Comparative Live-Cell Imaging Analyses of SPA-2, BUD-6 and BNI-1 in Neurospora crassa Reveal Novel Features of the Filamentous Fungal Polarisome

A key multiprotein complex involved in regulating the actin cytoskeleton and secretory machinery required for polarized growth in fungi, is the polarisome. Recognized core constituents in budding yeast are the proteins Spa2, Pea2, Aip3/Bud6, and the key effector Bni1. Multicellular fungi display a more complex polarized morphogenesis than yeasts, suggesting that the filamentous fungal polarisome might fulfill additional functions. In this study, we compared the subcellular organization and dynamics of the putative polarisome components BUD-6 and BNI-1 with those of the bona fide polarisome marker SPA-2 at various developmental stages of Neurospora crassa. All three proteins exhibited a yeast-like polarisome configuration during polarized germ tube growth, cell fusion, septal pore plugging and tip repolarization. However, the localization patterns of all three proteins showed spatiotemporally distinct characteristics during the establishment of new polar axes, septum formation and cytokinesis, and maintained hyphal tip growth. Most notably, in vegetative hyphal tips BUD-6 accumulated as a subapical cloud excluded from the Spitzenkörper (Spk), whereas BNI-1 and SPA-2 partially colocalized with the Spk and the tip apex. Novel roles during septal plugging and cytokinesis, connected to the reinitiation of tip growth upon physical injury and conidial maturation, were identified for BUD-6 and BNI-1, respectively. Phenotypic analyses of gene deletion mutants revealed additional functions for BUD-6 and BNI-1 in cell fusion regulation, and the maintenance of Spk integrity. Considered together, our findings reveal novel polarisome-independent functions of BUD-6 and BNI-1 in Neurospora, but also suggest that all three proteins cooperate at plugged septal pores, and their complex arrangement within the apical dome of mature hypha might represent a novel aspect of filamentous fungal polarisome architecture

Dinucleotide controlled null models for comparative RNA gene prediction

<p>Abstract</p> <p>Background</p> <p>Comparative prediction of RNA structures can be used to identify functional noncoding RNAs in genomic screens. It was shown recently by Babak <it>et al</it>. [BMC Bioinformatics. 8:33] that RNA gene prediction programs can be biased by the genomic dinucleotide content, in particular those programs using a thermodynamic folding model including stacking energies. As a consequence, there is need for dinucleotide-preserving control strategies to assess the significance of such predictions. While there have been randomization algorithms for single sequences for many years, the problem has remained challenging for multiple alignments and there is currently no algorithm available.</p> <p>Results</p> <p>We present a program called SISSIz that simulates multiple alignments of a given average dinucleotide content. Meeting additional requirements of an accurate null model, the randomized alignments are on average of the same sequence diversity and preserve local conservation and gap patterns. We make use of a phylogenetic substitution model that includes overlapping dependencies and site-specific rates. Using fast heuristics and a distance based approach, a tree is estimated under this model which is used to guide the simulations. The new algorithm is tested on vertebrate genomic alignments and the effect on RNA structure predictions is studied. In addition, we directly combined the new null model with the RNAalifold consensus folding algorithm giving a new variant of a thermodynamic structure based RNA gene finding program that is not biased by the dinucleotide content.</p> <p>Conclusion</p> <p>SISSIz implements an efficient algorithm to randomize multiple alignments preserving dinucleotide content. It can be used to get more accurate estimates of false positive rates of existing programs, to produce negative controls for the training of machine learning based programs, or as standalone RNA gene finding program. Other applications in comparative genomics that require randomization of multiple alignments can be considered.</p> <p>Availability</p> <p>SISSIz is available as open source C code that can be compiled for every major platform and downloaded here: <url>http://sourceforge.net/projects/sissiz</url>.</p

A Sensing Role of the Glutamine Synthetase in the Nitrogen Regulation Network in Fusarium fujikuroi

Contains fulltext : 125173.pdf (publisher's version ) (Open Access)In the plant pathogenic ascomycete Fusarium fujikuroi the synthesis of several economically important secondary metabolites (SM) depends on the nitrogen status of the cells. Of these SMs, gibberellin and bikaverin synthesis is subject to nitrogen catabolite repression (NCR) and is therefore only executed under nitrogen starvation conditions. How the signal of available nitrogen quantity and quality is sensed and transmitted to transcription factors is largely unknown. Earlier work revealed an essential regulatory role of the glutamine synthetase (GS) in the nitrogen regulation network and secondary metabolism as its deletion resulted in total loss of SM gene expression. Here we present extensive gene regulation studies of the wild type, the Deltagln1 mutant and complementation strains of the gln1 deletion mutant expressing heterologous GS-encoding genes of prokaryotic and eukaryotic origin or 14 different F. fujikuroi gln1 copies with site-directed mutations. All strains were grown under different nitrogen conditions and characterized regarding growth, expression of NCR-responsive genes and biosynthesis of SM. We provide evidence for distinct roles of the GS in sensing and transducing the signals to NCR-responsive genes. Three site directed mutations partially restored secondary metabolism and GS-dependent gene expression, but not glutamine formation, demonstrating for the first time that the catalytic and regulatory roles of GS can be separated. The distinct mutant phenotypes show that the GS (1) participates in NH4 (+)-sensing and transducing the signal towards NCR-responsive transcription factors and their subsequent target genes; (2) affects carbon catabolism and (3) activates the expression of a distinct set of non-NCR GS-dependent genes. These novel insights into the regulatory role of the GS provide fascinating perspectives for elucidating regulatory roles of GS proteins of different organism in general