Plant Molecular Biology 51: 991–999, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Double-stranded RNA replicons associated with chloroplasts of a green
alga, Bryopsis cinicola
, Hideki Horiuchi and Toshiyuki Fukuhara
Laboratory of Molecular and Cellular Biology, Faculty of Agriculture, Tokyo University of Agriculture
and Technology, Saiwaicho 3-5-8, Fuchu, Tokyo 183-8504, Japan (
Author for correspondence; e-mail
Present address: Research Institute of Biological Resources, National Institute of Advance
Industrial Science and Technology, Tsukuba, 305-8566, Japan
Received 30 November 2001; accepted in revised form 29 September 2002
Key words: Bryopsis cinicola, chloroplast, double-stranded RNA, green alga, replicon, RNA-dependent RNA
Double-stranded RNAs (dsRNAs) associated with chloroplasts and mitochondria have been found in the coenocytic
green alga Bryopsis cinicola. In this study we report molecular properties of the four chloroplast-associated dsR-
NAs (BDRC1 to BDRC4) The longest dsRNA molecule (BDRC1) was sequenced entirely (1959 bp) and a single
large ORF of 1722 bp was found within it. Database searches revealed similarities between the deduced amino acid
sequence of this ORF and RNA-dependent RNA polymerase (RdRp) sequences from several RNA viruses. The
most similar sequence in the database was the RdRp of beet cryptic virus 3. Phylogenetic analysis revealed that
the RdRp-like sequence of BDRC1 can be placed in the Partitiviridae clade. To detect autonomous replication of
these dsRNAs, RdRp assays were carried out with actinomycin D, which is an inhibitor of DNA-dependent RNA
synthesis. Incorporation of [α-
P]UTP was detected speciﬁcally in the chloroplast and mitochondrial dsRNAs,
indicating that both the chloroplast dsRNAs (BDRCs) and the mitochondrial dsRNA (BDRM) of B. cinicola are
RNA replicons. The green alga B. cinicola harbors different dsRNA replicons in its chloroplasts and mitochondria.
Abbreviations: BDRC, Bryopsis chloroplast-associated dsRNA; BDRM, Bryopsis mitochondria-associated
dsRNA; CTeV; carrot temperate alphacryptovirus; dsRNA, double-stranded RNA; ML, maximum likelihood;
MP, maximum parsimony; NJ, neighbor joining; ORF, open reading frame; RACE, rapid ampliﬁcation of cDNA
ends; RdRp, RNA-dependent RNA polymerase; RYEV; radish yellow edge virus; UTR, untranslated region; VLP,
Double-stranded RNA (dsRNA) replicons that have
properties of both viruses and plasmids can be found
in the plant kingdom (Brown and Finnegan, 1989).
Identiﬁed genes encoded by the dsRNA seem to have
the same functions of counterparts from conventional
RNA viruses. In addition, the plant dsRNA replicons
form virus-like particles or unusual vesicles in host
cells. However, their propagation is stringently reg-
ulated, with maintenance of deﬁned low levels, and
their transmission essentially relies on cell division
and fusion. Because of these properties, the terminol-
ogy describing these endogenous plasmid-like dsRNA
replicons has been confusing. Judging from their mor-
phology and molecular characteristics, they should be
termed viruses. However, their modes of propagation
and transmission allow us to consider them plasmids.
In many cases, the presence of the dsRNAs has little
effect on the phenotype of the host. These dsRNAs
have also been detected in fungi (Nuss and Koltin,
1990; Ghabrial, 1994), protozoa (Wang and Wang,
1991) and insects (Miyazaki et al., 1996).