Plant Molecular Biology 43: 439–450, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
Systemic induction of a Phytolacca insularis antiviral protein gene by
mechanical wounding, jasmonic acid, and abscisic acid
, Yeonhee Choi
, Young Ho Moon
, Sang-Gu Kim
Department of Molecular Biology and Research Center for Cell Differentiation, Seoul National University, Seoul
151-742, Korea (
author for correspondence) e-mail: firstname.lastname@example.org;
Department of Biology, Seoul
National University, Seoul 151-742, Korea;
Department of Agricultural Chemistry, Seoul National University,
Suwon 441-744, Korea
Received 17 August 1999; accepted in revised form 28 April 2000
Key words: jasmonic acid, Phytolacca insularis antiviral protein (PIP), ribosome-inactivating protein (RIP),
We have isolated a gene encoding a ribosome-inactivating protein (RIP) from Phytolacca insularis, designated as
P. insularis antiviral protein 2 (PIP2). The PIP2 gene contained an open reading frame encoding a polypeptide of
315 amino acids. The deduced amino acid sequence of PIP2 was similar to those of other RIPs from Phytolacca
plants. Recombinant PIP2 was expressed in Escherichia coli and was used to investigate its biological activities.
Recombinant PIP2 inhibited protein synthesis in rabbit reticulocyte lysate by inactivating ribosomes through N-
glycosidase activity. It also exhibited antiviral activity against tobacco mosaic virus (TMV). Expression of the PIP2
gene was developmentally regulated in leaves and roots of P. insularis. Furthermore, expression of the PIP2 gene
was induced in leaves by mechanical wounding. The wound induction of the PIP2 gene was systemic. Expression
of the PIP2 gene also increased in leaves in a systemic manner after treatment with jasmonic acid (JA) and abscisic
acid (ABA), but not with salicylic acid (SA). These results imply that plants have employed the systemic synthesis
of the defensive proteins to protect themselves more efﬁciently from infecting viruses.
Abbreviations: ABA, (±)-abscisic acid; IC
, concentration of RIP that gives 50% inhibition of in vitro translation
in rabbit reticulocyte lysate; IPTG, isopropyl-1-thio-β-D-galactoside; JA, (−)-jasmonic acid; OD
, optical den-
sity at 600 nm; PIP, Phytolacca insularis antiviral protein; RIP, ribosome-inactivating protein; SA, salicylic acid;
TMV, tobacco mosaic virus.
Plants have developed complex defense mechanisms
against pathogen attacks during evolution (Bowles,
1990). The defense mechanisms employed by a num-
ber of higher-plant species involve defensive chem-
icals, which include ribosome-inactivating proteins
(RIPs). RIPs are N-glycosidases that remove a speciﬁc
adenine base from ribosomal RNA of the large sub-
The nucleotide sequence data reported will appear in the EMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession number AF141331.
unit, thereby inhibiting protein synthesis irreversibly
(Hartley et al., 1996). RIPs could be divided into two
types according to their conformations. Most RIPs be-
long to type 1, existing as a single polypeptide chain,
whereas those of type 2 are composed of two chains
with an enzymatic domain and a galactose-binding
lectin domain, respectively, linked by hydrophobic
bonds and a disulﬁde bridge (Stirpe et al., 1992;
Barbieri et al., 1993; Hartley et al., 1996).
A variety of studies have indicated that when RIPs
are inoculated with pathogens or expressed in trans-
genic plants, they confer resistance on plants against