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Virus Research 153 (2010) 197–204

Contents lists available at ScienceDirect

Virus Research

journa l homepage: www.e lsev ier .com/ locate /v i rusres

omplementations and exclusions between mutated versions of a potato virus Yenotype during mixed infections of Nicotiana hosts

athieu Rollanda,b,1, Agnès Delaunaya, Thomas K. Baldwina, Camille Kerlana, Emmanuel Jacquota,∗

INRA-Agrocampus Ouest-Université Rennes 1, UMR1099 BiO3P (Biology of Organisms and Populations Applied to Plant Protection), F-35653 Le Rheu, FranceFNPPPT (Fédération Nationale des Producteurs de Plants de Pomme de Terre), 43-45 rue de Naples, F-75008 Paris, France

r t i c l e i n f o

rticle history:eceived 31 March 2010eceived in revised form 7 July 2010ccepted 3 August 2010vailable online 11 August 2010

eywords:ompetition

a b s t r a c t

Understanding the processes that have led to the recent prevalence of necrotic genotypes in PVY pop-ulations is an important challenge for research programs studying this virus. Non-necrotic PVYO-139,necrotic PVYN-605 and point mutated versions of PVYN-605 (PVYKRED, PVYKR and PVYED), were used inmixtures to inoculate two Nicotiana hosts which express (N. tabacum cv. Xanthi) or not (N. clevelandii)necrosis symptoms in response to infection by PVYN group members. The comparison during serial pas-sage experiments of proportions of PVY genotypes produced in mixed infected plants with those of theinocula was used to describe: (i) complementation between PVYKR and PVYN and between PVYKRED andPVYO genotypes; (ii) exclusion of the PVYKRED genotype, previously described as fitter, during mixed

itness

erial passage experimentlant-to-plant transmission

infections in the presence of one of the less fit PVYN, PVYED and PVYKR genotypes and (iii) the prevalenceof the non-necrotic PVYKR genotype in the presence of PVYN parental sequence. These results indicatethat the role of both A/G2213 and A/C2271 nucleotides in the fitness of PVY genotypes depends on othergenetic information in the viral genome that has not yet been identified. Moreover, the collected dataindicate that mutation of the nucleotide 2213 in the PVYN-605 sequence could lead to the prevalence,

nthi a

both in N. tabacum cv. Xa

. Introduction

Potato virus Y (PVY) is the type member of the genus Potyvirusfamily Potyviridae) (Kerlan and Moury, 2008a,b). The PVY genomeonsists of a single-stranded, positive-sense RNA molecule ofpproximately 10 kb, with a VPg protein covalently bonded to the 5′

nd and a poly-A tail at the 3′ end (Shukla et al., 1994). The viral RNAncodes a single large polypeptide, which is cleaved into nine prod-cts by three virus encoded proteases (Dougherty and Carrington,988). A recent study has reported the presence of a second shortRF (PIPO, Chung et al., 2008) embedded within the previouslyescribed large ORF. This plant virus is transmitted by numerousphid species in a non-persistent manner; the most important vec-or being the green peach aphid, Myzus persicae (Bokx and Cuperus,

987; Radcliffe and Ragsdale, 2002). PVY infects a wide host range

ncluding Solanaceae family members (Milne, 1988; Shukla et al.,994). Thus, it has been known for several decades that PVY ismajor threat for potato production (Bokx and Cuperus, 1987;

∗ Corresponding author at: INRA-Agrocampus Ouest-Université Rennes 1, UMRiO3P, Team « Biology and Evolution of Plant RNA Viruses », Domaine de la Motte,P 35327, 35653 Le Rheu Cedex, France. Tel.: +33 223 485817; fax: +33 223 485180.

E-mail address: emmanuel.jacquot@rennes.inra.fr (E. Jacquot).1 Cornell University, Dept. Plant Pathology, Ithaca NY 14853, USA.

168-1702/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2010.08.001

nd in N. clevelandii, of the non-necrotic PVYKR genotype.© 2010 Elsevier B.V. All rights reserved.

Loebenstein et al., 2001). In recent years, PVY has become the mostimportant virus for seed, ware, and processed potatoes. Indeed, PVYaffects most potato cultivars and causes major yield losses of up to80%. In addition to the yield reduction, PVY can seriously affect thequality of the harvested crop due to potato tuber necrotic ringspotdisease (PTNRD) (Kerlan, 2006). PVY is also the most damagingvirus in tobacco crops, causing height reductions and modifyingthe chemical composition of cured leaves, especially the nicotinecontent. Other major crop species affected by PVY include pepper,where infection rates of 100% have been observed, and tomato,where emerging strains of PVY cause serious damage to yieldsand fruit quality (Kerlan and Moury, 2008a,b). Other cultures thathave also been shown to be affected by PVY include petunia pro-duction in Europe (Boonham et al., 1999) and eggplants in India(Jeffries, 1998). Efficient control strategies have been developedfor the major crop species affected by PVY (Kerlan, 2006). How-ever, none of them take into account PVY evolution and they donot suppress the risks of new epidemics caused by emerging viruses(Kerlan and Moury, 2008a,b).

From the first description of PVY in 1931 (Smith, 1931) to the

latest PVY reports, biological, serological and molecular propertiesof PVY isolates have been extensively characterised and used tocreate a complex PVY classification (Fauquet et al., 2005) whichis still discussed by international experts working on this virus(Singh et al., 2008). Thus, PVY is subdivided in strains (according

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98 M. Rolland et al. / Virus

o the host from which isolates were originally collected), groupsbased mainly on symptoms induced in indicator hosts and onbilities to overcome selected resistance sources) and putative sub-roups (containing isolates with particular properties). PVY isolatesollected on potato plants have been classified into five groupsncluding the two main PVYN and PVYO groups in which isolateshat are either able to induce (PVYN) or not (PVYO) veinal necro-is symptoms on Nicotiana tabacum cv. Xanthi leaves are classified.n potato, isolates responsible of the PTNRD belong to the PVYN

roup (Beczner et al., 1984; Kerlan and Le Romancer, 1992). Non-ecrotic PVYO group members have long been predominant amongeld-collected PVY isolates. However, a progressive increase in theroportion of necrotic genotypes in natural populations infectingobacco and potato fields has been observed in several differentational PVY surveys in Europe during the last 10 years (Van derlugt, personal communication; Dedic et al., 2007; Lacroix et al.,010; Lindner, 2007; Rolot, 2007). Thus, in many areas, PVY necrotic

solates are now predominant and have become one of the primaryreoccupations of growers. The modification of the PVYO/PVYN

atios in viral populations could reflect either a possible change ofiotic and/or abiotic factors involved in the infection cycle of thislant pathogen or an increased fitness associated with some mod-

fication of the virus genotype. Among the different modificationsbserved (mutation and recombination), this paper addresses thempact of the acquisition of nucleotides known to be involved inVY necrosis capacity.

During virus infection of a healthy host, the few virions (theoret-cally one) present at the start of infection increase to several billionopies of the viral genome within a few days or weeks (Betancourtt al., 2008; Moury et al., 2007; Moya et al., 1993), which resultn the production of heterogeneous populations derived from thenitial genomic sequences (Domingo et al., 2001). Each individual

ithin these populations will compete with each other to colonizehe environment. According to the competitive exclusion principle,wo entities competing for the same resources cannot stably coex-st, and one of the two competitors will take over the other (Gause,934). Here, we hypothesize that such competitions could occur athe quasispecies level between individuals, but also for the simul-aneous presence of multiple viral quasispecies in a plant. Mixednfections are indeed frequently described for naturally infectedosts (Gagarinova et al., 2008; Rolland et al., 2008). The major

mportance of genetic drift during the colonization of a single hostas been extensively described (Ali et al., 2006; Choi et al., 2001;acristan et al., 2003). However, the study of numerous hosts duringultiple transmissions limits the stochastic effects. The structure

f the viral population present in the hosts is thus considered asn indicator of the relative fitness of the present quasispecies. Pro-edures have been described to determine the relative fitness ofenotypes (Carrasco et al., 2007). This should aid the understand-ng of the processes involved in the emergence, maintenance andpread of new variants, and allow a description of the steps leadingo isolates taking over from the previously prevalent viral entities.

Using a reverse genetic approach, two molecular determinantsinked to the necrotic ability of PVY on tobacco leaves have beendentified (Tribodet et al., 2005). Indeed, the polymorphism ofucleotides A/G2213 and A/C2271 (positions according to Jakab etl., 1997), located at the 3′ end of the region encoding the HC-Prorotein have been shown to be linked to the necrotic properties ofhe corresponding genotypes. The modifications of A2213 to G2213nd/or of A2271 to C2271, in the sequence of the necrotic PVYN-605nfectious clone lead to the loss of necrotic ability on N. tabacum

v. Xanthi (Tribodet et al., 2005). Moreover, when compared withhe parental PVYN-605 necrotic isolate, the resulting mutated non-ecrotic PVY genotypes are associated with an increased relativetness on two Nicotiana hosts (N. tabacum cv. Xanthi and N. cleve-

andii) (Rolland et al., 2009). Such a result does not seem to be in

ch 153 (2010) 197–204

favour of the emergence of necrotic PVY isolates on these hosts.However, this conclusion results from the analysis of data collectedafter a single infection cycle initiated with mixtures containingbalanced quantities of two PVY genotypes. Thus, to qualify thepreviously published results on PVY/tobacco interactions in thecontext of maintenance of necrotic PVY isolates in Nicotiana hosts,mechanical plant-to-plant transmissions of a range of viral mix-tures containing a PVY isolate (e.g. necrotic PVYN-605 genotype) inthe presence of a fitter one (e.g. non-necrotic mutated versions ofthe PVYN-605 genotype) were performed. The proportions of eachgenotype in resulting mixed infected plants and the correspond-ing inocula were quantified. The study of the impact of both thePVY genomic sequence and the nucleotides involved in necrosisproperties of PVY on the maintenance of necrotic PVY genotypesfrom heterogeneous viral populations infecting Nicotiana hosts isdiscussed.

2. Materials and methods

2.1. Viruses and host plants

Five PVY genotypes were used in this study. PVYN-605 (Jakabet al., 1997) and PVYO-139 (Singh and Singh, 1996) were usedas reference isolates for the PVYN and PVYO groups, respectively.Point mutated versions of the PVYN-605 infectious clone in whichnucleotides G2213 and/or C2271 are substituted for A2213 and/orA2271 (PVYKRED, PVYKR and PVYED) (Tribodet et al., 2005) were usedas non-necrotic PVY mutants with a necrotic (PVYN-605) geneticbackground. The five genotypes are equally infectious and close to100% of infected hosts are obtained by mechanical inoculation ofa suspension containing 107 copies of PVY RNA. Experiments wereperformed using two Nicotiana species able to respond (N. tabacumcv. Xanthi) or not (N. clevelandii) with vein leaf necrosis symptomsfollowing infection by PVYN group members. Isolates and mutantsused in the experiments were individually maintained on Nico-tiana hosts since 1998 (for PVYN-605 and PVYO-139) and 2003 (forPVYKR, PVYED and PVYKRED) by serial passages (in average every21 days) using mechanical inoculation procedure. The genome ofthese isolates and mutants has been regularly sequenced during themaintenance procedure to monitor possible nucleotide variations.Under our experimental conditions, there was neither modificationof the parental genomes nor second site mutation/reversion of themutant’s genome between initial (in 2003) and last (2009–2010)produced sequence data. In complement to viral sources used inthe experiments, infected N. tabacum cv. Xanthi plants were usedto monitor the necrotic capacity of the PVY genotypes used. Healthyand infected plants were grown in separated regulated insect proofgreenhouses at 20 ◦C.

2.2. Inoculation experiments and quantification of the viral RNA

Serial passage experiments (SPE) were based on the mechan-ical inoculation of test plants with the sap obtained from plantsinfected during a previous inoculation step. This plant-to-planttransmission procedure, initiated with 11–23 infected plants perPVY mixture, was performed three times. Two different hosts, cor-responding to N. tabacum cv. Xanthi and N. clevelandii were usedin each inoculation step throughout the study. The first seriesof inoculations were done with the saps extracted from mixedinfected plants obtained after mechanical inoculations of balanced

(ratio 1/1) mixtures of two PVY genotypes (Rolland et al., 2009).Plant-to-plant viral transmissions were performed every threeweeks. Twenty-one days after inoculation, leaf-discs were sam-pled (microtubes used as perforating devices) from three differentnon-inoculated leaves (two adjacent discs per leaf) of each infected

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lant. One set of three collected leaf-discs (i.e. one disc per sampledeaf) was ground by agitation for 2 min in microtubes using SO-20apparatus (Fast&Fuild, Sassenheim, Wheeling, IL, USA). Grindingas performed in the presence of glass balls (1 mm and 4 mm diam-

ters) and 300 �l inoculation buffer. One hundred microliters of theroduced sap was used to inoculate one test plant. The second setf three collected leaf-discs was used for the quantification of theenotypes present in the infected sampled plant. These leaf-discsere ground using the same grinding procedure but in the presence

f 100 �l SV Total RNA Isolation System® lysis buffer (Promega,adison WI, USA) and 200 �l dilution buffer (Promega, MadisonI, USA). Total RNA present in the produced sap was extractedith the SV Total RNA Isolation System® (Promega, Madison WI,SA) according to the manufacturer’s instructions. Specific quan-

ifications of PVY isolates and mutants putatively present in theample were performed using previously published real-time RT-CR assays (Balme-Sinibaldi et al., 2006; Rolland et al., 2009). Thesessays use specific TaqMan®-MGB probes (probeO, probeN androbeY419N, for nucleotide sequences of the probes, see Balme-inibaldi et al., 2006; Rolland et al., 2009) and specific primerairs surrounding the targeted sequences (FpO/RpO, FpN/RpN andp419N/Rp419N, for nucleotide sequences of the primers, seealme-Sinibaldi et al., 2006; Rolland et al., 2009). The assays areased on the quantification of PVY RNA according to the identityf the nucleotides A/G2213 and A2271. Real-time RT-PCR runs wereerformed using the ABI Prism 7700 Sequence Detection SystemApplied Biosystems, Carlsbad, CA, USA) using the One-Step RT-PCR

aster Mix Reagents Kit (Applied Biosystems, Carlsbad, CA, USA)ccording to the manufacturer’s instructions, and in a total volumef 25 �l containing a primer pair (800 nM of each primer), 200 nMf the appropriate probe and 2.5 �l of RNA extract. Quantificationsere calculated relative to standards of serially diluted plasmid

olutions containing 108–102 copies of the targeted PVYN or PVYO

equences prepared as previously described (Rolland et al., 2009).

.3. Standardized procedure for the inoculation of calibratednbalanced PVY mixtures

PVYKRED and PVYO sources used in this experiment were pre-ared by individually inoculating healthy N. tabacum cv. Xanthilants (4-leaf stage) with one of these two PVY genotypes, using atandard mechanical inoculation procedure. Two weeks later, sys-emically infected leaves from plants identified as infected in ELISAssays (as previously described Clark and Adams, 1977; Jacquot etl., 2005) were sampled, ground in the presence of liquid nitro-en, and stored (less than 24 h) at −20 ◦C before inoculation. Forach isolate, 50 mg of ground infected leaves were used to esti-ate the PVY RNA concentration of the viral sources. PVY RNAolecules present in the purified nucleic acids fractions were quan-

ified using the appropriate real-time RT-PCR as described above.ccording to the quantification results, ground materials (infected

eaves) were mixed with appropriate volumes of inoculation bufferNaHPO4·12(H2O)–KH2PO4 0.02 M, DIECA 0.2% (w/v), pH 7.2) toroduce suspensions containing 2 × 107 copies of PVY RNA/100 �l.hese fractions were used to prepare different mixtures contain-ng PVYKRED and PVYO genotypes at 1/9, 3/7 or 7/3 ratios (RNAopies/RNA copies). Two fully developed leaves of test plants (20. tabacum cv. Xanthi at four-leaf stage/mixture) were dusted withmix of carborundum and charcoal powder, then 100 �l of one of

he prepared fractions were used for mechanical inoculation.

.4. Statistical analyses

All statistical analyses, were performed using the CORR, GEN-OD and GLM procedures of SAS© version 8.01 (SAS Institute Inc.,

ary, NC, USA).

ch 153 (2010) 197–204 199

3. Results

3.1. Plant-to-plant transmission of PVY mixtures

In a previously published study, six mixtures containing bal-anced quantities (107 copies of each PVY genotype) of PVYN/PVYO,PVYN/PVYKR, PVYN/PVYKRED, PVYKRED/PVYO, PVYKRED/PVYKR andPVYKRED/PVYED were separately inoculated to 26 N. tabacum cv.Xanthi and 26 N. clevelandii (Rolland et al., 2009). In this exper-iment, the proportion of dual infections obtained 21 days afterinoculation ranged from 47.8% to 88.4% with an average per-centage of 71.2%. Quantification of each competitor present inmixed infected plants showed unbalanced proportions betweenPVY genotypes (Table 1). Each of the 210 available dually infectedplants was individually used as a viral source to inoculate ahealthy plant (Table 1). This plant-to-plant transmission proce-dure was repeated three times (described below as three passages).Each plant included in this experiment was analysed three weeksafter inoculation, using appropriate real-time RT-PCR assays tospecifically quantify each inoculated PVY genotype. As only mixedinfected plants produced at each passage were retained, the entirestudy involved the mechanical inoculation of 490 plants. Each ofthese plants was inoculated with a characterised PVY mixture (theidentity of the genotypes present and their respective proportions).The proportions of singly infected plants obtained after inoculationwith PVY mixtures were heterogeneous for the different testedmixture/host combinations. High proportions of single infectionswere observed for three mixtures (PVYN/PVYKRED, PVYKRED/PVYKR

and PVYKRED/PVYED). Inoculations of these mixtures were respon-sible for 97.5% (39/40) and 92% (23/25) of the recorded singleinfections at passages 1 and 2, respectively. Moreover, due to thelack of mixed infected plants at passage 2 for PVYN/PVYKRED onN. clevelandii and at passage 3 for PVYKRED/PVYED on N. tabacumcv. Xanthi, these PVY mixture/host combinations could not beperformed to completion. Consequently, the number of dual quan-tifications for the different PVY mixture/host combinations rangedfrom 4 (PVYN/PVYKRED in N. tabacum cv. Xanthi) to 57 (PVYN/PVYKR

in N. tabacum cv. Xanthi). The quantification results (the proportionof each isolate) obtained for each mixed infected plants were anal-ysed together with the data associated with the quantification ofthe corresponding inoculum (proportion of the isolates in the viralsource) (Fig. 1). According to the prevalence (>50%) or not (<50%) ofa reference isolate in inoculums and infected plants, graphs weredivided in four areas (Fig. 1, e.g. PVYKRED/PVYED on N. tabacum cv.Xanthi, a–d). To test for the random distribution of points (dataassociated to each tested plant) into these four areas, an exactmultinomial test was performed. The results of this statistical anal-ysis indicate that data collected for the different PVY mixture/hostcombinations are not randomly distributed (p < 0.01) except forPVYN/PVYKRED and PVYN/PVYO mixtures on N. tabacum cv. Xan-thi and N. clevelandii, respectively. However, the small number ofPVYN/PVYKRED mixed infected N. tabacum cv. Xanthi (4 plants) lim-its the relevance of the result obtained for this PVY mixture/hostcombination. Four different distributions were proposed for the dif-ferent PVY mixtures and host plants. For PVYN/PVYO in N. tabacumcv. Xanthi, and PVYN/PVYO and PVYKRED/PVYO in N. clevelandii, awide distribution of the data in the graph was observed (Fig. 1,pattern A). A partial correlation test was performed with these com-binations between the proportions of genotypes in the inocula andin the corresponding infected plant, excluding the influence of theproportions in the previous generation. Significant relationships

were found for PVYN/PVYO in N. tabacum cv. Xanthi (p = 0.0005) andfor PVYKRED/PVYO in N. clevelandii (p < 0.0001). However, as sug-gested by the random distribution of the data for PVYN/PVYO mixedinfections on N. clevelandii (see above), the test rejects the correla-tion (p = 0.1160) between the tested variables for this mixture/host

200 M. Rolland et al. / Virus Research 153 (2010) 197–204

Fig. 1. Schematic representation of the proportions of the referent PVY isolate during serial passage experiments performed using mixed-infected tobacco hosts as viralsources. Each PVY mixture contains referent (Ref) and challenger (Chal) PVY isolates. Each dot corresponds to an infected plant characterised for both the proportion of thereferent isolate in the used inoculums and the proportion of the referent isolate in the plant three week after inoculation. (a)–(d) The four areas used to test, according to theprevalence (>50%) or not (<50%) of referent isolate in inoculums and infected plants, and producing patterns (A–D) according to the distribution of data in these areas.

M. Rolland et al. / Virus Research 153 (2010) 197–204 201

Table 1Characterisation of infected N. tabacum and N. clevelandii plants obtained during passage experiments based on mechanical inoculations of PVY mixtures.

PVY mixturea Source plants obtained by balancedmixed inoculation

Passage Total of dually infectedplants/total ofinoculated plants

Ref Chal Host No. of duallyinfectedplants used asviral sources

Mean proportionof referent isolatein dually infectedplants (±�)

1 2 3

NI/SI/DI NI/SI/DI NI/SI/DI

N O X 20 29.75 (±18.40) —20→ 0/0/20 —20→ 0/1/19 —19→ 0/2/17 56/59C 15 64.79 (±22.64) —15→ 1/0/14 —14→ 2/1/11 —11→ 0/1/10 35/40

KR X 22 14.42 (±8.57) —22→ 0/1/21 —21→ 0/0/21 —21→ 6/0/15 57/64C 23 10.55 (±7.32) —23→ 1/0/22 —22→ 5/0/17 —17→ 1/0/16 55/62

KRED X 17 32.33 (±29.94) —17→ 0/15/2 —2→ 0/1/1 —1→ 0/0/1 4/20C 13 20.70 (±31.19) —13→ 0/5/8 —8→ 1/7/0 ——| – 8/21

KRED O X 17 82.74 (±16.72) —17→ 0/0/17 —17→ 2/0/15 —15→ 4/1/10 42/49C 17 82.81 (±18.49) —17→ 0/0/17 —17→ 0/0/17 —17→ 1/1/15 49/51

KR X 18 73.88 (±19.47) —18→ 0/3/15 —15→ 0/9/6 —6→ 4/1/1 22/39C 20 80.61 (±20.80) —20→ 0/4/16 —b—| – – – 18/20

ED X 17 75.15 (±22.01) —17→ 0/9/8 —8→ 3/5/0 ——| – 8/25C 11 74.00 (±33.07) —11→ 0/3/8 —8→ 2/1/5 —5→ 0/1/4 15/24

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a Each inoculated mixture contains a referent (Ref) and a challenger (Chal) isolatb Quantifications performed on PVYKRED/PVYKR mixed infected plants from passa

erformed for each PVY mixture at each passage is indicated by number on correspsolates), Dl: dually infected plant; X: N. tabacum cv. Xanthi; C: N. clevelandii.

ombination. A second pattern (Fig. 1, pattern B) was obtained foroth PVYKRED/PVYO in N. tabacum cv. Xanthi and PVYN/PVYKR in. clevelandii. For these two mixture/host combinations, the pro-ortions of the viral competitors in mixed infected plants were

ocated in a small area of the graph centred on viral proportionslose to 80–90% in favour of PVYKRED and PVYKR. Analyses of dataollected passage by passage could not be explained by the progres-ive exclusion of PVYN genotype from mixed infected plants whileean proportions of PVYN in plants associated with each passageere not significantly different from each other (data not shown).oreover, during these viral competition experiments, only one N.

abacum cv. Xanthi plant (out of the 43 inoculated plants) and no. clevelandii plants (among the 55 inoculated plants) were singly

nfected. This demonstrates that the prevalence of PVYKRED andVYKR did not result in the exclusion of the other genotype presentn the PVY mixture (PVYO for PVYKRED/PVYO mixed infections on N.abacum cv. Xanthi, and PVYN for PVYN/PVYKR mixed infections in N.levelandii). The data obtained for the PVYN/PVYKR mixed infectionsn N. tabacum cv. Xanthi (Fig. 1, pattern C) reveal a third pattern.ata associated with this PVY mixture present a modified pattern B,s the proportions of the prevalent isolate (PVYKR) present in mixednfected plants is more variable. It is important to note that in thexperiment with the PVYN/PVYKR mixture, 69 out of the 79 mixednfected plants were associated with quantifications of PVYN in the

ange 1–40% (mean proportion of PVYN = 24.68 ± 15.56%). Finally, aourth pattern, obtained for the PVYN/PVYKRED, PVYKRED/PVYKR andVYKRED/PVYED mixtures (Fig. 1, pattern D), was characterised byewer available data due to the high rates of single infections.

able 2mpact of PVYKRED/PVYO ratio present in PVY inoculum on both the infection efficiency aanthi plants.

PVYKRED/PVYO mixturea No. of infected plants

Single inf.

KRED O

1/9 4 23/7 0 01/1 7 27/3 4 1

a PVY mixtures were prepared using various quantities of ground PVYKRED- and PVYO

ypes/100 �l with the listed PVYKRED (referent isolate (Ref))/PVYO (challenger (Chal)) rarouping result.

nd 3 were not determined. The number of individual plant-to-plant transmissionsg arrow. NI: non-infected plant, SI: singly infected plant (by either Ref or Chal PVY

3.2. Inoculation of calibrated unbalanced PVY mixtures

Preliminary tests demonstrated that the inoculation of Nico-tiana plants (i.e. N. tabacum cv. Xanthi and N. clevelandii) using100 �l of a suspension containing 107 copies of viral RNA/100 �lresults in an average of 70% of infection (data not shown). When2 × 107 copies of PVY genotype were inoculated, most of the inoc-ulated plants (94.6%, 295/312) were associated with positive ELISAresults (Rolland et al., 2009), indicating that under our exper-imental conditions, the viral titre of the inoculums used wasappropriate to efficiently infect these susceptible hosts. However,it is important to note that some of these plants were infectedby only one of the two inoculated PVY genotypes. In order tocharacterise better the pattern associated with the proportion ofPVYKRED in N. tabacum cv. Xanthi plants inoculated in SPE proce-dures with PVYKRED/PVYO mixtures (Fig. 1, pattern B), inoculationswere performed with inocula corresponding to calibrated unbal-anced mixtures containing these two PVY genotypes at 1/9, 3/7 and7/3 ratios (Table 2). The resulting data were analysed together withdata from previous inoculations performed with the balanced mix-tures (PVYKRED/PVYO genotypes at a ratio 1/1, Rolland et al., 2009).As previously observed, some of the mixed inoculated plants werecharacterised as being healthy or infected by a single genotype. ASpearman’s Rank Correlation Test showed no correlation between

the ratio of the used inoculums and the proportion of each type ofsingle infection (not illustrated). Plants inoculated at ratios of 3/7and 7/3 resulted in a final proportion of approximately 80% PVYKRED

in infected plants. In contrast, inoculation of plants with a mixture

nd the mean proportion of each viral competitors in dually infected N. tabacum cv.

Mean proportion (%) ofreference isolate in duallyinfected plants (±�)

Dual inf.

KRED + O

12 44.04 (±42.27) (a)17 77.31 (±23.34) (b)17 82.74 (±16.72) (b)

9 82.29 (±22.15) (b)

-infected tobacco leaves in order to obtain fractions containing 2 × 107 PVY geno-tios. �: Standard deviation. a and b illustrate Student–Newman–Keuls (˛ = 0.05)

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ontaining only 10% PVYKRED (ratio 1/9) resulted in a final propor-ion of 44% PVYKRED. Statistical analysis (Student–Newman–Keuls,= 0.05) indicated a significant difference between inoculationserformed with fractions containing 10% (ratio 1/9) of PVYKRED

Table 2, group a) and fractions containing 30%, 50% and 70% (ratios/7, 1/1 and 7/3, respectively) of PVYKRED (Table 2, group b) in the

noculated mixture.

. Discussion

Mechanical inoculations of PVY mixtures containing relatedutants with different levels of fitness have been carried out

o observe maintenance of a PVY genotype in the presence ofcompetitor. This study was based on PVYN-605 and PVYO-139

solates, point mutated non-necrotic versions of the necrotic PVYN-05 isolate (PVYKRED, PVYKR and PVYED) and two Nicotiana hostsxhibiting (N. tabacum cv. Xanthi) or not (N. clevelandii) necro-is symptoms in response to infection by PVYN group members.VY isolates are known to be differentially transmitted by aphidsAlmasi et al., 2008; Basky and Almasi, 2005). The experimentsresented have then be performed using mechanical inoculation

n order to focus on the influence of both the PVY genetic back-round and the nucleotides (A/G2213 and A/C2271), known to beequired for PVY induced necrosis, on a range of fitness parame-ers. For example, PVYKRED and PVYO genomic RNAs both contain2213 and C2271, therefore fitness differences observed between

hese two PVY genotypes are due to the genetic backgrounds ofVYN-605 and PVYO-139 (82.89% sequence identity). The differenceetween necrotic PVYN and non-necrotic PVYKR, PVYED or PVYKRED

utants is limited to the polymorphism of nucleotides A/C2271r/and A/G2213. Thus, PVYN/PVYKR, PVYN/PVYKRED, PVYKRED/PVYKR

nd PVYKRED/PVYED mixtures allowed the analysis of the impactf the two nucleotides known to be involved in the PVY necroticroperty. During the experimental procedure, the proportions ofach genotype in resulting mixed infected plants and the corre-ponding inocula were quantified and compared. However, thepecific quantification of viral entities present in mixed infectedlants did not deal with the possible presence of recombinant(s) inhe mixed infected plants. Indeed, most of the mutants used in thexperiments correspond to point mutated versions of the PVYN-605enome. Thus, putative recombination events occurring betweenVYN/PVYKR, PVYKRED/PVYKR and PVYKRED/PVYED will not produceew genomic sequence. For the competition experiments basedith PVYN/PVYKRED mixture, recombination events could induce

he creation of the PVYKR and PVYED mutants. However, the dis-ance between A/G2213 and A/C2271 (54 nucleotides) reduces therobability to create PVYKR and PVYED through random recom-ination during PVYN/PVYKRED mixed infections. Moreover, it is

mportant to note that results associated to PVYN/PVYKRED mix-ure mainly correspond to exclusions of PVYKRED mutant illustratedy the detection of PVYN single infections. Thus, recombina-ion between these two competitors, if occurred, did not impairhe monitored exclusion phenomenon. Consequently, the puta-ive recombination events between PVYN, PVYKRED, PVYKR andVYED competitors is a phenomenon with no effect on the mon-tored parameters used to estimate the fitness of these PVY isolatend mutants. In experiments performed with PVYN/PVYO andVYKRED/PVYO mixtures, recombination, if occurred, will generateariants genetically different from the parental genomes. However,t is important to note that for each used PVY mixture, the work

as initiated with 11–23 plants; each initially inoculated with aalanced PVY mixture and considered as different sources of PVYixtures. As recombination events occurred independently in each

ost, recombinant genome with a different fitness generated inne plant will impact the distribution of the data set for the corre-

ch 153 (2010) 197–204

sponding PVY mixture/host pattern presented in Fig. 1. Accordingto the shape of the pattern associated with the PVYN/PVYO andPVYKRED/PVYO mixtures, the possible presence of recombinant inmixed infected plants should not be considered as a bias in the dataanalysis presented in this work. Thus, even if we cannot ruled outthe possible presence of recombinants in analysed infected plants,this parameter has not been considered in the presented study.

The analysis of the proportions of the two PVY genotypespresent in mixed infected plants (reference and challenger) high-lights host-dependent patterns for PVYKRED/PVYO and PVYN/PVYKR

mixtures whereas similar patterns were obtained on both N.tabacum and N. clevelandii for the other tested PVY mixtures. Thisobservation suggests that in complement to possible virus/viruscompetitions, virus/host interactions could impact on equilibriumsbetween viral entities present in mixed infected hosts. Moreover,the host-dependent patterns obtained for competitions betweentwo non-necrotic genotypes (PVYKRED/PVYO) indicate that necroticcapacity of PVY genotypes is not the only parameter to be involvedin the host effects on the PVY progenies produced during mixedinfections. Previous study performed using PVYKRED/PVYO balancedmixture inoculated on N. tabacum cv. Xanthi and PVYN/PVYKR bal-anced mixtures inoculated on both N. tabacum cv. Xanthi and N.clevelandii led to an increase of proportion of PVYKRED and PVYKR

in mixed infected plants to above 75%. In addition, analysis per-formed on N. tabacum cv. Xanthi with inoculums correspondingto artificially made unbalanced PVYKRED/PVYO mixtures showedthat proportions of PVYKRED increased during a single host infec-tion cycle (one passage) to reach values of up to 80%. These dataare in accordance with previous results indicating that PVYKRED andPVYKR are fitter than PVYO and PVYN, respectively (Rolland et al.,2009). The observed increased fitness of the PVY mutants in Nico-tiana plants could be the consequence of a faster viral replicationand/or a more efficient movement within their hosts. Indeed, thesesteps of the viral infection cycle are directly linked to the quantita-tive production of viral progeny and the qualitative distribution ofthe virus in infected hosts and have been described to be variableparameters in coinfected plants relative to single infections (Arendset al., 2005; Cicin-Sain et al., 2005; Pepin et al., 2008). Further exper-iments, such as the analyses of the time-dependent accumulationpatterns of viral isolates/mutants and the monitoring of their spa-tial progression from the inoculation site(s) to the distant tissuesin infected plants constitute examples of challenging perspectivesto better understand the consequences of mixed infection on boththe shape of viral populations and PVY evolution processes.

According to the competitive exclusion principle (Gause, 1934),fitter mutants should outcompete parental sequences (Fernández-Cuartero et al., 1994). Therefore plant-to-plant transmissions(serial passages) of viral mixtures should lead to a decrease in theproportion of the less fit genotype followed by its exclusion ininfected hosts (Dietrich et al., 2007). It is important to note thatdistinct potyviral entities have been shown to be spatially sepa-rated during mixed infection (Dietrich and Maiss, 2003). Under ourexperimental conditions only three plants out of the 157 infectedplants resulting from inoculations performed with PVYKRED/PVYO

or PVYN/PVYKR mixtures were infected with a single genotype.Thus for these PVY mixtures/hosts combinations, the fitter PVYgenotype accumulates efficiently in the infected plants but theless fit genotype is maintained in the viral populations. As theexpected exclusion of the less fit viral entity was not observedin PVYKRED/PVYO and PVYN/PVYKR mixed infections, the data col-lected suggest complementation processes occur between PVYKRED

and PVYO, and between PVYN and PVYKR genotypes. Moreover,in the case of the observed PVY/Nicotiana host interactions, thecomplementation seems to be host-dependant as no or weak com-plementation processes were observed for PVYKRED/PVYO in N.clevelandii and for PVYN/PVYKR in N. tabacum cv. Xanthi, respec-

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ively. Such complementation processes between less fit and fitterenotypes have been already reported for other plant/virus inter-ctions (Fernández-Cuartero et al., 1994; Moreno et al., 1997) andur results are in agreement with those obtained by Gomez et al.2009) on pepino mosaic virus/tomato pathosystem. Consequently,ur data strengthen the role of mixed infections in the dynamicsnd evolution of a viral species. However, as this complementationas observed between the dually mutated version of the PVYN-

05 HC-Pro sequence (PVYKRED: A2213 → G2213 and A2271 → C2271)nd the PVYO-139 genome, and between single point mutated ver-ion of the PVYN HC-Pro sequence (PVYKR: A2213 → G2213) and theon-mutated PVYN HC-Pro sequence (in PVYN/PVYKR mixed infec-ion assays), this suggests complex interactions between the twoucleotides A/G2213 and A/C2271 of the HC-Pro coding sequencend other molecular determinant(s) within the PVY genome. Inter-ctions between HC-Pro and all other Potyvirus encoded proteins,part from the recently described PIPO (Chung et al., 2008), havelready been described (Urcuqui-Inchima et al., 2001). Thus, nonef the currently available data allow the identification or the exclu-ion of candidate(s) in the PVY genome for the proposed interactionith the A/G2213 and A/C2271.

Serial passage experiments data associated with PVYKRED/PVYN,VYKRED/PVYKR and PVYKRED/PVYED mixed inoculations performedn both the tested hosts were associated with frequent single infec-ions. For example, inoculations with the PVYN/PVYKRED mixtureesulted in 28 single infections in 40 mixed inoculated plants.ingly infected plants obtained after mixed inoculations could beonsidered as exclusions of the less fit genotype. Previously pub-ished data on the relative fitness of PVY mutants, demonstratedhat PVYKRED was fitter than PVYN, PVYKR and PVYED (Rolland etl., 2009). Thus, any singly infected plants resulting from mixednoculums containing PVYKRED were expected to correspond toVYKRED-infected plants. However, the majority (57/67) of singlynfected plants contained the less fit isolate. This result could bexplained by the reversion of the dually mutated PVYKRED sequenceo either PVYKR, PVYED or PVYN sequences, however this hypothesisas rejected because mutations introduced into PVYN genotype to

reate PVYKRED have been proved to be genetically stable in long-erm maintenance (more than 100 serial mechanical inoculations,ee Section 2.1) on Nicotianeae hosts. Thus, in the viral competi-ions presented, the PVY genotype considered as the fittest afterne host generation is outcompeted by the viral genotype con-idered as the less fit. As the two challenger genotypes present inhese three PVY mixtures are connected mutants that differ by onlyne (PVYKRED/PVYKR and PVYKRED/PVYED) or two (PVYKRED/PVYN)oint mutations, the observed exclusion of PVYKRED genotypes fromVYKRED/PVYN, PVYKRED/PVYKR and PVYKRED/PVYED mixed infectedlants cannot yet be explained.

In conclusion, these analyses of the competition experimentserformed using mechanical plant-to-plant transmission of PVYixtures, indicate that among the tested point mutated versions of

he PVYN-605 isolate, the fittest genotype corresponds to the non-ecrotic PVYKR. Indeed, during competition experiments the latter

s able to outcompete PVYKRED, which was previously characterised,n single infection cycle experiment initiated with balanced mixedraction, as a mutant fitter than PVYKR. In addition, PVYKR accu-

ulates efficiently in infected Nicotiana hosts to become prevalentn the presence of the parental PVYN sequence. Thus, in comple-

ent to results obtained using single passage of PVY mixtures on. tabacum cv. Xanthi and N. clevelandii, necrotic PVY genotypeseem not to benefit from long-term maintenance (i.e. serial pas-

ages) on these Nicotiana hosts to become prevalent in naturalopulations. However, the efficient infection of Nicotiana hosts byhe non-necrotic PVYKR genotype seems to be favourable to the

aintenance (at a 20% estimated rate in the viral population) ofhe parental necrotic PVYN sequence. The impacts of the described

ch 153 (2010) 197–204 203

complementations between necrotic and non-necrotic PVY geno-types on the emergence and spread of necrotic PVY isolates innatural populations are currently under investigation.

Acknowledgements

We are grateful to Dr Maxime Trottet (INRA, Rennes, France)for statistical analyses, to Prof. Santiago F. Elena (IBMCP, Valencia,Spain) for critical reading of the manuscript, and to Michel Tribodetfor his technical support. The presented work has been supportedby the Institut National de la Recherche Agronomique (France), theFédération Nationale des Producteurs de Plants de Pomme de Terre(France) and the Association Nationale de la Recherche Technique(France).

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