Predicted SP sequences were removed by amplification using appropriate paired AttB1 recombination cloning primers; 3′ to the SP sequence and the 3′ end of the PbPE sequence. After removal of the SP sequence, in planta subcellular localization was determined for each ΔspPbPE. ΔspPbPE cDNA sequences or cellular marker gene sequences were cloned into plant expression binary vectors with (pH7XWG2) or without (pH7WG2) fluorescent tags, using Gateway cloning technology (Thermo Fisher Scientific; Karimi et al., 2002). The cDNA sequence of each ΔspPbPE, sandwiched between attB1 and attB2 recombination sites, was inserted into the entry vector pDONR221/207/Zeo via a BP reaction. From there the ΔspPbPE sequence was added to the C-terminal GFP tagged binary vector pH7FWG2, via an LR reaction, with expression driven by the CaMV 35S promoter.

The mCherry-tagged sub-cellular marker gene constructs, in the pBIN20 binary vector backbone, were purchased from the Arabidopsis Biological Resource Centre15. The GUS expression construct, pH7WG2-GUS, was created from pENTR-GUS provided in the gateway cloning kit, with the GUS sequence inserted into pH7WG2 via an LR reaction. The A. thaliana REMORIN 1.3 (AT2G45820.1) sequence was cloned into pH7RWG2, with a C-terminal mRFP fluorescence tag, for co-localization studies. A GFP construct, pH7WG2-GFP, was also generated for use as a negative control for the cell death assay and transient localization studies. All constructs were used to transform Agrobacterium tumefaciens, with positive transformants selected on LB medium supplemented with spectinomycin (100 mg/L), kanamycin (50 mg/L), or rifampicin (50 mg/L) and subsequently used to transform N. benthamiana for transient expression studies. All the constructs used and generated in this study are provided in Supplementary Table 2.

Subcellular localization of PbPEs was determined by transiently expressing the ΔspPbPE-GFP gene fusion-constructs, in A. tumefaciens at a final OD600 of 0.3, together with organelle-specific markers, in N. benthamiana leaves. Subcellular localization of the PEs was recorded 2-3 days after agroinfiltration. The localization of each ΔspPbPE-GFP was visualized with a LSM880 inverted confocal laser scanning microscope (Zeiss, MN, United States) using a 40X water objective at GFP-required wavelengths. GFP and chloroplast autofluorescence was monitored using an Argon laser at 488/500–530 and 488/580–620 nm excitation/emission wavelengths, respectively. The mRFP and mCherry fluorescence tags were monitored using a Helium-Neon laser at 561/600 and 561/630 nm, excitation/emission wavelengths, respectively.

To classify the localization of ΔspPbPE-GFPs at the cell periphery, N. benthamiana leaf segments (leaves) were plasmolyzed in 0.85 M KCl for 15 min before observation under the Zeiss LSM880 microscope using a 40X water objective as outlined above. Flg22 treatment was performed on leaves 2 days post infiltration and confocal images were taken 1 h after flg22 treatment.

Z-stack and time-lapse images were captured to provide further insight into the fluorescence distribution, association and dynamics of ΔspPbPE localization in N. benthamiana leaf epidermal cells. To verify the localization profile for each ΔspPbPE, multiple images were captured from different fluorescence-expressing cells. To avoid overexpression artifacts, transiently expressing cells, with comparatively low fluorescent signals, were imaged for analysis using FIJI ImageJ16. Fluorescence intensity plots were graphed based on the quantitative data measured in arbitrary units (a.u.), obtained from the region of interest of a confocal colocalized image represented by a blue line, using ImageJ. Each fluorescence channel in a colocalized confocal image represents the individual line graph in a fluorescence intensity plot.

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