A icle
Expe imen al E olu ion o Pseudogeniza ion and Gene Loss in a
Plan RNA Vi us
Ma k P. Zwa ,*
,1
Anouk Willemsen,
1
Jose
´-An onio Da o
`s,
1
and San iago F. Elena
1,2
1
Ins i u o de Biologı
´a Molecula y Celula de Plan as, Consejo Supe io de In es igaciones Cien ı
´ icas-UPV, Vale
`ncia, Spain
2
The San a Fe Ins i u e
*Co esponding au ho : E-mail: ma [email protected] .es.
Associa e edi o : Howa d Ochman
Abs ac
Vi uses ha e e ol ed highly s eamlined genomes and a a ie y o mechanisms o comp ess hem, sugges ing ha
genome size is unde s ong selec ion. Ho izon al gene ans e has, on he o he hand, played an impo an ole in
i us e olu ion. Howe e , e olu ion canno in eg a e ini ially non unc ional sequences in o he i al genome i hey a e
apidly pu ged by selec ion. He e we epo on he expe imen al e olu ion o pseudogeniza ion in i us genomes using a
plan RNA i us exp essing a he e ologous gene. When long 9-week passages we e pe o med, he added gene was los
in all lineages, whe eas i uses wi h la ge genomic dele ions we e ixed in only wo ou o en 3-week lineages and none in
1-week lineages. Illumina nex -gene a ion sequencing e ealed conside able con e gen e olu ion in he 9- and 3-week
lineages wi h genomic dele ions. Genome size was co ela ed o wi hin-hos compe i i e i ness, al hough he e was no
co ela ion wi h i us accumula ion o i ulence. Wi hin-hos compe i i e i ness o he 3-week i us lineages wi hou
genomic dele ions was highe han o he 1-week lineages. Ou esul s show ha he s eng h o selec ion o a educed
genome size and he a e o pseudogeniza ion depend on demog aphic condi ions. Mo eo e , o he 3-week passage
condi ion, we obse ed inc eases in wi hin-hos i ness, whe eas selec ion was no s ong enough o quickly emo e he
non unc ional he e ologous gene. These esul s sugges a demog aphically de e mined “swee spo ” migh exis , whe e
he e ologous inse ions a e no immedia ely los while e olu ion can ac o in eg a e hem in o he i al genome.
Key wo ds: genome e olu ion, plan i us, ho izon al gene ans e , pseudogeniza ion, i ness, nex -gene a ion sequencing.
In oduc ion
Vi us genomes a e highly s eamlined. Compa ed wi h mo e
complex o ganisms, i uses end o ha e small genomes wi h
1) a high pe cen age o coding sequences, 2) none o li le
in onic sequences, and 3) only sho s e ches o in e genic
sequences (Lynch 2006;Belshaw e al. 2007). Mo eo e ,
i uses ha e e ol ed s a egies o u he comp ess hei
genomes, such ameshi s and o e lapping open eading
ames (ORFs) (e.g., Belshaw e al. 2007;Chung e al. 2008).
Field obse a ions sugges ha genome sh inkage some imes
occu s du ing epidemic sp ead and migh be linked o in-
c eased wi hin-hos i ness and be adap i e o whi e spo
synd ome i us (WSSV), a la ge DNA i us (Ma ks e al. 2005;
Zwa , Dieu, e al. 2010). Mo eo e , i appea s o be a e y
gene al obse a ion ha i uses exp essing he e ologous
genes end o be uns able (Chapman e al. 1992;Dolja e al.
1993;Guo e al. 1998;Pijlman e al. 2001;Chung e al. 2007;
Paa e al. 2007). Fu he mo e, unde condi ions maximizing
selec ion o compe i i e i ness—exempli ied by undilu ed
se ial passage in cul u ed cells— i uses end o apidly
e ol e de ec i e in e e ing pa icles (DIPs): i uses wi h
la ge genomic dele ions a e unable o eplica e au onomously
bu wi h a eplica i e ad an age a high mul iplici ies o
in ec ion (Huang 1973;Simon e al. 2004;Zwa e al. 2008;
Pa hak and Nagy 2009). All hese obse a ions sugges ha
genome size is unde s ong selec ion o i uses and ha
ha ing unnecessa y genomic sequences has i ness cos s. By
con as , s iking cases o genome sh inkage ha e been ound
o obliga e hos -dependen species o bac e ia, bu his
sh inkage appea s o be he esul o a mu a ional bias owa d
dele ions and gene ic d i (Ochman and Da alos 2006;Kuo
and Ochman 2009).
Vi uses play an impo an e olu iona y ole as ec o s o
ho izon al gene ans e (HGT) in he genomes o hei hos s
(Canchaya e al. 2003;Belshaw e al. 2004;Rou h e al. 2012).
I is mo eo e becoming inc easingly appa en ha HGT is
also widesp ead in mos i uses and is a key mechanism in
hei e olu ion (Hughes and F iedman 2005;Dolja and
Koonin 2011;Koonin and Dolja 2012;Liu e al. 2011,2012;
Yu in and Koonin 2012). S iking inno a ions such as he
DNA-RNA i us hyb id (Dieme and S edman 2012)and
he coop ion o an en i e hos immuni y mechanism by a
phage (Seed e al. 2013) exempli y how HGT can empowe
he e olu iona y p ocess. Howe e , s ong selec ion o
genome size would in p inciple be an impedimen o HGT.
In o de o a he e ologous sequence o be bene icial o he
ecipien i us,i mus beaccommoda edin o he i us
genome, ansc ip ome, and p o eome. Mu a ion and selec-
ion mus he e o e ac on newly ans e ed he e ologous
sequences, bu o do so, hese sequences mus no be pu ged
igh upon acquisi ion because o selec ion o a s eamlined
genome. The mu a ional supply may a o he dele ion o
ßThe Au ho 2013. Published by Ox o d Uni e si y P ess on behal o he Socie y o Molecula Biology and E olu ion.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Non-Comme cial License (h p://
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Mol. Biol. E ol. 31(1):121–134 doi:10.1093/molbe /ms 175 Ad ance Access publica ion Oc obe 8, 2013 121
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he e ologous sequences; dele iono asequenceby ecombi-
na ion p obably has a g ea e likelihood han he occu ence
o bene icial mu a ions unc ionally in eg a ing his elemen
in he i us. I he e ec s o dele ions o he e ologous
sequences a e bene icial on he sho e m, how can HGT
be common in i uses?
One possible answe o his ques ion is ha selec ion
o genome size is no a e y s ong o ce. Fi s , i is no a
all clea ha he me abolic cos o addi ional gene ic ma e ial
is su icien o accoun o he expec ed i ness cos s (Lynch
2007). Second, expe imen al esul s on he ela ionship
be ween genome size and eplica i e i ness a e ambiguous.
When compa ing phages wi h di e en genome o ganiza-
ions adap ed o as eplica ion, he expec ed ela ionship
was no ound (Bull e al. 2004). When exp essing di e en -
size ma ke p o eins in Sendai i us, an in e se ela ionship
be ween inse size and eplica ion was ound in cul u ed
cells (Sakai e al. 1999). Howe e , his ela ionship was no
obse ed in i o (Sakai e al. 1999) and, u he mo e, he
use o sequences coding o di e en ma ke p o eins
makes he compa ison oublesome (Maje e al. 2013).
Mo eo e , ins abili y o i uses exp essing he e ologous
sequences (Chapman e al. 1992;Dolja e al. 1993;Guo
e al. 1998;Chung e al. 2007) may depend on many en i on-
men al ac o s (Paa e al. 2007), and e en sub le ies o he
he e ologous sequence, such as guanine-u acil con en (Lee
e al. 2002). Finally, expe imen s co obo a ing he ela ion-
ship be ween genome size and i ness o WSSV we e pe -
o med wi h ield isola es (Ma ks e al. 2005;Zwa , Dieu, e al.
2010), and hence, o he gene ic a ia ion could be a con-
ounding ac o . In conside ing whe he HGT is eally implau-
sible, we he e o e need o ask whe he inc eases in genome
size eally ha e app eciable i ness cos s and wha i ness
componen s migh be a ec ed.
He e we explo e he p ocess o pseudogeniza ion in i us
genomes by means o expe imen al e olu ion. As a model
sys em, we conside a plan RNA i us exp essing a non oxic
he e ologous gene, whose exp ession has been enginee ed
o minimally dis up he i al genome. We i s looked o
condi ions unde which he he e ologous gene would be
main ained in he genome. I has been shown ha he
ime pe iod be ween wo consecu i e ansmission e en s,
ha is, he ime a i al popula ion has o expand be ween wo
consecu i e bo lenecks, can be ins umen al in de e mining
genome s abili y in plan RNA i uses (Dolja e al. 1993).
Howe e , in his s udy, he he e ologous gene was exp essed
as a usion wi h one o he i al cis ons and, gi en he
s ong e ec s on i al accumula ion (Dolja e al. 1993),
mus he e o e be seen as a dele e ious, a he han a
me ely non unc ional addi ion. We hen conside whe he
he dele ion o he he e ologous gene was adap i e and
wha i us cha ac e is ics i modi ies. Finally, we conside
whe he he e a e condi ions ha ul ill wo equi emen s:
1) he he e ologous gene has a high p obabili y o being
main ained in he e ol ed i us popula ion and 2) he e is
e idence ha he i us popula ion is unde posi i e selec ion
and expe iences inc eases in i ness. We hink he combina-
ion o condi ions is ele an o he con ex o HGT in i uses.
I hese wo condi ions a e ul illed, hen in p inciple, a
he e ologous sequence can pe sis o long pe iods o ime
in he i us popula ion, while inc eases in i ness imply
ha na u al selec ion is ac ing on he popula ion and could
“ inke ” wi h he he e ologous gene, sequences egula ing
i s exp ession, and o he loci in e ac ing wi h he he e olo-
gous gene, po en ially and unc ionally in eg a ing i in o
he i al genome. On he o he hand, a he e ologous
gene may no be los in a i us popula ion subjec o high
le els o gene ic d i , bu i is hen also unlikely ha na u al
selec ion ac s o unc ionally in eg a e i . Simila ly, a i us
popula ion unde s ong posi i e selec ion in which he
he e ologous gene is los is also a dead end o HGT.
Howe e , simul aneously ha ing main enance o he he e ol-
ogous gene and inc eases in i al i ness sugges s he occu -
ence o a “swee spo ” ha could help explain how HGT
occu s in i uses.
Resul s and Discussion
Resul s o Se ial Passage Expe imen s
As a model sys em o i us exp essing a he e ologous gene
wi hou app eciable oxici y o dis up ion o i al eplica ion,
we used a a ian o Tobacco e ch i us (TEV; genus Po y i us,
amily Po y i idae). TEV is a posi i e-sense single-s anded
RNA i us ha encodes a polyp o ein au oca aly ically
clea ed in o en ma u e p o eins (Riechmann e al. 1992)
and a pa ially o e lapping ORF wi h a +2 ameshi
(Chung e al. 2008). The TEV a ian we used exp esses
enhanced g een luo escen p o ein (eGFP) as a sepa a e cis-
on be ween P1 and HC-P o by in oducing a second NIa-P o
p o eoly ic si e downs eam o he eGFP sequence while e-
aining he exis ing C- e minal si e in P1 ( ig. 1)(Zwa e al.
2011). E olu ion expe imen s we e pe o med in Nico iana
abacum L. c . Xan hi plan s. In b ie , 4-week-old plan s
we e inocula ed wi h high i us doses, and passages las ing
ei he 1 week ( o a o al o 27 consecu i e passages), 3 weeks
(nine passages) o 9 weeks ( h ee passages) we e pe o med.
Each “passage” is he in ec ion o a single plan and ha es ing
o issues a he designa ed ime (i.e., he “passage du a ion”).
The e o e, al hough he passage du a ion a ied among
FIG.1. Scheme o TEV-eGFP. Lines ep esen he i al 50-and3
0-un ansla ed egions (50-UTR and 30-UTR), he g ay box ep esen s eGFP, open boxes
ep esen he i al cis ons P1, HC-P o, P3, 6K1, CI, 6K2, VPg, NIa-P o, NIb, and CP, whe eas P3N-PIPO is indica ed by he lowe box.
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ea men s, each lineage e ol ed o he same o al ime (27
weeks) in N. abacum. A he end o a passage, all he lea es
abo e he inocula ed lea we e collec ed, pooled, and used o
ob ain he inoculum o he nex ound o se ial passaging.
Ten independen lineages we e gene a ed and main ained o
each passage du a ion (1, 3, o 9 weeks). An o e iew o he
expe imen al se up used is gi en in igu e 2,and u he de-
ails a e gi en in he Ma e ials and Me hods.
eGFP exp ession was eadily appa en in in ec ed plan s
( ig. 3A) and was used as a i s indica ion o whe he he
he e ologous gene was in ac . Pa ial losses o luo escence
( ig. 3B) almos always p eceded comple e losses o luo es-
cence ( ig. 3C). One ou o en 1-week lineages showed a
pa ial loss o luo escence, i s obse ed a passage 7 and
main ained un il passage 27 ( ig. 3Eand F). Two ou o en
3-week lineages showed a loss o luo escence. All bu one
9-week passage showed dec eased le els o luo escence a e
a single passage, and all lineages showed a comple e loss o
luo escence a e wo passages. Re e se ansc ip ion poly-
me ase chain eac ion (RT-PCR) wi h p ime s lanking eGFP
(Ma e ials and Me hods) con i med he occu ence o geno-
mic dele ions in all lineages wi h a pa ial o comple e loss o
luo escence ( ig. 3G). These esul s a e cong uen wi h p e-
ious wo k wi h TEV (Dolja e al. 1993), excep ha he de-
le ion o he he e ologous gene occu s much mo e slowly
he e, as an icipa ed.
We hen inocula ed N. abacum wi h TEV-eGFP and, a
9 weeks o in ec ion, ha es ed e e y i h lea up he s em.
Because he in ec ion p og esses linea ly as he plan g ows,
hese lea es enable us o moni o aquali a i e imecou seo
e olu ion wi hin he plan . All plan s had by hen eached he
45-lea s age, excep o one ha had only 40 lea es. We
pe o med RT-qPCR on indi idual lea es o asce ain a
wha lea le el dele ions occu ed, and i hey we e subse-
quen ly main ained in he popula ion. This analysis can
be pe o med since he i us mo es mainly upwa d in he
plan (Dolja e al. 1992). In mos cases, once a dele ion was
de ec ed in one lea , i was main ained and ixed in he
supe io lea es ( ig. 4). This obse a ion sugges s ha selec-
ion o dele ion a ian s is e y s ong in his expe imen ,
being a s onge e olu iona y o ce han gene ic d i wi hin
he hos . The i s lea in which a no el dele ion was de ec ed
was no uni o mly dis ibu ed o e all es ed lea es (lea es 5–
45; one-sample Kolmogo o –Smi no es : n= 13; P= 0.019);
new dele ion a ian s we e usually i s de ec ed in highe
lea es (mean ±s anda d de ia ion [SD] = 32.31 ±9.92). This
esul sugges s ha passage du a ion is impo an o he dy-
namics o he e ologous gene dele ion because i egula es he
amoun o expansion ha occu s be ween bo lenecks.
Du ing i us in ec ion o mechanically inocula ed plan s,
gene ic bo lenecks in he i us popula ion can occu du ing
p ima y in ec ion o he inocula ed lea and he subsequen
en y in o sys emically in ec ed lea es (Hall e al. 2001;
Sac is a
´n e al. 2003;Gu ie
´ ez e al. 2012). Fo TEV in ec ion
o obacco plan s, he numbe o p ima y in ec ion oci in
he inocula ed lea is a good es ima o o he numbe o
ounde s, whe eas he subsequen bo lenecks du ing en y
in o sys emically in ec ed lea es do no appea o be se e e
(Zwa e al. 2011). We he e o e inocula ed eigh plan s
wi h TEV-eGFP using he same p ocedu e and condi ions,
as du ing se ial passaging (see Ma e ials and Me hods;
homogenized issue o plan s in ec ed wi h TEV-eGFP was
used as an inoculum), and coun ed he numbe o p ima y
in ec ion oci (Zwa e al. 2011). The mean numbe o oci
obse ed ±SD was 417 ±140, sugges ing ha al hough he e
is a bo leneck a he s a o in ec ion, i is no oo se e e.
Ne e heless, his bo leneck could emo e a ia ion gene -
a ed de no o du ing he p e ious passage and he eby limi
he a ia ion upon which selec ion can ac . Longe passage
du a ion would allow bene icial a ia ion o inc ease in
equency, hus making i less likely o lose such a ian s
due o gene ic d i a he nex ansmission e en . The oc-
cu ence o gene ic bo lenecks he e o e ein o ces he idea
ha passage du a ion migh be impo an o he e olu iona y
dynamics in his sys em.
Genome Sequences o E ol ed Lines wi h Dele ions
All e ol ed lineages in which dele ions had been de ec ed by
RT-PCR we e ully sequenced by Illumina nex -gene a ion
sequencing (NGS). We de eloped an app oach (Ma e ials
and Me hods) o mapping la ge genomic dele ions (i.e., de-
le ions la ge han he ead size). We consis en ly saw pseu-
dogeniza ion o comple e loss o eGFP in all hese lineages
( ig. 5A). None o hese dele ions included he C- e minus o
P1, while o 7 ou o 13 lineages, hese dele ions included N-
e minal egions o HC-P o, simila o he p e ious esul s
(Dolja e al. 1993). The N- e minal egion o HC-P o is no
essen ial o eplica ion and mo emen (Dolja e al. 1993;
C onin e al. 1995) bu has been implica ed in ec o -bo ne
ansmission (Tho nbu y e al. 1990;A eya e al. 1992), which
is no a selec i e o ce in ou mechanical ansmission passage
expe imen s. In he se en lineages wi h dele ions ex ending
in o HC-P o, he emains o eGFP we e used wi h HC-P o,
while he p o eoly ic si e be ween P1 and eGFP emained
in ac . The s a o genomic dele ion (50end) was no uni-
o mly dis ibu ed (one-sample Kolmogo o –Smi no es :
n= 13; P= 0.006), and he e is clus e ing a he 50o he
eGFP cis on ( ig. 5Band C), sugges ing he exis ence o a
ho spo o ecombina ion o he un iabili y o any dele ions
in he P1-eGFP p o eoly ic si e. On he o he hand, he 30end
o he genomic dele ion was uni o mly dis ibu ed (one-
sample Kolmogo o –Smi no es : n= 13; P= 0.130).
We pe o med addi ional analyses o de ec mino i y
a ian s wi h di e en dele ion sizes in sequenced lineages
(Ma e ials and Me hods). Al hough mino i y a ian s we e
some imes de ec ed in he 3- and 9-week lineages, hese
we e always p esen a low equencies (<1.5%). Only in
he case o he 1-week lineage wi h a pa ial eGFP loss was
a mino i y a ian p esen : 47.1% o he popula ion was
composed o a a ian wi h in ac eGFP. The dele ion in
he majo i y a ian ex ends beyond he HC-P o N- e minal
egions nonessen ial o eplica ion and mo emen (C onin
e al. 1995), sugges ing his majo i y a ian is no able o
eplica e wi hou being complemen ed by he ull-leng h
a ian . When plan s we e inocula ed wi h low i us doses
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FIG.2. O e iew o he expe imen al se up employed in he s udy. A he s a o he se ial passage expe imen , 4-week-old N. abacum plan s we e
mechanically inocula ed wi h TEV-eGFP in he hi d ue lea (indica ed in ed abo e). A he end o he designa ed passage du a ion (1, 3, o 9 weeks),
all lea es abo e he inocula ed lea , which a e indica ed in g een abo e, we e collec ed and s o ed a 80 C. The ozen issue was hen homogenized,
and a sample o he homogenized issue was g ound o a ine powde . Fo inocula ion o subsequen passages, powde was esuspended in inocula ion
bu e and new N. abacum plan s we e inocula ed. Al hough he du a ion o he passages a ied (1, 3, and 9 weeks), he numbe o passages was se so
ha he o al ime each lineage was p esen in plan s was he same, being 27 weeks o all lineages. Fo each passage du a ion used, en independen
lineages we e aken. No e ha he igu e is only schema ic: he dis ance be ween lea laye s has been exagge a ed, and a e 9 weeks o in ec ion,
N. abacum plan s a e in eali y ela i ely alle han depic ed he e.
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FIG.3. Loss o eGFP luo escence and sequence du ing se ial passages: (A)AnN. abacum lea ha is comple ely symp oma ic, indica ing hea y i us
in ec ion, showed 1 week pos inocula ion wi h an e ol ed TEV-eGFP lineage wi h no loss o luo escence. (B) A comple ely symp oma ic lea showed
1 week pos inocula ion wi h an e ol ed TEV-eGFP lineage wi h a pa ial loss o luo escence (1-week passage lineage 7). (C) A comple ely symp oma ic
lea showed 1 week pos inocula ion wi h an e ol ed TEV-GFP lineage wi h a comple e loss o luo escence. (D)Obse ed luo escence du ing se ial
passage o TEV-eGFP o 1-week passages. Fo panels D–E, g een squa es indica e no loss o eGFP luo escence (as in panel A), ha ched g een squa es
indica e a pa ial loss o luo escence (as in panel B) in pa s o all o he plan , and whi e squa es indica e no luo escence was obse ed in he whole
plan (as in panel C). (E) Obse ed luo escence o 3-week passages. (F) Obse ed luo escence o 9-week passages. (G)Anaga osegelwi hRT-PCR
p oduc s o dele ions in he eGFP locus o TEV-eGFP is shown o he i s passage o he 9-week lineages. The alue 1 kb +indica es he lane wi h a
1kb+DNA ladde , NTC is he non empla e con ol, and NVC is he non i us con ol, a mock-inocula ed heal hy plan . TEV-eGFP, he ances al i us
o he e olu ion expe imen s, and TEV, he wild- ype i us wi hou he he e ologous gene inse ed, a e included o compa ison. No e ha in each
e ol ed lineage, dele ions o eGFP a e isible, al hough hei equency appea s o a y. In lineage 1, he band co esponding o he ances al i us is s ill
e y s ong, whe eas o lineage 4, i is no longe isible.
FIG.4. A single 9-week passage o TEV-eGFP was pe o med in eigh plan s, and he smalles obse ed inse size a he eGFP locus (o dina e) was
measu ed e e y i h lea (abscissa). Do ed lines indica e he ull-leng h eGFP sequence was s ill de ec ed, solid lines indica e i is no longe de ec ed, and
a ci cle indica es he poin a which he ull-leng h sequence is i s no longe de ec ed, and each eplica e has a di e en colo . Dele ions we e ixedin
ou ou o eigh eplica es, and only in a single eplica e we e no dele ions de ec ed h oughou in ec ion (yellow). Only in one case (pink) was a
dele ion no main ained a e i has i s been obse ed (a dele ion is obse ed in lea 40 bu no in lea 45).
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FIG.5. Genome sequences o e ol ed lineages: (A) NGS da a o he e ol ed lineages wi h dele ions om he se ial passage expe imen ( ig. 3Dand F)
a e gi en. The names on he le iden i y lineages (e.g., 9W L1 is he inal popula ion o 9-week passage, lineage 1). G ay boxes indica e genomic dele ions
in he majo i y a ian . Full ci cles and open ci cles a e nonsynonymous and synonymous subs i u ions, espec i ely. Black subs i u ions occu in only
one lineage, whe eas colo -coded subs i u ions a e epea ed in wo o mo e lineages. Fo lineage 1W L7, only a single sequence is ep esen ed. Howe e ,
no e ha in his lineage, ano he a ian wi h he ull-leng h TEV-eGFP genome is p esen a a equency 47.1%. None o he single-nucleo ide
mu a ions in 1W L7 we e ixed, sugges ing hey occu ed a e he genomic dele ion and a e p esen in only one o he wo a ian s p esen . Howe e ,
he sequence a ia ion is minimal and he wo a ian s a e p esen a app oxima ely same equencies in his lineage. The single-nucleo ide mu a ions
can he e o e no be assigned o he ull-leng h o he dele ed a ian , al hough hey a e ep esen ed on he dele ion a ian in he igu e. In all o he
lineages, he ull-leng h i us was no de ec ed and a ian s wi h o he genomic dele ions we e p esen only a e y low equencies (<1.5%).
(B) His og am o he posi ion o he s a o genomic dele ions in he e ol ed lines. Fo panels Band C, da k lines indica e egions in he eGFP cis on,
whi e lines indica e egions in he i al genome, and ha ched lines indica e he egions encompassing wo cis ons (i.e., P1 and eGFP in panel B).
(C) His og am o he posi ion o he end o he genomic dele ion in e ol ed lines.
Table 1. In Vi o Cloning o 1-Week Passage Lineage 7 (1W L7).
Expe imen Mean Foci Plan s
Unin ec ed eGFP Only Mix u e No eGFP
1 1.022 9 (0.360) 12 (0.480) 4 (0.160) 0 (0.000)
2 1.514 18 (0.243) 36 (0.486) 20 (0.270) 0 (0.000)
NOTE.–Two eplica e expe imen s we e pe o med in which Nico iana abacum plan s we e in ec ed wi h a 1:1,000 dilu ion o in ec ious sap o
he inal e ol ed popula ion o 1W L7. This popula ion had appa en ly ha bo ed a i us a ian ha had los luo escence ( ig. 3B), al hough
his a ian ne e wen o ixa ion ( ig. 3D). E en a low doses, all in ec ed plan s (as de e mined by symp oms 2 weeks a e inocula ion)
con ained only he eGFP-exp essing i us (“eGFP only”; luo escence pa e ns simila o ig. 3A) o bo h i us a ian s (“Mix u e”; ig. 3B).
A symp oma ic plan wi hou eGFP exp ession was ne e obse ed (“No eGFP”; ig. 3C), al hough he low dose esul ed in a low mean numbe
o p ima y in ec ion oci o TEV-eGFP (“mean oci”) and many unin ec edplan s.RT-PCRwaspe o medon i eplan sjudgedasbeing
unin ec ed, in ec ed only wi h he eGFP a ian , o in ec ed wi h a mix u e o he wo i uses, and he RT-PCR esul s we e always cong uen
wi h mic oscopic obse a ions. We he e o e conclude he i us a ian in he popula ion ha does no exp ess eGFP, has he e o e p obably
los in ec i i y, o i has a e y low in ec i i y. This a ian is ne e heless su p isingly s able. The 1W L7 popula ion was pu h ough h ee
1-week passages o one 3-week passage, wi h en eplica es each. The p esence o he i us a ian no exp essing eGFP could always be
deduced om eGFP exp ession pa e ns (i.e., ig. 3B).
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o his e ol ed lineage, we we e indeed unable o in i o clone
he majo i y a ian wi hou in ac eGFP ( able 1).
When single-base subs i u ions we e de ec ed in se-
quenced lineages, he e appea ed o be con e gen e olu ion
in he 3- and 9-week lineages ( ig. 5A). All hese lineages
con ained a leas one subs i u ion p esen in ano he lineage,
wo subs i u ions we e p esen in 8 ou o 12 lineages, and
one subs i u ion was p esen in 9 ou o 12 lineages. O e all,
mo e han hal o he subs i u ions ound we e p esen
in o he lineages, and some lineages con ained only subs i u-
ions also p esen in o he lineages (9-week lineages 1 and 2).
In he single 1-week passage sequenced, none o he epea ed
subs i u ions we e p esen , sugges ing ha con e gen
e olu ion did no occu unde hese condi ions.
O he subs i u ions ound he e, 28 we e nonsynonymous
and 53 we e synonymous. Ele en nonsynonymous subs i u-
ions we e con e gen , while 29 synonymous subs i u ions
we e con e gen . Synonymous subs i u ions we e he e o e
mo e common han nonsynonymous subs i u ions, al hough
bo h we e equally likely among cases o con e gence (Fishe ’s
exac es P= 0.244). Con e gen , nonsynonymous subs i u-
ions we e always ound in he P1 cis on (A872G, N!Sin9
ou o 13 lineages; all posi ions gi en ela i e o he o iginal
TEV-eGFP genome, GenBank KC918545), o he emaining
50end o he eGFP cis on (A1085U, E!V in 2 lineages).
Con e gen synonymous subs i u ions we e ound in P1
(C795U in six lineages), HC-P o (C1927A in i e lineages),
NIa-P o (U7092C in wo lineages and A7479C in eigh line-
ages), and NIb cis ons (A8253C in eigh lineages). The
A7479C and A8253C subs i u ions always occu ed oge he ,
sugges ing syne gis ic epis asis o possibly e en ecip ocal sign
epis asis, whe eas he A7479C and A8253C ne e occu ed
oge he wi h U7092C, sugges ing an agonis ic epis asis.
Howe e , nei he o hese wo e ec s was signi ican gi en
he numbe o obse a ions ( able 2). No a single mu a ion
was de ec ed in he coa p o ein (CP). The o e all d
N
/d
S
a io
was signi ican ly smalle han 1 (mean ±SD = 0.058 ±0.002;
z- es P<0.001), sugges ing he polyp o ein sequence is
unde pu i ying selec ion. Wi hin-popula ion single nucleo-
ide polymo phisms (SNPs) we e analyzed o e e y e ol ed
lineage agains hei co esponding consensus sequence.
O he SNPs ound, en we e synonymous and six we e non-
synonymous. Fi e ou o 13 e ol ed lineages con ained he
same synonymous SNP in he HC-P o cis on (C1879A). None
o he o he synonymous and nonsynonymous SNPs we e
epea ed in he e ol ed lineages.
P e ious e olu ion expe imen s wi h TEV ha e also shown
no genomic con e gences o 1-week passages in N. abacum:
a e 15 weeks o e olu ion, no subs i u ions we e epea ed in
di e en lineages (Bedhomme e al. 2012). These obse a ions
sugges ha li le adap i e e olu ion migh occu when
sho 1-week se ial passages a e pe o med. Fu he mo e,
he speci ic con e gen mu a ions obse ed he e we e no
obse ed in o he 1-week passage expe imen s (Bedhomme
e al. 2012; T omas N, Zwa MP, Elena SF, unpublished
manusc ip ). This sugges s ha hese con e gen mu a ions
may be linked o he inse ion o he eGFP gene. To es his
possibili y, we conside ed whe he he mos common mu a-
ions (C795U, A872G, A7479C, and A8253C) occu ed in lin-
eages o he wild- ype TEV pu h ough h ee 9-week passages
in N. abacum (Ma e ials and Me hods). In none o such
lineages we e any o hese mu a ions ound, s ongly sugges -
ing hey a e linked o he p esence o eGFP. Al hough we
sequenced only a small pa o hese e ol ed TEV genomes,
we did ind one mu a ion epea ed in 3 ou o 10 lineages
(A6806G, K!E), sugges ing he e is a leas some con e gen
e olu ion when he wild- ype TEV is pu h ough long
passages.
Accumula ion, Vi ulence, and Wi hin-Hos
Compe i i e Fi ness o E ol ed Lineages
We biologically cha ac e ized all e ol ed lineages, in e ms
o hei i ulence and i al accumula ion, and measu ed
wi hin-hos compe i i e i ness (W; Ma e ials and
Me hods). The e was no e ec o passage du a ion on
ei he i al accumula ion a 7 days pos inocula ion (dpi) o
i ulence ( ig. 6Aand B; able 3). On he o he hand, he e was
a highly signi ican e ec o passage du a ion on wi hin-hos
compe i i e i ness, which inc eased signi ican ly wi h passage
du a ion ( ig. 6Cand able 3).
Fo he cu en expe imen al se up, we a p io i expec ha
wi hin-hos compe i i e i ness will be unde selec ion, be-
cause we a e passaging a i us wi hin a single hos a high
inocula ion doses. Those i us a ian s ha exis a he high-
es equency a he end o in ec ion a e he e o e mos likely
o be ans e ed, i espec i e o accumula ion le els o he
en i e popula ion. We did no expec i ulence o change,
because we do no expec i o be unde selec ion and i is
p obably no linked o wi hin-hos i ness in ou model
sys em (Ca asco, de la Iglesia, e al. 2007). Gi en ha high
inocula ion doses a e used, i us accumula ion is no likely o
be e y impo an ei he , so long as i is enough o main ain
in ec ion in he nex ound o passaging. No e ha a 1,000-
old dilu ion o an inoculum s ill causes mode a e le els o
in ec ion, as shown by he in i o cloning esul s ( able 1).
Published da a (Ca asco, de la Iglesia, e al. 2007;Lalic
´e al.
Table 2. Cooccu ence o Single-Nucleo ide Subs i u ions.
Subs i u ion Combina ion P AðÞP BðÞ¼P A BðÞ.Obs A BðÞ.PValue
A7479C A8253C. (8/12)(8/12) = 0.444 8/12 = 0.667 0.1501
A7479C A8253CðÞ U7092C. (8/12)(2/12) = 0.111 0/12 = 0 0.3841
NOTE.–He e we es whe he he cooccu ence, o lack he eo , is s a is ically signi ican o wo g oups o single-nucleo ide subs i u ion (i.e., subs i u ion combina ion).
P AðÞP BðÞ¼P A BðÞis he p oduc o he equency o occu ence o he subs i u ions, he expec ed equency a which we expec o see bo h subs i u ions in he
absence o any epis a ic in e ac ions. We only conside ed he 3- and 9-week passages, because we do no hink selec ion is ac ing on he 1-week lineage.Obs A BðÞgi es he
equency a which he combina ion was obse ed, and P alue is he signi icance as de e mined by compa ison o p edic ed and obse ed alues wi h an exac binomial es .
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FIG.6. Vi ulence, accumula ion, and wi hin-hos i ness o e ol ed s ains a e gi en. (A) The heigh o con ol plan s (NVC), and plan s in ec ed wi h
TEV, TEV-eGFP ( he ances al i us o e ol ed lineages), and all lineages o e ol ed i uses a e gi en. We conside he in e se o plan heigh as a p oxy
o i ulence, hough in he absence o signi ican di e ences in he da a, we simply p esen he aw da a. Fo all panels, g een columns indica e no
dele ions in he he e ologous gene (eGFP) we e de ec ed, o ange indica es pa o all o he eGFP was no p esen , and ed indica es pa o eGFP and
he i al HC-P o cis on a e no p esen . The 1W L7 popula ion is ma ked magen a because i con ains a la ge dele ion esul ing in a i us appa en ly
unable o in ec on i s own. (B) Vi us accumula ion, as measu ed by RT-qPCR. (C) The eplica i e ad an age (W) o he es ed i us wi h espec o a
common compe i o , TEV-mChe y, is gi en, as de e mined by compe i ion expe imen s and RT-qPCR (Ma e ials and Me hods). We conside Was an
indica o o he wi hin-hos compe i i e i ness.
Table 3. Nes ed ANOVAs on Plan Heigh , Accumula ion, and Wi hin-Hos Fi ness o E ol ed Lineages.
T ai Sou ce o Va ia ion SS d MS FP
Plan heigh T ea men 66.040 2 33.020 0.791 0.463
Lineage wi hin ea men 1126.600 27 41.726 7.120 <0.001
E o 703.200 120 5.826
Accumula ion T ea men 41.440 2 20.720 0.391 0.680
Lineage wi hin ea men 1431.520 27 53.019 6.998 <0.001
E o 909.200 120 7.577
Fi ness
a
T ea men 1.881 2 0.940 48.534 <0.001
Lineage wi hin ea men 0.523 27 0.019 3.267 <0.001
E o 0.356 60 0.006
Fi ness
b
T ea men 0.100 1 0.100 7.868 0.013
Lineage wi hin ea men 0.203 16 0.013 4.096 <0.001
E o 0.112 36 0.003
a
Fi ness is a compa ison o all lineages.
b
Fi ness compa es only hose lineages ha ha e no ixed genomic dele ions, en o which a e om he 1-week ea men and eigh o which a e om he 3-week ea men (see
ig. 5). ANOVA was used o his compa ison o wo g oups o allow lineage o be nes ed wi hin ea men . T ea men is he passage du a ion (1, 3, o 9 weeks).
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2011) show ha he mu a ional e ec s on wi hin-hos i ness
and i us accumula ion a 7 dpi, exp essed as he Mal husian
g ow h a e pe day (Lalic
´e al. 2011), a e no co ela ed
(Spea man co ela ion: =0.034, 19 d ,P= 0.884; see also
supplemen a y ig. S1,Supplemen a y Ma e ial online).
The e o e, accumula ion was no expec ed o inc ease as a
pleio opic e ec o inc eases in wi hin-hos i ness ei he .
Accumula ion will be an impo an pa ame e when each
i us lineage is e ol ed in mul iple hos o ganisms, as
highe accumula ion can hen lead o a highe equency o
a i us a ian in he inal popula ion (e.g., Zwa , Van de
We , e al. 2010).
Fo a plan i us, i is plausible ha wi hin-hos i ness and
accumula ion a e la gely decoupled. Local in ec ion by cell- o-
cell mo emen can be achie ed by a small numbe o i ions
anspo ed o an adjacen cell (Miyashi a and Kishino 2010).
The e o e, hose i us a ian s ha sp ead apidly need no
necessa ily accumula e a high numbe o i ions pe cell.
Exclusion is mo eo e hough o play an impo an ole
in in ec ion (Die ich and Maiss 2003,Folimono a 2012),
po en ially allowing i uses ha sp ead quickly o each a
high equency and ye ha e ela i ely low accumula ion.
Fu he mo e, i should be no ed ha e en i wo i us a ian s
ha e he same le el o accumula ion la e in in ec ion (e.g.,
7 dpi), i uses wi h a high wi hin-hos compe i i e i ness may
each highe le els o accumula ion ea ly in in ec ion
(e.g., 3 dpi). When a i us apidly exi s he inocula ed lea ,
his can lead o highe le els o in ec ion be o e in ec ion
le els sa u a e (La o gue e al. 2012;Zwa e al. 2012).
Rapid eplica ion and mo emen migh he e o e be he
mechanism by which wi hin-hos i ness is inc eased in
he e ol ed lineages, especially i genome size was di ec ly
linked o eplica ion. Ne e heless, he key ai o measu e
om an e olu iona y pe spec i e—because i is expec ed o
be unde selec ion in his expe imen al se up—is compe i i e
wi hin-hos i ness.
We hen conside ed he ela ionship be ween genome
size and wi hin-hos i ness o he e ol ed lineages ( ig. 7)
and also ound a highly signi ican ela ionship (Spea man
co ela ion: =0.877, 28 d ,P<0.001). The mean i ness
o e ol ed lineages wi h genomic dele ions was highe
han ha o he ances al i us wi hou he he e ologous
gene (TEV) o 10 ou o 12 lineages. Howe e , when we
pe o med pai wise compa isons be ween TEV and he
e ol ed s ains ha ixed dele ions, no signi ican di e ences
we e ound ( - es wi h Holm–Bon e oni co ec ion on he
log- ans o med W alues). S a is ical powe is low when
compa ing indi idual lineages because indi idual-plan le el
a ia ion is high, a limi a ion o ou expe imen al sys em.
We mus he e o e conclude ha i ness o he wild- ype
TEV and e ol ed s ains is simila . This esul is, howe e ,
cong uen wi h he obse a ion ha he con e gen
single-nucleo ide mu a ions obse ed in he e ol ed TEV-
eGFP lineages we e no obse ed in wild- ype TEV in
his s udy and o he s (Bedhomme e al. 2012;T omasN,
Zwa MP, Elena SF, unpublished manusc ip ); i sup-
po s he sugges ion ha hese mu a ions a e speci ic
o accommoda ing changes in he TEV-eGFP backg ound
and will p obably no be bene icial in he wild- ype i us
backg ound.
We hen conside ed he wi hin-hos compe i i e i ness
o hose lineages wi hou genomic dele ions ( ig. 6C).
We ound ha 3-week lineages wi hou genomic dele ions
had a signi ican ly highe i ness han he 1-week lineages
( able 3). The 3-week lineages appea o be a he swee
spo whe e he he e ologous gene is main ained in many
i al lineages, while he e a e concomi an ly signi ican
inc eases in i al i ness. This obse a ion sugges s ha
demog aphy can play an impo an ole in modula ing he
e olu iona y ou come o HGT.
Al hough we ha e shown simul aneous main enance o
he he e ologous gene and inc eases in i ness, he inc eases in
FIG.7. The ela ionship be ween genome size (abscissa) and wi hin-hos compe i i e i ness (o dina e) is gi en. G een da a poin s indica e no dele ions
in he he e ologous gene (eGFP) we e de ec ed, o ange indica es pa o all o he eGFP was no p esen , and ed indica es pa o eGFP and o he i al
HC-P o cis on a e no p esen . The da a poin s o he ances al TEV-eGFP and TEV ha e been illed, and TEV-eGFP has been shi ed o he igh so
ha i can be easily iden i ied, e en hough i s genome size is he same as o he lineages wi hou dele ions. A linea eg ession line has been added only
o emphasize he end in he da a. No e ha mos o he e ol ed i uses wi h dele ions ha e a highe i ness han TEV, implica ing he obse ed
subs i u ions wi h inc eased i ness.
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