Prem P. Kandel, Hongyu Chen and Leonardo De La Fuente
6 July 2018
Applied and Environmental Microbiology: http://aem.asm.org/content/early/2018/07/03/AEM.01167-18.abstract
Twitching motility is one of the major virulence factors of the plant pathogenic bacterium Xylella fastidiosa, and is mediated by type IV pili (TFP) present at one of the cell poles. Genome analysis of X. fastidiosa showed the presence of at least four paralogs of the gene pilA that encodes TFP major pilin subunit. However, whether all these paralogs have a functional role in TFP structure and function is unknown. Here, using a short and reliable protocol based on overlap-extension PCR and natural transformation, deletion mutants of two pilA paralogs (pilA1 PD1924 and pilA2 PD1926) were generated in two X. fastidiosa subspecies fastidiosa strains WM1-1 and TemeculaL, followed by assessment of twitching motility and biofilm formation. Deletion of pilA2 caused loss of twitching motility, whereas deletion of pilA1 did not influence twitching motility but caused hyperpiliation and extended distribution of TFP along the sides of the cell. Loss of twitching motility due to pilA2 deletion was restored when a wild-type copy of the pilA2 gene was added at a neutral site in the genome of mutants in both wild-type backgrounds. This study demonstrates that PCR templates generated by overlap-extension PCR can be successfully used to rapidly generate gene knockouts and perform genetic complementation in X. fastidiosa, and that twitching motility in X. fastidiosa is controlled by regulating the transcription of the major pilin subunit pilA2.
Importance The bacterial plant pathogen Xylella fastidiosa causes incurable diseases in multiple hosts including grape, citrus, and blueberry. Historically restricted to the Americas, recently it was found to cause epidemics in olives in Italy, and infect other hosts in Europe and Asia. In this manuscript we report a short protocol to create deletion and complemented mutants using fusion PCR and natural transformation. We also determined the distinct function of two pilin paralogs, the main structural component of TFP involved in twitching motility which allows this bacterium to move inside the xylem vessels against the flow. One of the paralogs is needed for twitching movement while the other does not have an effect on motility, but influences the number and position of TFP. Since twitching motility is fundamental for the virulence of this xylem-limited bacterium, this study contributes to the understanding of the regulation of virulence by this pathogen.