Bioscience Reports 3, 767-774 (1983)
Printed in Great Britain
767
O l i g o m e r i c f o r m s of p o t a t o spindle t u b e r viroid (PSTV)
a n d of its c o m p l e m e n t a r y RNA a r e p r e s e n t in n u c l e i
isolated from viroid-infected potato cells
Ellen SPIESMACHER, Hans-Peter MUHLBACH, Martina SCHNOLZER,
Bernd HAAS, and Heinz L. S~NGER
Max-Planck-Institut fur Biochemie, Abteilung Viroidforschung,
D-8033 Planegg-Martinsried, Federal Republic of Germany
(Received 16 July 1983)
D i f f e r e n t oligomeric forms of PSTV are detected in
n u c l e i i s o l a t e d from PSTV-infected potato cells by
m e a n s of m o l e c u l a r h y b r i d i z a t i o n , using as probes
s y n t h e t i c o l i g o d e o x y r i b o n u c l e o t i d e s with s e q u e n c e
specificity for (+)PSTV and for (-)PSTV.
In addition
to several species of longer-than-unit-length (-)PSTV
m o l e c u l e s , two o l i g o m e r i c f o r m s os (+)PSTV are
detected, which correspond in size to RNA strands of
approximately two and three times viroid unit-length.
T h e y must be c o n s i d e r e d as the precursors os the
circular and linear (+)PSTV monomers accumulating in
the cell nucleus.
Viroids are molecular pathogens of higher plants. They are small,
single-stranded circular RNAs, which due to intramolecular base-pairing
e x i s t as l a r g e l y d o u b l e - s t r a n d e d r o d - l i k e structures (1).
Their
biological and structural properties have been elucidated in great detail
( 2 - # ) , but the m e c h a n i s m of viroid r e p l i c a t i o n is still largely
enigmatic.
Different lines of evidence suggest (i) that host enzymes
must be involved in viroid biosynthesis (5-8), (if) that it occurs most
probably within the cell nucleus (9,10), and (iii) that it proceeds via
c o m p l e m e n t a r y l o n g e r - t h a n - u n i t - l e n g t h RNA intermediates (11-16).
Attempts to follow the process of viroid replication in nuclei isolated
f r o m i n f e c t e d l e a v e s by the i n c o r p o r a t i o n of r a d i o a c t i v e RNA
precursors, however, did not allow detection of RNA species repres e n t i n g intermediates of viroid replication (17,18).
Therefore, we
searched for the presence of intermediates of viroid (PSTV) replication in highly purified nuclei isolated from potato cell suspension
cultures infected with PSTV.
We applied molecular hybridization on
Northern blots using as radioactive probes 'tailor-made' oligodeoxyribonucleotides with sequence complementarity for distinct regions only
p r e s e n t in (+)PSTV and (-)PSTV m o l e c u l e s , respectively.
The
rnonomeric circular and linear form of PSTV, which accumulates in
infected plants and which has been isolated and sequenced, is arbitrarily called (+)PSTV. With this approach we were able to establish
unequivocally that, in addition to (+)PSTV and (-)PSTV monomers,
different oligomeric forms of (+)PSTV and (-)PSTV are present in the
cell nucleus.
This finding clearly shows that not only longer-thanunit-length (-)PSTV intermediates, but also (+)PSTV oligomers, are
involved in viroid replication.
01983
The Biochemical Society
76g
SPIESMACHER
ET AL.
Materials and Methods
Isolation of nuclei and gel electrophoresis of nucleic acids
N u c l e i were i s o l a t e d from suspension cultures of either PSTVinfected or healthy potato cells (Solanum demissum L.) according to a
procedure originally described for tobacco cell suspensions ( ] 9 ) .
They
were purified by using two steps of Percol! density-gradient c e n t r i f u gation, with the fo!lowing modifications:
in the isolation buffer the
concentration of spermine had to be increased from 0.]5 mM to 0.3g
mM and that of spermidine from 0.5 mM to 2.5 mM. The content of
the detergent Nonidet P #0 was lowered from 0.6% to 0.2#% in order
to avoid damage of the nuclear membrane.
Nucleic acids were
extracted from the isolated nuclei with the aid of phenol/SDS (20).
A f t e r ethanol precipitation they were denatured by glyoxalation ( 2 ] )
and e / e c t r o p h o r e t i c a l l y separated on 3.5% p o l y a c r y l a m i d e gels
(0.0975% bisacrylamide) in l0 mM sodium phosphate buffer pH 6.5.
Northern blotting and hybridization analysis
T h e n u c l e i c a c i d s were e l e c t r o b l o t t e d (22) from the gel onto
t r a n s f e r m e m b r a n e s ( G e n e S c r e e n , NEN) and baked in a vacuum oven
a t g0~
for # h. The hybridization b u f f e r c o n t a i n e d 5 x SSC; 0.02%
e a c h of p o l y v i n y l p y r r o l i d o n e
( m o l . w t . 40 0 0 0 ) , F i c o l l ( m o l . w t .
#00 000), and bovine serum albumin (BRL); i % SDS; and 65 pg/ml
d e n a t u r e d salmon-sperm DNA. The azP-labeled probe was dissolved in
the hybridization b u f f e r to give a final a c t i v i t y of 1 x I06 c . p . m . / m l ,
and the hybridization was c a r r i e d out with 30 lal of b u f f e r per cm2
blotting area at # I ~
overnight.
The blots were washed in 2 x SSC
at room temperature, then in 2 x SSC containing 0.5% SDS at # I ~
and finally in 0.l x SSC at room temperature, air dried, and exposed
to Fuji X-ray film RX using DuPont intensifying screens.
Nuclease treatment of nucleic acid extracts
For the digestion with RNase, aliquots corresponding to the nucleic
acids extracted from 6 x 106 nuclei were incubated with # lag of
RNase A (Boehringer) for 30 min at 37~
For DNase digestion,
aliquots were incubated with g pg of DNase I (Miles Laboratories) for
30 rain at 37~ in the presence of 50 units RNasin (BRL) and 5 mM
dithiothreitol.
The samples were c h i l l e d on ice, t R N A from
Bscherdchia c o l i was added as carrier, and the remaining nucleic
acids were isolated by phenol-extraction and ethanol precipitation.
Synthesis of the hybridization probes
Synthesis of the tailor-made oligonucleotide probes pl#I, pl#II, and
p l 5 was carried out according to the phosphite-triester method on a
s i l i c a - g e l support (23,2#).
After their removal from the polymer
s u p p o r t they were purified by two h.p.l.c, runs, which have been
modified for our semipreparative purposes from a previously published
procedure (25), and finally sequenced by the m o b i l i t y - s h i f t analysis
(26).
They were p h o s p h o r y l a t e d at t h e i r Y-OH terminus with
[Y-32-p]ATP and T# polynucleotide kinase.
The labelled oligonucleotides were isolated free of ATP and other radioactive components by
c h r o m a t o g r a p h y on Sephadex G-50 using 100 mM triethylammonium
bicarbonate buffer (pH 7.6) as eluent.
The fractions containing the
PSTV OLIGOMER5 IN NUCLEI OF
POTATO
CELLS
769
Table i. Sequence and PSTV-specificity of the synthetic
oligodeoxyribonucleotides used as hybridization probes
It should be noted that due to the opposite polarity of the
(-)PSTV strand the numbering of its nucleotides is in reverse
order as compared to the (+)PSTV.
Probe
pl4I
p15
PI4II
Sequence
(5' + 3')
d(CGCTCCAGGTTTCC)
d(GTTTCCACCGGGTAG)
d(GGAAACCTGGAGCG)
Specific for
Binding region
of the PSTV
molecule
(nucleotide numbers)
(5' § 3')
(+)PSTV
(+)PSTV
(-)PSTV
97-110
259-273
250-263
l a b e l l e d otigonucleotide were pooled and lyophilized to remove the
volatile buffer.
The s p e c i f i c a c t i v i t y usually achieved by this
procedure was 2 x l07 c.p.m./IJg.
R e s u l t s and Discussion
Nuclei were isolated from a suspension culture of PSTV-infected
potato cells (•. dem~seum), which are characterized by an extensive
viroid replication (16).
For the detection of the intermediates of
PSTV replication three different 32p-labeled oligodeoxyribonucleotides
were used as hybridization probes (Table 1), because due to their high
degree of intramolecular base-pairing the entire 125I-labeled (+)PSTV
and cloned 32p-labeled PSTV cDNA molecules were found to be rather
u n r e l i a b l e for the unequivocal discrimination of (+) and (-)PSTV
strands. The oligodeoxyribonucleotide probes p14I and pl5 hybridize at
d i f f e r e n t regions to (+)PSTV, and pl~II hybridizes to (-)PSTV, as
indicated in Fig. 1.
Fig. 2 shows the hybridization pattern of these three probes to
nucleic acid extracts from nuclei and from intact cells, which were
d e n a t u r e d by g l y o x a l a t i o n (21), separated by gel electrophoresis,
e J e c t r o b l o t t e d (22) onto transfer membranes, and autoradiographed
after the hybridization step. An essential prerequisite for obtaining a
m a x i m u m h y b r i d i z a t i o n signal with the DNA probes is the exact
control of the temperature during hybridization. It was found to be
41~
in our e x p e r i m e n t s ; below and above this temperature the
hybridization is drastically reduced. At lower temperature this is most
probably due to the decreased accessibility of the DNA probe to the
partially renatured and consequently double-stranded viroid molecules
bound to the t r a n s f e r membrane.
Above 41~
the temperaturedependent dissociation of the hybridization complex becomes increasingly e f f e c t i v e .
The pattern obtained with the (+)PSTV-specific
probes plLtI and pl5 shows that in the isolated nuclei (Fig. 2a and 2b,
lane 3) three different classes of (+)PSTV exist: (i) the circular (C)
and the linear (L) (+)PSTV monomers consisting of 359 nucleotides,
which are visible as prominent bands; (ii) several bands which migrate
f a s t e r than the monomers; and (iii) two distinct slower-migrating
bands, indicated by arrows.
From the comparison with co-electrophoresed 32p-labeled HinfI restriction fragments of pBR322 DNA (Fig.
770
SPIESMACHER ET AL.
a
PSTV
(+)R NA
V
97
110
9
359
273
b
259
PSTV(-)RNA
263
359,~
I, F
;
I
250
I
t L
,
Fig. 1.
Hybridization of synthetic oligodeoxyribonucleotides to the (+)PSTV molecule (a) and to
the complementary (-)PSTV molecule (b).
2a and 2b, lane 1), the nucleotide numbers of these slower-migrating
(+)PSTV molecules can be estimated to be approx, g60 and 1150,
respectively. This would correspond to RNA chains of about two- and
t h r e e f o l d l e n g t h of the viroid unit-length.
The faster-migrating
v i r o i d - s p e c i f i c p r o d u c t s represent distinct bands in the size-range
between approx. 350 down to 60 nucleotides.
These bands could be
(+)PSTV chains, which have not been completed, or simply represent
a r t e f a c t u a l but specific cleavage products produced during the isolation
of nuclei. It should be noted that no (+)PSTV-related sequences are
detectable in nuclei isolated from healthy potato cells (Fig. 2a and 2b,
lane 2), whereas in nucleic acid extracts from PSTV-infected potato
c e l l s (Fig. 2a and 2b, lane 4) the circular and linear (+)PSTV
monomers are easily visible.
However, the bands representing the
(+)PSTV oligomers are difficult to identify in these lanes and (+)PSTV
molecules smaller than the PSTV monomers are not visible at all in
such preparations.
This is not too surprising, because for mechanistic
reasons the intermediates of (+)polarity should be short-lived, transient
p r o d u c t s p r e s e n t only in t r a c e amounts, whereas the unit-length
molecules accumulate as the end product of viroid replication.
The
faint hybridization signal in the total RNA from PSTV-infected cells
visible on top of the gel (Fig. 2a and b, lane 4) is due to unspecific
hybridization of our probes to I6S rRNA, which is not present in
nuclei (Fig. 2a and b, lanes 2 and 3).
When the (-)PSTV-specific probe pl#II is hybridized to the same
n u c l e i c acid preparations, only a very faint hybridization signal is
visible at the position of c i r c u l a r (C) and linear (L) (+)PSTV
monomers (Fig. 2c).
However, several hybridizing RNA species can
be detected in the range from monomeric up to t e t r a m e r i c forms in
the case of isolated nuclei (Fig. 2c, lane 2) or up to the sixfold
u n i t - v i r o i d - l e n g t h in the case of intact cells (Fig. 2c, lane #).
Numerous bands of (-)PSTV molecules smaller than unit-viroid-length
are visible in the range from about 350 down to 70 nucleotides (Fig.
PSTV OLIGOMERS IN NUCLEI OF
Hybridized
withDNA p r o b e
Specific
for
p15
PSTV('~')RNA
POTATO
~,~
PSTV(~-) RNA
~"
~
PSIV (--) RNA
~',~ ~,~,~
4~,~,~
~,>,~,~,~,
1,2,
1,2,3
1, 2 , 3 , 4 , 5
3,4,5
771
p14II
p14I
~
CELLS
,4,5
e. ~
,
Fig. 2. Northern blots of nucleic acids extracted
from isolated potato cell nuclei, hybridized to
different 32p-labeled synthetic oligodeoxyribonucleotide probes.
(a) (+)PSTV hybridization
with p15;
(b) (+)PSTV hybridization with pl4I;
(c) (-)PSTV hybridization with pl4II.
The size
marker in a~ b, and c is pBR322 DNA digested with
HinfI. This generates the following size fragments:
1631, 517/506, 396, 344, 298, 222/221, 154, and 75
nucleotides.
2% lane 2). The pattern of these small (-)PSTV strands differs from
that of the small (+)PSTV strands. Treatment with DNase I showed
t h a t the (+)PSTV p r o d u c t s are DNase-resistant (Fig. 3, lane 4,
whereas they are completely digested by RNase A (Fig. 3, lane 3).
The same result was obtained for (-)PSTV (not shown).
The reliability
of our s y n t h e t i c h y b r i d i z a t i o n probes for
u n a m b i g u o u s l y d i s c r i m i n a t i n g b e t w e e n (+)PSTV and (-)PSTV is
demonstrated by the fact that the (-)PSTV-specific probe pl~II does
not hybridize to the highly purified (+)PSTV used as marker (Fig. 2%
SPIESMACHER ET AL.
772
lane 5). However, in the same blot it recognizes several RNA bands,
especially the oligomeric forms, which cannot be visualized with the
(+)PSTV-specific probes plt~I and plS.
The (+)PSTV-specific probes,
on the other hand, produce strong signals by hybridizing to the highly
purified circular and linear (+)PSTV marker (Fig. 2a and 2b, lane 5).
In the region where the oligomeric forms migrate, both (+)PSTVspecific probes recognize only two distinct bands of approx, two- and
threefold viroid unit-length, but not the several other RNA species
that are detected by the (-)PSTV-specific probe pl#II. The specificity
1121314'5
C
~D-
L'-
Fig. 3. Northern blots of DNase- and RNase-treated
nucleic acids from isolated potato cell nuclei,
hybridized to the (+)PSTV-specific oligodeoxyribonucleotide probe p141.
PSTV
OLIGOMERS
IN
NUCLEI
OF
POTATO
CELLS
773
our probes has also been demonstrated independently in reverset r a n s c r i p t i o n experiments, in which viroid cDNA for cloning and
sequencing was produced.
With these DNA molecules as primers true
PSTV cDNA t r a n s c r i p t s are obtained from partially purified RNA
preparations, which contain trace amounts of PSTV only (SchnSlzer et
al., in preparation).
In this report we present evidence that in addition to the longerthan-unit-length (-)PSTV oligomers, two forms of oligomeric (+)PSTV
exist, the size of which is about twofold and threefold viroid-unitlength.
Oligomeric forms of viroid(+)RNA, which were considered as
p u t a t i v e i n t e r m e d i a t e s in viroid replication, have previously been
reported for avocado sunblotch viroid (ASBV) (27,2g) and coconut
cadang cadang viroid (CCCV) (29).
However, in contrast to PSTV
nothing is known about the mechanism and intracellular site of the
r e p l i c a t i o n of these viroids.
From the different oligomeric forms
observed, the following steps in viroid replication can be visualized:
in the first step of the cycle the infectious circular (+)PSTV molecule
is transcribed within the nucleus into longer-than-unit-length (-)PSTV
oligomers, for which a roiling-circle-like mechanism has been proposed
(13,14).
This t r a n s c r i p t i o n could be catalyzed by the nuclear
DNA-dependent RNA-polymerase I% because (i) this enzyme is capable
of transcribing in vitro viroid RNA into products including copies of
full-length (6) and even longer transcripts (unpublished results) and
because ( i i ) v i r o i d r e p l i c a t i o n is specifically inhibited by alphaarnanitin (5) as is polymerase H activity.
The (-)PSTV oligomers,
reaching a chain length up to seven times unit-viroid length (16), may
serve as template for the transcription into (+)PSTV oligomers as the
second step.
These oligomeric forms must be considered as the
precursors of the (+)PSTV monomers. The 'mature' circular (+)PSTV
molecules consisting of 359 nucleotides are then generated in a third
reaction step by specific endonucleolytic cleavage of the (+)PSTV
oligomers into unit-length linear strands, which must become modified
at their termini before they are finally ligated to covalently closed
circles. It is conceivable that the cleavage and ligating steps could be
carried out by enzymes normally involved in splicing of the host-cell
RNA.
In fact, a novel type of RNA ligase, which is capable of
Iigating in vitro linear viroid RNA to circular molecules, has been
f o u n d in tissue of higher plants (wheat germ) (7,g) and in
ChZamydomonas (g).
Therefore one may anticipate that viroid host
plants contain a similar type of RNA-ligating enzyme, which is
involved in viroid circularization in vivo.
Additional in vivo and in
vitro experiments are needed, however, to further substantiate the
proposed precursor-product relationship between the different PSTV
species, from which a more complete picture of the replication
mechanism of these unique plant pathogens will hopefully emerge.
of
Acknowledgement
We t h a n k Miss R. L u c k i n g e r for her e x c e l l e n t t e c h n i c a l a s s i s t a n c e .
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