Hydrogen Bonding Controls Excited-State Decay of the Photoactive Yellow Protein Chromophore

International audienceWe have performed excited-state dynamics simulations of a Photoactive Yellow Protein chromophore analogue in water. The results of the simulations demonstrate that in water the chromophore predominantly undergoes single-bond photoisomerization, rather than double-bond photoisomerization. Despite opposite charge distributions in the chromophore, excited-state decay takes place very efficiently from both single- and double-bond twisted minima in water. Radiationless decay is facilitated by ultrafast solvent reorganization, which stabilizes both minima by specific hydrogen bond interactions. Changing the solvent to the slightly more viscous D(2)O leads to an increase of the excited-state lifetime. Together with previous simulations, the present results provide a complete picture of the effect of the protein on the photoisomerization of the chromophore in PYP: the positive guanidinium group of Arg52 favors double-bond isomerization over single-bond isomerization by lowering the barrier for double-bond isomerization, while the hydrogen bonds with Tyr42 and Glu46 enhance deactivation from the double-bond twisted minimum

Genesis of a Fungal Non-Self Recognition Repertoire

Conspecific allorecognition, the ability for an organism to discriminate its own cells from those of another individual of the same species, has been developed by many organisms. Allorecognition specificities are determined by highly polymorphic genes. The processes by which this extreme polymorphism is generated remain largely unknown. Fungi are able to form heterokaryons by fusion of somatic cells, and somatic non self-recognition is controlled by heterokaryon incompatibility loci (het loci). Herein, we have analyzed the evolutionary features of the het-d and het-e fungal allorecognition genes. In these het genes, allorecognition specificity is determined by a polymorphic WD-repeat domain. We found that het-d and het-e belong to a large gene family with 10 members that all share the WD-repeat domain and show that repeats of all members of the family undergo concerted evolution. It follows that repeat units are constantly exchanged both within and between members of the gene family. As a consequence, high mutation supply in the repeat domain is ensured due to the high total copy number of repeats. We then show that in each repeat four residues located at the protein/protein interaction surface of the WD-repeat domain are under positive diversifying selection. Diversification of het-d and het-e is thus ensured by high mutation supply, followed by reshuffling of the repeats and positive selection for favourable variants. We also propose that RIP, a fungal specific hypermutation process acting specifically on repeated sequences might further enhance mutation supply. The combination of these evolutionary mechanisms constitutes an original process for generating extensive polymorphism at loci that require rapid diversification

Systematic Deletion of Homeobox Genes in Podospora anserina Uncovers Their Roles in Shaping the Fruiting Body

Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures

Biological Roles of the Podospora anserina Mitochondrial Lon Protease and the Importance of Its N-Domain

Mitochondria have their own ATP-dependent proteases that maintain the functional state of the organelle. All multicellular eukaryotes, including filamentous fungi, possess the same set of mitochondrial proteases, unlike in unicellular yeasts, where ClpXP, one of the two matricial proteases, is absent. Despite the presence of ClpXP in the filamentous fungus Podospora anserina, deletion of the gene encoding the other matricial protease, PaLon1, leads to lethality at high and low temperatures, indicating that PaLON1 plays a main role in protein quality control. Under normal physiological conditions, the PaLon1 deletion is viable but decreases life span. PaLon1 deletion also leads to defects in two steps during development, ascospore germination and sexual reproduction, which suggests that PaLON1 ensures important regulatory functions during fungal development. Mitochondrial Lon proteases are composed of a central ATPase domain flanked by a large non-catalytic N-domain and a C-terminal protease domain. We found that three mutations in the N-domain of PaLON1 affected fungal life cycle, PaLON1 protein expression and mitochondrial proteolytic activity, which reveals the functional importance of the N-domain of the mitochondrial Lon protease. All PaLon1 mutations affected the C-terminal part of the N-domain. Considering that the C-terminal part is predicted to have an α helical arrangement in which the number, length and position of the helices are conserved with the solved structure of its bacterial homologs, we propose that this all-helical structure participates in Lon substrate interaction

Wood Utilization Is Dependent on Catalase Activities in the Filamentous Fungus Podospora anserina

Catalases are enzymes that play critical roles in protecting cells against the toxic effects of hydrogen peroxide. They are implicated in various physiological and pathological conditions but some of their functions remain unclear. In order to decipher the role(s) of catalases during the life cycle of Podospora anserina, we analyzed the role of the four monofunctional catalases and one bifunctional catalase-peroxidase genes present in its genome. The five genes were deleted and the phenotypes of each single and all multiple mutants were investigated. Intriguingly, although the genes are differently expressed during the life cycle, catalase activity is dispensable during both vegetative growth and sexual reproduction in laboratory conditions. Catalases are also not essential for cellulose or fatty acid assimilation. In contrast, they are strictly required for efficient utilization of more complex biomass like wood shavings by allowing growth in the presence of lignin. The secreted CATB and cytosolic CAT2 are the major catalases implicated in peroxide resistance, while CAT2 is the major player during complex biomass assimilation. Our results suggest that P. anserina produces external H2O2 to assimilate complex biomass and that catalases are necessary to protect the cells during this process. In addition, the phenotypes of strains lacking only one catalase gene suggest that a decrease of catalase activity improves the capacity of the fungus to degrade complex biomass

Effector diversification within compartments of the Leptosphaeria maculans genome affected by Repeat-Induced Point mutations

Fungi are of primary ecological, biotechnological and economic importance. Many fundamental biological processes that are shared by animals and fungi are studied in fungi due to their experimental tractability. Many fungi are pathogens or mutualists and are model systems to analyse effector genes and their mechanisms of diversification. In this study, we report the genome sequence of the phytopathogenic ascomycete Leptosphaeria maculans and characterize its repertoire of protein effectors. The L. maculans genome has an unusual bipartite structure with alternating distinct guanine and cytosine-equilibrated and adenine and thymine (AT)-rich blocks of homogenous nucleotide composition. The AT-rich blocks comprise one-third of the genome and contain effector genes and families of transposable elements, both of which are affected by repeat-induced point mutation, a fungal-specific genome defence mechanism. This genomic environment for effectors promotes rapid sequence diversification and underpins the evolutionary potential of the fungus to adapt rapidly to novel host-derived constraints

Post-genomic approaches to understanding interactions between fungi and their environment

Fungi inhabit every natural and anthropogenic environment on Earth. They have highly varied life-styles including saprobes (using only dead biomass as a nutrient source), pathogens (feeding on living biomass), and symbionts (co-existing with other organisms). These distinctions are not absolute as many species employ several life styles (e.g. saprobe and opportunistic pathogen, saprobe and mycorrhiza). To efficiently survive in these different and often changing environments, fungi need to be able to modify their physiology and in some cases will even modify their local environment. Understanding the interaction between fungi and their environments has been a topic of study for many decades. However, recently these studies have reached a new dimension. The availability of fungal genomes and development of post-genomic technologies for fungi, such as transcriptomics, proteomics and metabolomics, have enabled more detailed studies into this topic resulting in new insights. Based on a Special Interest Group session held during IMC9, this paper provides examples of the recent advances in using (post-)genomic approaches to better understand fungal interactions with their environments

Analysis of Virion Structural Components Reveals Vestiges of the Ancestral Ichnovirus Genome

Many thousands of endoparasitic wasp species are known to inject polydnavirus (PDV) particles into their caterpillar host during oviposition, causing immune and developmental dysfunctions that benefit the wasp larva. PDVs associated with braconid and ichneumonid wasps, bracoviruses and ichnoviruses respectively, both deliver multiple circular dsDNA molecules to the caterpillar. These molecules contain virulence genes but lack core genes typically involved in particle production. This is not completely unexpected given that no PDV replication takes place in the caterpillar. Particle production is confined to the wasp ovary where viral DNAs are generated from proviral copies maintained within the wasp genome. We recently showed that the genes involved in bracovirus particle production reside within the wasp genome and are related to nudiviruses. In the present work we characterized genes involved in ichnovirus particle production by analyzing the components of purified Hyposoter didymator Ichnovirus particles by LC-MS/MS and studying their organization in the wasp genome. Their products are conserved among ichnovirus-associated wasps and constitute a specific set of proteins in the virosphere. Strikingly, these genes are clustered in specialized regions of the wasp genome which are amplified along with proviral DNA during virus particle replication, but are not packaged in the particles. Clearly our results show that ichnoviruses and bracoviruses particles originated from different viral entities, thus providing an example of convergent evolution where two groups of wasps have independently domesticated viruses to deliver genes into their hosts

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology

Array CGH Phylogeny: How accurate are Comparative Genomic Hybridization-based trees?

<p>Abstract</p> <p>Background</p> <p>Array-based Comparative Genomic Hybridization (CGH) data have been used to infer phylogenetic relationships. However, the reliability of array CGH analysis to determine evolutionary relationships has not been well established. In most CGH work, all species and strains are compared to a single reference species, whose genome was used to design the array. In the accompanying work, we critically evaluated CGH-based phylogeny using simulated competitive hybridization data. This work showed that a limited number of conditions, principally the tree topology and placement of the reference taxon in the tree, had a strong effect on the ability to recover the correct tree topology. Here, we add to our simulation study by testing the use of CGH as a phylogenetic tool with experimental CGH data from competitive hybridizations between <it>N. crassa </it>and other <it>Neurospora </it>species. In the discussion, we add to our empirical study of <it>Neurospora </it>by reanalyzing of data from a previous CGH phylogenetic analysis of the yeast <it>sensu stricto </it>complex.</p> <p>Results</p> <p>Array ratio data for <it>Neurospora </it>and related species were normalized with loess, robust spline, and linear ratio based methods, and then used to construct Neighbor-Joining and parsimony trees. These trees were compared to published phylogenetic analyses for <it>Neurospora </it>based on multilocus sequence analysis (MLSA). For the <it>Neurospora </it>dataset, the best combination of methods resulted in recovery of the MLSA tree topology less than half the time. Our reanalysis of a yeast dataset found that trees identical to established phylogeny were recovered only by pruning taxa - including the reference taxon - from the analysis.</p> <p>Conclusion</p> <p>Our results indicate that CGH data can be problematic for phylogenetic analysis. Success fluctuates based on the methods utilized to construct the tree and the taxa included. Selective pruning of the taxa improves the results - an impractical approach for normal phylogenetic analysis. From the more successful methods we make suggestions on the normalization and post-normalization methods that work best in estimating genetic distance between taxa.</p