© Benaki Phytopathological Institute
Abstracts - 16th Hellenic Phytopathological Congress
45
In vitro
template-dependent synthesis of
Pepino mosaic virus
positive-
and negative-strand RNA by its RNA-dependent RNA polymerase
T.A.M. O
SMAN
1,2
, R.C.L. O
LSTHOORN
3
AND
I.C. L
IVIERATOS
1
1
Plant Virology Laboratory, Department of Sustainable Agriculture, Mediterranean
Agronomic Institute of Chania, Alsylio Agrokepion, GR-731 00, Chania, Crete, Greece.
2
Department of Agricultural Botany, Faculty of Agriculture, Fayoum University,
Fayoum 63514, Egypt.
3
Department of Molecular Genetics, Leiden Institute of
Chemistry, PO Box 9502, 2300 RA Leiden, The Netherlands
Pepino mosaic virus
(PepMV)-infected toma-
to plants were used to develop an
in vitro
template-dependent system for the study
of viral RNA synthesis. Differential sedimen-
tation and sucrose-gradient purification of
PepMV-infected tomato extracts resulted in
fractions containing a transcriptionally ac-
tivemembrane-boundRNA-dependent RNA
polymerase (RdRp). In the presence of Mg
2+
ions,
32
P-labelled UTP and unlabelled nucle-
otides, the PepMV RdRp catalysed the con-
version of endogenous RNA templates into
single- and double-stranded (ds) genomic
RNAs and three 3’-co-terminal subgenomic
dsRNAs. Hybridisation experiments showed
that the genomic ssRNA was labelled only in
the plus strand, the genomic dsRNA mainly
in the plus strand and the three subgenom-
ic dsRNAs equally in both strands. Following
removal of the endogenous templates from
themembrane-bound complex, the purified
template-dependent RdRp could specifical-
ly catalyse transcription of PepMV virion
RNA,
in vitro
-synthesized full-length plus-
strand RNA and the 3’-termini of both the
plus- and minus-strand RNAs. Rabbit poly-
clonal antibodies against an immunogenic
epitope of the PepMV RdRp (anti-RdRp) de-
tected a protein of approximately 164 kDa
in the membrane-bound and template-de-
pendent RdRp preparations and specifically
inhibited PepMV RNA synthesis when add-
ed to the template-dependent
in vitro
tran-
scription system. The 300 nucleotide long
3’-terminal region of the PepMV genome
containing a stretch of at least 20 terminal
adenosine (A) residues, was an adequate ex-
ogenous RNA template for RdRp initiation
of the minus-strand synthesis but higher
transcription efficiency was observed as the
number of A residues increased. This obser-
vation might indicate a role for the poly(A)-
tail in the formation and stabilisation of
secondary structure(s) essential for the initi-
ation of transcription. The template-depen-
dent specific RdRp system described in this
article will facilitate identification of RNA el-
ements and host components required for
PepMV RNA synthesis.
Incidence of graft-transmissible pathogens in newly established peach
orchards
K. E
FTHIMIOU
, P.G. P
APPI
, L. L
OTOS
, A.T. K
ATSIANI
, S. G
AVALEKA
and N.I. K
ATIS
Aristotle University of Thessaloniki, Faculty of Agriculture, Forestry and Natural
Environment, School of Agriculture, Laboratory of Plant Pathology, GR-541 24
Thessaloniki, Greece
Production of certified plant propagation
material is regulated by the Hellenic Minis-
try of Rural Development and Food which
defines the requirements concerning its im-
port and export. Concerning the stone fruits,
certification includes, among others, the ab-
sence of
Plum pox virus
(PPV),
Apple chlorot-
ic leaf spot virus
(ACLSV),
Prunus necrotic ring-
spot virus
(PNRSV),
Prune dwarf virus
(PDV),
Peach latent mosaic viroid
(PLMVd) and the
European stone fruit yellows phytoplasma
(ESFY). In order to determine the presence
