Rnase a inhibitor

To remove RNAs or proteins of EVs, the EVs were put in liquid nitrogen for 1-2 minutes, and then dissolved in 37°C water bath for 2-3 minutes. EVs were underwent five freeze-thaw cycles as described previously (56 (link)). Thereafter, the EVs were treated with RNase A (Takara, 100 μg/mL, 37°C, 30 min) to degrade RNAs in EVs, followed by incubating with RNase A inhibitor (Takara, 2,000 units/mL, 37°C, 30 min) to inactivate RNase A. To degrade proteins, EVs were treated with proteinase K (Sigma, 25 μg/mL, 37°C, 30 min), followed by heating at 95°C for 5min to inactivate proteinase K.

RNA cleavage reactions were performed by incubating DIG-11-UTP-labeled RNA transcripts or DIG-labeled RNA complexed with N protein fractions from tospovirus-infected plants as described [12 (link)]. Five μg N proteins were mixed with 2 μg of DIG-labeled RNA probe in 10 μL volume and the mixtures were incubated for 10 min on ice to form N-RNA complexes. One μL of N-RNA mixtures was taken to incubate with 8 μL chromatography fractions. The mixtures were incubated for 10 min at room temperature to form N-RNA complexes. Cleavage reactions were incubated at 24°C for 60 minutes in presence of 15 U/mL RNase A inhibitor (TaKaRa, Japan) and 20 mM MgCl₂. Control reactions were performed with elution buffer or with chromatography fractions from mock-inoculated plants. DIG-labeled RNA transcripts were resolved by electrophoresis on a 1% agarose gel and transferred to a Hybond-N+ membranes (GE Healthcare, UK) using the Bio-Rad semi-dry transfer unit (Bio-Rad, Hercules, CA). DIG-labelled signals on the blots were detected by AP-conjugated anti-digoxigenin antibodies and visualized by incubation in the presence of BCIP/NBT (Sangon Biotech, Shanghai, China) staining.

KOD-plus DNA polymerase was purchased from Toyobo (Osaka, Japan). Nickel–nitrilotriacetic acid resin was purchased from Bio-Rad (Hercules, CA, USA). RNase A inhibitor was purchased from Takara (Shiga, Japan). Oligodeoxyribonucleotides and oligoribonucleotides (Table S1) were synthesized by Invitrogen (Carlsbad, CA, USA) and Takara (Shiga, Japan), respectively. The expression vectors of pDEST17 (Invitrogen) and pET28-sumo were used throughout this study. E. coli strain DH5α was used in the gene cloning and Rosetta 2(DE3)pLysS (Novagen) strain was used to express recombinant protein. All other chemicals and reagents were of analytic grade.