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INTRODUCTION |
Linear large (more than 10 kb1) double-stranded RNAs
(dsRNAs) have frequently been identified in healthy plants such
as alfalfa (1), barley (2, 3), broad bean (Vicia faba (4,
5)), cassava (6), common bean (Phaseolus vulgaris (7, 8)), pepper (9, 10) and rice (11-13). Most of these dsRNAs have no obvious
effect on the phenotype of the host plant, with the exception of the
dsRNA in V. faba, that is associated with cytoplasmic male
sterility (4, 14). These large dsRNAs are not associated with distinct
virus-like particles (2, 13). They are present at a (low) constant
concentration in host plants (15) and replicate using their own
RNA-dependent RNA polymerase (5). Although they are usually
found in the cytoplasm of host plant cells, they are effectively
transmitted to progeny plants via pollen as much as by ova (2, 15).
Because all attempts (mechanical inoculation, graft transmission, and
aphid transmission) other than by seeds failed to transmit dsRNA to
dsRNA-free plants, dsRNA is probably transmitted to progeny plants via
seeds alone (2, 9, 15). Thus, these endogenous dsRNAs have some
intriguing plasmid-like properties that differ from those of
conventional plant RNA viruses (16).
Double-stranded RNA (about 14 kb) is a feature of many strains of
temperate and tropical japonica rice (cultivated rice,
Oryza sativa) and of one strain of wild rice
(Oryza rufipogon W-1714, an ancestor of O. sativa). It is not found in any strains of indica rice (cultivated
rice, O. sativa), which rarely hybridizes with japonica rice
in the field (13). These dsRNAs occur in every tissue as well as at
every developmental stage, and they are transmitted very efficiently
(more than 98%) to progeny plants via seeds (15). The dsRNAs are
maintained at an almost constant concentration (100 copies/cell) by
host plants from generation to generation (15). However, dsRNA copy
number increases about 10-fold when host cells are grown in suspension
culture (15, 17). The entire sequence (13,952 nucleotides (nt)) of the
dsRNA from the temperate japonica rice (cv. Nipponbare, J-dsRNA) has
been determined. It consists of a long open reading frame (ORF; 13,716 nt, 4,572 amino acid residues) containing the conserved motifs of
RNA-dependent RNA polymerase (RdRp) and RNA helicase (18).
The coding strand of J-dsRNA contains a site-specific discontinuity
(nick) at position 1,211 nt from the 5'-end (19). This nick divides not
only the coding strand of the dsRNA molecule into 1,211-nt 5' and
12,741-nt 3' fragments but also the long ORF into 1,045-nt (348 amino
acids) 5' and 12,671-nt (4,224 amino acids) 3' segments.
The nucleotides of the dsRNA of tropical japonica rice (cv. Gendjah
Gempel, T-dsRNA) and the dsRNA of wild rice (O. rufipogon W-1714, W-dsRNA) have been partially sequenced (17). A comparison of
the nucleotide and deduced amino acid sequences of the core regions of
the RdRp domains found in these three dsRNAs indicates that J-dsRNA
(temperate japonica rice) is more similar to T-dsRNA (tropical japonica
rice) than to W-dsRNA (wild rice). The cytoplasmic inheritance of these
dsRNAs is unusual in some F1 hybrids when they are introduced into F1
hybrids by crossing japonica rice (O. sativa) and wild rice
(O. rufipogon). The evolutionarily related dsRNAs were
incompatible, and the resident dsRNA of an egg cell from cultivated
rice was excluded by the incoming dsRNA of a pollen cell from wild rice
in some F1 plants. Coexisting dsRNAs in the F1 hybrids segregated from
each other in the F2 plants. However, the total amounts of these dsRNAs
in the host cells remained constant (approximately 100 copies/cell)
even in interspecific hybrid rice (17). Stringent regulation of the
dsRNA copy number might be responsible for this unusual type of inheritance.
Here we determined the entire nucleotide sequence of the second dsRNA
from wild rice (W-dsRNA). We detected a site-specific discontinuity
(nick) in the coding strand, then compared it with the dsRNAs of
temperate (J-dsRNA) and tropical (T-dsRNA) japonica rice. Furthermore,
we discovered remarkable increases in the dsRNA copy number of pollen
grains and an unusual mode of dsRNA inheritance in interspecific F2 and
F3 hybrids between O. sativa and O. rufipogon.
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EXPERIMENTAL PROCEDURES |
Plant Materials--
The Nipponbare cultivar (temperate japonica
rice, O. sativa), the Gendjah Gempel BHB 721 cultivar
(tropical japonica rice, O. sativa), and W-1714 (wild rice,
O. rufipogon) were grown in a greenhouse at 28 °C or in
fields. Pollen grains of the three rice plants were harvested from
about 100 individual plants.
cDNA Cloning of dsRNA--
The dsRNA were extracted from
14-day-old seedlings or mature leaves using SDS-phenol, fractionated by
column chromatography on CF-11 cellulose (Whatman, Maidstone, UK) as
described by Morris and Dodds (20), then incubated with DNase I. A
series of overlapping cDNA clones that covered the entire sequence
of the dsRNA of wild rice (W-1714) was obtained by the method of Gubler
and Hoffman (21) using oligonucleotide primers synthesized with
reference to the J-dsRNA sequence (18). Complementary DNA clones
corresponding to the terminal regions of W-dsRNA and T-dsRNA were
generated by 5' rapid amplification of cDNA ends (22).
Northern Hybridization--
The dsRNAs were resolved by
electrophoresis on 1.2% denaturing agarose gels containing 20 mM MOPS buffer (pH 7.0), 5 mM sodium acetate, 1 mM EDTA, 660 mM formaldehyde, and 500 ng/ml
ethidium bromide. Resolved dsRNAs were transferred to nylon membranes
(Zeta-Probe GT membrane; Bio-Rad) and probed using cDNA clones
located at nt 770-1,723 (W161) and nt 6,554-8,215 (W149) from the 5'
end of the plus strand of the dsRNA (see Fig. 1). These probes were synthesized by using a BcaBESTTM labeling kit
(Takara, Kyoto, Japan) and [
-32P]dCTP (Amersham
Pharmacia Biotech). Hybridization was performed as described previously
(17, 19).
Reverse Transcriptase-Polymerase Chain Reaction (PCR)--
The
strand containing the site-specific nick was determined by reverse
transcriptase-PCR. Oligonucleotides, WN161
(5'-CCTTGGAGGTGTGGTGTATGT-3') located at nt 1,067-1087, WN184
(5'-GTCCTTAAATCTAGGGACAACA-3') complementary to nt
1,702-1,723, and W184 (5'-CTGTGCCAGTGTTATCCCTGA-3') complementary to
nt 1,734-1,754 from the 5' end of the coding strand of W-dsRNA were
used as PCR primers. When the coding strand was the template for
cDNA synthesis, W184 was used as the primer for cDNA synthesis,
then WN161 and WN184 were used as PCR primers. When the noncoding
strand was the template, WN161 was used as the primer for cDNA
synthesis, then WN161 and WN184 were used as PCR primers. Products of
PCR reactions were analyzed by agarose gel electrophoresis.
Primer Extension--
A 25-mer oligonucleotide
(5'-CTCTGGTTTGACGCTATTAAACTTG-3') complementary to nt 1,396-1,420 from
the 5' end of the coding strand of W-dsRNA was synthesized for use in
the primer extension reaction. The reaction proceeded in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 2.5 µM dATP, dGTP, and dTTP, 1 pmol/µl primer, 0.1 µg/µl heat-denatured W-dsRNA as a template, 10 units/µl
SuperScript II reverse-transcriptase (Life Technologies, Inc.), and
0.037 MBq/µl [
-32P]dCTP (Amersham Pharmacia Biotech)
for 2 min at 25 °C. One µl of 10 mM dATP, dCTP, dGTP,
and dTTP was added to 9 µl of the reaction mixture, then the reaction
was continued for 1 h at 37 °C. The reaction products were
resolved by electrophoresis on a 5% denaturing polyacrylamide gel
containing 89 mM Tris, 89 mM borate, 2 mM EDTA, and 50% urea.
DNA Sequencing--
DNA sequences were determined by
dideoxynucleotide chain termination using a 7-deaza Sequenase Ver.
2.0 DNA sequencing kit (U. S. Biochemicals Corp.) or the
BigDyeTM Cycle Primer Sequencing FS Ready reaction kit
(Applied Biosystems, Foster City, CA).
Analysis of Nucleotide Sequences--
Nucleotide and amino acid
sequences were analyzed using the SDC-GENETYX genetic
information-processing program (Software Development Co., Ltd., Tokyo,
Japan). Secondary structures of 5'- and 3'-noncoding regions of three
dsRNAs were predicted using the RNAdraw program (23).
Copy Number of dsRNA--
Seedlings and pollen grains were
thoroughly ground in liquid nitrogen using a mortar and pestle, then
total nucleic acids were extracted using SDS-phenol. Total nucleic
acids were resolved by agarose gel electrophoresis, then the gels were
stained with ethidium bromide (500 ng/ml). Band intensities of dsRNA
and DNA were analyzed using the NIH image program. The dsRNA copy
number was estimated from a comparison of the band intensity and genome size of dsRNA (14 × 103 nt) with those of rice DNA
(4.3 × 108 nt/haploid genome) (15, 24).
Inheritance of dsRNA--
Cultivars Nipponbare (temperate
japonica rice, O. sativa) and W-1714 (wild rice, O. rufipogon) were crossed as described (15, 17). The dsRNA (J-dsRNA
or W-dsRNA) in F1, F2, and F3 plants were identified by Northern
hybridization using J-dsRNA- or W-dsRNA-specific probes under high
stringency as described previously (17). The specific probes did not
cross-hybridize under these conditions.
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RESULTS |
Nucleotide Sequence of W-dsRNA--
The entire sequence (13, 936 nt) of the dsRNA of wild rice (O. rufipogon W-1714; W-dsRNA)
was determined from a series of independent and overlapping cDNA
and rapid amplification of cDNA end clones. A single long ORF of
13,719 nt (4, 573 amino acid) was found in one (coding) strand of
W-dsRNA. The noncoding region preceding the ORF was 166 nt long, and an
AUG codon was located at nt 167-169 from the 5'-end of the coding
strand. The termination codon at nucleotide positions 13,885-13,887
from the 5'-end of coding strand was followed by a noncoding region of
48 nt long (Fig. 1). W-dsRNA did not have
a poly(A) tail, and no ORF of significant size was found in the other
(noncoding) strand. The AU content of W-dsRNA was high (64.95%), and
analysis of the codon usage in the ORF indicated a significant bias
toward codons with A or U as the third letter (data not shown).

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Fig. 1.
Schematic representation of genetic
organization in W-dsRNA (wild rice, O. rufipogon,
W-1714). A long ORF of 13,719 base pair (bp), a 166- base pair 5'-noncoding leader sequence, and a 48- base pair
3'-noncoding sequence were found in the coding strand. Two cDNA
clones (W161 and W149) used as probes to detect nicks (see Fig. 2) are
represented by thick horizontal lines. Nucleotide sequence
data reported here will appear in the DDBJ, EMBL, and GenBankTM
nucleotide sequence data bases under the accession number AB014344.
kbp, kilobase pairs;aa, amino acids.
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Detection of the Nick in the Coding Strand of
W-dsRNA--
Purified W-dsRNA was resolved by electrophoresis on a
denaturing agarose gel, then hybridized using two cDNA probes
located between nt 770 and 1,723 (W161) and between nt 6,554 and 8,215 (W149) from the 5' end of the coding strand of dsRNA (Fig. 1). These
probes detected both (coding and noncoding) strands that were separated
by denaturing agarose gel electrophoresis. A 14-kb band was detected in
both experiments when either W161 or W149 was the probe (Fig.
2A, lanes 1 and
2). In addition, a band of about 1.2 kb was detected when
probed with W161 (Fig. 2A, lane 1) but not by
either Northern hybridization using W149 as a probe (Fig.
2A, lane 2) or by electrophoresis on
nondenaturing agarose gels stained with ethidium bromide (data not
shown). Because J-dsRNA contains a site-specific nick in the coding
strand (19), we considered that two bands (14 and 12.8 kb) should be
detected when probed with W149. However, these two bands could not be
completely separated by electrophoresis because of having similar
molecular masses (Fig. 2A, lane 2). Indeed two bands (14 and
12.8 kb) were detected using W149 when electrophoresis was prolonged
(data not shown). Any probes located within the 5' region from a
position nt 1,107 detected both the 1.2- and 14.0-kb bands but not the 12.8-kb band. Alternatively, any probes located in the 3' region from
nt 1,685 detected both the 14.0- and 12.8-kb bands but not the 1.2-kb
band (data not shown). These experimental results were very similar to
the findings of J-dsRNA, which has a site-specific nick on the coding
strand at nt 1,211 from the 5' end (19). The results indicated that
W-dsRNA has a site-specific nick around nt 1,200 from the 5' end.

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Fig. 2.
Detection of a nick in the coding strand of
W-dsRNA. A, Northern analyses of W-dsRNA. W-dsRNA was
resolved by electrophoresis on a denaturing agarose gel, transferred to
a membrane, and probed with either W161 (lane 1) or W149
(lane 2) (see Fig. 1). A band of about 1.2 kb was detected
only when probed with W161. B, the exact position of the
nick in the coding strand of W-dsRNA. The exact position of the nick
was determined by primer extension. Lanes C, T,
A, and G show sequencing ladders as size markers;
lane P shows products of the primer extension reaction.
Major products are indicated by the arrowhead.
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Reverse transcriptase-PCR experiments were carried out to determine
which strand contained the site-specific nick (see "Experimental Procedures"). When the template for cDNA synthesis was the
noncoding strand, PCR products 687 nt long between nt 1,067 and 1,754 were amplified. In contrast, no band was amplified when the coding strand was used as the template (data not shown). These results indicated that the cDNA could be synthesized through the nick point
on the noncoding strand as a template but not on the coding strand.
Therefore, the coding strand of W-dsRNA contained a site-specific discontinuity (nick).
To define the exact site of the nick on the coding strand of W-dsRNA,
we synthesized a 25-mer oligonucleotide primer that was complementary
to positions between nt 1,396 and 1,420 from the 5' end of the coding
strand and performed primer extension (see "Experimental
Procedures"). The main products of the reaction are shown in
lane P in Fig. 2B. This result indicates that the nick was located between U at nt 1,197 and G at nt 1,198 from the 5'
end of the coding strand. This nick divides not only the coding strand
of W-dsRNA into 1,197-nt 5' and 12,739-nt 3' fragments but also the
long ORF into 1,031-nt (343 amino acids) 5' and 12,688-nt (4, 230 amino
acids) 3' segments.
Comparison between W-dsRNA and J-dsRNA--
The entire sequences
of W-dsRNA (13,936 nt) and the deduced amino acid sequences of the ORF
in W-dsRNA (13,719 nt, 4,573 amino acids) were compared with those of
J-dsRNA (13,952 nt) and of the ORF in J-dsRNA (13,716 nt, 4,572 amino
acids) (Fig. 3). The identities of the
nucleotide sequences of these dsRNAs and the deduced amino acids
sequences of the ORFs were 75.5 and 79.6% (similarity: 96.4%),
respectively. The consensus motifs of RNA helicase and RdRp domains
were found between amino acids 1,505 and 1,748 (nt 4, 679 and 5410) and
between amino acids 4,234 and 4,476 (nt 12, 866 and 13, 594) from the
N-terminal end of the ORF, respectively. These locations were almost
identical to those of the RNA helicase and RdRp domains in J-dsRNA.
Identities of the amino acid sequences of the RdRp domain (94.7%) and
the RNA helicase domain (83.1%) were much higher than the average of
the entire ORF (79.6%). We divided the entire nucleotide sequences of
rice dsRNAs by 120 nt from the 5' end, then compared the nucleotide and
amino acid sequences between J-dsRNA and W-dsRNA every 120 nt (40 amino
acid; Fig. 3). The homology at the amino acid level between J-dsRNA and
W-dsRNA in the RdRp region was more conservative than that of other
regions except for the nick.

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Fig. 3.
Comparisons of entire structures and
nucleotide and amino acid sequences in J-dsRNA (temperate japonica
rice, O. sativa, accession number D32136) and W-dsRNA
(wild rice, O. rufipogon, accession number
AB014344). The entire structures of J-dsRNA and W-dsRNA and exact
positions of nicks (upper part of figure). Nucleotide and amino acid
sequences in J-dsRNA and W-dsRNA every 120 nt (40 amino acids) are
compared (lower part of figure). Identities of nucleotide and amino
acid sequences are graphically represented. Hel, RNA
helicase.
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The coding strands of J-dsRNA and W-dsRNA had site-specific nicks at nt
1,211 and at a nt 1,197 from their 5'-ends, respectively (Fig. 3). As
shown in Fig. 4A, the amino
acid sequences around each nick were highly conserved. We investigated
the conservation of the nucleotide and amino acid sequences around the
nick in detail by comparing the surrounding regions (nt 840-1,560 from the 5' end, amino acids 226-466 from the N-terminal), which were equivalent in size to the RdRp or RNA helicase domain, between J-dsRNA
and W-dsRNA. The nucleotide and amino acid sequences were 82.9 and
95.4% identical, respectively. These values were similar to those of
RdRp domain and higher than those of the RNA helicase domain, and the
highly conservative regions continued across the nick (Fig.
4A).

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Fig. 4.
Comparisons of nucleotide sequences in nick
regions (A), 5'-noncoding regions
(B), and 3'-noncoding regions (C) of
W-dsRNA (W), J-dsRNA (J), and T-dsRNA
(T; tropical japonica rice, O. sativa; accession number AB014345). Deduced amino acid
(aa) sequences (b) in nick regions (A)
are compared.
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On the contrary, two less conservative regions were found at nt
2,760-3,240 and nt 8040-8520 from the 5' end. The nucleotide (deduced
amino acid) sequences were 58.4% (43.1%) and 51.6% (26.9%) identical, respectively (Fig. 3). In these regions, not only
nonsynonymous base substitutions but also deletions and insertions of
nucleotides were found, which caused changes in the amino acid
sequences of the ORF (Table I). Although
many base substitutions and several deletions and insertions of
nucleotides were found, no termination codon within either ORF was
generated.
Secondary Structures of Noncoding Regions in Rice--
The
sequences of 5'-noncoding regions (166 nt) and the location of the
start codon (AUG) were conserved among the three dsRNAs (J-dsRNA,
W-dsRNA, and T-dsRNA; Fig. 4B). Five stem and loop
structures (SL1-SL5) in the 5'-noncoding region and one additional stem
and loop structure containing a start codon (SL6) of three dsRNAs are
shown in Fig. 5. The SL1 and SL2 were not
so conservative among the three dsRNAs, and the SL2 was not located in
J-dsRNA. The SL3, SL4, SL5, and SL6 were highly conserved among the
three dsRNAs. In particular, the SL3 (nt 55-82), which has a
remarkably GC-rich stem, was identical among the three dsRNAs. The SL3
must be the most stable among the six stem and loop structures, because the hydrogen bonds of the GC-rich stem are very strong. The
3'-noncoding region of W-dsRNA (48 nt) was shorter than that of J-dsRNA
(70 nt) (18). A comparison of the 3'-noncoding sequences between J-dsRNA and W-dsRNA revealed a long deletion (18 nt) in the
3'-noncoding region of W-dsRNA (Fig. 4C). No conservative
secondary structure was found in the 3'-noncoding regions.

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Fig. 5.
Secondary structures (stem and loop
structures) in the 5'-noncoding regions of W-dsRNA, T-dsRNA, and
J-dsRNA. SL, stem and loop structures. Start codons
(AUG) are indicated by shading.
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Increase of dsRNA Copy Number in Pollen Grains--
Rice dsRNA
(J-dsRNA) is efficiently transmitted to progeny plants via pollen
although it is localized in cytoplasm (15). We supposed that the dsRNA
copy number in pollen grains is responsible for the paternal
inheritance of J-dsRNA. A comparison of the amounts of dsRNA and rice
DNA showed that the estimated copy numbers of three dsRNAs (J-, T-, and
W-dsRNA) in leaves, roots, or seedlings (Fig.
6, lanes 1-3) were about 100 copies/cell (15). However, the copy numbers of these dsRNAs were
increased in pollen grains (Fig. 6, lanes 4-6). The band
intensity of dsRNA was compared with that of DNA in each sample
(lanes 4-6 in Fig. 6). From the genome sizes of rice DNA
(4.3 × 108 nt) (24) and dsRNA (14 × 103 nt), the estimated copy numbers of these dsRNAs in
pollen grains were more than 1,000 copies/cell. The concentration of
dsRNA was similarly in high samples extracted from the three rice
cultivars.

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Fig. 6.
Increases of dsRNA copy number in pollen
grains. The concentrations of dsRNAs in seedlings (lanes
1-3) and pollen grains (lanes 4-6) were compared
among temperate japonica rice, tropical japonica rice, and wild rice.
Total nucleic acids were extracted from seedlings and pollen grains,
then resolved by agarose gel electrophoresis. Lane M, DNA
size makers ( /EcoT14I, 0.3 µg); lanes 1 and
4, temperate japonica rice (cv. Nipponbare, O. sativa); lanes 2 and 5, tropical japonica
rice (cv. Gendjah Gempel BHB 721, O. sativa); lanes
3 and 6, wild rice (W-1714, O. rufipogon).
kbp, kilobase pairs.
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Inheritance of the dsRNAs in Interspecific Hybrids between
Cultivated (O. sativa) and Wild Rice (O. rufipogon)--
Reciprocal
crosses between temperate japonica rice (cv.
Nipponbare) and wild rice (W-1714) were performed to introduce the two
evolutionarily related dsRNAs (J-dsRNA and W-dsRNA) into F1 progeny
plants. To identify the dsRNA present in F1 plants, we performed
Northern hybridization studies under high stringency conditions
(65 °C) using J-dsRNA- or W-dsRNA-specific probes. Different rice
dsRNAs were efficiently transmitted to F1 hybrids between cultivated
and wild rice, most F1 progeny plants ((23 + 14 + 19)/(23 + 16 + 22) = 91.8%) contained either J-dsRNA, W-dsRNA, or both dsRNAs, and only 5 F1 plants did not contain any dsRNA (Table
II). To investigate the inheritance of
each dsRNA in hybrids from generation to generation, the dsRNAs in F2
and F3 plants, which were self-pollinated progenies of F1 hybrids, were
subjected to Northern analysis under high stringency conditions. When
the recipient (maternal parent) was Nipponbare and the pollen donor (paternal parent) was W-1714, the efficiency of seed-mediated transmission of dsRNAs from F1 plants was low in some F2 plants; the rates of dsRNA transmission from individual F1 plants to F2 plants
varied from 0 to 100% (Table II). For example, all 18 F2 plants from 1 F1 plant did not contain any dsRNA (0%). By contrast, all 14 F2 plants
from another F1 plant contained the dsRNA (100%). The type of dsRNA
(J- or W-dsRNA) in F1 plants was not responsible for the unstable
transmission to F2 plants (Table II). When the dsRNA-free plants of
Nipponbare were used as recipients, the efficiency of transmission was
also low in some F2 plants (Table II). Once the F2 plants harbored the
dsRNAs, the efficiency of dsRNA transmission to F3 plants increased
again (78/84 = 93%). When the recipient was W-1714 and the pollen
donor was Nipponbare, only W-dsRNA was detected in F1 plants and the
efficiency of seed-mediated transmission of dsRNA from these F1 plants
was high in all F2 plants (100%, Table II).
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Table II
Inheritance of J-dsRNA and W-dsRNA by F1, F2 and F3 hybrids between cv.
Nipponbare (O. sativa) and W-1714 (O. rufipogon)
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DISCUSSION |
W-dsRNA is very similar to J-dsRNA with respect to the sizes of
their genomes and their ORFs, locations of RNA helicase and RdRp
domains, and the sites of specific nicks (Fig. 3). Furthermore, the
stem and loop structures in the 5'-noncoding regions were conserved in
both dsRNAs (Fig. 5). Several characteristic stem and loop structures
found in the 5'-noncoding region may function as cis-acting elements
for their replication, transcription, and/or translation. By contrast,
the 3'-noncoding region of W-dsRNA has a long deletion in comparison
with J-dsRNA (18 nt, Fig. 4B). In our recent study of
J-dsRNA and W-dsRNA inheritance, the ability of W-dsRNA to replicate in
F1 plants between temperate japonica rice and wild rice seems to be
greater than that of J-dsRNA (17). The 3'-noncoding regions of J-dsRNA
and W-dsRNA might be responsible for the difference in their ability to
replicate, because noncoding (
) strand synthesis must start at the
3'-terminal region of the coding (+) strand. The two dsRNAs differ at
the nucleotide level by 3,418 nucleotides (24.5%) of the entire
sequence. Because cultivated rice (O. sativa) and wild rice
(O. rufipogon) diverged several thousand years ago (24),
about one nucleotide substitution each year has accumulated during the
evolution of these dsRNAs.
Because the amino acid sequences across the nick (nt 840-1,560 from
the 5' end, amino acids 226-466 from the N-terminal end) were highly
conserved, they might be a functional (important) domain of an unknown
translational product. The identity of amino acids sequences in this
region (95.4%) was similar to that of RdRp domain and higher than that
of the RNA helicase domain (Figs. 3 and 4A), an unknown
translational product containing this amino acid sequence may be
important as well as an RdRp. Once the nick occurs in the coding strand
of the dsRNA, the divided coding strand can no longer be used as a
template for noncoding (
) strand synthesis in its replication cycle
or as an mRNA for the full-length form of its putative functional protein.
Some endogenous RNA replicons, such as the yeast 20 S and 23 S RNAs (=
yeast T and W dsRNAs), encode single ORFs containing RdRps (25).
Although many base substitutions and several deletions and insertions
of nucleotides were found in two rice dsRNAs, no termination codon
within either ORF was generated (Table I and Fig. 3). It suggests that
a single long ORF is also important for the strategy of gene expression
(translation) in rice endogenous dsRNAs.
The dsRNAs isolated from mature leaves, roots, or seedlings (Fig. 6,
lanes 1-3) were detected at a concentration of
approximately 100 copies/cell (15). The dsRNA copy number increased
more than 10-fold in pollen grains of three rice plants (Fig. 6,
lanes 4-6). This remarkable increase may explain the high
efficiency of J-dsRNA transmission via pollen that we reported (15).
There is no evidence for the horizontal transmission of rice dsRNAs, so
their propagation seems to depend on steady replication before every
host cell division (not only mitosis but also meiosis) and their steady
(efficient) transmission to the next generation via not only eggs/ova
but also pollen. Mating of host plants must be an opportunity for dsRNA
propagation. An increase in the dsRNA copy number only in pollen grains
must be a reasonable strategy for their efficient transmission to
progenies despite their cytoplasmic localization (15). This phenomenon
may be reminiscent of meiotic derepression of dsRNA copy numbers seen
in the 20 S and 23 S RNA replicons of yeast (26, 27). These rice dsRNAs
could not have the property of horizontal transmission that almost all
viruses have; they have the ability of vertical transmission via both
eggs and pollen, which almost all viruses do not.
The two endogenous dsRNAs in rice (J-dsRNA and W-dsRNA) described here
have unique properties, such as the single large ORF and the
site-specific nick in the coding strand. The 16.7-kb dsRNA found in
V. faba contains the single long ORF and the site-specific nick (28). Furthermore, the deduced amino acid sequence of the ORF is
similar to those of the ORFs in rice dsRNAs (29). These three large
dsRNAs might constitute a novel virus family. Furthermore, large
endogenous dsRNAs with unknown sequences identified in other plants
(see the Introduction) may also belong to this family.