Unveiling the mechanisms determining tissue specificity in the plant pathogen Clavibacter michiganensis using experimental evolution

The plant pathogenic bacterium Clavibacter michiganensis (Cm) is a systemic vascular pathogen that thrives in both the host xylem vessels and intracellular apoplast tissues.To identify traits and loci associated with adaptation to each of these two microenvironments, we conducted tissue-specific experimental evolution, adapting twenty independent clone lineages to either vascular or apoplastic lifestyles through repeated passaging in tomato stems or leaves.Evolved Cm populations were characterized for aggression in the host and virulence-associated traits.These characterizations demonstrated clear differential associations of virulence-associated traits to adapted tissue.The majority of vascular-adapted clones displayed enhanced biofilm formation, reduced cellulase activity, reduced exopolysaccharides (EPS) production, and attenuated virulence compared to the parent clone.On the other hand, apoplast-adapted clones displayed reduced biofilm formation and enhanced EPS production while maintaining their aggression.To identify loci associated with tissue specificity, we determined the complete genome sequence of all plant-evolved clones and used comparative genomics to identify genome alterations.Interestingly, six out of the ten vascular-adapted clones harbored two independent mutations in a putative Cro/CI-type transcriptional regulator, suggesting that this regulator may play a role in vascular association.These experiments provide new insights into the tissue adaptation of plant pathogenic bacteria and suggest a role for cellulase activity and biofilm formation in tissue specificity.