A full-length infectious clone of beet soil-borne virus indicates the dispensability of the RNA-2 for virus survival in planta and symptom expression on Chenopodium quinoa leaves.

Abstract

For a better understanding of the functionality and pathogenicity of beet soil-borne virus (BSBV), full-length cDNA clones have been constructed for the three genomic RNAs. With the aim of assessing their effectiveness and relative contribution to the virus housekeeping functions, transcripts were inoculated on Chenopodium quinoa and Beta macrocarpa leaves using five genome combinations. Both RNAs-1 (putative replicase) and -3 (putative movement proteins) proved to be essential for virus replication in planta and symptom production on C. quinoa, whereas RNA-2 (putative coat protein, CP, and a read-through domain, RT) was not. No symptoms were recorded on B. macrocarpa, but viral RNAs were detected. In both host plants, the 19 kDa CP was detected by Western blotting as well as a 115 kDa protein corresponding to the CP-RT.

Full text

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Short
Communication
A full-length infectious clone of beet soil-borne
virus indicates the dispensability of the RNA-2 for
virus survival in planta and symptom expression on
Chenopodium quinoa leaves
Franc ¸ois Crutzen,1Mohsen Mehrvar,1,2David Gilmer3and Claude Bragard1
Correspondence
Franc ¸ois Crutzen
francois.crutzen@uclouvain.be
1Universite ´ catholique de Louvain, unite ´ de phytopathologie, Croix du Sud 2 bte 3, B-1348
Louvain-la-Neuve, Belgium
2Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad,
Iran
3Institut de Biologie Mole ´culaire des Plantes, Laboratoire propre du CNRS (UPR 2357)
conventionne ´ avec l’Universite ´ de Strasbourg, 12 rue du Ge ´ne ´ral Zimmer, 67084 Strasbourg,
France
Received 23 June 2009
Accepted 1 September 2009
For a better understanding of the functionality and pathogenicity of beet soil-borne virus (BSBV),
full-length cDNA clones have been constructed for the three genomic RNAs. With the aim of
assessing their effectiveness and relative contribution to the virus housekeeping functions,
transcripts were inoculated on Chenopodium quinoa and Beta macrocarpa leaves using five
genome combinations. Both RNAs-1 (putative replicase) and -3 (putative movement proteins)
proved to be essential for virus replication in planta and symptom production on C. quinoa,
whereas RNA-2 (putative coat protein, CP, and a read-through domain, RT) was not. No
symptoms were recorded on B. macrocarpa, but viral RNAs were detected. In both host plants,
the 19 kDa CP was detected by Western blotting as well as a 115 kDa protein corresponding to
the CP–RT.
Beet soil-borne virus (BSBV) is a pomovirus transmitted to
Chenopodiaceae by the protist Polymyxa betae (Ivanovic ´ et
al., 1983), which is also the vector of the aetiological agent
of the rhizomania syndrome of sugar beet, beet necrotic
yellow vein virus (BNYVV) (Tamada & Baba, 1973).
Originally reported in Italy (Canova, 1959), rhizomania
disease is now widespread in most countries where sugar
beet is grown (McGrann et al., 2009) and BSBV is often
found in beet infected with BNYVV (Meunier et al., 2003).
However, the pathogenicity of BSBV and its contribution
to the rhizomania syndrome remain unclear, with opinions
still divided on this (Prillwitz & Schlo ¨sser, 1992; Kaufmann
et al., 1993; Lindsten, 1993; Rush & Heidel, 1995).
The BSBV genome consists of three single-stranded RNAs
of positive polarity, packaged into rod-shaped particles
(Koenig et al., 1996, 1997; Koenig & Loss, 1997). RNA-1
(5.8 kb) encodes the putative viral replicase. The 19 kDa
coat protein (CP) and a putative 85 kDa read-through
(RT) domain are encoded by RNA-2 (3.5 kb). RNA-3
(3.0 kb) comprises three open reading frames (ORFs)
encoding three putative proteins (48, 13 and 22 kDa)
thought to be responsible for the viral cell-to-cell
movement, resembling the well-known triple gene block
proteins (TGBs) (Fig. 1).
In this study, the contribution of each RNA component to
virus survival and symptom expression was investigated
through the use of full-length cDNA clones on two host
plants, Chenopodium quinoa and Beta macrocarpa. This last
plant species was preferred to the natural host Beta vulgaris
with a view of developing the basis for further molecular
analysis of viral systemicity, following the example of
previous studies on BNYVV (Lauber et al., 1998).
Full-length cDNA sequences were generated from an
IranianBSBV isolate(Nyshabour,
Province), which was trapped from infested soil in roots
of B. vulgaris. After total RNA extraction from sugar beet
roots using the SV total RNA isolation kit (Promega), the
three genomic RNAs were reverse transcribed and
amplified by PCR (RT-PCR) using expand reverse
transcriptase and the expand long template PCR System
(Roche). Primers matching the extremities of the three
viral RNAs were designed following the only references
known to date (Koenig et al., 1996, 1997; Koenig & Loss,
1997). Forward primers were flanked with a T7 promoter
sequence (TAATACGACTCACTATAG) (Fig. 1). Ampli-
Khorasan Razavi
The GenBank/EMBL/DDBJ accession numbers for the sequences
reported in this paper are FN386612, FN386613 and FN386614.
Journal of General Virology (2009), 90, 3051–3056
DOI 10.1099/vir.0.014548-0
014548G2009 SGMPrinted in Great Britain3051

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cons were gel purified using QIAquick gel extraction kit
(Qiagen) and inserted into the pGEM-T vector (Promega).
Ligation products were transferred into the Escherichia coli
strain JM109. Full-length cDNA clones were then sub-
cloned into pUC19 and maintained in the E. coli strain
DH5-a, to obtain the final constructs pUBS-1-wt (RNA-1),
pUBS-2-wt (RNA-2) and pUBS-3-wt (RNA-3) (Fig. 1). A
full-length cDNA clone of RNA-2 containing a frame shift
mutation in the coding sequence for the putative CP–RT
domain (clone pUBS-2-rtBsiWI, Fig. 1) was obtained by
filling a BsiWI digestion (target site position 907–912) with
DNA polymerase I Klenow fragment (Promega) followed
by self-ligation with T4 DNA ligase (Roche).
The four full-length cDNA constructs in pUC19 were
linearized with SpeI or NcoI (Fig. 1) before run-off
transcription with the RiboMAX large-scale RNA produc-
tion system - SP6 and T7 (Promega) in the presence of Cap
analogue m7G(59)ppp(59)G (New England Biolabs). The
transcripts were mechanically inoculated in a 50 mM
KH2PO4(pH 4.2) and 0.04% bentonite buffer on both C.
quinoa and B. macrocarpa leaves. In vitro transcript
combinations of RNA-1 (1), -2 (2), -2 carrying a frame
shift mutation in the RT domain coding sequence (2*) and
-3 (3) were tested as follows: 1 + 2 + 3, 1 + 2* + 3, 1 + 2, 1 + 3 and
2 + 3. Inoculation buffer alone was applied for the negative
control. C. quinoa and B. macrocarpa were exposed to light
for 16 h per day at 15–20 uC and 20–25 uC for night and
day, respectively. The experiment was carried out on one C.
quinoa plant (three leaves inoculated) and two B. macro-
carpa plants (two leaves inoculated per plant) for each
condition and repeated twice to ensure reproducibility.
Symptomatic lesions appeared on all the C. quinoa leaves
inoculated with BSBV full-length transcripts of RNAs
1 +2 +3, 1 +2*+3 or 1+3 at 3 days post-inoculation (p.i.).
Small necrotic spots, necrotic ringspots (data not shown)
and chlorosis were clearly developed 7 days p.i. (Fig. 2).
However, the infection of the B. macrocarpa plants with the
same three combinations of BSBV full-length transcripts
did not produce any symptoms on the inoculated leaves
(data not shown). These observations indicated that the
presence of the BSBV RNA-2 was therefore not necessary
for symptom expression, which occurred only on C.
quinoa.
C. quinoa- and B. macrocarpa-inoculated leaves were
harvested at 7 and 10–17 days p.i., respectively. The
RNAs were extracted from the whole leaves using the
polysomes extraction protocol (Jupin et al., 1990) followed
by phenol extraction and ethanol precipitation of the
Fig. 1. Schematic representation of the BSBV tripartite genome: RNA-1 encodes the putative replicase with conserved motifs
methyl-transferase (MTR), helicase (Hel) and RNA-dependent RNA polymerase (Pol) translated as an RT domain; RNA-2
encodes the putative CP and an RT domain; RNA-3 encodes the putative TGBs-1, -2 and -3. The asterisks (*) represent the RT
stop codons. The molecular mass of the putative proteins is given beside each arrow beneath or above the corresponding ORF.
The full-length cDNA clones (pUBS-1-wt, -2-wt, -2-rtBsiWI, -3-wt; pUC19 backbone) are under the control of a T7 promoter
and possess a unique restriction site (X) for linearization and run-off transcription. pUBS-2-rtBsiWI clone has a frameshift at
the beginning of the CP–RT domain coding sequence as a result of the 4 nt insertion (underlined). The probes used for
Northern blot detection of the three viral RNAs are positioned above the corresponding full-length clone (NB-1, -2 and -3).
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RNAs. The viral RNAs were detected by Northern blot with
random DNA probes synthesized with the Prime-a-Gene
Labelling System (Promega) and labelled with [a-32P]dCTP
(1.1461014Bq mmol21; Perkin Elmer). The probes
matched nucleotide positions 3510–4015 (NB-1), 2281–
2711 (NB-2) and 1930–2330 (NB-3) of RNAs-1, -2 and -3,
respectively (Fig. 1).
Genomic combinations 1 +2* +3, 1 +2 +3 and 1+ 3 resulted in
corresponding viral RNA amplification on both host plants
(Fig. 3a, b, lanes 1, 2 and 4), whereas 1 +2 and 2 +3 did not
(Fig. 3a, b, lanes 3 and 5). The BSBV RNAs -1 and -3 full-
length transcripts were therefore required for the efficient
viral replication in planta, in contrast with BSBV RNA-2
that appeared dispensable for the viral RNA replication.
However, even if no viral RNAs were detected in plants
inoculated with the combinations 1 +2 and 2 +3 (Fig. 3a, b,
lanes 3 and 5), a potential undetected infection cannot be
ruled out for the mix 1 +2. Lacking the putative cell-to-cell
movement proteins encoded by RNA-3, the RNAs-1 and -2
might indeed replicate in the first inoculated cells but
couldn’t spread to the neighbouring ones (Hull, 2009).
This will be further characterized using protoplast
inoculation.
Fig. 2. C. quinoa leaves 7 days after inocu-
lation with BSBV full-length transcript mixes
1 + 2* + 3 (a), 1 + 2 (b), 1 + 2 + 3 (c), 2 + 3 (d) and
1 + 3 (e) and a negative control (f). Only
genome arrangements 1 + 2* + 3 (a), 1 + 2 + 3 (c)
and 1 + 3 (e) provoked the appearance of
symptoms on the inoculated leaves, with
necrotic spots and ringspots (data not shown)
and slight yellow chlorosis.
Fig. 3. Northern (a, b) and Western (c, d) blot analysis for the detection of BSBV RNAs and structural proteins in C. quinoa
(a,c)andB.macrocarpa(b,d) leaves,7and10–17 daysp.i.,respectively.Plantleaveswereeitherinoculatedwithoneofthefive
genome arrangements of BSBV full-length transcripts tested [combinations 1 + 2* + 3 (lane 1), 1 + 2 + 3 (2), 1 + 2 (3), 1 + 3 (4) and
2 + 3 (5)] or mock-inoculated (6). The three BSBVgenomic RNAs were detected in total RNAextracts using random DNA probes
labelled with [a-32P]dCTP and diluted transcripts constituted positive controls (+C) (a, b). The 19 kDa CP and 115 kDa CP–RT
proteins of BSBV were immuno-detected with primary polyclonal antibody directed against viral particles (c, d).
BSBV RNA-2 transcripts are dispensable in planta
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All three replicative combinations raised the infection rate
by 100% in both host plants, except the 1+2*+3 which
infected half of the inoculated B. macrocarpa.
BSBV CP and CP–RT proteins were detected by Western
blotting after SDS-PAGE separation of total protein
extracts from symptomatic samples and whole asympto-
matic leaves. Structural proteins were revealed using a
polyclonalantibodydirected
(DSMZ). The primary antibody was diluted 1:10000 in
TBS buffer containing 0.1% Tween 20, 5% powdered
skimmed milk, and supplemented with total protein
extracts from healthy C. quinoa or B. macrocarpa. Anti-
rabbit alkaline phosphatase-conjugated secondary IgG
(Sigma-Aldrich) was diluted 1:10000 in TBS–Tween–milk
buffer and the blots were revealed with premixed BCIP–
NBT solution (Sigma-Aldrich).
against viralparticles
A 19 kDa protein was detected in both C. quinoa and B.
macrocarpa plants inoculated with the 1 +2* +3 and 1 +2 +3
mixes of BSBV RNAs transcripts (Fig. 3c, d, lanes 1 and 2)
but not when RNA-2 was omitted (combination 1 +3; Fig.
3c, d, lane 4). Thus, the BSBV RNA-2 transcripts are
indeed responsible for the expression of the 19 kDa protein
in planta, which corresponds to the BSBV CP of the same
predicted molecular mass that is encoded by the BSBV
RNA-2 first ORF. Another protein with an estimated
molecular mass of 115 kDa was also observed in the leaves
of both plants inoculated with the genome combination
1 +2 +3 (Fig. 3c, d, lane 2) and not detected in the leaves
inoculated with the BSBV RNA-2 transcripts mutated for
the RT domain of the CP (genome combination 1 +2* +3;
Fig. 3c, d, lane 1). This product therefore corresponds to
the predicted 104 kDa BSBV CP–RT protein. Transcript
progeny analysis confirmed the stability of the frame shift
mutation (data not shown).
According to the different genomic combinations of BSBV
full-length transcripts inoculated, our study showed that
both the putative replicase (RNA-1) and TGB proteins
(RNA-3) of BSBV are essential and sufficient for the
replication of such RNAs in the inoculated leaves of C.
quinoa and B. macrocarpa. In addition, the symptom
expression on C. quinoa did not depend on the presence of
the BSBV RNA-2. The BSBV CP and CP–RT encoded by
the BSBV RNA-2 are therefore not required for viral
infection in the plant hosts or for symptom production on
C. quinoa leaves. The dispensability of the CP for viral
replication in planta is a common phenomenon, shared by
the closely related pomovirus potato mop-top virus
(PMTV) on Nicotiana benthamiana and Nicotiana cleve-
landii (McGeachy & Barker, 2000; Savenkov et al., 2003), as
well as other viruses such as the well-known tobamovirus
tobacco mosaic virus (TMV) on Nicotiana tabacum (Siegel
et al., 1962). However, whereas symptom production due
to TMV does not depend on the presence of the CP, no
symptoms are expressed when PMTV lacks the CP and
CP–RT. Thus, despite their taxonomic proximity and a
highly similar genome organization, the BSBV and PMTV
pomoviruses retain their own particularities. Nevertheless,
it should be borne in mind that PMTV is a potato virus
and symptom expression could be host-dependent.
It is also worth noting that, as the primers used to amplify
full-length cDNA sequences of BSBV were designed
according to a previously published nucleotide sequence
of a German isolate, the biological properties of the
resulting full-length cDNA clone might have been modi-
fied. However, viral terminal sequences are mostly highly
conserved for a virus species as they are involved in viral
replication (Duggal et al., 1994; Koenig et al., 2000), and
the BSBV full-length clone replicated efficiently.
Consistent with earlier findings reporting on foliar
symptoms on Chenopodium spp. and occasional lesions
on Beta spp. leaves when inoculating BSBV from the crude
sap of infected plants (Henry et al., 1986), transcripts from
the full-length clone of BSBV induced clear symptoms only
on the C. quinoa leaves. Following the example of BSBV,
the two first BNYVV RNAs, carrying the genetic material
homologous to one of the three BSBV RNAs plus a cystein-
richprotein(p14) with
(Bouzoubaa et al., 1986, 1987; Dunoyer et al., 2002), are
also unable to induce a systematic symptomatic foliar
reaction on Beta spp. However, lesions on Beta spp. leaves
are clearly produced when at least BNYVV RNAs-1, -2 and
-3 are present (Tamada et al., 1989; Rahim et al., 2007).
Although BSBV and BNYVV both naturally infect sugar
beet, BSBV lacks the genetic material necessary to induce
clear symptoms on B. macrocarpa leaves whereas BNYVV
has this through RNAs-3 and -4.
PTGSsuppressor activity
The use of a polyclonal antibody directed against BSBV
viral particles allowed two proteins expressed from BSBV
RNA-2 transcripts to be detected. As expected, the Western
blot analysis showed the production of the BSBV 19 kDa
CP in both C. quinoa and B. macrocarpa. However, whereas
the expected molecular mass of the CP–RT is 104 kDa, a
protein with an estimated molecular mass of 115 kDa
corresponding to the CP–RT was identified in both host
plants. It could be assumed that post-translational
modifications of the CP–RT domain, such as phosphor-
ylation or glycosylation events, are responsible for this
115 kDa observed molecular mass of CP–RT. Actually,
post-translational modifications can reduce the protein’s
electrophoretic mobility in SDS-PAGE and many viral
structural proteins are described as glycosylated and/or
phosphorylated (Hahn & Shepherd, 1980; Seddas &
Boissinot, 2006; Akamatsu et al., 2007; Hafre ´n &
Ma ¨kinen, 2008; Zayakina et al., 2008). Another hypothesis
would involve the intrinsic properties of the BSBV CP–RT,
knowing that highly hydrophilic domains containing many
charged residues, such as arginine (Hu & Ghabrial, 1995),
or the presence of transmembrane motifs (Rath et al.,
2009) in proteins might influence their migration on SDS-
PAGE. This is supported by the presence in the RT domain
of the BSBV CP of two predicted transmembrane helices, as
is also reported for the closely related beet virus Q (BVQ)
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3054 Journal of General Virology 90

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(Crutzen et al., 2009), which would be essential for viral
transmission by the vector P. betae (Diao et al., 1999;
Adams et al., 2001). Further experiments need to be
performed to determine the specificity of CP–RT, from the
post-translational modification or intrinsic properties,
which influences the molecular mass of the protein on
SDS-PAGE.
The rhizomania syndrome of sugar beet and its causal
agent, BNYVV, have been widely studied since the 1970s
because cropping losses, commonly about 50% and
sometimes up to 80%, have been attributed to the disease
(McGrann et al., 2009). However, the involvement of BVQ
and BSBV in the disease, often associated with beet infected
with BNYVV, has never been clearly established and no
particular interest in understanding their specific function-
ality was followed. Our study describing the first infectious
full-length cDNA clone available for a beet Pomovirus
therefore provides a useful tool for further investigating the
pathogenicity of BSBV in the complex rhizomania
syndrome, as well as its replication and infection mechan-
isms, and the potential viral interactions with other beny-
and pomoviruses on both susceptible and rhizomania-
resistant beets.
Acknowledgements
F.C. is a research fellow at the Belgian National Fund of Scientific
Research (FNRS) and M.M. is supported by grants from the Ministry
of Science, Research and Technology, Iran.
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