ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2013 Vol. 56, [t. 1: 85–90 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2013 Vol. 56, [t. 1: 85–90 
Secondary 
structures 
of 
Potato spindle tuber viroid variants 
detected 


in Slovenia 

Sekundarna struktura v Sloveniji najdenih genotipov viroida vretenatosti 

krompirjevih gomoljev 


Mojca Viršcek Marn*, Irena Mavric Pleško, Barbara Geric Stare 


Agricultural Institute of Slovenia, Plant Protection Department, Hacquetova ulica 17, 
1000 Ljubljana, Slovenia 
*correspondence: mojcavm@kis.si 


Abstract: 
A survey of Potato spindle tuber viroid 
was initiated in Slovenia in 
2006. Until the end of 2010, 
100 samples 
of ornamental plants were found to be 
infected. 
Analysis 
of 
96 
complete 
master 
sequences 
of 
Slovene 
samples 
revealed 
new genetic variants. Our sequence 
variants showed considerable variation in their 
predicted secondary structure. The variability was observed in the pathogenic, central 
and variable domains. In several cases even single mutations were sufficient for the 
change in predicted secondary structure. 

Keywords:Potato spindle tuber viroid, PSTVd,pospiviroid,predictedsecondary 
structure, sequence variants 


Izvlecek: 
Leta 2006 smo v Sloveniji uvedli posebni nadzor viroida vretenatosti 
krompirjevih gomoljev (Potato spindle tuber viroid). Do konca leta 2010 smo okužbo 
potrdili 
v 
100 
vzorcih 
okrasnih 
rastlin. 
Analizirali 
smo 
96 
celotnih 
prevladujocih 
nukleotidnih zaporedij in predvideli 
njihove sekundarne strukture. Odkrili smo nove 
genotipe in znatno variabilnost sekundarnih struktur in sicer v patogeni, osrednji in 
variabilnidomeniviroida.Vdolocenihprimerihježemutacijaenegasameganukleotida 
povzrocila spremembo predvidene sekundarne strukture viroida. 

Kljucne 
besede: 
Potato spindle tuber viroid, PSTVd, pospiviroid, sekundarna 
struktura, razlicice nukleotidnega zaporedja 


Introduction 
et al. 2003). The members of genus Pospiviroid 

show 
a 
rod-like 
secondary 
structure 
which 
is 

Viroids are circular, covalently closed RNA 
divided into 5 structural domains: terminal left, 
molecules with a high degree of self-complemen-pathogenic, central, variable and terminal right 
tationresultingincompactfolding.Approximately domain(KeeseandSymons1985).Thissecondary 
30viroidsareknowntoday,manyofwhichcause structure is assumed to be the key for biological 
serious losses of important crops. Potato spindle activity 
(replication, 
processing, 
transport, 
and 
tuber viroid 
(PSTVd) is the type species of the pathogenesis) by being functional as such or by 
genus Pospiviroid 
and can cause losses up to providing binding signals to host factors (Tabler 
64% in potato and up to 45% in tomato (Singh and Tsagris 2004, Verhoeven 2010). 



Acta Biologica Slovenica, 56 (1), 2013 


Viroidspropagate in their plant hostsaspopulations 
of closely related variants. In the family 
Pospiviroidae 
one genotype usually 
dominates 
the viroid population in host plant and is called 
the 
predominant 
genotype 
or 
master 
sequence 
(Verhoeven and Roenhorst 2010). 

Materials 
and 
methods 


In the years 2006-2010 389 samples of ornamental 
plants were tested for PSTVd infection 
as described in details by Viršcek Marn et al. 
(2013). 
Direct 
sequencing 
(Macrogen, 
Korea) 
of RT-PCR amplification products using primer 
pairs of Shamloul 
et al. (1997) and of Di Serio 
(2007) was performed to obtain whole PSTVd 
sequences. Additionally, amplification products 
fromthreeselectedsampleswerepurified,cloned 
into pGEM-T 
easy vector (Promega, WI, USA) 
and 
sequenced 
(Macrogen, 
Korea). 
Obtained 
sequences 
were 
analysed 
using 
the 
computer 
software 
BioEdit 
version 
7.0.5.3 
(Hall 
1999). 
Thermodynamic 
prediction 
of 
RNA 
secondary 
structurewasperformedusingcomputersoftware 
mfold 2.3 in circular mode at 25oC (http://mfold. 
rna.albany.edu/?q=mfold/RNA-Folding-Form2.3; 
Zuker 2003). 

Results 
anddiscussion 


PSTVdinfectionwasconfirmedin88samples 
of Solanum jasminoides, 5 samples of Solanum 
rantonnetii, 3 samples of Petunia spp., 3 samples 
of Solanum muricatum and one sample of 
Brugmansia cordata. Ninety-six 
whole 
viroid 
sequences 
were 
obtained. 
Determined 
PSTVd 
variants are presented in Table 1. Some of these 
variants weredetected in numerous samples and/ 
orvarioushosts.Ahotspotwasfoundonposition 

65. Multiple peaks of similar height for G and U; 
U and A; or G and U and Awere observed and/or 
sequences of the same sample amplified with differentprimershaddifferentnucleotides(
G,UorA) 
at position 65. The occurrence of the hotspot was 
confirmed by cloning tree samples showing ambiguity 
at this position. Apart from polymorphism 
at position 65, other substitutions or insertions 
were observed in 9 out of 33 clones (Table 1). 
Slovene 
sequences 
showed 
differences 
in 
predicted secondary structure models, calculated 
at 25oC (Fig. 1). The variability was observed in 
three out of five structural domains: 
pathogenic, 
central and variable domain. The replacement of 
G with U at position 65 enlarged the loop around 
this site (loop 10). Loop enlargement was also 
observed 
in 
some 
other 
sequences. 
Clone 
1F, 
which has an additional G at position 100, has 
an enlarged loop E (loop 16). The substitution at 
position 83 in clone 3M changed the secondary 
structure 
from 
loop 
11 
to 
loop 
14. 
Drastically 
changed secondary structure was observed from 
loop 17 to 23 due to the substitution at position 
136inclone2C.Otherpolymorphismsintheclone 
sequences do not affect the secondary structure. 
Master 
sequences 
HQ454933 
and 
HQ454936 
havethesamesecondarystructureastheprevalent 
mastersequencedetectedinSlovenia(HQ454914 
–HQ454916).AlloftheSlovenemastersequences 
with 360 nt have structural changes on the right 
sidefromtheloop17.Thesechangesaretheresults 
of several mutations on positions 121, 124, 126, 
127 and 238. In sequence variant HQ454937 the 
substitutionontheposition241contributestothe 
changes due to the before mentioned mutations, 
therefore the loop 18 is larger in this 360 nt long 
sequence.Differencesinthepositionandthesize 
of loop 10 were also observed among different 
360 nt long sequence. 


The study of secondary structures of genotypes 
detectedinSloveniaonornamentalplantsshowed 
several cases where single nucleotide mutation 
significantly 
changed 
the 
predicted 
secondary 
structure of the viroid. Apart from the single-site 
mutations, multiple mutation events in the same 
domain also resulted in the secondary structure 
changes.Inducedmutations,eitherofoneorseveral 
nucleotides, have been used to study the role of 
different loops for PSTVd characteristics. It was 
determined that disruption of nearly every loop 
had an impact on either replication 
or systemic 
trafficking(Qietal.2004,Zhongetal.2008).We 
havenottestedthefunctionalimpactofvariability 
in 
the 
secondary 
structure 
of 
PSTVd 
variants 
determined 
on 
ornamental 
plants 
in 
Slovenia, 
but these changes could have an impact on their 
characteristics. 


Marn et al.: PSTVd secondary structures 


Table 1: 
Characteristics of the Slovene PSTVd sequences. Differences that influence the predicted secondary 
structure are highlighted in bold. Mutations that influence the same domain of the secondary structure 
are shadowed. 


Tabela 1:Lastnosti slovenskih nukleotidnih zaporedij PSTVd. Razlike, ki vplivajo na predvideno sekundarno 
strukturo, so oznacene z odebeljenim tiskom. Mutacije, ki vplivajo na isto domeno sekundarne strukture, 
so sencene. 


Host 
Acc. 
No. 
No. 
of 
Sequence 
No. 
of 
Difference 
in 
comparison 
with 
HQ454914 
sequences 
name nt 
on 
position 
of 
the 
relevant 
sequence 
Master sequences 
Solanum HQ454914 
52 
G-type 
357 
– 
jasminoides 
S. rantonnetii HQ454915 
2 
G-type 
357 
– 
Petunia sp. 
HQ454916 
1 
G-type 
357 
– 
S. jasminoides 
HQ454917 
10 
U-type 
357 
G 
. 
U 
on 
65 
S. rantonnetii HQ454918 
2 
U-type 
357 
G . U on 65 
S. jasminoides 
HQ454919 
6 
K-type 357 
G . K on 65 
S. jasminoides 
HQ454920 
3 
W-type 357 
G . W on 65 
S. jasminoides 
HQ454921 
12 
D-type 357 
G . D on 65 
S. jasminoides 
HQ454933 
1 
B29, B140 
357 
A . U on 221 
Petunia sp. 
HQ454936 
1 
B141 
357 
A . U on 221 
S. jasminoides 
HQ454937 
2 
B14,B82 
360 
U 
.Aob 
121, 
G 
.Aon 
124, 
insertion 
of GAon 
126-127, 
insertion 
of 
C 
on 
238, 
U 
. 
C 
on 
241 
S. jasminoides 
HQ454934 
1 
B90 
360 
U 
.Aob 
121, 
G 
.Aon 
124, 
insertion 
of GAon 
126-127, 
insertion 
of 
C 
on 
238, 
C 
.Aon 
311,A. 
U 
on 
63, 
U 
.Aon 
64 
S. muricatum HQ454935 
2 
B313, 360 
U 
.Aob 
121, 
G 
.Aon 
124, 
insertion 
B314 
of GAon 
126-127, 
insertion 
of 
C 
on 
238 
S. muricatum HQ454932 
1 
B315 
360 
U 
.Aob 
121, 
G 
.Aon 
124, 
insertion 
of GAon 
126-127, 
insertion 
of 
C 
on 
238, 
G 
. 
U 
on 
65 
Clones 
S. jasminoides HQ454914 
7 
G-type 
357 
– 
S. jasminoides 
HQ454917 
10 
U-type 
357 
– 
S. jasminoides 
HQ454922 
7 
clone A 357 
G 
.Aon 
65 
S. jasminoides 
HQ454923 
1 
clone 1J 
357 
A . G on 30 
S. jasminoides 
HQ454924 
1 
clone 1D 
357 
A . G on 90, U . C on 258 
S. jasminoides 
HQ454925 
1 
clone 1F 
358 
G . A on 65, insertion 
of 
G 
on 
100 
S. jasminoides 
HQ454926 
1 
clone 1G 
357 
U . C on 35 
S. jasminoides 
HQ454927 
1 
clone 2C 
357 
G 
. 
U 
on 
65, 
U 
. 
C 
on 
136, U . C 
on 
302 
S. jasminoides 
HQ454928 
1 
clone 2J 
357 
U . C on 331 
S. jasminoides 
HQ454929 
1 
clone 3M 
357 
G 
. 
U 
on 
65, 
U 
. 
C 
on 
83 
S. jasminoides 
HQ454930 
1 
clone 3J 
357 
G 
. 
U 
on 
65, A . G on 122 
S. jasminoides 
HQ454931 
1 
clone 3L 
357 
G 
. 
U 
on 
65, A . G on 100 



88Acta Biologica Slovenica, 56 (1), 2013
clone 1FHQ454925clone 2CHQ454927clone 3MHQ454929U-typeHQ454917-
HQ454918G-typeHQ454914-
HQ454916 
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 E 17 18 19 20 21 22 23 24 25 26 27 28 29[ t e r m i n a l l e f t ][ pathogenicity ][ c e n t r a l ][ variable ][ terminal right ]
Loops ofG-typeB14/B82HQ454937B90HQ454934B313/B314HQ454935B315HQ45493211
clone 1FHQ454925clone 2CHQ454927clone 3MHQ454929U-typeHQ454917-
HQ454918G-typeHQ454914-
HQ454916 
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 E 17 18 19 20 21 22 23 24 25 26 27 28 29[ t e r m i n a l l e f t ][ pathogenicity ][ c e n t r a l ][ variable ][ terminal right ]
Loops ofG-typeB14/B82HQ454937B90HQ454934B313/B314HQ454935B315HQ45493211
Figure 1: 
Thermodynamic predictions of secondary structure for PSTVd sequences detected on ornamental plants. Changed secondary structures in comparison with the 
most prevalent PSTVd sequence (G-type, HQ454914- HQ454916) are shown above and below G-type. The term loop is used for both loops and bulges.

Slika 1: 
Termodinamicne napovedi sekundarnih struktur nukleotidnih zaporedij PSTVd potrjenih na okrasnih rastlinah v Sloveniji. Spremenjene sekundarne strukture 
v primerjavi z v Sloveniji najbolj pogosto najdenim nukleotidnim zaporedjem PSTVd (G-type, HQ454914- HQ454916) so prikazane nad in pod G-type nukleotidnim 
zaporedjem. 



Marn et al.: PSTVd secondary structures 


Conclusions 

Predicted 
secondary 
structure 
of 
PSTVd 
sequences 
detected 
on 
ornamental 
host 
plants 
in Slovenia showed considerable variation. The 
variabilitywasobservedinthepathogenic,central 
and variable domains. 


Povzetek 


Leta 
2006 
smo 
v 
Sloveniji 
uvedli 
posebni 
nadzor viroida vretenatosti krompirjevih go-
moljev (Potato spindle tuber viroid, 
PSTVd). 
Do konca leta 2010 smo analizirali 398 vzorcev 
vecinomaokrasnihrastlininokužbopotrdiliv88 
vzorcih Solanum 
jasminoides, 5 vzorcih Solanum 
rantonnetii, 3 vzorcih Petunia spp., 
3 
vzorcih 
Solanum muricatum in enem vzorcuBrugmansia 
cordata. Dolocili in analizirali smo 96 celotnih 
prevladujocihnukleotidnihzaporedij.Nekateraod 
teh nukleotidnih zaporedij smo našli v številnih 

References 

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Acknowledgements 


The work was financially supported by the 
MinistryofAgriculture,ForestryandFoodofthe 
Republic of Slovenia and the Slovenian Research 
Agency (Grant No. P4-0133). 

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