Abstract
The bacterium Herbaspirillum seropedicae is an endophytic diazotroph found in several plants, including economically important poaceous species. However, the mechanisms involved in the interaction between H. seropedicae and these plants are not completely characterized. We investigated the attachment of Herbaspirillum to maize roots and the invasion of the roots by this bacterium using H. seropedicae strain SMR1 transformed with the suicide plasmid pUTKandsRed, which carries a mini-Tn5 transposon containing the gene for the Discosoma red fluorescent protein (Dsred) constitutively expressed together with the kanamycin resistance gene. Integration of the mini-Tn5 into the bacterial chromosome yielded the mutant H. seropedicae strain RAM4 which was capable of expressing Dsred and could be observed on and inside fresh maize root samples. Confocal microscopy of maize roots inoculated with H. seropedicae three days after germination showed that H. seropedicae cell were attached to the root surface 30 min after inoculation, were visible in the internal tissues after twenty-four hours and in the endodermis, the central cylinder and xylem after three days.
Highlights
Bacterial colonization of plant internal tissue has been described in many plant species
The first studies on the interactions of diazotrophic bacteria with poaceous plants involved Azotobacter paspali isolated from the soil or from plant rhizospheres (Döbereiner, 1966) and later it was shown that nitrogenfixing Azospirillum brasilense colonized both the rhizosphere and the root interior of several forage grasses and cereals (Baldani et al, 1986a) of the family Poaceae
Microscopy and tagging bacteria with reporter genes such as gusA can be used to investigate colonization but these techniques can produce artifacts during sample preparation. Such problems can be avoided by marking endophytic bacteria with genes that allow the expression of fluorescent proteins which provide a unique visual marker in fresh plant tissue samples and the application of confocal microscopy, a technique which allows visualization of bacteria in thick samples and is tolerant to the background autofluorescence which often occurs with plant tissue
Summary
Bacterial colonization of plant internal tissue has been described in many plant species. Poaceae) roots to examine the progression of the early events of maize colonization by H. seropedicae strains expressing the coral-derived Discosoma red (DsRed) fluorescent protein (Matz et al, 1999), and to characterize the distribution and organizational patterns of the bacterial cells within the plant tissues.
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