Oligoanalyzer

Total RNA was cleaned up, cDNA synthesized, fungal biomass estimation and RT-qPCR assays for wheat genes were performed as described in Pan et al. [50 (link)]. Wheat gene selection for validation was done using similar criteria as in [50 (link)]. Gene-specific RT-qPCR primers were designed using the free online OligoAnalyzer (Integrated DNA Technologies [85 ]). All primers are listed in Additional file 22. The results were analysed as described in Pan et al. [50 (link)], except that GAPDH (TraesCS7A01G313100) was used instead of AOx (TraesCS2A01G327600) as the third reference wheat gene for normalisation of the data.

Complete pgi sequences (Table S2) were extracted from both non-public DuPont culture collection genomes and those in the National Centre for Biotechnology (NCBI, Bethesda, MD) and the Genomes Online Database (JGI, Walnut Creek, CA). Sequences were categorized by species or sub-species based upon whole genome alignments and full-length 16S identity. Pairwise alignment was then performed using Geneious alignment algorithm and resulted in a consensus sequence with degenerate nucleotides representing 100% sequence identity (Figures S1A,B). All alignments were made using the default input values. The consensus sequences were then compared to the top 100 matches using the Basic Local Alignment Search Tool (blastn; NCBI) to locate suitable priming targets compared to the closest related sequences. Primers were designed for each species or sub-species (Table S3) and then tested for hairpins and dimers using OligoAnalyzer (Integrated DNA Technologies, Coralville, IA). Further in silico analysis was performed using blastn to prevent possible amplification of undesired targets. The assays were grouped into four pentaplex reactions (rxnA-D) based on amplicon length (Figure S2). Oligos were obtained from IDT and rehydrated with 1X TE buffer to a stock concentration of 100 μM and stored at −20°C.

In all assays, a SuperSelective forward primer was paired with a conventional reverse primer unless mentioned otherwise. SuperSelective primer sequences were assessed using the Mfold web server [39 (link)] and the OligoAnalyzer computer program (Integrated DNA Technologies, Coralville, IA, USA) to ensure these sequences are unlikely to form internal secondary structures like hairpin loops, self-dimers, or heterodimers with conventional reverse primers. Conventional and SuperSelective primers were purchased from Integrated DNA Technologies. Molecular beacon probes were purchased from LGC Biosearch Technologies (Petaluma, CA, USA). Platinum Taq DNA Polymerase, deoxynucleotide triphosphates (dNTPs), and Sybr Green were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Nuclease-free water was purchased from Ambion, Inc. (Austin, TX, USA).

The total RNA was extracted after 48 h of incubation using an RNAiso plus Kit (Takara-Clontech, Kusatsu, Shiga, Japan) according to the manufacturer’s protocol and quantified using the BioPhotometer Plus (Eppendorf AG, Hamburg, Germany). A total of 1 μg of RNA was reverse transcribed with a high-capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Waltman, MA, USA) using the following thermal program: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min, and put in the hold at 4 °C. To analyze the gene expression of pluripotent markers (NANOG and SOX2), osteogenic markers (RunX2), cytoskeleton (Vimentin, Vinclulin, and Paxillin), and the proliferation marker (Ki67), the primers mentioned in Table 1 were used with SYBR Taq-II (Takara, Japan) as the master mix. The initial denaturation was set at 95 °C for 2 min and 30 s, followed by annealing at 60 °C for 30 s and an elongation at 72 °C for 30 s for 40 cycles. The relative mRNA levels of the gene of interest were normalized to the housekeeping gene, GAPDH, using the ΔΔCt method. All the primers were designed using an oligoanalyzer (Integrated DNA Technologies, Coralville, IA, USA) with default parameters (Integrated DNA Technologies, https://eu.idtdna.com/calc/analyzer) (accessed on 21 November 2020).

Peptide-coupled DNA strands (1–3) were combined in isotonic 0.5 X PBS pH 7.4 at a concentration of 150 µM and 140 µM of the compliment. The mixture was heated to 95°C followed by gradual cooling to 4°C (2 h). The annealed product was diluted to 1.2 OD at 260 nm in isotonic PBS, and the assembly was verified by the melting experiments using UV-Vis spectrophotometer Varian Cary 50 Bio with a temperature range of 10–85°C, data interval 0.1°C, temperature ramp rate 2°C/minute, signal averaging time 0.1. The melting temperature was compared to the theoretically melting temperature predicted by OligoAnalyzer™ from Integrated DNA Technologies (IDTDNA.com).