Differential expression of pathogen-responsive genes encoding two types of glycine-rich proteins in barley

Gene-specific probes (3' ends of cDNAs) were obtained from barley cDNAs encoding two types of glycine-rich proteins: HvGRP2, characterized by a cytokeratin-like and a cysteine-rich domain, and HvGRP3, whose main feature was an RNA-binding domain. Expression of genes Hvgrp2 and Hvgrp3, which are present at one (or two) copies per haploid genome, was ubiquitous and gene Hvgrp3 was under light/darkness modulation. Cold treatment increased Hvgrp2 and Hvgrp3 mRNA levels. Methyl jasmonate (10 M) switched off the two genes. Expression of Hvgrp2, but not that of Hvgrp3, was induced by ethylene treatment (100 ppm). Fungal pathogens Erysiphe graminis and Rhynchosporium secalis increased the mRNAs levels of the two genes, both in compatible and in incompatible interactions, while bacterial pathogens did not.     

Synthesis and processing of thionin precursors in developing endosperm from barley (Hordeum vulgare L.)

Thionin is a lysine-rich polypeptide (mol. wt. 5000) which is synthesized in developing barley endosperm from ˜8 days to ˜30 days after anthesis. Two thionin precursors (THP1 and THP2) have been identified using monospecific antibodies (A-TH) prepared against the mature protein. THP1, which is the only polypeptide recognized in vitro by A-TH, is encoded by a 7.5S mRNA obtained from membrane-bound polysomes, and its alkylated derivative has an apparent mol. wt. of 17 800. THP2, which is selected together with mature thionin by A-TH among labelled proteins in vivo, differs from THP1 in apparent mol. wt. (17 400 alkylated) and in electrophoretic mobility at pH 3.2. Both THP1 and THP2 are competed out of the antigen-antibody complex by purified thionin. The conversion of THP2 into thionin, which has been demonstrated in a pulse-chase experiment in vivo, is a post-translational process. As it has not been possible to detect THP1 in vivo it is assumed that it is converted co-translationally into THP2. Final deposition of thionin as an extrinsic membrane protein, possibly associated with the endoplasmic reticulum, has been tentatively established on the basis of subcellular fractionation experiments.     

A dimeric inhibitor or insect alpha-amylase from barley. Cloning of the cDNA and identification of the protein

A cDNA clone, designated pUP-44, whose longest open reading frame codes for a protein that is homologous to the wheat alpha-amylase inhibitors, has been isolated from a library obtained from developing barley endosperm. The deduced sequence for the mature protein, which is 122 residues long, is preceded by a sequence of 30 residues which has the typical features of a signal peptide. A closely corresponding protein, designated BDAI-1, has been isolated from mature endosperm. BDAI-1 behaves as a dimer and inhibits the alpha-amylase from the insect Tenebrio molitor at concentrations that have no effect on salivary or pancreatic alpha-amylases.     

Divergence across diet,time and populations rules out parallel evolution in the gut microbiomes of Trinidadian guppies

Diverse microbial consortia profoundly influence animal biology, necessitating an understanding of microbiome variation in studies of animal adaptation. Yet, little is known about such variability among fish, in spite of their importance in aquatic ecosystems. The Trinidadian guppy, Poecilia reticulata, is an intriguing candidate to test microbiome-related hypotheses on the drivers and consequences of animal adaptation, given the recent parallel origins of a similar ecotype across streams. To assess the relationships between the microbiome and host adaptation, we used 16S rRNA amplicon sequencing to characterize gut bacteria of two guppy ecotypes with known divergence in diet, life history, physiology and morphology collected from low-predation (LP) and high-predation (HP) habitats in four Trinidadian streams. Guts were populated by several recurring, core bacteria that are related to other fish associates and rarely detected in the environment. Although gut communities of lab-reared guppies differed from those in the wild, microbiome divergence between ecotypes from the same stream was evident under identical rearing conditions, suggesting host genetic divergence can affect associations with gut bacteria. In the field, gut communities varied over time, across streams and between ecotypes in a stream-specific manner. This latter finding, along with PICRUSt predictions of metagenome function, argues against strong parallelism of the gut microbiome in association with LP ecotype evolution. Thus, bacteria cannot be invoked in facilitating the heightened reliance of LP guppies on lower-quality diets. We argue that the macroevolutionary microbiome convergence seen across animals with similar diets may be a signature of secondary microbial shifts arising some time after host-driven adaptation.     

Exopolysaccharide from surface-liquid culture of <Emphasis Type="Italic">Clonostachys</Emphasis><Emphasis Type="Italic">rosea</Emphasis> originates from autolysis of the biomass

We describe the purification and chemical characterization of galactomannans that appear both in the biomass and the culture broth during surface-liquid culture of the fungus Clonostachys rosea, a common facultative saprophyte that has potential to be used as a biological control agent against several plant pathogenic fungi, insects and nematodes. The galactomannans from both sources had comparable ratios of Man, Gal and Glc and the similarity were confirmed by ¹H, ¹³C NMR, HMQC, and COSY spectra. We propose that the galactomannan in the culture broth originates from autolysis of the biomass, based not only on the similarity that it has with the galactomannan extracted from the biomass but also on the fact that its concentration increased rapidly after glucose depletion from the medium, when biomass concentration was falling. Polysaccharides from C. rosea have not previously been characterized; we show that the characteristics of the galactomannans are consistent with those that have been reported for other members of the Bionectriaceae, the family to which C. rosea belongs.     

Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum

Background

The identification of genetic changes that confer drug resistance or other phenotypic changes in pathogens can help optimize treatment strategies, support the development of new therapeutic agents, and provide information about the likely function of genes. Elucidating mechanisms of phenotypic drug resistance can also assist in identifying the mode of action of uncharacterized but potent antimalarial compounds identified in high-throughput chemical screening campaigns against Plasmodium falciparum.

Results

Here we show that tiling microarrays can detect de novo a large proportion of the genetic changes that differentiate one genome from another. We show that we detect most single nucleotide polymorphisms or small insertion deletion events and all known copy number variations that distinguish three laboratory isolates using readily accessible methods. We used the approach to discover mutations that occur during the selection process after transfection. We also elucidated a mechanism by which parasites acquire resistance to the antimalarial fosmidomycin, which targets the parasite isoprenoid synthesis pathway. Our microarray-based approach allowed us to attribute in vitro derived fosmidomycin resistance to a copy number variation event in the pfdxr gene, which enables the parasite to overcome fosmidomycin-mediated inhibition of isoprenoid biosynthesis.

Conclusions

We show that newly emerged single nucleotide polymorphisms can readily be detected and that malaria parasites can rapidly acquire gene amplifications in response to in vitro drug pressure. The ability to define comprehensively genetic variability in P. falciparum with a single overnight hybridization creates new opportunities to study parasite evolution and improve the treatment and control of malaria.     

Effects of light on cyanide‐resistant respiration and alternative oxidase function in Arabidopsis seedlings

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy wasteful cyanide (CN)‐resistant respiration and plays a role in optimizing photosynthesis. Although it has been demonstrated that leaf AOX is upregulated after illumination, the in vivo mechanism of AOX upregulation by light and its physiological significance are still unknown. In this report, red light and blue light‐induced AOX (especially AOX1a) expressions were characterized. Phytochromes, phototropins and cryptochromes, all these photoreceptors mediate the light‐response of AOX1a gene. When aox1a mutant seedlings were grown under a high‐light (HL) condition, photobleaching was more evident in the mutant than the wild‐type plants. More reactive oxygen species (ROS) accumulation and inefficient dissipation of chloroplast reducing‐equivalents in aox1a mutant may account for its worse adaptation to HL stress. When etiolated seedlings were exposed to illumination for 4 h, chlorophyll accumulation was largely delayed in aox1a plants. We first suggest that more reduction of the photosynthetic electron transport chain and more accumulation of reducing‐equivalents in the mutant during de‐etiolation might be the main reasons.