Identification and characterization of secreted and pathogenesis-related proteins in <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Interactions between plants and fungal pathogens require a complex interplay at the plant–fungus interface. Extracellular effector proteins are thought to play a crucial role in establishing a successful infection. To identify pathogenesis-related proteins in Ustilago maydis we combined the isolation of secreted proteins using a signal sequence trap approach with bioinformatic analyses and the subsequent characterization of knock-out mutants. We identified 29 secreted proteins including hydrophobins and proteins with a repetitive structure similar to the repellent protein Rep1. Hum3, a protein containing both, a hydrophobin domain and a repetitive Rep1-like region, is shown to be processed during passage through the secretory pathway. While single knock-outs of hydrophobin or repellent-like genes did not affect pathogenicity, we found a strong effect of a double knock-out of hum3 and the repetitive rsp1. Yeast-like growth, mating, aerial hyphae formation and surface hydrophobicity were unaffected in this double mutant. However, pathogenic development in planta stops early after penetration leading to a complete loss of pathogenicity. This indicates that Hum3 and Rsp1 are pathogenicity proteins that share an essential function in early stages of the infection. Our results demonstrate that focusing on secreted proteins is a promising way to discover novel pathogenicity proteins that might be broadly applied to a variety of fungal pathogens.     

Remote access to ACNUC nucleotide and protein sequence databases at PBIL

The ACNUC biological sequence database system provides powerful and fast query and extraction capabilities to a variety of nucleotide and protein sequence databases. The collection of ACNUC databases served by the Pôle Bio-Informatique Lyonnais includes the EMBL, GenBank, RefSeq and UniProt nucleotide and protein sequence databases and a series of other sequence databases that support comparative genomics analyses: HOVERGEN and HOGENOM containing families of homologous protein-coding genes from vertebrate and prokaryotic genomes, respectively; Ensembl and Genome Reviews for analyses of prokaryotic and of selected eukaryotic genomes. This report describes the main features of the ACNUC system and the access to ACNUC databases from any internet-connected computer. Such access was made possible by the definition of a remote ACNUC access protocol and the implementation of Application Programming Interfaces between the C, Python and R languages and this communication protocol. Two retrieval programs for ACNUC databases, Query_win, with a graphical user interface and raa_query, with a command line interface, are also described. Altogether, these bioinformatics tools provide users with either ready-to-use means of querying remote sequence databases through a variety of selection criteria, or a simple way to endow application programs with an extensive access to these databases. Remote access to ACNUC databases is open to all and fully documented (http://pbil.univ-lyon1.fr/databases/acnuc/acnuc.html).     

Phylogeny and biogeography of the genus Ainsliaea (Asteraceae) in the Sino-Japanese region based on nuclear rDNA and plastid DNA sequence data

BACKGROUND AND AIMS: The flora of the Sino-Japanese plant region of eastern Asia is distinctively rich compared with other floristic regions in the world. However, knowledge of its floristic evolution is fairly limited. The genus Ainsliaea is endemic to and distributed throughout the Sino-Japanese region. Its interspecific phylogenetic relationships have not been resolved. The aim is to provide insight into floristic evolution in eastern Asia on the basis of a molecular phylogenetic analysis of Ainsliaea species. METHODS: Cladistic analyses of the sequences of two nuclear (ITS, ETS) and one plastid (ndhF) regions were carried out individually and using the combined data from the three markers. KEY RESULTS: Phylogenetic analyses of three DNA regions confirmed that Ainsliaea is composed of three major clades that correspond to species distributions. Evolution of the three lineages was estimated to have occurred around 1.1 MYA during the early Pleistocene. CONCLUSIONS: The results suggest that Ainsliaea species evolved allopatrically and that the descendants were isolated in the eastern (between SE China and Japan, through Taiwan and the Ryukyu Islands) and western (Yunnan Province and its surrounding areas, including the Himalayas, the temperate region of Southeast Asia, and Sichuan Province) sides of the Sino-Japanese region. The results suggest that two distinct lineages of Ainsliaea have independently evolved in environmentally heterogeneous regions within the Sino-Japanese region. These regions have maintained rich and original floras due to their diverse climates and topographies.     

Root character evolution and systematics in Cranichidinae, Prescottiinae and Spiranthinae (Orchidaceae, Cranichideae)

Background and Aims: Previous studies have suggested that velamen characteristicsare useful as taxonomic markers in Orchidaceae. Members of tribeCranichideae have been assigned to two velamen types constructedbased on combinations of characters such as the presence ofsecondary cell-wall thickenings and pores. However, such charactershave not been analysed on an individual basis in explicit cladisticanalyses. Methods: The micromorphology of roots of 26 species of Cranichideae wasexamined through scanning electron microscopy and light microscopy,scoring the variation and distribution of four characters: numberof velamen cell layers, velamen cell-wall thickenings, presenceand type of tilosomes, and supraendodermal spaces. The lastthree characters were analysed cladistically in combinationwith DNA sequence data of plastid trnK/matK and nuclear ribosomalinternal transcribed spacer (ITS) regions and optimized on theresulting phylogenetic tree. Key Results: Thickenings of velamen cell walls group Prescottiinae with Spiranthinae,whereas tilosomes, documented here for the first time in Cranichideae,provide an unambiguous synapomorphy for subtribe Spiranthinae.Supraendodermal spaces occur mostly in species dwelling in seasonallydry habitats and appear to have evolved three times. Conclusions: Three of the four structural characters assessed are phylogeneticallyinformative, marking monophyletic groups recovered in the combinedmolecular–morphological analysis. This study highlightsthe need for conducting character-based structural studies toovercome analytical shortcomings of the typological approach.     

Evolution of rDNA in Nicotiana allopolyploids: a potential link between rDNA homogenization and epigenetics

Background: The evolution and biology of rDNA have interested biologistsfor many years, in part, because of two intriguing processes:(1) nucleolar dominance and (2) sequence homogenization. Wereview patterns of evolution in rDNA in the angiosperm genusNicotiana to determine consequences of allopolyploidy on theseprocesses. Scope: Allopolyploid species of Nicotiana are ideal for studying rDNAevolution because phylogenetic reconstruction of DNA sequenceshas revealed patterns of species divergence and their parents.From these studies we also know that polyploids formed overwidely different timeframes (thousands to millions of years),enabling comparative and temporal studies of rDNA structure,activity and chromosomal distribution. In addition studies onsynthetic polyploids enable the consequences of de novo polyploidyon rDNA activity to be determined. Conclusions: We propose that rDNA epigenetic expression patterns establishedeven in F₁ hybrids have a material influence on the likely patternsof divergence of rDNA. It is the active rDNA units that arevulnerable to homogenization, which probably acts to reducemutational load across the active array. Those rDNA units thatare epigenetically silenced may be less vulnerable to sequencehomogenization. Selection cannot act on these silenced genes,and they are likely to accumulate mutations and eventually beeliminated from the genome. It is likely that whole silencedarrays will be deleted in polyploids of 1 million years of ageand older.     

Improved sequence-based prediction of protein secondary structures by combining vacuum-ultraviolet circular dichroism spectroscopy with neural network

Synchrotron-radiation vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy can significantly improve the predictive accuracy of the contents and segment numbers of protein secondary structures by extending the short-wavelength limit of the spectra. In the present study, we combined VUVCD spectra down to 160 nm with neural-network (NN) method to improve the sequence-based prediction of protein secondary structures. The secondary structures of 30 target proteins (test set) were assigned into alpha-helices, beta-strands, and others by the DSSP program based on their X-ray crystal structures. Combining the alpha-helix and beta-strand contents estimated from the VUVCD spectra of the target proteins improved the overall sequence-based predictive accuracy Q(3) for three secondary-structure components from 59.5 to 60.7%. Incorporating the position-specific scoring matrix in the NN method improved the predictive accuracy from 70.9 to 72.1% when combining the secondary-structure contents, to 72.5% when combining the numbers of segments, and finally to 74.9% when filtering the VUVCD data. Improvement in the sequence-based prediction of secondary structures was also apparent in two other indices of the overall performance: the correlation coefficient (C) and the segment overlap value (SOV). These results suggest that VUVCD data could enhance the predictive accuracy to over 80% when combined with the currently best sequence-prediction algorithms, greatly expanding the applicability of VUVCD spectroscopy to protein structural biology.