Secretion and translocation signals and DspB/F-binding domains in the type III effector DspA/E of Erwinia amylovora

Abstract

DspA/E is a type III effector of Erwinia amylovora, the bacterial pathogen that causes fire blight disease in roseaceous plants. This effector is indispensable for disease development, and it is translocated into plant cells. A DspA/E-specific chaperone, DspB/F, is necessary for DspA/E secretion and possibly for its translocation. In this work, DspB/F-binding sites and secretion and translocation signals in the DspA/E protein were determined. Based on yeast two-hybrid assays, DspB/F was found to bind DspA/E within the first 210 amino acids of the protein. Surprisingly, both DspB/F and OrfA, the putative chaperone of Eop1, also interacted with the C-terminal 1059 amino acids of DspA/E; this suggests another chaperone-binding site. Secretion and translocation assays using serial N-terminal lengths of DspA/E fused with the active form of AvrRpt2 revealed that at least the first 109 amino acids, including the first N-terminal chaperone-binding motif and DspB/F, were required for efficient translocation of DspA/E, although the first 35 amino acids were sufficient for its secretion and the presence of DspB/F was not required. These results indicate that secretion and translocation signals are present in the N terminus of DspA/E, and that at least one DspB/F-binding motif is required for efficient translocation into plant cells.

Fire Blight: Applied Genomic Insights of the Pathogen and Host

“…These contradictory results indicate that the requirement for the DspB/F chaperone is influenced by the Cterminal part of the protein. In accordance with this, it was found that DspB/F also interacts with the C-terminal half of DspA/E (80).…”

“…Structural modeling of DspB/F and identification of candidate DspB/F residues involved in virulence and DspA/E interaction confirm that DspB/F functions as a classical T3SS chaperone (115). Neither disruption of the N-terminal DspA/E chaperonebinding domain nor deletion of the dspB/F gene had a significant impact on translocation levels of N-terminal DspA/E-CyaA fusions (114), but the presence of DspB/F strongly enhances the translocation of a DspA/E-AvrRpt2 fusion protein into plant cells (80). These contradictory results indicate that the requirement for the DspB/F chaperone is influenced by the Cterminal part of the protein.…”

“…Two independent groups identified DspA/E secretion and translocation signals in the Nterminal part of the DspA/E protein and DspB/F-binding sites within amino acids 51 through 100 of DspA/E (80,114). A binding site at this location is classically detected for almost all reported T3SS chaperones with their cognate T3Es (84,115).…”

“…DspA/E secretion depends on a specific chaperone called DspB/F, which prevents intrabacterial DspA/E degradation (35,80). Two independent groups identified DspA/E secretion and translocation signals in the Nterminal part of the DspA/E protein and DspB/F-binding sites within amino acids 51 through 100 of DspA/E (80,114).…”

Type III-Dependent Translocation of HrpB2 by a Nonpathogenic hpaABC Mutant of the Plant-Pathogenic Bacterium Xanthomonas campestris pv. vesicatoria

“…However, minimal transport signals as well as protein regions required for the HpaB-dependent translocation of XopA and HrpF have not yet been identified. To date, separate secretion and translocation signals have been described in effector proteins from the animal-pathogenic bacterium Yersinia and the plant pathogen Erwinia amylovora (69)(70)(71)(72). In the case of the Yersinia effector protein YopE, the secretion specificity is determined by the minimal secretion signal (73).…”