From endophytic to plant-pathogenic bacteria and return: unraveling evolution of pathogenicity and virulence

Endophytes are increasingly studied for their potential to protect plants from pathogenic diseases, particularly in agriculture. Their unique feature is their ability to colonize the internal tissues of plants without causing any symptoms. While Curtobacterium flaccumfaciens (Cf) has been identified as an endophyte, and has been found effective as a biocontrol agent against phytopathogenic bacteria like Xylella fastidiosa, Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) is recognized as a plant pathogen, causing bean bacterial wilt. Phytopathogenic bacteria possess sophisticated mechanisms enabling the successful colonization of host plants and initiating infection. Recent studies have highlighted the involvement of pathogenicity islands (PAI) and specific genes like cellulases, proteases, and pectinases in the pathogenicity processes. This suggests that horizontal gene transfer among phylogenetically related microorganisms may have led to the acquisition by Cf of genetic traits conferring pathogenicity towards Cff evolution. Here for the first time, a mutagenesis protocol has been developed for the EU quarantine bacterium Cff. Knock-out mutants for a protease, a cellulase, a pectinase, and a PAI, as well as for their combinations, were obtained through marker exchange mutagenesis. The mutants exhibited variable roles in Cff virulence when artificially inoculated on bean plants. In vitro tests revealed a significant reduction (50-70%) in proteolytic and cellulolytic activity for some mutants. However, reduced pectinase activity was shown using Citrus pectin, which needs to be further investigated. In addition, a more reduced degradative capacity was observed in double and triple mutants compared with singles, particularly in the protease activity assay. Hence, it appears that PAI and the identified genes may play a significant role in virulence mechanisms, which may have conferred at least some disease-causing capacity from Cf to Cff. In conclusion, this study raises new answers about virulence, serving as a valuable case study for understanding the acquisition of pathogenicity traits in bacteria.