Journal of General Virology (2005), 86, 797–801
Short
Communication
DOI 10.1099/vir.0.80689-0
An intact RBR-binding motif is not required for
infectivity of Maize streak virus in cereals, but is
required for invasion of mesophyll cells
David R. McGivern,13 Kim C. Findlay,2 Nicholas P. Montague1
and Margaret I. Boulton1
Correspondence
Margaret I. Boulton
[email protected]
Received 12 October 2004
Accepted 15 December 2004
Department of Disease and Stress Biology1 and Department of Cell and Developmental
Biology2, John Innes Centre, Colney, Norwich NR4 7UH, UK
The replication-associated protein (RepA) of Maize streak virus interacts in yeast with
retinoblastoma-related protein (RBR), the negative regulator of cell-cycle progression. This may
allow geminiviruses to subvert cell-cycle control to provide an environment that is suitable for
viral DNA replication. To determine the importance of this interaction for MSV infection, the
RBR-binding motif, LxCxE, was mutated to IxCxE or LxCxK. Whilst RBR binding in yeast could
not be detected for the LxCxK mutant, the IxCxE protein retained limited binding activity. Both
mutants were able to replicate in maize cultures and to infect maize plants. However, whereas
the wild-type virus invaded mesophyll cells of mature leaves, the LxCxK mutant was restricted to
the vasculature, which is invaded prior to leaf maturity. Mature leaves contain high levels of RBR
and it is suggested that the MSV RepA–RBR interaction is essential only in tissues with high
levels of active RBR.
Geminiviruses have small, single-stranded DNA (ssDNA)
genomes that replicate through double-stranded DNA
(dsDNA) intermediates in the nuclei of infected cells. The
viruses do not encode a DNA polymerase and must use the
host machinery for viral DNA replication. However, DNA
polymerases are not functional in mature leaf cells and, to
infect these cells, geminiviruses must deregulate cell-cycle
control to provide an environment that is permissive for
viral DNA replication. This is probably accomplished by
the interaction of geminivirus replication factors with the
negative regulator of G1–S phase progression, the retinoblastoma-related protein (RBR; Gutierrez, 2002). This
mechanism appears to be conserved throughout the
geminiviruses, irrespective of whether they infect monocots
[e.g. the mastreviruses Maize streak virus (MSV) and Wheat
dwarf virus (WDV) (Horvath et al., 1998; Xie et al., 1995)]
or dicots [Bean yellow dwarf virus (BeYDV; Liu et al., 1999)
and the begomovirus Tomato golden mosaic virus (TGMV;
Kong et al., 2000)]. For WDV and BeYDV, RBR binding
occurs via the conserved LxCxE motif in the replicationassociated protein RepA. The motif is not present in
begomovirus proteins and RBR binding is mediated in part
through a conserved a-helical region in the Rep protein
(Arguello-Astorga et al., 2004; Hanley-Bowdoin et al., 2004;
Kong et al., 2000).
Interactions with RBR have been shown in yeast, but not in
3Present address: Division of Pathology and Neuroscience, University
of Dundee Medical School, Dundee DD1 9SY, UK.
0008-0689 G 2005 SGM
plant cells, although RBR proteins have been cloned from
both monocots and dicots (Dewitte & Murray, 2003).
Genetic studies have not provided a definitive answer
regarding the requirement for RBR binding. Geminivirus
mutants with impaired RBR binding in yeast produce either
wild-type (wt) (BeYDV; Liu et al., 1999) or attenuated
symptoms (TGMV; Kong et al., 2000) or are unable to
replicate in wheat suspension cells (WDV; Xie et al., 1995).
To assess whether interaction of MSV RepA with RBR is
necessary for MSV replication and infection, we made
mutations in the RBR-binding motif that we predicted
would reduce, or abolish, the interaction.
Plasmid MB64 was used for mutagenesis of RepA; it contains a BamHI/BglII fragment (co-ordinates 1203–2686) of
the MSV Ns (renamed MSV-A[NG1]) genome (GenBank
accession no. X01633). Mutagenesis was performed by using
a QuikChange site-directed mutagenesis kit (Stratagene)
and the primers RbEKV (GGCTGGAGCCAATCATTGATTGACTtATTACAAAGTAAATCAGG) and RbEKC (CCTGATTTACTTTGTAATaAGTCAATCAATGATTGGCTCCAGCC) to replace the conserved glutamine (E) residue of
the RBR-binding motif with lysine (K), thereby producing
pMB64-LxCxK, which differs from the wt genome by only
1 nt. Primers RbLIV (CATCCACCCTCATCACCTGATaTcCTTTGTAATGAGTCAATC) and RbLIC (GATTGACTCATTACAAAGgAtATCAGGTGATGAGGGTGGATG)
were used to produce pMB64-IxCxE, in which isoleucine (I)
replaces the conserved leucine (L) residue. All constructs
were verified by sequencing.
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797
D. R. McGivern and others
The effect of these mutations on interaction with maize
RBR was assessed by using the Matchmaker yeast twohybrid system (Clontech) and the GAL4 binding domain
(BD) construct pGBT9ZmRb1 (Horvath et al., 1998). RepA
sequences were fused to the GAL4 activation domain
(AD) of pGAD10 following PCR amplification from
pMB64, pMB64-IxCxE and pMB64-LxCxK, thereby creating pGAD-LxCxE, pGAD-IxCxE and pGAD-LxCxK, which
were used to transform yeast strain Y187 (Gietz & Woods,
1994). After mating with strain CG1945 containing either
pGBT9ZmRb1 or control plasmids (pGBT9 or pLamC),
transformants containing both AD and BD plasmids were
selected as described in the Clontech manual.
Interaction between bait and prey proteins was evidenced
by histidine prototrophy on medium supplemented with
6 mM 3-amino-1,2,4-triazole (3-AT). RepA and RepAIxCxE, but not RepA-LxCxK, interacted with ZmRb1
(Table 1). The interaction of RepA and ZmRb1, assessed
by expression of the lac reporter gene, resulted in bgalactosidase expression approximately 10-fold higher
than the background activity associated with yeast transformed with pGBT9, pGAD10 or pLamC (Table 1).
However, RepA-IxCxE showed a markedly reduced affinity
for ZmRb1; approximately 10 % of that obtained with the
wt protein. This is consistent with the data reported for the
equivalent mutation in BeYDV RepA, where binding was
reduced by up to 85 % (Liu et al., 1999). The activity
obtained with RepA-LxCxK was not significantly different
from the background (Table 1). The equivalent mutation in
WDV RepA eliminated interaction with the human p130Rb
and ZmRb1 in yeast (Xie et al., 1995, 1996). Similarly, MSV
RepA proteins in which the latter two amino acids of the
RBR-binding motif were substituted (Shepherd et al.,
2005) do not interact with ZmRb1. Although the WDV
mutant was unable to replicate in suspension-cultured
wheat cells, the MSV mutants replicated efficiently.
To determine whether MSV carrying the LxCxK mutation
could replicate in maize cells, a dimeric copy of the MSVLxCxK genome was constructed. Plasmid MB64-LxCxK was
digested with BamHI and XhoI to release a 1 kb fragment
(MSV co-ordinates 1683–2686). An MSV-A[NG1] dimer
in pUC19 (Boulton et al., 1993), was similarly digested
and the fragment encompassing MSV co-ordinates 1–1682
was recovered. A three-way ligation was performed by using
BamHI-linearized pAHC17 (Christensen & Quail, 1996)
and the two MSV fragments. After confirming the sequence
of the 1 kb region, the mutant MSV monomer was released
from pAHC17 by digestion with BamHI and inserted into
pUC19 using a 15-fold molar excess of insert to vector. This
product was used to transform Escherichia coli DH5a. EcoRI
plus XbaI digestion identified the dimer construct.
Maize (BMS) suspension cells were inoculated (Boulton
et al., 1993) with dimeric copies of either MSV or MSVLxCxK as described by Liu et al. (2002). Samples were taken
0, 2, 5 and 8 days post-inoculation (p.i.) and extracted DNA
was subjected to Southern blot analysis as described by
Boulton et al. (1993). In two experiments, the wt and
mutant virus accumulated both ss- and dsDNA forms;
the time-course of mutant viral DNA accumulation was
similar to the wt in experiment 1, but was delayed in the
second experiment (shown in Fig. 1a).
The lack of requirement for RBR binding for virus replication was not unexpected, as dividing cells possess the
machinery for DNA synthesis and BMS cells contain low
levels of active RBR (Huntley et al., 1998). Our data contrast
with those obtained by Xie et al. (1995), who found that the
equivalent WDV mutant could not replicate in cultured
cells, but are consistent with the data of Shepherd et al.
(2005) for MSV in BMS cultures. Either wheat cells are
less competent for DNA replication, or the mutation in
WDV disables a function that is necessary for viral DNA
replication.
Table 1. Yeast two-hybrid analysis of MSV RepA–maize RBR protein (ZmRb1) binding affinity
Bait protein (GAL4
BD fusion)
Prey protein
(GAL4 AD fusion)
Growth
(His”, +3-AT)*
b-Galactosidase
None (pGBT9 only)
None (pGBT9)
LaminC
ZmRb1
ZmRb1
ZmRb1
ZmRb1
SV40 T
None (pGAD10 only)
RepA
RepA
None
RepA
RepA-IxCxE
RepA-LxCxK
p53
2
2
2
2
+++
+
2
+++++
0?26±0?11
0?26±0?23
0?34±0?16
0?35±0?16
3?80±0?78
0?72±0?11
0?39±0?21
83?28±15?65
activityD
*Growth of yeast was assessed visually on an incremental scale, from one to five colonies (+) to confluent
growth (+++++); 2, no growth.
DMean±SD b-galactosidase activity [relative light units (mg protein)21 min21] was calculated from three
individual yeast colonies for each construct.
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Journal of General Virology 86
Analysis of MSV RepA–RBR protein interactions
outcompeted in plants by a compensatory mutant (LxCIK).
These data confirm that an intact LxCxE motif is not
required for infection of maize by MSV.
Fig. 1. Southern hybridization analysis of the replication of
wild-type MSV (E) and MSV-LxCxK mutant (K) in Black
Mexican Sweet (BMS) maize suspension cultures (a) and
infected maize plants (b). Samples were taken at 0, 2, 5
and 8 days post-bombardment (dpb) for cell samples and
28 days p.i. for plants. The results from two plants infected
with each construct are shown in (b). The concentration of
plant DNA loaded is approximately 1 % of that loaded for the
BMS samples. IP, Infected maize plant DNA; oc, open circular
dsDNA; lin, linear dsDNA; ccc, covalently closed circular
(supercoiled) dsDNA; ss, ssDNA.
A mutation (KEE146) in TGMV Rep that reduced RBR
binding to 16 % of wt levels in yeast resulted in limitation
of the virus to the vascular cells (Kong et al., 2000). To
test whether MSV mutants with impaired RepA–ZmRb1
binding can infect plants, constructs were prepared for
agroinoculation of maize. An MSV monomer containing
the IxCxE mutation was constructed as described for
MSV-LxCxK. Dimeric copies of the genomes were inserted
into pBIN19 as described for pUC19 constructs. Agrobacterium tumefaciens strain pGV3850 was transformed
with pBINMSV, pBINMSV-IxCxE or pBINMSV-LxCxK
and agroinoculated to maize plants (cv. Golden Bantam) as
described previously (Boulton et al., 1989a, b). The mutant
and wt constructs were equally infectious (17/17, 20/21
and 21/22 plants were infected by pBINMSV, pBINMSVIxCxE and pBIN-LxCxK, respectively) and exhibited
identical time-courses of infection, with symptoms appearing 8–12 days p.i. Virus MSV-IxCxE produced symptoms
identical to those induced by wt MSV in maize plants.
However, plants infected with MSV-LxCxK contained less
viral DNA (Fig. 1b) and had narrower chlorotic streaks
(Fig. 2a). The milder phenotype persisted throughout the
experiment (32 days p.i.). To confirm that the mutation
was maintained in the progeny, total DNA was extracted
from the first infected (at 12 days p.i.) and the newly
emerging (28 days p.i.) leaves of each of four plants
infected with MSV-IxCxE or MSV-LxCxK. Analysis of the
RepA sequences amplified from these extracts confirmed
that the mutations were maintained; no compensatory
mutations were found. This is in contrast to the MSV
mutants made by Shepherd et al. (2005), where the MSV
RBR-binding domain mutant (LxCLK) was consistently
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MSV-A[NG1] is adapted to maize. To investigate whether
the LxCxE motif is a host-range determinant, Cicadulina
mbila was used to transmit the virus from MSV-LxCxKinfected maize to barley (cv. Igri) and wheat (cv. Chinese
Spring). All nine of the barley and four of the five wheat
plants became infected. Infectivity was equivalent to that
seen with the wt virus (10/10 and 3/4 infected, respectively),
although MSV-LxCxK caused less severe streaking and
stunting (Fig. 2b). Clearly, an intact LxCxE domain is not
required for insect transmission, encapsidation of viral
DNA or infection of hosts to which MSV-A[NG1] is not
adapted. Furthermore, infection is established irrespective
of whether cloned DNA is delivered to the meristematic
region of the plant (as ssDNA via agroinoculation) or
virions are introduced into the phloem of mature leaves
(via insect transmission). These data support the hypothesis
that the initiation of viral dsDNA synthesis must occur in a
cell that is competent for DNA replication.
As MSV infects most cells of the mature leaf, the narrowerstreak phenotype could indicate a restriction of the MSVLxCxK mutant to the vascular system, as shown for TGMV
mutant KEE146 (Kong et al., 2000). Immunocytochemical
staining of the MSV coat protein (Lucy et al., 1996) in
sections from MSV-LxCxK- or MSV-infected maize showed
that fewer vascular bundles contained virus in mutantcompared with wt-infected leaves (71 versus 88 %, respectively); this was consistent with the lower titre of viral DNA
in these plants (Fig. 1b). Furthermore, signal was detected in
the mesophyll cells of leaves infected with wt MSV, but not
in the MSV-LxCxK-infected leaves. However, staining in
mutant-infected plants was generally weaker and, because
maize has few mesophyll cells between the vascular bundles
(Fig. 2c), estimation of mesophyll invasion was difficult.
To further assess tissue tropism in the MSV-LxCxK-infected
leaves, sections were subjected to transmission electron
microscopy (TEM) (Wells, 1985) and immunogold staining. Four areas were sampled for each of the inocula. TEM
confirmed the decreased invasion of the vasculature by the
mutant compared with the wt virus, and suggested that the
infected cells contained fewer virions [evidenced as smaller
nuclear crystalline arrays; compare Fig. 2(d) and (e)]. The
lack of infection of cells outside the vasculature in mutantinfected leaves was also confirmed: out of 43 infected
bundles that were examined, only one infected mesophyll
cell and one infected epidermal cell were seen (Fig. 2f–i),
whereas virus was seen in over 80 % of mesophyll cells
adjoining vascular bundles infected with MSV (Fig. 2c).
The ability of MSV-LxCxK to replicate in the vasculature
of maize plants, despite its inability to bind RBR in yeast,
suggests that the vascular cells are DNA replicationcompetent. MSV replication occurs in the vascular tissue
of leaf primordia and immature leaves (Lucy et al., 1996)
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D. R. McGivern and others
Fig. 2. Infection of plants by wild-type MSV and MSV-LxCxK. (a) Symptoms obtained in maize leaves infected with wild-type
(WT) MSV and mutants MSV-IxCxE and MSV-LxCxK; H, uninfected leaf. (b) Symptoms obtained in barley plants inoculated
with the non-viruliferous vector C. mbila (H) or vector carrying wild-type MSV (WT) or mutant MSV-LxCxK. Note the
decreased stunting obtained in plants infected by the mutant virus. (c) Diagrammatic representation of sections showing cells
infected by WT MSV (left panel) or MSV-LxCxK (right panel). Tissue tropism for WT MSV is typical of that seen previously
(Lucy et al., 1996; Pinner et al., 1993; this study); that for MSV-LxCxK is typical of that seen by immunochemical staining and
electron microscopy (this study). Shaded areas represent infected cells. (d–i) Electron micrographs of leaf samples infected
with wild-type MSV (d) or mutant MSV-LxCxK (e–i); the arrows depict MSV aggregates within the nuclei of infected cells.
Comparatively more cells were infected in plants inoculated with MSV. The MSV-LxCxK-infected mesophyll (MES) cell is
shown in (f); MSV-specific immunogold labelling [panel (g)] confirmed the presence of virus in the aggregate arrowed in (f).
The MSV-LxCxK-infected epidermal cell (EP) is shown in panel (h); immunogold labelling [panel (i)] confirmed the presence of
virus in the aggregate arrowed in (h). All other MSV-LxCxK-infected cells were within the vasculature. M, Metaxylem; PX,
protoxylem; P, phloem; BS, bundle sheath.
800
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Journal of General Virology 86
Analysis of MSV RepA–RBR protein interactions
and DNA polymerases are likely to be present in these cells.
In contrast, invasion of the mesophyll occurs only as the
mature leaves unfold from the whorl. Mature mesophyll
cells do not incorporate [3H]thymidine (M. I. Boulton,
unpublished data) and, although RBR is present at low
levels in proliferating basal tissue, mature maize tissue
contains high levels of active pocket domain forms (Huntley
et al., 1998). Thus, it is likely that MSV RepA–RBR binding
is needed to sequester active (hypophosphorylated) RBR
and, thereby, to overcome the block to G1–S phase progression for efficient virus replication, only in mature leaf
cells. Taken together, our data, and those of Kong et al.
(2000) using TGMV KEE146, suggest that geminiviruses
that are naturally phloem-limited may not require RBR
binding and that disruption of RBR binding affects symptom production only in viruses that invade the mesophyll.
This conclusion is supported by the reduced symptom
severity that is seen in maize infected by other MSV RBRbinding domain mutants (Shepherd et al., 2005). Thus,
the wt symptoms produced by the BeYDV RBR-binding
mutants (Liu et al., 1999) may reflect vascular limitation of
BeYDV; this is currently under investigation.
The decreased immunochemical signal and size of crystalline arrays in cells infected by MSV-LxCxK suggest that
replication efficiency of this mutant is impaired slightly;
delay in accumulation of mutant viral DNA in BMS, in
one of two experiments, supports this premise. Decreased
replication could have pleiotropic effects on virus (or viral
DNA) transport to adjacent cells. For example, if infection
must occur during a developmental ‘window of opportunity’, it may not be established in cells receiving suboptimal amounts of viral DNA and uninfected vascular
bundles could result. Nevertheless, replication is not
impaired sufficiently to delay timing of symptom appearance or reduce infectivity.
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Acknowledgements
We thank Dr Jane Langdale (Oxford University, UK) for helpful
discussions with respect to maize-leaf morphology and Krisztina
Nikovics (BRC, Szeged, Hungary) for providing pGBT9ZmRb1. The
work was funded by the Biotechnology and Biological Sciences
Research Council (BBSRC) and D. R. M. was in receipt of a PhD
studentship from the BBSRC. MSV was maintained and manipulated
under DEFRA licences PHL185A/4538 and PHL185A/4513.
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