Lindsay R. Triplett,1,2
Maeli Melotto,3 and
George W. Sundin1,2
1Department of Plant Pathology and 2Center for Microbial Pathogenesis, Michigan State University, East Lansing, MI 48824, U.S.A.; 3Department of Biology, University of Texas at Arlington, Arlington, TX 76019, U.S.A.
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Accepted 28 May 2009.
DspA/E is a type III secreted effector protein required for pathogenicity in the apple and pear pathogen Erwinia amylovora, and DspB/F is a small chaperone protein involved in DspA/E secretion. While the secretion and translocation signals of many type III secretion effector proteins in human enteric pathogens have been characterized extensively, relatively little is known about the translocation requirements of many effectors in plant pathogens, including large DspE-like proteins. In this study, we report a functional analysis of the N terminus of DspE. The minimal requirements for secretion, translocation, and chaperone binding were characterized. Translocation assays using an adenylate cyclase (CyaA) reporter indicated that the first 51 amino acids of DspE were sufficient for translocation and that 150 amino acids were required for optimal translocation levels. The minimal translocation signal corresponded with the requirements for secretion into culture media. Mutations of conserved regions in amino acids 2 through 10 and 31 through 40 were found to influence translocation levels of an N-terminal DspE-CyaA fusion. Yeast two-hybrid and in-vitro pull-down assays revealed a chaperone-binding site within amino acids 51 through 100 of DspE and binding to DspF in this region was disrupted by specific mutations. However, neither disruption of the chaperone-binding domain nor deletion of the dspF gene had a significant impact on translocation levels of N-terminal DspE-CyaA fusions. Our results indicate that the minimal translocation signal of DspE is not coincident with the signal for DspF binding and that translocation of the N terminus of DspE is not dependent on the N-terminal DspF-binding domain.
© 2009 The American Phytopathological Society