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 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.
Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.
Laurène
L
Rochat
Maria
M
Péchy-Tarr
Eric
E
Baehler
Monika
M
Maurhofer
Christoph
C
Keel
D000935:Antifungal Agents / Q000378:metabolism*; D002523:Edible Grain / Q000382:microbiology*; D005434:Flow Cytometry; D005453:Fluorescence; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D010572:Pest Control, Biological; D010935:Plant Diseases / Q000382:microbiology; D018517:Plant Roots / Q000382:microbiology*; D011551:Pseudomonas fluorescens / Q000378:metabolism*
D000935:Antifungal Agents; D008164:Luminescent Proteins