Nanodrop

For mRNA extraction, the cells were disrupted using 500 mg of acid washed glass beads with a diameter of 425–600 µM on a Precellys FastPrep for 40 s with 7200 rpm. RNA was subsequently extracted using the RNeasy® Mini Kit from Qiagen, Hilden, Germany. RNA quality and quantity were assessed using a ThermoFischer, Waltham, MA, USA, NanoDrop and 2 µg of RNA were reverse transcribed into cDNA using the Qiagen QuantiTect Reverse transcription kit. Expression levels of LEU1 were measured using the ThermoFisher Dynamo Color Flash SYBR Green qPCR kit on an Agilent Technologies, Santa Clara, CA, USA, Stratagene Mx3005p (2 step qPCR protocol, 10 min initialization at 95°C, 40 cycles of: 30 s 95°C, 60 s 60°C). For control of input cDNA amounts the levels of LEU1 were normalized using the ΔΔCt method to measured levels of TAF10 (3 step qPCR protocol, 10 min initialization at 95°C, 40 cycles of: 30 s 95°C, 60 s 56°C, 30 s 72°C). The used primers are shown in Table 2. For both qPCR measurements the samples were analyzed in technical duplicates.

Total RNA was isolated from tissue samples (triplicates, n = 3) by using the RNeasy mini kit (Qiagen, Hilden, Germany). Before cDNA library construction, RNA purity and integrity were checked by using Nanodrop (Qiagen, Hilden, Germany). mRNA was enriched from total RNA by using polyA to generate cDNA libraries for sequencing. All libraries were sequenced on an Illumina HiSeq-2500 sequencer by using version 3 chemistry and flow cells with runs of 2 × 100 bases. Sequence reads were mapped to the genomes by using Hisat2 (v. 2.1.0) [65 (link)]. Previously published gene models were used as the basis for differential gene expression analysis [39 (link)]. Gene expression changes were calculated using Cuffdiff2 (v. 2.2.1) based on previously published gene models [66 (link)]. Differential expression was considered significant if the associated FDR-corrected significance value provided by the software was below the 0.05 threshold. Tobacco proteins were then identified by performing a BLAST search against a database of transcripts (e-value cutoff, 1e-80).
Affymetrix Tobacco exon array is a whole genome exon array available for research use through Affymetrix array catalogue [39 (link)]. We retrieved information about ASN gene expression from the Gene Expression Omnibus (GEO) accession viewers GSE42319 [67 ] and GPL16290 [68 ] as indicated in Martin et al., 2012 [39 (link)].

MDS was performed as previously described (Jee et al., 2016 (link)). In brief, samples of 10 ml from the last cultures of the four B. subtilis strains were spun down and resuspended in 1 ml Tris-EDTA buffer (pH7.5) and incubated −80°C overnight. Genomic DNA extraction was performed using Qiagen genomic tip (100G) and quantified using Nanodrop. To generate each library, 2 μg of genomic DNA were independently treated with NlaIII (leading strand) or Hpy166III (lagging strand) restriction enzyme, which cleaves and delimits the Region Of Interest (ROI). The primers design was performed considering the cut sequence of NlaIII or Hpy166III for leading or lagging strand, respectively. A single PCR cycle was performed with 500 ng of restriction enzyme treated genomic DNA, 500 μM barcoded forward adapter primers annealing to the 3′ end of the ROI and Q5 Hot Start polymerase (New England Biolabs; Ipswich, MA, United States). Unused barcodes were removed with ExoI. The forward library was amplified using a forward adapter (described below in “Primer schema”) and performing 14 cycles of PCR with Q5 Hot Start high-fidelity DNA polymerase. Reverse adapter primers were used to define the ROI. Finally reverse adapters were used to amplify the libraries in 15 cycles of PCR. All libraries were resolved in an 8% acrylamide gel and purified with Ampure XP beads (see Supplementary Figures S2, S9).