Geneelute kit

Total RNA samples were isolated using the GeneElute kit (#RTN350), following the manufacturer’s protocol (Sigma-Aldrich). cDNA synthesis was carried out using the qScript synthesis kit (#95048-100), following the manufacturer’s protocol (Quantabio). mRNA expression was measured by quantitative (Q)-PCR using SYBR Green Mastermix (#RT-SY2X-NRWOU+B, Eurogentec Ltd.) and the DNA Engine Opticon 2 system (BioRad). The Q-PCR primer sequences are included in Additional file 2.

P. aeruginosa strain MPAO1 (originating from the lab of Dr. Barbara Iglewski) was obtained from Prof. Colin Manoil, UW (Seattle, USA) together with the transposon insertion mutant collection of ~5000 mutated genes (9437 strains)^{21 (link)}. For DNA extraction, the MPAO1 cryoculture was streaked out on 20% BHI solid medium (7.4 g in 1 L water) containing 1.5% agar (both Sigma, Switzerland). Shaken 20% BHI fluid cultures were inoculated from a single colony and grown at 30 °C until the mid-exponential phase (OD600 = 0.5). Genomic DNA (gDNA) was extracted with the GeneElute kit (Sigma, Switzerland), following the Gram-negative protocol, including RNase treatment. A study that had analyzed 9331 complete bacterial genomes^{29 (link)} (NCBI RefSeq, assembly category: ‘complete genome’; status Feb. 23, 2018; see their TableS4) reported that 106 P. aeruginosa strains have been sequenced completely, which included only two PAO1 strains (and no complete genome of strain MPAO1). 38/106 (36%) had difficulty assemble genomes with repeat pairs >10 kilo base pairs (bp).

Root tissue from each time point was processed in parallel and care was taken to keep samples separate from one another in order to reduce cross contamination. The lyophilized root bundles were physically disrupted using a Qiagen TissueLyzer set at 30 Hz for 2 min with 3 mm tungsten-carbide beads. Total genomic DNA was isolated from the root tissue using the Sigma Gene-Elute kit (G2N70-1KT) according to the manufacturer’s protocol. The 200 μl eluted genomic DNA was ethanol precipitated using 100 μl of 7.5 M ammonium acetate and 600 μl of ice cold >95% ethanol and spinning for 20 min at 0°C. The DNA pellet was washed once with 70% ethanol, air-dried for ~10 min, resuspended in 10 μl of TE buffer and stored at 4°C. To isolate the high molecular weight DNA from lower weight DNA that may contain unintegrated T-DNA, the total DNA was loaded onto a 0.7% agarose gel and run at 90 volts for 1 hour. The band containing the high weight DNA was excised with a scalpel and the DNA extracted from the gel fragment on glasswool treated with Sigmacote in a nested microcentrifuge tube column. The DNA-containing liquid extracted from the column was ethanol precipitated as described above and resuspended in 20 μl of TE buffer. The DNA was stored at 4°C for short-term storage (<48 hours) and -80°C for longer storage.

Among all patients in whom both mutations were identified in our three labs (C.D, C.A. and M.M.), we selected 85 cases carrying the c.-32-13T > G on one allele.
The definition of “very severe (VS)” or “potentially less severe (PLS)” is as in http://www.pompecenter.nl; the severity rating of mutations is according to [21 (link)] based on size, amount and enzymatic activity of mutated protein expressed in transfected COS-7 cells.
The genetic polymorphisms were analysed by P. De F. and K.S.
DNA was extracted by routine methods using the “GENE ELUTE” kit by Sigma, amplification of all GAA exons was performed according to published methods [22 (link)].