Distribution of carnation viruses in the shoot tip Exclusion from the shoot apical meristem

Physiological and Molecular Plant Pathology 69(2006)43–51

Distribution of carnation viruses in the shoot tip:Exclusion

from the shoot apical meristem

B.Gosalvez-Bernal 1,S.Garcia-Castillo 1,V.Pallas 2,M.A.Sanchez-Pina ?

Departamento de Biolog?

′a del Estre ′s y Patolog?′a Vegetal,CEBAS (CSIC),Campus Universitario de Espinardo,30100Murcia,Spain Accepted 15December 2006

Abstract

Cell biological tools were used to localize the genomic RNAs/DNA and/or the coat protein (CP)of Carnation mottle ,Carnation vein

mottle ,Carnation latent ,and Carnation etched ring viruses in the carnation shoot tip to know whether any of them was capable of infecting the shoot apical meristem.Our results showed that all the viruses studied were excluded not only in single-infected but also in double-infected plants.Despite that,these viruses showed a different infection pattern inside the shoot tip,with different cell types being their main targets.For the mixed infection studied here,our results showed the existence of a spatial separation pattern within the carnation shoot tip which strongly suggests an exclusion phenomenon.r 2007Elsevier Ltd.All rights reserved.

Keywords:Carnation mottle virus ;Carnation vein mottle virus ;Carnation latent virus ;Carnation etched ring virus ;Dianthus caryophyllus ;cv.Dixie;In situ hybridization;Immunohistochemistry;Shoot tip;Shoot apical meristem;Single and mixed viral infection

1.Introduction

The systemic infection of a plant depends on the capacity of a given virus to replicate in the initially infected cells,move to adjacent cells through the plasmodesmata and reach distal parts of the plant through the vasculature [1,2].Although considerable progress has been made during recent few years to elucidate virus invasion patterns of plant organs and tissues,viral infection of the shoot tips have been less studied.The shoot apical meristem (SAM)of higher plants functions as a site of continuous organogenesis within which a small pool of pluripotent stem cells,replenishes the cells incorporated into lateral organs (leaves,stems and ?owers)[3].At present,a large body of evidence suggests that most viruses and viroids are unable to invade the SAM [4–8].One exception to this general rule is the work of Cohen et al.[9].These authors

used a chimera of Turnip crinkle virus (TCV)in which the coat protein (CP)gene was replaced by that of GFP.The consequent GFP expression was detected in the meriste-matic regions when this chimeric virus was inoculated into transgenic plants expressing TCV CP.

Carnation is susceptible to infection by many viruses,which in some cases may result in serious losses.Those caused by Carnation mottle (CarMV),Carnation etched ring (CERV),Carnation vein mottle (CVMV),Carnation ringspot (CRSV),Carnation Italian ringspot (CIRSV)and Carnation latent (CLV)viruses are the most important.These viruses occur as single and,more frequently,multiple infections.From all the viruses infecting carnation,we have focused on:(i)CarMV,which is the type member of the genus Carmovirus within the family Tombusviridae of plant viruses.This is a 34nm icosahedral plant virus consisting of a single-stranded,positive-sense 4.5kb RNA [10].CarMV is thought to be involved in synergistic reactions in mixed infections,with a negative impact on cut-?ower production [11–13].(ii)CVMV,which is a Potyvirus within the family Potyviridae of plant viruses.It has ?lamentous,usually ?exuous virion particles,790nm long and 12nm wide.Its genome consists of a

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0885-5765/$-see front matter r 2007Elsevier Ltd.All rights reserved.doi:10.1016/j.pmpp.2006.12.004

?Corresponding author.Tel.:+34968396307;fax:+34968396213.

E-mail address:spina@cebas.csic.es (M.A.Sanchez-Pina).1

These authors contributed equally to this manuscript.2

Permanent address:Instituto de Biolog?a Molecular y Celular de Plantas,UPV-CSIC,Avda.de los Naranjos s/n,46022Valencia,Spain.

single-stranded RNA[14].The symptoms include chlorotic and darker green spots,?ecks and mottling[15].(iii)CLV, which is the type member of the genus Carlavirus with ?lamentous,usually straight(or slightly curved)virions, 650nm long and12nm wide.Its genome consists of a single-stranded,positive-sense8.5kb RNA[14].The virus causes few or no symptoms;(iv)CERV is a Caulimovirus. It is a45nm isometric plant virus[16]with a genome of DNA(gDNA);double-stranded;circular and linear(also super coiled)with a size of7.9kb[14].Replication of the vDNA involves the reverse transcription of the largest transcribed RNA.The symptoms vary seasonally and it is often symptomless,although it sometimes causes necrotic ?ecks and lines.

In the case of many virus–shoot combinations[17],the meristem tip appears as a zone of variable length(usually about100m m but up to1000m m)near the shoot or root tip that is free or contains very little virus[18,19].This situation has already been exploited to obtain virus-free clones by growing excised shoot tips in tissue culture. Meristem tip culture has already been used to free carnation cultivars from the different viruses described, although,some of these,such as CERV,are very dif?cult to eliminate by this method.It is not always clear what the success of such a method depends on:the regular absence of virus from meristem tissue,some meristematic regions in the plant containing viruses and others containing none,or the inactivation of the virus present in the meristem during culture[17].

In this work,we studied the CP and/or genome distribution,in the carnation shoot tip,of the main viruses infecting carnation(CarMV,CVMV,CLV,and CERV), when present either in single or mixed(CarMV+CERV) infections,by in situ techniques(immunohistochemistry and in situ hybridization)at the light microscopy level, demonstrating that they are not able to reach the SAM of the infected plants.

2.Material and methods

2.1.Plants,growth conditions and CarMV inoculation Carnation plants(Dianthus caryophyllus L.)infected with the different viruses studied as well as virus-free carnations produced by meristem tip culture were grown and provided by Barberet&Blanc,S.A.(one of the main carnation breeders in Europe).Plants were grown and maintained,in a greenhouse at temperatures of35–401C day/12–151C night,with a day irradiance varying from300 to650m mol photons/m2s.

Some of the carnation plants studied were mechanically inoculated with a Dixie isolate of Carnation mottle virus (CarMV-Dix)obtained from naturally infected carnation plants(D.caryophyllus,cv.Dixie)[20].The CarMV-Dix was cloned,propagated in Chenopodium quinoa plants and puri?ed from leaf tissue as previously described[21].Thirty ?ve days after the rooting of the carnation cutting,one leaf per plant was mechanically inoculated with20m g/ml of puri?ed CarMV in20mM potassium phosphate buffer(pH 7.0),using carborundum as abrasive.The inoculation was carried out only on the apical part of the leaves.Mock-inoculated control plants were treated similarly.

2.2.Synthesis of digoxigenin-labelled RNA probes Digoxigenin-labelled RNA(dig-RNA)probes were synthesized from the corresponding plasmids previously obtained in the lab:pCarM.EC3[22–24];and pCVMV2; pCLV-1and pCERV-1[25]containing fragments of the genome of the viruses CarMV,CVMV,CLV and CERV, respectively,placed between the T3and T7RNA polymerase promoters.Plasmids were linearized with the corresponding restriction enzymes most suited to each one: pCarM.EC3,Eco RI;pCVMV2,Hind III;pCLV-1,Eco RI and pCERV-1,Bam HI.Then,the plasmids were digested and puri?ed by phenol-chloroform extraction and ethanol precipitation.The dig-RNA probes were synthesized as previously described[26].Transcript RNA was recovered by ethanol precipitation and resuspended in sterile water.

2.3.Tissue?xation and embedding

Shoot tip samples were harvested from carnation plants and processed as previously described[23,27].Brie?y, longitudinal sections through their center were obtained with a razor blade and both parts were?xed in a freshly made mixture of4%p-formaldehyde and2.5%glutar-aldehyde in0.2M phosphate buffer(pH7.2)at41C for4h. Samples were vacuum-in?ltrated for2min immediately after being put into the?xative.They were then washed in the same buffer,dehydrated in a tertiary butyl alcohol series,in?ltrated and embedded in paraf?n(Paraplast Plus, Sherwood Medical Co.,St Louis,MO,USA).Individual blocks of paraf?n were sectioned using a Reichert-Jung 2030(Leica Mycrosistems;Nusloch,Germany)rotary microtome set to8or10m m.The longitudinal sections were?xed to glass slides precoated with APTES(3-amino-propyl-triethoxy-silane;Sigma-Aldrich Co.,St Louis,MO, USA)by heating them at401C on a hot plate.The slides were allowed to stand overnight before further processing to make sure that the sections were tightly adhered to the slide.

2.4.In situ hybridization

In situ hybridization was performed as previously described[23,27,28].Brie?y,tissue sections on slides were dewaxed with xylene(2?10min each)and dehydrated with a series of decreasing ethanol concentrations from absolute ethanol to water.They were then incubated in 0.2M HCl for20min,washed in distilled water,2?SSC buffer(1?SSC is0.15M NaCl,0.015M sodium citrate, pH7.0)and again in distilled water,5min each time.Then, they were treated with proteinase K(1m g/ml,in100mM

B.Gosalvez-Bernal et al./Physiological and Molecular Plant Pathology69(2006)43–51 44

Tris–HCl,50mM EDTA,pH8.0)for30min at371C, followed by a wash on PBS(135mM NaCl, 1.5mM KH2PO4,8mM Na2HPO4, 2.7mM KCl,pH7.2),for 2min.After that,proteinase K was blocked by a glycine (2mg/ml)in PBS incubation and2?new PBS washes of 30s each.In order to detect the CERV DNA genome,the corresponding samples were treated by0.5M NaOH for 3min to denature the dsDNA and allow it to be hybridized to the dig-RNA probe.Hybridization was performed with100m l of hybridization solution consisting of50% deionized formamide,6?SSC,3%SDS,yeast t-RNA 0.1mg/ml and a probe concentration of200ng/ml.The dig-RNA probes used(pCarM Ec3,pCVMV2;pCLV-1and pCERV-1)were those described above.Before hybridiza-tion,the probes were denatured at801C for3min,and put back into ice.Hybridization was performed at551C overnight for the viruses with RNA genome(CarMV, CVMV,CLV)and at421C in the case of CERV with a DNA genome,while the stringent washes in0.2?SSC+ SDS0.1%were made at551C for the?rst group of viruses and at421C for CERV.

Hybridized probes were detected by antibody incubation using sheep anti-digoxigenin conjugated to alkaline phos-phatase(Roche)diluted1:500in0.5%bovine serum albumin(BSA)in buffer TBS for90min.For colorigenic detection,slides were incubated with nitroblue tetrazolium and5-bromo-4-cloro-3-indolyl phosphate(NBT/BICP) (Roche)in equilibration buffer(100mM Tris-HCl, 100mM NaCl,50mM MgCl2,pH9.5).The reaction was allowed to proceed for approximately30min and stopped with distilled water.Then,slides were dehydrated through a series of increasing ethanol concentrations,immersed in xylene and mounted using Merckoglass(Merck,Darmstad, Germany).Sections were examined using a DMRB LEITZ Microscope(Leica Microsistemas s.a,Barcelona,Spain) and photographed with a digital camera Leica DC500. Controls were made by performing the in situ hybridization on samples taken from healthy or mock-inoculated plants, as well as on infected samples incubated without the probe.

2.5.Immunohistochemistry

Immunohistochemistry was performed as previously described[27];using antiserum raised against whole CarMV virions(kindly provided by Drs.Daro s and Flores, Instituto de Biolog?a Molecular y Celular de Plantas,UPV-CSIC,Valencia,Spain).Brie?y,tissue slides were dewaxed and dehydrated as described above.They were then treated with PBS three times for5,10and15min,respectively. Tissue slides were pre-incubated for20min in a blocking solution consisting of5%BSA in PBS and transferred directly to a solution containing antiserum against CarMV (1:6000)in1%BSA/PBS,and incubated overnight at41C. After four5min washes with PBS,tissue samples were incubated for1h with goat anti-rabbit IgG conjugated with alkaline phosphatase(Roche)as secondary antibody (7U/ml)in1%BSA/PBS.Slides were washed four times,for5min each,with PBS,followed by one wash of15min with equilibration buffer.For colorigenic detection,the slides were treated as described above,before being dehydrated,immersed in xylene and mounted using Merckoglass.Sections were examined using a DMRB LEITZ Microscope(Leica Microsistemas s.a,Barcelona, Spain)and photographed with a digital camera Leica DC 500.

Controls were made by performing the immunohisto-chemistry on samples taken from healthy or mock-inoculated plants,as well as on an infected sample incubated without the?rst or second antibody.

3.Results

In this work,we used in situ techniques(immunohis-tochemistry and in situ hybridization)at tissue level,in order to know the exact distribution patterns of the virus CP and/or genome of different viruses in the carnation shoot tip and how close to the SAM they were located. Thus,longitudinal sections of shoot tips were taken from carnations mechanically inoculated with CarMV,plants naturally single infected by CarMV,CVMV,CLV or CERV and others mixed infected by CarMV plus CERV. In a longitudinal section of the shoot tip,SAM is located in the apical central area of the stem(Figs.1A–C;Figs.2A,F, J and Figs.3A,E,G,J–L)and is surrounded by the leaf primordial(Figs.1D–G and2B,G)as well as other leaves in different developmental stages(Figs.1–3). Immunolocalization showed that the distribution pattern of CarMV in the carnation shoot tip was similar in the inoculated(33dpi)(Fig.1A)and naturally infected (Fig.1B)plants,although the viral concentration was higher in naturally infected ones(compare Figs.1A and B). We know from other studies(Garc?a-Castillo,Palla s,& Sa nchez-Pina,paper in preparation)that inoculated plants at33dpi reach very similar infection levels as those naturally infected.In both,the stem located immediately below the shoot tip shows a high infection level,which affects most tissues(epidermis,cortex,phloem and pith) (Fig.1K,Garc?a-Castillo,Palla s,&Sa nchez-Pina,paper in preparation).However,in the present study,the shoot tip of the same naturally infected plants showed a gradual decrease in the infection signal as the SAM gets close.In none of the cases analyzed did CarMV infect the SAM or the leaf primordia that surrounds it(Figs.1D–F).CarMV was generally located in the developed vascular tissue of the shoot tip.Thus,in the stem,CarMV mainly infected its phloem connecting with the same tissue of the developed leaves,whereas the other tissues(epidermis,cortex and pith)were generally free of the virus(Fig.1).Exceptionally, in a CarMV natural infection(Figs.1B,F),the infection signal was located in one group of pith cells in the center of the stem very close to the SAM.In the same stem,and even closer to the SAM,another smaller group of infected cells was observed in the area connecting with a young leaf, where the phloem and parenchyma cells of the leaf as well

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as some cells of the stem cortex and pith were infected;however,even in this case,the SAM showed no infection (Fig.1F ).Immunolocalization of the CarMV CP (Fig.1E )revealed a quite similar distribution to that of the CarMV RNA (Fig.1D )in consecutive serial sections,indicating that CarMV probably reaches these tissues as virions.In the developed leaves of the shoot tip the virus was also located mainly in the phloem (Fig.1)and,later in development,the virus infected the rest of the leaf tissues (epidermis and mesophyll)(Figs.1B and 3G–I ).

Similar CarMV distribution pattern was observed when we studied shoot lateral meristems (Figs.1H,J ).CarMV never infected the meristematic tissue of them.The infection signal was mainly located in the phloem tissue

Fig.1.CarMV infection of carnation shoot tips.(A–C,E,F,H–K)CP immunohistochemical localization.(D,G)gRNA detection by in situ hybridization.Blue color infected area.Longitudinal sections of the shoot tip from mechanically inoculated plants (33dpi)(A)and naturally infected ones (B).(C),negative control of the immunohistochemistry done on sections from mock-inoculated plants.No signal is present.(D,E),high magni?cation of serial sections from the shoot apical meristem (SAM)in (A).The colocalization of the CarMV gRNA (D)and CP (E)is shown in these micrographs.Arrow-heads (c )point to colocalization areas.(F),high magni?cation of SAM in (B).(G),negative control of the in situ hybridization done on sections from healthy plants.No signal is present.(H),high magni?cation of one of the lateral meristems (m )in (A).(I),high magni?cation of the stem area connecting with a young leaf in (H).(J),serial longitudinal section of the lateral meristem (m )from the shoot tip in (B).SAMs in (D–F),and lateral meristems (m )in (H,J),are not infected by CarMV.The infection signal was mainly located in the phloem tissue (p )of developed leaves of the shoot tip and in the phloem tissue (p )connecting the stem of SAM and shoot lateral meristems (m )with the developed leaves that surrounds them ().(K),longitudinal section of the stem below the shoot tip to show its high CarMV infection.(Pi ):pith tissue;(M ):mesophyll;(Lp ):leaf primordium;(C),cortex of the stem;(X ),xylem.(A,B,C):Bars ?500m m;(D–K):Bars ?100m m.

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that connected the stem of shoot lateral meristems with the developed leaves that surrounds them(Figs.1H–J).In some cases we observed a highly infected area at the bottom of these shoot lateral meristems that corresponded to the phloem and pith cells of the stem as well as the mesophyll cells from the leaf,that is,in the connecting area (Figs.1H,I).The infection signal was also detected in the protophloem of the developing leaves located very close to the lateral meristem(second true leaves)(Fig.1J).There-fore,the protophloem is the route used by the virus to infect the leaves of the shoot tip.All the controls used for the immunohistochemistry and in situ hybridization experiments were always negative as shown(Figs.1C,G). The distribution pattern,in the carnation shoot tips infected by the other viruses studied(CVMV,CLV and CERV)was analyzed by in situ hybridization(Figs.2 and3).They all showed a pattern of virus accumulation very similar to that of CarMV described above,since none

Fig.2.CVMV and CLV infection of carnation shoot tips studied by in situ hybridization.(A–D),CVMV.(F–I),CLV.Blue color infected area.(E,J), negative controls;the in situ hybridization for CVMV and CLV,was performed on sections from healthy plants,and no signal was detected.(B,G),shoot apical meristems(SAM).Longitudinal sections of the shoot tips were used to study the viruses’distribution.(B–D),high magni?cations of different areas from the shoot tip in(A);(C)area inside the green square in(A);(D)area inside the red square in(A).(H)area inside the green square in(F)at higher magni?cation.Shoot apical meristems(SAM in A–B,F–H),and lateral meristems(m in F)are not infected by any of the viruses analyzed.Each virus shows a different pattern of shoot tip infection.(C,D),CVMV is located in vascular parenchyma cells(Vp)of the stem(C)and of the developed leaves (D).(H,I),CLV is located in the pith cells(Pi)of the stem,quite close to the SAM.(L):leaves at different developmental stages;(Lp):leaf primordium; (M):mesophyll;(X):xylem.(A,E,F,J):Bars?500m m;(B–D,G–I):Bars?100m m.

B.Gosalvez-Bernal et al./Physiological and Molecular Plant Pathology69(2006)43–5147

of the viruses studied is able to invade either the SAM or the leaf primordial that surrounds it (Figs.2and 3).All these viruses showed a speci?c distribution in the shoot tip (Figs.2and 3).CVMV infects some cells of the vascular parenchyma in the stem below the SAM (Figs.2A,C ),and the developed leaves (Figs.2A,D ).CLV infects cells of the stem pith as well as vascular cells of the developed leaves (Figs.2F,H,I ),while CERV infection in the stem is located in the phloem (Figs.3A,D ).In the developed leaves,phloem components were infected by CERV (Figs.3A,C ).Since samples infected with CERV have to be treated by NaOH in order to denature the gDNA and allow it to hybridize with the probe,those samples are not so well structurally preserved as the other samples (compare Figs.2and 3).

The viral distribution patterns observed in the shoot lateral meristems were similar to those observed in the SAM (not shown).In all the control experiments performed in single infected plants no signal was detected (Figs.2E,J and 3J ).

Fig.3.CERV gDNA and CarMV gRNA speci?c localization in carnation shoot tips of single and double infected plants studied by in situ hybridization.Blue color infected area.(A–D),CERV in single infected plants is located in the phloem components (p )of the leaves (C)and the stem (D).(C),area inside the red square in (A)at higher magni?cation.(J),negative control,the in situ hybridization for CERV was performed on sections from healthy plants,and no signal was detected.(E–I),mixed infected plants.Serial sections of the same shoot tip.(E,F,insert),CERV gDNA localization.CERV gDNA is scarcely present in the carnation shoot tip.Only a small group of pith cells (Pi )located just below the SAM are infected (F and insert).(F),high magni?cation of the area near the SAM in (E).(insert)higher magni?cation of the pith cells infected in (F).(G–I),CarMV gRNA localization.CarMV is mostly present in the developed leaves (L )of the shoot tip,where it infects the phloem (p ),mesophyll (M )and epidermal (E )cells.(H,I),are high magni?cations of different areas (green and red square,respectively)from the shoot tip in (G).(K,L)are negative controls,the in situ hybridization for CERV (K)and CarMV (L),were performed on sections from healthy plants and no signal was detected.(X ):xylem.(A,E,G,J–L):Bars ?500m m;(B–D,F,I):Bars ?100m m.(H):Bar ?50m m.

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In addition to the single infected plants,we studied carnation plants double infected by CarMV and CERV,by in situ hybridization,in serial sections of the same carnation shoot tip(Figs.3E–I).In this case,CERV infection was very much reduced(Fig.3E)compared with that seen in the single infected plants(Fig.3A)and the infection was not located in the phloem components of the shoot tip.On the contrary,in serial sections of the same shoot tips of mixed infected plants,CarMV infection showed a similar pattern to that of the single infected plants(compare Figs.3G–I with Fig.1B).Thus,in mixed infected plants CERV is not present in the phloem of either the stem or the leaves(Figs.3E,F),but it is present in a small group of pith cells located very close to the SAM (Fig.3F,and insert),although the SAM itself is still not infected.On the other hand,in serial sections of the same shoot tips,CarMV infection is located very abundantly in nearly all the tissues of the developed leaves,including the phloem(Figs.3G–I),similarly to the pattern of CarMV single infected plants(Fig.1).

In all the control experiments performed in mixed infected plants no signal was detected(Figs.3K,L).

4.Discussion

The in situ studies on the carnation shoot tip infection clearly showed that for all the viruses analyzed the SAM and lateral meristems,together with the?rst pair of leaf primordial,are free of the viruses CP and/or genome. Thus,all the viruses studied CarMV,CVMV,CLV and CERV,irrespective of their differences in their inter-actions with the carnation plants,and in both single and mixed infections,never invade the carnation SAM (Figs.1–3).These results re?ect the general situation observed in most other virus–host combinations studied (reviewed in[17]).However,some viruses have been shown to be able to invade the SAM,among them TCV [9],which is considered a model system for the Carmovirus genus and,has the same genome organization of CarMV. In this case,Cohen et al.[9]using different transgenic Arabidopsis plants showed that GFP-tagged TCV was detected throughout the plant,including meristematic regions.The discrepancy between their?ndings and ours could be due to the different approaches used,since we directly localized the viral genome or CP,while Cohen et al.[9]based their study on the detection of the GFP,as an indirect marker of the invasion pattern of the virus in the meristem.Indeed,previous results obtained by Imlau et al.[29]showed that GFP can move by passive diffusion through expanding tissues,which suggests that GFP expression might well be observed in meristematic regions of plants inoculated with a TCV chimera harboring the GFP gene.

Other viruses with a DNA genome have been detected in the shoot tip;such as the Bean dwarf mosaic geminivirus (BDMV)[7],that was localized in the protophloem and its surrounding cells but not in the SAM.These?ndings show the importance of the protophloem in the invasion process of the shoot tip and suggest the existence of a barrier against the viral transport in this organ[30].Of the different viruses we studied,only CarMV was in the protophloem of young leaves(second plastochron)sur-rounding lateral meristems of naturally single infected carnation shoot tips(Fig.1H).Some other viruses have also been located in different hosts shoot tips.For example Walkey and Webb[31]examined squashes of excised apical meristem tissue by electron microscopy and were able to observe the presence of several Nepoviruses in the apices of different hosts.Virus particles have been observed in the apical initials of tobacco shoots[32].Pepper ringspot virus (PepRSV)was identi?ed in meristematic cells of both root and shoot apices[33].Rods of Potato virus X(PVX)have been detected within or close to the potato shoot apex[34] and rods of Odontoglossum ringspot virus(ORSV)in mitotically active cells in the apical meristem of Cymbidium [35].Clusters of Barley stripe mosaic virus(BSMV)were also seen to be distributed quite unevenly in both infected roots and shoot apices of wheat[36].In the Nepoviruses mentioned above[31],the infection of the meristem was explained by the rapidity of the cell-to-cell spread of these viruses which,probably exceeded the rate at which the apex grew away from the infection,since these were very aggressive viral infections[37].

The reasons why the meristematic zone of the shoot apex frequently fails to support virus multiplication are gradu-ally becoming clearer[17].On the one hand,it could be due to the fact that a differentiated vasculature develops distal to that region,and then,access via phloem is restricted. Similarly to Maize streak virus(MSV)in wheat[5]and BDMV in Phaseolus vulgaris and Nicotiana benthamiana [7],CarMV and CERV were found in the phloem of the shoot tip in single infected carnation(Fig.1,Figs.3A–D and G–I).Furthermore,CarMV,CLV and CERV were able to infect the leaves that developed around the SAM from the moment they have a vascular region that connects with that of the stem allowing the virus to reach the leaves (Figs.1,2F and3A,C).It has been shown that MSV infects the developing leaves of the shoot apex from the?fth plastochron onwards[5].Among the viruses we studied here,the developing leaves of the shoot apex were infected from the second plastochron in the case of CarMV,but later with the others(Figs.1–3).In naturally infected carnations,CarMV,CLV,and CERV were located in a group of cells in the shoot tip pith quite close to the SAM (Fig.1;Figs.2F,H;Figs.3A–B).Furthermore,CERV was located in the stem phloem very close to the SAM (Figs.3A,B).However,none of these viruses were able to invade the SAM by either long-distance or cell-to-cell movement probably due to the high rate of cell division of the meristematic cells,which would hinders ef?cient viral replication,as several authors have already suggested [5,9,38].

It might be thought that symplasmic isolation is responsible for protecting the SAM from infection.

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However this is not very probable since,as already demonstrated,it is critical for the SAM to keep its cells in continuous communication within their own layer and in adjoining layers in order to asses their relative positions in the meristem and so behave accordingly[3].

For the mixed infection studied here(CarMV+CERV), we have shown the existence of a spatial separation pattern within the carnation shoot tip since CERV genome is not present in the shoot tip phloem in the double infected plants(Figs.3E,F),while it was present in the phloem of the shoot tip in the single infected plants(Figs.3A,D).It was previously described how the combination of CarMV with other viruses enhanced the symptomatology in carnation plants[25].It is interesting that in all tested combinations of the viruses which caused a synergistic increase in symptoms both viruses were present in the same cells throughout the infection[39].We had expected to localize both viral genomes(CarMV and CERV)in the same discrete areas corresponding,partly,to phloem components in the shoot tip but,as mentioned above, CERV genome was not localized in the phloem of mixed infected plants(Figs.3E,F).This result strongly suggests the presence of an exclusion phenomenon since CERV genome was localized in the phloem components of the single infected carnation shoot tips(Figs.3A–D).In this sense,we should mention that none of the sampled carnation plants showed visible symptoms.Spatial separa-tion patterns have been documented previously for three different virus genera from at least two families:three different Potyviruses(Potyviridae[39]),the type member of the Potexvirus,PVX[39,40]and the Alfamovirus AMV of the family Bromoviridae[41].All these viruses and those included in our investigations(Carmovirus,CarMV and Caulimovirus,CERV)exhibit different genome organiza-tion and expression strategy and differ widely in their host range,including herbaceous(PVX,AMV),woody(Plum pox virus,PPV)and ornamental(CarMV,CERV)plants. Thus,the spatial separation of different viral populations may be a common phenomenon[39].Further studies will be needed to clarify whether spatial separation is a silencing phenomenon or whether other models,which have been used to characterize cross-protection[42],might explain this virus distribution.The results of this paper are of great importance to the carnation producers since meristem tip culture is the method generally used in the carnation regeneration and with this information they can be sure that if they take only the SAM they will not take any risk of transmitting viral infections from one plant to another.

Our analysis on the distribution of the viral genomes of the main viruses infecting carnation plants in single (CarMV,CVMV,CLV,and CERV)and mixed (CarMV+CERV)infections within the shoot tip has provided fresh insights on the limitations of these viruses in the infection of the apical meristem since all the viruses studied were excluded from the SAM in both single or mixed infections(Figs.1–3).Further studies are needed to clarify the mechanism that protects the SAM from viral invasion.

Acknowledgements

We thank Mr.P.Thomas for checking the English grammar.This research was supported by Grants BIO96-0459and BIO2002-04099-C02-01both from the Spanish granting agency DGYCIT and FEDER.B.Gosalvez and S.Garc?a Castillo were the recipients of a fellowship from the Instituto de Fomento-Fundacio n Seneca from Comu-nidad de Murcia,Spain.

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