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 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 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 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 () 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 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, 2007) in the depth of plant tissues. 128C30(1819/9), was prepared by in vivo marking with a mini- A similar task of in planta monitoring of bacterial and plant transposon Tn1819 upon conjugative transfer on a suicidal nodule-specific gene (nodulin) activity led to similar approaches plasmid pFAJ1819 from Escherichia coli S17-1lpir according based on transcriptionally fused enzyme reporter genes (Sharma to Chovanec et al. (2008). The plasmid pFAJ1819 is a deriva- and Signer, 1990; Vijn et al., 1995). However, the fluorescent tive of pUT (Herrero et al., 1990) prepared by Xi et al. (1999) protein gene as a reporter of bacterial activity in the screen for while Tn1819 (Xi et al., 1999) is a mini-Tn5 derivative that nodule-expressed genes has also been successfully applied (Xi contains two copies of the optimized GFP gene put under et al., 2001). control of the constitutive promoter of nptII. The construct Although the fluorescent tracking of individual bacteria, also contains the promoterless gusA gene of E. coli as a pro- bacterial structures such as infection threads, infected cells moter trap for insertion site transcription activity. The cycle3 of symbiotic tissue and complementary plant molecules is allele of the GFP gene originating from Aequorea victoria is technically manageable by means of fluorescence microscopy optimized for fluorescence yield by recurrent mutagenesis in (Stuurman et al., 2000; Fournier et al., 2008), the application E. coli. The quantum yield of the cycle3 protein is increased of fluorescent tagging for whole nodule monitoring on the up to 18-fold compared with that of the wild-type. The plant level has not been reported. expression of cycle3 in Gram-negative bacteria is considered The present work concentrates on the determination of to be optimized as well (Crameri et al., 1995). nodule occupancy with fluorescently marked nodule bacteria The excitation and emission maxima of cycle3 GFP are 395 on a macro-scale using a fluorescence scanner designed for and 508 nm, respectively, thus corresponding to the published the documentation of electrophoretic gels. The choice of this wild-type GFP (Cubitt et al., 1995). The in vivo spectrum did apparatus is based on its high sensitivity, resolution and not change after cycle3 transfer from E. coli into the large scanning area, which all currently exceed the parameters Rhizobium background despite the sharp decrease in fluor- of charge coupled device (CCD)-based fluorescent cameras. escence yield (Chovanec et al., 2008). The bacteria were The powerful laser excitation source is a further advantage grown on yeast extract-mannitol (YM) agar with kanamycin of the scanner concept. The possibility of nodule occupancy at 50 mgmL21 as a transposon-stabilizing antibiotic scoring in planta using the non-shifted form of GFP, i.e. of (Chovanec et al., 2008) for 4 d at 25 8C. The inoculum was Nova´k — Determination of symbiotic nodule occupancy 711 prepared by scraping colonies off the surface of the YM agar direct light reaching the growing roots and nodules (Fig. 1A, plates, resuspending bacteria in distilled water and adjusting B). The root systems were harvested and evaluated for fluor- the titre by direct counting. escence traits 4 weeks after sowing, at the pre-flowering stage. The visibility of the GFP signal in the symbiotic nodules was verified by confocal microscopy. As can be seen in Confocal microscopy Fig. 1C, fluorescence was concentrated in centrally positioned Intact nodules attached to the root segments were mounted tissues. This nodule region corresponds to the invasion zone, in a drop of water and observed in the epifluorescence early symbiotic zone, LSZ and degradation zone according regime with a confocal laser scanning microscope (Leica to Newcomb (1976) and Franssen et al. (1992), where differ- SP2 AOBS MP; Leica Microsystems, Wetzlar, Germany) entiation of the early symbiotic zone is marked by endosym- equipped with a Fluotar 5× objective (NA ¼ 1.5). The exci- biotic rhizobia multiplication. The individual infected cells

tation wavelength of 810 nm from the tunable laser source were distinguishable from the interspersed uninfected cells Downloaded from https://academic.oup.com/aob/article/107/4/709/161997 by guest on 24 September 2021 allowed for two-photon excitation of GFP. The emission by a stronger GFP signal. Significant autofluorescence signal channels 500–530 nm (GFP) and 550–570 nm (presumably registered in the band 530–570 nm indicated the degradation autofluorescence) were used in combination with the trans- zone in the basal part of the nodule (Fig. 1C), which was mitted light channel which delineated the cell shapes so that distinguished by distorted cell walls. composite images were created using the ImageJ program Scanning of the root system was carried out on a group of (W. Rasband, National Institute of Health, Bethesda, MD, nine roots that fitted into the glass plate of the sample tray USA, http://rsbweb.nih.gov/ij/). Optical sections taken in of the FX imager (35 × 43 cm), when arranged in three rows 10-mm steps along the z-axis were processed using the of three roots. Scanning at 100-mm resolution lasted 12 min average intensity projection. for one wavelength, i.e. 2 min 40 s per root in a sequential two- channel scan. As expected, the excitation wavelength of 488 nm was the most effective for GFP signal generation. In Fluorescence spectrophotometry of root tissue spite of the availability of two excitation wavelengths of 488 Bundles of 3-cm root segments or root segments bearing and 532 nm and two long-wave reference channels (555-nm symbiotic nodules were mounted vertically in distilled H2O LP, 640-nm BP), the channel delimited by the 555-nm LP into 1-mL quartz cells of a fluorescence spectrophotometer filter upon 488-nm excitation provided the most uniform and (RF-540; Shimadzu, Kyoto, Japan). On searching for emission stable autofluorescence background for composite images. maxima with a wide UV excitation range, the excitation Comparison of the plants inoculated with control rhizobia maximum peak of the root/nodule tissue was specified at a with those inoculated with the fluorescent variant showed con- fixed maximum emission wavelength. sistent differences in signal distribution, provided that the specific and background channels (530-nm BP and 555-nm LP, respectively) were equilibrated in artificial colours. Fluorescence scanning Whereas the control nodules did not differ in false coloration The root systems were released from the agarized medium, from the part of the root to which they were attached repeatedly washed with distilled H2O and stretched in a 3-mm (Fig. 1D), the marked nodules exhibited a strong GFP signal layer of H2O on the glass sample tray designed for electrophor- (Fig. 1E). This was often confined to one or two transverse etic gels in the Molecular Imager FX-PRO Plus (Bio-Rad zones. The positive signal zones occupied either terminal or Laboratories, Hercules, CA, USA). Alternatively, the roots terminal and basal regions of the nodule. were placed in the water layer in 90-mm polystyrene Petri The intensity of the 555-nm LP channel decreased in the dishes without a cover to meet the technical limit of 8 mm distal parts of the tap and lateral roots, which produced the for the thickness of the objects placed in the sample tray. In prevalent GFP channel (Fig. 1D, E), although without speci- addition to the basic solid-state (diode-pumped) 532-nm ficity for the tagged Rhizobium variant. Importantly, this laser source, the apparatus was equipped with an external shift in favour of the GFP channel did not coincide with the laser module containing an argon–ion laser of 488 nm root zones bearing the established nodules, and therefore wavelength. does not interfere with nodule occupancy determination. The roots were scanned using the above excitation sources To examine the reasons for the fluorescence variation along combined with a 530-nm band-pass (BP), 555-nm long-pass the root as registered in the GFP (530-nm BP) and long-wave (LP) and 640 nm-BP emission filters. The scanning resolution background emission (555-nm LP and 640-nm BP) channels of was 50 or 100 mm. Images were acquired and initially edited the scanner, the fluorescence spectra of the intact root tissue with the program PDQuest (Bio-Rad) and, after having been were recorded (Fig. 2). Notably, the autofluorescence of the exported as files in TIF format, were processed with the root tissue was increased in the region below 550 nm, which ImageJ program. The minimum and maximum pixel values corresponds to a substantial part of the cycle3 GFP emission of the root tissue signal were used to standardize the brightness range. Moreover, the isolated peak at 737 nm in the root emis- of the false colour channels across the images. sion spectrum was linked to the prominent excitation peak at 482 nm. The spectrum of symbiotic nodules formed with the wild-type rhizobial strain was indistinguishable from the estab- RESULTS lished root spectrum. However, the root terminal segments had The vetch plants germinated within 1 week after sowing and markedly lower autofluorescence in the long-wave channels nodules appeared within 10 d after germination, in spite of (data not shown). 712 Nova´k — Determination of symbiotic nodule occupancy

A B Downloaded from https://academic.oup.com/aob/article/107/4/709/161997 by guest on 24 September 2021

C

DE

F IG. 1. Symbiotic nodule development in the model plant Vicia tetrasperma under the experimental conditions used here and green fluorescent protein (GFP) expression in the nodules formed with a tagged rhizobial strain. (A, B) Symbiotic nodule formation in the unshaded roots of V. tetrasperma grown in agarized medium after inoculation with a wild-type strain 128C30 of Rhizobium leguminosarum bv. viciae. (C) In-depth image of the V. tetrasperma nodule colonized with a GFP-tagged strain 128C30(1819/9) of R. leguminosarum bv. viciae. The intact nodule was viewed with a confocal microscope at 810 nm excitation wavelength and using the emission channels for GFP (500–530 nm, green), autofluorescence (550–570 nm, red) and the transmitted light channel (white). The image isa z-projection of six optical sections registered in 10-mm steps. (D, E) Fluorescence scans of V. tetrasperma root systems at the excitation wavelength of 488 nm combined with the 530-nm band-pass filter for GFP signal (green) and with the 555-nm long-pass filter for autofluorescence evaluation (red). The control plants (D) were inoculated with the wild-type strain 128C30 of R. leguminosarum bv. viciae and the variant (E) with the GFP-tagged strain 128C30(1819/9). Transverse quenching zones are indicated (arrows). In C: IC, infected cells; IZ, ESZ, LSZ, DZ denote invasion, early symbiotic, late symbiotic and degradation zones, respectively. Scale bars: (A, D, E) ¼ 1 cm; (B, E inset) ¼ 3 mm; (C) ¼ 200 mm.

Compared with the currently available CCD cameras for DISCUSSION biological fluorescence imaging, the fluorescence scanner provides a higher resolution up to 50 mm, which allows for a Fluorescence scanning for rhizobial marker monitoring 4-megapixel image from a 10 × 10-cm area at a dynamic Fluorescence scanning can be used to distinguish the fluor- range of five orders of magnitude. Only the most advanced escence signal of GFP-tagged rhizobia growing in plants on a low-noise fluorescent CCD cameras, e.g. LAS 4000 macro-scale. This enables this technique to be used for a (Fujifilm, Tokyo, Japan), might provide a comparable resol- number of traditional applications, for example the non- ution of 3.2 megapixels per image although only at four destructive determination of nodule occupancy with a particular orders of magnitude dynamic range. Moreover, the resolution bacterial strain. of the scanner image is independent of the scanned area, and Nova´k — Determination of symbiotic nodule occupancy 713

100 legumes of the pea inoculation group (Newcomb, 1976; Emission 737 Franssen et al., 1992) allows us to assign the observed zones Excitation of GFP signal reduction to the LSZ, consistent with Auriac 482 and Timmers (2007). These authors excluded changes in tran- scription activity and GFP degradation as the reason for the decreased fluorescence. Moreover, the quenching was indepen- dent of the type of fluorophore tested (GFP, SYTO9, acridine orange), consistent with the SYTO13 staining of symbiotic 50 structures (Haynes et al., 2004). Therefore, the most probable reason for GFP signal attenuation seems to be the presence of leghaemoglobin, which accumulates in the beginning of the % of maximum signal % of maximum

LSZ. This abundant, pink-coloured late nodulin can absorb Downloaded from https://academic.oup.com/aob/article/107/4/709/161997 by guest on 24 September 2021 excitation light at the 488-nm line of an Ar laser. The greenish product of Lb degradation in the degradation zone cannot quench fluorescence as efficiently in view of the bathochromic 0 400 500 600 700 800 shift in the degradation products (Jun et al., 1994). This might allow for the re-appearance of the GFP signal in the degra- Wavelength (nm) dation zone. Nevertheless, the zone-specific decrease in fluor- F IG. 2. Excitation and emission fluorescence spectra of the root tissue escence is not a principal constraint in the application of the of Vicia tetrasperma. The plant was inoculated with a wild-type strain of technique for discrimination of nodule occupancy. Rhizobium leguminosarum bv. viciae. The central part of the tap root free of nodules was assayed. The characteristic peaks at 482 nm (excitation) and 737 nm (emission) are indicated. Factors in root autofluorescence In the model of V. tetrasperma, further optimization of the thus of the root system size. This might be essential in acquir- method should reduce interference with the prominent auto- ing images of plants with a large and rich-branching root fluorescence, particularly in the region 500–550 nm overlap- system, such as alfalfa (Medicago sativa). The even excitation ping with the GFP signal. The nature of the autofluorescence beam intensity further increases the discriminating power in source in the roots and nodules remains unknown. Only the identifying areas with increased fluorescence. enhanced fluorescence in the nodule degradation zone, as The main disadvantage of the scanning procedure is the determined with whole-nodule microscopy, can be ascribed slow image acquisition of the root compared with fast pho- to the accumulation of secondary metabolites in the cell tography (within a range of seconds). However, the possibility walls. The intraradical differences in autofluorescence might of scanning a set of roots arranged in an object tray can be associated with the different composition of the developing substantially reduce the time for scanning one root to an and fully differentiated parts of the root system. The distal root acceptable level. zone showing increased GFP/long-wave channel ratio corre- The determination of root characteristics from the images sponds to the region of high nodulation-gene activity of rhizo- obtained by scanning with subsequent computer-assisted bia attached to the roots in the rhizoplane (Redmont et al., processing (program WinRhizo) has been reported earlier for 1986; Chovanec and Nova´k, 2005). However, the shift in the pea (McPhee, 2005). There is no doubt that the fluorescence terminal root signal was not specific for the tagged strain, variant of the optical scanner should provide additional possi- making the participation of rhizobia unlikely. On the other bilities for retrieving data about root biology. hand, the pair of excitation/emission peaks at 482 and 737 nm, respectively, coincided with the spectrum of the far-red chlorophyll form with a characteristic emission peak Limitations and improvement of the method at 735 nm as observed in leaves (Buschmann, 2007). Further optimization of the fluorescent markers, their The root system autofluorescence upon illumination with expression levels and the scanning wavelength as exemplified UV light is known to be cultivar-specific in soybean in monomeric red fluorescent protein (Gage, 2002; Limpens (Glycine max) and clover (Trifolium sp.) (Delannay and et al., 2004; Auriac and Timmers, 2007) should allow Palmer, 1982) and to be species-specific in ryegrass (Lolium) for the application of this technique to leguminous plants (Floyd and Barker, 2002). This property is strictly genetically with more robust roots than V. tetrasperma. Similarly, the determined by four genes in soybean (Delannay and Palmer, advance in the imaging techniques associated with the use of 1982). Both induced (Sawada and Palmer, 1987) and spon- far-red and near-infrared fluorophores has enabled in-depth taneous (Cubukcu et al., 2000) soybean mutants showing imaging of structures and processes in medical applications loss of fluorescence have been described. Although ryegrass (Ntziachristos et al., 2003). root fluorescence is associated with the production of a Another limitation may be due to the observed absence of secreted alkaloid annuloline (Floyd and Barker, 2002), the GFP signal in nodule transverse zones. This phenomenon biochemical nature of the soybean trait remains unknown. has been independently described by Auriac and Timmers The assumed presence of chlorophyll in the light-grown root (2007) for Medicago truncatula nodules and it has been of V. tetrasperma implies that the root undergoes light-induced shown to be limited to the LSZ (zone III according to the changes. This is contradictory to the constitutive nodulation authors). The known structure of indeterminate nodules of ability in the dark and light, which contrasts with the strong 714 Nova´k — Determination of symbiotic nodule occupancy light inhibition of nodulation present in related species such as LITERATURE CITED pea (Pisum sativum)(Lie, 1969; Lee and LaRue, 1992). In the Auriac MC, Timmers ACJ. 2007. 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