Variation in conservation of the cluster for biosynthesis of the phytotoxin phaseolotoxin in Pseudomonas syringae suggests at least two events of horizontal acquisition

Abstract

Certain strains of Pseudomonas syringae pathovars phaseolicola and actinidiae and P. syringae pv. syringae strain CFBP3388 produce the chlorosis-inducing phytotoxin phaseolotoxin, which inhibits biosynthesis of arginine and polyamines. The 25 kb Pht cluster, responsible for phaseolotoxin biosynthesis, is included in a putative pathogenicity island that is nearly identical in selected strains of the pathovars phaseolicola and actinidiae, suggesting that it has been recently acquired by horizontal transfer. The history of pathogenicity islands is pivotal for our understanding of the evolution of virulence in plant pathogenic bacteria; nevertheless, our knowledge of the origins, biology and genetics of this island is currently rather limited. The aim of this work was to explore the conservation of phaseolotoxin biosynthesis genes in a broader collection of isolates and in strain CFBP3388, in order to better understand its evolution and gene dynamics. PCR, hybridization and sequence analysis showed that the island is highly conserved among a diversity of strains of pathovars phaseolicola and actinidiae, suggesting that it was acquired only once by each pathovar. Strain CFBP3388 contained DNA homologous to the Pht cluster, and an insertional mutant in the regulatory gene phtL did not synthesize the toxin. A 6.5 kb fragment from strain CFBP3388 was syntenic to the Pht cluster, but showed nucleotide identity of only 85.3%. This contrasts with an identity higher than 99.8% among clusters of pathovars phaseolicola and actinidiae, in spite of the fact that pv. syringae is phylogenetically closer to pv. phaseolicola. In addition, strain CFBP3388 lacked the four integrases that are putatively responsible for the mobility of the pathogenicity island. These results indicate that genes for the biosynthesis of phaseolotoxin have a complex evolutionary history and were acquired by pathovars of P. syringae at least twice during evolution.