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1 Targeting MSV proteins for development of MSD resistance in tropical maize

Allan Jalemba Mgutu, 2Tara Nash, 2Linda Hanley-Bowdoin, 3 Dionne Shepherd and 1Jesse Machuka*

Department of Biochemistry and Biotechnology, Plant Transformation Department of Molecular and Structural Biochemistry, North Carolina State Department of Molecular and Cell Biology, University of Cape Town, Private

Laboratory, Kenyatta University, P.O .BOX 43844 - 00100, Nairobi, Kenya.


University, Raleigh, NC 27695-7622, North Carolina, USA.


Bag, Rondebosch, Cape Town 7701, South Africa Review perspective paper

Running Title: Protein strategies for creating MSD resistance

*Correspondingauthor: PlantTransformationLaboratory, DepartmentofBiochemistryandBiotechnology, KenyattaUniversity, P.O.Box4384400100,Nairobi,Kenya. Tel.+25402810245/813460;Fax.+25402811575 E-mail:

2 Targeting MSV proteins for development of MSD resistance in tropical maize ABSTRACT Maize streak virus (MSV) disease (MSD) causes a dramatic decrease in tropical maize yields in sub-Saharan Africa. The disease, characterised by yellow streaks along the maize leaf vasculature, limits photosynthesis leading to reduced or no grain filling, stunting and death in old and young plants, respectively. Control of MSD is mainly achieved via development of resistant maize varieties through conventional breeding. These varieties are compromised in other agronomical traits including yield, grain quality, abiotic, disease or pest tolerance caused by the linkage drag phenomenon. Although cultural control measures contain MSD in some regions, there are multiple impeding challenges including unpredictable weather patterns and limited genetic resistance among the farmer preferred maize cultivars. The scanty available genetic resistance is poorly understood at the molecular level. There is great need for alternative control strategies that compliment classical breeding based on a high-throughput molecular understanding of the disease coupled with comprehensible genetic manipulations. This is holistically termed molecular plant breeding. This approach has potential in facilitating brisk upgrading of the available parental maize germplasm to curb many production stresses including MSD. Here, we discuss molecular-based strategies tenable in understanding and controlling MSD in tropical maize using modern biotechnology. In particular, we examine progress towards generating MSD resistant transgenic tropical white maize in Africa by interfering with the hostpathogen protein interactome.

3 INTRODUCTION Improvement of tropical maize for stress tolerance: An overview Tropical white maize has been the principal food staple for communities in tropical sub-Saharan Africa (SSA) and Latin America for over a century (Pratt et al., 2003; Anami et al., 2009). As a consequence, there have been significant research efforts aimed at increasing tropical maize productivity by improved disease (Bosque-Perez, 2000; Pratt et al., 2003), drought (Anami et al., 2009) pest (Mugo et al., 2005; Stevens, 2008) parasitic (David-Schwartz et al., 2008) and general weed management (Kanampiu et al., 2002). These efforts have focused mainly on developing varieties that are tolerant or resistant to the abiotic and biotic production constraints through conventional breeding (Ribaut and Hoisington, 1998; Morris, 2002; Pratt et al., 2003) and marker assisted breeding (Welz et al., 1998; Moose and Mumm, 2008; Stevens, 2008; Ribaut et al., 2010). There exist maize varieties developed via conventional breeding that are resistant or tolerant to some of the stresses above (Efron et al., 1989; Barrow, 1992; Bosque-Perez, 2000; Pratt et al., 2003; Abalo et al., 2009). However, these varieties do not meet the prerequisites of growing across the vast agricultural land niches of Africa and retaining other beneficial biotic and abiotic adaptations while still maintaining high yields (Machuka, 2003; Barros et al., 2010). In addition, the capacity of pests, pathogens and parasites to rapidly evolve resistance to newly developed control strategies, natural or otherwise compounds these problems. Toovercometheseproblemsincludingamongothers,linkagedrag(Prattetal.,2000),astrategy holisticallytermedmolecularplantbreedingthatintegratesgeneticengineering,markerassisted breedingandconventionalbreedinghasbeenproposed(MooseandMumm,2008).Machuka (2004)andThomson(2008)havediscussedthebenefitssuchintegrationwouldbringtoAfrican agriculturalsystems,especiallyforthesmallandmiddlescalefarmerswhoarethemajorityfood producersinSSA.Webrieflydiscussthebiotechnologicalstrategiescurrentlyappliedtocontrol maize streakvirusdisease(MSD)becausethis hasrecentlybeenreviewed(Shepherdet al., 2009;2010).However,webeginbyreportingonthecurrentstatusofmolecularvirologyof MSD, which we believe underpins progress towards achieving the stable and durable MSD resistanceviamodernbiotechnology.Finally,weexaminerecentadvancesinmolecularbiology andpathogenesisandtheirpotentialapplicationtothedesignofmolecularresistancetoMSDvia geneticmanipulation,withafocusontargetingMSVproteins.

4 MAIZE STREAK VIRUS (MSV) DISEASE Maize streak virus (MSV) disease (MSD) is responsible for severe epidemics in maize in subSaharan Africa (Thottappilly et al., 1993; Harrison and Robinson, 1999). The disease, first described as MSD early 19th century (reviewed in Shepherd et al., 2010), is endemic in African savannah grasses. It is widely distributed in sub-Saharan Africa and its adjacent islands (Thottappilly et al., 1993) affecting many 'grain basket' regions of the continent. MSD is characterized by chlorotic yellow-white spots or streaks along the leaf vasculature (Figure 1). The streaks develop along the veins of the leaf and as the virus is systemic, symptoms appear only on the inoculated and subsequent emerging leaves (Thottappilly et al., 1993). In highly susceptible varieties, chlorotic striping often develops into chlorosis of the entire lamina followed by progressive necrosis and plant death (Bosque-Perez, 2000). Because of the preference for maize by African small and middle holder farmers, sometimes largely practicing monocropping (Efron et al., 1989), a high selection pressure for maize-adapted viruses was created and limited viral genetic diversity (reviewed in Shepherd et al., 2010). Overtime, frequent passages in maize may have allowed the maize-adapted viruses to become asymptomatic in some wild grasses, their original hosts (Martin et al., 2001; Owor et al., 2007). These maize-adapted strains, which are few in number, decimate maize yields all round the African continent (Martin et al., 2001). Studying the asymptomatic grasses may provide important breakthroughs for developing maize plants with durable broad-spectrum disease tolerance. The maize-adapted virus biotypes are designated MSV A and its subtypes MSV A1-A6 (Martin et al., 2001). Studies are under way using forced chimeric forms of the subtypes (Schnippenkoetter et al., 2001) and even of maize and grass type viruses (Martin et al., 2001; Martin and Rybicki, 2002), which are inoculated in maize to infer the role of viral genetic components to virus host specificity, virulence and pathogenesis (Varsani et al., 2008b). The studies are also set to unfold the pedigree of the viruses and what influences their recombination, which may underlie the development of MSV counter-resistance to control measures (van der Walt et al., 2009). Such studies are vital in designing new strategies to combat virus resistance either by limiting virus variants or targeting certain process that are vital to inter-biotype viral recombination (Owor et al., 2007; Varsani et al., 2008b). By taking advantage of this near homogeneity of MSV biotypes, genetic engineering (GE) offers new avenues for creating broad based molecular maize resistance effective over vast geographical regions and still keeps ahead or apace with the virus evolution.

5 MSVinfectioncycle MSV is transmitted by Cicadulina leafhoppers (Rose, 1972; Oluwafemi et al., 2007) in a circulative non-propagative and persistent manner (Hogenhout et al., 2008). MSV particles (virions) are acquired by leafhoppers through the stylet mouthparts during feeding and move through the fore gut, midgut, hindgut and finally the hemolymph, where the virions get to the salivary glands (Oluwafemi, 2006; Hohn, 2007; Hogenhout et al., 2008). The virus particles are acquired from the mesophyll cells of an infected maize leaf in a minimum 5 minutes (Hohn, 2007) although longer acquisition times are fit for subsequent transmission (Oluwafemi et al., 2007). Inoculation or transmission to other plants or leaves occurs from approximately 15 minutes after feeding and continues for life (Kairo et al., 1995; Hohn, 2007; Oluwafemi et al., 2007). The leafhoppers transmit the viruses back into maize mesophyll cells to restart the infection cycle (Lucy et al., 1996). Following infection, transcription of the early viral proteins, Rep and RepA, is induced in preparation for viral genome replication (Boulton, 2002). The proteins recruit host DNA replication enzymes/proteins (Hanley-Bowdoin et al., 1999; Gutierrez, 2000, 2002) notable among them plant retinoblastoma-related protein (RBR) that suppresses host cell cycle progression (Shepherd et al., 2005; McGivern et al., 2005). This situation permits a DNA synthesis environment in otherwise differentiated and non-replicating mesophyll cells (Kong et al., 2000; Nagar et al., 2002). Using the recruited host machinery, the virus amplifies its genome to high copy numbers through either rolling cycle replication (Stenger et al., 1991) or recombination-dependent replication (RDR) (Erdmann et al., 2010). One copy of the singlestranded viral DNA is encapsidated in each geminate viral particle and is ready for retransmission (Zhang et al., 2001). YieldlossduetoMSVdisease Reductions in maize grain yield due to MSV infestation depends on the age of plants at the time of infection, with younger plants suffering greater reductions than older ones (Mzira, 1984; Gibson and Page, 1997), even among resistant varieties (Bosque-Perez et al., 1998). Young plants infected at 2nd to 10th leaf can loose to 25%-55% in grain weight (Mzira, 1984). Yield loss is also a function of the relative level of susceptibility or resistance of the maize variety (Barrow, 1992; Asanzi et al., 1994). Simulatory studies by Martin et al. (2001) indicated that yield loss also directly correlate with virulence of the MSV strain or biotype. Disease severity is a function of viral inoculum size, with higher doses causing more rapid and severe symptoms. In the field, inoculums are influenced by the population size of viruliferous leafhoppers (Gibson and Page,

6 1997). Severe MSD outbreaks in West Africa are associated with drought and irregular rains and up to 100% crop losses (Efron et al., 1989; Bosque-Perez et al., 1998). MOLECULAR VIOLOGY OF MSV General introduction to structure of geminiviruses MSV belongs to the geminiviruses group of plant viruses that have single stranded DNA genomes encapsidated as twinned icosahedral virions (hence the name geminivirus) (Lazarowitz, 1992). The family contains four genera including Mastrevirus, Curtovirus, TopocuvirusandBegomovirusclassifiedaccordingtotheirhostrange,genomeorganizationand vector species (Fauquet et al., 2003). The Mastreviruses, typified by MSV, infect monocotyledonousplantsparticularlythe Gramineae familyexceptafewotherslikethebean yellow dwarf virus (BeYDV) and Tobacco yellow dwarf virus (TYDV) that infects dicotyledonousplants(Liuetal.,1999b;Bennettetal.,2006).Thegenomesofallgeminiviruses generally compriseacovalentlyclosed,circular,singlestrandedDNA(cccssDNA)ofabout 27003000nucleotides(Gutierrez,2000).TheMastrevirusgenomeisencapsidatedsinglytermed monopartite(Fauquetetal.,2003). The MSV particle, which is about 220 x 380 , consists of two joined but incomplete icosahedralheadscomposedof110CPsubunitsorganisedas22pentamericcapsomers(Zhang etal.,2001).Thegenomecodesfortwogenesonthevirionsensestrand,V1andV2,andtwo genesonthecomplementarysensestrand,C1andC2(Wrightetal.,1997).Italsocontainstwo intergenicregions,onelarge(LIR)andanothersmall(SIR)locatedatoppositesidesoftheviral genome.WithintheLIR,aconservedinvertedrepeatsequenceformsastemloopstructure.The loopcontainstheinvariant5'TAATATTAC3'sequencethatresemblescleavagesitesinssDNA phageX174andssDNAplasmidspC194andpT181(Grosetal.,1987).Cleavageoftheloop sequence results in 3'OH terminus used to prime initiation of plus strand DNA synthesis (Stengeretal.,1991).Asmallcomplementarysenseprimerlikeoligonucleotideannealedtothe genomicDNAwithintheSIRpurposelyprimesdsDNAsynthesispriortoreplication(Dryetal., 1997). FunctionandinteractionofMSVproteins Mastrevirus genomes encode four proteins the movement protein (MP, V1 gene) and the capsid protein (CP, V2 gene) on the V-sense strand (Morris-Krsinich et al., 1989; Mullineaux et al., 1988) and the replication associated RepA and Rep proteins on the C-sense strand (Gutierrez,

7 1999; Martin et al., 2001). The Rep protein is translated from C1:C2 spliced mRNA (Figure 1) whereas RepA is produced from the unspliced C1 open reading frame. The two proteins are identical in their N-termini (approximately 210 amino acid residues) but differ in their C-termini with the Rep protein being longer (372 amino acids) and RepA shorter (272 amino acid). The movement protein (MP) is involved in systemic and cell-to-cell movement of the virus (Boulton et al., 1989, 1993; Lazarowitz et al., 1989; Dickinson et al., 1996). The coat protein is essential for long distance movement, virus transmission (Boulton et al., 1993; Liu et al., 1998), and regulates inter-conversion of genomic ssDNA and dsDNA intermediates (Boulton et al., 1989; Krichevsky et al., 2006). The coat protein is essential for infection of whole plants but not the movement protein (Boulton et al., 1989). Rep (absolutely) and RepA proteins are essential for viral replication whereas, RepA alone reprograms the host cell and regulates viral gene expression (Lazarowitz, 1992). TranscriptionandexpressionofMSVgenes The genes of MSV are expressed bidirectionally from the long intergenic region (LIR) terminating in the short intergenic region (SIR), which contains polyadenylation signals (Mullineauxetal.,1984;MorrisKrsinichetal.,1985;Wrightetal.,1997;Boulton,1989;2002). Thepromoterelementsnecessaryforgeneexpressionofthesenseorantisensegenesarelocated inthelongintergenicregion(MorrisKrsinichetal.,1985;Fenolletal.,1990;Timmermannset al.,1994).TheLIRcontainstherightwardpromoter cisactingelement(rpe1)(Fenolletal., 1990)thatcontrolsthevirionsensegenesencodingtheMPandCP(Wrightetal.,1997).The rpe1 elementdrivesvascularspecificgeneexpressionintransgenicriceplants(Mazithulelaet al.,2000).TheLIRalsocontainspromoterelementsthatarehomologoustohexamermotifsof planthistonepromotersthatconferSphasespecificexpression(Morozovetal.,1994),implying thatthevirusgeneregulationisusuallyadaptedtotheSphaseoftheplantcellcycle(Nikovics et al., 2001). Differential splicing of the early complementarysense mRNAs contributes to regulationofRepandRepAexpression(Drugeon etal., 1999;HefferonandDugdale,2003). RepAisinvolvedinregulationofthesensetranscriptsV1andV2encodingforMPandCP, respectively(Timmermannsetal.,1994;HefferonandDugdale,2003). MSVgenomereplicationandroleofviralproteins Although information about MSV Rep-mediated origin recognition and DNA cleavage is not available, analysis of other geminiviruses may provide insight into these activities (Table 1) especially wheat dwarf virus -WDV (Kammann et al., 1991; Heyraud-Nitschke et al. 1995;

8 Laufs et al. 1995a). Geminiviruses replicate their ccc-ssDNA genomes in nuclei of infected plant cells (Accotto et al., 1993). Once the virions enter the cytoplasm, the CP alone (Mastreviruses, Krichevsky et al., 2006) or CP and MP components translocate the viral genome across the nuclear envelop into the nucleus (Liu et al., 1997; 1999a; 2001; Whittaker et al., 2000). Then the complementary sense small oligonucleotide located in the SIR serves as a primer for host DNA polymerase to synthesize a complimentary copy of the ccc-ssDNA resulting in a ccc-dsDNA replication intermediate (Donson et al., 1984; Liu et al., 1998). The host transcription machinery then assembles at the promoter elements within the LIR for expression of the early replication associated proteins Rep and RepA, in preparation for rolling cycle replication (Hanley-Bowdoin et al., 1999; Gutierrez, 2000). ThevirusesdonotencodetheentireproteinrepertoirerequiredforDNAsynthesisbutrecruit fromthehostcellthroughinteractionswiththeRepandRepAproteins(Boulton,2002).ssDNA synthesisisinitiatedatthe3'thymidineresidueaftersinglestrandcleavage(nick)oftheV strandofthecccdsDNAbytheRepproteinintheconservedTAATATTACsequenceinthe LIRhairpinmotif(Willmentetal.,2007).Followingcleavage,the5'endoftheDNAremains covalentlyboundtoRep,andafteraroundofreplication,aRepcatalysednucleotidyltransfer reactiontoreleaseacccssDNAformoftheviralgenome(Laufsetal.,1995a).Therecognition oftheoriginofreplicationfollowedbydsDNAbindingandcleavageisaccomplishedbytheN terminusoftheRepprotein(Willmentetal.,2007).AconservedTyrosineresiduelocatedwithin motifIIIinthisregion(Table1)isessentialindsDNAnickcleavage(Laufsetal.,1995b). Geminivirusesreplicateindifferentiatedcellsofthehostplants(Kongetal.,2000;Nagaretal., 2002).Somegeminivirusesinducetheendocycle(Kongetal., 2000;AscencioIbanezetal., 2008),whileothersareassociatedwithcellproliferation(HanleyBowdoinetal.,2004;Mills LujanandDeom,2010).Amongthebegomoviruses,infectionaffectstranscriptionofcellcycle genes(AscencioIbanezetal.,2008)butthereisnoevidencefordirectinteractionofreplication proteinswithanyofthecellcycleproteinfactors.Thereplicationassociatedproteinsinteract withtheretinoblastomarelated(RBR)protein(Nagaretal.,1995;Kongetal.,2000;Settlageet al., 2001; ArguelloAstorga et al., 2004; Shepherd et al., 2005) that represses cell cycle progressionpromotingcelldifferentiation(Sidleetal.,1996).ForMSV,RepAbutnotRepbinds RBRthroughaconservedLxCxEmotif(Hovarthetal.,1998;McGivernetal.,2005;Shepherd etal.,2005)whereasforbegomovirusesandprobablycurtovirusesandtopcoviruses,theRBR bindingproteinisRepthroughothersequences(ArguelloAstorgaetal.,2004).Theinteraction

9 withhostfactorsaltersthequiescentstateofthecell(G0phaseandG1phase)intotheDNA synthesisstate(Sphase)(HanleyBowdoinetal.,2004)thatallowsthevirustoreplicatetohigh copynumberinthehostcells(Nagaretal., 2002).TheanchoringofMSVreplicationtohost DNA synthesis makes Rep/RepA prime targets forcurtailing MSVreplication usinggenetic engineering. MutagenesisandrecombinationofMSV MSVadaptationtomaizeisduetoitspassagingthroughmaizeoveralongtime(Isnardetal., 1998).Monocroppingandpersistenttransmissionbottlenecks(Oluwafemi,2006)haveprovided selectivepressureforMSVtoovercomeanynativeresistanceinmaize.Thiscoevolutionis primarilyduetomechanistic(notfacilitated)recombinationbetweenvirusbiotypes(Martinet al.,2001,Oworetal.,2007;Varsanietal.,2008b;vanderWaltetal.,2009)andtheformationof an MSV quasispecies (Isnard et al., 1998). For maizeadapted viruses, intraspecific recombinationfrequencyishighcomparedtointertypespeciesrecombination,implyingthat mixed infections are not common or do not occur across the continent (Owor et al., 2007; Varsani etal.,2008b;Shepherdetal.,2010).Thequasispecies resultsfrompointmutations occurringlargelyinthelongintergenicregionsandtoalesserextentintheORFsoftheviral genome(Isnardetal.,1998;DuffyandHolmes,2009).MutationsintheORFsaregenerally singlesubstitutionsthatcanbesilentornonsynonymous(Isnardetal.,1998).Thevirusescan also develop mutations in response to new biological environments when passaged through differenthosts(ArguelloAstorgaetal.,2007).Forinstance,transmissionfrommaizetograsses orsugarcaneandbacktomaizemaycausemoremutationsthanpassagesineachofthesecrops alone(Isnardetal.,1998). CURRENT STRATEGIES FOR MSD CONTROL AND THE LIMITATIONS MSD epidemics are contained in the field by avoiding the vector influx periods through crop rotation, grass clearing and timing of planting during favourable rain-fed seasons. Because drought and erratic rains are associated with the build up of migrating leafhoppers and epidemics (Rose, 1972), planting time is crucial (Muhammad and Underwood, 2004; Gibson et al., 2005). However, for this to be effective, farmers must rely on precise weather predictions, which are not readily available in sub-Saharan Africa. In addition, the continuous presence of the crop and the savannah grasses associated with MSD ensures recurrence of the vector all year round. Frequent application of systemic insecticides eliminating leafhoppers effectively controls MSD, but is

10 generally inaccessible to the resource-poor subsistence farmers in Africa. In the long run, the only option that is accessible, easily applied, and environmentally friendly is the continued development of genetic resistance in maize cultivars (Ayliffe and Lagudah, 2004; Shepherd et al., 2009). Since the 18th century when MSD was first described (Harkins et al., 2009), no other avenues have existed for developing MSD resistance other than conventional breeding (Pratt et al., 2003; Golbach et al., 2003; Stevens, 2008). Although maize genotypes with varying degrees of MSD resistance are available, (Barrow, 1992; Rodier et al., 1995; Welz et al., 1998; Kyetere et al., 1999), their use is not widespread among resource-poor farmers in Africa. In addition, the mechanism(s) underlying the resistance are not known making it difficult to sustain or up-grade (Stevens, 2008). For example, it is not known whether conventional resistance to MSD reduces viral transmission, viral propagation or disease manifestation (Kairo et al., 1995; Morales, 2001). Page et al. (1999) demonstrated that intercropping maize with bean or millet decreased vector activity and/or vector numbers but this was not associated with reduction in MSD incidence. It is essential to better understand resistance mechanisms (especially at the molecular level) to increase resistance efficiency and delay viral counterresistance (Kang et al., 2005; Walters and Heil, 2007). Recombination and pseudorecombination among geminiviruses also pose imminent threats (Pita et al., 2001; Fargette et al., 2006), and newly emerging biotypes limit conventional resistance (Owor et al., 2007; Varsani et al., 2008b). Recent studies demonstrated that high mutation rates in geminiviruses contribute to the generation of new viral variants (sub-types) at high frequency that can stay ahead of conventional breeding resistance (Arguello-Astorga et al 2007; Duffy and Holmes, 2009). Conceptually, two types of resistance against MSD can be generated using modern biotechnology resistance to the virus (pathogen) or tolerance to the disease (symptom manifestation). In the latter case, the virus may move through the host in a manner that is indistinguishable from that in susceptible hosts with no visible disease symptoms. This happens in the fields to maize-adapted viruses transmitted to some grasses (Martins et al., 2001). Although disease tolerance occurs often in nature, the underlying biochemical and genetic mechanisms have not been explored (Kairo et al., 1995). Tolerance would be advantageous because selection pressure is not applied to the viral pathogen to counter the resistance. If tolerance to the pathogen is heritable and stable, the benefits to farmers would be enormous. DEVELOPING MSD RESISTANCE THROUGH MOLECULAR PLANT BREEDING Conserved and essential functional motifs have been identified in MSV gene products (Shepherd et al., 2010) that can be used to design molecular resistance or tolerance in maize that is stable

11 (Table 1, Vanderschuren et al., 2007). This resistance at the single cell level is a state where virus replication or subsistence occurs at essentially undetectable levels in inoculated cells (Shepherd et al., 2009). Successful development of maize with such resistance or tolerance to MSD requires thorough molecular understanding of MSD pathogenesis. Pioneering investigations into MSD epidemiology, aetiology and pathogenesis have come mainly from South Africa at the University of Cape Town (Shepherd et al., 2010) and the John Innes Center in the UK (Boulton, 2002). From the South African group, a thorough understanding of the virus evolution and recombination is finally unfolding (Harkins et al., 2009; van der Walt et al., 2009). Their studies also established that only a few MSV strains are highly virulent (Martins et al., 2001). Based on these studies, the group has used a pathogen derived resistance (PDR) strategy to develop a transgenic maize line with MSD resistance (Shepherd et al., 2007) that is currently undergoing field trials in South Africa (Shepherd et al., 2010). In the next section, we outline foreseeable studies in further understanding molecular pathogenesis of MSD and how the knowledge can assist design biotechnological control strategies particularly targeting the virus and host proteome. MOLECULAR PATHOGENESIS OF MSV AND MSD CONTROL MSVmolecularpathogenesis:Themissinggap The hallmark of geminiviruses infection (spots or streaks) occurs in combination with other symptoms depending on the virus strain and plant host. For example, MSV does not cause leaf curling like most dicot infecting geminiviruses (Levy and Tzfira, 2010). These differences reflect divergence in virus/host interactions and the factors contributing to the disease pathogenesis (Boulton et al., 1991a; 1991b; Liu et al., 1999b). MSV and its African streak virus counterparts (Varsani et al., 2007) induce characteristic streaks on plant leaves during infection. This collateral feature of pathogenesis is considered a co-evolution strategy to attract feeding insects to the 'attractive' yellow leaf of the infected plant guaranteeing subsequent transmission (Rojas et al., 2005). Whether streaking is solely because of chlorosis or it is an effect of viral ingenuity to guarantee transmission is speculative. Chlorosis signifies cell death, but the preceding biochemical events for the case of MSV infections are yet clear. As for leaf streaking, whether it reduces photosynthesis capacity of the plant (loss of chlorophyll) is also unknown. Plants infected when well established do not appear unhealthy (unchanged biomass) except for extensive yellowing of the leaves (Lukuyu et al., 2002) (Figure 1), suggesting that reduced photosynthesis may not be the only cause of stunting in infected young growing plants.

12 Plants infected with tobacco leaf curl geminivirus (TLCV) have reduced capacity to harvest light energy under low light intensity (Funayama et al., 1997). However, photosynthesis rate is unchanged under high light intensity (light saturation). Considering that the tropical climate is characterised by high light intensities, then photosynthesis may not be a core factor in MSD pathogenesis in tropical maize and grasses. Other factors including high turnover of light harvesting chlorophyll a and b binding proteins might be significant (Funayama et al., 1997). The carbon fixation enzyme rubisco (ribulose-1, 5-bisphosphate carboxylase) -and perhaps many other photosytem antennae proteins- are surprisingly unaffected by infection until programmed cell death sets in (Ishida et al., 2008). The role of such protein profiles on overall photosynthesis in maize under MSV attack remain unclear, given that maize is a C4 plant while the above observations were made in a C3 plant (Brautigam et al., 2008; Zhu et al., 2008). In the quest for controlling MSD, the fundamental question is whether the molecular/biochemical protein interactions involved in MSD pathogenesis have potential use in creating MSD resistance if they are known and well characterised. All MSV proteins interact with host factors changing the metabolic/physiological and ultimately morphological status of the host cell. Of these, the replication proteins (Rep and RepA) are better characterised regarding MSD pathogenesis (Gutierrez, 2002; Boulton, 2002). Nonetheless, fine details on biochemical elucidation of MSV replication proteins interaction with viral DNA is still lacking; although these are well characterized for begomoviruses (Fontes et al., 1994; Chatterji et al., 2001; Arguello-Astorga et al., 2004). Similarly, three different mutations in the N-terminus of begomovirus Rep protein spanning motif I, II and III (Table 1) inhibited DNA binding in vitro and DNA replication in vivo, demonstrating the importance of this region and further supporting the close relationship between DNA binding, cleavage and finally viral replication (Orozco et al., 1997; Orozco and Hanley-Bowdoin, 1998). For MSV, such information is inferred from begomovirus studies (Campos-Olivas et al., 2002; Vanderschuren et al., 2007; Vega-Rocha et al., 2007). Consequently, there exist many efforts to develop molecular viral resistance against global dicot infecting geminiviruses but only one for MSD (Shepherd et al., 2007). Implicationofadvancesinmolecularbiology:towardsunderstandingMSDpathogenesis Uptonow,infectionrates,chlorophylllevels,viralloads,leafchlorosisandstuntingareusedto infer MSDpathogenesis anddiseaseseverity(Boultonetal.,1991a;Funayamaetal.,1997; Isnardetal.,1998;Martinetal.,1999).Theseparametersdonotclearlyinformontherelevant biochemicaleventsleadingtotheobservedpathogenesisphenotypes(Boultonetal.,1991b).In thecurrentomicsera(MorenoRisuenoetal.,2009),theunprecedentedproliferationofhigh

13 throughput molecular biology tools and recombinant DNA technology should unravel the 'mystery'ofMSDpathogenesis.Severalapproachesarefeasibleandwediscussbelowunder functionalgenomics,metabolomics,transcriptomicsandproteomics. Functional genomics and unravelling MSD pathogenesis MicroarrayanalysisoftheArabidopsisthalianatranscriptomeshowedthatmanyhostgenesare differentially expressed in response to cabbage leaf curl geminivirus (CaLCuV) infection (AscencioIbanezetal.,2008).Thedifferentiallyexpressedgenesincludedgenesassociatedwith thesalicylicacid(SA)responsepathway,programmedcelldeath,DNArepairandthecellcycle. InfectionupregulatedgenesassociatedwithSandG2phasesofthecellcyclewhereasG1and Mphaseassociatedgenesweredownregulated.Itwouldbeinterestingtomonitorthesegene expressionpatternsinMSVinfectionsaswell,furtheridentifyingtheparticularproteinfactors involved.Theknownfactorswouldexplainthemanifestation(orlack)ofMSVpathogenesis comparativelyinsymptomaticandasymptomaticinfections. DifferentvirusesinducehostdefenceresponsesdifferentlyviaSApathway(Huangetal.,2005). AkeyquestioniswhetherMSVinfections thatinclude'mixed'viralsubtypesespecially the recombinant strains behave similarly and whether intra and interspecific interactions of the subtypes (MSV A1A6) engender analogous genetic responses. The possibility of synergism duringdiseasemanifestationcannotberuledout.Thecontributionofeachbiotypecomponents topathogenesisishoweverimportant.Mutational(andexpression)analysisoftheindividual MSVgenecomponentssinglyorinpredeterminedchimeraand/orfusioncombinationswill provide insight into the contribution of the specific viral proteins to pathogenesis (Hanley Bowdoinetal.,1990;Schnippenkoetteretal.,2001).Suchanalysesusingtruncatedviralgenes andnucleotidesubstitutionsordeletionsinmaizeplantshavebeenconsideredbefore(Schneider etal.,1992;Liuetal.,1997;1999a;2001).Thestudiesrevealedthemovementandcoatproteins arerequiredforsystemicspreadofthevirus,althoughonlythemovementproteinwasimplicated insymptomdevelopment(Dickinsonetal.,1996;HefferonandDugdale,2003).MutatingMSV RepA protein in the RBR binding motif reduces symptom severity but not viral replication (Shepherdetal.,(2005). Recently,Africancassavamosaicvirus(ACMV)Repproteinfusedtogreenfluorescentprotein (GFP)wasusedtoestablishinductionofhypersensitivityresponse(HR)byACMVRepprotein intransgenicNicotianabenthamianaplants(Jinetal.,2008).TheACMVRepdomaininvolved

14 wasmappedtotheparticularaminoacidresidueviaArgininetoSerinemutation(Jinetal., 2008).Suchmutationalstudiescanbedoneinmaizeasitsownmodel(Anamietal.,2009) probably more effectively using protoplasts or BMS (Black Mexican sweet corn) cell suspensions where possible (Boulton et al., 1993). When considering whole plant assays however, maize model plants that are easy to manipulate in vitro for example Digitaria sanguinaliscouldbeconsideredinstead,usingvirusstrainsadaptedbothtomaizeandgrasses (Chenetal.,1998;Shepherdetal.,2007).Someviralcomponentsmayworktogethertoachieve certainfunctions.Forexample,CPandMPtogetherfacilitateviralcelltocellmovement,but MPisabsolutelyrequiredforsymptomdevelopmentandCPforsuccessfulplantinfection. Non-protein molecular regulation of ORFs Small endogenous RNAs (microRNAs or miRNAs), regulate gene expression in both plant and animals directly (transcription and DNA hypermethylation) or indirectly (translation) (reviewed in Meins et al., 2005; Jones-Rhoades et al., 2006). These are readily detected using Northern blots (Reinhart et al., 2002; Gustafson et al., 2005) or micro arrays (Axtell and Bartel, 2005). Since their expression is amenable to environmental perturbations (Chellappan et al., 2005; Tuttle et al., 2008), miRNAs and siRNAs can monitor genomic, ultimately proteomic and metabolomic profiles during MSD pathogenesis. The accruing information provides insight into the role of miRNAs in disease development or resistance (tolerance) (Liu and Chen, 2010). In plants, immediate defence responses to viral attack are localised to the infection or inoculation area (Yang et al., 2007a). The role of miRNA in this scenario is unexplored; given that gene regulation is the primary function of miRNAs and that the viruses win the first battle against immediate host defences at these sites. Modulation of small interfering RNAs (siRNAs) in plants to avert viral (MSV) infection or disease manifestation could be an interesting exploration. For instance, using artificially designed micro RNAs (amiRNAs) (Schwab et al., 2006; Frizzi and Huang, 2010), MSV gene products can be targeted to avert viral abilities like replication, movement and encapsulation. These approaches can help unravel the genetic arms race between host plant and virus pathogen during disease manifestation when host factors are also targeted where probable (Miki et al., 2005). With the strategy of loss or gain of function used to disclose miRNA function (Jones-Rhoades and Bartel, 2004), the above propositions are realistic, especially if studies compare MSV infected and non-infected plants as well as symptomatic and asymptomatic infections.

15 Transcriptomics and unravelling MSD pathogenesis An extension of functional genomics involves studying transcription factors independently termed transcriptomics. Certain proteins (transcription factors) control gene expression at transcriptionlevelandarestudiedusingDNAmicroarrayanalysis(Gregoryetal.,2008).These studies involve genomewide locations of transcription factors (DNA binding peptides) and regions of histone modification sites involved in regulating DNA expression and even replication.TheuniquemaizeDNAbindingfactor(s)microarrayscanidentifyspecificelements involvedingeneticmodulation(replicationmodulatorschip)andgeneexpression(transcription factors) (TFs chip)duringinfection (Grotewold,2008).Upanddownregulated orinduced globalgeneregulatoryDNAbindingfactorsinvolvedinMSVdefenceresponseorMSVdisease progression can be detected. Analogous proteinprotein interaction studies would elucidate whether MSV protein factors directly interact with the host DNA elements involved in modulating DNA replication, gene expression (Akerfelt et al., 2006) and even signal transduction.Infact,geminivirusesmodulateinfectedhostcells(oftendifferentiatedandnon dividingcells)andhostDNAsynthesistosuittheirownreplication(Hanley-Bowdoin et al., 2004; Ascencio-Ibanez et al., 2008).Theknownhostproteinfactorsinteractingwithgeminiviral proteins include among others proliferating cell nuclear antigen (PCNA) protein key in assemblinghostDNAreplicationmachinery(Castilloetal.,2003;Bagewadietal., 2004),plant retinoblastomarelated protein (pRBR) that negatively regulates cell cycle progression and a differentiation (Weinberg, 1995; Ach et al., 1997; Kong et al., 2000) and protein kinases involved in signal cascading of host response (Shen and HanleyBowdoin 2006). Whether concomitantoverexpressionorsuppressionoftheidentifiedgeneregulatoryfactors(peptides) ininfected maizecanaidunderstanddiseaseprogressneedexplorationinMSD(Grotewold, 2008).Wherepossible,maizegeneregulatoryproteinprofilecanbegeneticallyremodelledtoo usingreversegeneticstostudyhostfactorcontributiontoMSVpathogenesis. OneobviousareathatneedssuchdiscreetremodellingregardingMSDtoleranceincludefactors involvedinfunctionalgenomicsofmaizeplastid,andchlorophyllturnover(Brautigametal., 2008;Covshoffetal.,2008;Rutschowetal.,2008).Theseorganelles(especiallychloroplasts) areresponsibleforphotosynthesis,whichisapparentlyimpairedduringgeminivirusesinfection (Funayamaetal.,1997).Thestudiesshouldunravelthefoundationalgeneticcauseofyellowing ininfectedleafcellsandwhatcanbedonetorestoreormaintainphotosynthesisduringinfection inastatesynonymouswithdiseasetolerance(Shimizuetal.,2007).Thisisinlightofnew

16 evidencethatrubiscoisselectivelydegradedbycellularautophagyduringcelldeathwhilethe chloroplasts are not affected (Ishida et al., 2008). Previously it has been found that genes contributingtoprogrammedcelldeath(autophagy)maybeupregulatedingeminivirusinfected plants(AscencioIbanezetal.,2008). Proteomics, metabolomics and unravelling MSD pathogenesis All living organisms control their cellular metabolism via protein functional associations or networkstermedcomplexome(BoganandThorn,1998;DrewesandBouwmeester,2003;Uhrig, 2006).Theseassociationsresultfrominterandintramolecularinteractionsofthewholeprotein repertoireofanorganism,comprisingtheproteininteractome(DrewesandBouwmeester,2003; Uhrig,2006).Thecomplexomeandinteractomeofanorganismconsistsofits proteome.The underlyingproteinproteinorproteinligandinteractionsarenumerousandthereforedifficultto interpret invivo. Notwithstanding,directdiscreetproteomicandmetabolomicanalysesofthe infectedmaizeduringdiseasedevelopmentcouldbedone invitro (Hall,2006).Inthiscase, metabolites(especiallysecondary),enzymaticactivityprofiles(Mgutu,2004),proteinandamino acidprofiles(turnover),andcarbonresourcepartitioning(Lehrer etal., 2007)amongmany otherintegratedphysiologicalparametersarecomparativelyinvestigated(Fiehn etal.,2008). The physiological condition of infected cells compared to uninfected controls may identify underlyingcausesofMSDpathogenesis(WhithamandWang,2004). Forexample,thegrowthrateofTYLCVinfectedplantsisafunctionofplantageatinfection andplantphotosyntheticcapacity(Funayamaetal.,1997).Theinfectedplantseventuallyhave reduced biomass than their uninfected counterparts, signifying importance of resource mobilisation during pathogenesis. Symptomatic sugarcane infected with a geminivirus has a reducedassimilateexportcomparedtoasymptomaticanduninfectedcounterparts(Lehreretal., 2007). Thus, viral infection interferes with photoassimilate translocation. With similar investigations in MSD, perhaps inherent physiological factors unique to symptomatic or asymptomaticphenotypesmayberevealed. Viral MPs interact with the host plasmodesmata impairing cellular integrity and perturbing cellularhomoeostasis(Dicksonetal.,1996;reviewedinKragleretal.,1998;Gilbertsonetal., 2003).Itispossiblethatmaizeresponsetoinfectioncontributestodiseaseprogressionthrough cellularperturbations(Gilbertson etal.,2003;WhithamandWang,2004).Whatisgenerally emerging is that host protein factors specifically facilitate systemic spread of viruses in

17 associationwiththeendoplasmicreticulum(ER).TheERplaysamajorroleinmacromolecular traffickinginplantsthroughtheplasmodesmata.Itiscontinuousbetweencellsinassociation with microfilaments and actin cytoskeleton (Heinlein and Epel, 2004). This association is suspectedinhelpingviralcelltocellmovement.However,usingtobaccomosaicvirus(TMV) MP, Hofmann et al. (2009) demonstrated that the actin cytoskeleton is not a player in this scenariobutERassociatedactinfilamentsandactinbindingcellularproteinsaretheculprits. Whether it is possible to target viral movement during MSD remains unexplored because understandingthehostinvolvementisnecessaryfirst(DawsonandHilf,1992;Whithamand Wang,2004).Uptonow,theparticularhostfactorscontributingtoMSVmovementinthehost (celltocellandsystemic)arenotknownandneedelucidation(Tzfiraetal.,2000;Liuetal., 2001;Hofmannetal.,2009).Screeningviralmovementandcoatproteinsagainstalibraryof hostcellmembraneproteinfactorsusingyeasttwohybridassayswillinferrelevantinteractions requiredforviriontranslocationmechanisms.Mutationalanddomainswapanalysestoelucidate theparticularmotifsresponsiblecanunlocknewtargetsfordevelopingresistancemechanism. Such efforts targeting viral cell to cell movement for disease resistance exist. Mutations in Arabidopsisthalianacum1andcum2genespreventcelltocellmovementofcucumbermosaic virus(CMV)inthemutants(Yoshiietal.,1998a;1998b).The CUM1 and CUM2 encodefor translation initiation factors (Yoshii et al., 2004). On the other hand, various silencing suppression activities of viral suppressors of RNA silencing (VSRs) facilitate celltocell movement and phloemdependent longdistance virus spread indirectly, intensifying disease symptomsinsystemicallyinfectedtissues(DiazPendonandDing,2008).Ifsuchunderstanding is thoroughly available in maize, perhaps new avenues for targeting MSD using GE at the molecularlevelcouldbeathand.Theinvestigativeconsiderationsaboveshouldcommencein symptomaticandasymptomaticphenotypescomparatively. Regarding global proteomic host-pathogen interaction, specific protein-protein interactions and modifications (Goritschnig et al., 2008) essential for viral subsistence and disease pathogenesis require exploration (Chien et al., 1991; Kelly and Stumpf, 2008). Genomic studies followed by knockout or site directed mutagenesis studies should precede such proteomic interactions however. The information obtained would set the stage for high-throughput protein-protein interaction assays of deduced protein interaction networks (Moreno-Risueno et al., 2009). Simple pair-wise protein-protein interactions and modifications can be dissected in two-hybrid yeast assays (Suter et al., 2008), mass spectroscopy (Zhu et al., 2003) then structure elucidation

18 through x-ray crystallography and cryo-electron microscopy (cryoEM) (ThumanCommike and Chiu, 2000). Once identification and characterization of proteomic functional motifs is completed, the proteins serve as targets for manipulation (viral proteins), over-expression, or silencing (maize proteins) in maize to confer disease resistance or tolerance. Already, comprehensive studies of such functional motifs that are highly conserved among geminivirus proteins (Orozco and Hanley-Bowdoin, 1998; Arguello-Astorga et al., 2004; Shen and Hanley-Bowdoin, 2006) are core to developing resistance in plants using peptide aptamers (Lopez-Ochoa et al., 2006). Genomics, transcriptomics, proteomics and perhaps metabolomics understanding established will fast-track design of integrated and durable systems for MSD resistance incorporating breeding and GE (Kelly and Stumpf, 2008; Saito and Matsuda, 2010). HARNESSING MSV PROTEINS FOR CREATING MSD RESISTANCE Earlytools:Thepathogenderivedresistance(PDR)strategy MostpopularmaizecultivarssownbysmallholderfarmersinAfricalackmappedspecificand nativegeneticresistanceagainstmanypestsandpathogensincludingMSV(Muhammadand Underwood, 2004). The resistance traits have to be introduced by introgression as in conventional(classical)breeding(Khush,2001)orbyGEbeforebreedingcommences.Several strategies employed to genetically engineering resistance to viruses in transgenic plants are predominantly pathogenderived resistance (PDR) (Sanford and Johnson, 1985). Some transgenicplantsexpresstruncateddefectiveviralproteinsormutantviralproteins(Bonflmet al., 2007; Shepherd et al., 2009). The products of the pathogenderived genes except transdominant negative viral proteins conferresistance through transacting small interfering RNAmoieties (tasiRNA)derivedfromdoublestrandedRNAgeneratedbyRNAdependent RNA(RDR)polymerase(Pandeyetal.,2008). 'Mixed' gene silencing using single viral derived proteins ThemodeofactionformostprecedingPDRstrategies isunclear,happeningeitherviagene silencing(JonesRhoadesetal.,2006)orbydysfunctioningthewildtypeproteininteractingwith the defective protein thus mixed silencing (Lucioli et al., 2003). The strategy is just beginningtobearfruitforcontrollingMSD(Shepherdetal.,2007).TruncatedNterminusof MSVreplicationprotein(Rep1219minusRBbindingdomain)wasconstitutivelyoverexpressed inthetransgenicmaizelines.TheCterminuswasexcludedbecausepartofitsmotif(s)leadto altered cell physiology that engender defective phenotypes (Shepherd et al., 2007). In

19 Begomoviruses,heterologous expressionofRepproteinswithmutatedCterminusintheRB bindingdomainrendersresistancetoTYLCVintomatoplants(Brunettietal.,1997;Lucioliet al.,2003).Expressingbeangoldenmosaicvirus(BGMV)Repproteinmutatedinthenucleoside triphosphate(NTP)bindingmotif(D262R),resultedinpartialresistancetoBGMVinbeans (Fariaetal.,2006).Genesusedagainstmanyothergeminivirusincludethoseencodingforthe CP(Sinisterraetal.,1999)andMP(Duanetal.,1997). Stacking of viral derived genes/proteins In another PDR approach, transdominant expression of negatively mutated multiple viral proteins (viagene stacking) engineered inmaize cangenerate strongresistance tothe virus (Halpin,2005). Inthisstrategy,targetingmultipleviralfunctionsincludingreplication,cellto cellandsystemicmovementorencapsidationispotentiallypossible.Heterologousexpressionof the defective proteins having minimal mutations in regions that otherwise would cause phenotypicdefectsinthehostormaypromoteviralsubsistenceordiseasemanifestationisdone. Mutationscanbedonewithintheregionresponsibleforinvasionofmesophyllcells(regionof RepAinteractionwithRBR)(Xieetal.,1995;McGivernetal.,2005),orwithinmotifsinvolved inDNAsequencerecognition(Fontesetal.,1994),DNAbinding(OrozcoandHanleyBowdoin 1996;Missichetal., 2000)andDNAcleavage(Willmentetal., 2007).Domainsinvolvedin helicase(Choudhuryetal.,2006)andATPaseactivity(ClerotandBernardi,2006)arepotential modificationsitesaswell.Thepermutationswilldependonthedesiredfunctionthatneedstobe eliminated.However,thedomainsshouldbemutatedsuchthattheheterologousproteinsinteract withthemselvesand/orwildtypemaintainingnormalhostcellcycleandoverallcellphysiology ofthetransgeniclines.Thecoatproteincanberemodeledsuchthatitisunabletoformthe necessaryDNAnucleoproteincomplexesneededforsystemicspreadofthevirusuponinfection. Theonlyhurdleisthatthecoatproteinsandmovementproteinsarenotwellcharacterizedtothe responsible interaction motifs. Only the CP seems to have the nuclear localisation signals characterized (Liu et al., 1999). MP is believed to block the (function of these) signals redirecting CP-DNA nucleoprotein complexes towards cell membrane and not nucleus (Liu et al., 2001). Direct gene silencing via RNAi without protein expression Expressing selfcomplementary RNA transcripts eventually results in dsRNA that triggers a

20 sequencespecificmRNAdegradationinaprocessknownasRNAi.Heterologousexpressionof antisense viral genes confers resistance to TGMV geminivirus (Bejarano and Lichtenstein, 1994).Bonflmetal.(2007)usedantisenseRNAsuccessfullytoproducetransgeniccommon beanshowingdelayedandattenuatedgoldenmosaicvirussymptomsuponwhiteflymediated infection. The potential of using selfcomplementary hairpin RNA (hpRNA) efficiently to silencegenesinplantshasbeendemonstrated(Wesleyetal.,2001).Thisapproachisapplicable indevelopingtransgenicmaizelinescontainingantisensehomologousviralgenesorhostgenes toimpedeMSVspreadforexample,bytargetingmovementproteinandperhapsdispensable host helper proteins. The viral coding sequences may be truncated and specific sequences employedforreasonsoutlinedabove.TheRNAistrategyfacesimminentchallengesstemming fromthefactthatplantRNAisystemsrequireperfectorverynearperfectnucleotidematches between thesiRNAandthetargeted mRNA(JonesRhoadesetal.,2006).ByshrewdRNAi designingandserendipitoustransitiveeffectofsiRNAproductionobservedinmaizeandrice, thisproblemcanbeavoidedifcommonsequencesaretargeted(Mazithulelaetal.,2000;Mikiet al.,2005;DietzgenandMitter,2006;FrizziandHuang,2009).Transcriptionalgenesilencingfor productionoftransgeniccropsisalsofeasible.Inthisstrategy,silencingofthepromoterofthe targeted gene using RNAi results in biologically insignificant activity of the gene (Bender, 2004).However,managingthecountersilencingofthevirusremainsanobviousimpediment giventhatgeminivirusesareabletoinduceviralsuppressionofRNAsilencing(VSRs). Emergingtools:thepeptideaptamerstrategyantibodiesforplants The ultimate field challenges to PDR is recombinant new viral strains, viral mutations, viral induced gene silencing (Li and Ding 2006) and phenotypic defects due to heterologous expression of viral derived proteins in the transgenic plants (Sharp et al., 2002; Shepherd et al., 2007). To overcome some of these problems, a strategy using synthetic peptide aptamers targeted against MSV proteins is under development (Lopez-Ochoa et al., 2006). The strategy uses non-pathogen derived peptides to bind and interfere with biological function of target proteins (Rudolph et al., 2003). The short peptides are designed such that they recognize and interact (bind) with high affinity to target protein motifs and domains interfering with the protein interactome (Figure 2). The peptide aptamers can be inserted within a loop domain of a protein scaffold for stability (Skerra, 2000), where they are presented in a constrained structural conformation akin to antibodies (Crawford et al., 2003; Hoppe-Seyler et al., 2004). Potentially, the peptide aptamers are candidates in developing broad-spectrum (cross) resistance against

21 geminiviruses and any of their emergent recombinant strains (Lopez-Ochoa et al., 2006). A select few are being introgressed into Kenyan tropical maize inbred lines via Agrobacterium mediated transformation, targeting MSV Rep/RepA hence MSV replication (Figure 3, Machuka JS personal communication). CONCLUSION Conventional breeding has shown some success in controlling MSD, but there are limitations that jeopardize its use as a stand-alone resistance mechanism. Genetic engineering offers an avenue to compliment the breeding efforts and is a useful tool for studies leading to understanding MSD pathogenesis. Through such studies, indispensable biochemical conditions for virus subsistence in the host cells will be unraveled. Such studies should focus on host contributory factors to MSD pathogenesis with the view of capitalizing on 'windows' (weakness or strength) in maize that can be tapped for designing stable MSD resistance. It is also important to build an MSV knowledge base before attacking it rather than relying on information about geminiviruses in other genera. Studies aimed at understanding MSV and maize host-pathogen interaction will serve as the basis for achieving durable resistance or tolerance and, thus, should be of high priority. We have highlighted various ways of tackling these questions and strongly support a collaborative effort involving shared resources and results by many scientists and laboratories in sub-Sahara Africa to expedite this effort. FUNDING The authors recognise funding from the Rockefeller Foundation (Grant FS 021) towards developing maize resistant to MSD using peptide aptamers. ACKNOWLEDGMENTS The authors appreciate Dr. Sylvester Anamis critical reviews during preparation of the manuscript.

22 FIGURE LEGENDS Figure 1. Morphological features of maize infected with MSV during grain filling stage in a peasants farm in Western Kenya. The infected maize plants are extensively yellow but of normal stature as uninfected ones. Figure 2. Schematic representation of the peptide aptamer strategy for developing immunity to MSV by targeting the replication associated proteins. The multi-functional domains in the replication proteins are active singly (e.g. mono-functional interactions with one interaction partner -IP1 or IP2) and on oligimerisation (for example bi-functional interaction with two partners -IP1 and IP2). Each domain becomes a target site for interference with peptide aptamers that irreversibly bind tightly to the domain preventing bio-functional interaction of the Rep protein with host factors. Apt1, 2, 3 Aptamer 1, 2, 3; IP1, 2-Interaction partner 1, 2; Repreplication associated protein; Red shades- constrained aptamer sequences within an exposed loop domain of the protein scaffolding. Figure 3. Agrobacterium-mediated transformation of tropical maize inbred lines via somatic embryogenesis. (A) Somatic embryogenesis of Kenyan tropical maize inbred line TL 23 showing proliferating somatic embryos from the underside of the scetullum (arrow) of the zygotic embryo. Plantlet establishment in culture bottles (B) and during acclimatization (C) before transferring to soil.

Table 1. Molecular function of protein and other genetic factors of MSV in relation to other geminiviruses.
Protein/geneti celement Replication associated proteins Rep (AL1, dsDNA Acronym Domain Functioncontributing motif* Nterminusresidues10112; Orirecognitionandbinding dsDNApriortoRCRinitiation recognitionand includesmotifI(F18LTYP 2)&II(H61LH4)(H61LH 4)andIII(V97XXYXXKE 2)andID1 ssDNAbinding Nterminusresidues1875 BindingssDNAduringinitiation Unmapped ofRCR DNAcleavage CamposOlivasetal.,2002;Missichetal., 2000;OrozcoandHanleyBowdoinetal., 1996 Nterminusresidues10112; NickingofssDNAofdsDNAto Begomoviruses CamposOlivasetal.,2002;Laufsetal., includesmotifI(F18LTYP 2)&II(H61LH4)(H61LH 4)andIII(V97XXYXXKE 2) NTPbindingP Consensussequenceinthe loop/oligomeris Cterminus ation Transcription activation domain Ligase Oligomerisatio n/helicase Consensus Circularisingnewlysynthesised WDV viralssDNA forMSVand134180for oligomers Begomoviruses Settlageetal.,2001,1996;Orozcoetal., Begomoviruses 2000;Hovarthetal.,1998 Laufsetal.,1995a;Heyraudetal.,1995 G230XXXXGKT/S DNAsynthesis;helicaseand ATPdependenttopoisomerase activity MSV Begomoviruses HansonandMaxwell1999;Clerotand Bernardietal.,2006;Desbiezetal.,1995; Choundhuryetal.,2006 Hovarthetal.,1998;EagleandHanley Bowdoin,1997 startRCR WDV 1995b;Willmentetal.,2007 Domainfunction Where mapped Begomoviruses CamposOlivasetal.,2002;Orozcoetal., WDV 1998;Fontesetal.,1994;Laufsetal., 1995a;Heyraudetal.,1995 References

AC1,C1) binding

Cterminusresidues250270 Activationofhostgene Unmappedinbegomoviruses expression

Nterminusresidues175187 Formationofinter/intraspecific MSV

Protein/geneti celement

Acronym Domain

Functioncontributing motif*


Where mapped


activity RepA pRBbinding domain

TGMV LXCXEintheCterminus otherMastrevirusesand begomoviruses) Interactionwithhost MSV Shepherdetal.,2005;Hovarthetal., 1998;ArguelloAstorgaetal.,2004 residues198202(missingin retinoblastomaprotein

Transcription activation domain Rep domains CDKbinding domain pRBbinding

Cterminusresidues250270 Activationofyeastreportergene MSV expression Umapped Interactionwithhost retinoblastomaprotein Unmapped InteractionwithhostCDKs InteractionwithhostCDK inhibitorfactors TGMV134180 Unmapped Interactionwithhostserine kinases Interactionwithhostcellcycle factors Encapsidationofvirions Begomoviruses Begomoviruses Begomoviruses Begomoviruses


unknown domain

CDKinhibitors Unmapped domain Serinekinase domain Cellcycle interaction domain Coatprotein CP Unknown Unknown

Begomoviruses Kongetal.,2002

Mastreviruses, Krichevskyetal.,2006;Boultonetal., Begomoviruses 1986 Krichevskyetal.,2006;Boultonetal., 1989

Systemicmovementthroughthe Mastreviruses hostplant

Protein/geneti celement

Acronym Domain

Functioncontributing motif*


Where mapped


Unknown Movement protein Functional genetic elements Histone hexamermotif Ori Stemloop repeat sequences SIR Polyadenylatio nsequences rNMPoligo sequences Iteron domain (ID) Nterminusresidues1017 (1) Unmapped Consensus 5'TAATATTAC3' Unmapped LIR rep1 MP Unknown

Translocatingviralgenome throughnuclearenvelope Celltocellmovementthrough theplasmodesmata


Krichevskyetal.,2006;Boultonetal., 1989

Mastreviruses, Dickinsonetal.,1996 Fenolletal.,1990;Wrightetal.,1997; Mazithulelaetal.,2000 Mastreviruses Morozovetal.,1994

Promotercisactingelementsfor Mastreviruses sensegenes Planthomologoushistonelike hexamerpromoterelements Replicationstartsite

Mastreviruses, Willmentetal.,2007;Stengeretal.,1991 Begomoviruses MSV, Begomoviruses Mastreviruses, Mullineauxetal.,1984;Wrightetal., 1997;MorrisKrsinichetal.,1985 Mastreviruses Donsonetal.,1984 McGivernetal.,Orozcoetal.,1996

5GCAGGAAAAGAAGG Formationofahairpinloop 3 necessaryforreplication initiation Terminationofviral transcription InitiationofviraldsDNA replicationintermediate

dsDNArecognitionandbinding Begomoviruses CamposOlivasetal.,2002;Arguello priortoRCRinitiation Astorgaetal.,2001;1994

LIRlongintergenicregion;SIRshortintergenicregion;RCRrollingcyclereplication;NTPnucleosidetriphosphate;pRBplantretinoblastoma;CDKcyclin dependentkinase;rNMPribosenucleosidemonophosphate;WDVwheatdwarfvirus.*IndicatedresiduepositionsareforMSVgenome(Martinetal.,2001). SeeotherhostfactorsinvolvedinholisticviralpathogenicityinWhithamandWang(2004).

REFERENCES Abalo, G., Tongoonaa, P., Derera, J., and Edema, R. (2009). A comparative analysis of conventional and marker-assisted selection methods in breeding maize streak virus resistance in maize. Crop Sci. 49, 509-520. Accotto, G.P., Mullineaux, P.M., Brown, S.C., and Marie, D. (1993). Digitaria streak geminivirus replicative forms are abundant in S-phase nuclei of infected cells. Virol. 195, 257-259. Ach, R.A., Durfee, T., Miller, A.B., Turanto, P., Hanley-Bowdoin, L., Zambryski, P.C., and Gruissem, W. (1997). RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein. Mol. Cell Biol. 17, 5077-5086. Achard, P., Herr, A., Baulcombe, D.C., and Harberd, N.P. (2004). Modulation of floral development by a gibberellin-regulated microRNA. Development 131, 3357-3367. Akerfelt, M., Henrikson, E., Laiho, A., Vihervaara, A., Rautoma, K., and Kotaja, N. (2006). Promoter ChIP-chip analysis in mouse testis reveals Y chromosome occupancy by HSF2. Proc. Natl. Acad. Sci. USA 105, 11224-11229. Anami, S., De Block, M., Machuka, J., and Van Lijsebettens, M. (2009). Molecular improvement of tropical maize for drought stress tolerance in sub-Saharan Africa. Crit. Rev. Plant Sci. 28, 16-35. Arguello-Astorga, G., Ascencio-Ibez, J. T., Dallas, M. B., Orozco, B. M., and HanleyBowdoin, L. (2007). High-frequency reversion of geminivirus replication protein mutants during infection. J. Virol. 81, 11005-11015. Arguello-Astorga, G.R., and Ruiz-Medrano, R. (2001). An iteron-related domain is associated to motif I in the replication proteins of geminiviruses: identification of potential interacting amino acid-base pairs by a comparative approach. Arch. Virol. 146, 1465-1485. Arguello-Astorga, G.R., Lopez-Ochoa, L., Kong, L.-J., Orozco, B.M., Settlage, S.B., and Hanley-Bowdoin, L. (2004). A novel motif in geminivirus replication proteins interacts with the plant retinoblastoma-related protein. J. Virol. 78, 4817-4826. Arguello-Astroga, G.R., Guevara-Gonzalez, R.G., Herrera-Estrella, L.R., and RiveraBustamante, R.F. (1994). Geminivirus replication origins have a group-specific organization of iterative elements: a model fro replication. Virol. 203, 90-100. Asanzi,C.M.,BosquePerez,N.A.,Buddenhagen,I.W.,Gordon,D.T.,andNault,L.R.(1994). Interactions among maize streak virus disease, leafhopper vector populations and maize cultivarsinforestandSavannahzonesofNigeria.PlantPathol.43,145157. Ascencio-Ibanez, J.T., Sozzani, R., Lee, T.-J., Chu, T.-M., Wolfinger, R.D., Cella, R., and Hanley-Bowdoin, L. (2008). Global analysis of Arabidopsis gene expression uncovers a complex array of changes impacting pathogen response and cell cycle during geminivirus infection. Plant Physiol. 148, 436-454. Axtell, M.J., and Bartel, D.P. (2006). Antiquity of microRNAs and their targets in land plants. Plant Cell 17, 1658-1673. Ayliffe, M.A., and Lagudah, E.S. (2004). Molecular genetics of disease resistance in cereals. Annal. Bot. 94, 765-773. Bagewadi, B., Chen, S., Lal, S.K., Choudhury, N.R., and Mukherjee, S.K. (2004). PCNA interacts with Indian mung bean yellow mosaic virus rep and down regulates Rep activity. J. Virol. 78, 11890-11903.

Barros, E., Lezar, S., Anttonen, M.J., van Dijk, J.P., Rhlig, R.M., Kok, E.J., and Engel, K.-H. (2010). Comparison of two GM maize varieties with a near isogenic non-GM variety using transcriptomics, proteomics and metabolomics. Plant Biotech. J. 8, 436-451. Barrow, M.R. (1992). Development of maize hybrids resistant to maize streak virus. Crop Prot. 11, 267-271. Bejarano, E.R., and Lichtenstein, C.P. (1994). Expression of TGMV anti-sense RNA in transgenic tobacco inhibits replication of BCTV but not ACMV geminiviruses. Plant Mol. Biol. 24, 241-248. Bender, J. (2004). DNA methylation and epigenetics. Annu. Rev. Plant Biol. 55, 41-68. Benett, A., McKenna, R., and Agbandje-McKenna, M. (2008). A comparative analysis of the structural architecture of ssDNA viruses. Comp. Math. Meth. In Med. 9, 183-196. Bogan A.A., and Thorn, K.S. (1998). Anatomy of hot spots in protein interfaces. J. Mol. Biol. 280, 1-9. Bonflm, K., Farla, J.C., Noguelra, E.O.P.L., Mendes, E.A., and Aragao, F.J.L. (2007). RNAimediated resistance to bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Mol. Plant-Microbe Interact. 20, 717-726. Bosque-Perez, N.A. (2000). Eight decades of maize streak virus research. Virus Res. 71, 107-121. Bosque-Perez, N.A., Olojede, S.O., and Buddenhagen, I.W. (1998). Effect of maize streak virus disease on the growth and yield of maize as influenced by varietal resistance levels and plant stage at the time of challenge. Euphytica 101, 307-317. Boulton, M.I. (2002). Functions and interactions of Mastrevirus gene products. Physiol Mol Plant Pathol. 60, 243-255. Boulton, M.I., King, D.I., Donson, J., and Davies, J.W. (1991a). Point substitutions in a promoterlike region and the V1 gene affect the host range and symptoms of maize streak virus. Virol. 183, 114-121. Boulton, M.I., King, D.I., Markham, P.G., Pinner, M.S., and Davies, J.W. (1991b). Host range and symptoms are determined by specific domains of the maize streak virus genome. Virol. 181, 312-318. Boulton, M.I., Pallaghy, C.K., Chatanmi, M., MacFarlane, S., and Davies, J.W. (1993). Replication of maize streak virus mutants in maize protoplasts: Evidence for a movement protein. Virol. 192, 85-93. Boulton, M.I., Steinkellner, H., Donson, J., Markham, P.G., King, D.I., and Davies, J.W. (1989). Mutational analysis of the virion sense genes of maize streak virus. J. Gen. Virol. 70, 2309-2323. Brautigam, A., Hofmann-Benning, S., and Weber, A.P.M. (2008). Comparative proteomics of chloroplast envelopes from C3 and C4 plants reveals specific adaptations of the plastid envelope to C4 photosynthesis and candidate proteins required for maintaining C4 metabolite fluxes. Plant Physiol. 148, 568-579. Brunetti, A., Tavazza, M., Noris, E., Tavazza, R., Caciagli, P., Ancora, G., Crespi, S., and Accotto, G.P. (1997). High expression of truncated viral Rep protein confers resistance to tomato yellow leaf curl virus in transgenic tomato plants. Mol. Plant-Microbe Interact. 10, 571-579. Campos-Olivas, R., Louis, J.M., Clerot, D., Gronenborn, B., and Gronenborn, A.M. (2002). The structure of a replication initiator unites diverse aspects of nucleic acid metabolism. Proc. Natl. Acad. Sci. USA 99, 10310-10315.

Castillo, A.G., Collinet, D., Deret, S., Kashoggi, A., and Bejarano, E.R. (2003). Dual interaction of plant PCNA with geminivirus replication accessory protein (Ren) and viral replication protein (Rep). Virology 312, 381-394. Chatterji,A.,Beachy,R.N.,Fauquet,andC.M.(2001).Expressionoftheoligomerisationdomain ofthereplicationassociatedprotein(Rep)of TomatoleafcurlNewDelhivirus interferes withDNAaccumulationofheterologousgeminiviruses.J.Biol.Chem.276,2563125638. Chellappan,P.,Vanitharani,R.,Ogbe,F.,andFauquet,C.M.(2005).Effectoftemperatureon geminivirusinducedRNAsilencinginplants.PlantPhysiol.138,18281841. Chen, M.H., and Citovsky, V. (2003). Systemic movement of a tobamovirus requires host cell pectin methylesterase. Plant J. 35, 386-392. Chen, W., Lennox, S.J., Palmer, K.E., and Thomson, J.A. (1998). Transformation of Digitaria sanguinalis: A model system for testing maize streak virus resistance in Poaceae. Euphytica 104, 25-31. Chien, C.-T., Bartel, P.L., Sternglanz, R., and Fields, S. (1991). The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA 88, 9578-9582. Choudhury, N.R., Malik, P.S., Singh, D.K., Islam, M.N., Kaliappan, K., and Mukherjee, S.K. (2006). The oligomeric Rep protein of mung bean yellow mosaic India virus (MYMIV) is a likely replicative helicase. Nucleic Acids Res. 34, 6362-6377. Clerot, D., and Bernardi, F. (2006). DNA Helicase activity is associated with the replication initiator protein Rep of tomato yellow leaf curl geminivirus. J. Virol. 80, 11322-11330. Corrado, G., and Karali, M. (2009). Inducible gene expression systems and plant biotechnology. Biotech. Adv. 27, 733-743. Covshoff, S., Majeran, W., Liu, P., Kolkman, J.M., van Wijk, K., and Brutnell, T.P. (2008). Deragulation of maize C4 photosynthetic development in a mesophyll cell-defective mutant. Plant Physiol. 146, 1469-1481. Crawford, M., Woodman, R., and Ferrigno, P.K. (2003). Peptide aptamers: Tools for biology and drug discovery. Brief. Funct. Genom. Proteom. 2, 72-79. Culver, J.N., and Padmanabhan, M.S. (2007). Virus-induced disease. Altering host physiology one interaction at a time. Annu. Rev. Phytopathol. 45, 10.1-10.3. David-Schwartz, R., Runo, S. Townsley, B., Machuka, J., and Sinha, N. (2008). Long-distance transport on mRNA via parenchyma cells and phloem across the host-parasite junction in Cuscuta. New Phytol. 179, 1131-1141. Dawson, W.O., and Hilf, M.E. (1992). Host-range determinants of plant viruses. Annu Rev Plant Physiol. Plant Mol. Biol. 43, 527-555. De Veylder, L., Joubes, J., and Inze, D. (2003). Plant cell cycle transitions. Curr. Opin. in Plant Biol. 6, 536-543. Desbiez, C., David, C., Mettouchi, A., Laufs, J., and Gronenborn, B. (1995). Rep protein of tomato yellow leaf curl geminivirus has an ATPase activity required for viral DNA replication. Proc. Natl. Acad. Sci. USA 92, 5640-5644. Diallo, A.O., Kikafunda, J., Wolde, L., Odongo, O., Mduruma, Z.O., Chivatsi, W.S., Friesen, D.K., Mugo, S., and Banziger, M. (2001). Drought and low nitrogen tolerant hybrids for the moist mid-altitude ecology of Eastern Africa. Seventh Eastern and Southern Africa Regional maize Conference, 11th-15th February, Nairobi Kenya pp. 206-212. Diaz-Pendon, J.A., and Ding, S.-W. (2008). Direct and indirect roles of viral suppressors of RNA silencing in pathogenesis. Annu. Rev. Phytopathol. 46, 303-326.

Dickinson, V.J., Halder, J., and Woolston, C.J. (1996). The product of maize streak virus ORF V1 is associated with secondary plasmodesmata and is first detected with the onset of viral lesions. Virol. 220, 51-59. Dietzgen, R.G., and Mitter, N. (2006). Transgene silencing strategies for viral control. Australasian Plant Pathol. 35, 605-618. Donson, J., Morris-Krsinich, B.A., Mullineaux, P.M., Boulton, M.I., and Davies, J.W. (1984). A putative primer for second-strand DNA synthesis of maize streak virus is virion associated. EMBO J. 3, 3069-3073. Drewes, G., and Bouwmeester, T. (2003). Global approaches to proteinprotein interactions. Curr. Opin. Cell Biol. 15, 199-205. Drugeon, G., Urcuqui-Inchima, S., Milner, M., Kadare, G., Valle, R.P.C., Voyatzakis, A., Haenni, A.-L, and Schirawski, J. (1999). The strategies of plant virus gene expression: models of economy. Plant Sci. 148, 77-88. Dry, I.B., Krake, L.R., Ridge, J.E., and Rezaian, M.A. (1997). A novel sub viral agent associated with a geminivirus: the first report of a DNA satellite. Proc. Natl. Acad. Sci. USA 94, 70887093. Duan, Y.P., Powell, C.A., Webb, S.E., Purcifull, D.E., and Hiebert, E. (1997). Geminivirus resistance in transgenic tobacco expressing mutated BC1 protein. Mol. Plant-Microbe Interact. 10, 617-623. Duffy, S., and Holmes, E.C. (2009). Validation of high rates of nucleotide substitution in geminiviruses: phylogenetic evidence from East African cassava mosaic viruses. J. Gen. Virol. 90, 1539-1547 Eagle, P.A., and Hanley-Bowdoin, L. (1997). cis Elements that contribute to geminivirus transcriptional regulation and efficiency of DNA replication. J. Virol. 71, 6947-6955. Efron, Y., Kim, S.K., Fajemisin, J.M., Mareck, J.H., Tang, C.Y., Dabrowski, Z.T., Rosell, H.W., Thottappilly, G., and Buddenhagen, I.W. (1989). Breeding for resistance to maize streak virus: A multidisciplinary team approach. Plant Breed. 103, 1-36. Erdmann, J.B., Shepherd, D.N., Martin, D.P., Varsani, A. Rybicki E.P., and Jeske, H. (2010). Replicative intermediates of maize streak virus found during leaf development. J. Gen. Virol. 91, 1077-1081. Fargette, D., Konate, G., Fauquet, C., Muller, E., Peterschmitt, M., and Thresh, J.M. (2006). Molecular ecology and emergence of tropical plant viruses. Annu. Rev. Phytopathol. 44, 235-260. Faria, J.C., Albino, M.M.C., Dias, B.B.A., Cancado, L.J., Cunha, N.B., Silva, L. de M., Vianna, G.R., and Aragao, F.J.L. (2006). Partial resistance to bean golden mosaic virus in a transgenic common bean (Phaseolus vulgaris L.) line expressing a mutated rep gene. Plant Sci. 171, 565-571. Fauquet, C.M., Bisaro, D.M., Briddon, R.W., Brown, J.K., Harrison, B.D., Rybicki, E.P., Stenger, D.C., and Stanley, J. (2003). Revision of taxonomic criteria for species demarcation in the family Geminiviridae and an updated list of Begomovirus species. Arch. Virol. 148, 405-421. Fenoll, C., Schwartz, J.J., Black, D.M., Schneider, M., and Howell, S.H. (1990). The intergenic region of maize streak virus contains a GC-rich element that activates rightward transcription and binds maize nuclear factors. Plant Mol. Biol. 15, 856-877. Ferry, N., Edwards, M.G., Gatehouse, J., Capell, P., Christou, P., and Gatehouse, A.M.R. (2006). Transgenic plants for insect pest control: a forward looking scientific perspective. Transgenic Res. 15, 13-19.

Fiehn, O., Wohlgemuth, G., Scholz, M., Kind, T., Lee, D.Y., Lu, Y., Moon, S., and Nikolau, B. (2008). Quality control for plant metabolomics: reporting MSI-compliant studies. The Plant J. 53, 691-704. Fontes, E.P.B., Gladfelter, H.J., Schaffer, R.L., Petty, I.T.D., and Hanley-Bowdoin, L. (1994). Geminivirus replication origins have a modular organization. Plant Cell 6, 405-416. Froissart, R., Doumayrou, J., Vullaume, F., Alizon, S., and Michalakis, Y. (2010). The virulence-transmission trade-off in vector-borne plant viruses: a review of (non-)existing studies. Phil. Trans. R. Soc. B 365, 1907-1918. Funayama, S., Hikosaka, K., and Yahara, T. (1997). Effects of virus infection and growth irradiance on fitness components and photosynthetic properties of Eupatorium makinoi (Compositae). American J. Bot. 84, 823-829. Gibson, R.W., and Page, W.W. (1997). The determination of when maize plants were infected with maize streak virus from the position of the lowest diseased leaf. African Crop. Sci. J. 5, 189-195. Gibson, R.W., Lyimo, N.G., Temu, A.E.M., Stathers, T.E., Page, W.W., Nsemwa, L.T.H, Acola, G., and Lamboll, R.I. (2005). Maize seed selection by East African small-holder farmers and resistance to maize streak virus. Annal. Appl. Biol. 147, 153-159. Gilbertson, R.L., Sudarshama, M., Jiang, H.M., Rojas, M.R., and Lucas, W.J. (2003). Limitations on geminivirus genome size imposed by plasmodesmata and virus-encoded movement protein: insights into DNA trafficking. Plant Cell 15, 2578-2591. Glazebrook, J. (2005). Contrasting mechanisms of defence against biotrophic and necrotrophic pathogens. Annu. Rev. Phytopathol. 43, 205-227. Golbach, R., Bucher, E., and Prins, M. (2003). Resistance mechanisms to plant viruses: an overview. Virus Res. 92, 207-212. Goritschnig, S., Weihmann, T., Zhang, Y., Fobert, P., McCourt, P., and Li, X. (2008). A novel role for protein farnesylation in plant innate immunity. Plant Physiol. 148, 348-357. Gregory, B.D., Yazaki, J., and Ecker, J.R. (2008). Utilizing tiling micro arrays for whole-genome analysis in plants. The Plant J. 53, 636-644. Gros, M.F., Te Riele, H., and Erlich, S.D. (1987). Rolling replication of the single stranded plasmid pC194. EMBO J. 6, 3863-3869. Grotewold, E. (2008). Transcription factors for predictive plant metabolic engineering: are we there yet? Curr. Opin. Biotechnol. 19, 138-144. Guo, H.S., Xie, Q., Fei, J., and Chua, N.H. (2005). MicroRNA directs mRNA cleavage of the transcription factor NAC1 to down regulate auxin signals for Arabidopsis lateral root development. Plant Cell, 17, 1376-1386. Gustafson, A.M., Allen, E., Givan, S., Smith, D., Carrington, J.C., and Kasschau, K.D. (2005). ASRP: the Arabidopsis small RNA project database. Nucleic Acids Res. 33, 637-640. Gutierrez, C. (1999). The retinoblastoma pathway in plant cell cycle and development. Curr. Opin. Plant. Biol. 1, 492-497. Gutierrez, C. (2000). DNA replication and cell cycle in plants: learning from geminiviruses. EMBO J. 19, 792-799. Gutierrez, C. (2002). Strategies for geminivirus DNA replication and cell cycle interference. Physiol. Mol. Plant. Pathol. 60, 219-230. Hall, R.D. (2006). Plant metabolomics: from holistic hope to hype, to hot topic. New Phytol. 169, 453-468. Halpin, C. (2005). Gene stacking in transgenic plants-the challenge for 21st century plant biotechnology. Plant Biotechnol. J. 3, 141-155.

Hanley-Bowdoin, L., Elmer, J.S., and Rogers, S. (1990). Expression of functional replication protein from tomato golden mosaic virus in transgenic tobacco plants. Proc. Natl. Acad. Sci. USA 87, 146-1450. Hanley-Bowdoin, L., Settlage, S.B., and Robertson, D. (2004). Reprogramming plant gene expression: a prerequisite to geminivirus DNA replication. Mol. Plant Pathol. 5, 149-156. Hanley-Bowdoin, L., Settlage, S.B., Orozco, B.M., Nagar, S., and Robertson, D. (1999). Geminiviruses: models for plant DNA replication, transcription and cell cycle regulation. Crit. Rev. Plant Sci. 18, 71-106. Hanson, S.F., and Maxwell, D.P. (1999). trans-Dominant inhibition of geminiviral DNA replication by bean golden mosaic geminivirus rep gene mutants. Virol. 89, 480-485. Harkins, G.W., Martin, D.P., Duffy, S., Monjane, A.L., Shepherd, D.N., Windram, O.P., Owor, B.E., Donaldson, L., van Antwerpen, T., Sayed, R.A., Flett, B., Ramusi, M., Rybicki, E.P., Petrschmitt, M., and Varsani, A. (2009). Dating the origins of the maizeadapted strain of maize streak virus, MSV-A. J. Gen. Virol. 90, 3066-3074. Hefferon, K.L., and Dugdale, B. (2003). Independent expression of Rep and RepA and their roles in regulating bean yellow dwarf virus replication. J. Gen. Virol. 84, 3465-3472. Heil, M., and Bostock, R.M. (2002). Induced systemic resistance (ISR) against pathogens in the context of induced plant defences. Annal. Bot. 89, 503-512. Heinlein, M., and Epel, B.L. (2004) Macromolecular transport and signaling through plasmodesmata. Int. Rev. Cytol. 235, 93-552. Herr, A.J. (2005). Pathways through the small RNA world of plants. FEBS Lett. 579, 5879-5888. Heyraud-Nitschke, F., Schumacher, S., Laufs, J., Schaefer, S., Schell, J., and Gronenborn, B. (1995). Determination of origin of cleavage and joining domain of geminivirus Rep proteins. Nucleic Acids Res. 23, 910-916. Hofmann, C., Niehl, A., Sambade, A., Steinmetz, A., and Heinlein, M. (2009) Inhibition of tobacco mosaic virus movement by expression of an actin-binding protein. Plant Physiol. 149, 1810-1823. Hogenhout, S.A., Ammar, E.-D., Whitfield, A.E., and Rendinbaugh, M.G. (2008). Insect vector interactions with persistently transmitted viruses. Annu. Rev. Phytopathol. 46, 327-359. Hohn, T. (2007). Plant virus transmission from the insect point of view. Proc. Natl. Acad. Sci. USA 104, 17905-17906. Horvath, G.V., Pettko-Szandtner, A., Nikovics, K., Bilgin, M., Boulton, M.I., Davies, J.W., and Dudits, D. (1998). Prediction of functional regions of the maize streak virus replicationassociated proteins by protein-protein interactions analysis. Plant Mol. Biol. 38, 699-712. Huang, Z., Yeakley, J.M., Garcia, E.W., Holdridge, J.D., Fan, J.B., and Whitham, S.A. (2005) Salicylic acid-dependent expression of host genes in compatible Arabidopsis virus interactions. Plant Physiol. 137, 1147-1159. Ilyina, T.V., and Koonin, E.V. (1992). Conserved sequence motifs in the initiator proteins for rolling cycle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucleic Acids Res. 20, 3279-3285. Ingelbrecht, I.L., Irvine, J.E., and Mirkov, T.E. (1999). Post transcriptional gene silencing in transgenic sugar cane. Dissection of homology-dependent virus resistance in a monocot that has a complex polyploid genome. Plant Physiol. 119, 1187-1198. Ishida, H., Yoshimoto, K., Izuni, M., Reisen, D., Yano, Y., Makino, A., Ohsimu, Y., Hanson, M.R., and Mae, T. (2008). Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process. Plant Physiol. 148, 142-155.

Isnard, M., Granier, M., Frutos, R., Reynaud, B., and Peterschmitt, M. (1998). Quasispecies nature of three maize streak virus isolates obtained through different modes of selection from a population used to assess response to infection of maize cultivars. J. Gen. Virol 79, 30913099 Jin,M.,Li,C.,Shi,Y.,Ryabov,E.,Huang,J.,Wu,Z.,Fan,Z.,andHong,Y.(2008).Asingle aminoacidchangeinageminiviralRepproteindifferentiatesbetweentriggering aplant defenceresponseandinitiatingviralDNAreplication.J.Gen.Virol.89,26362641. Jones-Rhoades, M.W., and Bartel, D.P. (2004). Computational identification of plant microRNAs and their targets including a stress-induced miRNA. Mol. Cell. 14, 787-799. Jones-Rhoades, M.W., Bartel, D.P., and Bartel, B. (2006). MicroRNAs and their regulatory roles in plants. Annu. Rev. Plant. Biol. 57, 19-53. Kairo, M.T.K., Kiduyu, P.K., Mutinda, C.J.M., and Empig, L.T. (1995). Maize streak virus: evidence for resistance against Cicadulina mbila Naude the main vector species. Euphatica 89, 109-114. Kammann, M., Schalk, H.-J., Matzeit, V., Schaefer, D., Schell, J., and Gronenborn, B. (1991). DNA replication of wheat dwarf virus, a geminivirus, requires two cis-acting signals. Virol. 184, 786-790. Kanampiu, F., Ransom, J., Gressel, J., Jewell, D., Friesen, D., Grimanelli, D., and Hoisington, D. (2002). Appropriateness of biotechnology to African agriculture: Striga and Maize as paradigms. Plant Cell Tiss. Org. Cult., 69,105-110. Kang, B.-C., Yeam, I., and Jahn, M.M. (2005). Genetics of plant virus resistance. Annu. Rev. Phytopathol. 43, 581-621. Kelly, W.P., and Stumpf, M.P.H. (2008). Protein-protein interactions: from global to local analyses. Curr. Opin. Biotechnol. 19, 396-403. Khush, G.S. (2001). Green revolution: the way forward. Nat. Genet. 2, 815-822. Kong, J.L., Orozco, B.M., Roe, J.L., Nagar, S., Miller, A.B., Gruissem, W., Robertson, D., and Hanley-Bowdoin, L. (2000). A geminivirus replication protein interacts with the retinoblastoma protein through a novel domain to determine symptoms and tissue specificity of infection in plants. EMBO J. 19, 3485-3495. Kragler, F., Lucas, W.J., and Monzer J. (1998). Plasmodesmata: Dynamics, domains and patterning. Annal. Bot. 81, 1-10. Krichevsky, A., Kozlovsky, S., Gafni, Y., and Citovsky, V. (2006). Nuclear import and export of plant virus proteins and genomes. Mol. Plant. Pathol. 7, 131-146. Kyetere, D.T., Ming, R., McMullen, M.D., Pratt, R.C., Brewbaker, J., and Musket, T. (1999). Genetic analysis of tolerance to maize streak virus. Genome 42, 20-26. Laufs, J., Schumacher, S., Geisler, N., Jupin, I., and Gronenborn, B. (1995b). Identification of the nicking tyrosine of geminivirus Rep protein. FEBS lett. 377, 258-262. Laufs, J., Traut, W., Heyraud, F., Matzeit, V., Rogers, S.G., Schell, J., and Gronenborn, B. (1995a). In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus. Proc. Natl. Acad. Sci. USA 92, 3879-3883. Lazarowitz, S.G. (1992). Geminiviruses: Genome structure and gene function. Crit. Rev. Plant. Sci. 11, 327-349. Lazarowitz, S.G., Pinder, A.J., Damsteegt, V.D., and Rogers, S.G. (1989). Maize streak virus genes essential for systemic spread and symptom development. EMBO J. 8, 1023-1032.

Le Provost, F., Lillico, S., Passet, B., Young, R., Whitelaw, B., and Vilotte, J.-L. (2009). Zinc finger nuclease technology heralds a new era in mammalian transgenesis. Trends Biotech. 28, 134-141. Lehrer, A.T., Moore, P.H., and Komor, E. (2007). Impact of sugar cane yellow leaf virus (ScYLV) on the carbohydrate status of sugar cane: Comparison of virus-free plants with symptomatic and asymptomatic virus-infected plants. Proc. Natl. Acad. Sci. USA 103, 11856-11861. Levy, A., and Tzfira, T. (2010). A Bean dwarf mosaic virus: a model system for the study of viral movement Mol. Plant Path. 11, 451-461. Li, F., and Ding, S.-W. (2006). Virus counter defence: Diverse strategies for evading the RNA silencing immunity. Annu. Rev. Microb. 60, 503-531. Liu, H., Boulton, M.I,, Oparka, K.J., and Davies, J.W. (2001). Interaction of the movement and coat proteins of Maize streak virus: implifications for the transport of viral DNA. J. Gen. Virol. 82, 35-44. Liu, H., Boulton, M.I., and Davies, J.W. (1997). Maize streak virus coat protein binds single and double stranded DNA in vitro. J. Gen. Virol. 78, 1265-1270. Liu, H., Davies, J.W., and Stanley, J. (1998). Mutational analysis of bean yellow dwarf virus, a geminivirus of the genus Mastrevirus that is adapted to dicotyledonous plants. J. Gen. Virol. 79, 2265-2274. Liu, H., Saunders, K., Thomas, C.L., Prior, D.A.M., Oparka, K.J., and Davies, J.W. (1999). Maize streak virus coat protein is karyophyllic and facilitates nuclear transport of viral DNA. Mol. Plant-Microbe Interact. 12, 894-900. Liu, L., Pinner, M.S., Davies, J.W., and Stanley, J. (1999). Adaptation of the geminivirus bean yellow dwarf virus to dicotyledonous hosts involves both virion-sense and complementarysense genes. J. Gen.Virol. 80, 501-506. Liu, Q., and Chen, Y.-G. (2010). A new mechanism in plant engineering: The potential roles of microRNAs in molecular breeding for crop improvement. Biotech. Advances 28, 301-307. Lopez-Ochoa, L., Ramirez-Prado, J., and Hanley-Bowdoin, L. (2006). Peptide aptamers that bind to a geminivirus replication protein interfere with viral replication in plant cells. J. Virol. 80, 5841-5853. Lucioli, A., Noris, E., Brunetti, A., Tavazza, R., Ruzza V., Castillo, A.G., Bejarano, E.R., Accotto, G.P., and Tavazza, M. (2003). Tomato yellow leaf curl sardinia virus Rep-derived resistance to homologous and heterologous geminiviruses occurs by different mechanisms and is overcome if virus-mediated transgene silencing is activated. J. Virol. 77, 6785-6798. Lucy, A.P., Boulton, M.I., Davies, J.W., and Maule, A.J. (1996). Tissue specificity of Zea mays infection by maize streak virus. Mol. Plant Microbe Interact. 9, 23-31. Lukuyu, B.A., Njuguna, J., Mwangi, D.M., Murdoch, A.J., Mould, F.L., Owen, E., and Romney, D.L. (2002). Effects of infection with maize streak virus, and cultivar, on yield and quality of maize forage, and on yield of grain. Machuka, J. (2003). Development of sustainable food production systems in Africa. In: Plants, Genes and Crop Biotechnology, 2nd Edition. Edited by M.J. Chrispeels and D. Sadava. Jones and Bartlett Publishers and the American Society of Plant Biologists. pp.100-123. Machuka, J. (2004). Agricultural genomics and sustainable development: perspectives and prospects for Africa. African J. Biotech. 3, 127-135. Martin, D.P., and Rybicki, E.P. (2002). Investigation of maize streak virus pathogenicity determinants using chimeric genomes. Virol. 300, 180-188.

Martin, D.P., Willment, J.A., and Rybicki, E.P. (1999). Evaluation of maize streak virus pathogenicity in differently resistant Zea mays genotypes. Virol. 89, 695-700. Martin, D.P., Willment, J.A., Billharz, R., Velders, R., Odhiambo, B., Njuguna, J., James, D., and Rybicki, E.P. (2001). Sequence diversity and virulence in Zea mays of maize streak virus isolates. Virol. 288, 247-255. Mazithulela, G., Sudhakar, D., Heckel, T., Mehlo, L., Christou, P., Davies, J.W., and Boulton, M.I. (2000). The maize streak virus protein transcription unit exhibits tissue specific expression in transgenic rice. Plant Sci. 155, 21-19. McGivern, D.R., Findlay, K.C., Montague, N.P., and Boulton, M.I. (2005). An intact RBRbinding motif is not required for infectivity of maize streak virus in cereals, but is required for invasion of mesophyll cells. J. Gen. Virol. 86, 797-801. Meins, F., Si-Ammour, A., and Blevins, T. (2005). RNA silencing systems and their relevance to plant development. Annu. Rev. Cell. Dev. 21, 297-318. Mgutu, A.J. (2004). Ascertaining metabolic pathway sites in Orobanche aegyptiaca that can be inhibited using anti-metabolites in vitro. Master of Science Thesis submitted to the School of Pure and Applied Sciences, Kenyatta University, Nairobi, Kenya. Miki, D., Itoh, R., and Shimamoto, K. (2005). RNA silencing of single and multiple members in a gene family of rice. Plant Physiol. 138, 1903-1913. Mills-Lujan, K., and Deom, C.M. (2010). Geminivirus C4 protein alters Arabidopsis development. Protoplasma 239, 95-110. Missich, R., Ramirez-Parra, E., and Gutierrez, C. (2000). Relationship of oligomerisation of DNA binding of wheat dwarf virus RepA and Rep proteins. Virol. 273, 178-188. Moose, S.P., and Mumm, R.H. (2008). Molecular plant breeding as the foundation for 21st century crop improvement. Plant Physiol. 147, 969-977. Morales, F.J. (2001). Conventional breeding for resistance to Bemisia tabaci-transmitted geminiviruses. Crop Prot. 20, 825-834. Moreno-Risueno, M.A., Busch, W., and Benfey, P.N. (2009). Omics meet networks using systems approaches to infer regulatory networks in plants. Curr. Opin. Plant Biol. 13, 126131. Morozov, S.Y., Merits, A., and Chernov, B.K. (1994). Computer search of transcription control sequences in small plant virus DNA reveals a sequence highly homologous to the enhancer element of histone promoters. DNA Seq. 4, 395-397. Morris, M.L. (2002). Impacts of international maize breeding research in developing countries, 1966-98. Mexico, D.F. CIMMYT. Morris-Krsinich, B.A.M., Mullineaux, P.M., Donson, J., Boulton, M.I., Markham, P.G., Short, M.N., and Davies J.W. (1985). Bidirectional transcription of maize streak virus DNA and identification of the coat protein gene. Nucleic Acids Res. 13, 7237-7256. Mugo, S., De Groote, H., Bergvinson, D., Mulaa, M., Songa, J., and Gichuki, S. (2005). Developing Bt maize for resource-poor farmers Recent advances in the IRMA project. African J. Biotech. 4, 1490-1504. Muhammad, L., and Underwood, E. (2004). The maize agricultural context in Kenya. In A Hilbeck and DA Andow ed. Environmental risk assessment of genetically modified organisms:AcasestudyofBtmaizeinKenya.pp.2156. Mullineaux, P.M., Boulton, M.I., Bowyer, P., van der Vlugt, R., Marks, M., Donson, J., and Davies, J.W. (1988). Detection of a non-structural protein of MW11000 encoded by the virion DNA of maize streak virus. Plant Mol. Biol 11, 57-66.

Mullineaux, P.M., Donson, J., Morris-Krsinich, B.A.M., Boulton, M.I., and Davies, J.W. (1984). The nucleotide sequence of maize streak virus DNA. EMBO J. 3, 3063-3068. Murdoch, A., Njuguna, J.M., Lukuyu, B., Musembi, F., Mwangi, D.M., Maina, J., Kivuva, B.M., Mburu, M.W.K., and McLeod, A. (2003). Intergrated pest management options to improve maize forage yield and quality for small-scale dairy farmers in central Kenya. Crop quality: its role in sustainable livestock production. Aspects App. Biol. 70, 1-7. Mzira,C.N. (1984).Assessmentofeffectsofmaizestreakvirusonyieldofmaize(Zeamays). ZimbabweJ.Agric.Res.22,141149. Nagar,S.,HanleyBowdoin,L.,andRobertson,D.(2002).HostDNAreplicationisinducedby geminivirusinfectionofdifferentiatedplantcells.PlantCell14,29953007. Nagar, S., Pedersen, T.J., Carrick, K.M., Hanley-Bowdoin, L., and Robertson, D. (1995). A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells. The Plant Cell 7, 705-719. Nikovics, K., Simidjieva, J., Peres, A., Ayaydin, F., Pasternak, T., Davies, J.W., Boulton, M.I., Dudits, D., and Horvth, G.V. (2001). Cell-cycle, phase-specific activation of maize streak virus promoters. M.P.M.I. 14, 609-617. Oluwafemi, S. (2006). Genetic variation among active and inactive transmitters of maize streak virus within a population of Cicadulina storeyi china (Homoptera: Cicadellidae). African J. Biotech. 5, 590-596. Oluwafemi, S., Jackai, L.E.N., and Alegbejo, M.D. (2007). Comparison of four Cicadulina species vectors of MSV from Nigeria. Entom. Exp. Appl. 124, 235-239. Orozco, B.M., and Hanley-Bowdoin, L. (1996). A DNA structure is required for geminivirus origin function. J. Virol. 270, 148-158. Orozco, B.M., and Hanley-Bowdoin, L. (1998). Conserved sequence and structural motifs contribute to the DNA binding and cleavage activities of a geminivirus replication protein. J. Biol. Chem. 273, 24448-24456. Orozco, B.M., Kong. L.-J., Batts, L.A., Elledge, S., and Hanley-Bowdoin, L. (2000). The multifunctional character of a geminivirus replication protein is reflected by its complex oligomerization properties. J. Biol. Chem. 275, 6114-6122. Orozco, B.M., Miller, A.B., Settlage, S.B., and Hanley-Bowdoin, L. (1997). Functional domains of a geminivirus replication protein. J. Biol. Chem. 272, 9840-9846. Owor, B.E., Martin D.P., Shepherd D.N., Edema R., Monjane A.L., and Rybicki, E.P., Thomson, J.A., and Varsani, A. (2007). Genetic analysis of maize streak virus isolates from Uganda reveals widespread distribution of a recombinant variant. J. Gen. Virol. 88, 3154-3165. Page, W.W., Smith, M.C., Holt, J., and Kyetere, D. (1999). Intercrops, Cicadulina spp. and maize streak virus disease. Ann. Appl. Biol. 135, 385-393. Pandey, S.P., Gaquel, E., Gase, K., and Baldwin, I.T. (2008). RNA-directed RNA polymerase3 from Nicotiana attenuata is required for competitive growth in natural environments. Plant Physiol. 147, 1212-1224. Pita, J.S., Fondong, V.N., Sangar, A., Otim-Nape, G.W., Ogwal, S., and Fauquet, C.M. (2001). Recombination, pseudorecombination and synergism of geminiviruses are determinant keys to the epidemic of severe cassava mosaic disease in Uganda. J. Gen. Virol. 82, 655-665.

Powell-Abel, P., Nelson, R.S., De, B., Hoffman, N., Rogers, S.G., Fraley, R.T., and Beachy, R.N. (1986). Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232, 738-743. Pratt, R., Gordon, S., Lipps, P., Asea, G., Bigirwa, G., and Pixley, K. (2003). Use of IPM in the control of multiple diseases in maize: strategies for selection of host resistance. African Crop Sci. J. 11, 189-198. Pratt, R.C., Lipps, P.E., Bigirwa, G., and Kyetere, D.T. (2000). Germplasm enhancement through cooperative research and breeding using elite tropical and U.S. Corn Belt maize germplasm. African Crop Sci. J. 8, 345-353. Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., Bartel, B., and Bartel, D.P. (2002). MicroRNA in plants. Gen. Dev. 16, 1616-1626. Ribaut J.-M., de Vicente, M.C., and Delannay, X. (2010). Molecular breeding in developing countries: challenges and perspectives. Curr. Opin. Plant. Biol. 13, 213-218. Ribaut, J.-M., and Hoisington, D.A. (1998). Marker assisted selection: new tools and strategies. Trends Plant Sci. 3, 236-239. Rodier, A., Assie, J., Marchand, J.-L., and Herve, Y. (1995). Breeding maize lines for complete and partial resistance to maize streak virus (MSV). Euphatica 81, 57-70. Rojas, M.R., Hagen, C., Lucas, W.J., and Gilbertson, R.L. (2005). Exploiting chinks in the plants armor: Evolution and emergence of geminiviruses. Annu. Rev. Phytopathol. 43, 361-394. Rose, D.J.W. (1972). Dispersal and quality in populations of Cicadulina species (Cicadellidae). J. Anim. Ecol. 41, 589-609. Rudolph, C., Schreier, P.H., and Uhrig, J.F. (2003). Peptide-mediated broad-spectrum plant resistance to tospoviruses. Proc. Natl. Acad. Sci. USA 100, 4429-443. Rutschow, H., Ytterberg, A.J., Friso, G., Nilsson, R., and van Wijk, K. (2008). Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis. Plant Physiol. 148, 156-175. Rybicki, E.P. (2010) Plant-made vaccines for humans and animals. Plant Biotech. J. 8, 620637 Saito, K., and Matsuda, F. (2010). Metabolomics for functional genomics, systems biology and biotechnology. Annu. Rev. Plant Biol.61, 463-89 Sanford, J.C., and Johnson, S.A. (1985). The concept of pathogen derived resistance: deriving resistance genes from the parasites own genome. J. Theor. Biol. 113, 395-405. Schneider, M., Jarchow, E., and Hohn B. (1992). Mutational analysis of the conserved region of maize streak virus suggests its involvement in replication. Plant Mol. Biol. 19, 601-610. Schnippenkoetter, W.H., Martin, D.P., Hughes, F., Fyvie, M., Willment, J.A., James, D., von Wechmar, B., and Rybicki, E.P. (2001). The biological and genomic characterisation of three Mastreviruses. Arch. Virol. 146, 075-1088. Schwab, R., Ossowski, S., Riester, M., Warthmann, N., and Weigel, D. (2006). Highly specific gene silencing by artificial microRNAs in Arabidopsis. The Plant Cell 18, 1121-1133. Settlage, S.B., Miller, A.B., and Hanley-Bowdoin, L. (1996). Interaction between geminivirus replication proteins. J. Virol. 70, 6790-6795. Settlage, S.B., Miller, A.B., Gruissem, W., and Hanley-Bowdoin, L. (2001). Dual interaction of a geminivirus replication accessory factor with a viral replication protein and a plant cell cycle regulator. Virol. 279, 570-576. Sharp, G.L., Martin, J.M., Lanning, S.P., Blake, N.K., Brey, C.W., Sivamani, E., Qu, R., and Talbert, L.E. (2002). Field evaluation of transgenic and classical sources of wheat streak mosaic virus resistance. Crop Sci. 42, 105-110.

Shen, W., and Hanley-Bowdoin, L. (2006). Geminivirus Infection up-regulates the expression of two Arabidopsis protein kinases related to yeast SNF1 and mammalian AMPK activating kinases. Plant Physiol. 142, 1642-1655. Shen,W.H.,Escudero,J.,Schlappi,M.,Ramos,C.,Hohn,B.,andNicola,Z.K.(1993)TDNA transfertomaizecells:Histochemicalinvestigationof glucoronidaseactivityinmaize tissues. Proc. Natl. Acad. Sci. USA 90, 1488-1492. Shepherd, D.N., Mangwende, T., Martin D.P., Bezuidenhout, M., Thomson, J.A., and Rybicki, E.P. (2007). Inhibition of maize streak virus replication by transient and transgenic expression of MSV replication-associated protein mutants. J. Gen. Virol. 88, 325-336. Shepherd, D.N., Martin, D.P., and Thomson, J.A. (2009). Transgenic strategies for developing crops resistant to geminiviruses. Plant Sci. 176, 1-11. Shepherd, D.N., Martin, D.P., Lefeuvre, P., Monjane, A.L., Owor, B.E., Rybicki, E.P., and Varsani, A. (2008a). A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue. J. Virol. Methods 149, 97-102. Shepherd, D.N., Martin, D.P., McGivern, D.R., Boulton, M.I., Thomson, J.A., and Rybicki, E.P. (2005). A three nucleotide mutation altering the maize streak virus Rep pRBRinteraction motif reduces symptom severity in maize and partially reverts at high frequency without restoring pRBR-Rep binding. J. Gen. Virol. 86, 803-813. Shepherd, D.N., Martin, D.P., van der Walt, E., Dent, K., Varsani, A., and Rybicki, E.P. (2010). Maize streak virus: an old and complex emergingpathogen. Mol. Plant Path. 11, 112. Shepherd, D.N., Varsani, A., Windram, O., Lefeuvre, P., Monjane, A.L., Owor, B., and Martin, D.P. (2008b). Novel sugarcane streak and sugarcane streak Runion mastrevirus from southern Africa and La Runion. Arch. Virol. 153, 605-609. Shimizu, T., Satoh, K., Kikuchi, S., and Omura, T. (2007). The repression of cell wall -and plastid- related genes and the induction of defence-related genes in rice plants infected with rice dwarf virus. Mol. Plant Microbe Interact. 20, 247-254. Sidle, A., Palaty, C., Dirks, P., Wiggan, O., Kiess, M., Gill, R.M., Wong, A.K., and Hamel, P.A. (1996). Activity of the retinoblastoma family proteins, pRB, p107, and p130, during cellular proliferation and differentiation. Crit. Rev. Biochem. Mol. Biol. 31, 237-271. Sinisterra, X.H., Polston, J.E., Abouzid, A.M., and Hiebert, E. (1999). Tobacco plants transformed with a modified coat protein of tomato mottle begomovirus show resistance to virus infection. Phytopathol. 89, 701-706. Skerra, A. (2000). Engineered protein scaffolds for molecular recognition. J. Mol. Recog. 13, 167187. Stenger, D.C., Revington, G.N., Stevenson, M.C., and Bisaro, D.M. (1991). Replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral DNA. Proc. Natl. Acad. Sci. USA 88, 8029-8033. Stevens, R. (2008). Prospects for using marker-assisted breeding to improve maize production in Africa. J. Sci. Food Agric. 88, 745-755. Suter, B., Kittanakom, S., and Stagljar, I. (2008). Two-hybrid technologies in proteomics research. Curr. Opin. Biotech. 19, 316-323. Thomson, J.A. (2008). The role of biotechnology for agricultural sustainability in Africa. Phil. Trans. R. Soc. B. 363, 905-913. Thottappilly, G., Bosque-Perez, N.A., and Rossel, H.W. (1993). Viruses and virus diseases of maize in tropical Africa. Plant Pathol. 42, 94-509.

Thuman-Commike, P.A., and Chiu, W. (2000). Reconstruction principles of icosahedral virus structure determination using electron cryomicroscopy. Micron. 31:6, 687-711. Timmermanns, M.C.P, Das, O.P., and Messing, J. (1994). Geminiviruses and their uses as extrachromosomal replicons. Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 45, 79-112. Tuttle, J.R., Idris, A.M., Brown, J.K., Haigler, C.H., and Robertson, D. (2008). Geminivirusmediated gene silencing from cotton leaf crumple virus is enhanced by low temperature in cotton. Plant Physiol. 148, 41-50. Tzfira, T., Rhee, Y., Chen, M.-H., Kunik, T., and Citovsky, V. (2000). Nucleic acid transport in plant-microbe interactions: The molecules that walk through the walls. Annu. Rev. Microb. 54, 187-219. Uhrig J.F. (2006). Protein interaction networks in plants. Planta 244, 771-781. van der Walt, E., Rybicki, E.P., Varsani, A., Polston, J.E., Billharz, R., Donaldson, L., Monjane, A.L., and Martin, D.P. (2009). Rapid host adaptation by extensive recombination. J. Gen. Virol. 90, 734-746. Vanderschuren, H., Stupak M., Futterer, J., Gruissem, W., and Zhang, P. (2007). Engineering resistance to geminiviruses -review and perspectives. Plant Biotech. J. 5, 207-220. Varsani, A., Oluwafemi S., Shepherd, D.N., Monjane, A.L., Owor, B., Windram, O., Rybicki, E.P., Lefeuvre P., and Martin D.P. (2008a). Panicum streak virus diversity is similar to that observed for maize streak virus. Arch. Virol. 153, 601-604. Varsani, A., Shepherd, D.N., Monjane, A.L., Owor, B.E., Erdmann, J.B., Rybicki, E.P., Peterschmitt, M., Briddon, R.W., Peter, G., Markham, P.G., Oluwafemi, S., Windram, O.P., Lefeuvre, P., Lett, J.-M., and Martin, D.P. (2008b). Recombination, decreased host specificity and increased mobility may have driven the emergence of maize streak virus as an agricultural pathogen. Virol. 89, 2063-2074. Vega-Rocha, S., Gronenborn, B., Gronenborn, A.M., and Campos-Olivas, R. (2007). Solution structure of the endonuclease domain from the master replication initiator protein of the nanovirus faba bean necrotic yellow virus and comparison with the corresponding geminivirus and circovirus structures. Biochem. 46, 6201-6212. Walters, D., and Heil, M. (2007). Costs and trade-offs associated with induced resistance. Physiol. Mol. Plant Pathol. 71, 3-17. Weinberg, R.A. (1995). The retinoblastoma protein and cell cycle control. Cell 81, 323-330. Welz, H.G., Schechert, A., Pernet, A., Pixley, K.V., and Geiger, H.H. (1998). A gene for resistance to the maize streak virus in the African CIMMYT maize inbred line CML202. Mol. Breed. 4, 147-154. Wesley, S.V., Helliwell, C.A., Smith, N.A., Wang, M.B., and Rouse, D.T. (2001). Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 27, 581590. Whitham, S.A., and Wang, Y. (2004). Roles for host factors in plant viral pathogenicity. Curr. Opin. Plant Biol. 7, 365-371. Whittaker, G.R., Kann, M., and Helenius, A. (2002). Viral entry into the nucleus. Annu. Rev. Cell. Dev. Biol. 16, 627-651. Willment, J.A., Martin, D.P., and Rybicki, E.P. (2001). Analysis of the diversity of African streak Mastreviruses using PCR-generated RFLPs and partial sequence data. J. Virol. Meth. 93, 7587.

Willment, J.A., Martin, D.P., Palmer, K.E., Schnippenkoetter, W.H., Shepherd, D.N., and Rybicki, E.P. (2007). Identification of long intergenic region sequences involved in maize streak virus replication. J. Gen. Virol. 88, 1831-1841. Wise, R.P., Moscou, M.J., Bogdanove, A.J., and Whitham, S.A. (2007). Transcript profiling in host-pathogen interactions. Annu. Rev. Phytopathol. 45, 329-269. Wright, E.A., Heckel, T., Groenendijk, J., Davies, J.W., and Boulton, M.I. (1997). Splicing features in maize streak virus virion and complementary-sense gene expression. Plant J. 12, 1285-1297. Xie, Q., Suarez-Lopez, P., and Gutierrez, C. (1995). Identification and analysis of a retinoblastoma binding motif in the replication protein of a plant DNA virus: requirement for efficient viral DNA replication. EMBO J. 14, 4073-4082. Yang, C., Guo, R., Jie, F., Nettleton, D., and Peng, J. (2007a). Spatial analysis of Arabidopsis thaliana gene expression in response to Turnip mosaic virus infection. Mol. Plant Microbe Interact. 20, 358-370. Yang, T., Xue, L., and An, L. (2007b). Functional diversity of miRNA in plants. Plant Sci. 172, 423-432. Yoshii, M., Nishikiori, M., Tomita, K., Yoshioka N., and Kozuka, R. (2004). The Arabidopsis cucumovirus multiplication 1 and 2 loci encode translation initiation factors 4E and 4G. J Virol. 78, 61012-61011. Yoshii, M., Yoshioka, N., Ishikawa, M., and Naito, S. (1998a). Isolation of an Arabidopsis thaliana mutant in which the multiplication of both cucumber mosaic virus and turnip crinkle virus is affected. J. Virol. 72, 8731-8737. Yoshii, M., Yoshioka, N., Ishikawa, M., and Naito, S. (1998b) Isolation of an Arabidopsis thaliana mutant in which accumulation of cucumber mosaic virus coat protein is delayed. Plant J. 13, 211-219. Zhang, W., Olson, N.H., Baker, T.S., Faulner, L., Agbandje-McKenna, M., Boulton, M.I., Davies, J.W., and McKenna, R. (2001). Structure of the maize streak virus germinate particle. Virol. 279, 471-477. Zheng, S.-J., and Dicke, M. (2008). Ecological genomics of plant-insect interactions: from gene to community. Plant Physiol. 146, 812-817. Zhu, H., Bilgin, M., and Snyder, M. (2003). Proteomics. Annu. Rev. Biochem. 72, 783-812. Zhu, X.-G., Long, S.P., and Ort, D.R. (2008). What is the maximum efficiency with which photosynthesis can convert solar energy into biomass? Curr. Opin. Biotech. 19, 153-159.