A conserved microtubule-binding region in Xanthomonas XopL is indispensable for induced plant cell death reactions

Pathogenic Xanthomonas bacteria cause disease on more than 400 plant species. These Gram-negative bacteria utilize the type III secretion system to inject type III effector proteins (T3Es) directly into the plant cell cytosol where they can manipulate plant pathways to promote virulence. The host range of a given Xanthomonas species is limited, and T3E repertoires are specialized during interactions with specific plant species. Some effectors, however, are retained across most strains, such as Xanthomonas Outer Protein L (XopL). As an ‘ancestral’ effector, XopL contributes to the virulence of multiple xanthomonads, infecting diverse plant species. XopL homologs harbor a combination of a leucine-rich-repeat (LRR) domain and an XL-box which has E3 ligase activity. Despite similar domain structure there is evidence to suggest that XopL function has diverged, exemplified by the finding that XopLs expressed in plants often display bacterial species-dependent differences in their sub-cellular localization and plant cell death reactions. We found that XopL from X. euvesicatoria (XopLXe) directly associates with plant microtubules (MTs) and causes strong cell death in agroinfection assays in N. benthamiana. Localization of XopLXe homologs from three additional Xanthomonas species, of diverse infection strategy and plant host, revealed that only the distantly related X. campestris pv. campestris harbors a XopL (XopLXcc) that fails to localize to MTs and to cause plant cell death. Comparative sequence analyses of MT-binding XopLs and XopLXcc identified a proline-rich-region (PRR)/α-helical region important for MT localization. Functional analyses of XopLXe truncations and amino acid exchanges within the PRR suggest that MT-localized XopL activity is required for plant cell death reactions. This study exemplifies how the study of a T3E within the context of a genus rather than a single species can shed light on how effector localization is linked to biochemical activity. Author summary Xanthomonas Outer Proteins (Xops) are type III effector proteins originating from bacterial plant pathogens of the Xanthomonas genus. Xanthomonas uses a needle-like structure to inject a cocktail of Xops directly into plant cells where they manipulate cellular processes to promote virulence. Previous studies of individual Xops have provided valuable insights into virulence strategies used by Xanthomonas, knowledge that can be exploited to fight plant disease. However, despite rapid progress in the field, there is much about effector activity we still do not understand. Our study focuses on the effector XopL, a protein with E3 ligase activity that is important for Xanthomonas virulence. In this study we expressed XopLs in leaves of the model plant N. benthamiana and found that XopLs from different Xanthomonas species differ in their subcellular localization. XopLs from closely related species associate with the microtubule cytoskeleton and disassemble it, whereas a XopL from a distantly related species did not. This prompted a comparative analysis of these proteins, which showed how microtubule binding is achieved and how it affects the plant response to XopL.