We have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twinarginine protein transport (Tat 1 ) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analyzed with the authentic Tat substrate pOEC16, reached routinely 20 -25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutant TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional mutant analyses. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport.
PathogenicXanthomonasbacteria 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 givenXanthomonasspecies 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 fromX. euvesicatoria(XopLXe) directly associates with plant microtubules (MTs) and causes strong cell death in agroinfection assays inN. benthamiana. Localization of XopLXehomologs from three additionalXanthomonasspecies, of diverse infection strategy and plant host, revealed that only the distantly relatedX. campestrispv.campestrisharbors a XopL (XopLXcc) that fails to localize to MTs and to cause plant cell death. Comparative sequence analyses of MT-binding XopLs and XopLXccidentified a proline-rich-region (PRR)/α-helical region important for MT localization. Functional analyses of XopLXetruncations 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 summaryXanthomonas Outer Proteins (Xops) are type III effector proteins originating from bacterial plant pathogens of theXanthomonasgenus.Xanthomonasuses 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 byXanthomonas,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 forXanthomonasvirulence. In this study we expressed XopLs in leaves of the model plantN. benthamianaand found that XopLs from differentXanthomonasspecies 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.
We have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twinarginine protein transport (Tat 1 ) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analyzed with the authentic Tat substrate pOEC16, reached routinely 20 -25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutant TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional mutant analyses. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport. Keywords Twin-arginine translocation, thylakoid membrane, transport reconstitution, heterologous overexpression, protein purification 1 The abbreviations used are: Tat, twin-arginine translocation; OEC16, 16 kDa subunit of the oxygen-evolving system associated with photosystem II; RTS, Rapid Translation System; Hepes, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; BrCN, cyanogen bromide -3 -
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