Overexpressing the ANR1 MADS-box gene in transgenic plants provides new insights into its role in the nitrate regulation of root development.

The expression of the ANR1 MADS-box gene was manipulated in transgenic plants to investigate its role in the NO(3)(-)-dependent regulation of root development in Arabidopsis thaliana. Constitutive overexpression of ANR1 in roots, achieved using GAL4 enhancer trap lines, resulted in more rapid early seedling development, increased lengths and numbers of lateral roots and increased shoot fresh weight. Based on results obtained with five different enhancer trap lines, the overexpression of ANR1 in the lateral root tips appears to be more important for this phenotype than its level of expression in the developing lateral root primordia. Dexamethasone-mediated induction of ANR1 in lines expressing an ANR1-GR (glucocorticoid receptor) fusion protein stimulated lateral root growth but not primary root growth. Short-term (24 h) dexamethasone treatments led to prolonged stimulation of lateral root growth, whether the lateral roots were already mature or still unemerged at the time of treatment. In split-root experiments, localized application of dexamethasone to half of the root system of an ANR1-GR line elicited a localized increase in both the length and numbers of lateral roots, mimicking the effect of a localized NO(3)(-) treatment. In both types of transgenic line, the root phenotype was strongly dependent on the presence of NO(3)(-), indicating that there are additional components involved in ANR1 function that are NO(3)(-) regulated. The implications of these results for our understanding of ANR1's mode of action in the root response to localized NO(3)(-) are discussed.

Molecular systematics of Keratinophyton: the inclusion of species formerly referred to Chrysosporium and description of four new species.

Four new Keratinophyton species (Ascomycota, Pezizomycotina, Onygenales), K. gollerae, K. lemmensii, K. straussii, and K. wagneri, isolated from soil samples originating from Europe (Austria, Italy, and Slovakia) are described and illustrated. The new taxa are well supported by phylogenetic analysis of the internal transcribed spacer region (ITS) region, the combined data analysis of ITS and the nuclear large subunit (LSU) rDNA, and their phenotype. Based on ITS phylogeny, within the Keratinophyton clade, K. lemmensii is clustered with K. durum, K. hubeiense, K. submersum, and K. siglerae, while K. gollerae, K. straussii and K. wagneri are resolved in a separate terminal cluster. All four new species can be well distinguished from other species in the genus based on phenotype characteristics alone. Ten new combinations are proposed for Chrysosporium species which are resolved in the monophyletic Keratinophyton clade. A new key to the recognized species is provided herein.

Nuclear export of the transcription factor NirA is a regulatory checkpoint for nitrate induction in Aspergillus nidulans.

NirA, the specific transcription factor of the nitrate assimilation pathway of Aspergillus nidulans, accumulates in the nucleus upon induction by nitrate. NirA interacts with the nuclear export factor KapK, which bridges an interaction with a protein of the nucleoporin-like family (NplA). Nitrate induction disrupts the NirA-KapK interaction in vivo, whereas KapK associates with NirA when this protein is exported from the nucleus. A KpaK leptomycin-sensitive mutation leads to inducer-independent NirA nuclear accumulation in the presence of the drug. However, this does not lead to constitutive expression of the genes controlled by NirA. A nirA(c)1 mutation leads to constitutive nuclear localization and activity, remodeling of chromatin, and in vivo binding to a NirA upstream activation sequence. The nirA(c)1 mutation maps in the nuclear export signal (NES) of the NirA protein. The NirA-KapK interaction is nearly abolished in NirA(c)1 and NirA proteins mutated in canonical leucine residues in the NirA NES. The latter do not result in constitutively active NirA protein, which implies that nuclear retention is necessary but not sufficient for NirA activity. The results are consistent with a model in which activation of NirA by nitrate disrupts the interaction of NirA with the NplA/KapK nuclear export complex, thus resulting in nuclear retention, leading to AreA-facilitated DNA binding of the NirA protein and subsequent chromatin remodeling and transcriptional activation.

Saksenaea dorisiae sp. nov., a New Opportunistic Pathogenic Fungus from Europe.

A new species, Saksenaea dorisiae (Mucoromycotina, Mucorales), isolated from a water sample originating from a private well in Manastirica, Petrovac, in the Republic of Serbia (Europe), is described and illustrated. The new taxon is well supported by multilocus phylogenetic analysis that included the internal transcribed spacer (ITS) region, domains D1 and D2 of the 28S rRNA gene (LSU), and translation elongation factor-1α gene (tef-1α), and it is resolved in a clade with S. oblongispora and S. trapezispora. This fungus is characterized by its moderately slow growth at 15 and 37°C, sparse rhizoids, conical-shaped sporangia, and short-cylindrical sporangiospores. Saksenaea dorisiae is a member of the opportunistic pathogenic genus often involved in severe human and animal mucormycoses encountered in tropical and subtropical regions. Despite its sensitivity to several conventional antifungals (terbinafine and ciclopirox), the fungus can potentially evoke clinically challenging infections. This is the first novel taxon of the genus Saksenaea described from the moderately continental climate area of Europe.

N-glycan modification in Aspergillus species.

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional alpha-1,2-mannosidase producing increased amounts of N-glycans of the Man5GlcNAc2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan5GlcNac2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man3GlcNAc2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi.