Spectrum plant total rna

RNA was isolated from Ler Arabidopsis plants with the Spectrum Plant Total RNA (Sigma-Aldrich) kit with on-column DNase I digestion (New England Biolabs). cDNA was synthesized from RNA using oligo (dT)18 primers and SuperScript III reverse transcriptase (Fisher Scientific). Splice forms were amplified with Phusion DNA polymerase (New England Biolabs), adding a CACC 5′ overhang to facilitate pENTR/D-TOPO cloning. RT-PCR amplified DNA was gel purified in a 1% agarose gel and extracted using a gel extraction kit (Bioneer). Purified DNA was used for TOPO reactions with pENTR/D-TOPO (Thermo Fisher), transformed into XL1-Blue E. coli chemically competent cells, and screened for resistance to kanamycin on LB agar. Plasmid was purified from positive colonies using a miniprep kit (Bioneer) and sequenced using Sanger technology with the M13F primer.
Oligonucleotides used for cloning were: JB1058: 5′-CACC ATG CCT CGT AAA GGA TTA TCC AAT TTC G-3′, JB1061: 5′-CACC ACA GTT GAG AGCAG ATG CAA AGA AC-3′, and JB1063: 5′-GCC TTT TGG ACC AGT TTT TGAGG ATG-3′. JB1058 + JB1063 surround the alternative exon, and amplified three majors products: the short splice form, the long splice form, and a small amount of the longer, minor splice form. JB1061 is specific to the 5′ end of the alternative exon, and in combination with JB1063, amplified both the long splice form and the longer, minor splice form.

RNA was extracted from 100 mg of fungal mycelium using Spectrum Plant Total RNA (Sigma-Aldrich, St. Louis, MO, United States), following the manufacturer’s instructions. Mycelium was placed in a 2-ml tube with two tungsten beads and tubes were immersed in liquid nitrogen for 1 min. Then, samples were immediately lysed using TissueLyser II at 20.00 Hz for 1 min.
DNase treatment and first-strand cDNA synthesis were performed according to Valente et al. (2020) (link) using a TURBO DNA-free^TM Kit (Thermo Fischer Scientific) and a High Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific).
RT-qPCR was performed with StepOne^TM and StepOnePlus^TM Real-Time PCR System with Power SYBR^TM Green PCR Master Mix (Thermo Fischer Scientific); cycling conditions were 5 min at 95°C, followed by 45 cycles of 10 s at 95°C, 30 s at 58°C, and 30 s at 72°C. In order to determine relative gene expression (RGE), the 2^ΔΔcq method (Pfaffl, 2001 (link)) was used with cDNA of samples, by comparing the amplification of β-tubulin gene with the amplification of the target gene. P. aurantiogriseum was used as reference strain for verrucosidin production, as it is reported to be a verrucosidin producer (Fink-Gremmels et al., 1991 ). All primer sequences used in RT-qPCR reactions are listed in Supplementary Table 1.

Total RNA was extracted from 0.2 g of apple skin with the ‘Spectrum Plant total RNA’ (Sigma-Aldrich, St. Louis, MO, USA). The manufacturer’s protocol was modified by the addition of a spatula tip (5-mm width × 2 mm length) of PVPP to the ground tissue at the beginning of the extraction. The RNA was cleaned using the RNeasy MinElute Cleanup (Qiagen, Leusden, The Netherlands). RNA quantity, integrity and purity were assessed by NanoDrop 1000 Spectrophotometer (Thermo Scientific, Villebon-sur-Yvette, France), and 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA).

Total RNA was extracted from callus fragments grown on selective medium for eight weeks, using a commercial kit (Sigma, SpectrumTM Plant Total RNA). First-strand cDNA was synthesised using a polyA primers and RevertAid First Strand cDNA synthesis kit (ThermoFisher Scientific). For each RT-PCR reaction, 500 ng of cDNA was used. The primer sequences and PCR conditions used to amplify GUS and UBIQUITIN (UBQ) fragments are provided in Table S1. Sequences of the UBIQUITIN gene (Contig980) were identified using BLAST tool for the F. excelsior genome [4 (link)].