Fig. 3: NbPip1 is a pseudogene, not required for immunity or HR signalling.

a Schematic representation of the NbPip1 gene model. The CRISPR/Cas9 frame-shift mutant of NbPip1 (pip1-1) carries a 1 bp deletion leading to an alternative open reading frame (ORF, red) and an untranslated ORF (white). Two fragments of NbPip1 used for VIGS are indicated below the sequence. b Avr4/Cf-4-triggered HR does not require NbPip1. Cf-4 and Avr4 were transiently co-expressed by agroinfiltration of both WT and pip1-1 mutant plants and pictures were taken 5 dpi. Numbers indicate the number of leaves showing HR. c The pip1-1 mutant line is not more susceptible to P. syringae pv. tomato DC3000 ΔhopQ1-1 (PtoDC3000ΔhQ). Plants were infiltrated with 10⁵ CFU/ml PtoDC3000ΔhQ and bacterial growth was measured by colony counting at 3 dpi. Error bars represent STDEV of n = 4 biological replicates. Probability values were calculated with Students t-test. d NbPip1 depletion by VIGS does not affect susceptibility to PtoDC3000ΔhQ. Young N. benthamiana plants were inoculated with TRV::GFP, TRV::NbPip1#1, or TRV2::NbPip1#2 and infiltrated with 10⁵ CFU/ml PtoDC3000ΔhQ and bacterial growth was measured by colony counting at 3 dpi. Error bars represent STDEV of n = 4 biological replicates. Probability values were calculated with ANOVA using the Tukey’s post-hoc test. e NbPip1 is a pseudogene in LAB and SA accessions. Two small deletions of 2 and 24 bp in the 3’end of NbPip1 results in an altered C-terminus of the encoded protein (red). Dotted line indicates unknown sequence; dashed line indicates putative intron. Protein sequences contain polymorphic residues (black), catalytic Asn (green) and Cys residues (blue). f NbPip1 can be resurrected into an active protease by correcting the 2 and 24 bp deletions. Pip1, φNbPip1, and an NbPip1 mutant with a corrected 3’ end (NbPip1*) were co-expressed with silencing inhibitor P19 by agroinfiltration. Apoplastic fluids were isolated at 4 dpi, preincubated for 45 min with or without 100 µM E-64, 2 µM Avr2 or Avr2Δ6, or DMSO, and labelled for 3 h with 2 µM MV201. Samples were separated on SDS-PAGE gels, scanned for fluorescence. g Pseudogenisation is common amongst PLCP subfamilies in N. benthamiana. The evolutionary history of the N. benthamiana PLCP gene family was inferred using the Maximum Likelihood method based on the Whelan and Goldman model⁶⁶. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analysed⁶⁷. Putative inactive PLCPs are indicated with gray dotted lines. PLCP clades were named according to²². See Supplementary Fig. 3 for all names.