Natural competence distribution among Xylella fastidiosa strains and construction of a mutant library for RB-TnSeq

Abstract

The plant pathogen Xylella fastidiosa is a gram-negative, fastidious, xylem-limited, vector-transmitted bacterium, causing severe diseases on many economically important plants such as grapes, citrus, coffee, olive and almond among others. The outbreak of X. fastidiosa in new geographic regions and new hosts is a serious problem. Extensive evidence indicates that homologous recombination (HR) may be one of the major forces contributing to the appearance of new genotypes, host adaptation and host shifts in X. fastidiosa. Natural competence has been considered one of the most important means of HR in X. fastidiosa. To date, natural competence among X. fastidiosa has been described experimentally only for a few strains. However, little is known about the distribution of natural competence among diverse X. fastidiosa strains, to what extent natural competence correlates with HR detected in the core-genome in silico, and genetic mechanisms of natural competence in X. fastidiosa. Therefore, we characterized natural competence in 71 X. fastidiosa strains by measuring plasmid recombination efficiency; analyzed correlation between natural competence in vitro and HR in the genomes; and constructed a mutant library to screen genetic elements essential for natural competence using Random-Barcoded Transposon Sequencing (RB-TnSeq). Results showed that recombination rates differed extensively among strains with diverse origins specially between different subspecies. Surprisingly, results demonstrated that natural competence in vitro was slightly but significantly negatively correlated with HR in the genomes. Genome-wide association analysis showed that quorum sensing and phage-related genes may be associated with natural competence, although there was a high likelihood of false-positive associations probably due to the small and skewed sample size. On the other hand, construction of a saturated RB-TnSeq mutant library is still ongoing based on the success of building a mini library despite its bias. Several steps of the library construction have been established, but additional troubleshooting is needed before an optimal library will be obtained for future research. Our findings suggest that natural competence may evolve differently among X. fastidiosa strains, and that other mechanisms in addition to natural competence may also be important for HR and host adaptation in this pathogen. Future efforts put on RB-TnSeq would help understand mechanisms of natural competence, and further clarify its role in HR and evolution in this pathogen.