Biochemical characterization of Bombyx mori Dicer-2 that dices double-stranded RNAs into 20-nt small RNA.

We detected enzymatic activity that generates 20-nucleotide (nt) RNA from double-stranded RNAs (dsRNAs) in crude extracts prepared from various silkworm (Bombyx mori) organs. The result using knocked-down cultured cells indicated that this dicing activity originated from B. mori Dicer-2 (BmDcr2). Biochemical analyses revealed that BmDcr2 preferentially cleaves 5'-phosphorylated dsRNAs at the 20-nt site-counted from the 5'-phosphorylated end-and required ATP and magnesium ions for the dicing reaction. This is the first report of the biochemical characterization of Dicer-2 in lepidopteran insects. This enzymatic property of BmDcr2 in vitro is consistent with the in vivo small interfering RNA profile in virus-infected silkworm cells.

The dicing activity of DCL3 and DCL4 is negatively affected by flavonoids.

KEY MESSAGE: The dicing activities of DCL3 and DCL4 are inhibited by accumulated metabolites in soybean leaves. Epicatechin and 7,4'-dihydroxyflavone inhibited Arabidopsis DCL3 and DCL4 in vitro. Flavonoids are major secondary metabolites in plants, and soybean (Glycine max L.) is a representative plant that accumulates flavonoids, including isoflavonoids, to high levels. Naturally-occurring RNA interference (RNAi) against the chalcone synthase (CHS) gene represses flavonoid (anthocyanin) biosynthesis in an organ-specific manner, resulting in a colorless (yellow) seed coat in many soybean cultivars. To better understand seed coat-specific naturally-occurring RNAi in soybean, we characterized soybean Dicer-like (DCL) 3 and 4, which play critical roles in RNAi. Using a previously established dicing assay, two dicing activities producing 24- and 21-nt siRNAs, corresponding to DCL3 and DCL4, respectively, were detected in soybean. Dicing activity was detected in colorless seed coats where RNAi against CHS genes was found, but no dicing activity was detected in leaves where CHS expression was prevalent. Biochemical analysis revealed that soybean leaves contained two types of inhibitors effective for Arabidopsis Dicers (AtDCL3 and AtDCL4), one of which was a heat-labile high molecular weight compound of 50 to 100 kD while another was a low molecular weight substance. We found that some flavonoids, such as epicatechin and 7,4'-dihydroxyflavone, inhibited both AtDCL3 and AtDCL4, but AtDCL4 was more sensitive to these flavonoids than AtDCL3. These results suggest that flavonoids inhibit the dicing activity of DCL4 and thereby attenuate RNAi in soybean leaves.

Structural features of T-DNA that induce transcriptional gene silencing during agroinfiltration.

Agrobacterium tumefaciens (Rhizobium radiobacter) is used for the transient expression of foreign genes by the agroinfiltration method, but the introduction of foreign genes often induces transcriptional and/or post-transcriptional gene silencing (TGS and/or PTGS). In this study, we characterized the structural features of T-DNA that induce TGS during agroinfiltration. When A. tumefaciens cells harboring an empty T-DNA plasmid containing the cauliflower mosaic virus (CaMV) 35S promoter were infiltrated into the leaves of Nicotiana benthamiana line 16c with a GFP gene over-expressed under the control of the same promoter, no small interfering RNAs (siRNAs) were derived from the GFP sequence. However, siRNAs derived from the CaMV 35S promoter were detected, indicating that TGS against the GFP gene was induced. When the GFP gene was inserted into the T-DNA plasmid, PTGS against the GFP gene was induced whereas TGS against the CaMV 35S promoter was suppressed. We also showed the importance of terminator sequences in T-DNA for gene silencing. Therefore, depending on the combination of promoter, terminator and coding sequences on T-DNA and the host nuclear genome, either or both TGS and/or PTGS could be induced by agroinfiltration. Furthermore, we showed the possible involvement of three siRNA-producing Dicers (DCL2, DCL3 and DCL4) in the induction of TGS by the co-agroinfiltration method. Especially, DCL2 was probably the most important among them in the initial step of TGS induction. These results are valuable for controlling gene expression by agroinfiltration.

The essential role of the quasi-long terminal repeat sequence for replication and gene expression of an endogenous pararetrovirus, petunia vein clearing virus.

Petunia vein clearing virus (PVCV) is a type member of the genus Petuvirus within the Caulimoviridae family and is defined as one viral unit consisting of a single open reading frame (ORF) encoding a viral polyprotein and one quasi-long terminal repeat (QTR) sequence. Since some full-length PVCV sequences are found in the petunia genome and a vector for horizontal transmission of PVCV has not been identified yet, PVCV is referred to as an endogenous pararetrovirus. Molecular mechanisms of replication, gene expression and horizontal transmission of endogenous pararetroviruses in plants are elusive. In this study, agroinfiltration experiments using various PVCV infectious clones indicated that the replication (episomal DNA synthesis) and gene expression of PVCV were efficient when the QTR sequences are present on both sides of the ORF. Whereas replacement of the QTR with another promoter and/or terminator is possible for gene expression, it is essential for QTR sequences to be on both sides for viral replication. Although horizontal transmission of PVCV by grafting and biolistic inoculation was previously reported, agroinfiltration is a useful and convenient method for studying its replication and gene expression.

Disturbance of floral colour pattern by activation of an endogenous pararetrovirus, petunia vein clearing virus, in aged petunia plants.

White areas of star-type bicolour petals of petunia (Petunia hybrida) are caused by post-transcriptional gene silencing (PTGS) of the key enzyme of anthocyanin biosynthesis. We observed blotched flowers and a vein-clearing symptom in aged petunia plants. To determine the cause of blotched flowers, we focused on an endogenous pararetrovirus, petunia vein clearing virus (PVCV), because this virus may have a suppressor of PTGS (VSR). Transcripts and episomal DNAs derived from proviral PVCVs accumulated in aged plants, indicating that PVCV was activated as the host plant aged. Furthermore, DNA methylation of CG and CHG sites in the promoter region of proviral PVCV decreased in aged plants, suggesting that poor maintenance of DNA methylation activates PVCV. In parallel, de novo DNA methylation of CHH sites in its promoter region was also detected. Therefore, both activation and inactivation of PVCV occurred in aged plants. The accumulation of PVCV transcripts and episomal DNAs in blotched regions and the detection of VSR activity support a mechanism in which suppression of PTGS by PVCV causes blotched flowers.