Opticon 2 system

For native gene expression, bacteria were grown for 4 hours in KB media from OD₆₀₀ = 0.2, washed twice with sterile 10 mM MgCl₂ and transferred into MM minimum media containing 10 mM mannitol for Pto_DC3000, Psy_B728a, Pla₁₀₇, Pja, Por strains or MM minimum media containing 10 mM fructose for Pph_1448A strain. Cells were collected after 5 hours of incubation shaking at 28°C and treated with RNAprotect reagent (Qiagen). Total RNA derived from cells grown in MM media or arabinose inducing media (as above) was extracted using the RNeasy minikit (Qiagen), DNase treated twice (Ambion Turbo DNase), and cleaned up with Qiagen RNeasy Mini kit. Reverse transcription was performed using SuperScript II (Invitrogen) with 2 µg total RNA. Diluted cDNA was analyzed with SYBR green (Applied Biosystem) using the Opticon 2 System (BioRad). Primers used are listed in Table S9.

To screen for the presence and amount of bacterial and archaeal amoA genes, quantitative PCR (qPCR) was employed. To identify bacterial amoA genes, primers amoA-1F (5’ GGGGTTTCTACTGGTGGT ‘3) and amoA-2R (5’ CCCCTCKGSAAAGCCTTCTTC ‘3) were used and genes amplified as previously described by Rotthauwe et al. [46 (link)]. To identify archaeal amoA genes, primers Crenamo1F (5’ AATGGTCTGGCTWAGACGC ‘3) and CrenAmo1R (5’ GACCARGCGGCCATCCA ‘3) were used and amplified as previously described by Könneke et al. [47 ]. The qPCR was performed in an Opticon2 system (BioRad) using the PeqLab KAPA Sybr FAST Kit, three replicates of each sample. Melting curve analysis (0.2°C s^-1) and agarose gel electrophoresis (1% agarose) revealed single amplicons for all samples.

Total RNA was isolated from Arabidopsis using the Trizol reagent (Promega, Madison, WI, USA), reversely transcribed into cDNA using the Primescript^TM RT reagent kit (Takara), and diluted with ultra-pure water (MilliQ) to 100 μl as the PCR template. The tubulin β-2 (Locus number AT5G62690) and Actin 7 (Locus number AT5G09810) genes were used as internal references. All primers used are shown in Supplementary Table S2. qRT-PCR was carried out on an Opticon 2 System (Bio-Rad). The PCR reaction mixture contained 10 μl of SYBR Green Real-time PCR Master Mix (Toyobo), 0.5 μM of each forward and reverse primers, and 2 μl of cDNA template (equivalent to 20 ng of total RNA) in a reaction volume of 20 μl. The PCR conditions were as follows: 94°C for 30 s; followed by 45 cycles at 94°C for 12 s, 60°C for 30 s, 72°C for 40 s; and 1 s at 82°C for plate reading. A melting curve of each sample was generated to evaluate the quality of the amplified product. Three biological replicates were conducted, and the expression levels were calculated using the 2^-ΔΔCt method.

Total RNA was isolated using the Spectrum^TM Plant Total RNA Kit (Sigma, Deisenhofen) according to the protocol of the manufacturer. The extracted RNA was treated with a DNA-free DNase (Qiagen, Hilden, Germany) to remove any potential contamination by genomic DNA. The mRNA was transcribed into cDNA using the M-MuLV cDNA Synthesis Kit (New England Biolabs; Frankfurt am Main, Germany) according to the instructions of the manufacturer. The RNase inhibitor (New England Biolabs; Frankfurt am Main, Germany) was used to protect the RNA from degradation. The amount of RNA template was 1 μg.
Quantitative real-time RT-PCR was performed on an Opticon 2 system (Bio-Rad, München) as described by Svyatyna et al. (2014) . To compare the mRNA expression levels between different samples, the C_t values from each sample were normalized to the value for the EF-1α internal standard obtained from the same sample. For each triplicate, these normalized C_t values were averaged. The difference between the C_t values of the target gene X and those for the EF-1α reference R were calculated as follows: △C_t(X)=C_t(X)–C_t(R). The final result was expressed as 2^–△Ct(X). Each experiment was repeated with three biological replicates.