Sublethal Cd exposure stimulates Laccaria bicolor x poplar symbiosis formation

Sublethal Cd exposure stimulates Laccaria bicolor x poplar symbiosis formation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sublethal Cd exposure stimulates Laccaria bicolor x poplar symbiosis formation Maarten Ottaway, Janne Swinnen, Joske Ruytinx This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8981216/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Soils have become increasingly polluted with Cd due to industrial and mining activities, as well as agricultural fertiliser usage. Because of its toxicity, plants face significant abiotic stress. Trees found in temperate and boreal forest ecosystems rely on their mutualistic relationship with ECM fungi to alleviate the toxic effects of Cd. In this study, we assessed the impact of Cd pollution on both L. bicolor and its symbiosis with P. tremula x alba . We investigated the impact of Cd pollution on fungal growth and mycorrhiza morphology, as well as the expression of symbiosis marker genes and ROS scavenging enzymes in presence and absence of a host plant. Results indicate that fungal growth is reduced by exposure to elevated Cd, however symbiosis formation is stimulated. Both symbiosis marker genes and ROS scavenging enzymes showed increased expression upon exposure to Cd, but only in the presence of a host plant. This data suggests that forming the ECM symbiosis is a key coping mechanism for both poplars and L. bicolor , and by stimulating the formation of the symbiotic structure, the reduced fungal growth can partially be mitigated. This research highlights the importance of the ECM symbiosis in both plant and fungal resilience in changing environmental conditions. Laccaria bicolor Poplar Cadmium toxicity Ectomycorrhiza antioxidative response Figures Figure 1 Figure 2 Figure 3 Introduction Cadmium (Cd) is a non-essential metal that is commonly found within ore deposits as a substitute for Zn (Yaciuk et al., 2022 ). Cd is considered highly toxic. Even at very low concentrations, it is known to cause stunted growth, chlorosis and wilting (Haider et al., 2021 ; Qin et al., 2020 ; Stahl & Smoliakova, 2007 ). Cd has seen an increase in industrial usage, as it is an important component for the production of batteries, electronics and paints (Angon et al., 2024 ). Due to these increased activities, soils surrounding mining operations, coal power plants and heavy industry have become polluted with Cd. Also agriculture has been affected by Cd pollution through the use of Cd-containing pesticides and phosphate fertilisers (Angon et al., 2024 ; Ballabio et al., 2024 ; Khan et al., 2017 ; Yuan et al., 2019 ; Zhao et al., 2023 ). As Cd is highly toxic, it renders polluted soils, and especially affected agricultural fields, economically unprofitable for plant and crop production. Ingestion of crops grown on Cd polluted soils can have a severe impact on human health. Cd toxicity can cause impaired heart function, neurodegenerative diseases and increases the risk of cancer (Angon et al., 2024 ; Charkiewicz et al., 2023 ; Khan et al., 2017 ). Remediation of Cd polluted soils is thus of great importance to benefit human health, but also to allow for contaminated areas to be used again for agriculture. Phytoremediation is a popular soil remediation strategy. By using fast-growing, metal tolerant and metal accumulating plants, metals such as Cd can safely and cheaply be removed from the soil (Aparicio et al., 2022 ). Poplars are commonly used for this because of their fast growth, but also due to their economic importance for the wood and paper industry (Komán et al., 2023 ; Pilipovic et al., 2019 ). The majority of trees found within temperate and boreal forest ecosystems form a symbiosis with ectomycorrhizal (ECM) fungi (Soudzilovskaia et al., 2019 ; Steidinger et al., 2019 ). This symbiotic relationship benefits both the plant and fungal partner. The fungus provides improved access to nutrients, as well as provides water and increased resistance to both biotic and abiotic stress (Becquer et al., 2019 ; Branzanti et al., 1999 ; Chot & Reddy, 2022 ; Ochoa-Hueso et al., 2023 ). For example, Laccaria bicolor will limit the uptake of soil pollutants, such as Cd, by their host plant by sequestering them to their cell wall (Quan et al., 2023 ). In exchange, the host plant provides carbon in the form of photosynthates (Genre et al., 2020 ). The ECM symbiotic structure is characterised by the formation of a tightly packed hyphal mantle surrounding host plant lateral roots (LR). Within colonised LRs a hartig net is formed, composed of hyphae that penetrate between root cortical and epidermal cells. The hyphae that extend outward from the plant root into the soil make up the extraradical mycelium (Garcia et al., 2015 ). So far, most research on the establishment of the ECM symbiosis has utilised the L. bicolor - Populus tremula x alba model co-culture system. Within this system, it was found that a complex chemical cross-talk takes place between both partners (Martin et al., 2016 ). Research has found this cross-talk to be crucial for fungal colonisation of the host root and the development of the symbiotic structure. One example of this is the fungal production of mycorrhiza induced small-secreted proteins. These will, for example, allow the fungus to regulate the plant defence response (Plett et al., 2014 ). Also the expression of fungal carbohydrate-active enzymes (CAZymes) is needed to modify the plant cell wall to allow for hyphal penetration (Zhang et al., 2018 ; F. Zhang et al., 2021 ). Reactive oxygen species (ROS) accumulate during stress, such as Cd toxicity, causing oxidative damage to cells (Choudhury et al., 2017 ; Cuypers et al., 2010 ). However, a role for ROS, and mainly H 2 O 2 , in stress response signalling was also discovered, allowing for the activation of the stress response pathway (Mittler et al., 2022 ). For example, in Arabidopsis thaliana , exposure to Cd and the accompanied increase in intracellular H 2 O 2 , will activate MAPK pathways as well as induce the jasmonic acid and salicylic acid pathways. In Brassica juncea , this exposure will rather activate the ethylene pathway (Cuypers et al., 2016 ). Additionally, it was also found that ROS produced throughout the ECM symbiosis is crucial for its development (Shi et al., 2024 ). How Cd pollution impacts the balance between ROS scavenging and ROS signalling throughout ECM symbiosis development has not yet been studied. In this study, we investigate the impact of Cd pollution on the formation of the ECM symbiosis between L. bicolor and poplar. We assessed the effect on both the ECM symbiotic structure, as well as the expression of symbiosis marker genes and ROS scavenging enzymes. Our data suggests that Cd exposure stimulates symbiosis formation in this model system, despite reducing fungal growth. Altogether, this study provides a new perspective on the impact of Cd pollution on the ECM symbiosis and further highlights its usefulness in soil remediation. Materials and Methods Biological strains and growth conditions Mycelium of Laccaria bicolor S238N (Maire) P.D. Orton was used throughout all experiments. Fungal cultures were grown and maintained at 23°C in the dark on Pachlewski P5 medium, consisting of 0.5 g l − 1 di-NH 4 -tartrate, 1 g l − 1 KH 2 PO 4 , 0.5 g l − 1 MgSO 4 .7H 2 O, 5 g l − 1 maltose, 20 g l − 1 glucose, 0.1 mg l − 1 thiamine-HCl, 5 mg l − 1 MnSO 4 .4H 2 O, 8.5 mg l − 1 H 3 BO 3 , 0.3 mg l − 1 (NH 4 ) 6 Mo 7 O 24 .4H 2 O, 6 mg l − 1 FeCl 3 , 0.6 mg l − 1 CuSO 4 .5H 2 O, 2.7 mg l − 1 ZnSO 4 .7H 2 O and 20 g l − 1 agar at pH 5.5 (Kemppainen & Pardo, 2011 ). The sensitivity of L. bicolor to Cd was determined through dose-response experiments. Mycelial plugs (0.5 cm 2 ) were placed on cellophane-covered P5 medium plates (9 cm diameter) supplemented with or without 3CdSO 4 .8H 2 O. Specifically, the following concentrations were tested: 0 (control), 0.1, 0.2, 0.4, 0.6, 0.7, 0.8, 0.9, 1, 2, 4, 8, 10, 20 and 100 µM Cd. Each concentration consisted of five biological replicates. Cultures were incubated for three weeks in the dark at 23°C, after which mycelium was harvested, weighed and lyophilised. Dry weights were subsequently determined and the data was fit using a non-linear regression with four parameter log-logistic model (Ritz et al., 2015 ) in “R” version 4.4.1 (R Core Team, 2024 ). The maximal effective concentration (EC50) was calculated and corresponds to the concentration that reduces growth by 50% compared to control conditions. Plant – fungal co-cultures Populus tremula x alba 717-1B4 plants were maintained by micropropagation on Murashige and Skoog medium (MS), supplemented with 10 µM IBA (Murashige & Skoog, 1962 ). Plants were subsequently used for in vitro co-culturing experiments between P. tremula x alba 717-1B4 and L. bicolor S238N as previously described (Felten et al., 2009 ). Mycelium grown in the presence or absence of a host plant was cultured on sugar reduced Pachlewski P20 medium supplemented with 0.1% MES at 23°C for 14 days with a photoperiod of 16h. P20 medium was either supplemented with 0 µM 3CdSO 4 .8H 2 O (control), or sublethal Cd (1 µM 3CdSO 4 .8H 2 O). Scans of the co-cultures were made after two weeks of incubation using a Perfection V600 Photo scanner (Epson). Roots were fixated in 4% paraformaldehyde (PFA) overnight at 4°C, after which they were washed and stored in phosphate buffered saline (PBS) before microscopic analysis. Six biological replicates (100 mg) of either mycelium grown in absence of a host plant or mixed plant-fungal material (composed of mycorrhized root tips and mycelium within 5 mm of the root) were harvested for RNA extractions, flash frozen in liquid nitrogen and stored at -80°C until further processing. Mycorrhizal morphology and root growth assessment Total root length per plant was measured using RhizoVision Explorer (Seethepalli et al., 2021 ). Both total and mycorrhized lateral roots (LR) were manually counted per plant, and the number of LR per cm root was calculated (total number of LR/root length). Mycorrhization percentage was also determined (number of mycorrhized LR/total number of LR). Normality of all data was assessed by means of the Shapiro-Wilk test, and statistical significances were determined using a Student’s t-test (P < 0.05) in Graphpad Prism V10.1.0. Overnight PFA-fixed roots were embedded in 4% agarose. Using a VT1000S vibratome (Leica), 25 µM thick cross-sections were made (0.225 mm/s; 40 Hz). Cross-sections located 275 µm from the start of the LR were stained for 1h using 10 µg/ml Wheat Germ Agglutinin Alexa Fluor 488 (WGA-488; Invitrogen; W11261). Sections were then counterstained for 30 min using 15 µM Propidium Iodide (PI; Invitrogen; P3566) and visualised using an Eclipse Ti2 inverted fluorescence microscope (Nikon). RNA extractions and Quantitative Real-Time PCR (qRT-PCR) RNA was extracted using the RNeasy Plant Mini kit (Qiagen). Using a pestle and mortar, 100 mg of material was ground in liquid nitrogen. Following this step, the manufacturers protocol was followed, and 2% PEG8000 was included in the RLC buffer. A DNase treatment was also included. The eluted RNA was flash frozen in liquid nitrogen and stored at -80°C. The integrity of the RNA was determined using a Bioanalyzer 2100 (Agilent Technologies), and the purity was assessed using a NanoDrop One Spectrophotometer (Thermo Fisher Scientific). Using the High-Capacity cDNA reverse transcription kit and protocol (Applied Biosystems), 250 ng of RNA was converted to cDNA. Expression of ectomycorrhizal symbiosis marker genes found in either P. tremula x alba or L. bicolor was determined. Specifically, expression of TPS16 (Terpene synthase; ID: PtXaTreH.01G253600.1), GH28a (endopolygalacturonase, ID: 613299), MiSSP7 (mycorrhiza induced small-secreted protein, ID: 298595) and MiSSP17 (ID: 332226) was measured. Gene expression of L. bicolor ROS decomposing enzymes was also measured, these being CAT (catalase, ID: 123238), Mn/Fe SOD1 (superoxide dismutase, ID: 635077), Mn/Fe SOD2 (ID: 192586), Mn/Fe SOD3 (ID: 291347), Mn/Fe SOD4 (ID: 295682) and Mn/Fe SOD5 (ID: 312019). Each condition consisted of a minimum of three biological replicates. A no-template control was also included. The qRT-PCR reactions consisted of 2x GoTaq qPCR Master Mix (Promega), 400 nM FW and RV primer, 2 µl cDNA and nuclease free H 2 O was added until a reaction volume of 20 µl. Reaction mixtures were pipetted into 96-well plates and run according to the following program on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad): 1 cycle of 95°C for 3 min and 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. Different reference genes, published previously, were also tested using the aforementioned protocol (Pellegrin et al., 2019 ). The obtained results were subjected to geNorm analysis using the qbase+ software (Biogazelle) to determine the stability of their expression in the growth conditions used. The software recommended genes (Mycocosm ID: 611151 and 313997) were subsequently used to calculate a normalization factor (NF), allowing for the relative expression of the genes of interest to be determined using the following formula: 2 (Ct−Ctmin) /NF (Vandesompele et al., 2002 ). The values were Log2 transformed and normality was tested using the Shapiro-Wilk test. All data was further analysed by two-way ANOVA with Tukey’s multiple comparisons test, except for TPS16 which was subjected to a Student’s t-test (P < 0.05). Statistical analysis were performed in Graphpad Prism V10.1.0. All primers used are listed in supplementary table S1 . Results Impact of sublethal Cd exposure on fungal growth and early mycorrhiza development To determine the impact of sublethal exposure to Cd on L. bicolor and its symbiosis with poplar, fungal growth and symbiosis formation were assessed. Exposure of L. bicolor mycelium to increasing concentrations of 3CdSO 4 .8H 2 O caused decreased growth, with fungal growth being almost completely inhibited at 20 µM. The EC50 value was calculated to be 0.9 µM. (Fig. 1 a; Fig. S1 ). Thus, for the following experiments, 1 µM 3CdSO 4 .8H 2 O was chosen as sublethal Cd concentration in order to induce stress but not cause severe growth issues. In co-culture, exposure to sublethal Cd did not significantly decrease LR formation by P. tremula x alba (Fig. 1 b). A significant increase in LR/cm root was observed under stress conditions (Fig. 1 c). This increase was accompanied by a significant increase in mycorrhization under stress conditions (Fig. 1 d). Microscopic analysis of mycorrhized LR revealed these to be in the early stages of development. A fully formed hyphal mantle was observed under control conditions, while this was delayed, showing only partial formation, under Cd stress (Fig. 1 e). Impact of sublethal Cd on symbiosis marker gene expression Cd exposure increases LR formation and mycorrhization. However, its impact on the expression of genes crucial for symbiosis development remains unclear. To further investigate this, the expression of four symbiosis marker genes from poplar and L. bicolor was measured. For poplar, the expression of terpene synthase TPS16 , which is not expressed in the fungus, was determined. A significant increase in the expression of TPS16 was found after exposure to Cd compared to control (Fig. 2 a). Within L. bicolor , the expression of three well established symbiosis marker genes was assessed. ANOVA analysis of these genes revealed their expression to be impacted by Cd, the presence of a host plant and their combined interaction (Cd x host plant) (Table S2). Specifically, expression of all three marker genes was only differentially expressed in co-culture when exposed to sublethal Cd. More so, they showed a similar expression profile under all conditions tested. (Fig. 2 b-d). Impact of sublethal Cd on ROS scavenging enzyme gene expression Exposure to elevated concentrations of Cd can cause oxidative damage. Thus, to better understand how the co-culture deals with Cd-induced ROS accumulation, the expression of ROS scavenging enzymes catalase ( CAT ) and Mn/Fe SODs was also investigated. Two-way ANOVA analysis indicated that CAT expression was impacted by Cd, presence of a host plant and their interaction (Table S2). Specifically, CAT was found to be significantly upregulated in co-culture upon exposure to Cd (Fig. 3 a). L. bicolor contains five predicted Mn/Fe SOD genes, of which the expression of two ( Mn/Fe SOD 1 and Mn/Fe SOD2 ) were found to not be affected by any condition (Fig. 3 b/c). Of the remaining three SOD genes, ANOVA results indicated an impact of the combined interaction (Cd x host plant) on the expression of all three. Cd alone was found to impact Mn/Fe SOD 3/4, with Mn/Fe SOD3 also being impacted by the presence of a host plant (Table S2). Despite these differences in ANOVA result, Mn/Fe SOD 3/4/5 all showed similar results. All were found to be upregulated upon exposure to Cd in the presence of a host plant (Fig. 3 d-f). Exposure of free-living mycelium to Cd did not significantly increase the expression of any tested ROS scavenging enzyme (Fig. 3 ). Discussion Exposure to sublethal Cd impacts fungal growth, root colonisation and mycorrhiza development As Cd is a non-essential metal, exposure to toxic amounts can lead to cellular damage, reduced growth and even cell death (Dachuan & Jinyu, 2021 ; De Oliveira & Tibbett, 2018b ). Certain species, including fungal species, have adapted to survive in environments rich in Cd pollution (Krznaric et al., 2009 ). This adaptation allows them to reduce their Cd uptake, and limit the damage that is inflicted by this pollutant. Certain isolates of the ectomycorrhizal fungus Suillus luteus , for example, were shown to be more tolerant towards high Cd concentrations compared to more Cd sensitive isolates. (Colpaert et al., 2011 ). Between different species, this tolerance towards Cd can also vary significantly. While Hebeloma cylindrosporum is highly sensitive towards Cd, Hebeloma subsaponaceum was found to have an increased tolerance towards it (De Oliveira & Tibbett, 2018a ). For the haploid L. bicolor S238 strain, a previous study found that at very high concentrations of Cd, such as 40–50 µM, growth was inhibited by ± 30% (Reddy et al., 2014 ). The dikaryotic L. bicolor S238N strain rather showed a gradual decrease in growth starting at concentrations of 1 µM Cd, with growth almost completely inhibited at 40 µM Cd (Liu et al., 2022 ). This difference in Cd sensitivity compared with our data might, however, be due to the use of Modified Melin Norkans (MMN) medium. The presence of malt extract might have an impact on the toxicity of Cd on L. bicolor . In P5 medium, which contains much less nutrients, L. bicolor was much more susceptible to Cd, as we calculated the EC50 value to be 0.9 µM (Fig. S1 ). This marks L. bicolor , grown on P5 medium, as a Cd-sensitive species (Colpaert et al., 2011 ). Not only fungi but also plants suffer from Cd pollution. Prolonged exposure of plants to elevated concentrations of Cd can cause stunted growth, decreased root length, chlorosis and even necrosis (He et al., 2017 ; Qin et al., 2020 ). For example, exposure of Populus deltoides x Populus nigra to 50 mg/kg Cd resulted in a significant decrease in biomass, root length and lateral root formation (Yi et al., 2022 ). Additionally, Cd also impacts the plant growth hormone auxin. In both Populus x canescens and Arabidopsis thaliana , Cd interfered with auxin metabolism and signalling, resulting in a decrease in auxin within the root apical meristem. This reduction in auxin negatively impacted root growth and resulted in stunted roots (Elobeid et al., 2012 ; Yuan & Huang, 2016 ). A common strategy to deal with Cd toxicity among tree species is to form a symbiosis with ECM fungi. Through this symbiosis, fungi can limit Cd uptake while maintaining sufficient nutrient influx (Reddy et al., 2016 ; K. Zhang et al., 2021 ). However, how Cd pollution specifically impacts the formation of the ECM symbiosis was never investigated. Throughout the early stages of ECM symbiosis development, chemical cross-talk will occur between the host plant and the fungus (Garcia et al., 2015 ). This cross-talk will stimulate both fungal growth as well as LR formation (Ditengou et al., 2015 ; Lagrange et al., 2001 ). Exposure of the co-culture to sublethal Cd did not result in a significant change in the number of LRs. Rather, it increased the LR/cm root ratio, indicating that while growth of the main root decreased, LR formation was stimulated. ECM fungi will induce LR formation, as was previously found between L. bicolor and poplar (Vayssières et al., 2015 ). Interestingly, in A. thaliana , exposure to Cd, specifically concentrations higher than 12.5 µM, was also found to induce LR formation (Hu et al., 2013 ). Furthermore, our results showed that Cd exposed co-cultures also exhibited higher mycorrhization rates. The increase in LRs upon exposure to Cd compared to control suggests that Cd, rather than the fungus itself, is responsible for the increase in LR development under Cd toxic conditions. As a consequence, as more LRs are formed by the plant, more points of contact become available for the fungus to colonise, thus increasing the mycorrhization rate. This response of the poplar x L. bicolor co-culture to Cd seems to differ based on the symbiotic partners. For example, poplar and Pinus sp. have a similar tolerance towards Cd (De Oliveira & Tibbett, 2018a ; Hartley-Whitaker et al., 2000 ; He et al., 2013 ; Sousa et al., 2012 ). However, exposure of Pinus pinaster co-cultures with either Suillus bovinus or Rhizopogon roseolus to Cd rather showed decreased LR formation and mycorrhization (Sousa et al., 2012 ). Similar results were also obtained with Pinus sylvestris , where decreased mycorrhization of LRs was found after Cd exposure (Hartley-Whitaker et al., 2000 ). As the symbiosis continues to develop, the fungus starts to form a hyphal mantle (Ruytinx et al., 2021 ). However, Cd still negatively impacts fungal growth. To understand the extent by which this reduced fungal growth impacts ectomycorrhiza development, mycorrhized LR cross-sections were analysed through fluorescence microscopy. Under control conditions the co-culture exhibited a hyphal mantle encompassing the complete lateral root, with the hartig net beginning to form. Cd-exposed co-cultures, on the other hand, showed incomplete hyphal mantles, as expected due to the slower fungal growth. A previous study investigating the ECM symbiosis between Paxillus involutus and Populus x canescens found that mycorrhized plants rather imported more Cd as compared to non-mycorrhized plants. However, mycorrhized plants also showed an increased defence response and Cd transport capabilities, thus increasing their Cd tolerance (Ma et al., 2014 ). On the other hand, other ECM partnerships, such as Hebeloma mesophaeum and Populus nigra , showed reduced Cd uptake by the plant when in symbiosis (Luo et al., 2014 ). While different strategies can be used, they illustrate the benefit of this symbiosis for the Cd tolerance of both partners. These results suggest that Cd stress stimulates symbiosis formation between L. bicolor and P. tremula x alba , despite also slowing down ectomycorrhiza development. Thus, promoting mycorrhization and increasing the contact points between both partners might be an adaptation strategy employed by the L. bicolor x poplar co-culture to increase their tolerance towards Cd pollution. Expression of symbiosis marker genes is upregulated by Cd exposure in presence of a host plant For successful colonisation of host roots, the plant defence response needs to be inactivated. Among the activated poplar defence genes are terpene synthases, such as TPS16 and TPS21 . Terpene synthases produce terpene mixtures which have been shown to reduce fungal growth and inhibit root colonisation. Furthermore, it was found that throughout symbiosis development, these genes are downregulated through cross-talk between L. bicolor and P. tremula x alba (Marqués-Galvez et al., 2024 ). Interestingly, a previous study discovered that TPS16 produces a monoterpene mix primarily composed of γ-terpinene (Lackus et al., 2018 ). Contrary to the other terpenes produced, γ-terpinene was found to stimulate growth of L. bicolor (Marqués-Galvez et al., 2024 ). Contrary to the downregulation of the TPS genes typically found, exposure of the co-culture to Cd rather caused a significant upregulation of TPS16 expression. As TPS16 can stimulate fungal growth, and Cd reduces fungal growth, this increased expression could assist L. bicolor in growing towards and colonise LRs under Cd polluted conditions. Besides attenuation of the plant defence response, major transcriptional changes within both plant and fungus need to occur to allow for ECM development. Most notably is the upregulation of MiSSPs, where MiSSP7 regulates the host defence response and MiSSP17 is one of the highest upregulated genes throughout symbiosis (Plett et al., 2014 ; Ruytinx et al., 2021 ). Not only are transcriptional changes required, the plant cell wall also needs to be modified to allow for hyphal penetration and hartig net formation. This can be achieved through the expression of specific CAZymes, such as the polygalacturonase GH28a, which will target the pectin within the plant cell wall (F. Zhang et al., 2021 ). Here, we assessed the impact of Cd on the expression of these three well studied symbiosis marker genes. Upon exposure of the co-culture to Cd, all three genes were found to be upregulated. Expression of these genes remained stable under every other condition tested. It is known that plants utilise their cell walls to bind Cd in order to limit the uptake of this pollutant. Specifically, pectin within plant cell walls serves as a binding site, as its negative charge attracts the positively charged Cd. Furthermore, under Cd stress plants produce more pectin to be able to sequester more labile Cd (Loix et al., 2017 ). This increase in pectin makes it more difficult for fungal hyphae to penetrate between plant cells, indicating that the increased expression of GH28a is needed for efficient root colonisation. JAZ6 is a repressor of the jasmonic acid (JA)-induced plant defence response. Upon activation of this response, JAZ6 will be degraded, allowing for the expression of defence response genes (Kazan & Manners, 2013 ; Y. Wang et al., 2021 ). Throughout symbiosis, MiSSP7 binds to JAZ6, preventing the activation of this response (Plett et al., 2014 ). It was previously found that exposure of Brassica juncea to elevated Cd increased the expression of JAZ proteins and of the JA-amino synthetase, responsible of producing isoleucine (Ile)-JA. Ile-Ja, in turn, promotes the degradation of JAZ proteins (Thakur et al., 2019 ). The increased expression of MiSSP7 indicates that Cd exposure further stimulates the activation of the JA defence response, requiring additional stabilisation of JAZ6 to prevent this response. ROS scavenging enzymes are only upregulated upon exposure to Cd in co-culture Cd is known to cause oxidative damage and induce the antioxidative response (Cuypers et al., 2010 ; Lin & Aarts, 2012 ). Furthermore, ROS have been found to play a role in establishing the ECM symbiosis within compatible hosts (Baptista et al., 2007 ; Gafur et al., 2004 ). For example, NADPH oxidase knock-down mutants of L. bicolor were found to be severely impaired in their ability to form a hartig net (Shi et al., 2024 ). However, how ROS signalling and scavenging are linked throughout symbiosis development is unclear. To gain a better understanding, we measured the effect of exposure to sublethal Cd on the expression of the two main ROS scavenging enzymes, SOD and CAT. While SOD catalyses the reduction of superoxide radicals into hydrogen peroxide (H 2 O 2 ), CAT facilitates the further reduction of H 2 O 2 into water and oxygen (Aebi, 1974 ; McCord & Fridovich, 1969 ). Exposure of the co-culture to sublethal Cd resulted in a significantly increased expression of CAT . In absence of a host plant, Cd exposure did not cause an increase in expression. At a protein level, CAT activity is known to be regulated through post-translational modifications, such as phosphorylation and acylation (Baker et al., 2023 ). This co-culture specific increase in expression suggests that post-translational modifications alone are not sufficient to counter the increased accumulation of H 2 O 2 . Two of the five predicted Mn/Fe SOD genes within L. bicolor ( SOD1 and SOD2 ) were not differentially expressed upon exposure to Cd, a host plant or both. The remaining three SOD genes ( SOD3-5 ) showed upregulation, but only in co-culture when exposed to Cd. Depending on the type of SOD or environmental condition, SODs can change their localisation and even their function. For example, Cu/Zn SODs can be found within the cytosol, but also within plastids as well as the extracellular environment (Kliebenstein et al., 1998 ). More so, SOD5 of Fusarium oxysporum localises primarily within phialides, sporogenous conidia-producing cells. However, under nutrient limiting conditions SOD5 rather localises to hyphae and septa (Sewall et al., 1990 ; Q. Wang et al., 2021 ). In human fibroblasts, H 2 O 2 bursts shift SOD1 from a cytosolic localisation to nuclear accumulation, additionally changing its functionality into that of a transcriptional regulator (Tsang et al., 2014 ). Within L. bicolor , there is no information available on the function or localisation of these predicted Mn/Fe SOD genes and further research on their activity and location is needed to fully understand these results. Despite this, the specific upregulation of three SOD genes in co-culture upon exposure to Cd suggests that they play an important role in the stress protection of the L. bicolor x poplar symbiosis. Additionally, prolonged exposure of Scots pine seedlings to 50 µM Cd resulted in decreased CAT and SOD activity (Schutzendubel et al., 2001 ). The significant increase in expression found for both CAT and SOD from L. bicolor might also be to compensate for a lower poplar CAT and SOD activity, similar to Scots pine. However, the expression and activity of these enzymes within poplar exposed to Cd would need to be investigated to test this hypothesis. Conclusion In this study, we found that L. bicolor and P. tremula x alba can still form the ECM symbiosis when exposed to sublethal Cd. Furthermore, Cd stress stimulated LR formation and mycorrhization, as supported by the increased expression of symbiotic marker genes. This suggests that both poplar and L. bicolor rely on their symbiosis as a Cd stress coping mechanism. However, formation of the symbiotic structure was slower compared to control. Under the same conditions, expression of ROS scavenging enzymes was also upregulated, indicating that post-translational modifications alone are not sufficient to overcome the increased H 2 O 2 accumulation. Elucidation of the exact role and subcellular localisation of the Mn/Fe SODs throughout ECM symbiosis is required to assess whether they also assist in ECM symbiosis formation or only provide superoxide radical protection. Declarations The authors declare that they have no conflict of interest. Funding Declaration This research was funded by Research Foundation Flanders through an FWO Fundamental Research PhD fellowship granted to MO (1193322N). Author Contribution The experiments were designed by **MO** , JS and JR. Dose-response experiments were performed by **MO** . Co-cultures were set up and harvested by **MO** and JS. Morphological and microscopic analyses were performed by **MO** . RNA extractions and qRT-PCR analysis were performed by JS. Statistical analyses were performed by **MO** . The manuscript was written by **MO** and edited by JR. Acknowledgement Sincere thanks to Joyce Garmyn for providing the poplar microcuttings, as well as to Karl Jonckheere for maintaining the fungal collections. Data Availability All raw data and metadata are publicly available on Zenodo ( [10.5281/zenodo.18792406](https:/doi.org/10.5281/zenodo.18792406) ). References Aebi H (1974) Catalase. In H. U. Bergmeyer (Ed.), Methods Of Enzymatic Analysis (Second Edition) (2 ed.). Academic Press. https://doi.org/10.1016/b978-0-12-091302-2.50032-3 Angon PB, Islam MS, Kc S, Das A, Anjum N, Poudel A, Suchi SA (2024) Sources, effects and present perspectives of heavy metals contamination: Soil, plants and human food chain. Heliyon 10(7):e28357. https://doi.org/10.1016/j.heliyon.2024.e28357 Aparicio JD, Raimondo EE, Saez JM, Costa-Gutierrez SB, Álvarez A, Benimeli CS, Polti MA (2022) The current approach to soil remediation: A review of physicochemical and biological technologies, and the potential of their strategic combination. J Environ Chem Eng 10(2). https://doi.org/10.1016/j.jece.2022.107141 Baker A, Lin CC, Lett C, Karpinska B, Wright MH, Foyer CH (2023) Catalase: A critical node in the regulation of cell fate. Free Radic Biol Med 199:56–66. https://doi.org/10.1016/j.freeradbiomed.2023.02.009 Ballabio C, Jones A, Panagos P (2024) Cadmium in topsoils of the European Union - An analysis based on LUCAS topsoil database. Sci Total Environ 912:168710. https://doi.org/10.1016/j.scitotenv.2023.168710 Baptista P, Martins A, Pais MS, Tavares RM, Lino-Neto T (2007) Involvement of reactive oxygen species during early stages of ectomycorrhiza establishment between Castanea sativa and Pisolithus tinctorius. Mycorrhiza 17(3):185–193. https://doi.org/10.1007/s00572-006-0091-4 Becquer A, Guerrero-Galán C, Eibensteiner JL, Houdinet G, Bücking H, Zimmermann SD, Garcia K (2019) The ectomycorrhizal contribution to tree nutrition. In Molecular Physiology and Biotechnology of Trees (pp. 77–126). https://doi.org/10.1016/bs.abr.2018.11.003 Branzanti MB, Rocca E, Pisi A (1999) Effect of ectomycorrhizal fungi on chestnut ink disease. Mycorrhiza 9:103–109. https://doi.org/10.1007/s005720050007 Charkiewicz AE, Omeljaniuk WJ, Nowak K, Garley M, Niklinski J (2023) Cadmium Toxicity and Health Effects-A Brief Summary. Molecules 28(18). https://doi.org/10.3390/molecules28186620 Chot E, Reddy MS (2022) Role of Ectomycorrhizal Symbiosis Behind the Host Plants Ameliorated Tolerance Against Heavy Metal Stress. Front Microbiol 13:855473. https://doi.org/10.3389/fmicb.2022.855473 Choudhury FK, Rivero RM, Blumwald E, Mittler R (2017) Reactive oxygen species, abiotic stress and stress combination. Plant J 90(5):856–867. https://doi.org/10.1111/tpj.13299 Colpaert JV, Wevers JHL, Krznaric E, Adriaensen K (2011) How metal-tolerant ecotypes of ectomycorrhizal fungi protect plants from heavy metal pollution. Ann For Sci 68(1):17–24. https://doi.org/10.1007/s13595-010-0003-9 Cuypers A, Hendrix S, Dos Reis A, De Smet R, Deckers S, Gielen J, Jozefczak H, Loix M, Vercampt C, Vangronsveld H, J., Keunen E (2016) Hydrogen Peroxide, Signaling in Disguise during Metal Phytotoxicity. Front Plant Sci 7:470. https://doi.org/10.3389/fpls.2016.00470 Cuypers A, Plusquin M, Remans T, Jozefczak M, Keunen E, Gielen H, Opdenakker K, Nair AR, Munters E, Artois TJ, Nawrot T, Vangronsveld J, Smeets K (2010) Cadmium stress: an oxidative challenge. Biometals 23(5):927–940. https://doi.org/10.1007/s10534-010-9329-x Dachuan Y, Jinyu Q (2021) The physiological response of Ectomycorrhizal fungus Lepista sordida to Cd and Cu stress. PeerJ 9:e11115. https://doi.org/10.7717/peerj.11115 De Oliveira VH, Tibbett M (2018a) Cd and Zn interactions and toxicity in ectomycorrhizal basidiomycetes in axenic culture. PeerJ 6:e4478. https://doi.org/10.7717/peerj.4478 De Oliveira VH, Tibbett M (2018b) Tolerance, toxicity and transport of Cd and Zn in Populus trichocarpa. Environ Exp Bot 155:281–292. https://doi.org/10.1016/j.envexpbot.2018.07.011 Ditengou FA, Muller A, Rosenkranz M, Felten J, Lasok H, van Doorn MM, Legue V, Palme K, Schnitzler JP, Polle A (2015) Volatile signalling by sesquiterpenes from ectomycorrhizal fungi reprogrammes root architecture. Nat Commun 6:6279. https://doi.org/10.1038/ncomms7279 Elobeid M, Gobel C, Feussner I, Polle A (2012) Cadmium interferes with auxin physiology and lignification in poplar. J Exp Bot 63(3):1413–1421. https://doi.org/10.1093/jxb/err384 Felten J, Kohler A, Morin E, Bhalerao RP, Palme K, Martin F, Ditengou FA, Legue V (2009) The ectomycorrhizal fungus Laccaria bicolor stimulates lateral root formation in poplar and Arabidopsis through auxin transport and signaling. Plant Physiol 151(4):1991–2005. https://doi.org/10.1104/pp.109.147231 Gafur A, Schutzendubel A, Langenfeld-Heyser R, Fritz E, Polle A (2004) Compatible and incompetent Paxillus involutus isolates for ectomycorrhiza formation in vitro with poplar (Populus x canescens) differ in H2O2 production. Plant Biol (Stuttg) 6(1):91–99. https://doi.org/10.1055/s-2003-44718 Garcia K, Delaux PM, Cope KR, Ane JM (2015) Molecular signals required for the establishment and maintenance of ectomycorrhizal symbioses. New Phytol 208(1):79–87. https://doi.org/10.1111/nph.13423 Genre A, Lanfranco L, Perotto S, Bonfante P (2020) Unique and common traits in mycorrhizal symbioses. Nat Rev Microbiol 18(11):649–660. https://doi.org/10.1038/s41579-020-0402-3 Haider FU, Liqun C, Coulter JA, Cheema SA, Wu J, Zhang R, Wenjun M, Farooq M (2021) Cadmium toxicity in plants: Impacts and remediation strategies. Ecotoxicol Environ Saf 211:111887. https://doi.org/10.1016/j.ecoenv.2020.111887 Hartley-Whitaker J, Cairney JWG, Meharg AA (2000) Toxic effects of cadmium and zinc on ectomycorrhizal colonization of scots pine (Pinus sylvestris L.) from soil inoculum. Environ Toxicol Chem 19(3):694–699. https://doi.org/10.1002/etc.5620190322 He J, Ma C, Ma Y, Li H, Kang J, Liu T, Polle A, Peng C, Luo ZB (2013) Cadmium tolerance in six poplar species. Environ Sci Pollut Res Int 20(1):163–174. https://doi.org/10.1007/s11356-012-1008-8 He S, Yang X, He Z, Baligar VC (2017) Morphological and Physiological Responses of Plants to Cadmium Toxicity: A Review. Pedosphere 27(3):421–438. https://doi.org/10.1016/s1002-0160(17)60339-4 Hu YF, Zhou G, Na XF, Yang L, Nan WB, Liu X, Zhang YQ, Li JL, Bi YR (2013) Cadmium interferes with maintenance of auxin homeostasis in Arabidopsis seedlings. J Plant Physiol 170(11):965–975. https://doi.org/10.1016/j.jplph.2013.02.008 Kazan K, Manners JM (2013) MYC2: the master in action. Mol Plant 6(3):686–703. https://doi.org/10.1093/mp/sss128 Kemppainen MJ, Pardo AG (2011) Transformation of the mycorrhizal fungus Laccaria bicolor using Agrobacterium tumefaciens. Bioeng Bugs 2(1):38–44. https://doi.org/10.4161/bbug.2.1.14394 Khan MA, Khan S, Khan A, Alam M (2017) Soil contamination with cadmium, consequences and remediation using organic amendments. Sci Total Environ 601–602:1591–1605. https://doi.org/10.1016/j.scitotenv.2017.06.030 Kliebenstein DJ, Monde R-A, Last RL (1998) Superoxide Dismutase in Arabidopsis: An Eclectic Enzyme Family with Disparate Regulation and Protein Localization. Plant Physiol 118(2):637–650. https://doi.org/10.1104/pp.118.2.637 Komán S, Németh R, Báder M (2023) An Overview of the Current Situation of European Poplar Cultures with a Main Focus on Hungary. Appl Sci 13(23). https://doi.org/10.3390/app132312922 Krznaric E, Verbruggen N, Wevers JH, Carleer R, Vangronsveld J, Colpaert JV (2009) Cd-tolerant Suillus luteus: a fungal insurance for pines exposed to Cd. Environ Pollut 157(5):1581–1588. https://doi.org/10.1016/j.envpol.2008.12.030 Lackus ND, Lackner S, Gershenzon J, Unsicker SB, Kollner TG (2018) The occurrence and formation of monoterpenes in herbivore-damaged poplar roots. Sci Rep 8(1):17936. https://doi.org/10.1038/s41598-018-36302-6 Lagrange H, Jay-Allgmand C, Lapeyrie F (2001) Rutin, the phenolglycoside from eucalyptus root exudates, stimulates Pisolithus hyphal growth at picomolar concentrations. New Phytol 149(2):349–355. https://doi.org/10.1046/j.1469-8137.2001.00027.x Lin YF, Aarts MG (2012) The molecular mechanism of zinc and cadmium stress response in plants. Cell Mol Life Sci 69(19):3187–3206. https://doi.org/10.1007/s00018-012-1089-z Liu B, Dong P, Zhang X, Feng Z, Wen Z, Shi L, Xia Y, Chen C, Shen Z, Lian C, Chen Y (2022) Identification and characterization of eight metallothionein genes involved in heavy metal tolerance from the ectomycorrhizal fungus Laccaria bicolor. Environ Sci Pollut Res Int 29(10):14430–14442. https://doi.org/10.1007/s11356-021-16776-0 Loix C, Huybrechts M, Vangronsveld J, Gielen M, Keunen E, Cuypers A (2017) Reciprocal Interactions between Cadmium-Induced Cell Wall Responses and Oxidative Stress in Plants. Front Plant Sci 8:1867. https://doi.org/10.3389/fpls.2017.01867 Luo Z-B, Wu C, Zhang C, Li H, Lipka U, Polle A (2014) The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ Exp Bot 108:47–62. https://doi.org/10.1016/j.envexpbot.2013.10.018 Ma Y, He J, Ma C, Luo J, Li H, Liu T, Polle A, Peng C, Luo ZB (2014) Ectomycorrhizas with Paxillus involutus enhance cadmium uptake and tolerance in Populus x canescens. Plant Cell Environ 37(3):627–642. https://doi.org/10.1111/pce.12183 Marqués-Galvez JE, Pandharikar G, Basso V, Kohler A, Lackus ND, Barry K, Keymanesh K, Johnson J, Singan V, Grigoriev IV, Vilgalys R, Martin F, Veneault-Fourrey C (2024) Populus MYC2 orchestrates root transcriptional reprogramming of defence pathway to impair Laccaria bicolor ectomycorrhizal development. New Phytol 242(2):658–674. https://doi.org/10.1111/nph.19609 Martin F, Kohler A, Murat C, Veneault-Fourrey C, Hibbett DS (2016) Unearthing the roots of ectomycorrhizal symbioses. Nat Rev Microbiol 14(12):760–773. https://doi.org/10.1038/nrmicro.2016.149 McCord JM, Fridovich I (1969) Superoxide Dismutase. J Biol Chem 244(22):6049–6055. https://doi.org/10.1016/s0021-9258(18)63504-5 Mittler R, Zandalinas SI, Fichman Y, Van Breusegem F (2022) Reactive oxygen species signalling in plant stress responses. Nat Rev Mol Cell Biol 23(10):663–679. https://doi.org/10.1038/s41580-022-00499-2 Murashige T, Skoog F (1962) A Revised Medium for Rapid Growth and Bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x Ochoa-Hueso R, Delgado‐Baquerizo M, Risch AC, Ashton L, Augustine D, Bélanger N, Bridgham S, Britton AJ, Bruckman VJ, Camarero JJ, Cornelissen G, Crawford JA, Dijkstra FA, Diochon A, Earl S, Edgerley J, Epstein H, Felton A, Fortier J, Bremer E (2023) Bioavailability of Macro and Micronutrients Across Global Topsoils: Main Drivers and Global Change Impacts. Glob Biogeochem Cycles 37(6). https://doi.org/10.1029/2022gb007680 Pellegrin C, Daguerre Y, Ruytinx J, Guinet F, Kemppainen M, Frey NFD, Puech-Pages V, Hecker A, Pardo AG, Martin FM, Veneault-Fourrey C (2019) Laccaria bicolor MiSSP8 is a small-secreted protein decisive for the establishment of the ectomycorrhizal symbiosis. Environ Microbiol 21(10):3765–3779. https://doi.org/10.1111/1462-2920.14727 Pilipovic A, Zalesny RS Jr., Roncevic S, Nikolic N, Orlovic S, Beljin J, Katanic M (2019) Growth, physiology, and phytoextraction potential of poplar and willow established in soils amended with heavy-metal contaminated, dredged river sediments. J Environ Manage 239:352–365. https://doi.org/10.1016/j.jenvman.2019.03.072 Plett JM, Daguerre Y, Wittulsky S, Vayssières A, Deveau A, Melton SJ, Kohler A, Morrell-Falvey JL, Brun A, Veneault-Fourrey C, Martin F (2014) Effector MiSSP7 of the mutualistic fungus Laccaria bicolor stabilizes the Populus JAZ6 protein and represses jasmonic acid (JA) responsive genes. Proc Natl Acad Sci U S A 111(22):8299–8304. https://doi.org/10.1073/pnas.1322671111 Qin S, Liu H, Nie Z, Rengel Z, Gao W, Li C, Zhao P (2020) Toxicity of cadmium and its competition with mineral nutrients for uptake by plants: A review. Pedosphere 30(2):168–180. https://doi.org/10.1016/s1002-0160(20)60002-9 Quan L, Shi L, Zhang S, Yao Q, Yang Q, Zhu Y, Liu Y, Lian C, Chen Y, Shen Z, Duan K, Xia Y (2023) Ectomycorrhizal fungi, two species of Laccaria, differentially block the migration and accumulation of cadmium and copper in Pinus densiflora. Chemosphere 334:138857. https://doi.org/10.1016/j.chemosphere.2023.138857 R Core Team (2024) R: A Language and Environment for Statistical Computing. In R Foundation for Statistical Computing https://www.R-project.org/ Reddy MS, Kour M, Aggarwal S, Ahuja S, Marmeisse R, Fraissinet-Tachet L (2016) Metal induction of a Pisolithus albus metallothionein and its potential involvement in heavy metal tolerance during mycorrhizal symbiosis. Environ Microbiol 18(8):2446–2454. https://doi.org/10.1111/1462-2920.13149 Reddy MS, Prasanna L, Marmeisse R, Fraissinet-Tachet L (2014) Differential expression of metallothioneins in response to heavy metals and their involvement in metal tolerance in the symbiotic basidiomycete Laccaria bicolor. Microbiol (Reading) 160(Pt 10):2235–2242. https://doi.org/10.1099/mic.0.080218-0 Ritz C, Baty F, Streibig JC, Gerhard D (2015) Dose-Response Analysis Using R. PLoS ONE 10(12):e0146021. https://doi.org/10.1371/journal.pone.0146021 Ruytinx J, Miyauchi S, Hartmann-Wittulsky S, de Freitas Pereira M, Guinet F, Churin JL, Put C, Le Tacon F, Veneault-Fourrey C, Martin F, Kohler A (2021) A Transcriptomic Atlas of the Ectomycorrhizal Fungus Laccaria bicolor. Microorganisms 9(12). https://doi.org/10.3390/microorganisms9122612 Schutzendubel A, Schwanz P, Teichmann T, Gross K, Langenfeld-Heyser R, Godbold DL, Polle A (2001) Cadmium-induced changes in antioxidative systems, hydrogen peroxide content, and differentiation in Scots pine roots. Plant Physiol 127(3):887–898. https://doi.org/10.1104/pp.010318 Seethepalli A, Dhakal K, Griffiths M, Guo H, Freschet GT, York LM (2021) RhizoVision Explorer: open-source software for root image analysis and measurement standardization. AoB Plants 13(6):plab056. https://doi.org/10.1093/aobpla/plab056 Sewall TC, Mims CW, Timberlake WE (1990) abaA controls phialide differentiation in Aspergillus nidulans. Plant Cell 2(8):731–739. https://doi.org/10.1105/tpc.2.8.731 Shi L, Wang Z, Chen JH, Qiu H, Liu WD, Zhang XY, Martin FM, Zhao MW (2024) LbSakA-mediated phosphorylation of the scaffolding protein LbNoxR in the ectomycorrhizal basidiomycete Laccaria bicolor regulates NADPH oxidase activity, ROS accumulation and symbiosis development. New Phytol 243(1):381–397. https://doi.org/10.1111/nph.19813 Soudzilovskaia NA, van Bodegom PM, Terrer C, Zelfde MV, McCallum I, Luke McCormack M, Fisher JB, Brundrett MC, de Sa NC, Tedersoo L (2019) Global mycorrhizal plant distribution linked to terrestrial carbon stocks. Nat Commun 10(1):5077. https://doi.org/10.1038/s41467-019-13019-2 Sousa NR, Ramos MA, Marques AP, Castro PM (2012) The effect of ectomycorrhizal fungi forming symbiosis with Pinus pinaster seedlings exposed to cadmium. Sci Total Environ 414:63–67. https://doi.org/10.1016/j.scitotenv.2011.10.053 Stahl L, Smoliakova IP (2007) Zinc Organometallics. In D. M. P. Mingos & R. H. Crabtree (Eds.), Comprehensive Organometallic Chemistry III (Vol. 2, pp. 309–418). Elsevier. https://doi.org/10.1016/B0-08-045047-4/00040-6 Steidinger BS, Crowther TW, Liang J, Van Nuland ME, Werner GDA, Reich PB, Nabuurs GJ, de-Miguel S, Zhou M, Picard N, Herault B, Zhao X, Zhang C, Routh D, Peay KG, consortium G (2019) Climatic controls of decomposition drive the global biogeography of forest-tree symbioses. Nature 569(7756):404–408. https://doi.org/10.1038/s41586-019-1128-0 Thakur S, Choudhary S, Bhardwaj P (2019) Comparative Transcriptome Profiling Under Cadmium Stress Reveals the Uptake and Tolerance Mechanism in Brassica juncea. J Plant Growth Regul 38(3):1141–1152. https://doi.org/10.1007/s00344-019-09919-8 Tsang CK, Liu Y, Thomas J, Zhang Y, Zheng XF (2014) Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance. Nat Commun 5:3446. https://doi.org/10.1038/ncomms4446 Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology , 3 (7). https://doi.org/research0034.1 Vayssières A, Pencik A, Felten J, Kohler A, Ljung K, Martin F, Legue V (2015) Development of the Poplar-Laccaria bicolor Ectomycorrhiza Modifies Root Auxin Metabolism, Signaling, and Response. Plant Physiol 169(1):890–902. https://doi.org/10.1104/pp.114.255620 Wang Q, Pokhrel A, Coleman JJ (2021) The Extracellular Superoxide Dismutase Sod5 From Fusarium oxysporum Is Localized in Response to External Stimuli and Contributes to Fungal Pathogenicity. Front Plant Sci 12:608861. https://doi.org/10.3389/fpls.2021.608861 Wang Y, Mostafa S, Zeng W, Jin B (2021) Function and Mechanism of Jasmonic Acid in Plant Responses to Abiotic and Biotic Stresses. Int J Mol Sci 22(16). https://doi.org/10.3390/ijms22168568 Yaciuk PA, Colombo F, Lecomte KL, De Micco G, Bohé AE (2022) Cadmium sources, mobility, and natural attenuation in contrasting environments (carbonate-rich and carbonate-poor) in the Capillitas polymetallic mineral deposit, NW Argentina. Appl Geochem 136. https://doi.org/10.1016/j.apgeochem.2021.105152 Yi L, Wu M, Yu F, Song Q, Zhao Z, Liao L, Tong J (2022) Enhanced cadmium phytoremediation capacity of poplar is associated with increased biomass and Cd accumulation under nitrogen deposition conditions. Ecotoxicol Environ Saf 246:114154. https://doi.org/10.1016/j.ecoenv.2022.114154 Yuan HM, Huang X (2016) Inhibition of root meristem growth by cadmium involves nitric oxide-mediated repression of auxin accumulation and signalling in Arabidopsis. Plant Cell Environ 39(1):120–135. https://doi.org/10.1111/pce.12597 Yuan Z, Luo T, Liu X, Hua H, Zhuang Y, Zhang X, Zhang L, Zhang Y, Xu W, Ren J (2019) Tracing anthropogenic cadmium emissions: From sources to pollution. Sci Total Environ 676:87–96. https://doi.org/10.1016/j.scitotenv.2019.04.250 Zhang F, Anasontzis GE, Labourel A, Champion C, Haon M, Kemppainen M, Commun C, Deveau A, Pardo A, Veneault-Fourrey C, Kohler A, Rosso MN, Henrissat B, Berrin JG, Martin F (2018) The ectomycorrhizal basidiomycete Laccaria bicolor releases a secreted beta-1,4 endoglucanase that plays a key role in symbiosis development. New Phytol 220(4):1309–1321. https://doi.org/10.1111/nph.15113 Zhang F, Labourel A, Haon M, Kemppainen M, Da Silva Machado E, Brouilly N, Veneault-Fourrey C, Kohler A, Rosso MN, Pardo A, Henrissat B, Berrin JG, Martin F (2021) The ectomycorrhizal basidiomycete Laccaria bicolor releases a GH28 polygalacturonase that plays a key role in symbiosis establishment. New Phytol 233(6):2534–2547. https://doi.org/10.1111/nph.17940 Zhang K, Tappero R, Ruytinx J, Branco S, Liao HL (2021) Disentangling the role of ectomycorrhizal fungi in plant nutrient acquisition along a Zn gradient using X-ray imaging. Sci Total Environ 801:149481. https://doi.org/10.1016/j.scitotenv.2021.149481 Zhao M, Wang H, Sun J, Tang R, Cai B, Song X, Huang X, Huang J, Fan Z (2023) Spatio-temporal characteristics of soil Cd pollution and its influencing factors: A Geographically and temporally weighted regression (GTWR) method. J Hazard Mater 446:130613. https://doi.org/10.1016/j.jhazmat.2022.130613 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8981216","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":602964682,"identity":"6f1a5180-a138-4ddf-abda-1b34a62cbaf0","order_by":0,"name":"Maarten Ottaway","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Maarten","middleName":"","lastName":"Ottaway","suffix":""},{"id":602964683,"identity":"e986af95-518f-4a6f-ac64-d0da12a680e5","order_by":1,"name":"Janne Swinnen","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Janne","middleName":"","lastName":"Swinnen","suffix":""},{"id":602964684,"identity":"e45d5d0a-5409-45f3-acd7-15d7ee492d98","order_by":2,"name":"Joske Ruytinx","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYBAC9gYGZiD1D4gZGxgYKhgYDBh48GthhGg5zMDABtJyhjQtIF4bMVra2x8bfGA4nLjhfnPbY955hxO3M/AefIBXS88Z48QZDP8SNxxjbDecue1w4s4GvmQDvFpm5DAf5mH4AdLSJvFxW1rihgM8ZhL4taQ/PvyH4QxES+IcsBbzH/i0CM5IME5mYDgGtaXBBmwLPh0M0jxnjA17DA4bzzyW2CY545iN8c5mvmS8DuNjb38s8aPisGzf4ePPpHlqJGS3s/ce/IDXGjAAhpDCARiHmbB6CJBvIFblKBgFo2AUjDgAAEV+ULk9rUENAAAAAElFTkSuQmCC","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":true,"prefix":"","firstName":"Joske","middleName":"","lastName":"Ruytinx","suffix":""}],"badges":[],"createdAt":"2026-02-26 20:08:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8981216/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8981216/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104311438,"identity":"4e6138c0-29dd-4016-88ef-4ba8ab07ae66","added_by":"auto","created_at":"2026-03-10 10:58:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":117176,"visible":true,"origin":"","legend":"\u003cp\u003eEarly stage ectomycorrhiza formed between P. tremula x alba 717-1B4 and L. bicolor S238N (14 dpi) grown under control (yellow) and 1 µM Cd (blue) conditions. \u003cstrong\u003e(a)\u003c/strong\u003e L. bicolor S238N mycelium cultured on P5 medium supplemented with 0 µM Cd/control \u003cstrong\u003e(Top)\u003c/strong\u003eand 1 µM Cd \u003cstrong\u003e(Bottom)\u003c/strong\u003e for three weeks. \u003cstrong\u003e(b)\u003c/strong\u003e Average number of lateral roots formed per plant grown under control or 1 µM Cd conditions (n ≥ 25). \u003cstrong\u003e(c)\u003c/strong\u003e Average number of lateral roots/cm root per plant (14dpi) grown under control or 1 µM Cd conditions (n ≥ 25). \u003cstrong\u003e(d)\u003c/strong\u003e Average degree of mycorrhization in co-cultures grown under control or 1 µM Cd conditions (n ≥ 25). \u003cstrong\u003e(e)\u003c/strong\u003e Fluorescent microscopy image of early stage mycorrhiza cross-sections grown under control \u003cstrong\u003e(Left) \u003c/strong\u003eor 1 µM Cd \u003cstrong\u003e(Right)\u003c/strong\u003econditions. Samples are stained with WGA-488 and counterstained with PI. Scale bar = 100 µm. Statistical analyses were performed in Graphpad Prism. All data was analysed using a Student’s t-test. * = p\u0026lt;0.05 and ** = p\u0026lt;0.01. Means of all replicates ± SE are shown.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8981216/v1/1f95497731b27c76e40ecbec.jpg"},{"id":104311439,"identity":"81589ba1-537f-47b0-a27a-70a3214d4afb","added_by":"auto","created_at":"2026-03-10 10:58:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68561,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression analysis of symbiosis marker genes from P. tremula x alba 717-1B4 and L. bicolor S238N (14 dpi) grown under control (yellow) or 1 µM Cd (blue) conditions. Log2 relative expression of \u003cstrong\u003e(a) \u003c/strong\u003epoplar TPS16 (n = 3) and fungal \u003cstrong\u003e(b)\u003c/strong\u003e GH28a (n = 3-5), \u003cstrong\u003e(c)\u003c/strong\u003e MiSSP7 (n = 3-4) and \u003cstrong\u003e(d)\u003c/strong\u003e MiSSP17 (n = 3-4). Statistical analysis was performed in Graphpad Prism. TPS16 data was analysed using a Student’s t-test. All other data was analysed using a two-way ANOVA with Tukey’s multiple comparisons test. * = p\u0026lt;0.05, ** = p\u0026lt;0.01 and *** = p\u0026lt;0.001. Means of all replicates ± SE are shown.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8981216/v1/aea6ed4c63e065110045b490.jpg"},{"id":104405327,"identity":"18ab5b0e-0cdd-46af-815e-6e20e90d1a62","added_by":"auto","created_at":"2026-03-11 12:22:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":115097,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression analysis of ROS scavenging genes from L. bicolor S238N (14dpi) grown under control (yellow) or 1 µM Cd (blue) conditions. Log2 relative expression of \u003cstrong\u003e(a)\u003c/strong\u003e Catalase (n = 3-5), \u003cstrong\u003e(b)\u003c/strong\u003e Mn/Fe SOD1 (n = 3-5), \u003cstrong\u003e(c)\u003c/strong\u003e Mn/Fe SOD2 (n = 4-5), \u003cstrong\u003e(d)\u003c/strong\u003e Mn/Fe SOD3 (n = 3-5), \u003cstrong\u003e(e)\u003c/strong\u003e Mn/Fe SOD4 (n = 3-5) and \u003cstrong\u003e(f)\u003c/strong\u003e Mn/Fe SOD5 (n = 3-5). Statistical analysis were performed in Graphpad Prism. All data was analysed using a two-way ANOVA with Tukey’s multiple comparisons test. * = p\u0026lt;0.05, ** = p\u0026lt;0.01 and *** = p\u0026lt;0.001. Mean of all replicates ± SE are shown.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8981216/v1/1b84eede0829165088b9eb64.jpg"},{"id":104408874,"identity":"1e92fb44-318e-4ebc-a71e-da62828500b9","added_by":"auto","created_at":"2026-03-11 12:43:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1181391,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8981216/v1/a91175aa-1e21-4632-ae49-ea156742e639.pdf"},{"id":104311440,"identity":"79342c87-1227-4a7f-9277-964ce259e733","added_by":"auto","created_at":"2026-03-10 10:58:49","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":136284,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8981216/v1/cafd7ec87e01c1f13798aebd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sublethal Cd exposure stimulates Laccaria bicolor x poplar symbiosis formation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCadmium (Cd) is a non-essential metal that is commonly found within ore deposits as a substitute for Zn (Yaciuk et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Cd is considered highly toxic. Even at very low concentrations, it is known to cause stunted growth, chlorosis and wilting (Haider et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Qin et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Stahl \u0026amp; Smoliakova, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Cd has seen an increase in industrial usage, as it is an important component for the production of batteries, electronics and paints (Angon et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Due to these increased activities, soils surrounding mining operations, coal power plants and heavy industry have become polluted with Cd. Also agriculture has been affected by Cd pollution through the use of Cd-containing pesticides and phosphate fertilisers (Angon et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ballabio et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Khan et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yuan et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As Cd is highly toxic, it renders polluted soils, and especially affected agricultural fields, economically unprofitable for plant and crop production.\u003c/p\u003e \u003cp\u003eIngestion of crops grown on Cd polluted soils can have a severe impact on human health. Cd toxicity can cause impaired heart function, neurodegenerative diseases and increases the risk of cancer (Angon et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Charkiewicz et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Khan et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Remediation of Cd polluted soils is thus of great importance to benefit human health, but also to allow for contaminated areas to be used again for agriculture. Phytoremediation is a popular soil remediation strategy. By using fast-growing, metal tolerant and metal accumulating plants, metals such as Cd can safely and cheaply be removed from the soil (Aparicio et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Poplars are commonly used for this because of their fast growth, but also due to their economic importance for the wood and paper industry (Kom\u0026aacute;n et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pilipovic et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe majority of trees found within temperate and boreal forest ecosystems form a symbiosis with ectomycorrhizal (ECM) fungi (Soudzilovskaia et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Steidinger et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This symbiotic relationship benefits both the plant and fungal partner. The fungus provides improved access to nutrients, as well as provides water and increased resistance to both biotic and abiotic stress (Becquer et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Branzanti et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Chot \u0026amp; Reddy, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ochoa-Hueso et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For example, \u003cem\u003eLaccaria bicolor\u003c/em\u003e will limit the uptake of soil pollutants, such as Cd, by their host plant by sequestering them to their cell wall (Quan et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In exchange, the host plant provides carbon in the form of photosynthates (Genre et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The ECM symbiotic structure is characterised by the formation of a tightly packed hyphal mantle surrounding host plant lateral roots (LR). Within colonised LRs a hartig net is formed, composed of hyphae that penetrate between root cortical and epidermal cells. The hyphae that extend outward from the plant root into the soil make up the extraradical mycelium (Garcia et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). So far, most research on the establishment of the ECM symbiosis has utilised the \u003cem\u003eL. bicolor\u003c/em\u003e - \u003cem\u003ePopulus tremula x alba\u003c/em\u003e model co-culture system. Within this system, it was found that a complex chemical cross-talk takes place between both partners (Martin et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Research has found this cross-talk to be crucial for fungal colonisation of the host root and the development of the symbiotic structure. One example of this is the fungal production of mycorrhiza induced small-secreted proteins. These will, for example, allow the fungus to regulate the plant defence response (Plett et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Also the expression of fungal carbohydrate-active enzymes (CAZymes) is needed to modify the plant cell wall to allow for hyphal penetration (Zhang et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; F. Zhang et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eReactive oxygen species (ROS) accumulate during stress, such as Cd toxicity, causing oxidative damage to cells (Choudhury et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Cuypers et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, a role for ROS, and mainly H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, in stress response signalling was also discovered, allowing for the activation of the stress response pathway (Mittler et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For example, in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, exposure to Cd and the accompanied increase in intracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, will activate MAPK pathways as well as induce the jasmonic acid and salicylic acid pathways. In \u003cem\u003eBrassica juncea\u003c/em\u003e, this exposure will rather activate the ethylene pathway (Cuypers et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, it was also found that ROS produced throughout the ECM symbiosis is crucial for its development (Shi et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). How Cd pollution impacts the balance between ROS scavenging and ROS signalling throughout ECM symbiosis development has not yet been studied.\u003c/p\u003e \u003cp\u003eIn this study, we investigate the impact of Cd pollution on the formation of the ECM symbiosis between \u003cem\u003eL. bicolor\u003c/em\u003e and poplar. We assessed the effect on both the ECM symbiotic structure, as well as the expression of symbiosis marker genes and ROS scavenging enzymes. Our data suggests that Cd exposure stimulates symbiosis formation in this model system, despite reducing fungal growth. Altogether, this study provides a new perspective on the impact of Cd pollution on the ECM symbiosis and further highlights its usefulness in soil remediation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBiological strains and growth conditions\u003c/h2\u003e \u003cp\u003eMycelium of \u003cem\u003eLaccaria bicolor\u003c/em\u003e S238N (Maire) P.D. Orton was used throughout all experiments. Fungal cultures were grown and maintained at 23\u0026deg;C in the dark on Pachlewski P5 medium, consisting of 0.5 g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e di-NH\u003csub\u003e4\u003c/sub\u003e-tartrate, 1 g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.5 g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, 5 g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e maltose, 20 g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose, 0.1 mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e thiamine-HCl, 5 mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MnSO\u003csub\u003e4\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO, 8.5 mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, 0.3 mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003eMo\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e24\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO, 6 mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FeCl\u003csub\u003e3\u003c/sub\u003e, 0.6 mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e CuSO\u003csub\u003e4\u003c/sub\u003e.5H\u003csub\u003e2\u003c/sub\u003eO, 2.7 mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ZnSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO and 20 g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e agar at pH 5.5 (Kemppainen \u0026amp; Pardo, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The sensitivity of \u003cem\u003eL. bicolor\u003c/em\u003e to Cd was determined through dose-response experiments. Mycelial plugs (0.5 cm\u003csup\u003e2\u003c/sup\u003e) were placed on cellophane-covered P5 medium plates (9 cm diameter) supplemented with or without 3CdSO\u003csub\u003e4\u003c/sub\u003e.8H\u003csub\u003e2\u003c/sub\u003eO. Specifically, the following concentrations were tested: 0 (control), 0.1, 0.2, 0.4, 0.6, 0.7, 0.8, 0.9, 1, 2, 4, 8, 10, 20 and 100 \u0026micro;M Cd. Each concentration consisted of five biological replicates. Cultures were incubated for three weeks in the dark at 23\u0026deg;C, after which mycelium was harvested, weighed and lyophilised. Dry weights were subsequently determined and the data was fit using a non-linear regression with four parameter log-logistic model (Ritz et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) in \u0026ldquo;R\u0026rdquo; version 4.4.1 (R Core Team, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The maximal effective concentration (EC50) was calculated and corresponds to the concentration that reduces growth by 50% compared to control conditions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlant – fungal co-cultures\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003ePopulus tremula\u003c/em\u003e x \u003cem\u003ealba\u003c/em\u003e 717-1B4 plants were maintained by micropropagation on Murashige and Skoog medium (MS), supplemented with 10 \u0026micro;M IBA (Murashige \u0026amp; Skoog, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). Plants were subsequently used for \u003cem\u003ein vitro\u003c/em\u003e co-culturing experiments between \u003cem\u003eP. tremula\u003c/em\u003e x \u003cem\u003ealba\u003c/em\u003e 717-1B4 and \u003cem\u003eL. bicolor\u003c/em\u003e S238N as previously described (Felten et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Mycelium grown in the presence or absence of a host plant was cultured on sugar reduced Pachlewski P20 medium supplemented with 0.1% MES at 23\u0026deg;C for 14 days with a photoperiod of 16h. P20 medium was either supplemented with 0 \u0026micro;M 3CdSO\u003csub\u003e4\u003c/sub\u003e.8H\u003csub\u003e2\u003c/sub\u003eO (control), or sublethal Cd (1 \u0026micro;M 3CdSO\u003csub\u003e4\u003c/sub\u003e.8H\u003csub\u003e2\u003c/sub\u003eO).\u003c/p\u003e \u003cp\u003eScans of the co-cultures were made after two weeks of incubation using a Perfection V600 Photo scanner (Epson). Roots were fixated in 4% paraformaldehyde (PFA) overnight at 4\u0026deg;C, after which they were washed and stored in phosphate buffered saline (PBS) before microscopic analysis. Six biological replicates (100 mg) of either mycelium grown in absence of a host plant or mixed plant-fungal material (composed of mycorrhized root tips and mycelium within 5 mm of the root) were harvested for RNA extractions, flash frozen in liquid nitrogen and stored at -80\u0026deg;C until further processing.\u003c/p\u003e\n\u003ch3\u003eMycorrhizal morphology and root growth assessment\u003c/h3\u003e\n\u003cp\u003eTotal root length per plant was measured using RhizoVision Explorer (Seethepalli et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Both total and mycorrhized lateral roots (LR) were manually counted per plant, and the number of LR per cm root was calculated (total number of LR/root length). Mycorrhization percentage was also determined (number of mycorrhized LR/total number of LR). Normality of all data was assessed by means of the Shapiro-Wilk test, and statistical significances were determined using a Student\u0026rsquo;s t-test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in Graphpad Prism V10.1.0. Overnight PFA-fixed roots were embedded in 4% agarose. Using a VT1000S vibratome (Leica), 25 \u0026micro;M thick cross-sections were made (0.225 mm/s; 40 Hz). Cross-sections located 275 \u0026micro;m from the start of the LR were stained for 1h using 10 \u0026micro;g/ml Wheat Germ Agglutinin Alexa Fluor 488 (WGA-488; Invitrogen; W11261). Sections were then counterstained for 30 min using 15 \u0026micro;M Propidium Iodide (PI; Invitrogen; P3566) and visualised using an Eclipse Ti2 inverted fluorescence microscope (Nikon).\u003c/p\u003e\n\u003ch3\u003eRNA extractions and Quantitative Real-Time PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eRNA was extracted using the RNeasy Plant Mini kit (Qiagen). Using a pestle and mortar, 100 mg of material was ground in liquid nitrogen. Following this step, the manufacturers protocol was followed, and 2% PEG8000 was included in the RLC buffer. A DNase treatment was also included. The eluted RNA was flash frozen in liquid nitrogen and stored at -80\u0026deg;C. The integrity of the RNA was determined using a Bioanalyzer 2100 (Agilent Technologies), and the purity was assessed using a NanoDrop One Spectrophotometer (Thermo Fisher Scientific). Using the High-Capacity cDNA reverse transcription kit and protocol (Applied Biosystems), 250 ng of RNA was converted to cDNA.\u003c/p\u003e \u003cp\u003eExpression of ectomycorrhizal symbiosis marker genes found in either \u003cem\u003eP. tremula\u003c/em\u003e x \u003cem\u003ealba\u003c/em\u003e or \u003cem\u003eL. bicolor\u003c/em\u003e was determined. Specifically, expression of \u003cem\u003eTPS16\u003c/em\u003e (Terpene synthase; ID: PtXaTreH.01G253600.1), \u003cem\u003eGH28a\u003c/em\u003e (endopolygalacturonase, ID: 613299), \u003cem\u003eMiSSP7\u003c/em\u003e (mycorrhiza induced small-secreted protein, ID: 298595) and \u003cem\u003eMiSSP17\u003c/em\u003e (ID: 332226) was measured. Gene expression of \u003cem\u003eL. bicolor\u003c/em\u003e ROS decomposing enzymes was also measured, these being \u003cem\u003eCAT\u003c/em\u003e (catalase, ID: 123238), \u003cem\u003eMn/Fe SOD1\u003c/em\u003e (superoxide dismutase, ID: 635077), \u003cem\u003eMn/Fe SOD2\u003c/em\u003e (ID: 192586), \u003cem\u003eMn/Fe SOD3\u003c/em\u003e (ID: 291347), \u003cem\u003eMn/Fe SOD4\u003c/em\u003e (ID: 295682) and \u003cem\u003eMn/Fe SOD5\u003c/em\u003e (ID: 312019). Each condition consisted of a minimum of three biological replicates. A no-template control was also included. The qRT-PCR reactions consisted of 2x GoTaq qPCR Master Mix (Promega), 400 nM FW and RV primer, 2 \u0026micro;l cDNA and nuclease free H\u003csub\u003e2\u003c/sub\u003eO was added until a reaction volume of 20 \u0026micro;l. Reaction mixtures were pipetted into 96-well plates and run according to the following program on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad): 1 cycle of 95\u0026deg;C for 3 min and 40 cycles of 95\u0026deg;C for 10 seconds and 60\u0026deg;C for 30 seconds.\u003c/p\u003e \u003cp\u003eDifferent reference genes, published previously, were also tested using the aforementioned protocol (Pellegrin et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The obtained results were subjected to geNorm analysis using the qbase+ software (Biogazelle) to determine the stability of their expression in the growth conditions used. The software recommended genes (Mycocosm ID: 611151 and 313997) were subsequently used to calculate a normalization factor (NF), allowing for the relative expression of the genes of interest to be determined using the following formula: 2\u003csup\u003e(Ct\u0026minus;Ctmin)\u003c/sup\u003e/NF (Vandesompele et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The values were Log2 transformed and normality was tested using the Shapiro-Wilk test. All data was further analysed by two-way ANOVA with Tukey\u0026rsquo;s multiple comparisons test, except for \u003cem\u003eTPS16\u003c/em\u003e which was subjected to a Student\u0026rsquo;s t-test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Statistical analysis were performed in Graphpad Prism V10.1.0. All primers used are listed in supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImpact of sublethal Cd exposure on fungal growth and early mycorrhiza development\u003c/h2\u003e \u003cp\u003eTo determine the impact of sublethal exposure to Cd on \u003cem\u003eL. bicolor\u003c/em\u003e and its symbiosis with poplar, fungal growth and symbiosis formation were assessed. Exposure of \u003cem\u003eL. bicolor\u003c/em\u003e mycelium to increasing concentrations of 3CdSO\u003csub\u003e4\u003c/sub\u003e.8H\u003csub\u003e2\u003c/sub\u003eO caused decreased growth, with fungal growth being almost completely inhibited at 20 \u0026micro;M. The EC50 value was calculated to be 0.9 \u0026micro;M. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea; Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Thus, for the following experiments, 1 \u0026micro;M 3CdSO\u003csub\u003e4\u003c/sub\u003e.8H\u003csub\u003e2\u003c/sub\u003eO was chosen as sublethal Cd concentration in order to induce stress but not cause severe growth issues. In co-culture, exposure to sublethal Cd did not significantly decrease LR formation by \u003cem\u003eP. tremula x alba\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). A significant increase in LR/cm root was observed under stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). This increase was accompanied by a significant increase in mycorrhization under stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Microscopic analysis of mycorrhized LR revealed these to be in the early stages of development. A fully formed hyphal mantle was observed under control conditions, while this was delayed, showing only partial formation, under Cd stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImpact of sublethal Cd on symbiosis marker gene expression\u003c/h3\u003e\n\u003cp\u003eCd exposure increases LR formation and mycorrhization. However, its impact on the expression of genes crucial for symbiosis development remains unclear. To further investigate this, the expression of four symbiosis marker genes from poplar and \u003cem\u003eL. bicolor\u003c/em\u003e was measured. For poplar, the expression of terpene synthase \u003cem\u003eTPS16\u003c/em\u003e, which is not expressed in the fungus, was determined. A significant increase in the expression of \u003cem\u003eTPS16\u003c/em\u003e was found after exposure to Cd compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Within \u003cem\u003eL. bicolor\u003c/em\u003e, the expression of three well established symbiosis marker genes was assessed. ANOVA analysis of these genes revealed their expression to be impacted by Cd, the presence of a host plant and their combined interaction (Cd x host plant) (Table S2). Specifically, expression of all three marker genes was only differentially expressed in co-culture when exposed to sublethal Cd. More so, they showed a similar expression profile under all conditions tested. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eImpact of sublethal Cd on ROS scavenging enzyme gene expression\u003c/h3\u003e\n\u003cp\u003eExposure to elevated concentrations of Cd can cause oxidative damage. Thus, to better understand how the co-culture deals with Cd-induced ROS accumulation, the expression of ROS scavenging enzymes \u003cem\u003ecatalase\u003c/em\u003e (\u003cem\u003eCAT\u003c/em\u003e) and \u003cem\u003eMn/Fe SODs\u003c/em\u003e was also investigated. Two-way ANOVA analysis indicated that \u003cem\u003eCAT\u003c/em\u003e expression was impacted by Cd, presence of a host plant and their interaction (Table S2). Specifically, \u003cem\u003eCAT\u003c/em\u003e was found to be significantly upregulated in co-culture upon exposure to Cd (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). \u003cem\u003eL. bicolor\u003c/em\u003e contains five predicted \u003cem\u003eMn/Fe SOD\u003c/em\u003e genes, of which the expression of two (\u003cem\u003eMn/Fe SOD 1\u003c/em\u003e and \u003cem\u003eMn/Fe SOD2\u003c/em\u003e) were found to not be affected by any condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb/c). Of the remaining three \u003cem\u003eSOD\u003c/em\u003e genes, ANOVA results indicated an impact of the combined interaction (Cd x host plant) on the expression of all three. Cd alone was found to impact \u003cem\u003eMn/Fe SOD\u003c/em\u003e 3/4, with \u003cem\u003eMn/Fe SOD3\u003c/em\u003e also being impacted by the presence of a host plant (Table S2). Despite these differences in ANOVA result, \u003cem\u003eMn/Fe SOD\u003c/em\u003e3/4/5 all showed similar results. All were found to be upregulated upon exposure to Cd in the presence of a host plant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f). Exposure of free-living mycelium to Cd did not significantly increase the expression of any tested ROS scavenging enzyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExposure to sublethal Cd impacts fungal growth, root colonisation and mycorrhiza development\u003c/h2\u003e \u003cp\u003eAs Cd is a non-essential metal, exposure to toxic amounts can lead to cellular damage, reduced growth and even cell death (Dachuan \u0026amp; Jinyu, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; De Oliveira \u0026amp; Tibbett, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e). Certain species, including fungal species, have adapted to survive in environments rich in Cd pollution (Krznaric et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). This adaptation allows them to reduce their Cd uptake, and limit the damage that is inflicted by this pollutant. Certain isolates of the ectomycorrhizal fungus \u003cem\u003eSuillus luteus\u003c/em\u003e, for example, were shown to be more tolerant towards high Cd concentrations compared to more Cd sensitive isolates. (Colpaert et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Between different species, this tolerance towards Cd can also vary significantly. While \u003cem\u003eHebeloma cylindrosporum\u003c/em\u003e is highly sensitive towards Cd, \u003cem\u003eHebeloma subsaponaceum\u003c/em\u003e was found to have an increased tolerance towards it (De Oliveira \u0026amp; Tibbett, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e). For the haploid \u003cem\u003eL. bicolor\u003c/em\u003e S238 strain, a previous study found that at very high concentrations of Cd, such as 40\u0026ndash;50 \u0026micro;M, growth was inhibited by \u0026plusmn;\u0026thinsp;30% (Reddy et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The dikaryotic \u003cem\u003eL. bicolor\u003c/em\u003e S238N strain rather showed a gradual decrease in growth starting at concentrations of 1 \u0026micro;M Cd, with growth almost completely inhibited at 40 \u0026micro;M Cd (Liu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This difference in Cd sensitivity compared with our data might, however, be due to the use of Modified Melin Norkans (MMN) medium. The presence of malt extract might have an impact on the toxicity of Cd on \u003cem\u003eL. bicolor\u003c/em\u003e. In P5 medium, which contains much less nutrients, \u003cem\u003eL. bicolor\u003c/em\u003e was much more susceptible to Cd, as we calculated the EC50 value to be 0.9 \u0026micro;M (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This marks \u003cem\u003eL. bicolor\u003c/em\u003e, grown on P5 medium, as a Cd-sensitive species (Colpaert et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNot only fungi but also plants suffer from Cd pollution. Prolonged exposure of plants to elevated concentrations of Cd can cause stunted growth, decreased root length, chlorosis and even necrosis (He et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Qin et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For example, exposure of \u003cem\u003ePopulus deltoides\u003c/em\u003e x \u003cem\u003ePopulus nigra\u003c/em\u003e to 50 mg/kg Cd resulted in a significant decrease in biomass, root length and lateral root formation (Yi et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, Cd also impacts the plant growth hormone auxin. In both \u003cem\u003ePopulus x canescens\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, Cd interfered with auxin metabolism and signalling, resulting in a decrease in auxin within the root apical meristem. This reduction in auxin negatively impacted root growth and resulted in stunted roots (Elobeid et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yuan \u0026amp; Huang, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A common strategy to deal with Cd toxicity among tree species is to form a symbiosis with ECM fungi. Through this symbiosis, fungi can limit Cd uptake while maintaining sufficient nutrient influx (Reddy et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; K. Zhang et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, how Cd pollution specifically impacts the formation of the ECM symbiosis was never investigated. Throughout the early stages of ECM symbiosis development, chemical cross-talk will occur between the host plant and the fungus (Garcia et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This cross-talk will stimulate both fungal growth as well as LR formation (Ditengou et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lagrange et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Exposure of the co-culture to sublethal Cd did not result in a significant change in the number of LRs. Rather, it increased the LR/cm root ratio, indicating that while growth of the main root decreased, LR formation was stimulated. ECM fungi will induce LR formation, as was previously found between \u003cem\u003eL. bicolor\u003c/em\u003e and poplar (Vayssi\u0026egrave;res et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Interestingly, in \u003cem\u003eA. thaliana\u003c/em\u003e, exposure to Cd, specifically concentrations higher than 12.5 \u0026micro;M, was also found to induce LR formation (Hu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Furthermore, our results showed that Cd exposed co-cultures also exhibited higher mycorrhization rates. The increase in LRs upon exposure to Cd compared to control suggests that Cd, rather than the fungus itself, is responsible for the increase in LR development under Cd toxic conditions. As a consequence, as more LRs are formed by the plant, more points of contact become available for the fungus to colonise, thus increasing the mycorrhization rate. This response of the poplar x \u003cem\u003eL. bicolor\u003c/em\u003e co-culture to Cd seems to differ based on the symbiotic partners. For example, poplar and \u003cem\u003ePinus sp.\u003c/em\u003e have a similar tolerance towards Cd (De Oliveira \u0026amp; Tibbett, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e; Hartley-Whitaker et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; He et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sousa et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, exposure of \u003cem\u003ePinus pinaster\u003c/em\u003e co-cultures with either \u003cem\u003eSuillus bovinus\u003c/em\u003e or \u003cem\u003eRhizopogon roseolus\u003c/em\u003e to Cd rather showed decreased LR formation and mycorrhization (Sousa et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Similar results were also obtained with \u003cem\u003ePinus sylvestris\u003c/em\u003e, where decreased mycorrhization of LRs was found after Cd exposure (Hartley-Whitaker et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs the symbiosis continues to develop, the fungus starts to form a hyphal mantle (Ruytinx et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, Cd still negatively impacts fungal growth. To understand the extent by which this reduced fungal growth impacts ectomycorrhiza development, mycorrhized LR cross-sections were analysed through fluorescence microscopy. Under control conditions the co-culture exhibited a hyphal mantle encompassing the complete lateral root, with the hartig net beginning to form. Cd-exposed co-cultures, on the other hand, showed incomplete hyphal mantles, as expected due to the slower fungal growth. A previous study investigating the ECM symbiosis between \u003cem\u003ePaxillus involutus\u003c/em\u003e and \u003cem\u003ePopulus\u003c/em\u003e x \u003cem\u003ecanescens\u003c/em\u003e found that mycorrhized plants rather imported more Cd as compared to non-mycorrhized plants. However, mycorrhized plants also showed an increased defence response and Cd transport capabilities, thus increasing their Cd tolerance (Ma et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). On the other hand, other ECM partnerships, such as \u003cem\u003eHebeloma mesophaeum\u003c/em\u003e and \u003cem\u003ePopulus nigra\u003c/em\u003e, showed reduced Cd uptake by the plant when in symbiosis (Luo et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). While different strategies can be used, they illustrate the benefit of this symbiosis for the Cd tolerance of both partners. These results suggest that Cd stress stimulates symbiosis formation between \u003cem\u003eL. bicolor\u003c/em\u003e and \u003cem\u003eP. tremula\u003c/em\u003e x \u003cem\u003ealba\u003c/em\u003e, despite also slowing down ectomycorrhiza development. Thus, promoting mycorrhization and increasing the contact points between both partners might be an adaptation strategy employed by the \u003cem\u003eL. bicolor\u003c/em\u003e x poplar co-culture to increase their tolerance towards Cd pollution.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of symbiosis marker genes is upregulated by Cd exposure in presence of a host plant\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor successful colonisation of host roots, the plant defence response needs to be inactivated. Among the activated poplar defence genes are terpene synthases, such as \u003cem\u003eTPS16\u003c/em\u003e and \u003cem\u003eTPS21\u003c/em\u003e. Terpene synthases produce terpene mixtures which have been shown to reduce fungal growth and inhibit root colonisation. Furthermore, it was found that throughout symbiosis development, these genes are downregulated through cross-talk between \u003cem\u003eL. bicolor\u003c/em\u003e and \u003cem\u003eP. tremula x alba\u003c/em\u003e (Marqu\u0026eacute;s-Galvez et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Interestingly, a previous study discovered that TPS16 produces a monoterpene mix primarily composed of γ-terpinene (Lackus et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Contrary to the other terpenes produced, γ-terpinene was found to stimulate growth of \u003cem\u003eL. bicolor\u003c/em\u003e (Marqu\u0026eacute;s-Galvez et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Contrary to the downregulation of the \u003cem\u003eTPS\u003c/em\u003e genes typically found, exposure of the co-culture to Cd rather caused a significant upregulation of \u003cem\u003eTPS16\u003c/em\u003e expression. As TPS16 can stimulate fungal growth, and Cd reduces fungal growth, this increased expression could assist \u003cem\u003eL. bicolor\u003c/em\u003e in growing towards and colonise LRs under Cd polluted conditions.\u003c/p\u003e \u003cp\u003eBesides attenuation of the plant defence response, major transcriptional changes within both plant and fungus need to occur to allow for ECM development. Most notably is the upregulation of MiSSPs, where MiSSP7 regulates the host defence response and MiSSP17 is one of the highest upregulated genes throughout symbiosis (Plett et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ruytinx et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Not only are transcriptional changes required, the plant cell wall also needs to be modified to allow for hyphal penetration and hartig net formation. This can be achieved through the expression of specific CAZymes, such as the polygalacturonase GH28a, which will target the pectin within the plant cell wall (F. Zhang et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Here, we assessed the impact of Cd on the expression of these three well studied symbiosis marker genes. Upon exposure of the co-culture to Cd, all three genes were found to be upregulated. Expression of these genes remained stable under every other condition tested. It is known that plants utilise their cell walls to bind Cd in order to limit the uptake of this pollutant. Specifically, pectin within plant cell walls serves as a binding site, as its negative charge attracts the positively charged Cd. Furthermore, under Cd stress plants produce more pectin to be able to sequester more labile Cd (Loix et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This increase in pectin makes it more difficult for fungal hyphae to penetrate between plant cells, indicating that the increased expression of \u003cem\u003eGH28a\u003c/em\u003e is needed for efficient root colonisation. JAZ6 is a repressor of the jasmonic acid (JA)-induced plant defence response. Upon activation of this response, JAZ6 will be degraded, allowing for the expression of defence response genes (Kazan \u0026amp; Manners, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Y. Wang et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Throughout symbiosis, MiSSP7 binds to JAZ6, preventing the activation of this response (Plett et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It was previously found that exposure of \u003cem\u003eBrassica juncea\u003c/em\u003e to elevated Cd increased the expression of JAZ proteins and of the JA-amino synthetase, responsible of producing isoleucine (Ile)-JA. Ile-Ja, in turn, promotes the degradation of JAZ proteins (Thakur et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The increased expression of \u003cem\u003eMiSSP7\u003c/em\u003e indicates that Cd exposure further stimulates the activation of the JA defence response, requiring additional stabilisation of JAZ6 to prevent this response.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eROS scavenging enzymes are only upregulated upon exposure to Cd in co-culture\u003c/h2\u003e \u003cp\u003eCd is known to cause oxidative damage and induce the antioxidative response (Cuypers et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Lin \u0026amp; Aarts, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, ROS have been found to play a role in establishing the ECM symbiosis within compatible hosts (Baptista et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Gafur et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). For example, NADPH oxidase knock-down mutants of \u003cem\u003eL. bicolor\u003c/em\u003e were found to be severely impaired in their ability to form a hartig net (Shi et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, how ROS signalling and scavenging are linked throughout symbiosis development is unclear. To gain a better understanding, we measured the effect of exposure to sublethal Cd on the expression of the two main ROS scavenging enzymes, SOD and CAT. While SOD catalyses the reduction of superoxide radicals into hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), CAT facilitates the further reduction of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into water and oxygen (Aebi, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; McCord \u0026amp; Fridovich, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1969\u003c/span\u003e). Exposure of the co-culture to sublethal Cd resulted in a significantly increased expression of \u003cem\u003eCAT\u003c/em\u003e. In absence of a host plant, Cd exposure did not cause an increase in expression. At a protein level, CAT activity is known to be regulated through post-translational modifications, such as phosphorylation and acylation (Baker et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This co-culture specific increase in expression suggests that post-translational modifications alone are not sufficient to counter the increased accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eTwo of the five predicted Mn/Fe SOD genes within \u003cem\u003eL. bicolor\u003c/em\u003e (\u003cem\u003eSOD1\u003c/em\u003e and \u003cem\u003eSOD2\u003c/em\u003e) were not differentially expressed upon exposure to Cd, a host plant or both. The remaining three SOD genes (\u003cem\u003eSOD3-5\u003c/em\u003e) showed upregulation, but only in co-culture when exposed to Cd. Depending on the type of SOD or environmental condition, SODs can change their localisation and even their function. For example, Cu/Zn SODs can be found within the cytosol, but also within plastids as well as the extracellular environment (Kliebenstein et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). More so, SOD5 of \u003cem\u003eFusarium oxysporum\u003c/em\u003e localises primarily within phialides, sporogenous conidia-producing cells. However, under nutrient limiting conditions SOD5 rather localises to hyphae and septa (Sewall et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Q. Wang et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In human fibroblasts, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e bursts shift SOD1 from a cytosolic localisation to nuclear accumulation, additionally changing its functionality into that of a transcriptional regulator (Tsang et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Within \u003cem\u003eL. bicolor\u003c/em\u003e, there is no information available on the function or localisation of these predicted Mn/Fe SOD genes and further research on their activity and location is needed to fully understand these results. Despite this, the specific upregulation of three SOD genes in co-culture upon exposure to Cd suggests that they play an important role in the stress protection of the \u003cem\u003eL. bicolor\u003c/em\u003e x poplar symbiosis. Additionally, prolonged exposure of Scots pine seedlings to 50 \u0026micro;M Cd resulted in decreased CAT and SOD activity (Schutzendubel et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The significant increase in expression found for both CAT and SOD from \u003cem\u003eL. bicolor\u003c/em\u003e might also be to compensate for a lower poplar CAT and SOD activity, similar to Scots pine. However, the expression and activity of these enzymes within poplar exposed to Cd would need to be investigated to test this hypothesis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we found that \u003cem\u003eL. bicolor\u003c/em\u003e and \u003cem\u003eP. tremula x alba\u003c/em\u003e can still form the ECM symbiosis when exposed to sublethal Cd. Furthermore, Cd stress stimulated LR formation and mycorrhization, as supported by the increased expression of symbiotic marker genes. This suggests that both poplar and \u003cem\u003eL. bicolor\u003c/em\u003e rely on their symbiosis as a Cd stress coping mechanism. However, formation of the symbiotic structure was slower compared to control. Under the same conditions, expression of ROS scavenging enzymes was also upregulated, indicating that post-translational modifications alone are not sufficient to overcome the increased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation. Elucidation of the exact role and subcellular localisation of the Mn/Fe SODs throughout ECM symbiosis is required to assess whether they also assist in ECM symbiosis formation or only provide superoxide radical protection.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\u003ch2\u003eFunding Declaration\u003c/h2\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eThis research was funded by Research Foundation Flanders through an FWO Fundamental Research PhD fellowship granted to MO (1193322N).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe experiments were designed by **MO** , JS and JR. Dose-response experiments were performed by **MO** . Co-cultures were set up and harvested by **MO** and JS. Morphological and microscopic analyses were performed by **MO** . RNA extractions and qRT-PCR analysis were performed by JS. Statistical analyses were performed by **MO** . The manuscript was written by **MO** and edited by JR.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eSincere thanks to Joyce Garmyn for providing the poplar microcuttings, as well as to Karl Jonckheere for maintaining the fungal collections.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll raw data and metadata are publicly available on Zenodo ( [10.5281/zenodo.18792406](https:/doi.org/10.5281/zenodo.18792406) ).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAebi H (1974) Catalase. In H. U. Bergmeyer (Ed.), \u003cem\u003eMethods Of Enzymatic Analysis (Second Edition)\u003c/em\u003e (2 ed.). Academic Press. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/b978-0-12-091302-2.50032-3\u003c/span\u003e\u003cspan address=\"10.1016/b978-0-12-091302-2.50032-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAngon PB, Islam MS, Kc S, Das A, Anjum N, Poudel A, Suchi SA (2024) Sources, effects and present perspectives of heavy metals contamination: Soil, plants and human food chain. Heliyon 10(7):e28357. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.heliyon.2024.e28357\u003c/span\u003e\u003cspan address=\"10.1016/j.heliyon.2024.e28357\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAparicio JD, Raimondo EE, Saez JM, Costa-Gutierrez SB, \u0026Aacute;lvarez A, Benimeli CS, Polti MA (2022) The current approach to soil remediation: A review of physicochemical and biological technologies, and the potential of their strategic combination. J Environ Chem Eng 10(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jece.2022.107141\u003c/span\u003e\u003cspan address=\"10.1016/j.jece.2022.107141\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaker A, Lin CC, Lett C, Karpinska B, Wright MH, Foyer CH (2023) Catalase: A critical node in the regulation of cell fate. Free Radic Biol Med 199:56\u0026ndash;66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.freeradbiomed.2023.02.009\u003c/span\u003e\u003cspan address=\"10.1016/j.freeradbiomed.2023.02.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBallabio C, Jones A, Panagos P (2024) Cadmium in topsoils of the European Union - An analysis based on LUCAS topsoil database. Sci Total Environ 912:168710. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2023.168710\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2023.168710\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaptista P, Martins A, Pais MS, Tavares RM, Lino-Neto T (2007) Involvement of reactive oxygen species during early stages of ectomycorrhiza establishment between Castanea sativa and Pisolithus tinctorius. Mycorrhiza 17(3):185\u0026ndash;193. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00572-006-0091-4\u003c/span\u003e\u003cspan address=\"10.1007/s00572-006-0091-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBecquer A, Guerrero-Gal\u0026aacute;n C, Eibensteiner JL, Houdinet G, B\u0026uuml;cking H, Zimmermann SD, Garcia K (2019) The ectomycorrhizal contribution to tree nutrition. In \u003cem\u003eMolecular Physiology and Biotechnology of Trees\u003c/em\u003e (pp. 77\u0026ndash;126). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/bs.abr.2018.11.003\u003c/span\u003e\u003cspan address=\"10.1016/bs.abr.2018.11.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBranzanti MB, Rocca E, Pisi A (1999) Effect of ectomycorrhizal fungi on chestnut ink disease. Mycorrhiza 9:103\u0026ndash;109. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s005720050007\u003c/span\u003e\u003cspan address=\"10.1007/s005720050007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCharkiewicz AE, Omeljaniuk WJ, Nowak K, Garley M, Niklinski J (2023) Cadmium Toxicity and Health Effects-A Brief Summary. Molecules 28(18). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules28186620\u003c/span\u003e\u003cspan address=\"10.3390/molecules28186620\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChot E, Reddy MS (2022) Role of Ectomycorrhizal Symbiosis Behind the Host Plants Ameliorated Tolerance Against Heavy Metal Stress. Front Microbiol 13:855473. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2022.855473\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2022.855473\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoudhury FK, Rivero RM, Blumwald E, Mittler R (2017) Reactive oxygen species, abiotic stress and stress combination. Plant J 90(5):856\u0026ndash;867. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/tpj.13299\u003c/span\u003e\u003cspan address=\"10.1111/tpj.13299\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eColpaert JV, Wevers JHL, Krznaric E, Adriaensen K (2011) How metal-tolerant ecotypes of ectomycorrhizal fungi protect plants from heavy metal pollution. Ann For Sci 68(1):17\u0026ndash;24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13595-010-0003-9\u003c/span\u003e\u003cspan address=\"10.1007/s13595-010-0003-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuypers A, Hendrix S, Dos Reis A, De Smet R, Deckers S, Gielen J, Jozefczak H, Loix M, Vercampt C, Vangronsveld H, J., Keunen E (2016) Hydrogen Peroxide, Signaling in Disguise during Metal Phytotoxicity. Front Plant Sci 7:470. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2016.00470\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2016.00470\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCuypers A, Plusquin M, Remans T, Jozefczak M, Keunen E, Gielen H, Opdenakker K, Nair AR, Munters E, Artois TJ, Nawrot T, Vangronsveld J, Smeets K (2010) Cadmium stress: an oxidative challenge. Biometals 23(5):927\u0026ndash;940. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10534-010-9329-x\u003c/span\u003e\u003cspan address=\"10.1007/s10534-010-9329-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDachuan Y, Jinyu Q (2021) The physiological response of Ectomycorrhizal fungus Lepista sordida to Cd and Cu stress. PeerJ 9:e11115. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7717/peerj.11115\u003c/span\u003e\u003cspan address=\"10.7717/peerj.11115\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Oliveira VH, Tibbett M (2018a) Cd and Zn interactions and toxicity in ectomycorrhizal basidiomycetes in axenic culture. PeerJ 6:e4478. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7717/peerj.4478\u003c/span\u003e\u003cspan address=\"10.7717/peerj.4478\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Oliveira VH, Tibbett M (2018b) Tolerance, toxicity and transport of Cd and Zn in Populus trichocarpa. Environ Exp Bot 155:281\u0026ndash;292. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envexpbot.2018.07.011\u003c/span\u003e\u003cspan address=\"10.1016/j.envexpbot.2018.07.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDitengou FA, Muller A, Rosenkranz M, Felten J, Lasok H, van Doorn MM, Legue V, Palme K, Schnitzler JP, Polle A (2015) Volatile signalling by sesquiterpenes from ectomycorrhizal fungi reprogrammes root architecture. Nat Commun 6:6279. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ncomms7279\u003c/span\u003e\u003cspan address=\"10.1038/ncomms7279\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElobeid M, Gobel C, Feussner I, Polle A (2012) Cadmium interferes with auxin physiology and lignification in poplar. J Exp Bot 63(3):1413\u0026ndash;1421. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jxb/err384\u003c/span\u003e\u003cspan address=\"10.1093/jxb/err384\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFelten J, Kohler A, Morin E, Bhalerao RP, Palme K, Martin F, Ditengou FA, Legue V (2009) The ectomycorrhizal fungus Laccaria bicolor stimulates lateral root formation in poplar and Arabidopsis through auxin transport and signaling. Plant Physiol 151(4):1991\u0026ndash;2005. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.109.147231\u003c/span\u003e\u003cspan address=\"10.1104/pp.109.147231\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGafur A, Schutzendubel A, Langenfeld-Heyser R, Fritz E, Polle A (2004) Compatible and incompetent Paxillus involutus isolates for ectomycorrhiza formation in vitro with poplar (Populus x canescens) differ in H2O2 production. Plant Biol (Stuttg) 6(1):91\u0026ndash;99. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1055/s-2003-44718\u003c/span\u003e\u003cspan address=\"10.1055/s-2003-44718\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarcia K, Delaux PM, Cope KR, Ane JM (2015) Molecular signals required for the establishment and maintenance of ectomycorrhizal symbioses. New Phytol 208(1):79\u0026ndash;87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.13423\u003c/span\u003e\u003cspan address=\"10.1111/nph.13423\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGenre A, Lanfranco L, Perotto S, Bonfante P (2020) Unique and common traits in mycorrhizal symbioses. Nat Rev Microbiol 18(11):649\u0026ndash;660. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41579-020-0402-3\u003c/span\u003e\u003cspan address=\"10.1038/s41579-020-0402-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaider FU, Liqun C, Coulter JA, Cheema SA, Wu J, Zhang R, Wenjun M, Farooq M (2021) Cadmium toxicity in plants: Impacts and remediation strategies. Ecotoxicol Environ Saf 211:111887. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ecoenv.2020.111887\u003c/span\u003e\u003cspan address=\"10.1016/j.ecoenv.2020.111887\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHartley-Whitaker J, Cairney JWG, Meharg AA (2000) Toxic effects of cadmium and zinc on ectomycorrhizal colonization of scots pine (Pinus sylvestris L.) from soil inoculum. Environ Toxicol Chem 19(3):694\u0026ndash;699. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/etc.5620190322\u003c/span\u003e\u003cspan address=\"10.1002/etc.5620190322\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe J, Ma C, Ma Y, Li H, Kang J, Liu T, Polle A, Peng C, Luo ZB (2013) Cadmium tolerance in six poplar species. Environ Sci Pollut Res Int 20(1):163\u0026ndash;174. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11356-012-1008-8\u003c/span\u003e\u003cspan address=\"10.1007/s11356-012-1008-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe S, Yang X, He Z, Baligar VC (2017) Morphological and Physiological Responses of Plants to Cadmium Toxicity: A Review. Pedosphere 27(3):421\u0026ndash;438. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s1002-0160(17)60339-4\u003c/span\u003e\u003cspan address=\"10.1016/s1002-0160(17)60339-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu YF, Zhou G, Na XF, Yang L, Nan WB, Liu X, Zhang YQ, Li JL, Bi YR (2013) Cadmium interferes with maintenance of auxin homeostasis in Arabidopsis seedlings. J Plant Physiol 170(11):965\u0026ndash;975. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jplph.2013.02.008\u003c/span\u003e\u003cspan address=\"10.1016/j.jplph.2013.02.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKazan K, Manners JM (2013) MYC2: the master in action. Mol Plant 6(3):686\u0026ndash;703. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/mp/sss128\u003c/span\u003e\u003cspan address=\"10.1093/mp/sss128\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKemppainen MJ, Pardo AG (2011) Transformation of the mycorrhizal fungus Laccaria bicolor using Agrobacterium tumefaciens. Bioeng Bugs 2(1):38\u0026ndash;44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4161/bbug.2.1.14394\u003c/span\u003e\u003cspan address=\"10.4161/bbug.2.1.14394\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan MA, Khan S, Khan A, Alam M (2017) Soil contamination with cadmium, consequences and remediation using organic amendments. Sci Total Environ 601\u0026ndash;602:1591\u0026ndash;1605. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2017.06.030\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2017.06.030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKliebenstein DJ, Monde R-A, Last RL (1998) Superoxide Dismutase in Arabidopsis: An Eclectic Enzyme Family with Disparate Regulation and Protein Localization. Plant Physiol 118(2):637\u0026ndash;650. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.118.2.637\u003c/span\u003e\u003cspan address=\"10.1104/pp.118.2.637\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKom\u0026aacute;n S, N\u0026eacute;meth R, B\u0026aacute;der M (2023) An Overview of the Current Situation of European Poplar Cultures with a Main Focus on Hungary. Appl Sci 13(23). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/app132312922\u003c/span\u003e\u003cspan address=\"10.3390/app132312922\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrznaric E, Verbruggen N, Wevers JH, Carleer R, Vangronsveld J, Colpaert JV (2009) Cd-tolerant Suillus luteus: a fungal insurance for pines exposed to Cd. Environ Pollut 157(5):1581\u0026ndash;1588. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envpol.2008.12.030\u003c/span\u003e\u003cspan address=\"10.1016/j.envpol.2008.12.030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLackus ND, Lackner S, Gershenzon J, Unsicker SB, Kollner TG (2018) The occurrence and formation of monoterpenes in herbivore-damaged poplar roots. Sci Rep 8(1):17936. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-018-36302-6\u003c/span\u003e\u003cspan address=\"10.1038/s41598-018-36302-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLagrange H, Jay-Allgmand C, Lapeyrie F (2001) Rutin, the phenolglycoside from eucalyptus root exudates, stimulates Pisolithus hyphal growth at picomolar concentrations. New Phytol 149(2):349\u0026ndash;355. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1469-8137.2001.00027.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1469-8137.2001.00027.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin YF, Aarts MG (2012) The molecular mechanism of zinc and cadmium stress response in plants. Cell Mol Life Sci 69(19):3187\u0026ndash;3206. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00018-012-1089-z\u003c/span\u003e\u003cspan address=\"10.1007/s00018-012-1089-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu B, Dong P, Zhang X, Feng Z, Wen Z, Shi L, Xia Y, Chen C, Shen Z, Lian C, Chen Y (2022) Identification and characterization of eight metallothionein genes involved in heavy metal tolerance from the ectomycorrhizal fungus Laccaria bicolor. Environ Sci Pollut Res Int 29(10):14430\u0026ndash;14442. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11356-021-16776-0\u003c/span\u003e\u003cspan address=\"10.1007/s11356-021-16776-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoix C, Huybrechts M, Vangronsveld J, Gielen M, Keunen E, Cuypers A (2017) Reciprocal Interactions between Cadmium-Induced Cell Wall Responses and Oxidative Stress in Plants. Front Plant Sci 8:1867. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2017.01867\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2017.01867\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo Z-B, Wu C, Zhang C, Li H, Lipka U, Polle A (2014) The role of ectomycorrhizas in heavy metal stress tolerance of host plants. Environ Exp Bot 108:47\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envexpbot.2013.10.018\u003c/span\u003e\u003cspan address=\"10.1016/j.envexpbot.2013.10.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa Y, He J, Ma C, Luo J, Li H, Liu T, Polle A, Peng C, Luo ZB (2014) Ectomycorrhizas with Paxillus involutus enhance cadmium uptake and tolerance in Populus x canescens. Plant Cell Environ 37(3):627\u0026ndash;642. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pce.12183\u003c/span\u003e\u003cspan address=\"10.1111/pce.12183\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarqu\u0026eacute;s-Galvez JE, Pandharikar G, Basso V, Kohler A, Lackus ND, Barry K, Keymanesh K, Johnson J, Singan V, Grigoriev IV, Vilgalys R, Martin F, Veneault-Fourrey C (2024) Populus MYC2 orchestrates root transcriptional reprogramming of defence pathway to impair Laccaria bicolor ectomycorrhizal development. New Phytol 242(2):658\u0026ndash;674. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.19609\u003c/span\u003e\u003cspan address=\"10.1111/nph.19609\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin F, Kohler A, Murat C, Veneault-Fourrey C, Hibbett DS (2016) Unearthing the roots of ectomycorrhizal symbioses. Nat Rev Microbiol 14(12):760\u0026ndash;773. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrmicro.2016.149\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro.2016.149\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCord JM, Fridovich I (1969) Superoxide Dismutase. J Biol Chem 244(22):6049\u0026ndash;6055. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0021-9258(18)63504-5\u003c/span\u003e\u003cspan address=\"10.1016/s0021-9258(18)63504-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMittler R, Zandalinas SI, Fichman Y, Van Breusegem F (2022) Reactive oxygen species signalling in plant stress responses. Nat Rev Mol Cell Biol 23(10):663\u0026ndash;679. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41580-022-00499-2\u003c/span\u003e\u003cspan address=\"10.1038/s41580-022-00499-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurashige T, Skoog F (1962) A Revised Medium for Rapid Growth and Bio assays with tobacco tissue cultures. Physiol Plant 15:473\u0026ndash;497. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1399-3054.1962.tb08052.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1399-3054.1962.tb08052.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOchoa-Hueso R, Delgado‐Baquerizo M, Risch AC, Ashton L, Augustine D, B\u0026eacute;langer N, Bridgham S, Britton AJ, Bruckman VJ, Camarero JJ, Cornelissen G, Crawford JA, Dijkstra FA, Diochon A, Earl S, Edgerley J, Epstein H, Felton A, Fortier J, Bremer E (2023) Bioavailability of Macro and Micronutrients Across Global Topsoils: Main Drivers and Global Change Impacts. Glob Biogeochem Cycles 37(6). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1029/2022gb007680\u003c/span\u003e\u003cspan address=\"10.1029/2022gb007680\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePellegrin C, Daguerre Y, Ruytinx J, Guinet F, Kemppainen M, Frey NFD, Puech-Pages V, Hecker A, Pardo AG, Martin FM, Veneault-Fourrey C (2019) Laccaria bicolor MiSSP8 is a small-secreted protein decisive for the establishment of the ectomycorrhizal symbiosis. Environ Microbiol 21(10):3765\u0026ndash;3779. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1462-2920.14727\u003c/span\u003e\u003cspan address=\"10.1111/1462-2920.14727\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePilipovic A, Zalesny RS Jr., Roncevic S, Nikolic N, Orlovic S, Beljin J, Katanic M (2019) Growth, physiology, and phytoextraction potential of poplar and willow established in soils amended with heavy-metal contaminated, dredged river sediments. J Environ Manage 239:352\u0026ndash;365. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jenvman.2019.03.072\u003c/span\u003e\u003cspan address=\"10.1016/j.jenvman.2019.03.072\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlett JM, Daguerre Y, Wittulsky S, Vayssi\u0026egrave;res A, Deveau A, Melton SJ, Kohler A, Morrell-Falvey JL, Brun A, Veneault-Fourrey C, Martin F (2014) Effector MiSSP7 of the mutualistic fungus Laccaria bicolor stabilizes the Populus JAZ6 protein and represses jasmonic acid (JA) responsive genes. Proc Natl Acad Sci U S A 111(22):8299\u0026ndash;8304. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1322671111\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1322671111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin S, Liu H, Nie Z, Rengel Z, Gao W, Li C, Zhao P (2020) Toxicity of cadmium and its competition with mineral nutrients for uptake by plants: A review. Pedosphere 30(2):168\u0026ndash;180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s1002-0160(20)60002-9\u003c/span\u003e\u003cspan address=\"10.1016/s1002-0160(20)60002-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuan L, Shi L, Zhang S, Yao Q, Yang Q, Zhu Y, Liu Y, Lian C, Chen Y, Shen Z, Duan K, Xia Y (2023) Ectomycorrhizal fungi, two species of Laccaria, differentially block the migration and accumulation of cadmium and copper in Pinus densiflora. Chemosphere 334:138857. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chemosphere.2023.138857\u003c/span\u003e\u003cspan address=\"10.1016/j.chemosphere.2023.138857\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR Core Team (2024) \u003cem\u003eR: A Language and Environment for Statistical Computing.\u003c/em\u003e In \u003cem\u003eR Foundation for Statistical Computing\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.R-project.org/\u003c/span\u003e\u003cspan address=\"https://www.R-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReddy MS, Kour M, Aggarwal S, Ahuja S, Marmeisse R, Fraissinet-Tachet L (2016) Metal induction of a Pisolithus albus metallothionein and its potential involvement in heavy metal tolerance during mycorrhizal symbiosis. Environ Microbiol 18(8):2446\u0026ndash;2454. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1462-2920.13149\u003c/span\u003e\u003cspan address=\"10.1111/1462-2920.13149\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReddy MS, Prasanna L, Marmeisse R, Fraissinet-Tachet L (2014) Differential expression of metallothioneins in response to heavy metals and their involvement in metal tolerance in the symbiotic basidiomycete Laccaria bicolor. Microbiol (Reading) 160(Pt 10):2235\u0026ndash;2242. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/mic.0.080218-0\u003c/span\u003e\u003cspan address=\"10.1099/mic.0.080218-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRitz C, Baty F, Streibig JC, Gerhard D (2015) Dose-Response Analysis Using R. PLoS ONE 10(12):e0146021. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0146021\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0146021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuytinx J, Miyauchi S, Hartmann-Wittulsky S, de Freitas Pereira M, Guinet F, Churin JL, Put C, Le Tacon F, Veneault-Fourrey C, Martin F, Kohler A (2021) A Transcriptomic Atlas of the Ectomycorrhizal Fungus Laccaria bicolor. Microorganisms 9(12). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms9122612\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms9122612\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchutzendubel A, Schwanz P, Teichmann T, Gross K, Langenfeld-Heyser R, Godbold DL, Polle A (2001) Cadmium-induced changes in antioxidative systems, hydrogen peroxide content, and differentiation in Scots pine roots. Plant Physiol 127(3):887\u0026ndash;898. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.010318\u003c/span\u003e\u003cspan address=\"10.1104/pp.010318\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeethepalli A, Dhakal K, Griffiths M, Guo H, Freschet GT, York LM (2021) RhizoVision Explorer: open-source software for root image analysis and measurement standardization. AoB Plants 13(6):plab056. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/aobpla/plab056\u003c/span\u003e\u003cspan address=\"10.1093/aobpla/plab056\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSewall TC, Mims CW, Timberlake WE (1990) abaA controls phialide differentiation in Aspergillus nidulans. Plant Cell 2(8):731\u0026ndash;739. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.2.8.731\u003c/span\u003e\u003cspan address=\"10.1105/tpc.2.8.731\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi L, Wang Z, Chen JH, Qiu H, Liu WD, Zhang XY, Martin FM, Zhao MW (2024) LbSakA-mediated phosphorylation of the scaffolding protein LbNoxR in the ectomycorrhizal basidiomycete Laccaria bicolor regulates NADPH oxidase activity, ROS accumulation and symbiosis development. New Phytol 243(1):381\u0026ndash;397. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.19813\u003c/span\u003e\u003cspan address=\"10.1111/nph.19813\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoudzilovskaia NA, van Bodegom PM, Terrer C, Zelfde MV, McCallum I, Luke McCormack M, Fisher JB, Brundrett MC, de Sa NC, Tedersoo L (2019) Global mycorrhizal plant distribution linked to terrestrial carbon stocks. Nat Commun 10(1):5077. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-019-13019-2\u003c/span\u003e\u003cspan address=\"10.1038/s41467-019-13019-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSousa NR, Ramos MA, Marques AP, Castro PM (2012) The effect of ectomycorrhizal fungi forming symbiosis with Pinus pinaster seedlings exposed to cadmium. Sci Total Environ 414:63\u0026ndash;67. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2011.10.053\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2011.10.053\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStahl L, Smoliakova IP (2007) Zinc Organometallics. In D. M. P. Mingos \u0026amp; R. H. Crabtree (Eds.), \u003cem\u003eComprehensive Organometallic Chemistry III\u003c/em\u003e (Vol. 2, pp. 309\u0026ndash;418). Elsevier. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/B0-08-045047-4/00040-6\u003c/span\u003e\u003cspan address=\"10.1016/B0-08-045047-4/00040-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteidinger BS, Crowther TW, Liang J, Van Nuland ME, Werner GDA, Reich PB, Nabuurs GJ, de-Miguel S, Zhou M, Picard N, Herault B, Zhao X, Zhang C, Routh D, Peay KG, consortium G (2019) Climatic controls of decomposition drive the global biogeography of forest-tree symbioses. Nature 569(7756):404\u0026ndash;408. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-019-1128-0\u003c/span\u003e\u003cspan address=\"10.1038/s41586-019-1128-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThakur S, Choudhary S, Bhardwaj P (2019) Comparative Transcriptome Profiling Under Cadmium Stress Reveals the Uptake and Tolerance Mechanism in Brassica juncea. J Plant Growth Regul 38(3):1141\u0026ndash;1152. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00344-019-09919-8\u003c/span\u003e\u003cspan address=\"10.1007/s00344-019-09919-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsang CK, Liu Y, Thomas J, Zhang Y, Zheng XF (2014) Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance. Nat Commun 5:3446. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ncomms4446\u003c/span\u003e\u003cspan address=\"10.1038/ncomms4446\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. \u003cem\u003eGenome Biology\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(7). https://doi.org/research0034.1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVayssi\u0026egrave;res A, Pencik A, Felten J, Kohler A, Ljung K, Martin F, Legue V (2015) Development of the Poplar-Laccaria bicolor Ectomycorrhiza Modifies Root Auxin Metabolism, Signaling, and Response. Plant Physiol 169(1):890\u0026ndash;902. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.114.255620\u003c/span\u003e\u003cspan address=\"10.1104/pp.114.255620\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Q, Pokhrel A, Coleman JJ (2021) The Extracellular Superoxide Dismutase Sod5 From Fusarium oxysporum Is Localized in Response to External Stimuli and Contributes to Fungal Pathogenicity. Front Plant Sci 12:608861. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2021.608861\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2021.608861\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Mostafa S, Zeng W, Jin B (2021) Function and Mechanism of Jasmonic Acid in Plant Responses to Abiotic and Biotic Stresses. Int J Mol Sci 22(16). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms22168568\u003c/span\u003e\u003cspan address=\"10.3390/ijms22168568\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYaciuk PA, Colombo F, Lecomte KL, De Micco G, Boh\u0026eacute; AE (2022) Cadmium sources, mobility, and natural attenuation in contrasting environments (carbonate-rich and carbonate-poor) in the Capillitas polymetallic mineral deposit, NW Argentina. Appl Geochem 136. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.apgeochem.2021.105152\u003c/span\u003e\u003cspan address=\"10.1016/j.apgeochem.2021.105152\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi L, Wu M, Yu F, Song Q, Zhao Z, Liao L, Tong J (2022) Enhanced cadmium phytoremediation capacity of poplar is associated with increased biomass and Cd accumulation under nitrogen deposition conditions. Ecotoxicol Environ Saf 246:114154. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ecoenv.2022.114154\u003c/span\u003e\u003cspan address=\"10.1016/j.ecoenv.2022.114154\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan HM, Huang X (2016) Inhibition of root meristem growth by cadmium involves nitric oxide-mediated repression of auxin accumulation and signalling in Arabidopsis. Plant Cell Environ 39(1):120\u0026ndash;135. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pce.12597\u003c/span\u003e\u003cspan address=\"10.1111/pce.12597\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan Z, Luo T, Liu X, Hua H, Zhuang Y, Zhang X, Zhang L, Zhang Y, Xu W, Ren J (2019) Tracing anthropogenic cadmium emissions: From sources to pollution. Sci Total Environ 676:87\u0026ndash;96. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2019.04.250\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2019.04.250\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang F, Anasontzis GE, Labourel A, Champion C, Haon M, Kemppainen M, Commun C, Deveau A, Pardo A, Veneault-Fourrey C, Kohler A, Rosso MN, Henrissat B, Berrin JG, Martin F (2018) The ectomycorrhizal basidiomycete Laccaria bicolor releases a secreted beta-1,4 endoglucanase that plays a key role in symbiosis development. New Phytol 220(4):1309\u0026ndash;1321. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.15113\u003c/span\u003e\u003cspan address=\"10.1111/nph.15113\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang F, Labourel A, Haon M, Kemppainen M, Da Silva Machado E, Brouilly N, Veneault-Fourrey C, Kohler A, Rosso MN, Pardo A, Henrissat B, Berrin JG, Martin F (2021) The ectomycorrhizal basidiomycete Laccaria bicolor releases a GH28 polygalacturonase that plays a key role in symbiosis establishment. New Phytol 233(6):2534\u0026ndash;2547. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/nph.17940\u003c/span\u003e\u003cspan address=\"10.1111/nph.17940\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang K, Tappero R, Ruytinx J, Branco S, Liao HL (2021) Disentangling the role of ectomycorrhizal fungi in plant nutrient acquisition along a Zn gradient using X-ray imaging. Sci Total Environ 801:149481. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2021.149481\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2021.149481\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao M, Wang H, Sun J, Tang R, Cai B, Song X, Huang X, Huang J, Fan Z (2023) Spatio-temporal characteristics of soil Cd pollution and its influencing factors: A Geographically and temporally weighted regression (GTWR) method. J Hazard Mater 446:130613. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jhazmat.2022.130613\u003c/span\u003e\u003cspan address=\"10.1016/j.jhazmat.2022.130613\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mycorrhiza","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcor","sideBox":"Learn more about [Mycorrhiza](http://link.springer.com/journal/572)","snPcode":"572","submissionUrl":"https://submission.nature.com/new-submission/572/3","title":"Mycorrhiza","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Laccaria bicolor, Poplar, Cadmium toxicity, Ectomycorrhiza, antioxidative response","lastPublishedDoi":"10.21203/rs.3.rs-8981216/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8981216/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSoils have become increasingly polluted with Cd due to industrial and mining activities, as well as agricultural fertiliser usage. Because of its toxicity, plants face significant abiotic stress. Trees found in temperate and boreal forest ecosystems rely on their mutualistic relationship with ECM fungi to alleviate the toxic effects of Cd. In this study, we assessed the impact of Cd pollution on both \u003cem\u003eL. bicolor\u003c/em\u003e and its symbiosis with \u003cem\u003eP. tremula x alba\u003c/em\u003e. We investigated the impact of Cd pollution on fungal growth and mycorrhiza morphology, as well as the expression of symbiosis marker genes and ROS scavenging enzymes in presence and absence of a host plant. Results indicate that fungal growth is reduced by exposure to elevated Cd, however symbiosis formation is stimulated. Both symbiosis marker genes and ROS scavenging enzymes showed increased expression upon exposure to Cd, but only in the presence of a host plant. This data suggests that forming the ECM symbiosis is a key coping mechanism for both poplars and \u003cem\u003eL. bicolor\u003c/em\u003e, and by stimulating the formation of the symbiotic structure, the reduced fungal growth can partially be mitigated. This research highlights the importance of the ECM symbiosis in both plant and fungal resilience in changing environmental conditions.\u003c/p\u003e","manuscriptTitle":"Sublethal Cd exposure stimulates Laccaria bicolor x poplar symbiosis formation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-10 10:58:42","doi":"10.21203/rs.3.rs-8981216/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-13T14:10:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T13:51:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77679912854122246222306436615595334481","date":"2026-03-23T17:31:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-23T13:07:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"202587813803904498747488168401248680336","date":"2026-03-05T11:14:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-05T10:13:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-04T23:11:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-04T22:53:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mycorrhiza","date":"2026-02-26T19:57:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mycorrhiza","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcor","sideBox":"Learn more about [Mycorrhiza](http://link.springer.com/journal/572)","snPcode":"572","submissionUrl":"https://submission.nature.com/new-submission/572/3","title":"Mycorrhiza","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4e04367a-9242-4e8b-8ad8-3de7f456a6fe","owner":[],"postedDate":"March 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-05T13:08:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-10 10:58:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8981216","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8981216","identity":"rs-8981216","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}