A self-assembling split Nano luciferase-based assay for investigating Pseudomonas syringae effector secretion
Abstract Many Gram-negative pathogens employ the type III secretion system (T3SS) to deliver effector proteins into host cells, thereby modulating host cellular processes and suppressing host immunity to facilitate pathogenesis and colonization. In this study, we developed a straightforward, rapid,...
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| Format: | Article |
| Language: | English |
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Springer
2024-02-01
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| Series: | Stress Biology |
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| Online Access: | https://doi.org/10.1007/s44154-024-00152-2 |
| _version_ | 1797275855875997696 |
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| author | Pei Miao Jian-Min Zhou Wei Wang |
| author_facet | Pei Miao Jian-Min Zhou Wei Wang |
| author_sort | Pei Miao |
| collection | DOAJ |
| description | Abstract Many Gram-negative pathogens employ the type III secretion system (T3SS) to deliver effector proteins into host cells, thereby modulating host cellular processes and suppressing host immunity to facilitate pathogenesis and colonization. In this study, we developed a straightforward, rapid, and quantitative method for detecting T3SS-mediated translocation of Pseudomonas syringae effectors using a self-assembling split Nano luciferase (Nluc)-based reporter system. It was demonstrated that this system can detect effector secretion in vitro with an exceptionally high signal-to-noise ratio and sensitivity, attributed to the strong affinity between the split domains of Nluc and the intense luminescence generated by functional Nluc. During natural infections, effectors fused to a small C-terminal fragment of Nluc were successfully translocated into plant cells and retained their virulence functions. Furthermore, upon infection of plants expressing the N-terminal fragment of Nluc with these P. syringae strains, functional Nluc proteins were spontaneously assembled and produced bright luminescence, demonstrating that this system enables the straightforward and rapid assessment of P. syringae T3SS-mediated effector translocation during natural infections. In conclusion, the self-assembling split Nluc-based reporting system developed in this study is suitable for efficient in vitro and in planta detection of effectors secreted via T3SS. |
| first_indexed | 2024-03-07T15:20:01Z |
| format | Article |
| id | doaj.art-7b12f6ee592840919405f7aa2ec48e29 |
| institution | Directory Open Access Journal |
| issn | 2731-0450 |
| language | English |
| last_indexed | 2024-03-07T15:20:01Z |
| publishDate | 2024-02-01 |
| publisher | Springer |
| record_format | Article |
| series | Stress Biology |
| spelling | doaj.art-7b12f6ee592840919405f7aa2ec48e292024-03-05T17:43:34ZengSpringerStress Biology2731-04502024-02-01411610.1007/s44154-024-00152-2A self-assembling split Nano luciferase-based assay for investigating Pseudomonas syringae effector secretionPei Miao0Jian-Min Zhou1Wei Wang2State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of SciencesState Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of SciencesState Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of SciencesAbstract Many Gram-negative pathogens employ the type III secretion system (T3SS) to deliver effector proteins into host cells, thereby modulating host cellular processes and suppressing host immunity to facilitate pathogenesis and colonization. In this study, we developed a straightforward, rapid, and quantitative method for detecting T3SS-mediated translocation of Pseudomonas syringae effectors using a self-assembling split Nano luciferase (Nluc)-based reporter system. It was demonstrated that this system can detect effector secretion in vitro with an exceptionally high signal-to-noise ratio and sensitivity, attributed to the strong affinity between the split domains of Nluc and the intense luminescence generated by functional Nluc. During natural infections, effectors fused to a small C-terminal fragment of Nluc were successfully translocated into plant cells and retained their virulence functions. Furthermore, upon infection of plants expressing the N-terminal fragment of Nluc with these P. syringae strains, functional Nluc proteins were spontaneously assembled and produced bright luminescence, demonstrating that this system enables the straightforward and rapid assessment of P. syringae T3SS-mediated effector translocation during natural infections. In conclusion, the self-assembling split Nluc-based reporting system developed in this study is suitable for efficient in vitro and in planta detection of effectors secreted via T3SS.https://doi.org/10.1007/s44154-024-00152-2Pseudomonas syringaeT3SS effectorsSecretionNano luciferase |
| spellingShingle | Pei Miao Jian-Min Zhou Wei Wang A self-assembling split Nano luciferase-based assay for investigating Pseudomonas syringae effector secretion Stress Biology Pseudomonas syringae T3SS effectors Secretion Nano luciferase |
| title | A self-assembling split Nano luciferase-based assay for investigating Pseudomonas syringae effector secretion |
| title_full | A self-assembling split Nano luciferase-based assay for investigating Pseudomonas syringae effector secretion |
| title_fullStr | A self-assembling split Nano luciferase-based assay for investigating Pseudomonas syringae effector secretion |
| title_full_unstemmed | A self-assembling split Nano luciferase-based assay for investigating Pseudomonas syringae effector secretion |
| title_short | A self-assembling split Nano luciferase-based assay for investigating Pseudomonas syringae effector secretion |
| title_sort | self assembling split nano luciferase based assay for investigating pseudomonas syringae effector secretion |
| topic | Pseudomonas syringae T3SS effectors Secretion Nano luciferase |
| url | https://doi.org/10.1007/s44154-024-00152-2 |
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