HcZnT2 is a highly mycorrhiza-induced zinc transporter from Hebeloma cylindrosporum in association with pine.

Zinc (Zn) shortage is a common micronutrient deficiency affecting plants worldwide, while Zn toxicity may occur when this metal is in excess. Ectomycorrhizal (ECM) fungi are known to be able to modulate the transfer of macro- and microelements, among them Zn, to the plant. However, the underlying mechanisms are not well understood. We identified the HcZnT2 gene from the ECM fungus Hebeloma cylindrosporum, encoding a member of the Cation Diffusion Facilitator (CDF) family including Zn transporters, and analyzed its transcriptional regulation, the transport function by yeast complementation experiments, and its subcellular localization using a GFP fusion protein in yeast. HcZnT2 is highly induced during mycorrhization of Pinus pinaster, and upregulated in presence of the host plant root even without any direct contact. However, HcZnT2 is repressed by Zn excess conditions. By functional expression in yeast, our results strongly support the ability of HcZnT2 to transport Zn and, to a lesser extent, manganese. HcZnT2 localization was associated with the endoplasmic reticulum of yeast. Mycorrhizal gene activation at low external Zn suggests that the Zn transporter HcZnT2 might be important for the early establishment of the ECM symbiosis during Zn deficiency, rather than under Zn excess. HcZnT2 arises as an extremely remarkable candidate playing a key role in Zn homeostasis and regulation in ectomycorrhiza.

Comparative transcriptomics provides insights into molecular mechanisms of zinc tolerance in the ectomycorrhizal fungus Suillus luteus.

Zinc (Zn) is a major soil contaminant and high Zn levels can disrupt growth, survival, and reproduction of fungi. Some fungal species evolved Zn tolerance through cell processes mitigating Zn toxicity, although the genes and detailed mechanisms underlying mycorrhizal fungal Zn tolerance remain unexplored. To fill this gap in knowledge, we investigated the gene expression of Zn tolerance in the ectomycorrhizal fungus Suillus luteus. We found that Zn tolerance in this species is mainly a constitutive trait that can also be environmentally dependent. Zinc tolerance in S. luteus is associated with differences in the expression of genes involved in metal exclusion and immobilization, as well as recognition and mitigation of metal-induced oxidative stress. Differentially expressed genes were predicted to be involved in transmembrane transport, metal chelation, oxidoreductase activity, and signal transduction. Some of these genes were previously reported as candidates for S. luteus Zn tolerance, while others are reported here for the first time. Our results contribute to understanding the mechanisms of fungal metal tolerance and pave the way for further research on the role of fungal metal tolerance in mycorrhizal associations.

Antibiotic dose and nutrient availability differentially drive the evolution of antibiotic resistance and persistence.

Effective treatment of bacterial infections proves increasingly challenging due to the emergence of bacterial variants that endure antibiotic exposure. Antibiotic resistance and persistence have been identified as two major bacterial survival mechanisms, and several studies have shown a rapid and strong selection of resistance or persistence mutants under repeated drug treatment. Yet, little is known about the impact of the environmental conditions on resistance and persistence evolution and the potential interplay between both phenotypes. Based on the distinct growth and survival characteristics of resistance and persistence mutants, we hypothesized that the antibiotic dose and availability of nutrients during treatment might play a key role in the evolutionary adaptation to antibiotic stress. To test this hypothesis, we combined high-throughput experimental evolution with a mathematical model of bacterial evolution under intermittent antibiotic exposure. We show that high nutrient levels during antibiotic treatment promote selection of high-level resistance, but that resistance mainly emerges independently of persistence when the antibiotic concentration is sufficiently low. At higher doses, resistance evolution is facilitated by the preceding or concurrent selection of persistence mutants, which ensures survival of populations in harsh conditions. Collectively, our experimental data and mathematical model elucidate the evolutionary routes toward increased bacterial survival under different antibiotic treatment schedules, which is key to designing effective antibiotic therapies.

Population Bottlenecks Strongly Affect the Evolutionary Dynamics of Antibiotic Persistence.

Bacterial persistence is a potential cause of antibiotic therapy failure. Antibiotic-tolerant persisters originate from phenotypic differentiation within a susceptible population, occurring with a frequency that can be altered by mutations. Recent studies have proven that persistence is a highly evolvable trait and, consequently, an important evolutionary strategy of bacterial populations to adapt to high-dose antibiotic therapy. Yet, the factors that govern the evolutionary dynamics of persistence are currently poorly understood. Theoretical studies predict far-reaching effects of bottlenecking on the evolutionary adaption of bacterial populations, but these effects have never been investigated in the context of persistence. Bottlenecking events are frequently encountered by infecting pathogens during host-to-host transmission and antibiotic treatment. In this study, we used a combination of experimental evolution and barcoded knockout libraries to examine how population bottlenecking affects the evolutionary dynamics of persistence. In accordance with existing hypotheses, small bottlenecks were found to restrict the adaptive potential of populations and result in more heterogeneous evolutionary outcomes. Evolutionary trajectories followed in small-bottlenecking regimes additionally suggest that the fitness landscape associated with persistence has a rugged topography, with distinct trajectories toward increased persistence that are accessible to evolving populations. Furthermore, sequencing data of evolved populations and knockout libraries after selection reveal various genes that are potentially involved in persistence, including previously known as well as novel targets. Together, our results do not only provide experimental evidence for evolutionary theories, but also contribute to a better understanding of the environmental and genetic factors that guide bacterial adaptation to antibiotic treatment.