Annals of Botany 107: 709–715, 2011 doi:10.1093/aob/mcr002, available online at www.aob.oxfordjournals.org TECHNICAL ARTICLE Determination of symbiotic nodule occupancy in the model Vicia tetrasperma using a fluorescence scanner Karel Nova´k* Department of Ecology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vı´denˇska´ 1083, 142 20 Prague 4, Czech Republic *For correspondence. E-mail: [email protected] Downloaded from https://academic.oup.com/aob/article/107/4/709/161997 by guest on 24 September 2021 Received: 6 September 2010 Returned for revision: 18 October 2010 Accepted: 3 December 2010 Published electronically: 24 January 2011 † 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 con- stitutive 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 photo- multiplier 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. Key words: Green fluorescent protein, in-depth imaging, nodule, Rhizobium, symbiosis, Vicia tetrasperma. INTRODUCTION 2002). Upon release into the cytoplasm of the infected host cells, bacteria multiply and differentiate into bacteroids, The symbiosis between nodule bacteria (rhizobia) and their which contribute to the nutrition of the host plants by the legume (Fabaceae) host plants provides a model for under- fixation of atmospheric nitrogen. standing the general principles of plant–microbe recognition. Nodules of the indeterminate type have characteristic trans- At the same time, rhizobial symbiosis provides the main verse zonation of the central tissues (Franssen et al., 1992). input of nitrogen for agriculture worldwide, and is highly pre- Behind the nodule apical meristem is the infection zone har- ferable for both economic and ecological reasons to industrial bouring permanent infection threads, followed by the invasion nitrogen fertilizers (Bohlool et al., 1992). (infection) zone where the internalization of rhizobia occurs. Although nodule bacteria have been shown to belong to at Whereas the early symbiotic zone is characterized by rhizobia least 13 (sometimes unrelated) genera thanks to the widespread multiplying intracellularly, the late symbiotic zone (LSZ) con- molecular taxonomy applied in the last decade (Willems, tains the differentiated bacteroids with enzyme activities 2006), the most studied remain the microsymbionts of tra- required for the nitrogen fixation process. The concurrent ditional legume crops belonging to the genera Rhizobium, differentiation of the host cells is characterized by a spectrum Ensifer, Mesorhizobium and Bradyrhizobium. Their host of late nodulins (nodule-specific proteins), the most prominent plants show two nodule development types: an indeterminate being leghaemoglobin (Lb). type with a persisting apical meristem; and a determinate The study of symbiosis has always involved monitoring type, growth of which is stopped after nodule symbiotic bacterial strains and their specific activities inside the host tissue differentiation (Franssen et al., 1992). The nodules are tissues. Determination of nodule occupancy is a key approach induced inside the root pericycle or the adjacent layers of to the study of the nodulation competitiveness of rhizobial primary cortex by the action of bacterial Nod factors, which strains and the associated quality of commercial bacterial prep- represent the terminal products of the bacterial nodulation arations (Dowling and Broughton, 1986). The task has been (nod, noe, nol) genes (Spaink, 2000). In parallel, the epidermal addressed by the re-isolation of strains (Vincent et al., 1973; symbiotic programme starts from the intracellular growth of Duodu et al., 2009), immunological detection using strain- bacteria organized into infection threads (Guinel and Geil, specific antibodies (Vincent et al., 1973) and the detection # The Author 2011. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 710 Nova´k — Determination of symbiotic nodule occupancy of strain-specific DNA sequences upon re-isolation (Svenning the wild-type emission maximum, produced by a tracked bac- et al., 2001). Genetic marking with specific enzyme-coding terial strain is demonstrated. Additionally, the fine root system genes using rhizobial suicidal plasmids as a transposon deliv- in the model plant Vicia tetrasperma reveals the suitability of ery system (Wilson, 1995) represents an almost perfect sol- this species for laboratory studies of rhizobial symbiosis. ution for detection; however, the technique still includes the enzymatic reaction step, which is time-consuming. Moreover, the use of sometimes expensive substrates might be limiting MATERIALS AND METHODS in large-scale experiments. Many enzymatic reactions, such Plants and symbiosis establishment as hydrolysis by b-galactosidase, do not allow for vital detec- tion or for detection without disturbing nodule physiology. Seeds of Vicia tetrasperma (L.) Schreb. (smooth tare, slender, The fluorescence marking of rhizobia with genes coding for sparrow or lentil vetch) were collected from the local popu- fluorescent proteins of Cnidaria appeared to have solved this lation. The dry seeds were stored at room temperature for up Downloaded from https://academic.oup.com/aob/article/107/4/709/161997 by guest on 24 September 2021 problem (Gage et al., 1996; Gage, 2002). Nevertheless, the to 10 years without a change in germinability. The seeds high autofluorescence of plant tissues (Wang et al., 2007) were surface-sterilized with 2 % Chloramine B for 40 min and the fluorescence quenching described for the infected and sown without rinsing into 15-mL glass tubes containing nodule cells (Auriac and Timmers, 2007) limit the efficiency 7 mL of autoclaved agarized (1.7 %) nutrient solution of Van of the method. To overcome these constraints, two-photon Egeraat and Lie (Lie, 1969) as previously modified (Nova´k excitation confocal microscopy has been successfully used et al., 1994). Mineral nitrogen forms were omitted from the (Stuurman et al., 2000). In this technique, a longer wavelength nutrient solution. The tubes were covered with inverted allows (1) the excitation light to travel deeper into nodule 20-mL glass vials to prevent contamination. After 3 d of incu- tissues and decrease the light losses on the peripheral nodule bation at 4 8C in the dark to synchronize germination, each seed was inoculated with 100 mL of bacterial suspension con- tissues, (2) damage to the object cells and the fluorophore to 7 21 be reduced and (3) the intensity of the exciting light beam to taining 10 cells mL prepared as described below. The be increased. assemblies were placed in laboratory tube racks and transferred to a growth chamber where germination and growth occurred In parallel, the use of red-shifted forms of green fluorescent 22 21 protein (GFP) allowed for plant-associated bacteria in general at 500 mmol m s irradiance with photosynthetically (Miller et al., 2000) and nodule bacteria in particular active radiation, 16/8-h day/night light regime and temperature (Stuurman et al., 2000; Gage, 2002) to use longer emission of 20/16 8C. wavelengths. This reduces the interference from non-specific autofluorescence, the intensity of which drops with increasing Bacteria wavelength. The use of the DsRed protein from Discosoma sp. solved both facets of the problem, moving both the excitation Rhizobium leguminosarum 128C30 (EMD Crop Bioscence, and the emission wavelengths towards the far red region. This formerly Nitragin, Milwaukee, WI, USA) is a wild-type strain was efficient for tracking both the host plant transgenes containing symbiotic megaplasmid pSym128C30 of 212 MDa (Limpens et al., 2004) and rhizobial symbionts (Gage, 2002; (¼ 326 kbp; Leyva et al., 1987). Its fluorescent variant strain, Auriac and Timmers,