Paola Ballario

Périgord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis

The Périgord black truffle (Tuber melanosporum Vittad.) and the Piedmont white truffle dominate today’s truffle market. The hypogeous fruiting body of T. melanosporum is a gastronomic delicacy produced by an ectomycorrhizal symbiont endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal Laccaria bicolor, the expansion of gene families may have acted as a ‘symbiosis toolbox’. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of T. melanosporum, which at ∼125u2009megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for ∼58% of the genome. In contrast, this genome only contains ∼7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that T. melanosporum degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both L. bicolor and T. melanosporum, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis—‘the symbiosis toolbox’—evolved along different ways in ascomycetes and basidiomycetes.

The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase Gcn5p

The bromodomain is an ∼110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. We report the crystal structure at 1.9 Å resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15–29 of histone H4 acetylated at the ζ‐N of lysine 16. We show that this bromodomain preferentially binds to peptides containing an N‐acetyl lysine residue. Only residues 16–19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N‐acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water‐mediated H‐bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.

White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein.

The Neurospora crassa blind mutant white collar‐1 (wc‐1) is pleiotropically defective in all blue light‐induced phenomena, establishing a role for the wc‐1 gene product in the signal transduction pathway. We report the cloning of the wc‐1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc‐1 gene product provides a key piece of the blue light signal transduction puzzle. The wc‐1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine‐rich putative transcription activation domain. We demonstrate that the wc‐1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light‐regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc‐1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation.

Role of a white collar-1-white collar-2 complex in blue-light signal transduction

Mutations in either white collar‐1 (wc‐1) or white collar‐2 (wc‐2) lead to a loss of most blue‐light‐induced phenomena in Neurospora crassa. Sequence analysis and in vitro experiments show that wc‐1 and wc‐2 are transcription factors regulating the expression of light‐induced genes. The WC proteins form homo‐ and heterodimers in vitro; this interaction could represent a fundamental step in the control of their activity. We demonstrate in vivo that the WC proteins are assembled in a white collar complex (WCC) and that wc‐1 undergoes a change in mobility due to light‐induced phosphorylation events. The phosphorylation level increases progressively upon light exposure, producing a hyperphosphorylated form that is degraded and apparently replaced in the complex by a newly synthesized wc‐1. wc‐2 is unmodified and also does not change quantitatively in the time frame examined. Light‐dependent phosphorylation of wc‐1 also occurs in a wc‐2 mutant, suggesting that a functional wc‐2 is dispensable for this light‐specific event. These results suggest that light‐induced phosphorylation and degradation of wc‐1 could play a role in the transient expression of blue‐light‐regulated genes. Our findings suggest a mechanism by which wc‐1 and wc‐2 mediate light responses in Neurospora.

Roles in dimerization and blue light photoresponse of the PAS and LOV domains of Neurospora crassa white collar proteins.

The genes coding for white collar‐1 and white collar‐2 (wc‐1 and wc‐2u200a) have been isolated previously, and their products characterized as Zn‐finger transcription factors involved in the control of blue light‐induced genes. Here, we show that the PAS dimerization domains present in both proteins enable the WC‐1 and WC‐2 proteins to dimerize in vitro. Homodimers and heterodimers are formed between the white collar (WC) proteins. A computer analysis of WC‐1 reveals a second domain, called LOV, also identified in NPH1, a putative blue light photoreceptor in plants and conserved in redox‐sensitive proteins and in the phytochromes. The WC‐1 LOV domain does not dimerize with canonical PAS domains, but it is able to self‐dimerize. The isolation of three blind wc‐1 strains, each with a single amino acid substitution only in the LOV domain, reveals that this region is essential for blue light responses in Neurospora. The demonstration that the WC‐1 proteins in these LOV mutants are still able to self‐dimerize suggests that this domain plays an additional role, essential in blue light signal transduction.

White collar proteins: PASsing the light signal in Neurospora crassa

The filamentous fungus Neurospora crassa is an excellent paradigm for the study of blue light signal transduction. The isolation and characterization of the genes for two central regulators of the blue light response, white collar-1 and white collar-2, have begun to shed light on the mechanism of blue light signal transduction in fungi. These proteins are not only proposed to encode blue-light-activated transcription factors but also to be elements of the blue light signal transduction pathway.

A novel histone acetyltransferase inhibitor modulating Gcn5 network: cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl)hydrazone.

Acetylation is a key modulator of genome accessibility through decondensation of the chromatin structure. The balance between acetylation and opposite deacetylation is, in fact, a prerequisite for several cell functions and differentiation. To find modulators of the histone acetyltransferase Gcn5p, we performed a phenotypic screening on a set of newly synthesized molecules derived from thiazole in budding yeast Saccharomyces cerevisiae. We selected compounds that induce growth inhibition in yeast strains deleted in genes encoding known histone acetyltransferases. A novel molecule CPTH2, cyclopentylidene-[4-(4-chlorophenyl)thiazol-2-yl)hydrazone, was selected based on its inhibitory effect on the growth of a gcn5Delta strain. We demonstrated a specific chemical-genetic interaction between CPTH2 and HAT Gcn5p, indicating that CPTH2 inhibits the Gcn5p dependent functional network. CPTH2 inhibited an in vitro HAT reaction, which is reverted by increasing concentration of histone H3. In vivo, it decreased acetylation of bulk histone H3 at the specific H3-AcK14 site. On the whole, our results demonstrate that CPTH2 is a novel HAT inhibitor modulating Gcn5p network in vitro and in vivo.

Genomic distribution of copia-like elements in laboratory stocks of Drosophila melanogaster

The genomic location of five copia-like transposable elements has been compared by the Southern technique in laboratory lines of Oregon R and Canton S strains of Drosophila melanogaster. The results show that extensive rearrangements have taken place in the few decades of separation between the stocks and suggest that transposition occurs at a sizable rate.

Agrobacterium-mediated gene transfer and enhanced green fluorescent protein visualization in the mycorrhizal ascomycete Tuber borchii: a first step towards truffle genetics

Mycorrhizal ascomycetes are ecologically and commercially important fungi that have proved impervious to genetic transformation so far. We report here on the successful transient transformation of Tuber borchii, an ectomycorrhizal ascomycete that colonizes a variety of trees and produces highly prized hypogeous fruitbodies known as “truffles”. A hypervirulent Agrobacterium tumefaciens strain bearing the binary plasmid pBGgHg was used for transformation. The genes for hygromycin resistance and the enhanced green fluorescent protein (EGFP), both under the control of vector-borne promoters, were employed as selection markers. Patches of dark and fluorescent hyphae were observed upon fluorescence microscopic examination of hygromycin-resistant mycelia. The presence of EGFP was confirmed by both confocal microscopy and PCR analysis. The lack in the transformed mycelia of the DNA coding for kanamicin resistance (a trait encoded by a vector-borne gene located outside of the T-DNA region) indicates that Agrobacterium-mediated gene transfer correctly occurred in T. borchii.

Sequence of the GLT1 gene from Saccharomyces cerevisiae reveals the domain structure of yeast glutamate synthase

Glutamate synthase (GOGAT) and glutamine synthetase play a crucial role in ammonium assimilation and glutamate biosynthesis in the yeast Saccharomyces cerevisiae. The GOGAT enzyme has been purified and the GOGAT structural gene (GLT1) has been cloned, showing that this enzyme is a homotrimeric protein with a monomeric size of 199kDa.