Pureyield plasmid miniprep

DNA was extracted using the Wizard genomic DNA purification kit (Promega). Plasmid and PCR/gel extractions were done using PureYield Plasmid Miniprep and Wizard SV Gel and PCR clean-up systems respectively (Promega). Purified DNA from V. cholerae S24 was sequenced at the Wellcome Trust Sanger Institute using Illumina-based technology.
All primers used in this study are shown in Table 1. Standard PCR was performed using the PCR master mix (Promega) containing 25 units ml⁻¹ of Taq DNA polymerase, 800 μM dNTPs and 1.5 mM MgCl₂. Primers were used at a final concentration of 0.5 μM each. All PCRs were performed with 30 cycles of denaturation at 94°C for 30 s, the appropriate annealing temperature for 30 s and an extension of 72°C (1 min kb⁻¹) and sequencing performed at Macrogen. From whole genome sequencing (Wellcome Trust Sanger Institute) it became evident that the host recA had been disrupted and was present on two separate contigs. The two contigs (contigs 675 and 708) were pieced together by PCR and joined to an intervening third contig to produce a contig of 262,869 bp (contig 367) using primers described in Table 1. The accession number for RME is KJ123688.

PCR products generated in obg-FR PCR (841-bp) were separated through 1% agarose gels, bands of expected size were excised, purified using Wizard® SV Gel and PCR clean-Up System (Promega) and cloned into pGEM®-T Easy vector (Promega) using instructions provided by the manufacturer. The resultant constructs were propagated in α-select competent cells, silver efficiency (Bioline, Alexandria, New South Wales, Australia) and extracted using PureYield^™ Plasmid Miniprep (Promega). All purified PCR products or plasmid extracts were subjected to automated sequencing (BigDye Terminator v3.1; Applied Biosystems, Foster City, California, USA) in both directions using primers obg-F and obg-R, or M13 forward and reverse sequencing primers for purified PCR products or cloned PCR products, respectively. Nucleotide sequences were edited using SeqMan^™ II and EditSeq programs in DNASTAR. Multiple sequences were aligned using computer program ClustalW2. Nucleotide sequence of complete obg of 25 strains including MS-H and its related strains, belonging to four different genotypes, has been described in our previous study [18] (link). Nucleotide sequence of partial obg of additional 10 M. synoviae strains, including 94041/12a, 4GPH3, F10-2AS, K1938, K870, K1858, YA, K1968, K1723 and WVU-1853, has been submitted to GenBank under accession numbers KF875990 to KF875999.

The total RNA was extracted from the leaves of chickpea cultivar Dwelley using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from 100 mg samples in accordance with the manufacturer’s protocol. First strand cDNA synthesis and genomic DNA elimination were performed simultaneously using 5X All-In-One RT MasterMix, containing AccurT Genomic DNA Removal (Applied Biological Materials Inc., Richmond, Canada). cDNA samples were stored at−80 °C until use. Full-length ORFs were amplified with Phusion^® High-Fidelity DNA Polymerase (NEB, Ipswich, MA, United States) using gene-specific primer pairs (Supplementary Table S1) using the following protocol: initial denaturation at 98°C for 30 s and 35 cycles of 98°C for 10 s, 60°C and 72°C for 30 s each and followed by a final elongation at 72°C for 8 min. Amplified PCR products with appropriate expected sizes were purified with the Monarch^® DNA Gel Extraction Kit (NEB). Purified PCR products were cloned into the pMiniT 2.0 vector (NEB) and transformed into DH10B high-efficiency E. coli competent cells (NEB). The plasmids were recovered from E. coli using PureYield™ Plasmid Miniprep (Promega, Madison, WI, United States), verified using Sanger sequencing (Laboratory of Biotechnology & Bioanalysis, Pullman, WA, United States), and compared to the GenBank sequences of CaPGIP1 (XM_004504675), CaPGIP3 (XM_004493500), and CaPGIP4 (XM_012713804).

The genes plsB and plsC were kindly provided by Dr. Yutetsu Kuruma [15 (link)] in the form of circular plasmids. The plasmids carrying an ampicillin selection marker were amplified in E. coli (TOP10) cells and purified with a PureYield^™ plasmid miniprep (Promega). Linear DNA templates were generated by PCR using the primers reported in Table C in S1 File. For the lipid headgroup modification experiments the DNA templates containing the genes cdsA, pgsA, pgpA, pgpC, psd or pssA were all constructed from E. coli MG1655 (K12) genomic DNA that was extracted with GenElute^™ Bacterial Genomic DNA Kit (Sigma Aldrich). The genes were cloned into a pET11a backbone by Gibson assembly [45 (link),46 (link)]. Amplification of the target genes was performed using two primers. A forward primer with the following regions (from 5’ to 3’): a region overlapping the pET11a sequence and a region overlapping the gene of interest. A reverse primer with the same sequence design (pET11a homology, gene of interest homology) was employed. The assembled plasmids were then amplified in E coli cells (TOP10), purified and linear PCR products were generated as described above. All sequences were confirmed by sequencing. The list of primers is reported in Table C in S1 File. Proteins were then synthesized in vitro using either the plasmid or linear DNA as a template for gene expression.