ABSTRACT
Introduction. Pseudomonas syringae pv. actinidiae (Psa) has emerged as a major bacterial pathogen of kiwifruit cultivation throughout the world.Aim. We aim to introduce a CRISPR-Cas9 system, a commonly used genome editing tool, into Psa. The protocols may also be useful in other Pseudomonas species.Methodology. Using standard molecular biology techniques, we modified plasmid pCas9, which carries the CRISPR-Cas9 sequences from Streptococcus pyogenes, for use in Psa. The final plasmid, pJH1, was produced in a series of steps and is maintained with selection in both Escherichia coli and Psa.Results. We have constructed plasmids carrying a CRISPR-Cas9 system based on that of S. pyogenes, which can be maintained, under selection, in Psa. We have shown that the gene targeting capacity of the CRISPR-Cas9 system is active and that the Cas9 protein is able to cleave the targeted sites. The Cas9 was directed to several different sites in the P. syringae genome. Using Cas9 we have generated Psa transformants that no longer carry the native plasmid present in Psa, and other transformants that lack the integrative, conjugative element, Pac_ICE1. Targeting of a specific gene, a chromosomal non-ribosomal peptide synthetase, led to gene knockouts with the transformants having deletions encompassing the target site.Conclusion. We have constructed shuttle plasmids carrying a CRISPR-Cas9 system that are maintained in both E. coli and P. syringae pv. actinidiae. We have used this gene editing system to eliminate features of the accessory genome (plasmids or ICEs) from Psa and to target a single chromosomal gene.
Subject(s)
CRISPR-Cas Systems/physiology , Pseudomonas syringae/physiology , Actinidia/microbiology , CRISPR-Cas Systems/genetics , Escherichia coli/physiology , Fruit/microbiology , Gene Deletion , Gene Knockout Techniques , Gene Targeting , Genetic Engineering , Peptide Synthases/genetics , Plasmids , Pseudomonas syringae/genetics , Sequence Analysis, DNA , Streptococcus pyogenes/physiology , Whole Genome SequencingABSTRACT
We present here the complete genome sequence of M228, a Chinese biovar 3 strain of Pseudomonas syringae pv. actinidiae, a bacterial pathogen of kiwifruit. A comparison of the insertion sequence (IS) profile of M228 with that of ICMP18708, a New Zealand isolate of P. syringae pv. actinidiae, provided insight into the evolutionary history of IS elements within biovar 3.
ABSTRACT
The modern pandemic of the bacterial kiwifruit pathogen Pseudomonas syringae pv actinidiae (Psa) is caused by a particular Psa lineage. To better understand the genetic basis of the virulence of this lineage, we compare the completely assembled genome of a pandemic New Zealand strain with that of the Psa type strain first isolated in Japan in 1983. Aligning the two genomes shows numerous translocations, constrained so as to retain the appropriate orientation of the Architecture Imparting Sequences (AIMs). There are several large horizontally acquired regions, some of which include Type I, Type II or Type III restriction systems. The activity of these systems is reflected in the methylation patterns of the two strains. The pandemic strain carries an Integrative Conjugative Element (ICE) located at a tRNA-Lys site. Two other complex elements are also present at tRNA-Lys sites in the genome. These elements are derived from ICE but have now acquired some alternative secretion function. There are numerous types of mobile element in the two genomes. Analysis of these elements reveals no evidence of recombination between the two Psa lineages.
