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.
Highlights
Pseudomonas syringae is a highly successful species complex that comprises important plant pathogens and epiphytes, and has recently been found in non-agricultural environments such as waterways [1, 2]
We have demonstrated the utility of both libraries through the identification of several auxotrophic and motility mutants (POI library), as well as a mutant disrupted in a predicted component of the Psa lipopolysaccharide (LPS) biosynthesis pathway (MOI library)
Preliminary characterization of Psa mutants generated by transposon mutagenesis As a starting point for transposon mutagenesis experiments involving Psa, the transformation efficiencies of two plasmids, pKRCPN1 and pKRCPN2, which carry the mini-Tn5-derived transposons Tn-DS1028uidAKm and Tn-DS1028lacZKm, respectively [23], were tested by electroporation
Summary
Pseudomonas syringae is a highly successful species complex that comprises important plant pathogens and epiphytes, and has recently been found in non-agricultural environments such as waterways [1, 2]. The isolates from New Zealand, Italy, Chile and China possessed different members of a family of integrative conjugative elements [8, 10, 11] Another canker-causing biovar of Psa has been found in Japan, which appears to be most closely related to biovar 2 from Korea [12]. Despite the relatively small number of SNPs in the core genomes of the canker-causing biovars, each has a surprisingly varied accessory genome, with different complements of genes encoding effectors and toxins [8, 13] Many of these genes are coded on active mobile genetic elements [8, 10]
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