Abstract
We completely sequenced 13,936 nucleotides (nt) of a double-stranded RNA (dsRNA) of wild rice (W-dsRNA). A single long open reading frame (13,719 nt) containing the conserved motifs of RNA-dependent RNA polymerase and RNA helicase was located in the coding strand. The identity between entire nucleotide sequence of W-dsRNA and that of the dsRNA of temperate japonica rice (J-dsRNA, 13,952 nt) was 75.5%. A site-specific discontinuity (nick) was identified at nt 1,197 from the 5' end of the coding strand of W-dsRNA. This nick is also located at nt 1,211 from the 5' end in the coding strand of J-dsRNA. The dsRNA copy number was increased more than 10-fold in pollen grains of both rice plants. This remarkable increase may be responsible for the highly efficient transmission of J-dsRNA via pollen that we already reported. J-dsRNA and W-dsRNA were also efficiently transmitted to interspecific F1 hybrids. Seed-mediated dsRNA transmission to F2 plants was also highly efficient when the maternal parent was wild rice. The efficiency of dsRNA transmission to F2 plants was reduced when the maternal parent was temperate japonica rice; however, the reduced rates in F2 plants were returned to high levels in F3 plants.
Highlights
Linear large double-stranded RNAs 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 [11,12,13]
Two Endogenous double-stranded RNA (dsRNA) in Rice ing the open reading frame (ORF) was 166 nt long, and an AUG codon was located at nt 167–169 from the 5Ј-end of the coding strand
We investigated the conservation of the nucleotide and amino acid sequences around the nick in detail by comparing the surrounding regions, which were equivalent in size to the RNA-dependent RNA polymerase (RdRp) or RNA helicase domain, between J-dsRNA and W-dsRNA
Summary
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. 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 5Ј 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). Primer Extension—A 25-mer oligonucleotide (5Ј-CTCTGGTTTGACGCTATTAAACTTG-3Ј) complementary to nt 1,396 –1,420 from the 5Ј end of the coding strand of W-dsRNA was synthesized for use in the primer extension reaction. The specific probes did not cross-hybridize under these conditions
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