The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer, cytological staining and 31 P NMR spectroscopy study.

Ectomycorrhizal (ECM) association can improve plant phosphorus (P) nutrition. Polyphosphates (polyP) synthesized in distant fungal cells after P uptake may contribute to P supply from the fungus to the host plant if they are hydrolyzed to phosphate in ECM roots then transferred to the host plant when required. In this study, we addressed this hypothesis for the ECM fungus Hebeloma cylindrosporum grown in vitro and incubated without plant or with host (Pinus pinaster) and non-host (Zea mays) plants, using an experimental system simulating the symbiotic interface. We used 32 P labelling to quantify P accumulation and P efflux and in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and cytological staining to follow the fate of fungal polyP. Phosphate supply triggered a massive P accumulation as newly synthesized long-chain polyP in H. cylindrosporum if previously grown under P-deficient conditions. P efflux from H. cylindrosporum towards the roots was stimulated by both host and non-host plants. However, the host plant enhanced 32 P release compared with the non-host plant and specifically increased the proportion of short-chain polyP in the interacting mycelia. These results support the existence of specific host plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots.

Strategies utilized by trophically diverse fungal species for Pinus sylvestris root colonization.

Physiological changes in host plants in response to the broad spectrum of fungal modes of infection are still not well understood. The current study was conducted to better understand the infection of in vitro cultures of Pinus sylvestris L. seedlings by three trophically diverse fungal species, Fusarium oxysporum E. F. Sm. & Swingle, Trichoderma harzianum Rifai and Hebeloma crustuliniforme (Bull.) Quél. Biochemical methods and microscopy were utilized to determine (i) which factors (apoplastic and cellular pH, reactive oxygen species, glutathione and cell death) play a role in the establishment of pathogenic, saprotrophic and mycorrhizal fungi, and (ii) whether cell death is a common response of conifer seedling tissues when they are exposed to trophically diverse fungi. Establishment of the pathogen, F. oxysporum, was observed more frequently in the meristematic region of root tips than in the elongation zone, which was in contrast to T. harzianum and H. crustuliniforme. Ectomycorrhizal (ECM) hyphae, however, were occasionally observed in the studied root zone and caused small changes in the studied factors. Colonization of the meristematic zone occurred due to host cell death. Independently of the zone, changes in cellular pH resulting in an acidic cytoplasm conditioned the establishment of F. oxysporum. Additionally, cell death was negatively correlated with hydrogen peroxide (H2O2) in roots challenged by a pathogenic fungus. Cell death was the only factor uniquely associated with the colonization of host roots by a saprotrophic fungus. The mechanism may differ, however, between the zones since apoplastic pH was negatively correlated with cell death in the elongation zone, whereas in the meristematic zone, none of the studied factors explained cell death. Colonization by the ECM fungus, H. crustuliniforme, was associated with a decreasing number of cells with acidic apoplast and by production of H2O2 in the elongation zone resulting in cell death. Saprotrophic and ECM fungi had a greater effect on cell acidification in the meristematic zone than the pathogenic fungus.

Different patterns of regulation for the copper and cadmium metallothioneins of the ectomycorrhizal fungus Hebeloma cylindrosporum.

Metallothioneins (MTs) are small cysteine-rich peptides involved in metal homeostasis and detoxification. We have characterized two MT genes, HcMT1 and HcMT2, from the ectomycorrhizal fungus Hebeloma cylindrosporum in this study. Expression of HcMT1 and HcMT2 in H. cylindrosporum under metal stress conditions was studied by competitive reverse transcription-PCR analysis. The full-length cDNAs were used to perform functional complementation in mutant strains of Saccharomyces cerevisiae. As revealed by heterologous complementation assays in yeast, HcMT1 and HcMT2 each encode a functional polypeptide capable of conferring increased tolerance against Cd and Cu, respectively. The expression levels of HcMT1 were observed to be at their maximum at 24 h, and they increased as a function of Cu concentration. HcMT2 was also induced by Cu, but the expression levels were lower than those for HcMT1. The mRNA accumulation of HcMT1 was not influenced by Cd, whereas Cd induced the transcription of HcMT2. Zn, Pb, and Ni did not affect the transcription of HcMT1 or of HcMT2. Southern blot analysis revealed that both of these genes are present as a single copy in H. cylindrosporum. While the promoters of both HcMT1 and HcMT2 contained the standard stress response elements implicated in the metal response, the numbers and varieties of potential regulatory elements were different in these promoters. These results show that ectomycorrhizal fungi encode different MTs and that each of them has a particular pattern of expression, suggesting that they play critical specific roles in improving the survival and growth of ectomycorrhizal trees in ecosystems contaminated by heavy metals.