Transformation of Xylella fastidiosa subspecies pauca strain De Donno

Xylella fastidiosa subsp. pauca strain ‘De Donno’ has been recently identified as the causal agent of a severe disease affecting olive trees in a wide area of the Apulia Region (Italy). While insights on the genetics and epidemiology of this virulent strain have been gained, the complex network of interactions with the main susceptible host remains to be explored. A fundamental tool for understanding such interactions is the development of bacterial mutants for functional analysis of genes involved in the host recognition, pathogenicity and insect transmission. Experimental studies have demonstrated natural competence of X. fastidiosa in the uptake of exogenous genetic material, a feature exploited for site-specific introduction or deletion of genes through homologous recombination. Nevertheless, numerous studies have shown that several factors may affect X. fastidiosa transformation efficiency, including growth rate, twitching motility, sequence similarity, and the presence of Restriction-Modification systems that cleave incoming DNA. On this basis, two different plasmids containing the chromosomal replication origin (oriC) of X. fastidiosa and E. coli were used to transform X. fastidiosa ‘De Donno’ in order to produce a GFP-expressing and a knockout strain for the rpfF gene, a crotonase producing a diffusible signal factor (DSF), involved in the quorum-sensing system. Repeated attempts to exploit natural competence, introducing the donor plasmids into X. fastidiosa ‘De Donno’ failed, highlighting the critical role of genetic diversity in recombination performances of this pathogen. Conversely, GFP and RpfF- mutants were successfully obtained by co-electroporation in the presence of an inhibitor of the Type I R-M system, that had been proved to impact the stable acquisition of foreign DNA by X. fastidiosa subsp. fastidiosa. Availability of mutants for one of the most virulent strains of X. fastidiosa opens for new explorations of host-microbe interactions, important to elucidate mechanisms underpinning the differential responses recorded upon infections of different olive cultivars and toward the implementation of strategies to mitigate the impact of the disease