Four powdery mildew species with catenate conidia infect Galium: molecular and morphological evidence

The Erysiphaceae are a group of obligately biotrophic fungi that cause powdery mildew disease of angiosperms. Due to their inability to be cultured on artificial media, the taxonomy of the Erysiphaceae has generally been based on the morphological characteristics of fresh and herbarium specimens. Thus, several morphological species with wide host ranges have long been maintained in this family, even though they clearly consist of several biological species. Erysiphe galii has been known as a powdery mildew of Galium spp. Recently, the former E. galii var. galii has been reassessed as Neoerysiphe galii and E. galii var. riedliana as Golovinomyces riedlianus, along with a taxonomic revision of the generic concept of the Erysiphaceae. The present study was conducted to evaluate the validity of the taxonomic revision of the two varieties of E. galii. During the course of this study, we found that the Galium powdery mildews consist of at least four different species, viz. Neoerysiphe galii, Golovinomyces orontii, G. riedlianus, and an unknown species collected in Argentina. The latter species is described as a new species, Golovinomyces calceolariae. The three species belonging to Golovinomyces are morphologically very similar to each other, i.e. the discrimination between them is rather difficult. The morphological differences of the three Golovinomyces species of Galium are discussed.     

Amino acid substitutions in the C-terminal AAA+ module of Hsp104 prevent substrate recognition by disrupting oligomerization and cause high temperature inactivation

Hsp104 is an important determinant of thermotolerance in yeast and is an unusual molecular chaperone that specializes in the remodeling of aggregated proteins. The structural requirements for Hsp104-substrate interactions remain unclear. Upon mild heat shock Hsp104 formed cytosolic foci in live cells that indicated co-localization of the chaperone with aggregates of thermally denatured proteins. We generated random amino acid substitutions in the C-terminal 199 amino acid residues of a GFP-Hsp104 fusion protein, and we used a visual screen to identify mutants that remained diffusely distributed immediately after heat shock. Multiple amino acid substitutions were required for loss of heat-inducible redistribution, and this correlated with complete loss of nucleotide-dependent oligomerization. Based on the multiply substituted proteins, several single amino acid substitutions were generated by site-directed mutagenesis. The singly substituted proteins retained the ability to oligomerize and detect substrates. Intriguingly, some derivatives of Hsp104 functioned well in prion propagation and multiple stress tolerance but failed to protect yeast from extreme thermal stress. We demonstrate that these proteins co-aggregate in the presence of other thermolabile proteins during heat treatment both in vitro and in vivo suggesting a novel mechanism for uncoupling the function of Hsp104 in acute severe heat shock from its functions at moderate temperatures.     

Evidence for an unfolding/threading mechanism for protein disaggregation by Saccharomyces cerevisiae Hsp104

Saccharomyces cerevisiae Hsp104, a hexameric member of the Hsp100/Clp subfamily of AAA+ ATPases with two nucleotide binding domains (NBD1 and 2), refolds aggregated proteins in conjunction with Hsp70 molecular chaperones. Hsp104 may act as a "molecular crowbar" to pry aggregates apart and/or may extract proteins from aggregates by unfolding and threading them through the axial channel of the Hsp104 hexamer. Targeting Tyr-662, located in a Gly-Tyr-Val-Gly motif that forms part of the axial channel loop in NBD2, we created conservative (Phe and Trp) and non-conservative (Ala and Lys) amino acid substitutions. Each of these Hsp104 derivatives was comparable to the wild type protein in their ability to hydrolyze ATP, assemble into hexamers, and associate with heat-shock-induced aggregates in living cells. However, only those with conservative substitutions complemented the thermotolerance defect of a Deltahsp104 yeast strain and promoted refolding of aggregated protein in vitro. Monitoring fluorescence from Trp-662 showed that titration of fully assembled molecules with either ATP or ADP progressively quenches fluorescence, suggesting that nucleotide binding determines the position of the loop within the axial channel. A Glu to Lys substitution at residue 645 in the NBD2 axial channel strongly alters the nucleotide-induced change in fluorescence of Trp-662 and specifically impairs in protein refolding. These data establish that the structural integrity of the axial channel through NBD2 is required for Hsp104 function and support the proposal that Hsp104 and ClpB use analogous unfolding/threading mechanisms to promote disaggregation and refolding that other Hsp100s use to promote protein degradation.     

Determination of protein rotational correlation time from NMR relaxation data at various solvent viscosities

An accurate determination of the overall rotation of a protein plays a crucial role in the investigation of its internal motions by NMR. In the present work, an innovative approach to the determination of the protein rotational correlation time _R from the heteronuclear relaxation data is proposed. The approach is based on a joint fit of relaxation data acquired at several viscosities of a protein solution. The method has been tested on computer simulated relaxation data as compared to the traditional _R determination method from T₁/T₂ ratio. The approach has been applied to ribonuclease barnase from Bacillus amyloliquefaciens dissolved in an aqueous solution and deuterated glycerol as a viscous component. The resulting rotational correlation time of 5.56 ± 0.01 ns and other rotational diffusion tensor parameters are in good agreement with those determined from T₁/T₂ ratio.     

Modulation of dual fluorescence in a 3-hydroxyquinolone dye by perturbation of its intramolecular proton transfer with solvent polarity and basicity.

A representative of a new class of dyes with dual fluorescence due to an excited state intramolecular proton transfer (ESIPT) reaction, namely 1-methyl-2-(4-methoxy)phenyl-3-hydroxy-4(1H)-quinolone (QMOM), has been studied in a series of solvents covering a large range of polarity and basicity. A linear dependence of the logarithm of its two bands intensity ratio, log(I(N*)/I(T*)), upon the solvent polarity expressed as a function of the dielectric constant, (epsilon- 1)/(2epsilon + 1), is observed for a series of protic solvents. A linear dependence for log(I(N*)/I(T*)) is also found in aprotic solvents after taking into account the solvent basicity. In contrast, the positions of the absorption and the two emission bands of QMOM do not noticeably depend on the solvent polarity and basicity, indicating relatively small changes in the transition moment of QMOM upon excitation and emission. Time-resolved experiments in acetonitrile, ethyl acetate and dimethylformamide suggest an irreversible ESIPT reaction for this dye. According to the time-resolved data, an increase of solvent basicity results in a dramatic decrease of the ESIPT rate constant, probably due to the disruption of the intramolecular H-bond of the dye by the basic solvent. Due to this new sensor property, 3-hydroxyquinolones are promising candidates for the development of a new generation of environment-sensitive fluorescence dyes for probing interactions of biomolecules.     

Phylogenetic analysis of the suborder plagiorchiata (Platyhelminthes, Digenea) based on partial lsrDNA sequences

The phylogenetic relationships and systematic position of the members of the suborder Plagiorchiata, one of the derived and most diverse groups of Digenea, have always been controversial. Here, we present a phylogeny of this group based on the analysis of partial sequences of the lsrDNA in 28 species representing 13 families of Plagiorchiata, as well as four outgroups. Our results show that the Plagiorchiata, as considered by most authors, is not monophyletic, and that the superfamilies Opecoeloidea, and most probably Dicrocoelioidea and Gorgoderoidea, may have to be removed from this suborder. According to our results, the Plagiorchiata includes only parasites of terrestrial vertebrates. We find the Plagiorchiata to be composed of two well-supported clades which can be ranked as superfamilies: (1) Plagiorchioidea, including the Plagiorchiidae, Haematoloechidae, Telorchiidae, Brachycoeliidae and Leptophallidae; and (2) Microphalloidea containing the Microphallidae, Prosthogonimidae, Lecithodendriidae and Pleurogenidae. The genetic analysis also allowed revision of the position of several taxa of Plagiorchiata, including: (1) a confirmation of the position of the Brachycoeliidae within the Plagiorchiata; (2) a close phylogenetic relationships of Macrodera with Paralepoderma, Leptophallus and Metaleptophallus; (3) the grouping of Opisthioglyphe and Telorchis within a distinct and strongly supported clade; and (4) the placement of Allassogonoporus amphoraeformis within the Pleurogenidae, and not close to Lecithodendriidae. Some systematic changes, corresponding to these results, are proposed.