Qx200 evagreen supermix

Quantitative reverse transcription−PCR was performed using QX200 EvaGreen Supermix (Bio Rad, Catalog No. 1864034). Three biological replicates were performed for each reverse transcription-PCR experiment. The Sorghum GAPDH gene is used as the internal reference to normalize the expression data (GAPDH-f: 5′-AAGGCCGGCATTGCTTTGAAT-3′, GAPDH-r: 5′-ACATGTGGCAGATCAGGTCGA-3′). The primers used for Streptomyces quantitation are Actino235 (5′-CGCGGCCTATCAGCTTGTTG-3’) and Eub518 (5’-ATTACCGCGGCTGCTGG-3’), and the primers for Pseudomonas quantitation are Eub338 (5′-ACTCCTACGGGAGGCAGCAG-3′) and Bet680 (5′-TCACTGCTACACGYG-3′). Relative expression levels were calculated according to the 2 − 2∆∆CT (cycle threshold) method¹⁰³ , and the standard deviation was calculated among the three biological replicates. The PCR conditions for the amplification were 95 °C for 2 min, and 50 two-step cycles (95 °C for 10 s, 60 °Cs for 25 s) followed by a plate read; a melt curve was generated by heating from 72 to 95 °C with 0.2 °C increments. The starting DNA template amount for taxa-specific qPCR is 10 ng per sample. The p-value of the relative bacterial amount between treatments under each condition was calculated by function t.test in R.

Full-length 16S amplifications were performed using: 1 μL of total DNA as template; 0.25 μM of the universal 16S primers F27 and R1492 with four different sets of asymmetric barcodes at (Additional file 1: Table S9). and GoTaq Hot Start Master Mix (Promega) in a 50 μL final volume. Cycling conditions were: 94 °C, 3 min; 35 cycles of 94 °C 30 s, 54 °C 30 s, 72 °C 2 min; following by a 5 min final elongation at 72 °C. PCR products were cleaned with AxyPrep™ MagPCR (Corning Life Sciences) according to the manufacturer’s protocol and eluted in 40 μL of water. Cleaned PCR products were quantified using the Bio-Rad QX200 droplet digital PCR (Bio-Rad) and QX200 EvaGreen® Supermix with primers F357 and R534 (Additional file 1: Table S10) targeting the V3 variable region of 16S rDNA. Based on the results, amplicon libraries were normalized to the same concentration before pooling. Pooling was always performed using amplicon libraries with distinct barcodes. Multiplexing was performed with 2–4 libraries per pool.

Genomic DNA of N. benthamiana and Arabidopsis plants was extracted with cetyl trimethyl ammonium bromide (CTAB) buffer. PPR was used as the internal control for the qPCR assays.
A QX200 droplet digital PCR system (Bio-Rad) was used for droplet digital PCR. Genomic DNA samples of 2 μl (20 ng/μl) were added to 20-μl reaction mixes of BioRad QX200 EvaGreen supermix. The primers were present at final concentrations of 100 nM. The reaction mixes were briefly vortexed, avoiding the formation of bubbles. Each 20 μl reaction mix was loaded into a cell of a BioRad DG8 cartridge followed by addition of a 70-μl droplet generation oil. The cartridge was then placed into the droplet generator for droplet generation. Droplets were transferred to a 96-well PCR plate, heat-sealed with a pierceable foil seal, and PCR amplified. Amplification was conducted under the following standard cycling conditions: 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s; 60 °C for 60 s, 4 °C for 5 min, and 90 °C for 5 min, after which the plate was held at 4 °C; ramp rate 2 °C/s. Following PCR, the plate was put onto the QX200 Droplet Digital reader. Data were collected with Quantasoft™ Software.

Specific regions of 24 ERCC spike-ins with a T7 promoter attachment were synthesized (IDT) and amplified using a forward primer annealing to the T7 promoter and a reverse primer containing 60 Ts (Additional file 2: Table S4). The resulting dsDNA templates were subject to T7 IVT, and the transcribed RNAs were quantified using the Qubit RNA HS Assay Kit (Invitrogen) and the Bioanalyzer RNA 6000 Pico Kit (Agilent). The 24 RNA spike-in species were then diluted and mixed into the A60 RNA spike-in mix (Additional file 2: Table S5). For digital quantification, the A60 RNA spike-in mix was converted to cDNA by oligo-dT₆₀ primed reverse transcription and quantified by the QX200 Droplet Digital (ddPCR) system using the QX200 EvaGreen Supermix (Bio-Rad), Droplet Generation Oil (Bio-Rad), and the corresponding primers (Additional file 2: Table S4). The copy number of each A60 RNA spike-in species is reported in Additional file 2: Table S5.