Functional Analysis of the N Terminus of the Erwinia amylovora Secreted Effector DspA/E Reveals Features Required for Secretion, Translocation, and Binding to the Chaperone DspB/F
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 enteri...
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| Format: | Article |
| Language: | English |
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The American Phytopathological Society
2009-10-01
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| Series: | Molecular Plant-Microbe Interactions |
| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/MPMI-22-10-1282 |
| _version_ | 1811287362375778304 |
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| author | Lindsay R. Triplett Maeli Melotto George W. Sundin |
| author_facet | Lindsay R. Triplett Maeli Melotto George W. Sundin |
| author_sort | Lindsay R. Triplett |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T03:17:10Z |
| format | Article |
| id | doaj.art-202900a8508f4f42967bef237d6021de |
| institution | Directory Open Access Journal |
| issn | 0894-0282 1943-7706 |
| language | English |
| last_indexed | 2024-04-13T03:17:10Z |
| publishDate | 2009-10-01 |
| publisher | The American Phytopathological Society |
| record_format | Article |
| series | Molecular Plant-Microbe Interactions |
| spelling | doaj.art-202900a8508f4f42967bef237d6021de2022-12-22T03:04:53ZengThe American Phytopathological SocietyMolecular Plant-Microbe Interactions0894-02821943-77062009-10-0122101282129210.1094/MPMI-22-10-1282Functional Analysis of the N Terminus of the Erwinia amylovora Secreted Effector DspA/E Reveals Features Required for Secretion, Translocation, and Binding to the Chaperone DspB/FLindsay R. TriplettMaeli MelottoGeorge W. SundinDspA/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.https://apsjournals.apsnet.org/doi/10.1094/MPMI-22-10-1282 |
| spellingShingle | Lindsay R. Triplett Maeli Melotto George W. Sundin Functional Analysis of the N Terminus of the Erwinia amylovora Secreted Effector DspA/E Reveals Features Required for Secretion, Translocation, and Binding to the Chaperone DspB/F Molecular Plant-Microbe Interactions |
| title | Functional Analysis of the N Terminus of the Erwinia amylovora Secreted Effector DspA/E Reveals Features Required for Secretion, Translocation, and Binding to the Chaperone DspB/F |
| title_full | Functional Analysis of the N Terminus of the Erwinia amylovora Secreted Effector DspA/E Reveals Features Required for Secretion, Translocation, and Binding to the Chaperone DspB/F |
| title_fullStr | Functional Analysis of the N Terminus of the Erwinia amylovora Secreted Effector DspA/E Reveals Features Required for Secretion, Translocation, and Binding to the Chaperone DspB/F |
| title_full_unstemmed | Functional Analysis of the N Terminus of the Erwinia amylovora Secreted Effector DspA/E Reveals Features Required for Secretion, Translocation, and Binding to the Chaperone DspB/F |
| title_short | Functional Analysis of the N Terminus of the Erwinia amylovora Secreted Effector DspA/E Reveals Features Required for Secretion, Translocation, and Binding to the Chaperone DspB/F |
| title_sort | functional analysis of the n terminus of the erwinia amylovora secreted effector dspa e reveals features required for secretion translocation and binding to the chaperone dspb f |
| url | https://apsjournals.apsnet.org/doi/10.1094/MPMI-22-10-1282 |
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