A nutrient-regulated, dual localization phospholipase A(2) in the symbiotic fungus Tuber borchii

Important morphogenetic transitions in fungi are triggered by starvation-induced changes in the expression of structural surface proteins. Here, we report that nutrient deprivation causes a strong and reversible up-regulation of TbSP1, a surface-associated, Ca(2+)-dependent phospholipase from the mycorrhizal fungus Tuber borchii. TbSP1 is the first phospholipase A(2) to be described in fungi and identifies a novel class of phospholipid-hydrolyzing enzymes. The TbSP1 phospholipase, which is synthesized initially as a pre-protein, is processed efficiently and secreted during the mycelial phase. The mature protein, however, also localizes to the inner cell wall layer, close to the plasma membrane, in both free-living and symbiosis-engaged hyphae. It thus appears that a dual localization phospholipase A(2) is involved in the adaptation of a symbiotic fungus to conditions of persistent nutritional limitation. Moreover, the fact that TbSP1-related sequences are present in Streptomyces and Neurospora, and not in wholly sequenced non-filamentous microorganisms, points to a general role for TbSP1 phospholipases A(2) in the organization of multicellular filamentous structures in bacteria and fungi.     

Molecular markers for the identification of the ectomycorrhizal fungus Tuber borchii

Five species of white truffle were classified using PCR-based techniques. RAPD (random amplified polymorphic DNA) fingerprints and specific pairs of primers were used. A RAPD fragment was constant in Tuber borchii Vittad. isolates and polymorphic among the other species. Two molecular markers specific for T. borchii were developed from the sequence of the non-polymorphic RAPD fragment and from regions flanking the 5'-3' ends of a truffle gene. These markers were applied in the identification of T. borchii fruit bodies, mycelia and mycorrhizas, allowing us to monitor the development of this fungus during its entire life cycle.     

A high-affinity ammonium transporter from the mycorrhizal ascomycete Tuber borchii

An ammonium transporter cDNA, named TbAMT1, was isolated from the ectomycorrhizal ascomycetous truffle Tuber borchii. The polypeptide encoded by TbAMT1 (52 kDa) functionally complements ammonium uptake-defective yeast mutants and shares sequence similarity with previously characterized ammonium transporters from Saccharomyces (Mep) and Arabidopsis (AtAMT1). Structural characteristics common to the Mep/Amt family and peculiar features of the Tuber transporter have been evidenced by a detailed topological model of the TbAMT1 protein, which predicts 11 transmembrane helices with an N terminus(OUT)/C terminus(IN) orientation. As revealed by uptake/competition experiments conducted in yeast, TbAMT1 is a high-affinity transporter with an apparent K(m) for ammonium of 2 microM. The TbAMT1 mRNA was very slowly, yet specifically upregulated in nitrogen-deprived T. borchii mycelia. Instead, a much faster return to basal expression levels was observed upon resupplementation of either ammonium or nitrate, which thus appear to be utilized as equally effective nitrogen sources by Tuber mycelia.     

Enolase from the ectomycorrhizal fungus Tuber borchii Vittad.: biochemical characterization, molecular cloning, and localization

Enolase from Tuber borchii mycelium was purified to electrophoretical homogeneity using an anion-exchange and a gel permeation chromatography. Furthermore, the corresponding gene (eno-1) was cloned and characterized. The purified enzyme showed a higher affinity for 2-PGA (0.26 mM) with respect to PEP; the stability and activity of enolase were dependent of the divalent cation Mg2+. T. borchii eno-1 has an ORF of 1323 bp coding for a putative protein of 440 amino acids and Southern blotting analysis revealed that the gene is present as a single copy in T. borchii. The enzymatic activity and the mRNA expression level evaluated in mycelia grown either in different carbon sources, in pyruvate or during starvation were the same in all the conditions tested, while biochemical and Northern blotting analyses performed with mycelia at different days of growth showed T. borchii eno-1 regulation in response to the growth phase. Finally, Western blotting analysis demonstrated that enolase is localized only in the cytosolic fraction confirming its important role in glycolysis.     

Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2

Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A₂ (sPLA₂s). TbSP1, the sPLA₂ primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A₂, whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA₂ overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation.     

Identification of differentially expressed cDNA clones in Tilia platyphyllos-Tuber borchii ectomycorrhizae using a differential screening approach

No information is presently available on the molecular mechanisms that control the morphogenesis of the truffle, an ectomycorrhizal ascomycetous fungus of great economic interest not only for forestry and agronomy but also for the organoleptic properties of its hypogeous fruitbodies. A Tilia platyphyllos- Tuber borchii model system was used in order to identify genes induced or up-regulated during symbiosis, since their isolation is a prerequisite for the understanding of the molecular bases of mycorrhizal development and regulation. The strategy applied involved the construction of an ectomycorrhizal cDNA library and random selection of clones, followed by a differential screening procedure to analyse cDNA expression in uninfected roots, ectomycorrhizae and free-living mycelia. The results revealed that many genes - and more plant genes than fungal genes - are expressed at higher levels during the symbiotic phase. Several clones were also investigated in order to understand their biological function. This study represents the first attempt to extend our knowledge of the molecular mechanisms underlying the establishment of ectomycorrhiza in Tuber species.     

Green fluorescent protein expression in the symbiotic basidiomycete fungus Hebeloma cylindrosporum

The symbiotic basidiomycete Hebeloma cylindrosporum is a model fungal species used to study ectomycorrhizal symbiosis at the molecular level. In order to have a vital marker, we developed a green fluorescent protein (GFP) reporter system efficiently expressed in H. cylindrosporum using the sgfp coding region bordered by two introns fused to the saprophytic basidiomycete Coprinopsis cinerea cgl1 promoter. Expression of this reporter system was tested under different environmental conditions in two transformants, and glucose was shown to repress gfp expression. Such a reporter system will be used in plant-fungus interaction to evaluate sugar supply by the plant to the compatible mycorrhizal symbiont and to compare the expression of various genes of interest in the free-living mycelia, in the symbiotic (mycorrhizas) and the reproductive (fruit bodies) structures formed by H. cylindrosporum.     

A multiple-plant module for aseptic nutritional studies and the synthesis of mycorrhizas

Summary A general design of seedling chambers, set into sections controlling root temperature and supplying nutrients and within which aseptic conditions are maintained by membrane filters, is described. The designed module was used to study the formation of mycorrhizas and their effect on seedling growth at various levels of nutrient supply and root temperature.     

Rapid typing of truffle mycorrhizal roots by PCR amplification of the ribosomal DNA spacers

DNA analyses were developed to type mycorrhizas of two Tuber species of commercial value (T. melanosporum, T. borchii) and a competitive fungus (Sphaerosporella brunnea) which forms ectomycorrhizas with plants usually considered hosts for truffles. Polymerase chain reaction (PCR) amplification of DNA isolated from fruitbodies, mycelia, mycorrhizas and leaves of host plants, was performed with a primer pair for an internal transcribed spacer ITS1-4. ITS amplification followed by restriction fragment length polymorphism (RFLP) analysis of the amplified products clearly distinguished the two Tuber species at the fruitbody, mycorrhiza and mycelium levels. Accepted: 6 September 1996     

Two genetically related strains of<Emphasis Type="Italic"> Tuber borchii</Emphasis> produce<Emphasis Type="Italic"> Tilia</Emphasis> mycorrhizas with different morphological traits

Two genetically related strains of Tuber borchii Vittad. (1BO and 43BO) produce mycorrhizas with Tilia platyphyllos Scop. with a different degree of efficiency. The aim of this work was to characterize the morphology of the fungal symbiotic structures in order to examine potential relationships between the anatomical traits of the mycorrhiza, the mycorrhizal capacities of the fungal strains and their effect on the host plants. Some morphological features of mantle hyphae (small size, intense staining, vacuolization, abundance of mitochondria) led to a mantle with morphological features that were isolate-specific. There were unexpected differences, at least under our experimental conditions: 1BO strain mantle cells were larger, less reactive to staining, more highly vacuolated and poorer in mitochondria than those of 43BO. These features were found throughout the mantle in 1BO, while the inner mantle hyphae of 43BO were significantly smaller and more intensely stained than the outer cells. In the 43BO strain there was a positive relation between these features and higher infectivity (evaluated as percentage of mycorrhizal tips) as well as a slightly more effective stimulation of plant growth. These observations suggest that genetically related truffle strains produce mycorrhizas with different morphologies, which may be related to a more efficient response of the host plant to inoculation.     

Differential expression of chitin synthase III and IV mRNAs in ascomata of Tuber borchii Vittad

A full-length genomic clone encoding a class III chitin synthase (CHS) and one DNA fragment corresponding to a class IV CHS were isolated from the mycorrhizal fungus Tuber borchii and used for an extensive expression analysis, together with a previously identified DNA fragment corresponding to a class II CHS. All three Chs mRNAs are constitutively expressed in vegetative mycelia, regardless of the age, mode of growth, and proliferation capacity of the hyphae. A strikingly different situation was observed in ascomata, where class III and IV, but not class II, mRNAs are differentially expressed in a maturation stage-dependent manner and accumulate, respectively, in sporogenic and vegetative hyphae. These data, the first on the expression of distinct Chs mRNAs during fruitbody development, point to the different cellular roles that can be played by distinct chitin synthases in the differentiation of spores of sexual origin (CHS III) or in ascoma enlargement promoted by the growth of vegetative hyphae (CHS IV).     

Diversity of culturable bacterial populations associated to Tuber borchii ectomycorrhizas and their activity on T. borchii mycelial growth

Isolation and physiological and molecular characterisation of culturable bacterial strains belonging to actinomycetes, pseudomonads and aerobic spore-forming bacteria were carried out on mycorrhizal root tips of Quercus robur var. peduncolata infected by Tuber borchii. Cellular density of the three bacterial groups in ectomycorrhizal root tips was estimated to be 1.3+/-0.11 x 10(6) cfu g(-1) dry weight for total heterotrophic bacteria and 1.08+/-0.6 x 10(5) (mean+/-S.E.), 1.3+/-0.3 x 10(5) and 1.4+/-0.2 x 10(5) cfu g(-1) dry weight for pseudomonads, actinomycetes and spore-forming bacteria respectively. Identification of pseudomonads by the Biolog system indicated, besides the most represented species Pseudomonas fluorescens (biotypes B, F and G), the occurrence of strains belonging to Pseudomonas corrugata. Amplified ribosomal DNA restriction analysis of actinomycetes and spore formers revealed at least three and six different groups of patterns, respectively. Many bacterial isolates were able to induce variations in growth rates of T. borchii mycelium; among these, 101 strains showed antifungal activity, whereas 17 isolates, belonging to spore formers, were able to increase mycelial growth up to 78% when compared to uninoculated mycelial growth. The potential role of these populations in the development and establishment of mycorrhizas is discussed.     

Opposing developmental functions of Agrocybe aegerita galectin (AAL) during mycelia differentiation

Mycelia of basidiomycetes differentiating into fruiting body is a controlled developmental process, however the underlying molecular mechanism remains unknown. In previous work, a novel fungal Agrocybe aegerita galectin (AAL) was isolated from A. aegerita in our laboratory. AAL was shown to promote mycelial differentiation in A. aegerita and Auricularia polytricha, indicating that AAL might function as a conserved fruiting initiator during basidiomycete mycelia development. In the current work, we investigate the role of AAL in mycelia differentiation and fruiting body formation. First, the expression and localization of AAL in mycelia, primordium and fruiting body were assessed by Western blotting and immunohistochemistry. AAL was found to be ubiquitously expressed in the primordium and fruiting body but not in the mycelia. AAL facilitated mycelia congregation and promoted fruiting body production when AAL was applied on mycelia. At the same time, when AAL was spread on potato dextrose agar (PDA) medium prior to mycelia inoculation, mycelia exhibited slowed growth rates, resulting in mycelia cords formation and inhibition of fruiting body formation. The 5' regulatory sequence of aal was cloned by 'genome walking'. Here, we show that aal lack introns in the coding region and the upstream 740 bp sequence was characterized by the existence of core promoter elements, which included: two CCAAT boxes (-535/-280), a GC box (-145), a TATA box (-30) and a fungal leader intron within the 5' UTR. The identification of regulatory expression elements may provide an explanation to the stage-specific and high-level expression of aal during fruiting development.     

Molecular characterisation of a Tuber borchii Smt3 gene.

Tbsmt3 gene from the ectomychorrizal fungus Tuber borchii was identified and sequenced. The Tbsmt3 gene encodes for a protein sharing significant amino acid homology with the yeast SMT3, a ubiquitin-like protein that is post-translationally attached to several proteins involved in many cellular processes. The comparison between the Tbsmt3 genomic and cDNA sequences established that the encoding sequence is interrupted by an intron of 312 bp. Southern blot analysis revealed only one copy of Tbsmt3 gene in the T. borchii genome. Tbsmt3 is expressed in all phases of T. borchii life cycle: mycelium, ectomycorrhiza and ascoma. However, the Tbsmt3 mRNA decreased during fruit body maturation.     

Regulation of photoassimilate allocation in Pinus sylvestris seedlings by the nutritional status of the mycorrhizal fungus Suillus variegatus

Summary Seedlings of Pinus sylvestris L. were grown on defined nutrient solutions on carbon filters, either sterile or infected with the basidiomycete Suillus variegatus O. Kuntze. After mycorrhizas were established, the shoot of the seedling was subjected to ¹⁴CO₂ photosynthesis. ¹⁴C-labelled photoassimilates were translocated to both mycorrhizas and non-infected root tips. Microautoradiographs of mycorrhizas indicated that omission of external sugars did not affect the formation of mycorrhizas; ¹⁴C-photoassimilates were supplied to cortex, Hartig net and the mantle of hyphae surrounding the rootlet. Nutrient solution containing sugars (malt extract, glucose) enhanced the growth of the fungus. As a consequence, ¹⁴C-photoassimilates from the seedling were accumulated in the mantle, but defence mechanisms of the host cannot be excluded. When soluble nitrogen was omitted from the nutrient solution and replaced by chitin precipitated on the filter-bearing mycorrhizas, the fungus appeared strongly labelled in the mantle, where the fungal chitinase provided soluble nitrogen compounds, necessary for the growth of the seedling.