Plant rna reagent

Total RNA was extracted with plant RNA Reagent (Invitrogen) following the manufacturer’s protocol. RNA purity was checked using the kaiaoK5500®Spectrophotometer (Kaiao, Beijing, China). RNA integrity and concentration was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA) at Annoroad Gene Technology (Beijing) co., LTD. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. RNA concentration of library was measured using Qubit® RNA Assay Kit in Qubit® 3.0. Insert size was assessed by the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The clustering was performed on a cBot cluster generation system using HiSeq PE Cluster Kit v4-cBot-HS (Illumina). After cluster generation, the libraries were sequenced on an Illumina HiSeq 2500 platform and 150 bp paired-end reads were generated. Low-quality RNA-Seq reads (Qscore < Q30) were discarded.

Total RNA was isolated from wheat samples using Plant RNA Reagent (Invitrogen, California, USA), according to the manufacturer’s instructions. RNA was further purified through a Qiagen RNeasy column (Qiagen, Australia) after pre-treatment with RNase-free DNase I (Xue and Loveridge, 2004 (link)).
TaHsfA6f cDNA was isolated from the leaves of heat-treated wheat plants using 3ʹ-RACE with primers designed from the partial TaHsfA6f cDNA containing the N-terminal sequence. The PCR-amplified product was cloned into pGEM-T Easy vector (Promega) and sequenced using a BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, USA). The TaHsfA6f cDNA sequence was deposited in GenBank (KJ774108).

Genomic DNA was extracted from cacao leaves 5 days after Agrobacterium infiltration using a modified CTAB protocol (Helliwell et al., 2016 (link)), followed by a 0.3 M final concentration sodium acetate/ethanol precipitation. RNA was extracted using Invitrogen Plant RNA Reagent (Invitrogen, Carlsbad, CA) with minor modifications of the recommended protocols. These include: 1 ml of Plant RNA Reagent was added to each ground tissue sample, 0.2 ml of 5 M NaCl were added to samples prior to chloroform extraction, and all centrifugations were performed at 14,000 rpm.

The wild-type (UMAvr7) and mutant (umavr7) isolates were inoculated on cotyledons of 7-day-old seedlings of B. napus genotypes Westar (No R gene) and 01-23-2-1 (Rlm7 gene). RNA was extracted from 100 mg of cotyledons sampled from 1 day and 11 days post inoculation using Plant RNA Reagent (Invitrogen, Carlsbad, CA, United States). The first strand cDNA was synthesized from one microgram of total RNA from each sample using RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions (Thermo Scientific, Waltham, MA, United States). Reverse transcription PCR (RT-PCR) was performed with the primers designed from coding region of AvrLm7 gene by 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s. Three biological replicates were sampled for RT-PCR analysis. B. napus actin gene was used as a control.

For real‐time PCR analysis, total RNA was extracted from internodes with the Plant RNA Reagent (Invitrogen, UK) and cleaned‐up by DNAase treatment on an RNeasy column (Qiagen, UK) before further DNase treatment of the eluent with Turbo DNase (Ambion, CA). RNA was checked via Nanodrop and the Bioanalyzer 2100 (Agilent, UK). cDNA was synthesised from 600 ng RNA with random primers using iscript reverse transcriptase (Bio‐Rad, UK). Barley homologues of wheat genes TaSnRK1 (Gene Index TC253257) and TaRPII36 (Gene Index TC235230) (Kam et al., 2007) named here as HvSnRK1 and HvRPII36 were used as reference genes. Tables S2 and S3 give primer sequences and reaction set up. PCR products were validated by sequencing. Three technical replicates were performed for each gene and sample. Relative expression was calculated with the Pfaffl efficiency equation (Pfaffl, 2001) using the primer efficiency determined by LinRegPCR in the equation.

Mature flowers and buds were dissected on three independent plants using micro-dissecting forceps. Carpels, stamens, and tepals were removed separately and immediately frozen in liquid nitrogen. Forceps were cleaned with 100% ethanol between each tissue and flower. RNA for qPCR was extracted using Plant RNA Reagent (Invitrogen™), treated with Ambion TURBO DNA-free™, and converted to cDNA using Invitrogen Superscript III, primed using oligo(dT)₂₀ and a random hexamer. CcActin was selected as a reference gene based on successful preliminary trials demonstrating stable expression across tissues. Primer sequences are provided in Supplementary Table S1. Forty cycles of PCR were performed using either a BioRad DNA Engine Thermocycler or a CFX Connect Real-Time PCR Detections System (185-5200). A melting curve was performed from 60 °C to 95 °C with readings taken at 0.5 °C intervals. Relative gene expression was quantified using an Opticon Monitor 3 and CFX Manager software (both BioRad Laboratories, Inc.). Ct values were exported to Microsoft Excel, and ∆Ct values were calculated by subtracting the Ct of the reference gene, actin. Each dataset was statistically analysed in Excel using a t-test.

Floral buds from FC and NC experiments were subjected to RNA isolation. Buds were cut off from branches immediately before the stick heat activation process, immediately frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted from flower buds using the Plant RNA Reagent (Invitrogen) according to the manufacturer’s instructions. RNA was quantified using the Qubit system (Invitrogen), and RNA integrity was verified on a 1.5% MOPS denaturing gel and by the Fragment Analyzer system (DNF-472; Advanced Analytical Technologies), using the criterion of RNA quality number (RQN) > 8.0 for the sRNA sequencing libraries and RQN > 7.0 for qRT-PCR analysis.