Investigation of the potential of Maize streak virus to act as an infectious gene vector in maize plants

Arch Virol (2001) 146: 1089–1104

Investigation of the potential ofMaize streak virusto actas an infectious gene vector in maize plants

K. E. Palmer∗ andE. P. Rybicki

Department of Molecular and Cell Biology, University of Cape Town,Rondebosch, Western Cape, South Africa

Accepted December 6, 2000

Summary. There has been a great deal of interest in the possibility that gemini-viruses might be used as infectious gene vectors for expression of foreign proteinsin plants. However, generic mastreviruses such asMaize streak virus(MSV) haveno sequences which are dispensable for systemic infection of plants, and thereis a strict limitation on the size of viral DNA which can be moved systemically.We attempted to complement the movement functions deleted from a wild-type-sized, replication-proficient gene replacement vector, by co-infecting plants withit and either wild type MSV, or a replication-deficient but putatively movement-proficient viral construct. While ssDNA formation by the gene replacement vectorcould be complemented intransby co-transfected wild type virus, true systemicmovement of either the vector, or of co-complementing constructs, did not oc-cur. However, recombination between the two complementing viral constructsfrequently occurred to generate wild-type virus genomes. The results thereforesuggest that formation of ssDNA and size of the viral replicon are not the sole de-terminants of whether the MSV movement proteins can mobilise viral sequencesand move them systemically in plants.

Introduction

The taxonomic familyGeminiviridaeis a large diverse group of viruses withsmall ssDNA circular genomes and geminate virions, which replicate in the nu-clei of infected plant cells via a rolling circle mechanism. Geminiviruses havea minichromosome-like dsDNA replicative intermediate form that is the tem-plate for transcription of the viral genes. Viruses in the genusMastrevirushavemonopartite genomes of approximately 2.7 kb, with four open reading frames

∗Current address: Large Scale Biology Corporation, 3333 Vaca Valley Parkway, VacavilleCA 95688, USA.

1090 K. E. Palmer and E. P. Rybicki

(ORFs) encoding three genes [5]: for a review of mastrevirus molecular biology,see [23].Maize streak virus(MSV) is the type member of the genus [28, 29,40], the majority of which are the only geminiviruses capable of infecting cerealcrops.

There has been much interest in the prospect of developing infectiousgeminivirus-derived gene vectors for the systemic expression of non-viral se-quences in plants (reviewed by [22]). Bipartite geminiviruses (genus:Bego-movirus) such asAfrican cassava mosaic virusandTomato golden mosaic virus(ACMV and TGMV) have a DNA A which encodes replication-related proteins,and a DNA B which encodes movement proteins (MPs) [9]. They have consider-able potential as vectors as they do not require the coat protein gene for systemicinfections in permissive hosts [10, 11, 41]. In contrast, mastreviruses require func-tional coat and movement proteins for systemic movement [4, 18] and tolerateonly very small insertions into the viral genome, before wild-type systemic move-ment is abolished [33–35, 37]. We have previously shown that the MSV genomecan be used as a high-level, stable expression vector in a cell suspension culturesystem [25]; however, these constructs were not usable as agroinfectious vectorsin plants [21].

Although movement of mastreviruses in plants is not well understood, it ishypothesised that the coat and movement proteins interact to move the viralgenome out of the nucleus [17], and from cell-to-cell, in a similar fashion tothe movement protein complex of begomoviruses [15, 31]. The MP of MSV (MPgene= V1 ORF; [3]), like the begomovirus BC1-encoded MP, localises to thecell wall [7, 26]. The coat protein (CP) is essential for viral movement and isrequired for accumulation of ssDNA in protoplasts and around the inoculationsite in plants [2–4]. The CP of MSV, like the BV1 ORF-encoded nuclear shuttleprotein (NSP) of begomoviruses, binds both ssDNA and dsDNA without sequencespecificity in vitro [17, 19, 27]. It is possible that the mastrevirus MP is a functionalhomologue of the begomovirus MP, and that it performs similar functions toboth the begomoviral NSP and CP proteins: that is, it acts as a nuclear shuttleprotein, and causes ssDNA accumulation. The mechanisms involved in specificrecognition of the viral genome by its movement proteins are unknown.

We wished to test whether MSV could be made into an infectious “binary”systemic gene vector system, where sequences required for movement, and po-tentially also for replication, would be supplied intransbetween two components.The investigations described in this paper were therefore aimed at establishingwhether wild-type or mutant MSV could complement the movement-deficientphenotype of an MSV gene replacement construct in agroinfections, currentlythe only reliable way available to transfer MSV DNA into intact plants [8].

Materials and methods

All standard molecular biological techniques were performed as described in [30], and ac-cording to the specifications of enzyme manufacturers (Boehringer Mannheim, Promega andAmersham).

Movement of MSV vector plasmids 1091

MSV clones and gene replacement vectors

Partially dimeric clones of the sequenced infectious MSV-Kom isolate (Genbank accessionAF003952) in pUC19 or pBI121 were from FL Hughes and WH Schnippenkoetter of this lab-oratory (see Table 1 and Fig. 1); these were pMSV1.4mer, pMSV1.1mer and pMSV1.1merBI.Construct pMSV35S-bar was made by replacing a 1005 bpBamHI-NcoI fragment (V1 ORFand most of the V2 ORF) in the monomeric MSV genomic clone pMSVKom500 [14], with a1045-bp CaMV 35S promoter andbargene cassette from pKEP7503 [6, 25, 39]. The recombi-nant viral genome was dimerised in pUC19 (pMSV35S-bar-dim), with a 40-bpSmaI-BamHIdeletion of the V1 proximal region of the LIR: this was for introduction into theAgrobac-terium binary vector pBI121 (Clontech, Palo Alto, CA). The composition of recombinantviral constructs is described in Fig. 1 and Table 1. Inserts were excised from pUC19-basedconstructs withEcoRI andXbaI (flanking the insert), and introduced into similarly-digestedBI121 so that theXbaI site was proximal to the T-DNA right border. Clones inAgrobacteriumbinary vectors have the suffix “BI” added to the name of the clone in pUC19.

Site-directed mutagenesis

The Chameleon Site Directed Mutagenesis kit (Stratagene, La Jolla, CA) was used to intro-duce mutations into the MSV genome in clone pMSVKom500. Two additional nucleotideswere inserted into the C1 ORF at position 2304, creating a newPstI site, and inducing a+2frameshift and truncation of the C1 ORF in construct pMSV-PstI.

Agroinfection

The nopaline strain ofAgrobacterium tumefaciens, C58C1(pMP90) [16], was used for agroin-fections. Transformation ofA. tumefacienswas by the freeze-thaw method of Holsters etal. [13]. Transformants were selected and maintained on LA plates containing rifampicin(100mg/ml), gentamycin (40mg/ml) and kanamycin (100mg/ml). An overnight culture ofA. tumefacienswas concentrated by centrifugation and resuspended in one-fifth volume ofsterile distilled water. Different cultures were mixed in a 1:1 ratio prior to co-agroinfectionexperiments. Kernels of the sweetcorn cv. “Jubilee” were germinated at 30◦C in damp ster-ile vermiculite. Agroinfection of three day-old maize seedlings was by side injection at thecoleoptilar node, with 2–3ml of the Agrobacteriumsuspension in a 10ml Hamilton syringe[8]. Injected seedlings were transferred to soil in a plant growth room, at 25◦C with a 16/8hour day/night cycle. Infection was scored from 5 days post-inoculation.

Transient replication assay

An assay for replication of virus and virus-derived DNA replicons was done as describedelsewhere [25, 43]. Black Mexican Sweetcorn (BMS) suspension cultured cells were bom-barded with gold microprojectiles carrying pMSV35S-bar-dim, pMSV::MSV35S-bar-dimor pMSV1.1mer DNA (see Table 1), using a BioRad PDS-1000/He particle gun. After 3 days,low molecular weight nucleic acids were isolated from two of the plates from each set [1],run on an agarose gel, blotted and hybridised with a probe homologous to thebar gene.

DNA manipulations

Total plant DNA or DNA from bombarded callus was isolated as described [24, 25]. Poly-merase chain reaction (PCR) amplification of MSV sequences was with primers derivedfrom the MSV-Kom sequence positions 1770–1792 and 215–234: these amplify 1304 bpin the MSV-Kom genome, from near the 5′ end of the C2 ORF to within the V1 ORF(see Fig. 1). The PCR cycle conditions were 95◦C/120 sec, 30× (94◦C/45 sec, 54◦C/45 sec


and 72◦C/90 sec), and final elongation at 72◦C for 180 seconds. PCRs for detection ofthebar gene were with primers BARP1 (5′-CGTCAACCACTACATCGAG-3′) and BARP2(5′-GAAACCCACGTCATGCCAG-3′), which amplify a 413 bp fragment. PCR cycle con-ditions were 94◦C/60 sec, 30× (94◦C/45 sec, 53◦C/30 sec, 72◦C/45 sec), and 72◦C for 300seconds. MSV genome andbar gene fragments were labelled with digoxygenin-labelleddUTP by PCR (Boehringer Mannheim, “Dig User’s Guide to Filter Hybridisation”).

Results

Construction ofAgrobacteriumbinary vectors for agroinfection

The various constructs used in agroinfection experiments are explained in Table 1and Fig. 1. When “trimeric” constructs were cloned thebar construct dimer wasalways cloned at the right border side of theAgrobacteriumT-DNA. As the rightborder is transferred first into plant cells [44], this ensured that the movementdefective virus was always present in infected cells before the complementingvirus.

Release of monomeric replication-competent replicons is explained in Table1. Replication-defective constructs on the same T-DNA as a replication-proficientconstruct, or in the same cell as a replication-proficient replicon, could be releasedeither by homologous replication or intransby Rep-mediated replicative release[38]. The bar gene replicons should be the same size as wild type MSV-Kom(2690 bp) while the replication-deficient MSV-PstI (“ PstI mutant”) should betwo bases larger than the wild type genome (Table 1).

Transient replication assay in BMS cells

Cells bombarded with pMSV35S-bar-dim (CaMV35S-bar vector dimer) con-tained replicative form DNAs, but with no typical ssDNA forms (Fig. 2, lanes 5and 6). In contrast, cells bombarded with pMSV35S-bar-dim::MSV (vector dimerplus wt MSV) clearly contained extra bands running at the position expectedfor ssDNA (Fig. 2, lanes 3 and 4; see also Fig. 4B; [25]). Thebar gene probedid not hybridise to DNA from cells bombarded with pMSV1.1mer (wt MSV;Fig. 2, lanes 1 and 2). Extracts from cells bombarded with pMSV1.1mer andpMSV35S-bar-dim::MSV, but not pMSV35S-bar-dim, contained small numbersof typical geminate particles when tested by immunoelectron microscopy (resultnot shown).

Agroinfection of sweetcorn cv. Jubilee

Seedlings agroinfected with the vectors described in Table 1 were scored for in-fection from 5 days after injection to 21 days after injection. Injection with any ofpMSV1.4mer, pMSV1.1merBI (both wt MSV), pMSV35S-bar-dim::MSVBI andpMSV35S-bar-dim::MSV-PstIBI (vector dimer plusPstI mutant MSV) yieldedplants with typical symptoms of MSV-Kom infection (not shown). Plants in-oculated with pMSV35S-bar-dimBI and pMSV-PstI-dimBI (PstI mutant MSVdimer) remained asymptomatic. In three separate experiments a total of five outof 90 plants inoculated became wild-type MSV symptomatic after injection with


Fig. 1. Map representations of MSV-Kom-derived constructs.A pMSVKom500. This isshown with the MSV genome insert only, linearised at the uniqueBamHI site: V1, V2 arevirion-sense ORFs, coding for movement (MP) and coat (CP) proteins respectively;C1andC2ORFs are exons of theRepgene;LIR is the long intergenic region containing virion- andcomplementary-sense promoters;SIRis the short intergenic region containing polyadenyla-tion sites in both senses. Restriction sites of relevance to cloning are shown, as are markersshowing how agroinfectious constructs were derived. Sites of binding and directions of elon-gation of the MSV-specific PCR primers (MSV215, MSV1792) are shown by small arrowsand dotted lines.B Replicative constructs. The different sizes and organisations of the MSVgenome-derived constructs are shown. Genome designations are as shown inA. 35S-baris the CaMV 35S Pr with adjacentbar gene from pHP7503. The designation pMSV-35S-bar::MSV indicates a clone with the MSV-35S-bar chimaera cloned 5′ of a native MSV-Kom

genome monomer

a mixture ofAgrobacteriumstrains carrying pMSV35S-bar-dimBI and pMSV-PstI-dimBI. Table 2 summarises the results of three separate agroinfection ex-periments withAgrobacteriumstrains carrying the seven binary vector constructsdescribed in Table 1.

The time courses of infection of plants injected with each of these agroin-fectious constructs are presented in Fig. 3. There was a clear difference in thekinetics of the rate of infection associated with plasmids from which the wild-



Fig. 2. Complementation of ssDNA formation. BMScells were bombarded as described with pMSV1.1mer(releases infectious wild-type MSV), pMSV35S-bar-dim::MSV (trimer which releases wild-type and CaMV35S-bar MP/CP replacement vector), and pMSV35S-bar-dim (CaMV 35S-bar MP/CP replacement vectordimer). LMW DNA was isolated and run undigestedon a 0.8% agarose gel, blotted to nylon membrane,and probed with a Dig-labelled bar gene probe whichwas detected with a chromogenic substrate.1, 2pMSV1.1mer; 3, 4 pMSV35S-bar-dim::MSV; 5, 6pMSV35S-bar-dim. Labels down the side of the blotindicate replicative and other DNA forms detected.ocOpen circular dsDNA;ccc covalently closed circular(= plasmid-like) dsDNA;ss single-stranded circular(= genomic) DNA

Table 2. Summary of the results of agroinoculation experiments

Binary vector in Experiment I Experiment II Experiment III TotalA. tumefaciens no. symptomatic/ no. symptomatic/ no. symptomatic/ percentage

no. surviving no. surviving no. surviving symptomatic

pMSV1.4mer 17/28 8/12 N.D.b 63%pMSV1.1merBI 13/13 10/10 N.D.b 100%pMSV35S-bar- 0/26 0/22 0/11 0dimBIpMSV35S-bar- 33/35 49/52 14/14 95%dim::MSVBIpMSV35S-bar::MSVBI 21/24 12/12 N.D.b 92%pMSV-PstI-dimBI 0/24 0/10 0/12 0pMSV35S-bar- 46/58 22/25 12/13 83%dim::MSV-PstIBIpMSV35S-bar- 1/51 3/27 1/12 6%dimBI+pMSV-PstI-dimBIa

aAgrobacteriumcultures were mixed in a 1:1 ratio before injectionbOnly two repeats of this experiment were doneN.D. Not determined

type virus could escape by replicative release (pMSV1.1merBI; pMSV35S-bar-dim::MSVBI and pMSV35S-bar::MSVBI; see Table 1), compared with wherethe wild-type virus could escape only by recombination (pMSV1.4mer andpMSV35S-bar-dim::MSV-PstIBI). The main difference between infections withpMSV1.4mer and pMSV35S-bar-dim::MSV-PstIBI was that inoculation withpMSV35S-bar-dim::MSV-PstIBI usually resulted in a higher percentage of in-fected plants.


Fig. 3. Infection kinetics after agroinfection of sweetcorn seedlings with five different agroin-fectious constructs. The rate of symptom development in sweetcorn seedlings agroinfectedwith Agrobacteriumcontaining wild-type agroinfectious MSV constructs pMSV1.4mer andpMSV1.1merBI, and constructs carrying MSV35Sbar CP and MP gene replacement con-structs. Day 0 is the day of injection. The results shown are for one experiment series (thesecond column in Table 2), but other repeats of the same experiment showed virtually identi-cal results. Data for agroinfection with pMSV35S-bar-dimBI and pMSV-PstI-dimBI are not

shown, as these constructs did not induce symptoms in agroinfected seedlings

The first symptoms on plants infected with pMSV1.1merBI, pMSV35S-bar::MSVBI (vector plus wt MSV heterodimer) and pMSV35S-bar-dim::MSVBI(= “replicative release” constructs) were observed very occasionally on the firstleaf, but most seedlings first showed symptoms on the second leaves, and allinfected seedlings showed symptoms on their third leaves. In contrast, plantsagroinfected with pMSV1.4mer and pMSV35S-bar-dim::MSV-PstIBI (= “re-combinational release” constructs) never showed symptoms on the first leaf:10 to 20% showed symptoms on the second leaf; most showed symptoms onthe third leaves, but occasionally only on the fourth leaf. Of five plants withsymptoms after co-agroinfection with a mixture of pMSV35S-bar-dimBI andpMSV-PstI-dimBI, one showed symptoms from its third leaf, and four plantsshowed symptoms first on their fourth leaves.

Detection ofbarreplicons in agroinfected sweetcornby Southern hybridisation

Figure 4 shows a Southern blot of undigested total DNA isolated from the firstthree leaves of plants agroinfected with pMSV35S-bar-dimBI and pMSV35S-bar-dim::MSV-PstIBI. The membrane was hybridised first with abar gene probe


Fig. 4. Southern blot analysis ofthe spread ofbar-gene replacementconstructs and MSV in plants. DNAisolated from the first three leavesof six randomly selected plants (in-dicated above the lane numbersin the figure) agroinfected withApMSV35S-bar-dimBI (vector dimer)and B pMSV35S-bar-dim::MSV-PstIBI (trimer with PstI mutantwild type and vector dimer) waselectrophoresed in a 0.8% agarosegel, and transferred to nylon mem-branes. In both cases, the top blotwas probed with a digoxygenin-labelledbar probe, then stripped andprobed with an MSV-homologousprobe (bottom). Plant 4 inB didnot show MSV symptoms.1 in thetwo blots in B contained DNA iso-lated from a plant agroinfected withpMSV1.1merBI (wild type virus only)

to detect the MSV-CaMV35Sbar construct only, then stripped and hybridisedwith an MSV-specific probe which would detect all replicons. Low MWbar-homologous DNA forms were seen in four of six plants agroinfected withpMSV35S-bar-dimBI (Fig. 4A, plants 1, 2, 3 and 6). Replicon DNAs werepresent in the second leaves of all four plants, and in 3 of 4 plants were mostconcentrated in the first or second leaves. Only plant 1 (Fig. 4A) had detectablebar-homologous DNA present in its third leaf. Of 6 plants agroinfected withpMSV35S-bar-dim::MSV-PstIBI, bar gene DNA was present in the second leafof one plant (plant 3, Fig. 4B), but replicating viral DNAs were detected with anMSV probe in the third leaves of 5 of 6 plants (Fig. 4B).

Detection of vector constructs in agroinfected plants by PCR

PCR was used to detectbar sequences in agroinfected plants because its sensitiv-ity would allow detection of replicatingbar constructs present at very low levels.This was necessary as the long exposure times required in Southern blots to detectbarsequences usually resulted in low-level non-specific hybridisation to the muchhigher copy number MSV sequences (not shown), unless such high stringency


Fig. 5. Spread ofbargene replacement construct and MSVRepframeshift mutants in agroin-fected plants detected by PCR. Gels inA contains PCR products amplified from five or sixleaves of each of three plants agroinfected with pMSV35S-bar-dim::MSV-PstIBI; primersused in each case are indicated alongside. The MSV-specific products were digested withPstIto distinguish between normal andPstI mutant replicating genomes. The gels inB show PCRproducts from amplification with primers as indicated; MSV products were digested withPstI. 1–7contain DNA isolated from each of the first seven individual leaves from one plant

which was agroinfected with a mixture of pMSV35S-bar-dimBI and pMSV-PstI-dimBI

washes were done that the detection of thebarsequences became relatively insen-sitive (see Fig. 4). PCR with primers MSV1770–1792 and MSV215–234 ampli-fied a 1304 bp fragment: digestion of these withPstI did not affect products fromwild-type genomes, but produced two fragments of 709 and 595 bp from PCRproducts containing theRepgenePstI mutation (eg: derived from pMSV-PstI).The primers would not amplify a product from the input DNA except from con-structs where the priming sites were duplicated, for example in a largely dimericor trimeric construct.

Total DNA was isolated from the first 5–7 leaves of plants agroinfected withpMSV35S-bar-dimBI, pMSV35S-bar-dim::MSVBI and pMSV35S-bar-dim::MSV-PstIBI alone, and from plants showing symptoms after co-agroinfectionwith pMSV35S-bar-dimBI and pMSV-PstI-dimBI. DNA isolated from the thirdleaves of plants infected with MSV-Kom by agroinfection with pMSV1.1merBI,and uninfected plants injected with plasmids pMSV-PstI-dimBI and pMSV35S-bar-dimBI, served as controls for the PCR.


Thebar gene fragment was amplified from all but one of the plants agroin-fected with the simple vector dimer construct pMSV35S-bar-dimBI (see Table 2).Similarly to plants agroinfected with pMSV35S-bar-dim::MSVBI (vector dimerplus wild-type MSV),bar DNA was present in the first two or three leaves, andoccasionally in the fourth leaf (not shown). This showed that the presence of a“helper” wild-type genome did not noticeably assist the spread of vector DNA.

Figure 5 shows the results of PCR amplification of a 418-bp fragment ofthebar gene and of a 1304 bp fragment of the MSV genome from the same leafsamples of plants agroinfected with pMSV35S-bar-dim::MSV-PstIBI alone (vec-tor dimer plus PstI mutant MSV), or with pMSV35S-bar-dimBI (vector dimer)and pMSV-PstI-dimBI (PstI mutant MSV dimer). For plants agroinfected withpMSV35S-bar-dim::MSV-PstIBI, with one exception thebarDNA did not spreadhigher than the fourth leaf; when it did, it was apparently present at very low copynumber (Fig. 5A, plant 2). In plants agroinfected with pMSV35S-bar-dim::MSV-PstIBI, or with both pMSV-PstI-dimBI and pMSV35S-bar-dimBI, DNA withthePstI-associated frameshift was always present, but its concentration droppedsteadily in higher leaves, often paralleling the decline in concentration of thebarconstruct (Fig. 5A and B). However, thePstI mutant virus usually persisted longerthan the autonomously replicatingbar construct (Fig. 5A). In allPstI-digestedsamples, the presence of a full-length 1304-bp PCR product in addition to twosmaller fragments indicated the presence of a wild-type viral DNA that had lostthePstI mutation, as well as of thePstI mutant virus. In all cases, the PCR prod-ucts generated from control pMSV35S-bar-dim::MSVBI DNA (vector dimer plusMSV) did not digest to any extent withPstI, while products of pMSV-PstI-dimBIamplification digested completely withPstI (not shown).

Discussion

Work by others has previously indicated that “engineered” MSV genomescannot move systemically to the same extent as wild-type virus in agroinocu-lated plants [18, 37]. To eliminate the possibility that lack of systemic movementwas due to even minor differences in size between the gene replacement constructand the wild type virus, we constructed an MSV vector exactly the same size asthe wild type viral genome. This had a CaMV35S-barexpression cassette in placeof the virion-sense ORFs: in previous studies, Shen and Hohn [37] used a similarcassette in substantially larger recombinant viruses (4.6 and 5.6 kb). Despite thedifferent vector sizes, however, our results reiterate those of Lazarowitz et al.[18] and Shen and Hohn [36, 37], who showed that MSV-based vectors are usefulonly as transient expression vectors in the first two or three leaves of agroin-fected seedlings. Thus, it appears that there is little potential for development ofmastrevirus-based gene vectors for systemic expression of foreign genes in cerealplants, unlike their potential for high-level long-term protein expression in cellculture systems, previously shown by us [25].

The requirement for coat protein for movement of monopartite geminivirusesremains largely uninvestigated; however, it is well established that the formation


of ssDNA is strongly linked to the expression of the coat protein gene: mastre-viruses with null mutations or deletions of the coat protein gene do not accumu-late detectable levels of ssDNA ([3, 4, 18, 42]; this study). In this study, we haveshown complementation of the ssDNA-negative phenotype of the coat proteingene replacement vector construct by co-infecting with wt MSV (Fig. 2). Thiscould either have represented sequestration of the recombinant ssDNA genomeby encapsidation, or a coat protein-induced switch from accumulation of predom-inantly dsDNA genome forms, to production of ssDNA forms. It was possible todetect geminate particles in these same cells; however it was not possible to showconclusively that these particles containedbar-specific DNA. Although prelimi-nary antibody-capture PCR results indicated they did, the experiments could notbe satisfactorily repeated (KE Palmer and EP Rybicki, unpubl.).

Given that CP expression complemented the formation of ssDNA in theCaMV35S-bar vector, it is difficult to explain why movement of the vector wasnot significantly complemented by the wild type virus, as our cloning strategy en-sured that every cell which became infected with MSV also had the vector present.Replicative release of a monomeric replicon is far more efficient than release byhomologous recombination from partial multimers [12, 38]; thus, both the vectorand the wild type (orPstI mutant) MSV genomes should escape by replicativerelease from any “trimer” construct (see Table 1). However, the infection kinet-ics of pMSV35S-bar-dim::MSV-PstIBI (Fig. 3) most closely resembled those ofpMSV1.4mer, which produces wild type virus only by homologous recombina-tion, and were markedly delayed compared to plants infected with pMSV35S-bar-dim::MSVBI, where both virus genomes presumably escape by replicativerelease.

This phenomenon is well documented in our laboratory for agroinfectiousconstructs of several MSV strains [20, 32]. Thus, it can be assumed either that com-plementation of functions between the movement-deficientbar vector constructand replication-deficientPstI mutant did not occur, and that infection-competentvirus arose by homologous recombination in the original T-DNA, or that replica-tive release of both virus constructs occurred, and that homologous recombinationbetween the replicatingbar construct and thetrans-replicatedPstI construct oc-curred subsequently. We favour the second possibility, because replicative releaseis a more efficient process. In addition, the proportion of plants infected with MSVafter pMSV35S-bar-dim::MSV-PstIBI inoculation was higher than is usual with“homologous recombination escape” constructs (Fig. 3; WH Schnippenkoetter,DP Martin and EP Rybicki, unpublished results). The fact that genomes carryingthePstI frameshift persisted into later leaves in some plants shows that replicationfunctions can betrans-complemented in MSV infections. These results providethe first evidence fortransreplication of MSV mutantsin planta. Plants carryingthe replicating virus mutant did not show any associated symptom amelioration,so the mutant did not function as a DI-DNA, at least in sweetcorn, which is ahighly susceptible host.

In the case of co-agroinoculation of pMSV35S-bar-dimBI and pMSV-PstI-dimBI, only a small proportion of plants developed symptoms. In this case, both


constructs would presumably have to have been delivered to the same cell, asthebar construct in pMSV35S-bar-dimBI did not contain the movement or coatprotein genes to move and pMSV-PstI-dimBI contained a frameshift in theRepgene. Thus, recombination to form wt virus would probably have occurred post-release, as the constructs were on different T-DNAs.

Our results suggest that the formation of a genome competent for both repli-cation and movement appears to be a prerequisite for symptomatic infection withMSV. Boulton et al. [4] found that they could achieve infection by complemen-tation of virus movement functions intrans, but in the context of genomes whichwere otherwise intact. However, these authors also found, as we did, that wildtype virus eventually arose by homologous recombination between complement-ing mutants, in every case. In the light of the results presented here, it is reasonableto propose that genome size and formation of ssDNA are not the sole factors thatdetermine whether a virus genome is capable of systemic movement with coatprotein and movement protein provided intrans.

In their DNA binding studies, Liu et al. [19] found no evidence for sequencespecificity in binding of MSV CP or MP to viral ss- or dsDNAs, nor is there anyother evidence for specificity with other mastreviruses. However, Pascal et al.[27] did note some affinity of the NSP for the begomoviral common region, andFrischmuth et al. [9] found some apparent sequence specificity associated withbegomovirus B component-encoded movement proteins: ACMV could comple-ment movement of bothAbutilon mosaic virus(AbMV) and TGMV DNA A;however, although these were capable of mobilising each other’s DNA A, nei-ther could move DNA A of ACMV. This implies an element of specificity ingenome recognition, by one or both of the begomoviral B component movementproteins.

Evidence for sequence-specific interactions from our results is that replication-defectivePstI RepAgene mutants were essentially fully complemented in bothreplication and movement by wild-type genomes, whereas identically-sizedbargene replacement constructs were only effectively complemented in replicationand ssDNA formation. It is possible that sequences essential for interaction ofthe recombinant viral genome with a “movement complex” – possibly consist-ing of CP and MP – were deleted in the 1045-bpSmaI-NcoI fragment of theMSV-Kom genome absent in ourbar gene replacement vector. Geminivirus en-capsidation/movement protein interaction signals may not be specific sequences,but rather secondary or tertiary ssDNA structure(s). It is also possible that for-mation of ssDNA and encapsidation can be uncoupled, so that although thebarconstructs could be made into ssDNA, they could not be encapsidated, thus ad-versely affecting their extracellular movement. These possibilities remain to beinvestigated.

Acknowledgements

KEP acknowledges support from AECI Ltd, and KEP and EPR are grateful for supportfrom the Foundation for Research Development. We thank Mohammed Jaffer for electronmicroscopy.


References

1. Anat S, Subramanian KN (1992) Isolation of low molecular weight DNA from bacteriaand animal cells. Methods Enzymol 216: 20–29

2. Azzam O, Frazer J, de la Rosa D, Beaver JS, Ahlquist P, Maxwell DP (1994) White-fly transmission and efficient ssDNA accumulation of bean golden mosaic geminivirusrequire functional coat protein. Virology 204: 289–296

3. Boulton MI, Pallaghy CK, Chatani M, Macfarlane S, Davies JW (1993) Replicationof maize streak virus mutants in maize protoplasts – evidence for a movement protein.Virology 192: 85–93

4. Boulton MI, Steinkellner H, Donson J, Markham PG, King DI, Davies JW (1989) Muta-tional analysis of the virion-sense genes of maize streak virus. J Gen Virol 70: 2309–2323

5. Briddon RW, Markham PG (1995)Geminiviridae. In: Murphy FA, Fauquet CM, BishopDHL, Ghabrial SA, Jarvis AW, Martelli GP, Mayo MA, Summers MD (eds) Virus Tax-onomy. Classification and Nomenclature of Viruses. Sixth Report of the InternationalCommittee on Taxonomy of Viruses. Springer, Wien New York, pp 158–165 (Arch Virol[Suppl] 10)

6. D’Halluin K, De Block M, Denecke J, Janssens J, Leemans J, Botterman J (1992) Thebar gene as a selectable and screenable marker in plant engineering. Methods Enzymol216: 415–426

7. Dickinson VJ, Halder J, Woolston CJ (1996) The product of maize streak virus ORF V1is associated with secondary plasmodesmata and is first detected with the onset of virallesions. Virology 220: 51–59

8. Escudero J, Hohn B (1994) Agroinfection. In: Freeling M, Walbot V (eds) The maizehandbook. Springer, New York, pp 599–603

9. Frischmuth T, Roberts S, von Arnim A, Stanley J (1993) Specificity of bipartite gemi-nivirus movement proteins. Virology 196: 666–673

10. Hayes RJ, Coutts RH, Buck KW (1989) Stability and expression of bacterial genes inreplicating geminivirus vectors in plants. Nucleic Acids Res 17: 2391–2403

11. Hayes RJ, Petty IT, Coutts RH, Buck KW (1988) Gene amplification and expression inplants by a replicating geminivirus vector. Nature 334: 179–182

12. Heyraud F, Matzeit V, Schaefer S, Schell J, Gronenborn B (1993) The conserved nonanu-cleotide motif of the geminivirus stem-loop sequence promotes replicational release ofvirus molecules from redundant copies. Biochimie 75: 605–615

13. Holsters M, De Waele D, Depicker A, Messens E, Van Montagu M, Schell J (1978)Transfection and transformation ofAgrobacterium tumefaciens. Mol Gen Genet 163:181–187

14. Hughes FL, Rybicki EP, von Wechmar MB (1992) Genome typing of southern Africansubgroup-1 geminiviruses. J Gen Virol 73: 1031–1040

15. Jeffrey JL, Pooma W, Petty IT (1996) Genetic requirements for local and systemicmovement of tomato golden mosaic virus in infected plants. Virology 223: 208–218

16. Koncz C, Schell J (1986) The promoter of TL-DNA gene5 controls the tissue-specificexpression of chimaeric genes carried by a novel type ofAgrobacteriumbinary vector.Mol Gen Genet 204: 383–396

17. Kotlizky G, Boulton MI, Pitaksutheepong C, Davies JW, Epel BL (2000) Intracellularand intercellular movement of maize streak geminivirus V1 and V2 proteins transientlyexpressed as green fluorescent protein fusions. Virology 274: 32–38

18. Lazarowitz SG, Pinder AJ, Damsteegt VD, Rogers SG (1989) Maize streak virus genesessential for systemic spread and symptom development. EMBO J 8: 1023–1032

19. Liu H, Boulton MI, Davies JW (1997) Maize streak virus coat protein binds single- anddouble-stranded DNA in vitro. J Gen Virol 78: 1265–1270


20. Martin DP, Willment JA, Rybicki EP (1999) Evaluation of maize streak virus pathogenic-ity in differentially resistantZea maysgenotypes. Phytopathology 89: 695–700

21. Palmer KE (1997) Investigations into the use of maize streak virus as a gene vector. PhDThesis, University of Cape Town

22. Palmer KE, Rybicki EP (1997) The use of geminiviruses in biotechnology andplant molecular biology, with particular focus on Mastreviruses. Plant Sci 129:115–130

23. Palmer KE, Rybicki EP (1998) The molecular biology of mastreviruses. Adv Virus Res50: 183–234

24. Palmer KE, Schnippenkoetter WH, Rybicki EP (1998) Geminivirus isolation and DNAextraction. In: Foster G, Taylor S (eds) Plant virology protocols: from virus isolation totransgenic resistance. Humana Press, Totowa, pp 41–52

25. Palmer KE, Thomson JA, Rybicki EP (1999) Generation of maize cell lines containingautonomously replicating maize streak virus-based gene vectors. Arch Virol 237: 1345–1360

26. Pascal E, Goodlove PE, Wu LC, Lazarowitz SG (1993) Transgenic tobacco plants ex-pressing the geminivirus BL1 protein exhibit symptoms of viral disease. Plant Cell 5:795–807

27. Pascal E, Sanderfoot AA, Ward BM, Medville R, Turgeon R, Lazarowitz SG (1994) Thegeminivirus BR1 movement protein binds single-stranded DNA and localizes to the cellnucleus. Plant Cell 6: 995–1006

28. Pringle CR (1999) Virus Taxonomy – 1999. The Universal System of Virus Taxon-omy, updated to include the new proposals ratified by the International Committee onTaxonomy of Viruses during 1998. Arch Virol 144: 421–429

29. Pringle CR, Fauquet CM (1998) ICTV announcement – ratification of new taxonomicproposals. Arch Virol 143: 2504

30. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nded. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

31. Sanderfoot AA, Ingham DJ, Lazarowitz SG (1996) A viral movement protein as a nu-clear shuttle. The geminivirus BR1 movement protein contains domains essential forinteraction with BL1 and nuclear localization. Plant Physiol 110: 23–33

32. Schnippenkoetter WH (1998) The use of agroinfectious clones to investigate recom-bination between distinct maize streak virus strains. PhD Thesis, University of CapeTown

33. Shen W-H, Hohn B (1994) Amplification and expression of theb-glucuronidase genein maize plants by vectors based on maize streak virus. Plant J 5: 227–236

34. Shen WH, Hohn B (1991) Mutational analysis of the small intergenic region of maizestreak virus. Virology 183: 721–730

35. Shen WH, Hohn B (1992) Excision of a transposable element from a viral vector intro-duced into maize plants by agroinfection. Plant J 2: 35–42

36. Shen WH, Hohn B (1994) Amplification and expression of the beta-glucuronidase genein maize plants by vectors based on maize streak virus. Plant J 5: 227–236

37. Shen WH, Hohn B (1995) Vectors based on maize streak virus can replicate to high copynumbers in maize plants. J Gen Virol 76: 965–969

38. Stenger DC, Revington GN, Stevenson MC, Bisaro DM (1991) Replicational release ofgeminivirus genomes from tandemly repeated copies: evidence for rolling-circle repli-cation of a plant viral DNA. Proc Natl Acad Sci USA 88: 8029–8033

39. Thompson CJ, Movva NR, Tizard R, Crameri R, Davies JE, Lauwereys M, Botterman J(1987) Characterization of the herbicide-resistance genebar from Streptomyces hygro-scopicus. EMBO J 6: 2519–2523

1104 K. E. Palmer and E. P. Rybicki: Movement of MSV vector plasmids

40. van Regenmortel MHV, Bishop DH, Fauquet CM, Mayo MA, Maniloff J, Calisher CH(1997) Guidelines to the demarcation of virus species [news]. Arch Virol 142: 1505–1518

41. Ward A, Etessami P, Stanley J (1988) Expression of a bacterial gene in plants mediatedby infectious geminivirus DNA. EMBO J 7: 1583–1587

42. Woolston CJ, Reynolds HV, Stacey NJ, Mullineaux PM (1989) Replication of wheatdwarf virus DNA in protoplasts and analysis of coat protein mutants in protoplasts andplants. Nucleic Acids Res 17: 6029–6041

43. Xie Q, Suarez-Lopez P, Gutierrez C (1995) Identification and analysis of a retinoblastomabinding motif in the replication protein of a plant DNA virus: requirement for efficientviral DNA replication. EMBO J 14: 4073–4082

44. Zupan J, Zambryski P (1995) Transfer of T-DNA fromAgrobacteriumto the plant cell.Plant Physiol 107: 1041–1047

Author’s address: Dr. E. P. Rybicki, Department of Molecular and Cell Biology,University of Cape Town, Private Bag, Rondebosch 7701, Western Cape, South Africa;e-mail: [email protected]

Received December 6, 2000

Documents

Investigation of the potential of Maize streak virus to act as an infectious gene vector in maize plants