Combination of Fluorescent Reporters for Simultaneous Monitoring of Root Colonization and Antifungal Gene Expression by a Biocontrol Pseudomonad on Cereals with Flow Cytometry
Some root-associated pseudomonads sustain plant growth by suppressing root diseases caused by pathogenic fungi. We investigated to which extent select cereal cultivars influence expression of relevant biocontrol traits (i.e., root colonization efficacy and antifungal activity) in Pseudomonas fluores...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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The American Phytopathological Society
2010-07-01
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| Series: | Molecular Plant-Microbe Interactions |
| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/MPMI-23-7-0949 |
| _version_ | 1811278055625195520 |
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| author | Laurène Rochat Maria Péchy-Tarr Eric Baehler Monika Maurhofer Christoph Keel |
| author_facet | Laurène Rochat Maria Péchy-Tarr Eric Baehler Monika Maurhofer Christoph Keel |
| author_sort | Laurène Rochat |
| collection | DOAJ |
| description | Some root-associated pseudomonads sustain plant growth by suppressing root diseases caused by pathogenic fungi. We investigated to which extent select cereal cultivars influence expression of relevant biocontrol traits (i.e., root colonization efficacy and antifungal activity) in Pseudomonas fluorescens CHA0. In this representative plant-beneficial bacterium, the antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN), pyoluteorin (PLT), and hydrogen cyanide (HCN) are required for biocontrol. To monitor host plant effects on the expression of biosynthetic genes for these compounds on roots, we developed fluorescent dual-color reporters suited for flow cytometric analysis using fluorescence-activated cell sorting (FACS). In the dual-label strains, the constitutively expressed red fluorescent protein mCherry served as a cell tag and marker for root colonization, whereas reporter fusions based on the green fluorescent protein allowed simultaneous recording of antifungal gene expression within the same cell. FACS analysis revealed that expression of DAPG and PRN biosynthetic genes was promoted in a cereal rhizosphere, whereas expression of PLT and HCN biosynthetic genes was markedly less sustained. When analyzing the response of the bacterial reporters on roots of a selection of wheat, spelt, and triticale cultivars, we were able to detect subtle species- and cultivar-dependent differences in colonization and DAPG and HCN gene expression levels. The expression of these biocontrol traits was particularly favored on roots of one spelt cultivar, suggesting that a careful choice of pseudomonad–cereal combinations might be beneficial to biocontrol. Our approach may be useful for selective single-cell level analysis of plant effects in other bacteria–root interactions. |
| first_indexed | 2024-04-13T00:27:49Z |
| format | Article |
| id | doaj.art-7425ae4f79e2499082a3df24e80347d9 |
| institution | Directory Open Access Journal |
| issn | 0894-0282 1943-7706 |
| language | English |
| last_indexed | 2024-04-13T00:27:49Z |
| publishDate | 2010-07-01 |
| publisher | The American Phytopathological Society |
| record_format | Article |
| series | Molecular Plant-Microbe Interactions |
| spelling | doaj.art-7425ae4f79e2499082a3df24e80347d92022-12-22T03:10:33ZengThe American Phytopathological SocietyMolecular Plant-Microbe Interactions0894-02821943-77062010-07-0123794996110.1094/MPMI-23-7-0949Combination of Fluorescent Reporters for Simultaneous Monitoring of Root Colonization and Antifungal Gene Expression by a Biocontrol Pseudomonad on Cereals with Flow CytometryLaurène RochatMaria Péchy-TarrEric BaehlerMonika MaurhoferChristoph KeelSome root-associated pseudomonads sustain plant growth by suppressing root diseases caused by pathogenic fungi. We investigated to which extent select cereal cultivars influence expression of relevant biocontrol traits (i.e., root colonization efficacy and antifungal activity) in Pseudomonas fluorescens CHA0. In this representative plant-beneficial bacterium, the antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN), pyoluteorin (PLT), and hydrogen cyanide (HCN) are required for biocontrol. To monitor host plant effects on the expression of biosynthetic genes for these compounds on roots, we developed fluorescent dual-color reporters suited for flow cytometric analysis using fluorescence-activated cell sorting (FACS). In the dual-label strains, the constitutively expressed red fluorescent protein mCherry served as a cell tag and marker for root colonization, whereas reporter fusions based on the green fluorescent protein allowed simultaneous recording of antifungal gene expression within the same cell. FACS analysis revealed that expression of DAPG and PRN biosynthetic genes was promoted in a cereal rhizosphere, whereas expression of PLT and HCN biosynthetic genes was markedly less sustained. When analyzing the response of the bacterial reporters on roots of a selection of wheat, spelt, and triticale cultivars, we were able to detect subtle species- and cultivar-dependent differences in colonization and DAPG and HCN gene expression levels. The expression of these biocontrol traits was particularly favored on roots of one spelt cultivar, suggesting that a careful choice of pseudomonad–cereal combinations might be beneficial to biocontrol. Our approach may be useful for selective single-cell level analysis of plant effects in other bacteria–root interactions.https://apsjournals.apsnet.org/doi/10.1094/MPMI-23-7-0949 |
| spellingShingle | Laurène Rochat Maria Péchy-Tarr Eric Baehler Monika Maurhofer Christoph Keel Combination of Fluorescent Reporters for Simultaneous Monitoring of Root Colonization and Antifungal Gene Expression by a Biocontrol Pseudomonad on Cereals with Flow Cytometry Molecular Plant-Microbe Interactions |
| title | Combination of Fluorescent Reporters for Simultaneous Monitoring of Root Colonization and Antifungal Gene Expression by a Biocontrol Pseudomonad on Cereals with Flow Cytometry |
| title_full | Combination of Fluorescent Reporters for Simultaneous Monitoring of Root Colonization and Antifungal Gene Expression by a Biocontrol Pseudomonad on Cereals with Flow Cytometry |
| title_fullStr | Combination of Fluorescent Reporters for Simultaneous Monitoring of Root Colonization and Antifungal Gene Expression by a Biocontrol Pseudomonad on Cereals with Flow Cytometry |
| title_full_unstemmed | Combination of Fluorescent Reporters for Simultaneous Monitoring of Root Colonization and Antifungal Gene Expression by a Biocontrol Pseudomonad on Cereals with Flow Cytometry |
| title_short | Combination of Fluorescent Reporters for Simultaneous Monitoring of Root Colonization and Antifungal Gene Expression by a Biocontrol Pseudomonad on Cereals with Flow Cytometry |
| title_sort | combination of fluorescent reporters for simultaneous monitoring of root colonization and antifungal gene expression by a biocontrol pseudomonad on cereals with flow cytometry |
| url | https://apsjournals.apsnet.org/doi/10.1094/MPMI-23-7-0949 |
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