title: Monitoring Azospirillum-wheat interactions using the gfp and gusA genes constitutively expressed from a new broad-host range vector.
abstract: To monitor the colonization of wheat roots by Azospirillum brasilense, we constructed several plasmids based on the pBBR1 replicon expressing the gfp and gusA genes constitutively. Both genes were placed under control of the gentamycin resistance gene promoter resulting in high levels of expression in Escherichia coli and A. brasilense. The constructed plasmids were stably maintained in A. brasilense strains even in the absence of selective pressure. The colonization of wheat plants grown under controlled conditions in sterilized vermiculite by A. brasilense strain FP2 (a Sp7-derivative) transconjugants containing these plasmids was monitored. Bacteria expressing GFP were easily observed in fresh plant material by fluorescence microscopy. Cell aggregates and single bacteria were visualized on the surfaces of young root zones, such as roots hairs and lateral roots. Large cellular clumps were observed at the points of lateral root emergence or at intercellular spaces of root epidermal cells 30 days after inoculation. Although we failed to detected bacteria in internal cortical and xylem tissues of wheat roots, the initial stage of endophytic colonization by A. brasilense may involve the sites detected in this work.
authors.affiliation: Department of Pharmacology, Biochemistry and Molecular Biology, UFPR, C. Postal 19046, CEP 81531-990 Curitiba, PR, Brazil.
authors.forename: Humberto J O
authors.initials: HJ
authors.lastname: Ramos
authors.forename: Lauren D B
authors.initials: LD
authors.lastname: Roncato-Maccari
authors.forename: Emanuel M
authors.initials: EM
authors.lastname: Souza
authors.forename: Juliana R L
authors.initials: JR
authors.lastname: Soares-Ramos
authors.forename: Mariangela
authors.initials: M
authors.lastname: Hungria
authors.forename: Fábio O
authors.initials: FO
authors.lastname: Pedrosa
mesh_terms: D000538:Aluminum Silicates / Q000494:pharmacology; D015806:Azospirillum brasilense / Q000166:cytology / Q000187:drug effects / Q000235:genetics* / Q000472:pathogenicity*; D001422:Bacterial Adhesion / Q000235:genetics / Q000502:physiology; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics*; D008856:Microscopy, Fluorescence / Q000379:methods; D018517:Plant Roots / Q000382:microbiology; D010957:Plasmids / Q000235:genetics*; D012680:Sensitivity and Specificity; D013559:Symbiosis; D014908:Triticum / Q000382:microbiology*
chemical_list: D000538:Aluminum Silicates; D005819:Genetic Markers; D007202:Indicators and Reagents; D008164:Luminescent Proteins; C003760:vermiculite; D049452:Green Fluorescent Proteins; D005966:Glucuronidase