Promoter-trap identification of wheat seed extract-induced genes in the plant-growth-promoting rhizobacterium Azospirillum brasilense Sp245.
Azospirillum strains have been used as plant-growth-promoting rhizobacteria (PGPR) of cereal crops, but their adaptation to the root remains poorly understood. Here, we used a global approach based on differential fluorescence induction (DFI) promoter trapping to identify genes of the wheat isolate Azospirillum brasilense Sp245 that are induced in the presence of spring wheat seed extracts. Fluorescence-based flow cytometry sorting of Sp245 cells was validated using PlacZ, PsbpA and PnifH promoters and egfp. A random promoter library was constructed by cloning 1-3 kb Sp245 fragments upstream of a promoterless version of egfp in the promoter-trap plasmid pOT1e (genome coverage estimated at threefold). Exposure to spring wheat seed extracts obtained using a methanol solution led to the detection of 300 induced DFI clones, and upregulation by seed extracts was confirmed in vitro for 46 clones. Sequencing of 21 clones enabled identification of seven promoter regions. Five of them displayed upregulation once inoculated onto spring wheat seedlings. Their downstream sequence was similar to (i) a predicted transcriptional regulator, (ii) a serine/threonine protein kinase, (iii) two conserved hypothetical proteins, or (iv) the copper-containing dissimilatory nitrite reductase NirK. Two of them were also upregulated when inoculated on winter wheat and pea but not on maize, whereas the three others (including PnirK) were upregulated on the three hosts. The amounts of nitrate and/or nitrite present in spring wheat seed extracts were sufficient for PnirK upregulation. Overall, DFI promoter trapping was useful to reveal Azospirillum genes involved in the interaction with the plant.
Pothier
Joël F
JF
CNRS, UMR 5557, Ecologie Microbienne, Villeurbanne, F-69622, France.|Université Lyon 1, Lyon, F-69003, France.|Université de Lyon, Lyon, F-69003, France.
Wisniewski-Dyé
Florence
F
CNRS, UMR 5557, Ecologie Microbienne, Villeurbanne, F-69622, France.|Université Lyon 1, Lyon, F-69003, France.|Université de Lyon, Lyon, F-69003, France.
Weiss-Gayet
Michèle
M
CNRS, UMR 5534, Centre de Génétique Moléculaire et Cellulaire, Villeurbanne, F-69622, France.|Université Lyon 1, Lyon, F-69003, France.|Université de Lyon, Lyon, F-69003, France.
Moënne-Loccoz
Yvan
Y
CNRS, UMR 5557, Ecologie Microbienne, Villeurbanne, F-69622, France.|Université Lyon 1, Lyon, F-69003, France.|Université de Lyon, Lyon, F-69003, France.
Prigent-Combaret
Claire
C
CNRS, UMR 5557, Ecologie Microbienne, Villeurbanne, F-69622, France.|Université Lyon 1, Lyon, F-69003, France.|Université de Lyon, Lyon, F-69003, France.
D015806:Azospirillum brasilense / Q000235:genetics* / Q000502:physiology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000382:microbiology; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D012639:Seeds / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D014908:Triticum / Q000382:microbiology*; D003313:Zea mays / Q000382:microbiology
D004269:DNA, Bacterial; D010936:Plant Extracts