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J Gen Virol 88 (2007), 325-336; DOI 10.1099/vir.0.82338-0

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© 2007 Society for General Microbiology

Inhibition of maize streak virus (MSV) replication by transient and transgenic expression of MSV replication-associated protein mutants

Dionne N. Shepherd1, Tichaona Mangwende1,2, Darren P. Martin3, Marion Bezuidenhout1, Jennifer A. Thomson1,{dagger} and Edward P. Rybicki1,3,{dagger}

1 Department of Molecular and Cell Biology, University of Cape Town, Private Bag, Rondebosch, Cape Town 7701, South Africa
2 Division of Pharmacology, University of Cape Town, Cape Town, South Africa
3 Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, Anzio Road, Cape Town 7925, South Africa

Correspondence
Edward P. Rybicki
ed{at}science.uct.ac.za


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Maize streak disease is a severe agricultural problem in Africa and the development of maize genotypes resistant to the causal agent, Maize streak virus (MSV), is a priority. A transgenic approach to engineering MSV-resistant maize was developed and tested in this study. A pathogen-derived resistance strategy was adopted by using targeted deletions and nucleotide-substitution mutants of the multifunctional MSV replication-associated protein gene (rep). Various rep gene constructs were tested for their efficacy in limiting replication of wild-type MSV by co-bombardment of maize suspension cells together with an infectious genomic clone of MSV and assaying replicative forms of DNA by quantitative PCR. Digitaria sanguinalis, an MSV-sensitive grass species used as a model monocot, was then transformed with constructs that had inhibited virus replication in the transient-expression system. Challenge experiments using leafhopper-transmitted MSV indicated significant MSV resistance – from highly resistant to immune – in regenerated transgenic D. sanguinalis lines. Whereas regenerated lines containing a mutated full-length rep gene displayed developmental and growth defects, those containing a truncated rep gene both were fertile and displayed no growth defects, making the truncated gene a suitable candidate for the development of transgenic MSV-resistant maize.

{dagger}These authors contributed equally to this work. Back


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Maize (Zea mays L.) is Africa's most important staple food crop and is the mainstay of most of the continent's rural economies. Despite this, mean maize yields per hectare in Africa are extremely low compared with the developed world and, as a result, food shortages are a perpetual problem in most sub-Saharan countries (see www.fao.org and www.wfp.org). Whilst lack of access to modern farming techniques and provisions are the main reasons for low yields, maize pathogen epidemics can decrease yields further. Of the many viral pathogens infecting maize, the leafhopper-vectored Maize streak virus (MSV; family Geminiviridae, genus Mastrevirus) is considered the most important and widespread (Thottappilly et al., 1993Down; Bosque-Perez, 2000Down). In epidemic years, maize streak disease (MSD) can result in up to 100 % yield losses (Wambugu & Wafula, 1999Down; Bosque-Perez, 2000Down). The most effective control strategy for MSD prevention is frequent application of systemic insecticides for the Cicadulina spp. vector leafhoppers; this option is not generally open to poorer farmers. Although maize genotypes with varying degrees of MSV resistance have been developed (Kim et al., 1989Down; Van Rensburg et al., 1991Down; Barrow, 1992Down, 1993Down; Rodier et al., 1995Down; Welz et al., 1998Down; Buddenhagen & Bosque-Pérez, 1999Down; Kyetere et al., 1999Down), their use has not been widespread among resource-poor farmers, due either to cost or their inability to yield as productively as MSV-sensitive genotypes in non-epidemic years.

MSV replicates in the nuclei of infected cells by rolling-circle replication (Saunders et al., 1991Down; Stenger et al., 1991Down) and possibly via recombination-dependent mechanisms (Jeske et al., 2001Down). Rolling-circle replication is initiated by binding of the virus replication-associated protein (Rep) to the virion-strand origin of replication, where the protein initiates and terminates virion-strand DNA synthesis. MSV Rep is the translation product of two complementary-sense open reading frames (ORFs), C1 and C2. The C1–C2 transcript, which contains an intron, is translated to yield either Rep (from the spliced transcript) or RepA (from the unspliced transcript). Rep and RepA share several distinct domains with diverse biochemical activities (Fig. 1Down), such as sequence-specific DNA binding, virion-sense origin cleavage and ligation (motif III; Heyraud-Nitschke et al., 1995Down) and potential transactivation of viral promoters (Hofer et al., 1992Down; Zhan et al., 1993Down; Collin et al., 1996Down). However, only RepA interacts detectably with the host retinoblastoma-related protein (pRBR) (Horvath et al., 1998Down; Liu et al., 1999Down; Gutierrez et al., 2004Down) and a group of host NAC (non-apical-meristem, ATAF and CUC2 genes) domain-containing GRAB (geminivirus RepA-binding) proteins (Xie et al., 1999Down) and only Rep is potentially involved in ATP-dependent unwinding of viral DNA (Pant et al., 2001Down).


Figure 1
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  		Fig. 1. MSV rep genes and gene products, after Gutierrez (1999)Down and Boulton (2002)Down. (a) The MSV-Kom repA (C1) and C2 ORFs, including selected nucleotide co-ordinates (relative to the rep start codon) and the positions of restriction-enzyme sites used in the cloning of rep gene variants. Also indicated are the co-ordinates of the rep gene in relation to the last A of the conserved TAATATTAC sequence of the MSV-Kom genome. (b) Known sequence motifs and functional domains of the mastrevirus Rep protein (expressed from the spliced C1/C2 ORFs). Amino acid numbering is relative to the N-terminal methionine. Potential DNA-binding and GRAB-interaction domains are shown in positions analogous to their locations inbegomovirus Rep proteins and WDV RepA, respectively. (c) Wild-type and truncated RepA variants used in the trans-replication assays.

 
As Rep is the only viral protein essential for virus replication and is required early in the infection process, it is an ideal target for genetic engineering of MSV resistance in maize. Pathogen-derived resistance (PDR) approaches have previously been successful against a wide range of dicot-infecting begomoviruses (Stanley et al., 1990Down; Day et al., 1991Down; Frischmuth & Stanley, 1991Down, 1994Down; von Arnim & Stanley, 1992Down; Kunik et al., 1994Down; Hong & Stanley, 1996Down; Hong et al., 1996Down; Noris et al., 1996Down; Bendahmane & Gronenborn, 1997Down; Sangaré et al., 1999Down; Hou et al., 2000Down; Brunetti et al., 2001Down; Chatterji et al., 2001Down; Asad et al., 2003Down; Chellappan et al., 2004Down; Zhang et al., 2005Down). To our knowledge, however, there are no published reports of transgenic geminivirus resistance in monocots.

In this study, our aim was to develop dominant-negative rep mutants that could be used to engineer MSV resistance in maize. Quantitative transient-replication assays in maize suspension cells co-bombarded with an infectious MSV genomic plasmid and various rep constructs were used to identify trans-dominant mutations that might inhibit MSV replication in maize. Digitaria sanguinalis, an MSV-sensitive grass (Chen et al., 1998Down), was then transformed with the most promising of these mutant rep constructs and a number of MSV-resistant lines, some of which were both phenotypically normal and fertile, were characterized.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids.
All mutations and truncations were performed by using the wild-type (wt) MSV-Kom rep construct pSKrep. pSKrepRb–, containing a pRBR-interaction motif triple mutation, has been described previously, as have all basic techniques used here (Shepherd et al., 2005Down).

PCR site-directed mutagenesis.
pSKrep was used as template DNA for abutting primer PCR site-directed mutagenesis as described previously (Shepherd et al., 2005Down). The rep gene in pSKrep has a BamHI site inserted at its 5' end and a BglII site at its 3' end (see Fig. 1Up). The mutagenic primer set used to generate mutations in motif III (the nicking/ligation motif) was C1D99A,I101F [5'-AGAGTGAGGGccTAttTTCTCAAGGAAC-3' (MSV-Kom co-ordinates 2243–2216)] and C1Univ1 [5'-GTTAACTGACTTGGCACTCTG-3' (MSV-Kom co-ordinates 2244–2264]. Numbering starts from the last A of the conserved TAATATTAC sequence of MSV-Kom, in the 5'->3' direction of the primer. Lower-case letters denote changes from wt rep; an engineered HaeIII site is underlined. These primers resulted in amino acid changes in Rep of D99 to A and I101 to F (numbering relative to the Rep N-terminal methionine). The same motif III mutation was made by using pSKrepRb– as a template to create a double mutant. Mutated rep genes were identified by HaeIII (motif III mutation) and BfrI (pRBR-interaction motif mutation) cleavage; mutations were confirmed by sequencing. Mutated plasmids were named pSKrepIII– and pSKrepIII–Rb–.

Truncated rep gene constructs.
The 3'-truncated rep genes were constructed by cloning BamHI–HindIII fragments of pSKrep and pSKrepIII– into pSK to yield pSKrep1–179 and pSKrep1–179III–, respectively (Fig. 1aUp). For the plasmids and transgenes, the numbers in superscript refer to the codons of the first and last amino acids of the encoded Rep protein variants. They were named Rep1–179 and Rep1–179III– according to the amino acids that they contained (Fig. 1cUp). In addition, BamHI–HindIII fragments of pSKrep and pSKrepRb– created by a partial HindIII digest were cloned into pSK to yield pSKrep1–219 and pSKrep1–219Rb– (Fig. 1aUp), expressing Rep1–219 and Rep1–219Rb–, respectively (Fig. 1cUp). The correctness of all constructs was confirmed by sequencing.

Construction of plant expression cassettes.
A 1.2 kb BamHI–BglII fragment containing a full-length rep gene (Fig. 1Up) from pSKrepIII– and pSKrepIII–Rb– was inserted into the BamHI site of pAHC17 (Christensen & Quail, 1996Down) downstream of the maize ubiquitin promoter to yield prepIII– and prepIII–Rb–, respectively. Construction of prep, prepRb–, prepA and prepARb– (rep gene variants cloned in pAHC17) has been described previously (Shepherd et al., 2005Down). Truncated rep genes were cloned into pAHC17 by using BamHI sites added at their 3' ends. Plasmids with rep in the sense orientation were selected and designated prep1–X or prep1–XMut (1–X, portion of rep contained in the construct; and Mut, Rb– or III– mutations). Truncated antisense rep genes were made by cloning rep1–219 and rep1–179 in the BamHI site of pAHC17 in the antisense orientation to give prep1–219(AS) and prep1–179(AS), respectively.

Construction of rep mutants in MSV-Kom.
Full-length rep mutants were introduced into pKom602 (a 1.1-mer infectious clone of the MSV-Kom genome; Schnippenkoetter et al., 2001Down) by replacing a BglII–NsiI fragment from wt pKom602 with the same fragment from the mutated rep genes. The MSV-Kom mutant plasmids (pKomMut) were used to investigate the effect of the mutations on the replication of MSV-Kom in rapidly dividing black Mexican sweetcorn (BMS) suspension culture cells.

Transient-replication assays.
Replication of MSV DNA in BMS cells was assayed by a biolistic bombardment-mediated transient assay system as described previously (Shepherd et al., 2005Down). BMS was first bombarded with each MSV-KomMut replicon to determine whether the mutations interfered with Rep function. The potential for the mutant Rep proteins to interfere with viral DNA replication was determined by a replicative form (RF)-specific quantitative PCR assay (Shepherd et al., 2005Down). In each experiment (which was repeated at least once), up to nine BMS samples were bombarded with pKom602 alone or with pKom602 plus each mutant rep construct.

Construction of MSV-Kom rep mutants for in planta replication analysis.
DNAs of wt and mutant viruses were inserted into the EcoRI and XbaI sites of the binary vector pBI121 (Clontech) to obtain pBIKom602 (wt) and pBIKomMut, which were used to transform Agrobacterium tumefaciens C58C1 (Koncz & Schell, 1986Down) by the method of An et al. (1988)Down. The constructs were used to investigate the effect of the mutations on the viability of MSV-Kom in planta. Maize seedlings (Z. mays L. ‘Jubilee’) were inoculated with agroinfectious constructs as described previously (Martin et al., 1999Down).

D. sanguinalis tissue culture and transformation.
The tissue culture, transformation and regeneration of D. sanguinalis, including details of tissue-culture media used, have been described previously (Chen et al., 1998Down). For this work, embryogenic D. sanguinalis calli were transformed by particle bombardment using the Bio-Rad/DuPont PDS1000-He system. For each bombardment, 2 µg plasmid DNA was precipitated onto gold particles as described previously for the transfection of BMS (Shepherd et al., 2005Down). Each mutated or truncated rep construct was co-bombarded with pAHC25 (Ubi-Bar/Ubi-Gus; Christensen & Quail, 1996Down) at a 1 : 1 weight ratio. rep constructs chosen to transform D. sanguinalis were prepIII–, prepIII–Rb– and prep1–219Rb–. Non-bombarded calli and calli bombarded with pAHC25 alone were used as controls in all experiments. The settings on the biolistics device were as follows: gap distance, 6 mm; macrocarrier travel distance, 5 mm; target distance, 6 cm. Each target plate was bombarded twice at a pressure of 900 p.s.i. (6.2 MPa), each shot delivering ~333 ng per plasmid. Seven days after bombardment, calli were placed on regeneration medium with selection (3 mg bialophos l–1) and cultured as described by Chen et al. (1998)Down. Shooting calli were transferred to rooting medium with selection and plantlets were hardened off in a 33 : 33 : 33 mix of sand, compost and palm peat and finally transferred to potting soil.

Extraction of plant DNA and molecular analysis by PCR and Southern hybridization.
Total DNA was extracted from transgenic D. sanguinalis as described previously (Shepherd et al., 2005Down) for extraction of DNA from BMS cells. The presence of the transgene was confirmed by PCR. Primers were designed that could amplify all rep transgenes from the different samples: TrepF, 5'-ATGGCCTCCTCCTCATCCAAC-3' (MSV-Kom co-ordinates 2528–2508; rep nt positions 1–21 in Fig. 1aUp); and TrepR, 5'-AAGCTTCGGGACTAACCT-3' (MSV-Kom co-ordinates 1871–1888; rep nt positions 641–658 in Fig. 1aUp). Southern analysis was carried out following standard protocols (Sambrook et al., 1989Down). Genomic DNA (5 µg) was digested with BglII, which cuts once in the expression cassette upstream of the transgene. The hybridization probe specific to the rep transgene was created by using a PCR DIG probe synthesis kit (Roche Diagnostics) according to the manufacturer's instructions, with TrepF and TrepR primers.

D. sanguinalis MSV challenge experiments.
Transgenic plants were challenged with MSV by using viruliferous leafhoppers (Cicadulina mbila Naudé), obtained from M. Barrow (Pannar Pty Ltd, Greytown, South Africa). Leafhoppers were placed in small vials containing ~1x10 mm slits, through which a single leaf was inserted. Three vials, each containing four leafhoppers, were placed at different positions on each plant (see Fig. 5aDown). Symptom development was monitored daily with chlorotic areas on symptomatic leaves being quantified on a six-point scale (0, no symptoms; 5, >95 % chlorosis) by using a symptom key (Fig. 3Down). Viral DNA levels in the leaves of challenged plants were assessed by using MSV RF-specific degenerate primers (Willment et al., 2001Down; Shepherd et al., 2005Down), which are capable of amplifying all MSV strains used in this study, but do not amplify the transgene.


Figure 5
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  		Fig. 5. Virus challenge of D. sanguinalis. (a) In challenge experiments, viruliferous leafhoppers were placed in vials attached to three leaves per plant. (b) Severe streaking on a control plant from challenge 1. It can be seen that some leaves are almost entirely chlorotic (photograph courtesy of R. Kloppers, Pannar Pty, Greytown, South Africa). (c–e) Comparison of a mildly symptomatic D. sanguinalis line containing prep1–219Rb– (Tr1.1) with its matched control plant (3.1). In (c), a symptomatic leaf of one tiller of Tr1.1 is shown (see arrowhead), whilst in (d), newly emerged leaves of the same plant are non-symptomatic. In contrast (e), control plant 3.1 has severe symptoms in all of its newly emerged leaves (see arrowheads) and is close to dying. Photographs were taken 35 days p.i. (f–g) Virus replication in plants of line Tr1 (f) and matched control plants (g) was analysed by RF-specific degenerate-primer PCR (Willment et al., 2001Down) 47 days p.i. Lanes: A, symptomatic leaf (except in the case of non-symptomatic plants); B, newly emerged leaf adjacent to the symptomatic leaf; C, newly emerged leaf distant from the symptomatic leaf.

 

Figure 3
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  		Fig. 3. (a) Symptom key for quantification of disease symptoms. Symptom severities in Table 2Up were analysed by using a rating scale based on percentage of chlorotic leaf area according to Martin & Rybicki (1998)Down. (b) Virus DNA levels present in leaves were assessed qualitatively by using PCR amplification.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of transiently expressed MSV Rep mutants on virus replication in vitro and in planta
BMS cells were bombarded with MSV-Kom genomes containing no mutations (wt), a mutation in motif III (III– mutation) or mutations in both motif III and the pRBR-interaction motif (III–Rb– mutations). Virus replication was assayed by quantitative RF-specific PCR. Whilst wt MSV-Kom replicated efficiently, no replication was detectable for either mutant virus. The two mutant viruses also could not complement the replication functions of a rep-deficient MSV-Kom genome [pMSVPstI, described previously (Shepherd et al., 2005Down)]. The rep-deficient virus, however, replicated successfully when wt prep was provided in trans (data not shown). The effects of the mutations on virus infectivity were analysed by agroinoculation of 3-day-old maize plants (‘Jubilee’). Whereas all of the 28 maize plants receiving the wt virus developed symptoms, none of 28 plants agroinoculated with either mutant became symptomatically infected.

Having established that the rep mutations abolished the replication functions of the expressed protein, truncated versions of the mutant and wt genes were produced. The C2 ORF portions of the wt and the III– mutant rep genes, including sequences encoding the pRBR-interaction motif, were deleted to produce prep1–179 and prep1–179III– (Fig. 1Up). Another pair of 3' rep truncations, prep1–219 and prep1–219Rb–, containing sequences encoding either the wt or mutated pRBR-interaction motif, was also constructed (Fig. 1Up). It has been shown previously that the pRBR-interaction motif mutation (Rb–) abolishes detectable pRBR binding to RepA (Shepherd et al., 2005Down). Here, the influence of the pRBR-interaction motif in both RepA and C-terminal Rep truncations on MSV-Kom replication in maize suspension cells was analysed. In addition, the potential trans-dominant-negative effects of the mutant rep constructs on virus replication were assayed with a view to using these constructs to engineer MSV resistance in maize.

The effects of the various mutant rep genes on MSV-Kom replication varied as follows: total inhibition of replication (prepIII–Rb–, prepIII–, prepARb–, prepA and prep1–219Rb–); ~80 % inhibition of replication (prep1–219); enhancement of replication (prep1–179III– and prep1–179); and no significant effect on replication (prep1–219(AS) and prep1–179(AS)) (Fig. 2Down). A striking correlation can be seen between the size of the truncated Reps and their effects on MSV-Kom replication. The two 179 aa Reps enhanced virus replication when supplied in trans, whereas the opposite was true of the 219 aa Reps, which inhibited replication. The antisense versions of both the shorter and the longer truncated rep genes (prep1–179(AS) and prep1–219(AS)) had no effect on virus replication, suggesting that the inhibition and enhancement of replication observed for the sense versions of these genes are a consequence of their expression. Surprisingly, there was no significant difference between the enhancement of replication observed with Rep1–179 and Rep1–179III–, suggesting that an intact motif III in Rep1–179 plays no part in the replication-enhancing property of this protein. The effect of mutating the pRBR-interaction motifs in RepA and RepIII– could not be determined, as all constructs, both with and without the mutation, inhibited replication completely. It is apparent, however, that the pRBR-interaction motif mutation in Rep1–219Rb– contributed significantly to this protein's ability to inhibit MSV-Kom replication totally, in that Rep1–219 only afforded ~80 % inhibition of MSV-Kom replication. Whereas complete removal of the pRBR-interaction motif (in Rep1–179III– and Rep1–179) resulted in an enhancement of virus replication, this effect may also have been due to removal of sequences between residues 179 and 219, including the Rep oligomerization domain.


Figure 2
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  		Fig. 2. Viral DNA replication levels following co-bombardment of maize suspension cells with mutant rep plasmids and MSV-Kom DNA. From individual replicate data calculated by using quantitative PCR (Shepherd et al., 2005Down), the mean amount of replicated virus DNA (in pg) in the presence and absence of each rep construct was calculated. Mean amounts of DNA for MSV-Kom plus each rep construct are expressed relative to MSV-Kom replicating alone. Error bars represent 95 % confidence intervals. Sequence motifs present in each expressed Rep mutant are as shown in Fig. 1Up.

 
rep transgenes in a monocot model system
Based on results from the transient-replication assays, prepIII–Rb–, prepIII– and prep1–219Rb– were chosen to transform D. sanguinalis. Table 1Down provides a summary description of transgenic lines obtained. To determine whether the intact pRBR-interaction motif sequence in the repIII– transgene might interfere with plant regeneration through interaction of RepIII– with the cell-cycle regulator pRBR (Huntley et al., 1998Down), after selecting the bombarded calli for 6 weeks on bialophos-containing medium (MS-Bi), each bialophos-resistant callus piece was divided into two and one half was plated onto regeneration medium, whereas the other half was replica-plated and maintained on MS-Bi. Some of the calli on regeneration medium formed leaves and shoots, but none regenerated into plants. After 5 months maintenance on MS-Bi, the replica-plated calli were tested for the presence of the transgene. Of 27 calli, 22 were positive for repIII– and all were positive for the bar gene.


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  		Table 1. Properties of transgenic Digitaria sanguinalis

 
Conversely, D. sanguinalis repIII–Rb– transformants did regenerate. However, the regeneration efficiency of all bombardments with this construct was low and, as with the III– mutant rep, the III–Rb– mutant appeared to have a negative effect on various aspects of plant development. Most prepIII–Rb– transgenic lines were severely stunted (Fig. 4aDown) and all were infertile. At the same time, identical-source calli transformed with pAHC25 (Ubi-Bar/Ubi-Gus) alone regenerated with higher efficiency into phenotypically normal, mature plants (Fig. 4aDown; Table 1Up). This indicates that the stunting and infertility of prepIII–Rb– transgenic plants are caused by the transgene and are not a property of the D. sanguinalis cell line used. Although the pRBR-interaction motif was rendered non-functional in RepIII–Rb–, other motifs potentially affecting plant development are encoded in the C2 ORF and the C terminus of RepA (Hofer et al., 1992Down; Xie et al., 1999Down). Therefore, rep genes with different 3' deletions were constructed (Fig. 1Up). One of these, prep1–219Rb– (expressing Rep1–219Rb–), inhibited virus replication completely in maize suspension cells (Fig. 2Up). As this mutated/truncated Rep cannot interact detectably with pRBR (confirmed in yeast two-hybrid studies; data not shown) and is missing the C2 ORF and C terminus of RepA, it was considered to be the best choice for the potential generation of phenotypically normal transgenic plants.


Figure 4
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  		Fig. 4. Transgenic D. sanguinalis. (a) Three representative D. sanguinalis lines transformed with prep1–219Rb– (left), prepIII–Rb– (middle) or pAHC25 (right), illustrating the phenotypic effects of the transgenes. (b) Southern blot analysis of transgenic lines used in challenge experiments described in Table 2Up, showing transgene-integration patterns in BglII-digested genomic DNA using a rep-specific probe. FC8, repIII–Rb– transgenic line; Tr1 and Tr2, rep1–219Rb– transgenic lines; control, non-transgenic D. sanguinalis.

 
Indeed, a high percentage of plants containing prep1–219Rb– did regenerate into phenotypically normal, fertile adults (Fig. 4aUp). Additionally, whilst five out of six plants regenerated from calli co-bombarded with prepIII–Rb–+pAHC25 contained the bar gene but not the repIII–Rb– gene, 20 out of 22 transgenic lines regenerated from calli bombarded with prep1–219Rb–+pAHC25 contained both rep1–219Rb– and bar genes (Table 1Up). This indicates that, whereas transgenic cells that received only the bar gene had a large selective advantage over repIII–Rb+bar transgenics, they did not have a significant advantage over the rep1–219Rb–+bar transgenics.

Mutated rep confers MSV resistance to transgenic D. sanguinalis
Several D. sanguinalis transgenic lines were tested for MSV resistance in a preliminary challenge using leafhopper transmission of clonal MSV-Kom, a moderately severe isolate (data not shown). Based on these results, several plants of the potentially resistant line FC8 (F line, containing the full-length rep gene of prepIII–Rb–) and Tr1 and Tr2 (Tr lines, for truncated Rep, both containing prep1–219Rb–) were given a more severe challenge with MSV, using leafhopper transmission of a field isolate and an extremely severe cloned isolate, MSV-MatB (Martin et al., 1999Down, 2001Down). Southern analysis on these lines indicated low-copy integration of the transgene in lines FC8 and Tr2 and high-copy integration in line Tr1 (Fig. 4bUp). Because all F lines were infertile, callus was initiated from the parent (T0) transgenic lines. Plants were then regenerated from bulked-up stock transgenic callus as an alternative to T1 offspring and were tested for the presence of the transgene by PCR (data not shown). The transgene was inherited in both the callus initiated from T0 plants and plants regenerated from this callus, thus showing that the transgene was probably integrated stably. Stock callus kept on MS maintenance medium has remained positive for the transgene for over 6 years.

The first of four challenges was carried out on three plants of line FC8 and a non-transgenic D. sanguinalis control plant (challenge 1; see Table 2Down). Whereas the non-transgenic control plant developed symptoms 10 days post-inoculation (p.i.) (see Fig. 5bUp), no symptoms were observed on any transgenic plant throughout the 2 month trial. Virus replication was inhibited completely in two and markedly reduced in one out of the three challenged FC8 plants (Table 2Down).


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  		Table 2. Summary of challenge experiments of transgenic Digitaria sanguinalis

 
In a second challenge, 10 plants of line Tr2 (prep1–219Rb–), one plant of line FC8 and 11 control plants containing the bar transgene were challenged with an MSV field isolate (Table 2Up). Viruliferous leafhoppers in vials were fed first on the bar control plants for 2 days and then transferred to the rep-transgenic plants and allowed to feed for 2 days. Plants were monitored for symptoms for 5 weeks, after which they were tested for the presence of viral DNA.

bar controls 2.1, 2.3 and 2.6 developed symptoms 13 days p.i., whereas the corresponding Tr2.1, Tr2.3 and Tr2.6 plants never developed a symptomatic infection. Although Tr2.2, Tr2.7 and Tr2.9 did develop symptomatic infections, they were all obviously milder than those of their matched controls. In addition, Tr2.7 and Tr2.9 developed symptoms in fewer tillers than their matched controls; in particular, symptom development in Tr2.9 was limited to three tillers, compared with ten tillers in the severely infected control. Tr2.4 and Tr2.5 also developed mild symptoms and their matched controls were also only mildly symptomatic (severity score 1.5). The absence of symptoms on Tr2.8 by day 35 p.i. was most likely due to escape, as its matched control plant (fed on previously by the same leafhoppers) also remained symptomless.

As in challenge 1, FC8 (prepIII–Rb–) developed no symptoms and no viral DNA was detected, whereas the corresponding control plant showed symptoms and had high levels of viral DNA.

In a third challenge, five plants from transgenic line Tr1 (prep1–219Rb–) and five non-transgenic plants were treated as in challenge 2. Symptoms were monitored daily, whilst symptom severity was analysed on day 35 p.i. All controls developed symptoms after 15–17 days p.i., whereas transgenic Tr1 plant responses were variable (Table 2Up). Compared with matched controls, all Tr1 plants showed some resistance: this ranged from immunity (Tr1.5) to delayed symptom development (Tr1.2) to attenuated symptoms (Tr1.1 and Tr1.3). Only in the case of Tr1.4 was the severity of symptoms similar to that of the matched control. However, whereas the control plant developed symptoms in all leaves that emerged after MSV transmission, only the leaves of one tiller of Tr1.4 developed symptoms. Although Tr1.1 developed mild symptoms in leaves of three tillers, many newly emerged leaves remained non-symptomatic (see Fig. 5c, dUp), indicating that the spread of the virus from initially infected leaves may have been inhibited.

To better characterize some of these infections, DNA of three leaves taken from each plant 47 days p.i. was analysed by PCR (Fig. 5f, gUp). From the Tr1 plants showing symptoms, a symptomatic leaf (A), a newly emerged leaf immediately adjacent to the symptomatic leaf (B) and a newly emerged leaf on a different tiller distant from the symptomatic leaf (C) were tested to determine whether virus had spread from the symptomatic leaf. From non-transgenic controls, three young leaves (that had emerged after inoculation) were taken from three different areas of the plant. Viral DNA was present only in the symptomatic leaves of the transgenic plants Tr1.1 (leaf A), Tr1.2 (leaf A) and Tr1.3 (leaf A) (Fig. 5fUp), and even newly emerged leaves immediately adjacent to the symptomatic leaves (leaf B) contained no viral DNA. Plant Tr1.4 at 47 days p.i. had no new symptomatic leaves: the one tiller containing an infected leaf had died and no new infection emerged, correlating with the lack of viral DNA in any of the leaves analysed. Visible symptoms on Tr1.2 only emerged on one leaf on day 47 p.i.; viral DNA was subsequently amplified from this leaf (leaf A) by PCR, despite this plant having a symptom-severity score of 0 at day 35 p.i. (Table 2Up).

In a final challenge, two plants of line FC8 were leafhopper-inoculated with MSV. Leafhoppers were first fed on the transgenic plants and then transferred to non-transgenic control plants, to test the possibility that the resistance seen in this line was due to inoculum reduction in the leafhoppers during feeding on the control plants prior to transfer to the rep-transgenic plants in the previous challenges. Transgenic plants developed no symptoms, whereas the matched controls developed severe symptoms 12 days p.i. (Table 2Up). This indicates that inoculum levels are maintained at a sufficiently high level after the first feed to produce a severe infection following the subsequent inoculation period and that it is very probable that line FC8 is highly resistant, if not immune, to MSV infection.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Historically, attempts at controlling MSD have focused on evasive measures such as insecticide use and cultural practices or the breeding of resistant maize. A major problem facing breeders is the introduction of resistance traits without compromising crop quality and yield (Frischmuth & Stanley, 1993Down): natural genetic resistance is not usually available in varieties with improved agronomic qualities and transferring resistance traits without also transferring undesirable characteristics can be difficult. The transfer problem can become nearly insurmountable when resistance is polygenic, which appears to be the case with MSV resistance (Kim et al., 1989Down; Van Rensburg et al., 1991Down; Rodier et al., 1995Down; Welz et al., 1998Down). Although one major quantitative trait locus (QTL) for tolerance to MSV has been identified in Tzi4 (Msv1; Kyetere et al., 1995Down, 1999Down), an inbred line developed at the International Institute of Tropical Agriculture (Kim et al., 1987Down), improved resistance has been reported in lines containing QTLs on several additional chromosomes (Welz et al., 1998Down; Pernet et al., 1999aDown, bDown), which have not been detected in Tzi4 and probably contributed to the superior resistance in these lines.

According to Harrison (2002)Down, the most durable virus resistance will protect against a wide range of virus strains, require multiple mutations in the viral genome to overcome host resistance and involve a resistance mechanism that confines the virus to the inoculated cell. Examples of the latter include blocking replication or cell-to-cell movement of the virus. For this reason, we attempted to engineer MSV resistance by using trans-dominant-negative Rep protein variants that interfere with MSV replication. Various rep mutants were constructed and their ability to interfere with virus replication when provided in trans was tested (Fig. 2Up).

It is likely that the rep constructs inhibiting wt MSV-Kom replication behave in a dominant-negative manner. Whilst wt Rep and a pRBR-interaction motif (RepRb–) Rep variant both trans-replicate MSV-Kom efficiently (Shepherd et al., 2005Down), the addition of the III– mutation to both led to complete trans-inhibition of virus replication. This inhibition could occur via several routes, including the competitive occupation of Rep-binding sites on viral DNA and the assembly of defective Rep hetero-oligomers that could interfere with both virus transcription and replication. Rep activities are determined in part by the protein's aggregation state (Orozco et al., 2000Down) or, for mastreviruses, the aggregation state of Rep and/or RepA homo- and hetero-oligomers (Horvath et al., 1998Down; Missich et al., 2000Down). Horvath et al. (1998)Down identified the domains in MSV necessary for Rep and RepA homo- and hetero-oligomerization (Fig. 1Up). Of the rep constructs shown in Fig. 2Up, the only ones that inhibited virus replication were those expressing intact oligomerization domains. Interference with virus replication through transgene expression of Rep oligomerization domains has actually been suggested before as a mechanism for achieving African cassava mosaic virus and tomato leaf curl virus (ToLCV) resistance (Hong & Stanley, 1996Down; Chatterji et al., 2001Down). A truncated ToLCV Rep with sites for DNA cleavage, DNA binding and protein oligomerization interfered with DNA binding and oligomerization of Rep during virus infection (Chatterji et al., 2001Down). It is therefore probable that formation of hetero-oligomers with wt and defective mutant MSV Rep subunits would interfere with the functionality of Rep oligomers.

Whilst hetero-oligomerization with wt Rep could explain the inhibition of replication by RepA and RepARb–, alternative mechanisms could also be involved. Because Rep and RepA have different activities in mastrevirus replication, mechanisms altering their relative concentrations may control the progression of the infection cycle from replication initiation through to movement and encapsidation. It has been suggested that the mastrevirus wheat dwarf virus (WDV) RepA activates virion-sense gene expression and downregulates virus replication (Collin et al., 1996Down), which could explain why overexpression of MSV RepA inhibited virus replication in our assays. As RepARb– also inhibited virus replication completely, it is apparent that, as is the case with the mastrevirus Bean yellow dwarf virus (Hefferon & Dugdale, 2003Down), an intact pRBR-interaction motif is not required for MSV RepA-mediated replication inhibition.

The enhancement of virus replication by the truncated Rep1–179 and Rep1–179III– is intriguing. These Reps contain no oligomerization domain (Fig. 1Up). It has been shown in WDV that the formation of Rep oligomers occurs in a stepwise manner, the first being binding of a Rep monomer to DNA and the second being the sequential addition of Rep monomers (Missich et al., 2000Down). It has been suggested that the formation of a large oligomeric complex near the rep TATA box in the long intergenic region of WDV could inhibit rep transcription by interfering with the assembly or activity of the transcription pre-initiation complex (Castellano et al., 1999Down). By analogy with Rep DNA-binding domains mapped in other geminiviruses, Rep1–179 and Rep1–179III– are probably capable of binding near the rep TATA box and preventing Rep oligomerization there. This may either prevent the assembly of a replication-inhibiting transcription pre-initiation complex or encourage the replication-enhancing formation of a replication-initiation complex at the virion-sense origin of replication, as has been observed in WDV (Castellano et al., 1999Down).

Three of the constructs that inhibited MSV replication (Fig. 2Up) were chosen for transformation of D. sanguinalis as a test of their efficacy. These experiments provided some insights into potential MSV Rep–host factor interactions. None of the calli containing repIII– and bar regenerated; this inhibition of regeneration was possibly due to interaction of the expressed repIII– transgene with pRBR and disruption of cell-cycle regulation. The fact that calli transformed with the pRBR-interaction motif mutant prepIII–Rb– did regenerate provides evidence in favour of this hypothesis. Importantly, prepIII–Rb– also induced growth and developmental defects in transgenic plants, indicating that other Rep motifs interacting with host regulatory molecules are possibly expressed by this construct. These might include an MSV homologue of the WDV RepA GRAB-interaction domain (Xie et al., 1999Down) and/or the Rep myb-related motif that has been shown to have transcription-activation activity in yeast (Horvath et al., 1998Down). Either or both of these may have been responsible for the stunting and infertility phenotypes observed in regenerated prepIII–Rb– lines, as shown by the fact that prep1–219Rb– transgenics expressing truncated Reps missing these domains are phenotypically normal.

Challenges of prepIII–Rb– and prep1–219Rb– transgenic D. sanguinalis lines indicate that the trans-dominant-negative mutant strategy is effective. Whilst the mutated full-length repIII–Rb– conferred excellent MSV resistance, particularly in line FC8, the phenotypic side effects of this transgene will prevent its use in maize improvement. The truncated/mutated transgene rep1–219Rb– conferred, without any obvious phenotypic side effects, varying degrees of resistance in individual plants, including delayed symptom development, reduced symptom severity and decreased virus loads. The prep1–219Rb– construct has therefore been used to transform maize, and phenotypically normal, fertile transgenic offspring have been obtained (D. N. Shepherd, T. Mangwende, D. P. Martin, R. Kloppers, M. Bezuidenhout, C. H. Titus, A. L. Monjane, E. P. Rybicki and J. A. Thomson, unpublished).

We have also shown that the rep1–219Rb– transgene is effective against a range of MSV strains. Three different MSV variants were used in different challenges: the moderately severe cognate clonal MSV-Kom isolate, the extremely severe clonal MSV-MatB isolate and a field MSV isolate predominantly comprising viruses in the extremely severe and widely distributed A5 subgroup (data not shown) described by Martin et al. (2001)Down. This indicates that any resistance achieved in maize should be effective in sub-Saharan Africa, against even the most severe MSV isolates.


   ACKNOWLEDGEMENTS
 
Research performed at the University of Cape Town was partially supported by a grant from the South African National Research Foundation (D. N. S.) and from the Claude Harris Leon Foundation (T. M.). We thank Di James for sequencing, Rikus Kloppers (Pannar Pty Ltd) for performing the first challenge of D. sanguinalis line FC8 and Eric van der Walt for his work in identifying the viral population present in viruliferous leafhoppers used to challenge transgenic D. sanguinalis.


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Received 28 June 2006; accepted 7 September 2006.