A Bacterial Type III Secretion Assay for Delivery of Fungal Effector Proteins into Wheat
Large numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effec...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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The American Phytopathological Society
2014-03-01
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| Series: | Molecular Plant-Microbe Interactions |
| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/MPMI-07-13-0187-FI |
| _version_ | 1811261223795163136 |
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| author | Narayana M. Upadhyaya Rohit Mago Brian J. Staskawicz Michael A. Ayliffe Jeffrey G. Ellis Peter N. Dodds |
| author_facet | Narayana M. Upadhyaya Rohit Mago Brian J. Staskawicz Michael A. Ayliffe Jeffrey G. Ellis Peter N. Dodds |
| author_sort | Narayana M. Upadhyaya |
| collection | DOAJ |
| description | Large numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effective in cereal leaves. Here, we show that a nonpathogenic Pseudomonas fluorescens engineered to express the type III secretion system (T3SS) of P. syringae and the wheat pathogen Xanthomonas translucens can deliver fusion proteins containing T3SS signals from P. syringae (AvrRpm1) and X. campestris (AvrBs2) avirulence (Avr) proteins, respectively, into wheat leaf cells. A calmodulin-dependent adenylate cyclase reporter protein was delivered effectively into wheat and barley by both bacteria. Absence of any disease symptoms with P. fluorescens makes it more suitable than X. translucens for detecting a hypersensitive response (HR) induced by an effector protein with avirulence activity. We further modified the delivery system by removal of the myristoylation site from the AvrRpm1 fusion to prevent its localization to the plasma membrane which could inhibit recognition of an Avr protein. Delivery of the flax rust AvrM protein by the modified delivery system into transgenic tobacco leaves expressing the corresponding M resistance protein induced a strong HR, indicating that the system is capable of delivering a functional rust Avr protein. In a preliminary screen of effectors from the stem rust fungus Puccinia graminis f. sp. tritici, we identified one effector that induced a host genotype-specific HR in wheat. Thus, the modified AvrRpm1:effector–Pseudomonas fluorescens system is an effective tool for large-scale screening of pathogen effectors for recognition in wheat. |
| first_indexed | 2024-04-12T18:59:21Z |
| format | Article |
| id | doaj.art-1e16b1a7c1e74ae3a19eb23122c4180e |
| institution | Directory Open Access Journal |
| issn | 0894-0282 1943-7706 |
| language | English |
| last_indexed | 2024-04-12T18:59:21Z |
| publishDate | 2014-03-01 |
| publisher | The American Phytopathological Society |
| record_format | Article |
| series | Molecular Plant-Microbe Interactions |
| spelling | doaj.art-1e16b1a7c1e74ae3a19eb23122c4180e2022-12-22T03:20:12ZengThe American Phytopathological SocietyMolecular Plant-Microbe Interactions0894-02821943-77062014-03-0127325526410.1094/MPMI-07-13-0187-FIA Bacterial Type III Secretion Assay for Delivery of Fungal Effector Proteins into WheatNarayana M. UpadhyayaRohit MagoBrian J. StaskawiczMichael A. AyliffeJeffrey G. EllisPeter N. DoddsLarge numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effective in cereal leaves. Here, we show that a nonpathogenic Pseudomonas fluorescens engineered to express the type III secretion system (T3SS) of P. syringae and the wheat pathogen Xanthomonas translucens can deliver fusion proteins containing T3SS signals from P. syringae (AvrRpm1) and X. campestris (AvrBs2) avirulence (Avr) proteins, respectively, into wheat leaf cells. A calmodulin-dependent adenylate cyclase reporter protein was delivered effectively into wheat and barley by both bacteria. Absence of any disease symptoms with P. fluorescens makes it more suitable than X. translucens for detecting a hypersensitive response (HR) induced by an effector protein with avirulence activity. We further modified the delivery system by removal of the myristoylation site from the AvrRpm1 fusion to prevent its localization to the plasma membrane which could inhibit recognition of an Avr protein. Delivery of the flax rust AvrM protein by the modified delivery system into transgenic tobacco leaves expressing the corresponding M resistance protein induced a strong HR, indicating that the system is capable of delivering a functional rust Avr protein. In a preliminary screen of effectors from the stem rust fungus Puccinia graminis f. sp. tritici, we identified one effector that induced a host genotype-specific HR in wheat. Thus, the modified AvrRpm1:effector–Pseudomonas fluorescens system is an effective tool for large-scale screening of pathogen effectors for recognition in wheat.https://apsjournals.apsnet.org/doi/10.1094/MPMI-07-13-0187-FI |
| spellingShingle | Narayana M. Upadhyaya Rohit Mago Brian J. Staskawicz Michael A. Ayliffe Jeffrey G. Ellis Peter N. Dodds A Bacterial Type III Secretion Assay for Delivery of Fungal Effector Proteins into Wheat Molecular Plant-Microbe Interactions |
| title | A Bacterial Type III Secretion Assay for Delivery of Fungal Effector Proteins into Wheat |
| title_full | A Bacterial Type III Secretion Assay for Delivery of Fungal Effector Proteins into Wheat |
| title_fullStr | A Bacterial Type III Secretion Assay for Delivery of Fungal Effector Proteins into Wheat |
| title_full_unstemmed | A Bacterial Type III Secretion Assay for Delivery of Fungal Effector Proteins into Wheat |
| title_short | A Bacterial Type III Secretion Assay for Delivery of Fungal Effector Proteins into Wheat |
| title_sort | bacterial type iii secretion assay for delivery of fungal effector proteins into wheat |
| url | https://apsjournals.apsnet.org/doi/10.1094/MPMI-07-13-0187-FI |
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