## 2 Production of Recombinant dsRNA in Microorganisms
### Pseudomonas syringae  
For in vivo dsRNA production, a novel method that uses the RNA-dependent RNA polymerase (RdRp) function of bacteriophage phito, a dsRNA virus infecting _Pseudomonas syringae_ cells, was reported by [PERSON] et al. [55] RdRp in phito performs de novo initiation of transcription and produces full-length dsRNAs from given template ssRNAs. _P. syringae_ is a Gram-negative bacillus with subpolar pili, to which phie phages typically attach. Many _P. syringae_ strains are plant pathogens, but [PERSON] et al. constructed a dsRNA production system using nonpathogenic _P. syringae_ strain Cit7 [56] (containing plasmid pLM1086 constitutively expressing T7 RNA polymerase) as the host microbe. The phie genome consists of three segments (L, M, and S), and the genes on each genomic segment are clustered into functional groups, which are flanked by 5'-packaging and 3'-replication signals [57]. The L-segment encodes the components of the polymerase complex (to form a procapsid (PC) with RNA polymerase activity), and the PC particles formed in the host microbe incorporate ssRNAs with 5'-packaging signals and convert them to dsRNAs. pLM991 is designed to produce phage PC components and kanamycin resistance (as a selection marker) under the control of the T7 promoter [58]. [PERSON] et al. [55] employed the _egf_ gene (encoding green fluorescent protein) as a target dsRNA example for genetic knockdown experiments, allowing the _egf_ gene flanked by 5'-packaging and 3'-replication signals to be expressed by T7 RNA polymerase on plasmid pPS9, and introduced these into the host microbial cells. _egf_-ssRNAs were converted to dsRNAs inside empty PCs, and finally, the production of capsids containing _egf_-dsRNAs (1.6 mg dsRNA/g wet cells) was achieved in _P. syringae_.  
The dsRNA production system consisting of _P. syringae_ and phie was subsequently reported to produce dsRNA (named dsRNArep-MP) against TMV genomic DNA [59] (Figure 4). Two dsRNAs corresponding to sequences encoding the replicase (Rep) and movement protein (MP) partial regions of TMV were produced. By inserting the DNA fragments for dsRNA production into the DNA regions corresponding to the M- and S-segments of the phie genome, two plasmids were constructed in which the ssRNAs were expressed under the control of the T7 promoter, and the plasmids were introduced into _P. syringae_ strain LM2691 expressing T7 RNA polymerase along with a plasmid expressing the L segment of the phie-polymerase complex. The resulting strain was cultured to produce dsRNAs, and accumulation of approximately 700 ug dsRNArep-MP per 100 mL of culture (4 \(\times\) 10\({}^{9}\) cells/mL) was achieved. The TMV-derived dsRNAs produced in this way, when applied to TMV-infected _N. benthamiana_ plants, were shown to inhibit the propagation of TMV and to provide efficient protection against local spread of the pathogen. Compared with other in vivo dsRNA production systems, this system allows the efficient production of very long dsRNAs (>2.6 kbp). Such long dsRNAs have the advantage of giving a large pool of siRNAs for a single target (i.e., a viral genome), although the issue of off-target effects should be considered. Long dsRNAs will also be able to link multiple target sequences to protect plants against several pathogens simultaneously. In addition, the formation of target dsRNAs inside the procapsid structure is expected to efficiently avoid degradation by ribonucleases present in the producing host microbial cells, which is advantageous for the preparation of structurally more homogeneous dsRNAs and will contribute to stable dsRNA storage.