title: Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae.
abstract: Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a 'phenotype of interest' (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with 'Fuzzy-Spreader'-like morphologies were also identified through a visual screen. The second, a 'mutant of interest' (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either by DNA-binding proteins or by the architecture of the nucleoid.
authors.affiliation: Bioprotection Portfolio, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.|Laboratory of Molecular Plant Pathology, Institute of Agriculture and Environment, Massey University, Palmerston North, New Zealand.|Bio-Protection Research Centre, New Zealand.
authors.forename: Carl H
authors.initials: CH
authors.lastname: Mesarich
authors.affiliation: Bioprotection Portfolio, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Rees-George
authors.affiliation: Bio-Protection Research Centre, New Zealand.|School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.
authors.forename: Paul P
authors.initials: PP
authors.lastname: Gardner
authors.affiliation: Bio-Protection Research Centre, New Zealand.|School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.
authors.forename: Fatemeh Ashari
authors.initials: FA
authors.lastname: Ghomi
authors.affiliation: Department of Biochemistry, University of Otago, Dunedin, New Zealand.
authors.forename: Monica L
authors.initials: ML
authors.lastname: Gerth
authors.affiliation: Bioprotection Portfolio, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.
authors.forename: Mark T
authors.initials: MT
authors.lastname: Andersen
authors.affiliation: Bioprotection Portfolio, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.
authors.forename: Erik H A
authors.initials: EH
authors.lastname: Rikkerink
authors.affiliation: Bio-Protection Research Centre, New Zealand.|Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Fineran
authors.affiliation: Bioprotection Portfolio, The New Zealand Institute for Plant & Food Research Limited, Auckland, New Zealand.|Bio-Protection Research Centre, New Zealand.|School of Biological Sciences, University of Auckland, Auckland, New Zealand.
authors.forename: Matthew D
authors.identifier: http://orcid.org/0000-0003-0192-9041
authors.initials: MD
authors.lastname: Templeton
mesh_terms: D029042:Actinidia / Q000382:microbiology*; D004251:DNA Transposable Elements; D005638:Fruit / Q000382:microbiology*; D015723:Gene Library; D054643:INDEL Mutation; D016254:Mutagenesis, Insertional; D010935:Plant Diseases / Q000382:microbiology*; D044224:Pseudomonas syringae / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements