Plant Molecular Biology 33: 235–243, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
Characterization of RNA-mediated resistance to tomato spotted wilt virus in
transgenic tobacco plants expressing NS
, Marjolein Kikkert
, Cahya Ismayadi
, William de Graauw
Peter de Haan
and Rob Goldbach
Department of Virology, Wageningen Agricultural University, Binnenhaven 11, P.O. Box 8045, 6709 PD
Wageningen, Netherlands (
author for correspondence);
S&G Seeds, Division of Technology, Westeinde 62, P.O.
Box 26, 1600 AA Enkhuizen, Netherlands.
Received 29 September 1995; accepted in revised form 16 September 1996
Key words: movement protein, protoplasts, resistance, tomato spotted wilt virus (TSWV), tospovirus, transgenic
Transgenic Nicotiana tabacum plants expressing RNA sequences of the tomato spotted wilt virus NS
which encodes the putative viral movement protein, were found to be highly resistant to infection with the virus.
Expression of untranslatable as well as anti-sense RNA of the NS
gene resulted in resistance levels as high as
those in plants expressing translatable RNA sequences. For all three types of transgenic plants resistance levels of
up to 100% were reached in the S2 progeny. These results indicate that the resistance mediated by the NS
accomplished by expression of transcripts rather than protein in transgenic plants, similar to previously observed N
gene-mediatedresistance. Protoplast inoculations revealed that resistant plants expressing NS
are, in contrast to N
transgenic resistant plants, not resistant at the cellular level. This suggests the RNA-mediated resistance mechanism
against TSWV targets viral mRNAs rather than the viral genome.
The tospoviruses form a distinct genus of phytopatho-
genic viruses within the arthropod-borne family of
Bunyaviridae [16, 37] which is otherwise restric-
ted to animals. Based on serological differences and
sequence divergence of the nucleoprotein gene, six
distinct tospovirus species have so far been estab-
lished: tomato spotted wilt virus (TSWV) , tomato
chlorotic spot virus (TCSV) , groundnut ring-
spot virus (GRSV) , impatiens necrotic spot virus
(INSV) , groundnut bud necrosis virus (GBNV)
 and watermelon silver mottle virus (WSMV)
. Tospoviruses are the only plant viruses that are
transmitted by thrips species (Thysanoptera)  in a
propagative manner [49, 55].
The type species of the genus Tospovirus,TSWV,
has a very broad host range, encompassing more than
650 plant species belonging to 70 different families
, including many importantcropsandornamentals.
The TSWV particle consists of a nucleocapsid core,
in which the three genomic RNAs are tightly associ-
ated with the nucleoprotein (N), surrounded by a lipid
membrane containing two types of glycoprotein pro-
trusions, G1 and G2 . In addition, several copies of
the putativeviralRNA-dependent RNA polymerase are
present in the virus particle . Nucleotide sequence
determination revealed ﬁve open reading frames in the
TSWV genome, specifying six mature viral proteins
[10, 11, 23]. The L RNA is of complete negative
polarity and encodes the putative viral polymerase of
331.5 kDa. The M and S RNAs both have an ambisense
coding arrangement and are translationally expressed
via subgenomic messenger RNAs . The M RNA
codes for the common precursor to the membrane gly-
coproteins G1 and G2 (of 78 kDa and 58 kDa respect-
ively) and a non-structural protein (NS
The S RNA codes for the N protein of 28.8 kDa and
another non-structural protein (NS