Topvision

The pool of candidate reference targets comprised 11 bacterial genes. Nine of them (16S rRNA, 23S rRNA, dnaK, groEL, gyrB, recA, rho, rpoB, rpoD) were chosen based on literature data. Homologues of these sequences were derived from the complete genome of O. quorumnocens A44 (CP022602.1–CP022605.1) (detailed list of loci in Table 2). The two other genes were: gfp, carried on the artificially introduced vector pPROBE-GTkan^{45 (link)} and CES85_4722, a hypothetical A44 gene with no data concerning its stability (a blind control). All PCR primers (Supplementary Table S1) were designed using the Primer3Plus software^{78 (link),79 (link)} (http://primer3plus.com/). Specificity of the primers was verified using real-time PCR followed by melt curve analysis and electrophoresis in 1.2% agarose gel (TopVision, Thermo Scientific).

gDNA samples were used for the following validation and segregation analysis, which was performed by Sanger sequencing. Polymerase chain reactions (PCR) of gDNA sequences flanking the pathogenic or likely pathogenic variants of the PIGN gene were performed using specific primers (Supplementary Table S3) designed with the Primer Blast tool. PCR was performed using Phusion High-Fidelity PCR Master Mix (Thermo Fisher Scientific, USA). PCR products were fractioned by 1.5% agarose gel (TopVision, Thermo Fisher Scientific, USA) electrophoresis and visualized under ultraviolet light. The PCR products were sequenced with BigDye^® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA) and ABI 3130xL Genetic Analyser (Thermo Fisher Scientific, USA). The resulting sequences were aligned with the reference sequence of PIGN (NCBI: NG_033144.1; NM_176787.5) containing 31 exons.

Seedlings were arranged with minimal perturbation and immobilized with 2% low melting agarose (TopVision, Thermoscientific) on the agar plate on which the plants were grown. The ablation was performed as in [29 ], under a binocular with a fine Minutiens needle (0.15 mm diameter) set on a pin holder to apply a small pressure at the surface of the hypocotyl to physically rupture a few cells (Supplemental Fig. S5). For an experiment, in general, 15 to 20 seedlings were placed on the plate and ablation was attempted for each sample. Due to the variability in precision of manual ablation, some samples did not appear to have ablation while others had ablations that were too large. Only those that appeared to have resulted in the ablation of approximately 4 cells were kept for imaging and further analysis. In the case of the mock experiments, the samples were prepared as described above without performing the ablation.