Fig. 7: A systematic analysis of the distribution and virulence role of a novel Non-Ribosomal Peptide Synthase (NRPS-PKS) from Pseudomonas plant pathogens.

a Subset Sequence similarity network of the NRPSs present in Pseudomonas constructed with BiG-SCAPE⁴² using a threshold of 0.3. Nodes are representing the BGCs, each BGC is colored based on the Pseudomonas specie where the BGC was detected. The novel NRPS-PKS is present in three BGC clusters, two of them significantly assigned to the phytopathogenic bacteria P. syringae and P. viridiflava and one associated with environmental species such as P. fluorescens (highlighted with a black square). Well-studied NRPSs from the MIBiG database⁸³ involved in Pseudomonas plant pathogenesis such as syringofactin⁸⁰, syringopeptin¹¹¹, syringomycin⁷⁹, brabantamide¹¹², nunapeptin¹¹³, corpeptin¹¹⁴ and pseudomonine¹¹⁵ are highlighted in the network. b Phylogenetic analysis of the plant pathogen Pseudomonas spp. (n = 332), harboring this NRPS family, based on concatenated alignment (11001 positions) 77 ubiquitously conserved proteins identified with PhyloPhlAn 3.0¹⁰² and visualized/annotated using iTOL¹⁰⁶. The phylogenetic tree is coupled with a colored strip showing the species and a heatmap indicating the distribution of syringomycin (syr), syringofactin (syf), and the novel NRPS. Light gray indicates the absence, whereas black indicates presence. This NRPS family is highly conserved in the phytopathogenic bacteria P. syringae, P. viridiflava having the same BGC product prediction by antiSMASH⁴¹. c CORASON⁴² phylogenetic reconstruction of the novel NRPS using a random representative of each of the three different clusters associated with this NRPS detected in the network shown in panel (a). Colors indicate the domains predicted for each gene. d Shown below the NRPS and PKS genes are the module and domain organizations of the encoded proteins. The domains are labeled as follows: C condensation, A adenylation, T thiolation, TE thioesterase, KS ketosynthase, and KR ketoreductase. Predicted substrates of the NRPS and PKS modules in novel NRPS-PKS are arginine (Arg), proline (Pro), cysteine (Cys), 2 unknown amino acids, and, again, cysteine. e Symptoms induced by P. syringae pv. phaseolica 1448A (Pph 1448A) and Pph NRPS mutant in bean leaves (Phaseolus vulgaris cv. Canadian Wonder) at 4 dpi and 7 dpi after inoculation with suspensions of 10⁶ CFU mL⁻¹. f Quantification of necrotic areas as a percentage of necrotic lesion per infiltrated area in the inoculated leaves shown in panel (e). Error bars correspond to the standard deviation of five different leaves from two independent experiments. The mean serves as the measure to define the center of the error bars. Asterisks indicate significantly differences between wild-type and mutant strains using Student’s t-test two-sided (P ≤ 0.05). g Competitive index (CI) assay in leaves of beans cv. Canadian Wonder after inoculation of mixed suspensions of wild-type strain and NRPS mutant strain at 10⁴ CFU mL⁻¹. Error bars correspond to the standard deviation of three biological replicates. The mean serves as the measure to define the center of the error bars. Asterisks indicate indices that deviate significantly from unity using Student’s t-test two-sided (P < 0.05).