RESUMO
The relative quantitative real-time expression of two expressed sequence tags (ESTs) codifying for key enzymes in nitrogen metabolism in maize, nitrate reductase (ZmNR), and glutamine synthetase (ZmGln1-3) was performed for genotypes inoculated with Azospirillum brasilense. Two commercial single-cross hybrids (AG7098 and 2B707) and two experimental synthetic varieties (V2 and V4) were raised under controlled greenhouse conditions, in six treatment groups corresponding to different forms of inoculation and different levels of nitrogen application by top-dressing. The genotypes presented distinct responses to inoculation with A. brasilense. Increases in the expression of ZmNR were observed for the hybrids, while V4 only displayed a greater level of expression when the plants received nitrogenous fertilization by top-dressing and there was no inoculation. The expression of the ZmGln1-3EST was induced by A. brasilense in the hybrids and the variety V4. In contrast, the variety V2 did not respond to inoculation.
Assuntos
Azospirillum brasilense/patogenicidade , Etiquetas de Sequências Expressas , Nitrogênio/metabolismo , Zea mays/genética , Genótipo , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Zea mays/microbiologiaRESUMO
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.
Assuntos
Azospirillum brasilense/genética , Azospirillum brasilense/patogenicidade , Glucuronidase/genética , Proteínas Luminescentes/genética , Plasmídeos/genética , Triticum/microbiologia , Silicatos de Alumínio/farmacologia , Azospirillum brasilense/citologia , Azospirillum brasilense/efeitos dos fármacos , Aderência Bacteriana/genética , Aderência Bacteriana/fisiologia , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Marcadores Genéticos/genética , Glucuronidase/metabolismo , Proteínas de Fluorescência Verde , Indicadores e Reagentes , Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Raízes de Plantas/microbiologia , Sensibilidade e Especificidade , SimbioseRESUMO
Bacteria of the genus Azospirillum are able to colonize plant roots. Using the beta-glucuronidase (GUS) reporter system, various Azospirillum mutants, including mutants affected in chemotactic motility or extracellular polysaccharide biosynthesis, were investigated for their capacity to initiate wheat root colonization at the root hair zones. Only non-flagellated mutants and a generally non-chemotactic mutant exhibited a strongly reduced colonization ability as compared to the wild-type. No role of the Azospirillum calcofluor-binding polysaccharide in primary wheat root colonization could be observed. This is the first report demonstrating directly, by using different motility mutants, the requirement of bacterial motility in the establishment of the Azospirillum-plant root association.