Transient RNAi by in vitro transcribed dsRNA in protoplasts

In-vitro synthesis of dsRNADNA templates for the production of dsRNA (~250 bp) were produced by PCR usingprimers containing the 28-nucleotide minimal T7 promoter sequence(5’-TAATACGACTCACTATAGGGAGACCAC-3’) at the 5’ terminal followed by a
21-nucleotide gene-specific sequence.
After PCR, the dsDNA were purified and used as templates for in-vitro synthesis ofdsRNA using the Ribomax Express kit (Promega).
The dsRNA were purified by phenol-chloroform-isopropanol extraction, dissolved inRNase-free water and quantified by UV spectrophotometry.

Protoplast transformation
Protoplast preparation methods should be optimized for the plant species used in theexperiment. For example, protoplasts from C. japonica were prepared by cell-walldigestion solution containing 0.4% Onozuka Cellulase R10 (Yakult Pharmaceutical Ind.
Co.), 0.2% Macerozyme R10 (Yakult Pharmaceutical Ind. Co.), 0.01% Pectolyase, 0.6
M sorbitol, 20 mM MES at pH 5.5, and 5 mM MgCl2, which was pre-treated at 50o C
for 30 minutes before filter-sterilization.
The introduction of dsRNA into the protoplasts was performed essentially as describedby Chiu et al. Engineered GFP as a vital reporter in plants. Curr. Biol., 6, 325-330(1996). See also Sheen, J., A transient expression assay using Arabidopsis mesophyll
protoplasts. http://genetics.mgh.harvard.edu/sheenweb/protocols_reg.html.
After being washed three times in W5 solution (154 mM NaCl, 125 mM CaCl2.2H2O, 5

mM KCl and 5 mM glucose), the protoplasts were suspended in MaCaM solution (0.4Mmannitol, 20 mM CaCl2.2H2O and 5 mM MES at pH 5.8) at 2 x 106 protoplasts/ml.
For the model RNAi experiments for luciferase gene, 1 µg of CaMV35S promoter
driven Photinus LUC expression plasmid, 0.1 µg of CaMV35S promoter driven Renilla
LUC expression plasmid, and 1-10 µg of Photinus LUC dsRNA were used for every 105
protoplasts. The amount of dsRNA should be optimized for the silencing of theendogenous genes.
The expression plasmids and dsRNA were gently mixed with the protoplasts in MaCaM
solution.An equal volume of polyethylene glycol (PEG) solution [40% PEG indissolved in 0.4 M mannitol and 0.1 M Ca(NO3)2 ] was then gradually added.
After gentle mixing, the protoplasts were incubated at 28˚C for 30 min., spun down, andre-suspended in 4ml of W5 solution.
After incubation at 28˚C at 40 rpm for 24 or 72 hours, the protoplasts were recovered tomeasure LUC activity.

Quantitation of luciferase activity (for the model experiment)
LUC activity was measured using a Dual-Luciferase assay system (Promega).
Cells were harvested by centrifugation and homogenized in 100 µl Passive Lysis Buffer(Promega). From 0.2 - 0.5 µl of the extracts were mixed with 100 µl LUC Assay
Reagent II and the apparent luminescence was measured.
After the measurement of Photinus LUC acitivity, 50 µl of Stop & Glo reagent wereadded and the resultant Renilla activity was measured. Data were expressed as a ratio of
the luminescence from Photinus divided by the luminescence value from Renilla.
<References>

１．Dubouzet JG, Morishige T, Fujii N, An CI, Fukusaki E, Ifuku K, Sato F. Transient
RNA silencing of scoulerine 9-O-methyltransferase expression by double stranded
RNA in Coptis japonica protoplasts. Biosci Biotechnol Biochem. 2005
Jan;69(1):63-70.
２．Kato N, Dubouzet E, Kokabu Y, Yoshida S, Taniguchi Y, Dubouzet JG, Yazaki K,Sato F. Identification of a WRKY protein as a transcriptional regulator ofbenzylisoquinoline alkaloid biosynthesis in Coptis japonica. Plant Cell Physiol.
2007 Jan;48(1):8-18.