Angelika Landgraf

The pathogenicity of many bacteria depends on the injection of effector proteins via type III secretion into eukaryotic cells in order to manipulate cellular processes. TAL (transcription activator-like) effectors from plant pathogenic Xanthomonas are important virulence factors that act as transcriptional activators in the plant cell nucleus, where they directly bind to DNA via a central domain of tandem repeats. Here, we show how target DNA specificity of TAL effectors is encoded. Two hypervariable amino acid residues in each repeat recognize one base pair in the target DNA. Recognition sequences of TAL effectors were predicted and experimentally confirmed. The modular protein architecture enabled the construction of artificial effectors with new specificities. Our study describes the functionality of a distinct type of DNA binding domain and allows the design of DNA binding domains for biotechnology.

SUMMARY The plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000 is a key model organism to study plant-pathogen interactions. We realized that two versions of this strain, which carry plasmids of different sizes, exist in our strain collections. The difference was located to a 9.4-kb deletion within the larger of the two endogenous plasmids encompassing the partitioning genes parA and parB and a putative mobile element encoding the type III effector hopAM1-2 (formerly avrPpiB2). Both plasmid variants are lost in similar frequency, indicating that the partitioning genes are not essential for stability of the plasmid. In addition, the deletion derivative strain DC3001 exhibited the same virulence towards Arabidopsis as strain DC3000. The deletion site in DC3001 is located immediately adjacent to a putative transposon that carries the effector hopX1 (formerly avrPphE), suggesting that the deletion originated from an aberrant transposition event of this element. By tagging the hopX1 transposon with an antibiotic resistance cassette on a suicide plasmid it was shown that the element is functional and produces a target site duplication of 5 bp. The plasmid also integrated into the chromosome in several cases, possibly mediated by one-ended transposition of the hopX1 transposon. This is the first report that describes an active effector-transposon. Comparison of DC3000 strains from several sources revealed that strains exist with differences in the endogenous plasmid composition.

The hrp-type III secretion (TTS) system is a key pathogenicity factor of the plant pathogen Pseudomonas syringae pv. tomato DC3000 that translocates effector proteins into the cytosol of the eukaryotic host cell. The translocation of a subset of effectors is dependent on specific chaperones. In this study an operon encoding a TTS chaperone (ShcS1) and the truncated effector HopS1' was characterized. Yeast two-hybrid analysis and pull-down assays demonstrated that these proteins interact. Using protein fusions to AvrRpt2 it was shown that ShcS1 facilitates the translocation of HopS1', suggesting that ShcS1 is a TTS chaperone for HopS1' and that amino acids 1 to 118 of HopS1' are required for translocation. P. syringae pv. tomato DC3000 carries two shcS1 homologues, shcO1 and shcS2, which are located in different operons, and both operons include additional putative effector genes. Transcomplementation experiments showed that ShcS1 and ShcO1, but not ShcS2, can facilitate the translocation of HopS1' :: AvrRpt2. To characterize the specificities of the putative chaperones, yeast two-hybrid interaction studies were performed between the three chaperones and putative target effectors. These experiments showed that both ShcS1 and ShcO1 bind to two different effectors, HopS1' and HopO1-1, that share only 16% amino acid sequence identity. Using gel filtration it was shown that ShcS1 forms homodimers, and this was confirmed by yeast two-hybrid experiments. In addition, ShcS1 is also able to form heterodimers with ShcO1. These data demonstrate that ShcS1 and ShcO1 are exceptional class IA TTS chaperones because they can bind more than one target effector.