## 2 Production of Recombinant dsRNA in Microorganisms  
### Escherichia coli  
The combination of _E. coli_ strain HT115(DE3) (which has a transposon inserted into the _rnc_ gene that encodes the dsRNA-degrading enzyme RNase III) and expression plasmid L4440, which was first used for a successful RNAi experiment in the nematode _Caenorhabditis elegans_ in 1998 [20,21], has since become one of the standard methods for dsRNA preparation in the laboratory. Deletion of the gene encoding RNase III was a prerequisite for dsRNA of interest to accumulate in the bacterium. The HT115(DE3) strain carries the \(\lambda\)DE3 lysogen to allow the T7 RNA polymerase gene to be expressed under the control of the _lac_UV5 promoter. Therefore, the addition of IPTG (isopropyl \(\beta\)-D-1-thiogalactopyranoside), an inducer of gene expression from the _lac_UV5 promoter, results in the production of T7 RNA polymerase, which in turn leads to expression of the target RNA gene from the T7 promoter in the RNA expression unit carried on plasmid L4440, thus resulting in the accumulation of target RNA in the cells. In general, there are two modes of expression in dsRNA production in microorganisms (Figure 2). One is to insert the target dsRNA coding region between two oppositely directed promoters (usually T7 promoters are used) and produce the target dsRNA molecule by convergent transcription, as in the case of RNA expression in L4440. The other expression mode is to design the nucleotide sequence to form a hairpin-loop structure within a single-stranded RNA transcript after transcription from a single strong promoter to produce an hpRNA containing the dsRNA segment of interest. One of the pioneering works using in vivo-produced dsRNA against plant viral diseases was performed by [PERSON] et al. in 2003 [22]. To suppress the infectivity of _Pepper mild mottle virus_ (PMMoV) that infects the plant _Nicotiana benthamiana_, they constructed a system in which dsRNA (hp-type RNA, 977-bp long) corresponding to the partial sequence of the PMMoV replicase gene was highly expressed under the control of the T7 promoter using _E. coli_ HT115(DE3) as the host. The productivity of the hpRNA was 4 ug per milliliter of culture of the producer strain. The producing _E. coli_ cells were disrupted using a French press, and the centrifugal supernatant (as crude extract) was tested for its ability to interfere with viral infection. It was shown that the infectivity of the virus was significantly decreased when plants were sprayed with the lysate several days before viral inoculation [22]. There are now numerous reports on preparation of various dsRNAs or hpRNAs using the combination of strain HT115(DE3) and the T7 promoter-directed expression system [23; 24; 25; 26; 27; 28]. For example, the productivity of recombinant hpRNA (including a 400-bp dsRNA region) targeted to silence the expression of a protease gene in yellow head virus (YHV), which infects the edible shrimp _Penaetus monodon_, was 1.5 mg per 5 mL of culture medium [29]. The productivity of dsRNA for silencing the wing development vestigial (_cg_) gene in _Aedes aegypti_ mosquitoes was reported to be 500 ug per 25 mL of culture medium [30]. RNAi-mediated silencing targeting the _HvRPS18_ and _HvRPL13_ genes encoding ribosomal proteins [31] and V-ATPase subunit A and E genes [32] has been investigated for application to the 28-spotted ladybeetle _Henosipilachna viginitioctopunctata_; in these studies,  
Figure 2: Two typical transcriptional modes of dsRNA molecular species produced by microorganisms. In convergent transcription mode (**left panel**), RNA polymerase from a pair of promoters transcribes sense and antisense strands coding for the target RNA, and then the two transcripts anneal with each other to form dsRNA species after the single-stranded portions are trimmed in vivo. A transcription unit that uses repeats of the target sequence transcribes single-stranded RNA, which self-associates to form hairpin RNA (hpRNA) with an intramolecular dsRNA structure.