Combination of Fluorescent Reporters for Simultaneous Monitoring of Root Colonization and Antifungal Gene Expression by a Biocontrol Pseudomonad on Cereals with Flow Cytometry

Rochat, Laurène; Péchy-Tarr, Maria; Baehler, Eric; Maurhofer, M.; Keel, Christoph

doi:10.1094/mpmi-23-7-0949

Cited by 67 publications

(57 citation statements)

References 51 publications

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“…However, very little information is available on rice root colonization by this bacterium though it has been known to have desirable characteristics such as nitrogen fixation, P-solubilization, and IAA production [5]. We used fluorescent protein as a reporter which allows easy detection of tagged bacteria in a nondestructive manner and does not require the addition of extraneous substrates [27]. Root colonization by Enterobacter agglomerans in rice and wheat has been studied earlier by electron microscopy [28].…”

Section: Discussionmentioning

confidence: 99%

Root colonization of a rice growth promoting strain of Enterobacter cloacae

Shankar

Ponraj

Ilakkiam

et al. 2011

J. Basic Microbiol.

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Enterobacter cloacae GS1 was isolated by in-planta enrichment of a rice rhizoplane bacterial community. It displayed strong seed adherence ability (2.5 × 10(5) cfu/seed) and colonized rice roots reaching up to 1.65 × 10(9) cfu/g of fresh root weight in a gnotobiotic root colonization system. E. cloacae GS1 was motile, able to solubilize tricalcium phosphate, and produced indole acetic acid like substances (15 μg/ml). As an introduced bioinoculant in non-sterile soil, E. cloacae GS1 colonized rice roots and significantly improved the fresh weight, root length, shoot length, and nitrogen content in inoculated rice seedlings as compared to uninoculated controls. This isolate was tagged with green fluorescent protein and various stages of root colonization in gnotobiotic hydroponic environment and non-sterile soil environment were followed by fluorescence microscopy. Owing to its effective root colonizing ability and growth promoting potential, Enterobacter cloacae GS1 is a promising symbiotic bioinoculant for rice.

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Section: Discussionmentioning

confidence: 99%

Root colonization of a rice growth promoting strain of Enterobacter cloacae

Shankar

Ponraj

Ilakkiam

et al. 2011

J. Basic Microbiol.

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“…This enzyme causes bacteria expressing the gene to appear blue when grown on a medium that contains the substrate analog X-gal (an inducer molecule such as IPTG is also needed under the native promoter). An example of a selectable-marker reporter in bacteria is the chloramphenicol acetyltransferase (CAT) gene, which confers resistance to the antibiotic chloramphenicol (Sørensen and Nybroe 2006;Rochat et al 2010). …”

Section: Use Of Reporter Genesmentioning

confidence: 99%

Rhizosphere and Root Colonization by Bacterial Inoculants and Their Monitoring Methods: A Critical Area in PGPR Research

Ahmad

Husain²,

Ahmad³

2011

Microbes and Microbial Technology

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Roots serve a multitude of functions in plants including anchorage, acquisition of nutrients and water, and production of exudates with growth regulatory properties. The root-soil interface, or rhizosphere, is the site of greatest biological and chemical activity within the soil matrix. Plant growth-promoting rhizobacteria (PGPR) are known to influence plant health by controlling plant pathogens or via direct enhancement of plant development in the laboratory and in greenhouse experiments. Unfortunately, however, results in the field have been less consistent. The colonization of roots by inoculated bacteria is an important step in the interaction between beneficial bacteria and the host plant. However, colonization is a complex phenomenon influenced by many biotic and abiotic parameters, some of which are only now apparent. Monitoring fate and metabolic activity of microbial inoculants as well as their impact on rhizosphere and soil microbial communities are needed to guarantee safe and reliable application, independent of whether they are genetically modified or not. The first and most crucial prerequisite for effective use of PGPRs is that strain identity and activity are continuously confirmed. A combination of both classical and molecular techniques must be perfected for more effective monitoring of inoculants strain (both genetically modified and unmodified) after release into the soil. Recent developments in techniques for studying rhizobacterial communities and detection and tracking systems of inoculated bacteria are important in future application and assessment of effectiveness and consistent performance of microbial inoculants in crop production and protection.

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“…We investigated the effects of predator-prey interactions on the regulation of the production of the extracellular toxins DAPG, PLT, PRN, and HCN using a set of autofluorescent green fluorescent protein (GFP)-and mCherry-based reporter fusions (2,32). Autofluorescent reporter fusions allow in situ measurement of gene expression patterns and have been ap-plied to monitor the expression of antifungal genes in the rhizosphere (10,32).…”

mentioning

confidence: 99%

“…Autofluorescent reporter fusions allow in situ measurement of gene expression patterns and have been ap-plied to monitor the expression of antifungal genes in the rhizosphere (10,32). The response of the bacteria to predators or associated signal molecules was investigated in batch experiments and on barley roots.…”

mentioning

confidence: 99%

“…In order to distinguish between variations of the global activity state of the bacteria and modulations of the secondary metabolism, two reporter fusions were used; a mCherry-based reporter fusion of the DAPG biosynthetic gene phlA (phlAmcherry), i.e., the most strongly expressed toxin gene of P. fluorescens CHA0 in the rhizosphere (10), and a GFP-based reporter fusion of the expression of rRNA (rrnBP1-gfp). The diverging excitation and emission spectra of GFP and mCherry avoid cross excitation of the fluorophores, allowing the simultaneous detection of both signals by flow cytometry (32).…”

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confidence: 99%

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Predator-Prey Chemical Warfare Determines the Expression of Biocontrol Genes by Rhizosphere-Associated Pseudomonas fluorescens

Jousset

Rochat

Scheu

et al. 2010

Appl Environ Microbiol

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Soil bacteria are heavily consumed by protozoan predators, and many bacteria have evolved defense strategies such as the production of toxic exometabolites. However, the production of toxins is energetically costly and therefore is likely to be adjusted according to the predation risk to balance the costs and benefits of predator defense. We investigated the response of the biocontrol bacterium Pseudomonas fluorescens CHA0 to a common predator, the free-living amoeba Acanthamoeba castellanii. We monitored the effect of the exposure to predator cues or direct contact with the predators on the expression of the phlA, prnA, hcnA, and pltA genes, which are involved in the synthesis of the toxins, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, hydrogen cyanide, and pyoluteorin, respectively. Predator chemical cues led to 2.2-, 2.0-, and 1.2-fold increases in prnA, phlA, and hcnA expression, respectively, and to a 25% increase in bacterial toxicity. The upregulation of the tested genes was related to the antiprotozoan toxicity of the corresponding toxins. Pyrrolnitrin and DAPG had the highest toxicity, suggesting that bacteria secrete a predator-specific toxin cocktail. The response of the bacteria was elicited by supernatants of amoeba cultures, indicating that water-soluble chemical compounds were responsible for induction of the bacterial defense response. In contrast, direct contact of bacteria with living amoebae reduced the expression of the four bacterial toxin genes by up to 50%, suggesting that protozoa can repress bacterial toxicity. The results indicate that predator-prey interactions are a determinant of toxin production by rhizosphere P. fluorescens and may have an impact on its biocontrol potential.

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Combination of Fluorescent Reporters for Simultaneous Monitoring of Root Colonization and Antifungal Gene Expression by a Biocontrol Pseudomonad on Cereals with Flow Cytometry

Cited by 67 publications

References 51 publications

Root colonization of a rice growth promoting strain of Enterobacter cloacae

Root colonization of a rice growth promoting strain of Enterobacter cloacae

Rhizosphere and Root Colonization by Bacterial Inoculants and Their Monitoring Methods: A Critical Area in PGPR Research

Predator-Prey Chemical Warfare Determines the Expression of Biocontrol Genes by Rhizosphere-Associated Pseudomonas fluorescens

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