1 Department of Disease and Stress Biology, John Innes Centre, Colney, Norwich NR4 7UH, UK
2 Department of Cell and Developmental Biology, John Innes Centre, Colney, Norwich NR4 7UH, UK
Correspondence
Margaret I. Boulton
margaret.boulton{at}bbsrc.ac.uk
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ABSTRACT |
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Present address: Division of Pathology and Neuroscience, University of Dundee Medical School, Dundee DD1 9SY, UK.
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MAIN TEXT |
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Interactions with RBR have been shown in yeast, but not in 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 12032686) 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.
The effect of these mutations on interaction with maize RBR was assessed by using the Matchmaker yeast two-hybrid 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 RepA-IxCxE, 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
-galactosidase 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.
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Maize (BMS) suspension cells were inoculated (Boulton et al., 1993) with dimeric copies of either MSV or MSV-LxCxK 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
).
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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 RepAZmRb1 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, pBINMSV-IxCxE and pBIN-LxCxK, respectively) and exhibited identical time-courses of infection, with symptoms appearing 812 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 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.
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As MSV infects most cells of the mature leaf, the narrower-streak phenotype could indicate a restriction of the MSV-LxCxK 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 mutant- compared 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 mutant-infected 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. 2fi
), 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 replication-competent. MSV replication occurs in the vascular tissue of leaf primordia and immature leaves (Lucy et al., 1996) 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 RepARBR binding is needed to sequester active (hypophosphorylated) RBR and, thereby, to overcome the block to G1S 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 RBR-binding 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 |
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Received 12 October 2004;
accepted 15 December 2004.