RNA Blot Analysis of Plant Samples

Leaf samples were collected from the top two fully expanded leaves at 28 days post-inoculation (dpi). Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). RNA blot was performed as previously described [16 (link)]. Roughly 10 mg of RNA samples were loaded onto a 5% polyacrylamide gel containing 8 M urea followed by electrophoresis using Bio-Rad Mini-PROTEAN Tetra Cell. These samples were then transferred onto a Hybond-XL nylon membrane (Amersham Biosciences, Piscataway, NJ, USA) using a Trans-Blot^® SD Semi-Dry Transfer Cell (Bio-Rad, Shanghai, China) and immobilized through UV-cross-linking. Hybridization was conducted at 65 °C using DIG-11-UTP-labeled riboprobes. These riboprobes were prepared through in vitro transcription using the T7 Maxiscript kit (ThermoFisher Scientific), with the SpeI-linearized pInter(–) plasmid serving as the template [19 (link)]. Following an overnight hybridization, the membranes were subjected to two rounds of washing at 65 °C, first in a solution of 2× SSC (where 1× SSC contains 0.15 M NaCl and 0.015 M sodium citrate) with 0.1% sodium dodecyl sulfate (SDS) and then in 0.2× SSC-0.1% SDS. Signal detection was accomplished using the MYECL™ Imager (Thermo Fisher Scientific, Shanghai, China).