Highest scoring region to lowest scoring region: Preetinanda Panda Preetinanda.Panda@lincolnuni.ac.nz Bio-Protection Research Centre Lincoln University Christchurch, New Zealand Andrew Pitman1,2, Mark Fiers2, Karen Armstrong1, Ian Toth3 1 Bio-Protection Research Centre, Lincoln University, Christchurch, New Zealand 2 The New Zealand Institute for Plant & Food Research, Lincoln, New Zealand 3 James Hutton Institute, Dundee, Scotland Identification of a novel Non-Ribosomal Peptide Synthetase required for pathogenicity of Pectobacterium on potatoes Introduction Pectobacterium carotovorum subspecies brasiliensis (Pbr) is an aggressive phytopathogen of potato causing blackleg of stems and soft rot of tubers in numerous potato growing regions worldwide (1). Genome sequencing of Pbr NZEC1, a highly virulent isolate collected from potatoes in New Zealand, has revealed several putative virulence factors encoded on Horizontally Acquired Islands (HAIs). Comparative genomics of Pbr NZEC1 with other Pectobacterium strains (Fig. 1) showed that one HAI carries a novel non-ribosomal peptide synthetase (NRPS) cluster, which was present in all other blackleg-causing Pectobacterium strains in this study (e.g. P. atrosepticum SCRI1043 (Pba SCRI1043)), but absent in those unable to cause blackleg (e.g. P. carotovorum subsp. carotovorum PC1). The NRPS had similarity to the synthetase genes in Pseudomonas syringae pv. syringae that encode syringomycin, a putative phytotoxin involved in causing necrosis by plant pathogens in host plants (2,3). Here, genes encoding the synthetase and its related ABC transporter were inactivated in Pbr NZEC1 and Pba SCRI1043. The aim of this experiment was to assess the role of the NRPS in virulence of both Pbr and Pba species by comparing the virulence of the resulting knockout mutants with wild type strains using subsequent pathogenicity assays on potato stems and tubers. Methods The genome of Pbr NZEC1 was sequenced using 454 pyrosequencing (Liverpool University Sequencing Facility, UK). Reciprocal best hit (RBH) analyses were carried out against five genomes (ftp.ncbi.nih.gov/genomes/Bacteria) for each coding sequence in Pbr NZEC1 using blastx and based on the sequence identity and percentage alignment a BIT score was assigned to each hit. The results of the RBH analyses were visualised using the Circos package (4). Knockout mutants were constructed using Gene Allelic Mutagenesis and Triparental Conjugation. Blackleg and soft rot assays were conducted on potato plants and tubers (cv. Ilam Hardy) using the resulting mutants. 12 replicates were used for each treatment. Results NRPS knockout mutants of Pbr NZEC1 and Pba SCRI1043 exhibited significantly reduced virulence on stems and tubers (Fig. 2 and 3). Results for Pba SCRI1043 not shown. Highest scoring region to lowest scoring region: Red - Green - Blue A B C Figure 3. Blackleg lesion development on potato plant stems 3 days after infection with (A) wild type Pbr NZEC1 and (B) a NRPS knockout mutant. (C) Bar graph showing the mean lesion length for infected Pbr NZEC1 and the NRPS mutant. C A B Figure 1. Comparison of Pbr NZEC1 with other blackleg and non-blackleg causing Pectobacterium strains. Colour coded regions are regions of similarity between the Pectobacterium genomes; red coloured regions are highly conserved. White gaps represent the unique regions associated with each genome. Pba – P. atrosepticum, Pcc – P. carotovorum subsp. carotovorum, Pecwa – P. wasabiae. Figure 2. Soft rotting of potato tubers 3 days after infection with (A) wild type Pbr NZEC1 and (B) a NRPS knockout mutant. (C) Bar graph showing the mean weight of macerated tissue obtained from tubers infected with Pbr NZEC1 and the NRPS mutant. Conclusions Comparative genomics led to the identification of a novel NRPS present in all blackleg-causing Pectobacterium species. Inactivation of the NRPS abolished the ability of both Pectobacterium species (Pbr NZEC1 and Pba SCRI1043) to cause blackleg and soft rot. Since the NRPS cluster is in all blackleg strains compared in this study, we hypothesise that this peptide synthetase is an important virulence factor in all blackleg-causing Pectobacterium species. The role of NRPS in causing infection and manipulating plant defences is yet to be studied. References (1) Duarte et al. (2004). Journal of Applied Microbiology, 96, 535-45 (2) Bell et al. (2004). PNAS, 30, 11105–10 (3) Bender et al. (1999). Microbiol. Mol. Biol. Rev, 63, 266-92 (4) Krzywinski et al. (2009). Genome Research, 19, 1639-4 Acknowledgements This research was funded by the New Zealand Tertiary Education Commission