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*
1

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.

2

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

3

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: machuka.jesse@ku.ac.ke

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*

Domainfunction

Where mapped

References

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

Hovarthetal.,1998

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*

Domainfunction

Where mapped

References

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

Mastreviruses

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).

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