High salt solution for precipitation plant

Strain D40 was isolated from diseased seedlings of sugar beet with the symptom of damping-off and identified to be R. zeae using previously reported method [19 (link),20 (link),24 (link)]. Samples were collected from Qiqihaer city, Heilongjiang province of China, in 2010. Mycelia of D40 strain was cultured on potato dextrose agar (PDA) plates with cellophane film membranes (PDA-CF) at 25 °C in the dark for five days before extracting total RNA and dsRNA. Total RNA was extracted using RNAiso Plus (TaKaRa, Dalian, China) according to the manufacturer’s instructions. DsRNA of strain D40 was extracted using the CF-11 cellulose (Sigma-Aldrich, St. Louis, MI, USA) chromatography method, as previously described [25 (link)]. In order to remove polysaccharides and pigment from mycelia, addition of Fruit-mate™ for RNA Purification (TaKaRa) and High-Salt Solution for Precipitation (Plant) (TaKaRa) was conducted during extraction of both total RNA and dsRNA according to the manufacturer’s instructions.

Total RNA was extracted from expanded leaves using Seprasol RNA 1 Super G (Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s instructions. Extracted RNAs were purified once using a commercial agent [High-Salt Solution for Precipitation (Plant), TaKaRa Bio Inc.] according to the manufacturer’s instructions. RT was performed in a 5 μL volume containing 25 units of ReverTra Ace (Toyobo, Osaka, Japan), 0.5 μL RT buffer, 1 mM dNTPs (10 mM), 10 units of RNase inhibitor (Toyobo), and 1 μM reverse primer CSVd-R [5′-AGGATTACTCCTGTCTCGCA-3′; Hosokawa et al., 2004b (link)]. Total RNA was added to the reaction mixture to a final concentration of 10–20 ng⋅μL^-1, and then, this mixture was incubated at 42°C for 30 min and 99°C for 5 min. One μL of RT product was added to 9 μL of the PCR mixture to obtain a final volume of 10 μL. RT-PCR was performed in a 10 μL mixture containing 0.25 units of Blend Taq polymerase (Toyobo), reaction buffer for the Blend Taq polymerase, 0.2 mM dNTPs, and 0.2 μM forward and reverse primers. One cycle of 3 min at 98°C, 35 cycles of 30 s at 98°C, 10 s at 58°C, and 30 s at 74°C, and one cycle of 5 min at 74°C were performed. The primers CSVd-R and CSVd-F (5′-CAACTGAAGCTTCAACGCCTT-3′; Hosokawa et al., 2004b (link)) were used. The RT-PCR products were separated by electrophoresis on a 1.0% agarose gel and visualized by ethidium bromide staining.