-stained gel is placed at the bottom to show

equal loading of RNA in all lanes. Sizes of the RNA standard

(Ambion, Austin, U.S.A.) are marked on the left. b Southern blot

analysis of DNA (5 lg) from leaf discs of AC4-sense transgenic

tobacco plants agroinoculated with the partial dimers of MYMV DNA

A and DNA B. The blot was hybridized to the full-length MYMV

DNA A probe. NI: DNA from non-agroinoculated leaf discs of a non-

transformed control plant; C: DNA from leaf discs of a non-

transgenic plant agroinoculated with MYMV. Leaf discs from four

AC4-sense transgenic plants AC4-1, -2, -5 and -6 (1, 2, 5 and 6) were

agroinoculated. The positions of open circular (oc), linear (lin),

single-stranded (ss) and super-coiled (sc) forms [36] of MYMV and

Agrobacterium binary vector (Bi) with the partial dimers of MYMV

are marked

Virus Genes (2013) 46:496–504 501

123

effective transgenically expressed MYMV genes are at

inhibiting viral DNA accumulation [39, 47]. Pathogen-

derived resistance (PDR), trans-dominant inhibition by

mutant viral genes and RNA silencing-based resistance

using viral hpRNA genes are the major approaches

deployed to generate geminivirus resistance [48–50].

The effect of transgenic expression of MYMV AC4-

sense and AC4 hpRNA genes on viral DNA replication was

evaluated using the tobacco leaf disc agroinoculation assay.

Northern blot analysis indicated that two plants (AC4-1 and

AC4-5) express the AC4 transcript (Fig. 3a), whereas plants

AC4-2 and AC4-6 do not. Although the coding sequence of

AC4 is only 0.4-kb long, the AC4 transcript was approxi-

mately 0.7 kb (Fig. 3a and Fig. S1Bb). RT-PCR analysis

with AC4-specific primers confirmed that the AC4 tran-

script accumulates in plants AC4-1 and AC4-5, but not in

plants AC4-2 and AC4-6 (Fig. S1Ba). Read through tran-

scription of CaMV 35S terminator, when used with a

transgene driven by a strong CaMV 35S promoter, has

been reported earlier [51, 52]. Northern blotting analysis

with the 35S terminator as probe displayed hybridization to

the 0.7-kb transcript (which hybridized to AC4) in plants

AC4-1 and AC4-5 (Fig. S1Bc). This observation substan-

tiated the hypothesis that read through transcription of the

35S terminator resulted in an increase in the transcript

length. Upon agroinoculation of leaf discs of four AC4-

sense transgenic plants with the partial dimers of MYMV

DNA A and DNA B, no reduction of MYMV DNA was

seen in any of the four plants. On the contrary, an increase

in the viral DNA level was observed in the leaf discs of

transgenic plants which accumulate the AC4 transcript

(Fig. 3b, AC4-1 and AC4-5). The leaf discs of transgenic

plants which do not express the AC4 transcript (AC4-2 and

AC4-6) and those of the non-transgenic control plant

accumulated comparable levels of MYMV DNA. The

observed promotion of MYMV DNA accumulation in the

presence of AC4 agrees well with the observations made in

other geminiviruses; for example, the transient expression

of ACMV-AC4 in BY-2 protoplasts increased EACMCV

accumulation by eight fold [22]. In addition, the replication

efficiency of a viral amplicon encompassing the common

region (CR)-AC3 region of ToLCV was reduced to 50 %

when a premature translation termination mutation was

introduced in the AC4 ORF [53]. Even so, because

Fig. 4 The T-DNA of pML-hpAC4 [31]. The AC4 gene was cloned

in sense and antisense orientations with an intervening intron of the

pyruvate orthophosphate dikinase gene [64] in the binary vector

pMLBART [65]. RB T-DNA border-right; P35S Cauliflower mosaicvirus (CaMV) 35S promoter; Pnos nopaline synthase gene promoter;

ocs 30 octopine synthase polyadenylation signal; LB T-DNA border-

left. Probes used for Southern blotting have been marked with boldlines. The junction fragment ([2.9-kb) and internal T-DNA fragment

(0.5-kb, 3.6-kb) have been marked with dashed arrow and lines,

respectively

Fig. 5 Analysis of tobacco plants transformed with the binary vector

pML-hpAC4. a siRNA analysis of pML-hpAC4-transformed plants.

siRNAs from the non-transgenic control plant (C) and six transgenic

plants hpAC4-1 to -6 (1–6) were probed with the AC4 coding

sequence. The tRNA portion of the ethidium bromide-stained gel

(tRNA) is placed at the bottom to show equal loading of RNA in all

lanes. b Southern blot analysis of MYMV-agroinoculated leaf discs of

non-transformed and hpAC4 transgenic tobacco plants. A blot with

the total DNA (5 lg) from non-agroinoculated leaf discs (NI) of the

control plant, agroinoculated leaf discs of the control plant (C) and six

hpAC4 transgenic plants (1–6) was hybridized to the full-length

MYMV DNA A probe. The positions of linear (lin), open circular

(oc), single-stranded (ss) and super-coiled (sc) forms [36] of MYMV

DNA and Agrobacterium binary vector (Bi) with the partial dimers of

MYMV are marked

502 Virus Genes (2013) 46:496–504

123

transgenic plants expressing the MYMV AC4-sense gene

do not reduce MYMV DNA accumulation, this molecular

strategy does not hold promise for pathogen-derived

resistance. Silencing suppressors frequently induce abnor-

mal phenotypes when expressed in transgenic plants [54,

55]. Upon transformation of MYMV TrAP in N. tabacum,

only truncated T-DNA integrations were recovered, pos-

sibly due to toxicity manifested by TrAP [30]. Four of the

six MYMV AC4-sense transgenic plants, which are phe-

notypically normal and contain the complete AC4 gene

(Fig. 2c), indicate that AC4 is not toxic to the plant and

does not manifest typical abnormalities associated with

silencing suppressor genes.

RNA silencing-mediated resistance against many gem-

iniviruses has been achieved by targeting the Rep gene

[56–59], the bidirectional promoter [60, 61] and the CP

gene [62]. ToLCV resistance in tomato was engineered by

transforming tomato with the AC1 hpRNA construct which

included the AC4 gene [58, 63]. In our study, we evaluated

whether the MYMV AC4 hpRNA gene expression in

transgenic tobacco blocked MYMV DNA accumulation. Six

hpAC4 transgenic tobacco plants (hpAC4-1 to hpAC4-6)

from our previous report [31] produce varying levels of the

AC4 siRNAs (Fig. 5a). Agroinoculated leaf discs of plants

which express high levels of the AC4 siRNAs (hpAC4-2,

hpAC4-3 and hpAC4-5) and plants with moderate levels of

the AC4 siRNAs (hpAC4-1 and hpAC4-4) inhibit the accu-

mulation of MYMV DNA by almost 100 %. The plant

hpAC4-6, which has a low level of the AC4 siRNAs, only

partially inhibited MYMV DNA accumulation (Fig. 5b).

Therefore, the extent of inhibition of MYMV DNA is

directly proportional to the level of siRNAs. Similar dose-

dependent resistance has been observed when Rep hpRNAs

of TYLCV [57] and ACMV [59] were expressed in trans-

genic tomato and cassava, respectively.

It should be noted that the reduction in MYMV DNA

accumulation in the hpAC4 transgenic plants cannot be

solely attributed to the silencing of the AC4 gene. AC4 is

translated from a polycistronic mRNA comprising Rep,

TrAP, AC3 and AC4. Therefore, the AC4 siRNAs might

target and degrade the Rep transcript as well. If this is the

case, there is a potential that siRNAs generated from the

hpAC4 construct may in turn generate secondary siRNAs

against TrAP and AC3 through transitivity. The presence of

the AC4 ORF within Rep makes it difficult to separate the

silencing of the Rep gene from the silencing of the AC4

gene. The resistance observed in transgenic tomato

expressing a hpRNA of TYLCV-Rep was also attributed to

the overlapping nature of the hpRNA gene with the

silencing suppressor TrAP [57]. However, siRNAs corre-

sponding to the region outside the target of hpRep were not

observed when this transgene was used to confer BGMV

resistance in common bean [56].

Transgenic expression of MYMV-T-Rep [39], MYMV–

TrAP–DAD and Agrobacterium VirE2 [47] in tobacco and

particle bombardment of blackgram with the MYMV

common region (CR) hpRNA gene [60] were reported

earlier to cause a pronounced reduction in MYMV DNA

accumulation. The work presented here indicates that the

AC4 hpRNA has the ability to inhibit MYMV DNA

accumulation which makes AC4 an attractive candidate

gene for engineering MYMV resistance.

Acknowledgments We thank Prof. Thomas Hohn, University of

Basel for providing Nicotiana benthamiana 16c seeds. We thank

Dr. Donna Bond, University of Cambridge for her comments on the

manuscript. We are thankful to Dr. K. Dharmalingam, Madurai

Kamaraj University for permitting us to use the Liquid Scintillation

Counter. Ms. Jasvinder Kaur and Ms. K. Bhagyalakshmi are

acknowledged for the construction of pCAM-AC4-gus. This study

was financially supported by the Department of Biotechnology

(Project Number-BT/PR9823/AGR/36/10/2007 dt 7/12/2007), Gov-

ernment of India and University Grants Commission, Government of

India. S.S. acknowledges the Council of Scientific and Industrial

Research, Government of India for the research fellowship.

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