Journal Article

Determination of symbiotic nodule occupancy in the model <i>Vicia tetrasperma</i> using a fluorescence scanner

Karel Novák

in Annals of Botany

Published on behalf of The Annals of Botany Company

Volume 107, issue 4, pages 709-715
Published in print April 2011 | ISSN: 0305-7364
Published online January 2011 | e-ISSN: 1095-8290 | DOI: http://dx.doi.org/10.1093/aob/mcr002
Determination of symbiotic nodule occupancy in the model Vicia tetrasperma using a fluorescence scanner

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  • Ecology and Conservation
  • Evolutionary Biology
  • Plant Sciences and Forestry

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Background

Fluorescent tagging of nodule bacteria forming symbioses with legume host plants represents a tool for vital tracking of bacteria inside the symbiotic root nodules and monitoring changes in gene activity. The constitutive expression of heterologous fluorescent proteins, such as green fluorescent protein (GFP), also allows screening for nodule occupancy by a particular strain. Imaging of the fluorescence signal on a macro-scale is associated with technical problems due to the robustness of nodule tissues and a high level of autofluorescence.

Scope

These limitations can be reduced by the use of a model species with a fine root system, such as Vicia tetrasperma. Further increases in the sensitivity and specificity of the detection and in image resolution can be attained by the use of a fluorescence scanner. Compared with the standard CCD-type cameras, the availability of a laser source of a specified excitation wavelength decreases non-specific autofluorescence while the photomultiplier tubes in emission detection significantly increase sensitivity. The large scanning area combined with a high resolution allow us to visualize individual nodules during the scan of whole root systems. Using a fluorescence scanner with excitation wavelength of 488 nm, a band-pass specific emission channel of 532 nm and a long-pass background channel of 555 nm, it was possible to distinguish nodules occupied by a rhizobial strain marked with one copy of cycle3 GFP from nodules colonized by the wild-type strain.

Conclusions

The main limitation of the current plant model and GFP with the wild-type emission peak at 409 nm is a sharp increase in root autofluorescence below 550 nm. The selectivity of the technique can be enhanced by the use of red-shifted fluorophores and the contrasting labelling of the variants, provided that the excitation (482 nm) and emission (737 nm) maxima corresponding to root chlorophyll are respected.

Keywords: Green fluorescent protein; in-depth imaging; nodule; Rhizobium; symbiosis; Vicia tetrasperma

Journal Article.  4726 words.  Illustrated.

Subjects: Ecology and Conservation ; Evolutionary Biology ; Plant Sciences and Forestry

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