Zinc exposure primes Suillus tomentosus to tolerate cadmium

Zinc exposure primes Suillus tomentosus to tolerate cadmium | 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 Zinc exposure primes Suillus tomentosus to tolerate cadmium Jessica Fletcher, Sara Branco This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5357023/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Metal toxicity is detrimental and can lead to death. However, some fungi display high metal tolerance and it is not clear how the trait arises. Here we used an in vitro experimental evolution experiment to test whether metal exposure induces metal tolerance in Suillus tomentosus , a widespread ectomycorrhizal fungus with a wide range of tolerance to several metals. We exposed a zinc (Zn) and cadmium (Cd) sensitive S. tomentosus isolate to both these metals and assessed whether prolonged exposure induced metal tolerance. We found that even though Zn or Cd exposure did not lead to Zn or Cd tolerance, Zn exposure increased S. tomentosus Cd tolerance. Our findings show metal exposure does not explain the existence of metal tolerance but suggest the existence of shared mechanisms for metal homeostasis and provide a framework for understanding how fungi can inhabit soils contaminated with multiple metals. Suillus heavy metals ectomycorrhizal fungi priming adaptation Figures Figure 1 Figure 2 Introduction Soil heavy metal contamination due to anthropogenic activity is widespread and can be detrimental to organisms. Metal toxicity is often mutagenic and genotoxic, leading to cellular defects, metabolism impairment and death (Narayanan and Ma 2023 ). Some heavy metals are toxic even in small amounts, such as cadmium, mercury and lead, while others are micronutrients essential for cell function (e.g., copper, iron and zinc) but lethal at high concentrations (Singh 2016). Previous work showed Suillus , a widespread conifer-associated genus of ectomycorrhizal fungi, displays inter- and intra-specific variation regrading heavy metal tolerance (Branco 2022, Lofgren 2024). For example, S. luteus , S. bovinus , S. brevipes and S. tomentosus display different average tolerance to distinct metals and all include isolates displaying high and low metal tolerance (Colpaert and van Assche 1987 ; Colpaert and van Assche 1992 ; Colpaert 2000; Colpaert 2004; Krznaric 2009, Branco 2022, Lofgren 2024, Fletcher 2024). In some cases, such as in S. luteus , metal tolerance is largely associated with soil contamination levels and metal tolerant isolates are mainly found in soils with high metal levels (Krznaric 2009; Colpaert 2004; Colpaert 2000; Colpaert and Vanassche 1992; Colpaert and van Assche 1987 ). In other species, including S. tomentosus and S. brevipes , metal tolerant and sensitive isolates are found living in both contaminated and non-contaminated soils, likely due to low metal bioavailability, soil heterogeneity, and/or localized pockets with low metal content (Fletcher 2024). In addition, metal contaminated sites tend to be polluted with a broad suite of heavy metals making it reasonable to expect fungal isolates to be tolerant to multiple metals. However, this is often not the case. For example, it is common to find Suillus isolates that are tolerant to Zn are not also tolerant to Cd and vice versa (Colpaert 2000; Fletcher 2024). These findings suggest that tolerance to different metals in Suillus stems from distinct mechanisms that have little overlap (Fletcher 2024). There has been increasing interest in understanding the genetic and physiological bases of metal tolerance in Suillus (Lofgren 2024; Branco 2022). Genomic studies in S. luteus showed metal tolerance is a highly polygenic trait that stems from genes involved in signaling and metal homeostasis. Interestingly, metal tolerance in this species arises from genetic standing variation and is not associated with population structure, with tolerant and sensitive isolates having very few genomic differences (Bazzicalupo 2020). However, S. luteus metal-tolerant and sensitive isolates show distinct gene expression profiles, including differences in transmembrane transporters, chelators, antioxidants, and cellular signaling, indicating metal tolerance is under transcriptomic and possibly epigenetic control (Smith 2024). Here, we use an in vitro experimental evolution approach to determine whether metal exposure induces fungal metal tolerance. Experimental evolution has been successfully applied to investigate tolerance to a wide range of stressors across a large suite of organisms, showing for example that Zn tolerance in plants can evolve in a single generation (Nowak 2018) and that Cd tolerance in Drosophila melanogaster is associated with adaptive evolution of gene expression across multiple generations (Huang and Agrawal 2016 ). Even though the use of experimental evolution has been limited in fungi (Fisher and Lang 2016 ), it has contributed to understanding the patterns and mechanisms of antifungal resistance in Candida (Selmecki 2009) and Aspergillus (Ferreira 2004), salt tolerance in Saccharomyces (Dhar 2011), thermotolerance in the filamentous fungal insect-pathogen Melanoplus sanguinipes (de Crecy 2009), as well as heavy metal tolerance in yeasts (Gorter 2017; Gorter 2016) and filamentous fungi (Le 2006; Anahid, Yaghmaei, and Ghobadinejad 2011 ). Experimental evolution has also shown that an isolate of the decomposing basidiomycete Schizophyllum commune developed tolerance to multiple heavy metals within a relatively short time in the absence of sexual reproduction. This evolved isolate also displayed metal cross tolerance, growing better than the control when exposed to some but not all metals (Traxler 2022). We studied S. tomentosus , a common ectomycorrhizal species in the pine forests of the American West and known for including individuals sensitive and tolerant to Cd and Zn (Fletcher 2024). We exposed a Cd- and Zn-sensitive S. tomentosus isolate to prolonged Cd and Zn and hypothesized exposure would lead to increased tolerance to these metals. Given we previously showed that in S. tomentosus Cd and Zn tolerance are not correlated (Fletcher 2024), we also hypothesized that increased tolerance to one metal would not lead to increased tolerance to the other metal. We found Cd or Zn exposure did not induce Cd or Zn tolerance in S. tomentosus , but prolonged Zn exposure primed the isolate to tolerate Cd. Materials and Methods Isolate selection We selected S. tomentosus 220 (hereafter referred to as St 0 ), a Zn and Cd sensitive S. tomentosus isolate previously described in (Fletcher 2024). St 0 was collected from Colorado (USA), confirmed as S. tomentosus by ITS sequencing (GenBank OR665202) and phylogenetic analyses, and vouchered in the Sam Mitchell Herbarium at the Denver Botanic Gardens (DBG-F-034794). Evolution experiment We routinely cultured St 0 on Fries agar at pH 4.5 (Lofgren 2024). For the in vitro evolution experiment, we subcultured St 0 in triplicate on either plain Fries agar (control), or Fries agar amended with 1.5 mM Zn or 3.5 µM Cd (added as ZnSO 4 ·7H 2 O and CdSO 4 , respectively). These concentrations inhibit ~50% of St 0 growth (Zn inhibited 47.6% of growth and Cd inhibited 49.2% of growth). We serially subcultured the isolate for 10 weeks on control (St ctr ), high Zn (St Zn ), and high Cd (St Cd ) conditions and measured culture growth every week, as described in (Fletcher 2024). To determine if subculturing impacted growth on control and metal conditions (cross checks), we further measured St ctr , St Zn and St Cd 1-week growth on all metal/control conditions. We compared growth across treatments using ANOVA and post-hoc Tukey tests (after assumptions of normality, equal variance and independence were met). Significance was considered when p ≤ 0.05. We analyzed growth data in RStudio (R v4.2.1) with packages ggplot2 (v3.4.4), rstatix (v0.7.2), tidyverse (v2.0.0) and ggpubr (v0.6.0). Results Zn and Cd exposure did not increase S. tomentosus Zn and Cd tolerance Subculturing the metal sensitive S. tomentosus isolate for 10 weeks in high Cd or high Zn did not result in increased Cd or Zn tolerance (Fig. 1). Growth was always higher in control conditions and consistently low in both metals throughout the experiment (Fig. 1A). Notably, St ctr grew significantly more than St 0 on control media (p = 2.1e-3) (Fig 1B). Zn and Cd exposure did not change growth, as there were no significant differences between St 0 and St Zn on Zn (p = 5.8 e-1) or St 0 and St Cd on Cd (p = 1.62e-1) (Fig. 1B). Prolonged Zn exposure primes S. tomentosus tolerance to Cd Cross tolerance checks of St ctr , St Cd and St Zn showed growth differences across control, Cd, and Zn conditions. We found significant growth differences across serially passaged isolates in control Fries agar (ANOVA = F(3, 8) = 15.131, p = 0.001). St ctr and St Zn grew significantly more than St 0 (p = 0.00187 and 0.00449 respectively), while St Cd did not (p = 0.428) (Fig 2A). We also found significant growth differences across serially passaged isolates in high Zn (ANOVA = F(3, 8) = 4.623, p = 0.037), with St Zn growing less than St ctr (p = 0.0492), but not differently than St 0 and St Cd . Finally, there were differences between growth on Cd (F(3, 8) = 29.945, p = 0.000106), with St Cd showing the lowest growth (p = 0.0138 vs. St 0 ; p = 0.0393 vs. St H2O ; p = 0.0001 vs. St Zn ) and St Zn the highest (p = 0.00357 vs. St 0 ; p = 0.00148 vs. St H2O ; p = 0.0001 vs. St Cd ). Discussion We found that overall, prolonged metal exposure does not lead to metal tolerance in S. tomentosus. However, Zn exposure resulted in higher growth under Cd. Such priming effect provides a framework for understanding how fungi can inhabit soils contaminated with multiple metals. Our results differ from other similar experiments that found metal exposure to induce fungal metal tolerance. For example, exposure to Cd in the yeast Saccharomyces cerevisiae led to Cd tolerance (Gorter 2016), and continuous Cd, cesium (Cs) and strontium (Sr) exposure of the filamentous fungus Schizophyllum commune promoted tolerance to these metals (Traxler 2022). The latter study used metal stressors comparable to ours, with Cd and Zn levels leading to ~ 50% growth inhibition and reported 10-week exposure to Cd increased Cd tolerance, but exposure to Zn did not lead to Zn tolerance. In addition, Traxler (2022) found that continued exposure to Sr primed tolerance to Cs and Cd, but exposure to Zn did not prime isolates for Cd exposure. Such differences indicate variation in metal exposure effect across fungi, ranging from inducing metal tolerance to not having an effect. We did not anticipate Zn exposure to prime S. tomentosus for Cd tolerance, as there is no evidence for correlation between tolerance to Zn and Cd in Suillus (Colpaert 2000; Fletcher 2024) (Fletcher 2024). However, organisms rarely encounter single metal stress in the environment and the interaction and overlap of detoxification pathways is not unexpected. In fact, Cd and Zn toxicity are known to trigger both independent and shared metal detoxification pathways in other eukaryotic cells (Earley 2021). Notably, previous research has shown that the presence of Zn can alleviate Cd toxicity in S. luteus . When exposed to a combination of these metals, S. luteus tolerates increased concentrations of Cd. The reverse is not true, and Zn tolerance does not increase in the presence of Cd (Krznaric 2009; Colpaert and Vanassche 1993). It is possible that Zn exposure triggers metal exclusion or detoxification mechanisms which can also protect cells from Cd exposure. This might be due to the role of Zn as a micronutrient, and the existence of specific uptake and homeostasis pathways for Zn in Suillus species while no such specific pathways exist for Cd (Coninx 2019; Coninx 2017; Ruytinx 2017). In addition, it is possible for Cd to activate different pathways to combat toxicity that do not carry over to Zn toxicity. Zinc and Cd are chemically similar, both falling into group 12 of the periodic table, and forming divalent ions (Zn 2+ and Cd 2+ respectively). Such similarity allows Cd to hijack many of the cellular transporters involved in Zn homeostasis (Yu 2021). Cadmium can then take the place of Zn in enzymes and cause disfunction (Singh 2016). Because Zn is an essential micronutrient, cells have mechanisms that function to maintain optimum levels of Zn for cellular function that also act to limit toxicity. These include transporters that move Zn into and out of the cell as well as into vesicles that maintain homeostasis. In other eukaryotic systems, Zn transporters can be hijacked by Cd and allow this metal to enter the cell. For example, ZIP transporters can allow for Cd uptake into Xenopus cells (Liu 2008), and ZnT proteins have been implicated in the uptake of Cd into rat neurons (Ohana 2006). Zinc transporters have been well described in Suillus luteus and include ZIP transporters localized in the plasma membrane ( SlZRT1 and SlZRT2; (Coninx 2017; Coninx 2019), and in vacuoles ( SlZnT2; (Ruytinx 2017). Differential expression of Zn transporters has been implicated in the Zn tolerance phenotype, and metal tolerant Suillus isolates can exclude metals from the cell more efficiently than sensitive isolates, likely through modulated transporter regulation (Fletcher 2024; Colpaert 2005; Krznaric 2009; Smith 2024). It is likely that Cd hijacks Suillus Zn transporters to enter cells, and these transporters may also be implicated in the Cd toxicity response and transcriptomic analysis of the Suillus Cd response would allow determining if genes implicated in Cd tolerance overlap with those involved in the Zn response. In a broader sense, priming of cells to one stressor through exposure to another stress has been documented in several eukaryotes. For example, exogenous H 2 O 2 primes rice plants ( Oryza sativa ) for exposure to Cd, likely from increased antioxidant activity induced by H 2 O 2 that might prepare cells for the increased ROS under Cd toxicity (Hu 2009). In fungi, osmotic stress is known to prime Saccharomyces cerevisiae yeast cells for exposure to heat shock (Trollmo 1988), exposure to Sr confers Cs and Cd tolerance in Sch. commune (Traxler 2022), and riboflavin primes Agaricus bisporus , another decomposing basidiomycete, to drought tolerance. In the latter, riboflavin might activate similar transcriptional pathways that are also involved in the response to drought (Guhr, Horn, and Weig 2017 ). We are unsure of how exactly Zn exposure triggers increased Cd tolerance in our experiment, though we speculate that exposure to Zn may activate antioxidants that may carry over, priming cells for Cd exposure. Further investigation into transcriptomic and biological pathways involved should help unravel how these two tolerance phenotypes are connected. We have no good explanation for that fact that St ctr was able to grow significantly better in control conditions than the St 0 parent strain, however, this phenomenon was also observed in Sch. commune (Traxler 2022) and might be common in filamentous basidiomycetes. It is also puzzling that St Zn performed significantly worse in Zn and better than all other strains in the Cd cross-tolerance tests. Given there was no increased tolerance to the primary metal stressors, we did not expect to find any metal cross-tolerance. However, we speculate that a buildup of Zn toxicity within the cells may be behind this response. We show that prolonged metal exposure does not lead to fungal metal tolerance, but that exposure to high levels of a micronutrient metal can prime tolerance to a highly toxic metal. These suggest the existence of shared mechanisms for metal homeostasis and provide a framework for explaining how fungi can inhabit soils contaminated with multiple metals. Uncovering the transcriptomic basis of metal priming will unveil if shared metal detoxification pathways underlying this phenomenon. Declarations The authors declare that they have no conflict of interest. Funding Declaration This work was supported by National Science Foundation NSF IOS-PBI (2029168) awarded to S.B. 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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-5357023","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":372718346,"identity":"83c33e52-7b80-474f-8284-c5e148614284","order_by":0,"name":"Jessica Fletcher","email":"data:image/png;base64,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","orcid":"","institution":"University of Colorado Denver","correspondingAuthor":true,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Fletcher","suffix":""},{"id":372718348,"identity":"97d3a31f-0fc4-4bc0-b349-77f9c243bdcd","order_by":1,"name":"Sara Branco","email":"","orcid":"","institution":"University of Colorado Denver","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Branco","suffix":""}],"badges":[],"createdAt":"2024-10-29 21:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5357023/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5357023/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68783871,"identity":"01d950c2-f252-40a9-8ec9-633bc6f35446","added_by":"auto","created_at":"2024-11-12 03:26:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":104519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCd and Zn exposure did not increase \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. tomentosus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e metal tolerance\u003c/strong\u003e. A) St\u003csup\u003ectr\u003c/sup\u003e, St\u003csup\u003eCd\u003c/sup\u003e and St\u003csup\u003eZn\u003c/sup\u003e weekly mean radial growth on control, 1.5 mM Zn, and 3.5 µM Cd treatments (in triplicate). Error bars represent standard deviation. B) Comparison of St\u003csup\u003e0 \u003c/sup\u003e(the original isolate) growth at week 1 and St\u003csup\u003ectr\u003c/sup\u003e, St\u003csup\u003eCd\u003c/sup\u003e and St\u003csup\u003eZn\u003c/sup\u003e (grown on control, Zn, and Cd treatments) at week 10. Error bars represent standard error of the mean. Asterisks indicate statistical significance between indicated groups (** = \u003cem\u003ep\u003c/em\u003e \u0026lt;= 0.01, ns = not significant). St\u003csup\u003e0 \u003c/sup\u003e= original \u003cem\u003eS. tomentosus\u003c/em\u003e isolate; St\u003csup\u003ectr\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e=\u003cem\u003e S. tomentosus\u003c/em\u003e isolate subcultured for 10 weeks in control conditions; St\u003csup\u003eCd\u003c/sup\u003e = \u003cem\u003eS. tomentosus\u003c/em\u003e isolate subcultured for 10 weeks in 3.5 µM Cd; St\u003csup\u003eZn\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e=\u003cem\u003e S. tomentosus\u003c/em\u003e isolate subcultured for 10 weeks in 1.5 mM Zn.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5357023/v1/92fea1710e1e9265a5e3ead8.png"},{"id":68783990,"identity":"1e1f3eeb-ad78-4062-a009-6c16b278ca61","added_by":"auto","created_at":"2024-11-12 03:34:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTolerance crosschecks showed growth differences and that Zn exposure primes \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. tomentosus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e to tolerate Cd\u003c/strong\u003e. A) St\u003csup\u003e0\u003c/sup\u003e, St\u003csup\u003ectr\u003c/sup\u003e, St\u003csup\u003eCd\u003c/sup\u003e and St\u003csup\u003eZn\u003c/sup\u003e growth on control conditions. B) St\u003csup\u003e0\u003c/sup\u003e, St\u003csup\u003ectr\u003c/sup\u003e, St\u003csup\u003eCd\u003c/sup\u003e and St\u003csup\u003eZn\u003c/sup\u003e growth on 1.5 mM Zn. C) St\u003csup\u003e0\u003c/sup\u003e, St\u003csup\u003ectr\u003c/sup\u003e, St\u003csup\u003eCd\u003c/sup\u003e and St\u003csup\u003eZn\u003c/sup\u003e growth on 3.5 µM Cd. Letter groups indicate significant differences. St\u003csup\u003e0 \u003c/sup\u003e– original \u003cem\u003eS. tomentosus\u003c/em\u003e isolate; St\u003csup\u003ectr\u003c/sup\u003e\u003cem\u003e - S. tomentosus\u003c/em\u003e isolate subcultured for 10 weeks in control conditions; St\u003csup\u003eCd\u003c/sup\u003e - \u003cem\u003eS. tomentosus\u003c/em\u003e isolate subcultured for 10 weeks in 3.5 µM Cd; St\u003csup\u003eZn\u003c/sup\u003e\u003cem\u003e - S. tomentosus\u003c/em\u003e isolate subcultured for 10 weeks in 1.5 mM Zn.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5357023/v1/425b1c666ea1558cc2dc5330.png"},{"id":74955021,"identity":"82510406-a544-4da0-bd41-b398f7c65065","added_by":"auto","created_at":"2025-01-28 17:16:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":725007,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5357023/v1/00001f2a-690a-450a-b59f-e3e86b562748.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Zinc exposure primes Suillus tomentosus to tolerate cadmium","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoil heavy metal contamination due to anthropogenic activity is widespread and can be detrimental to organisms. Metal toxicity is often mutagenic and genotoxic, leading to cellular defects, metabolism impairment and death (Narayanan and Ma \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Some heavy metals are toxic even in small amounts, such as cadmium, mercury and lead, while others are micronutrients essential for cell function (e.g., copper, iron and zinc) but lethal at high concentrations (Singh 2016).\u003c/p\u003e \u003cp\u003ePrevious work showed \u003cem\u003eSuillus\u003c/em\u003e, a widespread conifer-associated genus of ectomycorrhizal fungi, displays inter- and intra-specific variation regrading heavy metal tolerance (Branco 2022, Lofgren 2024). For example, \u003cem\u003eS. luteus\u003c/em\u003e, \u003cem\u003eS. bovinus\u003c/em\u003e, \u003cem\u003eS. brevipes\u003c/em\u003e and \u003cem\u003eS. tomentosus\u003c/em\u003e display different average tolerance to distinct metals and all include isolates displaying high and low metal tolerance (Colpaert and van Assche \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Colpaert and van Assche \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Colpaert 2000; Colpaert 2004; Krznaric 2009, Branco 2022, Lofgren 2024, Fletcher 2024). In some cases, such as in \u003cem\u003eS. luteus\u003c/em\u003e, metal tolerance is largely associated with soil contamination levels and metal tolerant isolates are mainly found in soils with high metal levels (Krznaric 2009; Colpaert 2004; Colpaert 2000; Colpaert and Vanassche 1992; Colpaert and van Assche \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). In other species, including \u003cem\u003eS. tomentosus\u003c/em\u003e and \u003cem\u003eS. brevipes\u003c/em\u003e, metal tolerant and sensitive isolates are found living in both contaminated and non-contaminated soils, likely due to low metal bioavailability, soil heterogeneity, and/or localized pockets with low metal content (Fletcher 2024). In addition, metal contaminated sites tend to be polluted with a broad suite of heavy metals making it reasonable to expect fungal isolates to be tolerant to multiple metals. However, this is often not the case. For example, it is common to find \u003cem\u003eSuillus\u003c/em\u003e isolates that are tolerant to Zn are not also tolerant to Cd and vice versa (Colpaert 2000; Fletcher 2024). These findings suggest that tolerance to different metals in \u003cem\u003eSuillus\u003c/em\u003e stems from distinct mechanisms that have little overlap (Fletcher 2024).\u003c/p\u003e \u003cp\u003eThere has been increasing interest in understanding the genetic and physiological bases of metal tolerance in \u003cem\u003eSuillus\u003c/em\u003e (Lofgren 2024; Branco 2022). Genomic studies in \u003cem\u003eS. luteus\u003c/em\u003e showed metal tolerance is a highly polygenic trait that stems from genes involved in signaling and metal homeostasis. Interestingly, metal tolerance in this species arises from genetic standing variation and is not associated with population structure, with tolerant and sensitive isolates having very few genomic differences (Bazzicalupo 2020). However, \u003cem\u003eS. luteus\u003c/em\u003e metal-tolerant and sensitive isolates show distinct gene expression profiles, including differences in transmembrane transporters, chelators, antioxidants, and cellular signaling, indicating metal tolerance is under transcriptomic and possibly epigenetic control (Smith 2024).\u003c/p\u003e \u003cp\u003eHere, we use an \u003cem\u003ein vitro\u003c/em\u003e experimental evolution approach to determine whether metal exposure induces fungal metal tolerance. Experimental evolution has been successfully applied to investigate tolerance to a wide range of stressors across a large suite of organisms, showing for example that Zn tolerance in plants can evolve in a single generation (Nowak 2018) and that Cd tolerance in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e is associated with adaptive evolution of gene expression across multiple generations (Huang and Agrawal \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Even though the use of experimental evolution has been limited in fungi (Fisher and Lang \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), it has contributed to understanding the patterns and mechanisms of antifungal resistance in \u003cem\u003eCandida\u003c/em\u003e (Selmecki 2009) and \u003cem\u003eAspergillus\u003c/em\u003e (Ferreira 2004), salt tolerance in \u003cem\u003eSaccharomyces\u003c/em\u003e (Dhar 2011), thermotolerance in the filamentous fungal insect-pathogen \u003cem\u003eMelanoplus sanguinipes\u003c/em\u003e (de Crecy 2009), as well as heavy metal tolerance in yeasts (Gorter 2017; Gorter 2016) and filamentous fungi (Le 2006; Anahid, Yaghmaei, and Ghobadinejad \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Experimental evolution has also shown that an isolate of the decomposing basidiomycete \u003cem\u003eSchizophyllum commune\u003c/em\u003e developed tolerance to multiple heavy metals within a relatively short time in the absence of sexual reproduction. This evolved isolate also displayed metal cross tolerance, growing better than the control when exposed to some but not all metals (Traxler 2022).\u003c/p\u003e \u003cp\u003eWe studied \u003cem\u003eS. tomentosus\u003c/em\u003e, a common ectomycorrhizal species in the pine forests of the American West and known for including individuals sensitive and tolerant to Cd and Zn (Fletcher 2024). We exposed a Cd- and Zn-sensitive \u003cem\u003eS. tomentosus\u003c/em\u003e isolate to prolonged Cd and Zn and hypothesized exposure would lead to increased tolerance to these metals. Given we previously showed that in \u003cem\u003eS. tomentosus\u003c/em\u003e Cd and Zn tolerance are not correlated (Fletcher 2024), we also hypothesized that increased tolerance to one metal would not lead to increased tolerance to the other metal. We found Cd or Zn exposure did not induce Cd or Zn tolerance in \u003cem\u003eS. tomentosus\u003c/em\u003e, but prolonged Zn exposure primed the isolate to tolerate Cd.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003eIsolate selection\u003c/h2\u003e\n\u003cp\u003eWe selected \u003cem\u003eS. tomentosus\u0026nbsp;\u003c/em\u003e220 (hereafter referred to as St\u003csup\u003e0\u003c/sup\u003e), a Zn and Cd sensitive \u003cem\u003eS. tomentosus\u003c/em\u003e isolate previously described in (Fletcher 2024). St\u003csup\u003e0\u0026nbsp;\u003c/sup\u003ewas collected from Colorado (USA), confirmed as \u003cem\u003eS. tomentosus\u0026nbsp;\u003c/em\u003eby ITS sequencing (GenBank OR665202) and phylogenetic analyses, and vouchered in the Sam Mitchell Herbarium at the Denver Botanic Gardens (DBG-F-034794). \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eEvolution experiment\u003c/h2\u003e\n\u003cp\u003eWe routinely cultured St\u003csup\u003e0\u0026nbsp;\u003c/sup\u003eon Fries agar at pH 4.5\u0026nbsp;(Lofgren 2024). For the \u003cem\u003ein vitro\u003c/em\u003e evolution experiment, we subcultured St\u003csup\u003e0\u0026nbsp;\u003c/sup\u003ein triplicate on either plain Fries agar (control), or Fries agar amended with 1.5 mM Zn or 3.5 \u0026micro;M Cd (added as ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO and CdSO\u003csub\u003e4\u003c/sub\u003e, respectively). These concentrations inhibit ~50% of St\u003csup\u003e0\u0026nbsp;\u003c/sup\u003egrowth (Zn inhibited 47.6% of growth and Cd inhibited 49.2% of growth). We serially subcultured the isolate for 10 weeks on control (St\u003csup\u003ectr\u003c/sup\u003e), high Zn (St\u003csup\u003eZn\u003c/sup\u003e), and high Cd (St\u003csup\u003eCd\u003c/sup\u003e) conditions and measured culture growth every week, as described in\u0026nbsp;(Fletcher 2024). To determine if subculturing impacted growth on control and metal conditions (cross checks), we further measured St\u003csup\u003ectr\u003c/sup\u003e, St\u003csup\u003eZn\u003c/sup\u003e and St\u003csup\u003eCd\u003c/sup\u003e 1-week growth on all metal/control conditions. We compared growth across treatments using ANOVA and post-hoc Tukey tests (after assumptions of normality, equal variance and independence were met). Significance was considered when p \u0026le; 0.05. We analyzed growth data in RStudio (R v4.2.1) with packages ggplot2 (v3.4.4), rstatix (v0.7.2), tidyverse (v2.0.0) and ggpubr (v0.6.0).\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eZn and Cd exposure did not increase \u003cem\u003eS. tomentosus\u003c/em\u003e Zn and Cd tolerance\u003c/h2\u003e\n\u003cp\u003eSubculturing the metal sensitive \u003cem\u003eS. tomentosus\u003c/em\u003e isolate for 10 weeks in high Cd or high Zn did not result in increased Cd or Zn tolerance (Fig. 1). Growth was always higher in control conditions\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand consistently low in both metals throughout the experiment (Fig. 1A). Notably, St\u003csup\u003ectr\u003c/sup\u003e grew significantly more than St\u003csup\u003e0\u003c/sup\u003e on control media (p = 2.1e-3) (Fig 1B). Zn and Cd exposure did not change growth, as there were no significant differences between St\u003csup\u003e0\u0026nbsp;\u003c/sup\u003eand St\u003csup\u003eZn\u003c/sup\u003e on Zn (p = 5.8 e-1) or St\u003csup\u003e0\u0026nbsp;\u003c/sup\u003eand St\u003csup\u003eCd\u0026nbsp;\u003c/sup\u003eon Cd (p = 1.62e-1) (Fig. 1B).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eProlonged Zn exposure primes \u003cem\u003eS. tomentosus\u003c/em\u003e tolerance to Cd\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eCross tolerance checks of St\u003csup\u003ectr\u003c/sup\u003e, St\u003csup\u003eCd\u003c/sup\u003e and St\u003csup\u003eZn\u003c/sup\u003e showed growth differences across control, Cd, and Zn conditions. We found significant growth differences across serially passaged isolates in control Fries agar (ANOVA = F(3, 8) = 15.131, p = 0.001). St\u003csup\u003ectr\u003c/sup\u003e and St\u003csup\u003eZn\u003c/sup\u003e grew significantly more than St\u003csup\u003e0\u0026nbsp;\u003c/sup\u003e(p = 0.00187 and 0.00449 respectively), while St\u003csup\u003eCd\u0026nbsp;\u003c/sup\u003edid not (p = 0.428) (Fig 2A). We also found significant growth differences across serially passaged isolates in high Zn (ANOVA = F(3, 8) = 4.623, p = 0.037), with St\u003csup\u003eZn\u003c/sup\u003e growing less than St\u003csup\u003ectr\u003c/sup\u003e (p = 0.0492), but not differently than St\u003csup\u003e0\u003c/sup\u003e and St\u003csup\u003eCd\u003c/sup\u003e. Finally, there were differences between growth on Cd (F(3, 8) = 29.945, p = 0.000106), with St\u003csup\u003eCd\u0026nbsp;\u003c/sup\u003eshowing the lowest growth (p = 0.0138 vs. St\u003csup\u003e0\u003c/sup\u003e; p = 0.0393 vs. St\u003csup\u003eH2O\u003c/sup\u003e; p = 0.0001 vs. St\u003csup\u003eZn\u003c/sup\u003e) and St\u003csup\u003eZn\u0026nbsp;\u003c/sup\u003ethe highest (p = 0.00357 vs. St\u003csup\u003e0\u003c/sup\u003e; p = 0.00148 vs. St\u003csup\u003eH2O\u003c/sup\u003e; p = 0.0001 vs. St\u003csup\u003eCd\u003c/sup\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe found that overall, prolonged metal exposure does not lead to metal tolerance in \u003cem\u003eS. tomentosus.\u003c/em\u003e However, Zn exposure resulted in higher growth under Cd. Such priming effect provides a framework for understanding how fungi can inhabit soils contaminated with multiple metals.\u003c/p\u003e \u003cp\u003eOur results differ from other similar experiments that found metal exposure to induce fungal metal tolerance. For example, exposure to Cd in the yeast \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e led to Cd tolerance (Gorter 2016), and continuous Cd, cesium (Cs) and strontium (Sr) exposure of the filamentous fungus \u003cem\u003eSchizophyllum commune\u003c/em\u003e promoted tolerance to these metals (Traxler 2022). The latter study used metal stressors comparable to ours, with Cd and Zn levels leading to ~\u0026thinsp;50% growth inhibition and reported 10-week exposure to Cd increased Cd tolerance, but exposure to Zn did not lead to Zn tolerance. In addition, Traxler (2022) found that continued exposure to Sr primed tolerance to Cs and Cd, but exposure to Zn did not prime isolates for Cd exposure. Such differences indicate variation in metal exposure effect across fungi, ranging from inducing metal tolerance to not having an effect.\u003c/p\u003e \u003cp\u003eWe did not anticipate Zn exposure to prime \u003cem\u003eS. tomentosus\u003c/em\u003e for Cd tolerance, as there is no evidence for correlation between tolerance to Zn and Cd in \u003cem\u003eSuillus\u003c/em\u003e (Colpaert 2000; Fletcher 2024) (Fletcher 2024). However, organisms rarely encounter single metal stress in the environment and the interaction and overlap of detoxification pathways is not unexpected.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn fact, Cd and Zn toxicity are known to trigger both independent and shared metal detoxification pathways in other eukaryotic cells (Earley 2021). Notably, previous research has shown that the presence of Zn can alleviate Cd toxicity in \u003cem\u003eS. luteus\u003c/em\u003e. When exposed to a combination of these metals, \u003cem\u003eS. luteus\u003c/em\u003e tolerates increased concentrations of Cd. The reverse is not true, and Zn tolerance does not increase in the presence of Cd (Krznaric 2009; Colpaert and Vanassche 1993). It is possible that Zn exposure triggers metal exclusion or detoxification mechanisms which can also protect cells from Cd exposure. This might be due to the role of Zn as a micronutrient, and the existence of specific uptake and homeostasis pathways for Zn in \u003cem\u003eSuillus\u003c/em\u003e species while no such specific pathways exist for Cd (Coninx 2019; Coninx 2017; Ruytinx 2017). In addition, it is possible for Cd to activate different pathways to combat toxicity that do not carry over to Zn toxicity.\u003c/p\u003e \u003cp\u003eZinc and Cd are chemically similar, both falling into group 12 of the periodic table, and forming divalent ions (Zn\u003csup\u003e2+\u003c/sup\u003e and Cd\u003csup\u003e2+\u003c/sup\u003e respectively). Such similarity allows Cd to hijack many of the cellular transporters involved in Zn homeostasis (Yu 2021). Cadmium can then take the place of Zn in enzymes and cause disfunction (Singh 2016). Because Zn is an essential micronutrient, cells have mechanisms that function to maintain optimum levels of Zn for cellular function that also act to limit toxicity. These include transporters that move Zn into and out of the cell as well as into vesicles that maintain homeostasis. In other eukaryotic systems, Zn transporters can be hijacked by Cd and allow this metal to enter the cell. For example, ZIP transporters can allow for Cd uptake into \u003cem\u003eXenopus\u003c/em\u003e cells (Liu 2008), and ZnT proteins have been implicated in the uptake of Cd into rat neurons (Ohana 2006). Zinc transporters have been well described in \u003cem\u003eSuillus luteus\u003c/em\u003e and include ZIP transporters localized in the plasma membrane (\u003cem\u003eSlZRT1\u003c/em\u003e and \u003cem\u003eSlZRT2;\u003c/em\u003e (Coninx 2017; Coninx 2019), and in vacuoles (\u003cem\u003eSlZnT2;\u003c/em\u003e (Ruytinx 2017). Differential expression of Zn transporters has been implicated in the Zn tolerance phenotype, and metal tolerant \u003cem\u003eSuillus\u003c/em\u003e isolates can exclude metals from the cell more efficiently than sensitive isolates, likely through modulated transporter regulation (Fletcher 2024; Colpaert 2005; Krznaric 2009; Smith 2024). It is likely that Cd hijacks \u003cem\u003eSuillus\u003c/em\u003e Zn transporters to enter cells, and these transporters may also be implicated in the Cd toxicity response and transcriptomic analysis of the \u003cem\u003eSuillus\u003c/em\u003e Cd response would allow determining if genes implicated in Cd tolerance overlap with those involved in the Zn response.\u003c/p\u003e \u003cp\u003eIn a broader sense, priming of cells to one stressor through exposure to another stress has been documented in several eukaryotes. For example, exogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e primes rice plants (\u003cem\u003eOryza sativa\u003c/em\u003e) for exposure to Cd, likely from increased antioxidant activity induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e that might prepare cells for the increased ROS under Cd toxicity (Hu 2009). In fungi, osmotic stress is known to prime \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e yeast cells for exposure to heat shock (Trollmo 1988), exposure to Sr confers Cs and Cd tolerance in \u003cem\u003eSch. commune\u003c/em\u003e (Traxler 2022), and riboflavin primes \u003cem\u003eAgaricus bisporus\u003c/em\u003e, another decomposing basidiomycete, to drought tolerance. In the latter, riboflavin might activate similar transcriptional pathways that are also involved in the response to drought (Guhr, Horn, and Weig \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). We are unsure of how exactly Zn exposure triggers increased Cd tolerance in our experiment, though we speculate that exposure to Zn may activate antioxidants that may carry over, priming cells for Cd exposure. Further investigation into transcriptomic and biological pathways involved should help unravel how these two tolerance phenotypes are connected.\u003c/p\u003e \u003cp\u003eWe have no good explanation for that fact that St\u003csup\u003ectr\u003c/sup\u003e was able to grow significantly better in control conditions than the St\u003csup\u003e0\u003c/sup\u003e parent strain, however, this phenomenon was also observed in \u003cem\u003eSch. commune\u003c/em\u003e (Traxler 2022) and might be common in filamentous basidiomycetes. It is also puzzling that St\u003csup\u003eZn\u003c/sup\u003e performed significantly worse in Zn and better than all other strains in the Cd cross-tolerance tests. Given there was no increased tolerance to the primary metal stressors, we did not expect to find any metal cross-tolerance. However, we speculate that a buildup of Zn toxicity within the cells may be behind this response.\u003c/p\u003e \u003cp\u003eWe show that prolonged metal exposure does not lead to fungal metal tolerance, but that exposure to high levels of a micronutrient metal can prime tolerance to a highly toxic metal. These suggest the existence of shared mechanisms for metal homeostasis and provide a framework for explaining how fungi can inhabit soils contaminated with multiple metals. Uncovering the transcriptomic basis of metal priming will unveil if shared metal detoxification pathways underlying this phenomenon.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Science Foundation NSF IOS-PBI (2029168) awarded to S.B.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSB \u0026amp; JF designed study; JF performed research; JF analyzed data; JF \u0026amp; SB wrote the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnahid S, Yaghmaei S, Ghobadinejad Z (2011) Heavy metal tolerance of fungi. \u003cem\u003eSci Iran\u003c/em\u003e 18 (3): 502-508. https://doi.org/10.1016/j.scient.2011.05.015\u003c/li\u003e\n\u003cli\u003eBazzicalupo AL, Ruytinx J, Ke YH, Coninx L, Colpaert JV, Nguyen NH, Vilgalys R, Branco S (2020) Fungal heavy metal adaptation through single nucleotide polymorphisms and copy-number variation. \u003cem\u003eMol Ecol\u003c/em\u003e 29 (21): 4157-4169. https://doi.org/10.1111/mec.15618\u003c/li\u003e\n\u003cli\u003eBranco S, Schauster A, Liao HL, Ruytinx J. 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However, some fungi display high metal tolerance and it is not clear how the trait arises. Here we used an \u003cem\u003ein vitro\u003c/em\u003e experimental evolution experiment to test whether metal exposure induces metal tolerance in \u003cem\u003eSuillus tomentosus\u003c/em\u003e, a widespread ectomycorrhizal fungus with a wide range of tolerance to several metals. We exposed a zinc (Zn) and cadmium (Cd) sensitive \u003cem\u003eS. tomentosus\u003c/em\u003e isolate to both these metals and assessed whether prolonged exposure induced metal tolerance. We found that even though Zn or Cd exposure did not lead to Zn or Cd tolerance, Zn exposure increased \u003cem\u003eS. tomentosus\u003c/em\u003e Cd tolerance. Our findings show metal exposure does not explain the existence of metal tolerance but suggest the existence of shared mechanisms for metal homeostasis and provide a framework for understanding how fungi can inhabit soils contaminated with multiple metals.\u003c/p\u003e","manuscriptTitle":"Zinc exposure primes Suillus tomentosus to tolerate cadmium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-12 03:26:38","doi":"10.21203/rs.3.rs-5357023/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"723dae6a-ab70-44c5-b79f-57b7b7e5ee92","owner":[],"postedDate":"November 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-10T13:08:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-12 03:26:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5357023","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5357023","identity":"rs-5357023","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}