Pb Stress and Ectomycorrhizas: Strong Protective Proteomic Responses in Poplar Roots Inoculated with Paxillus involutus Isolate and Characterized by Low Root Colonization Intensity

The commonly observed increased heavy metal tolerance of ectomycorrhized plants is usually linked with the protective role of the fungal hyphae covering colonized plant root tips. However, the molecular tolerance mechanisms in heavy metal stressed low-colonized ectormyocrrhizal plants characterized by an ectomycorrhiza-triggered increases in growth are unknown. Here, we examined Populus × canescens microcuttings inoculated with the Paxillus involutus isolate, which triggered an increase in poplar growth despite successful colonization of only 1.9% ± 0.8 of root tips. The analyzed plants, lacking a mantle—a protective fungal biofilter—were grown for 6 weeks in agar medium enriched with 0.75 mM Pb(NO3)2. In minimally colonized ‘bare’ roots, the proteome response to Pb was similar to that in noninoculated plants (e.g., higher abundances of PM- and V-type H+ ATPases and lower abundance of ribosomal proteins). However, the more intensive activation of molecular processes leading to Pb sequestration or redirection of the root metabolic flux into amino acid and Pb chelate (phenolics and citrate) biosynthesis coexisted with lower Pb uptake compared to that in controls. The molecular Pb response of inoculated roots was more intense and effective than that of noninoculated roots in poplars.

Fungal taste for minerals: the ectomycorrhizal fungus Paxillus involutus triggers specific genes when extracting potassium from different silicates

Silicates make up about 90% of the Earth’s crust and constitute the main source of mineral nutrients for microorganisms and plants. Fungi can actively weather silicates to extract nutrients. However, it is unclear whether they are able to obtain the same amounts of nutrients and use the same mechanisms when tapping into different mineral sources. We performed a microcosm experiment using the ectomycorrhizal basidiomycetes Paxillus involutus and the silicates K-vermiculite, muscovite and phlogopite as only potassium sources, as they show a different resistance for the removal of K cations from the mineral structure. A combination of transcriptomic, elemental and SEM analyses showed that different minerals stimulated specific weathering mechanisms and led to a change in fungal genes expression. The differential expression of the fungal genes generated alternative chemical attacks on the minerals, resulting in a tailored dissolution and selective uptake of chemical elements according to the leachability of K from the silicate mineral. The K uptake capacity of the fungus was highest with vermiculite in comparison to growth on phlogopite and muscovite. The findings provide new insights into fungal-mineral interactions that will help to interpret key processes for the homeostasis of soil environments.

Secretion of Iron(III)-Reducing Metabolites during Protein Acquisition by the Ectomycorrhizal Fungus Paxillus involutus

The ectomycorrhizal fungus Paxillus involutus decomposes proteins using a two-step mechanism, including oxidation and proteolysis. Oxidation involves the action of extracellular hydroxyl radicals (•OH) generated by the Fenton reaction. This reaction requires the presence of iron(II). Here, we monitored the speciation of extracellular iron and the secretion of iron(III)-reducing metabolites during the decomposition of proteins by P. involutus. X-ray absorption spectroscopy showed that extracellular iron was mainly present as solid iron(III) phosphates and oxides. Within 1 to 2 days, these compounds were reductively dissolved, and iron(II) complexes were formed, which remained in the medium throughout the incubation. HPLC and mass spectrometry detected five extracellular iron(III)-reducing metabolites. Four of them were also secreted when the fungus grew on a medium containing ammonium as the sole nitrogen source. NMR identified the unique iron(III)-reductant as the diarylcyclopentenone involutin. Involutin was produced from day 2, just before the elevated •OH production, preceding the oxidation of BSA. The other, not yet fully characterized iron(III)-reductants likely participate in the rapid reduction and dissolution of solid iron(III) complexes observed on day one. The production of these metabolites is induced by other environmental cues than for involutin, suggesting that they play a role beyond the Fenton chemistry associated with protein oxidation.

Phosphate availability and ectomycorrhizal symbiosis with Pinus sylvestris have independent effects on the Paxillus involutus transcriptome

Many plant species form symbioses with ectomycorrhizal fungi, which help them forage for limiting nutrients in the soil such as inorganic phosphate (Pi). The transcriptional responses to symbiosis and nutrient-limiting conditions in ectomycorrhizal fungal hyphae, however, are largely unknown. An artificial system was developed to study ectomycorrhizal basidiomycete Paxillus involutus growth in symbiosis with its host tree Pinus sylvestris at different Pi concentrations. RNA-seq analysis was performed on P. involutus hyphae growing under Pi-limiting conditions, either in symbiosis or alone. We show that Pi starvation and ectomycorrhizal symbiosis have an independent effect on the P. involutus transcriptome. Notably, low Pi availability induces expression of newly identified putative high-affinity Pi transporter genes, while reducing the expression of putative organic acid transporters. Additionally, low Pi availability induces a close transcriptional interplay between P and N metabolism. GTP-related signalling was found to have a positive effect in the maintenance of ectomycorrhizal symbiosis, whereas multiple putative cytochrome P450 genes were found to be downregulated, unlike arbuscular mycorrhizal fungi. We provide the first evidence of global transcriptional changes induced by low Pi availability and ectomycorrhizal symbiosis in the hyphae of P. involutus, revealing both similarities and differences with better-characterized arbuscular mycorrhizal fungi.

Metabolome adjustments in ectomycorrhizal Populus × canescens associated with strong promotion of plant growth by Paxillus involutus despite a very low root colonization rate

It is believed that resource exchange, which is responsible for intensified growth of ectomycorrhizal plants, occurs in the fungus–plant interface. However, increasing evidence indicates that such intensified plant growth, especially root growth promotion, may be independent of root colonization. Nevertheless, the molecular adjustments in low-colonized plants remain poorly understood. Here, we analysed the metabolome of Populus × canescens microcuttings characterized by significantly increased growth triggered by inoculation with Paxillus involutus, which successfully colonized only 2.1 ± 0.3% of root tips. High-throughput metabolomic analyses of leaves, stems and roots of Populus × canescens microcuttings supplemented with leaf proteome data were performed to determine ectomycorrhiza-triggered changes in N-, P- and C-compounds. The molecular adjustments were relatively low in low-colonized (M) plants. Nevertheless, the levels of foliar phenolic compounds were significantly increased in M plants. Increases of total soluble carbohydrates, starch as well as P concentrations were also observed in M leaves along with the increased abundance of the majority of glycerophosphocholines detected in M roots. However, compared with the leaves of the non-inoculated controls, M leaves presented lower concentrations of both N and most photosynthesis-related proteins and all individual mono- and disaccharides. In M stems, only a few compounds with different abundances were detected, including a decrease in carbohydrates, which was also detected in M roots. Thus, these results suggest that the growth improvement of low-colonized poplar trees is independent of an increased photosynthesis rate, massively increased resource (C:N) exchange and delivery of most nutrients to leaves. The mechanism responsible for poplar growth promotion remains unknown but may be related to increased P uptake, subtle leaf pigment changes, the abundance of certain photosynthetic proteins, slight increases in stem and root amino acid levels and the increase in flavonoids (increasing the antioxidant capacity in poplar), all of which improve the fitness of low-colonized poplars.

Fatal Immunohaemolysis after the Consumption of the Poison Pax Mushroom: A Focus on the Diagnosis of the Paxillus Syndrome with the Aid of Two Case Reports

This retrospective report focuses on the diagnosis of the Paxillus syndrome, based on two fatal cases of haemolysis following the consumption of Paxillus involutus. These mushrooms are still consumed regularly, despite earlier reports of life-threatening autoimmune haemolytic anaemia. Such cases are nevertheless rare, and thus far no toxin could be identified that causes this unusual form of mushroom poisoning. All these factors contribute to the difficulty in diagnosing the Paxillus syndrome. The following aspects support the diagnosis in the two cases presented here: Both patients consumed the mushroom oftentimes before, yet allegedly without ill effects. Symptoms occurred 2–3 h after the last consumption, exacerbating into circulatory collapse, multiorgan failure, and death. Disseminated intravascular coagulation was identified as cause of death by autopsy of patient 1. Patient 2 died of multiorgan failure, mainly hepatic. Our mycological analyses could identify the consumed mushroom in both cases as Paxillus involutus. Furthermore, we could exclude anticoagulants and several other drugs as trigger for the haemolysis by post-mortem toxicological analysis. However, findings in each of the two cases may have led to the haemolysis, independent of the consumption of Paxillus involutus. Patient 1 carried the anti-erythrocytic antibody, auto-anti-e. Patient 2 contracted chronic hepatitis C years prior to the current incident. Considering the rarity of the Paxillus syndrome, our findings suggest that these patients were particularly susceptible for haemolysis after consuming this mushroom over a prolonged period. Occurrence of the Paxillus syndrome may thus be restricted to regular consumers of Paxillus involutus mushrooms with an existing predisposition for haemolysis.