Quantitative Assessment of Hormogonia Induction in Nostoc punctiforme by a Fluorescent Reporter Strain

Journal of Experimental Botany, Vol. 00, No. 0 pp. 1–11, 2025 https://doi.org/10.1093/jxb/eraf197 Advance Access Publication 13 May 2025 TECHNICAL INNOVATION Quantitative assessment of hormogonia induction in Nostoc punctiforme by a fluorescent reporter strain Anna Neubauer 1,2,†, , Macarena Iniesta-Pallarés 3,†, , Consolación Álvarez 3, , Aurélien Bailly 2,4, , Péter Szövényi 1,2, * , , and Vicente Mariscal 3, * , 1 Department of Systematic and Evolutionary Botany, University of Zurich, 8008 Zurich, Switzerland 2 Zurich-Basel Plant Science Center, ETH Zurich, 8092 Zurich, Switzerland 3 Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Américo Vespucio 49, 41092 Sevilla, Spain 4 Department of Plant and Microbial Biology, University of Zurich, 8008 Zurich, Switzerland † These authors contributed equally to this work. * Correspondence: [email protected] or [email protected] Received 6 January 2025; Editorial decision 11 May 2025; Accepted 11 May 2025 Editor: Oswaldo Valdés-López, Universidad Nacional Autonoma de Mexico, Mexico Abstract While symbiotic plant–cyanobacteria interactions hold significant potential for revolutionizing agricultural practices by reducing the application of artificial nitrogen fertilizers, the genetic underpinnings of the symbiotic interaction between the plant host and the cyanobiont remain poorly understood. In particular, the molecular mechanisms through which host plants induce the formation of motile cyanobacterial filaments (hormogonia), essential for colonization and initiation of symbiosis, are not well characterized. In this study, we present a novel yet objective method for quantifying hormogonia induction, addressing limitations of traditional qualitative approaches. We have developed a reporter strain of Nostoc punctiforme PCC 73102 capable of quantifying hormogonia induction in response to diverse biotic and abiotic stimuli. This reporter strain, generated via triparental mating conjugation transformation, contains the promoter sequence of prepilin pilA fused to a green fluorescent protein (GFP) and enables quantitative and high-throughput monitoring of hormogonia induction using a microplate reader. Our innovative approach, employing a cyanobacterial hormogonia reporter strain, allows high-throughput screening of the hormogonia-inducing effect of a wide array of environmental and plant signals. This method is expected to greatly advance our understanding of the genetic determinants underpinning plant–cyanobacteria symbioses. Keywords: Anthoceros agrestis, biological nitrogen fixation, cyanobiont, hormogonia, Nostoc punctiforme, plant–cyanobacteria symbiosis. © The Author(s) 2025. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/ licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact [email protected] for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact [email protected]. Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf197/8129438 by guest on 09 July 2025 Introduction Nostocales cyanobacteria are phototrophic soil bacteria capable of establishing facultative, diazotrophic symbiosis with a wide variety of eukaryotic organisms, including diatoms, fungi, and plants (Nilsson et al., 2002; Adams et al., 2006; Usher et al., 2007; Álvarez et al., 2023). The most extensively studied cyanobacterial symbiont (cyanobiont) is Nostoc punctiforme (Meeks et al., 2001; Meeks, 2003). Nostoc punctiforme PCC 73102 (also known as ATCC 29133) serves as a model organism for plant–cyanobacteria symbioses due to its ability to form endophytic association with isolated species across all major taxonomic groups of land plants (Bonnett and Silvester, 1981; Bergman et al., 1996; Costa et al., 2004; Álvarez et al., 2020; Frangedakis et al., 2023). The establishment of endosymbiosis between plants and N. punctiforme can be broadly divided into three phases (Johansson and Bergman, 1992; Meeks, 2003; Alvarenga et al., 2022). In the initial phase, the host plant secretes hormogonia-inducing factors (HIFs), prompting the differentiation of vegetative Nostoc cells into motile hormogonia filaments (Enderlin and Meeks, 1983; Knight and Adams, 1996; Nilsson et al., 2006). During the subsequent phase, the plant host is colonized by motile hormogonia which are guided by chemoattractants produced by the host plant. Studies in Oryza sativa have demonstrated that during colonization, core components of the common symbiosis signaling pathway (CSSP; also known as common symbiosis pathway) are activated in the plant host (Álvarez et al., 2022). This pathway is essential for establishing symbiotic interactions not only with arbuscular mycorrhizal fungi but also with nodule-forming bacteria (Parniske, 2000; Delaux et al., 2014; Li et al., 2020). Ultimately, a stable symbiosis is established as the plant entraps the cyanobiont and enhances its nitrogen fixation rate by increasing the heterocyst frequency within the plant tissue (Steinberg and Meeks, 1991; Cohen and Meeks, 1997; Meeks and Elhai, 2002; Alvarenga et al., 2022). Nostoc punctiforme is able to differentiate into three specialized cell types: heterocysts, hormogonia, and akinetes (Rippka et al., 1979; Strunecký et al., 2023). While heterocysts and akinetes (dormant cells) function in nitrogen fixation and survival under harsh environmental conditions, respectively, hormogonia are essential for the cyanobiont to establish symbiosis with the host plant and thus are referred to as the ‘plant infection units’ (Bergman et al., 2007). Hormogonia differentiate from the vegetative filament and can be identified by their small cell size, the absence of heterocysts, the presence of arrow-like cells in the poles, and—in certain taxa—the presence of gas vesicles (Herdman and Rippka, 1988; Meeks and Elhai, 2002). Additionally, hormogonia exhibit pilus-like appendages on their cell surface and are covered in mucilage, which facilitates movement and host recognition (Duggan et al., 2007; Khayatan et al., 2015). The differentiation of vegetative cells into hormogonia involves the activation of >1800 genes and can be induced by specific wavelengths of light, nutrient stress, or phytochemicals (Rasmussen, 1994; Rai et al., 2000; Schüßler, 2000; Nilsson et al., 2006; Campbell et al., 2007, 2008; Harwood and Risser, 2021). Upon stimulation, 90–100% of cells can differentiate into hormogonia within 18–24 h (Herdman and Rippka, 1988), with a life span averaging 72–96 h (Campbell et al., 1998). Stimuli such as symbiotic plant hosts, exposure to green light, and the addition of sucrose or sucralose can suppress hormogonia formation (Herdman and Rippka, 1988; Campbell et al., 1997; Splitt and Risser, 2016). While transcriptional regulation of hormogonia differentiation has been extensively investigated, the biotic and abiotic factors activating hormogonia formation in N. punctiforme are poorly understood (Campbell et al., 2007, 2008; Harwood and Risser, 2021; Álvarez et al., 2023). Remarkably, despite decades of research, the HIFs secreted by the host plant remain largely uncharacterized (Nilsson et al., 2002; Duggan et al., 2013; Hashidoko et al., 2019). A comprehensive understanding of the chemical composition of HIFs is pivotal to deciphering the communication between the plant host and the cyanobiont during the initial phase of the symbiotic interaction (Álvarez et al., 2022). The recent advent of genetically tractable model host systems makes it possible to carry out large-scale forward genetic experiments, facilitating the inference of the molecular mechanisms underpinning plant–cyanobacteria symbiotic interactions (Warshan et al., 2018; Cassier-Chauvat et al., 2021). These efforts aim to shed light specifically on the molecular underpinnings of HIF production and its influence on the infection unit of Nostoc spp., namely the motile hormogonia. However, such experiments demand a methodology that supports swift, automated, and quantitative screening of hormogonia formation. Currently, hormogonia induction is evaluated predominantly qualitatively using plate mobility assays (Liaimer et al., 2011; Splitt and Risser, 2016; Nishizuka and Hashidoko, 2018; Hashidoko et al., 2019). More labor-intensive quantitative assays employ light microscopy and manual counting (Campbell and Meeks, 1989; Knight and Adams, 1996) or, alternatively, use reverse transcription real-time PCR (RT–PCR) to quantify the expression of genes involved in hormogonia formation (Liaimer et al., 2011). However, none of the existing techniques offers the capability for rapid screening across a vast array of samples in a quantitative manner. In this study, we present a novel approach employing cyanobacterial hormogonia reporter strains, facilitating the quantitative assessment of hormogonia formation in N. punctiforme. Our methodology supports high-throughput screening across many different external conditions and plant exudates that potentially induce hormogonia. Consequently, this approach holds significant potential to enhance our understanding of the genetic underpinnings of plant–cyanobacteria symbiotic interactions. 2| Neubauer et al. Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf197/8129438 by guest on 09 July 2025 Materials and methods Cyanobacterial culture conditions The laboratory model strain N. punctiforme PCC 73102 (also known as ATCC 29133 and UCD 154) was obtained from the culture service of the Institute of Plant Biochemistry and Photosynthesis, Sevilla, Spain. It was routinely maintained in solid BG11 (Rippka et al., 1979) medium containing 1% w/v Bacto Agar (Becton, Dickinson and Company, France). Solid cultures were kept on a shelf at 25 °C under reduced illumination (20–25 µmol m −2 s −1 ) in a growth chamber. Liquid cultures were prepared in BG11 from solid cultures and incubated at 20–25 °C, with a continuous light exposure of 45–55 µmol m −2 s −1 on an orbital shaker (100–120 rpm). Mutant strains derived from N. punctiforme were maintained under the same conditions, but growth media were supplemented with 25 µg ml –1 neomycin to preserve the genetic modification. Plant culture Thallus tissue of the hornwort Anthoceros agrestis Bonn isolate (Szövényi et al., 2015) was cultivated in liquid BCD medium (Cove et al., 1996) under the same conditions as described above. To induce the secretion of the HIF, 1 g (FW) of thallus tissue was transferred to BG11 0 (BG11 without nitrogen; Rippka et al., 1979) and supplemented with 5 mM MES (Merck KGaA, Germany) to stabilize the pH at 7.4. The cultures were grown in 500 ml Erlenmeyer flasks containing 200 ml of BG11 0 sealed with a sponge plug for 1 week. Gunnera tinctoria plants were cultivated in a greenhouse in pots. Gunnera mucilage containing the HIF was obtained from the emerging leaves, using a sterile spoon. Rice (Oryza sativa) and wheat (Triticum aestivum) plants were germinated axenically and grown hydroponically in BG11. To obtain HIF, they were maintained for 1 week in BG11 0 under illumination. This BG11 0 medium was used in the experiments to induce hormogonia. Construction of N. punctiforme reporter strains Reporter strains were created by introducing a genetic fusion cassette into the N. punctiforme genome carrying the putative promoter sequences of the N. punctiforme genes pilT2 and pilA fused to a green fluorescent protein (GFP; Supplementary Fig. S1). Using the genomic sequence of N. punctiforme (GCA_000020025.1, (Fujisawa et al., 2017), ∼700 bp upstream of the predicted transcription start of pilT2 and pilA were amplified with the primers listed in Supplementary Table S1. Two plasmids were created by cloning the putative promoter sequences into pCSAM135 (Flores et al., 2007) in Escherichia coli DH5α (Supplementary Table S2). The plasmid pCSAM135 bears the GFP-mut2 gene and represents a common chassis vector for GFP transcriptional fusions in cyanobacteria. The absence of mutations in the amplified genomic regions was verified by Sanger sequencing at Eurofins Genomics Sequencing GmbH, Germany. The constructs were subsequently cloned into the conjugative plasmid pRL424 (Elhai and Wolk, 1988), producing the plasmids pAN2 and pMIP22 bearing the p pilA –GFP and p pilT2 –GFP fusions, respectively (Supplementary Table S2). The plasmids pAN2 and pMIP22 were methylated in E. coli HB101 containing the conjugation-enabling plasmid pRL623 (Elhai et al., 1997). The plasmids were introduced into the N. punctiforme genome by homologous recombination using the triparental conjugation method (Elhai and Wolk, 1988). The triparental mating included E. coli HB101 bearing the methylated target conjugal plasmids, E. coli bearing the helper plasmid pRL443 (Elhai and Wolk, 1988), and N. punctiforme. The incubated bacterial mix was subsequently plated [BG11 medium, 5% LB, 1% w/v Bacto Agar, topped with a Mixed Cellulose Filter (0.45 µm, REF HATF08550, Merck Millipore Ltd, USA)]. After 24 h at 25 °C under LED light tubes (45–55 µmol, 4000 K, 840), the filters were transferred to solid BG11 plates (1% w/v Bacto Agar). The selection started 48 h post-transformation by transferring the filter to BG11 plates supplemented with 25 µM neomycin (1% w/v Bacto Agar). This transfer onto selection plates was repeated every 3 d for 2 weeks until the appearance of colonies. The exconjugants were picked and streaked on selection plates until axenic cultures of the transformed Nostoc strains were obtained. Integration of the exogenous DNA and full segregation (absence of wild-type DNA copies) was validated via PCR using the segregation primers (Supplementary Table S2; Fig. 1). Hormogonia induction For the induction of hormogonia, liquid cultures of the cyanobacterial strains were prepared in BG11. Six days prior to hormogonia induction, the culture was transferred to antibiotic-free BG11 0 that was supplemented with 4 mM sucralose (Merck KGaA, Germany) to repress the formation of hormogonia (Splitt and Risser, 2016). Additionally, the culture was homogenized by passing through a needle (0.8×50 mm), and the density was adjusted to a Chl a concentration of 0.5 µg ml –1 . The concentration of Chl a was assessed by a 1:10 dilution of N. punctiforme in methanol (Mackinney, 1941). Prior to hormogonia induction, the cultures were washed twice with BG11 0 . Hormogonia induction was carried out by exposing the reporter strains to red light (three panels of a far-red LED module, 50–60 μmol; Green Power LED module HF far red, IONC 9290004645, Koninklijke Philips NV, the Netherlands), or the different plant exudates. As controls, we used cultures in BG11 0 without any additional treatment. Quantification of hormogonia To measure the induction of hormogonia, a VARIOSKAN LUX microplate reader was used. For this, 100 ml of each culture was quickly homogenized by passing through a needle (0.8×50 mm), and 200 μl aliquots were added into each well of the black 96-well microplate (Nunc, Denmark) to measure the GFP fluorescence, turbidity, and Chl a autofluorescence. We carried out at least 12 measurements (12 wells, technical replicates) for each biological replicate (four flasks of 100 ml culture per condition). A blank sample with BG11 0 for the correction of autofluorescence and turbidity was used in each measurement (measurement was made in at least eight wells). Turbidity was measured at 730 nm. Chl a autofluorescence and GFP were measured at 650–700 nm and 485–520 nm, after excitation at 650 nm and 485 nm, respectively. To test the effect of abiotic and biotic factors on GFP fluorescence, a one-way ANOVA in combination with the Tukey’s honestly significant difference (HSD) post-hoc test was used in R (R Core Team, 2008). Microscopy information Microscopy images were acquired using a Leica TCS SP2 confocal microscope (Leica Microsystems, Germany). GFP was excited at 488 nm. Fluorescent emission was monitored by collection across windows of 500–540 nm (GFP imaging) and 630–700 nm or 670–720 nm (cyanobacterial autofluorescence). The images were processed using the Fiji package (Schindelin et al., 2012). Results Construction of N. punctiforme strains expressing green fluorescent protein in hormogonia We aimed at creating two reporter strains expressing GFP in mature hormogonia. To obtain these genetically modified N. punctatus strains, we designed a construct bearing a transcriptional fusion of the promoter regions of pilA and pilT2 to the gfp-mut2 gene. We selected these genes because they Quantitative assessment of hormogonia induction | 3 Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf197/8129438 by guest on 09 July 2025 have been previously shown to be strongly induced specifically in hormogonia (Duggan et al., 2007; Campbell et al., 2008; Harwood and Risser, 2021). The promoter fusions were inserted in the N. punctiforme chromosome by homologous recombination (Fig. 1), generating strains NpPilA-GFP and NpPilT2-GFP. Firstly, given that a single N. punctiforme cell often contains multiple chromosomal copies (Meeks et al., 2001), we confirmed the absence of wild-type copies and verified the correct genomic integration for both reporter lines via PCR (Fig. 1). Subsequently, we examined GFP expression in these reporter lines. To do so, we visualized GFP expression in filaments A B C D Fig. 1. Construction of N. punctiforme reporter strains and their verification by PCR. (A and B) The promoter regions of the pilA (A) and pilT2 (B) genes are shown in gray, and the coding sequences in black. Primer 1 binds to the promoter region, primer 2 to the GFP sequence, and primer 3 to the gene of interest, respectively. In a wild-type genome, primers 1 and 3 will be able to anneal and amplify the respective intervening sequence stretch. Primer 3 has no target region to bind to. In the transformed genome, primers 1 and 3 will be able to anneal but amplification will fail because the intervening sequence is too long. By contrast, the primer pair 1+2 will lead to successful amplification. Each putative transformant strain/line is tested with both primer pairs (1+2 and 1+3). The cross refers to a failed PCR elongation due to the product being too long. (C and D) Gel images show the verification of correct integration into the N. punctiforme genome. Wild-type strains will only show an amplification product for primer pair 1+3, whereas strains carrying the promoter–GFP insertion will only produce a band in the gel for the primer pair combination 1+2. Nostoc punctiforme has multiple genome copies per cell. Thus, if incomplete segregation is present, the sample will be positive for both primer pair combinations, and further selection treatment is needed. 4| Neubauer et al. Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf197/8129438 by guest on 09 July 2025 Fig. 2. Confocal microscopy of N. punctiforme strains. Filaments from the indicated strains were taken from cultures grown in BG11 0 medium (not induced; A) or after incubation for 24 h in the presence of Anthoceros agrestis exudates (hormogonia induction; B) and visualized by confocal microscopy. Merged (2 channels) show the overlay of autofluorescence (magenta) and GFP fluorescence (green). Merged (3 channels) represent the overlay of magenta autofluorescence, bright field, and green GFP fluorescence. Hormogonia are identified as short filaments with small cells. Scale bar=20 μm. Quantitative assessment of hormogonia induction | 5 Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf197/8129438 by guest on 09 July 2025 grown in BG11 0 medium after a 24 h incubation with A. agrestis exudates known to induce hormogonia using a confocal microscope (Fig. 2). Hormogonia were induced in the presence of Anthoceros exudates, as detailed in the Materials and methods. As expected, we did not detect GFP signal in samples that were not induced, and visual inspection confirmed that they contained only vegetative cells. However, we observed strong GFP fluorescence in hormogonia from the strain NpPilA-GFP, indicative of hormogonia-specific expression of the transcriptional fusion (Fig. 2). The strain NpPilT2-GFP exhibited faint GFP expression, suggesting low promoter activity. This result is consistent with previous observations reporting strong and weak expression of the PilA and PilT2 genes, respectively, in Nostoc cells undergoing hormogonia formation (Harwood and Risser, 2021). Consequently, we selected the strain NpPilA-GFP for subsequent analyses. Green fluorescent protein expression reaches a maximum at 18–21 h post-induction We monitored GFP expression of the NpPilA-GFP strain under inductive conditions to determine the time point at which GFP reaches maximum expression (Fig. 3). A pre-culture grown in the presence of hormogonia-repressing sucralose was exposed with Anthoceros exudates (Fig. 3) after thorough washing to induce hormogonia differentiation. As a negative control, part of the pre-culture was maintained in the presence of sucralose. We found that GFP expression measured by a microplate reader (see the Materials and methods) increased, reaching a plateau at 18–21 h post-induction, indicating full hormogonia differentiation (Fig. 3). As the expression signal stayed stable until 30 h, we defined 24 h post-induction as the optimal time point which we used in all subsequent analyses. While sucralose treatment inhibits, its removal is known to induce massive hormogonia differentiation (Splitt and Risser, 2016). Therefore, to separate the effect of Anthoceros exudates from that of sucralose removal on hormogonia induction, we also assessed the sole effect of sucralose removal after 24 h (Fig. 4). While sucralose removal significantly increased the GFP signal compared with the sucralose-treated control, confirming previous observations (Splitt and Risser, 2016), exposure to Anthoceros exudates led to a much more dramatic expression increase (see treatment ‘Repressed’ in Fig. 4). Abiotic factors influencing hormogonia formation Hormogonia differentiation in N. punctiforme can be induced by various abiotic stimuli, including changes in light quality and quantity, as well as transfer to new growth media (Campbell and Meeks, 1989; Katoh et al., 2003). To test our Fig. 3. Time course of GFP expression during hormogonia induction in the NpPilA-GFP strain. GFP fluorescence intensity was measured at various time points post-induction using a microplate reader in the NpPilA-GFP strain grown with sucralose (repressed) or induced with A. agrestis exudate. To correct for varying numbers of cells in the wells, GFP fluorescence was normalized by chlorophyll autofluorescence. 6| Neubauer et al. Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf197/8129438 by guest on 09 July 2025 reporter strain, we induced hormogonia in the NpPilA-GFP strain which was previously grown in BG11 0 supplemented with sucralose by (i) transferring it to a new medium without sucralose, and (ii) exposing it to red light. As negative controls, we also measured GFP expression in the NpPilA-GFP strain grown in sucralose-containing BG11 0 . As expected, we found that the sucralose-treated samples showed weak GFP expression in comparison with the wild-type strain (Fig. 5). Transfer to a new medium without sucralose resulted in a significant increase in GFP expression compared with the repressed samples, although the levels were much lower than those observed with red light exposure (Fig. 5). Biotic factors influencing hormogonia differentiation Hormogonia differentiation can be significantly influenced by biotic factors, particularly the presence of plant exudates. Previous studies have demonstrated the role of plant-derived compounds in modulating hormogonia differentiation, enhancing our understanding of cyanobacteria–plant symbiosis (Meeks and Elhai, 2002; Risser and Meeks, 2013). To investigate the impact of specific plant exudates on hormogonia differentiation, we treated N. punctiforme cultures with exudates from A. agrestis, Gunnera tinctoria, Oryza sativa, and Triticum aestivum. All four plants are known hosts of N. punctiforme (Neubauer et al., 2024). The control treatment included cultures maintained with hormogonia-repressing sucralose. Our results demonstrate that exudates from G. tinctoria and T. aestivum induced the highest levels of GFP expression, indicating robust hormogonia differentiation. The induction by A. agrestis exudate was moderate, while O. sativa exudate resulted in the lowest induction among the plant exudates tested. Nonetheless, all plant exudate treatments led to significantly higher GFP expression compared with the sucralose-treated control (repressed) (Fig. 6). Discussion By integrating a high-throughput GFP fluorescence measurement, we have developed a simple, yet effective, method for quantifying hormogonia formation in the model cyanobiont N. punctiforme. This technique not only facilitates rapid and accurate quantification but also ensures reproducibility and scalability for large-scale studies. The use of a microplate reader to measure GFP fluorescence allows for high-throughput analysis, which is essential for screening multiple samples and conditions simultaneously. This methodological advancement addresses the limitations of previous techniques (Splitt and Risser, 2016) by providing a quick and straightforward means to assess hormogonia induction across various experimental conditions. Fig. 4. Effect of sucralose removal and Anthoceros exudates on GFP expression in the pilA reporter strain of N. punctiforme (NpPilA-GFP) 24 h postinduction. Repressed: NpPilA-GFP grown in BG11 0 supplemented with sucralose; subculture: NpPilA-GFP transferred to BG11 0 without sucralose; Anthoceros: NpPilA-GFP transferred to Anthoceros agrestis exudate. GFP fluorescence is normalized by chlorophyll autofluorescence. ns, not significant, ***P ≤ 0.001 (one-way ANOVA, with Tukey’s HSD post-hoc test). Quantitative assessment of hormogonia induction | 7 Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf197/8129438 by guest on 09 July 2025 Our methodology offers a quantitative and high-throughput approach for screening the effects of various environmental and plant-derived stimuli. Traditional methods for evaluating hormogonia induction, such as plate mobility assays and light microscopy, are predominantly qualitative and labor-intensive (Campbell and Meeks, 1989; Liaimer et al., 2011). These methods require significant time and manual effort, limiting their applicability for large-scale studies. Other quantitative approaches, such as RT– PCR, provide precise measurements but are also time-consuming and require specialized equipment and expertise (Knight and Adams, 1996; Campbell et al., 2007). It is important to note that all our procedures use Nostoc grown with sucralose, a non-metabolizable sucrose analog, to inhibit hormogonia formation prior to testing the effect of biotic or abiotic factors (Splitt and Risser, 2016). This treatment was necessary because Nostoc cultures grown without sucralose contained highly variable amounts of hormogonia, thus making measurements hardly comparable. While sucralose treatment provides a convenient way of keeping the amount of hormogonia low, previous studies reported that sucralose removal triggers massive formation of hormogonia (Splitt and Risser, 2016). Therefore, when sucralose-treated Nostoc is exposed to abiotic or biotic factors in media lacking sucralose, hormogonia induction is expected to be caused in part by the effect of sucralose removal. Consequently, all our experiments include a control by transferring Nostoc to sucralosefree medium and comparing this with a culture exposed to additional biotic or abiotic factors (Figs 4–6). In our experimental set up, hormogonia formation was much more strongly induced by the tested abiotic and biotic factors than by sucralose removal alone, allowing us to separate these two effects. Nevertheless, the inclusion of a control assessing the sole effect of sucralose removal is essential to accurately assess the effect of abiotic and biotic factors on hormogonia formation. As shown here, this methodology can be applied to study the varying influences of biotic and abiotic factors on hormogonia differentiation in N. punctiforme. Abiotic factors, such as light quality and nutrient availability, also play a crucial role in hormogonia differentiation. Our results show that exposure to red light significantly induced hormogonia differentiation (Fig. 5). This is consistent with previous studies indicating that specific wavelengths of light can trigger hormogonia formation in cyanobacteria (Katoh et al., 2003; Campbell et al., 2007). Regarding biotic factors, our results show that exudates from G. tinctoria and T. aestivum are particularly potent in inducing hormogonia differentiation, as evidenced by the highest levels of GFP expression (Fig. 6). This aligns with previous findings that plant-derived compounds can significantly modulate cyanobacterial development, enhancing symbiotic interactions (Meeks and Elhai, 2002; Risser and Meeks, 2013). Fig. 5. Effect of abiotic factors (transfer to new media and exposure to red light) on GFP expression in the pilA reporter strain of N. punctiforme (NpPilA-GFP) 24 h post-induction. WT: wild type N. punctifome (without promoter–gene fusion insertion); repressed: NpPilA-GFP grown in BG11 0 supplemented with sucralose; subculture: NpPilA-GFP transferred to BG11 0 without sucralose; red light: NpPilA-GFP grown under red light. GFP fluorescence is normalized by chlorophyll autofluorescence. ns, not significant, ***P ≤ 0.001 (one-way ANOVA, with Tukey’s HSD post-hoc test). 8| Neubauer et al. Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf197/8129438 by guest on 09 July 2025 The moderate induction observed with Anthoceros exudates and the comparatively lower induction by O. sativa exudates suggest a spectrum of hormogonia-inducing activity across different plant species (Fig. 6). The reduced induction by O. sativa may also be due to lower concentrations of bioactive compounds, or requirement for a longer induction time, reflecting differences in the timing or mechanism of its interaction with cyanobacteria. This variability underscores the specificity of cyanobacterial responses to distinct phytochemicals, reflecting the evolutionary adaptations of N. punctiforme to its symbiotic partners (Rai et al., 2000; Nilsson et al., 2002). Despite the lower induction by rice exudates, all plant treatments resulted in significantly higher GFP expression compared with sucralose-treated controls, confirming the effectiveness of these biotic factors in promoting hormogonia differentiation. The robust response to G. tinctoria and T. aestivum exudates could be attributed to the presence of specific hormones or secondary metabolites that act as strong HIFs. The precise chemical nature of these HIFs remains to be elucidated, but their identification could provide valuable insights into the molecular mechanisms underlying plant–cyanobacteria symbioses (Bergman et al., 2007; Adams et al., 2013; Duggan et al., 2013). Future studies should focus on isolating and characterizing these compounds to understand their roles in the symbiotic signaling pathways. Conclusion Altogether, the differential responses to plant exudates observed in this study highlight the complex and specific interactions between N. punctiforme and its plant hosts. Understanding these interactions at the molecular level will not only advance our knowledge of cyanobacterial symbioses but also pave the way for potential agricultural applications, such as biofertilization and sustainable crop management. Supplementary data The following supplementary data are available at JXB online. Fig. S1. Sequence of the green fluorescent protein used in the reporter strains. Table S1. Plasmids used in the transformation of N. punctiforme. Table S2. Primers used to amplify the putative promoter sequences and validate the segregation. Acknowledgements We thank Laura Frías (Institute of Plant Biochemistry and Photosynthesis, CSIC and University of Seville, Sevilla, Spain) for her technical assistance. Fig. 6. Effect of plant exudates (biotic factors) on GFP expression in the pilA reporter strain of N. punctiforme (NpPilA-GFP) 24 h post-induction. The reporter strain was exposed to BG11 0 medium containing exudates of four plants, the hornwort Anthoceros agrestis, rice Oryza sativa, wheat Triticum aestivum, as well as mucilage collected from the giant rhubarb Gunnera tinctoria. GFP fluorescence is normalized by chlorophyll autofluorescence. WT: GFP fluorescence of wild-type N. punctiforme; repressed: NpPilA-GFP grown in BG11 0 supplemented with sucralose is shown as control. For clarity, only statistically significant differences (one-way ANOVA, with Tukey’s HSD post-hoc test) to the repressed (sucralose) control of the pilA reporter are indicated: ns, not significant, ***P ≤ 0.001. Quantitative assessment of hormogonia induction | 9 Downloaded from https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/eraf197/8129438 by guest on 09 July 2025