BRIEF REPORT

bC1 is a pathogenicity determinant: not only for begomovirusesbut also for a mastrevirus

Jitendra Kumar • Jitesh Kumar • Sudhir P. Singh •

Rakesh Tuli

Received: 13 March 2014 / Accepted: 8 June 2014

� Springer-Verlag Wien 2014

Abstract bC1 proteins, encoded by betasatellites, are

known to be pathogenicity determinants, and they are

responsible for symptom expression in many devastating

diseases caused by begomoviruses. We report the

involvement of bC1 in pathogenicity determination of a

mastrevirus. Analysis of field samples of wheat plants

containing wheat dwarf India virus (WDIV) revealed the

presence of a full-length and several defective betasatellite

molecules. The detected betasatellite was identified as

ageratum yellow leaf curl betasatellite (AYLCB). No be-

gomovirus was detected in any of the samples. The full-

length AYLCB contained an intact bC1 gene, whereas the

defective molecules contained complete or partial deletions

of bC1. Agroinoculation of wheat with the full-length

AYLCB and WDIV or of tobacco with ageratum enation

virus enhanced the pathogenicity and accumulation of the

respective viruses, whereas the defective molecules could

not. This study indicates that bC1 is a pathogenicity

determinant for WDIV and can interact functionally not

only with begomoviruses but also with a mastrevirus.

Keywords AYLCB � WDIV � AEV � Trans-replication �Virus accumulation

Betasatellites are single-stranded DNA molecules associ-

ated with begomoviruses (family Geminiviridae) [1–3].

They are approximately half the size (*1.3 kb) of their

helper viruses and encode a single protein, bC1, which is a

pathogenicity determinant [4–6]. bC1 suppresses silencing,

up-regulates viral DNA levels in plants, helps in virus

movement, and modulates virus symptoms and host range

[4, 6–13]. Betasatellites share only the nonanucleotide

(TAATATTAC) sequence with the helper begomovirus,

which is predicted to serve as the origin of replication [1–

3]. Betasatellites depend on their helper viruses for repli-

cation, encapsidation and transmission by the insect vector

and are often required by their helper viruses for symptom

induction in their original hosts [14, 15]. Some monopartite

begomoviruses are either poorly infectious or induce

atypical symptoms in the absence of the betasatellite [6–8,

12, 14].

The association of betasatellites with monopartite be-

gomoviruses has been reported in numerous diseases of

plants [1–3], and in select cases, with bipartite begomovi-

ruses [16–18]. In a recent study, the association of a

betasatellite, ageratum yellow leaf curl betasatellite (AY-

LCB), with a mastrevirus, wheat dwarf India virus

(WDIV), which infects wheat, was detected [19]. AYLCB

enhanced WDIV accumulation and symptom (dwarfing)

severity and remained capable of interacting with ageratum

enation virus (AEV) [19]. The present study shows that

AYLCB with a full-length bC1 gene enhanced virus

(WDIV or AEV) accumulation and symptom severity,

whereas defective molecules with bC1 deleted could not.

Leaf samples from 50 symptomatic wheat plants were

collected during 2011–12 from fields at Mohali, India. The

symptomatic plants were selected on the basis of dwarfing

and yellowing (Fig. 1A-C). Leaves from five asymptomatic

plants were taken as negative controls. Total DNA was

isolated using a DNeasy Plant Mini Kit column (QIAGEN

GmbH, Germany). WDIV and satellite DNAs were

amplified by rolling-circle amplification (RCA) and

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00705-014-2149-5) contains supplementarymaterial, which is available to authorized users.

J. Kumar � J. Kumar � S. P. Singh � R. Tuli (&)

Department of Biotechnology, National Agri-Food

Biotechnology Institute, Mohali 160071, Punjab, India

e-mail: rakeshtuli@hotmail.com

123

Arch Virol

DOI 10.1007/s00705-014-2149-5

polymerase chain reaction (PCR). RCA was performed

using a TempliPhi Amplification Kit (GE Healthcare,

USA), and the RCA products were partially digested with

NdeI restriction endonuclease to obtain DNA fragments of

WDIV and the satellite. The RCA products were also

digested separately with the BamHI, EcoRI, HindIII, KpnI

and PstI restriction endonucleases to detect the presence of

a begomovirus. The results showed identical products for

all samples and thus suggested the presence of identical

viruses in all diseased plant samples (data not shown).

MF1_FOR/REV was used in PCR assays to amplify WDIV

DNA from wheat samples, whereas b01/04 was used to

amplify betasatellite DNA (Supplementary Table 1) as

described earlier [19]. The RCA and PCR assays were

performed on all 55 wheat samples (50 symptomatic and 5

asymptomatic). Both RCA and PCR yielded fragments of

*2.7 and 1.3 kb, the sizes expected for WDIV and beta-

satellite, respectively, whereas the asymptomatic plants did

not (Fig. 1D-F). In addition to a fragment of *1.3 kb,

smaller amplicons of *657 and *900 bp were obtained

using the betasatellite-specific primers (Fig. 1F).

Sequencing of the RCA and PCR products revealed the

presence of WDIV (accession no. JF781306) and AYLCB

(accession no. KC305085) in the symptomatic wheat

samples. Additional fragments of smaller sizes were

detected using the betasatellite-specific primers. These

represented defective molecules of AYLCB with smaller

size (accession nos. KC305086, KC305087, and

KC305089-KC305091). Detailed analysis of the sequence

revealed that all of the betasatellite molecules, including

the full-length (*1.3 kb) and smaller (less than *1.3 kb

in length) molecules, retained three highly conserved fea-

tures: a predicted stem-loop structure with the loop

sequence TAATATTAC as the origin of replication (the

nonanucleotide sequence), a region of high sequence sim-

ilarity known as the ‘‘satellite conserved region’’ (SCR),

and an adenine (A)-rich region. The full-length betasatel-

lite molecule (1365 bp) contained the positionally and

Fig. 1 Wheat plants suspected of being infected with WDIV and

detection of the virus and satellite DNAs. Wheat plants (red circle)

showing dwarfing with yellowing (A, B) and reddening with

yellowing (C). NdeI-digested RCA products showing the fragments

of *1.3, *1.6, *2.8 and *5.6 kb in the diseased wheat samples

(D). PCR amplicon of *2.8 kb representing the genome size of

WDIV (E). PCR products of the betasatellite showing multiple

amplicons of *700, *900 bp and *1.3 kb (F). Lanes 1, 2 and 3 in

panels D-F represent the symptomatic samples of panels A, B and C,

whereas lane 4 represents an asymptomatic plant sample. M, EcoRI/

HindIII-digested kDNA. The dimeric (WD) and monomeric (WM)

forms of WDIV and the partial digestion products of WDIV (WP),

betasatellite (beta) and defective betasatellite (M beta) are shown

J. Kumar et al.

123

sequence-conserved full-length bC1 gene, while the

defective molecules contained partial deletions of bC1,

except the 657-bp molecule, in which all of bC1 was

absent (Fig. 2).

Infectious clones of WDIV, AEV and AYLCB (full-

length as well as defective AYLCB) were prepared in the

binary vector pCAMBIA1301 (CAMBIA, Canberra, Aus-

tralia) as described previously [19]. The full-length and

defective AYLCBs of 1365, 1084, 1046, 929, 891 and

657 bp are represented by AYLCB_1365, AYLCB_1084,

AYLCB_1046, AYLCB_929, AYLCB_891 and AY-

LCB_657, respectively. Derivatives of the binary vector

pCAMBIA1301, containing the infectious constructs were

introduced separately into Agrobacterium tumefaciens

strain GV3101 by transformation. Leaves of seven-day-old

seedlings of 200 wheat plants (25 plants for each of the

eight constructs; control, WDIV, WDIV and AY-

LCB_1365, WDIV and AYLCB_1084, WDIV and AY-

LCB_1046, WDIV and AYLCB_929, WDIV and

AYLCB_891, WDIV and AYLCB_657) at the two- to

three-leaf stage were agroinoculated using a needleless

syringe. A total of 48 Nicotiana tabacum plants (six plants

for each of the eight constructs: control, AEV, AEV and

AYLCB_1365, AEV and AYLCB_1084, AEV and AY-

LCB_1046, AEV and AYLCB_929, AEV and AY-

LCB_891, AEV and AYLCB_657) at the four-leaf stage

were inoculated. The inoculated wheat and tobacco plants

were maintained in separate plant growth chambers

(PGR14, Conviron, Canada) at 22 and 25 �C, respectively.

The presence of WDIV or AEV in the agroinoculated

wheat or tobacco plants was investigated using the primer

pairs CP01/02 or CPB1/2 (Supplementary Table 1),

respectively, as described previously [19]. The presence of

the full-length or defective AYLCB in the agroinoculated,

infected plants was investigated using the BSCRF/R primer

pair (Supplementary Table 1), which amplifies the

sequence (Supplementary Fig. 1) conserved in all of the

betasatellites without discriminating between the full-

length and defective molecules. One hundred fifty wheat

plants out of the 200 infectious-clone-inoculated plants

showed the presence of WDIV (Fig. 3K) and/or AYLCB

(Fig. 3N). A total of 38 N. tabacum plants out of the 48

infectious-clone-inoculated plants, showed the presence of

AEV (Fig. 3L) and/or AYLCB (Fig. 3O). The PCR-

amplified products from the inoculated plant samples were

sequenced to confirm their identity to the sequence of the

initially inoculated clones.

Wheat plants harboring WDIV showed dwarfing in

comparison to the mock-inoculated control (Fig. 3A, B),

whereas the plants containing WDIV and full-length AY-

LCB (1365 bp; Fig. 2) showed more-severe stunting

(Fig. 3E). Wheat plants with WDIV and defective mole-

cules of AYLCB showed a phenotype similar to that of the

plants inoculated with WDIV in the absence of AYLCB

(Fig. 3B, C, D). The average height of five wheat plants at

42 days postinfection (dpi), in the case of the control and

those infected with WDIV and full-length AYLCB, WDIV

and defective AYLCB and WDIV, alone was 70.3 ± 2.51,

35.3 ± 1.52, 53.3 ± 3.05 and 54.3 ± 1.52 cm, respec-

tively. Tobacco plants containing AEV showed leaf curling

and stunting in comparison to mock-inoculated control

(Fig. 3F, G). The viral symptoms, leaf curling and stunting,

were more severe in the presence of AEV and full-length

AYLCB (Fig. 3J). Plants with AEV and defective AYLCB

Fig. 2 Representation of the genomic organization of the full-length

and the defective molecules of AYLCB. The sequences deleted in

defective AYLCB molecules are indicated by dashed lines within the

solid lines. Size variants of AYLCBs are indicated, with their length

given in bp: 657, 891, 929, 1046, 1084 and 1365 bp. The positions of

the bC1 gene, the A-rich region and the satellite conserved region

(SCR), are also shown. The region suggested to be responsible for

trans-replication of a betasatellite [20] is also shown

Role of betasatellite bC1 in mastrevirus pathogenicity

123

molecules showed viral symptoms, leaf curling and stun-

ting, similar to the plants inoculated with AEV in the

absence of AYLCB (3G-I). The average height of three

tobacco plants at 35 dpi, in the case of the control and

those infected with AEV and full-length AYLCB, AEV

and defective AYLCB and AEV alone, was 50.41 ± 3.51,

12.4 ± 3.02, 28.15 ± 4.05 and 30.2 ± 4.44 cm,

respectively.

The coat protein genes of WDIV and AEV were

amplified by real-time qPCR and semi-quantitative PCR

for quantification of virus accumulation in the presence of

full-length and defective AYLCB molecules. The concen-

tration of total DNA from the inoculated, infected plants

was adjusted to 50 ng ll-1, and the primer pairs CP01/02

and CPB1/2 (Supplementary Table 1) were used for WDIV

and AEV, respectively. Elongation factor-1 alpha (EF-

1afor/rev; Supplementary Table 1) and actin (TbAct.F/R;

Supplementary Table 1) genes were amplified as internal

controls for wheat and tobacco, respectively. Three bio-

logical replicates were amplified separately in real-time

Fig. 3 Effects of the AYLCB on the symptom severity and

accumulation of WDIV and AEV in wheat and tobacco, respectively.

Mock-inoculated wheat plants and those inoculated with (A), WDIV

(B), WDIV and 1084-bp AYLCB (C), WDIV and 657-bp AYLCB

(D) and WDIV and 1365-bp AYLCB (E) at 35 dpi are shown. Mock-

inoculated tobacco plants (A), and those inocuated with AEV (B),

AEV and 1084-bp AYLCB (C), AEV and 657-bp AYLCB (D), and

AEV and 1365-bp AYLCB (E) at 30 dpi are shown. Semi-quanti-

tative PCR (K, L) and real-time qPCR (M, N) – based on three

replicates – were used for analysis of WDIV (K, M) or AEV (L,

N) from agroinoculated, infected wheat and tobacco plants,

respectively. The CP genes of WDIV (WCP) and AEV (ACP) were

amplified by semi-quantitative PCR (K, L) and real-time qPCR (M,

N). The samples used for semi-quantitative PCR (K, L) and real-time

PCR (M, N) were as follows: virus alone (WDIV or AEV; lane 1),

virus and 657-bp AYLCB (lane 2), virus and 891-bp AYLCB (lane 3),

virus and 929-bp AYLCB (lane 4), virus and 1046-bp AYLCB (lane

5), virus and 1084-bp AYLCB (lane 6), virus and 1365-bp AYLCB

(lane 7). The results of semi-quantitative PCR-based quantification of

AYLCB in wheat (O) and tobacco (P) are shown. The same samples

used for panels (K) and (L) were used for the quantification of

AYLCB (O, P)

J. Kumar et al.

123

PCR assays for each satellite-virus combination, as

described earlier [19]. Semi-quantitative and real-time qRT

PCR revealed that the accumulation of WDIV or AEV was

highest in plants inoculated with WDIV and AYLCB_1365

or AEV and AYLCB_1365, (Fig. 3K-N). Accumulation of

WDIV or AEV was lower in the plants inoculated with a

defective AYLCB molecule together with WDIV or AEV,

similar to the plants inoculated with WDIV or AEV alone

(Fig. 3K-N).

Detection and sequencing of full-length (1365 bp) and

defective AYLCB molecules (657, 891, 929, 1046 and

1084 bp) from systemically infected wheat and tobacco

leaves showed that they were maintained by WDIV as well

as AEV in their respective hosts. The region predicted to be

responsible for the trans-replication of a betasatellite [20]

was present in all the betasatellites – full-length as well as

defective molecules. Conservation of the trans-replication

region among the AYLCB sequences (Fig. 2) and the

maintenance of bC1-less/truncated defective AYLCB

molecules by WDIV and AEV (Fig. 3O and P), indicates

that the region between the A-rich region and the SCR, as

predicted earlier [20], is responsible for trans-replication of

the betasatellite. This also suggests that the bC1 gene is not

required for trans-replication of betasatellite with the

helper viruses, AEV and WDIV. However, more-severe

viral disease symptoms (Fig. 3A-J) and the higher accu-

mulation of helper viruses (WDIV and AEV) in the pre-

sence of full-length AYLCB in comparison to the defective

AYLCB (Fig. 3K-N) show that bC1 is a pathogenicity

determinant and helps in virus accumulation.

The presence of defective AYLCB molecules in a field

population of wheat plants is presumably the result of

errors during DNA replication (polymerase ‘jumping’) or

some other mechanism [21]. The fact that the defective

AYLCB detected by us are maintained in the infection,

suggests that the deletions are not in the region essential for

replication. In fact, in some cases, deletions have been

predicted to give a replicative advantage to such defective

molecules [22]. The defective molecules have been sug-

gested to compete with full-length molecules for replica-

tion and, as a consequence, lead to delay or attenuation of

symptoms [22–24].

bC1 is known as a pathogenicity determinant for be-

gomoviruses in infection of permissive host plants [4–6].

However, this is the first study in which the involvement of

bC1 in the determination of pathogenicity of a mastrevirus

has been reported. The trans-replication ability and

involvement of a betasatellite, AYLCB, in symptom

severity of a mastrevirus can have serious implications for

the economic impact of the virus on crop yield.

Acknowledgements The authors are grateful to the Department of

Biotechnology, Government of India for supporting the present work

at National Agri-Food Biotechnology Institute, Mohali, India; to the

Council of Scientific and Industrial Research for Senior Research

Fellowship to JK and JK; and to the Department of Science and

Technology, Government of India, for the JC Bose Fellowship to RT.

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