A hormone and proteome approach to picturing the initial metabolic events during Plasmodiophora brassicae infection on Arabidopsis

We report on the early response of Arabidopsis thaliana to the obligate biotrophic pathogen Plasmodiophora brassicae at the hormone and proteome level. Using a CYCB1;1::GUS construct, the re-initiation of infection-related cell division is shown from 4 days after inoculation on. Sensitivity to cytokinins and auxins as well as the endogenous hormone levels are evaluated. Both an enhanced cytokinin gene response and an accumulation of isopentenyl adenine and adenosine precede this re-initiation of cell division, whereas an enhanced auxin gene response is observed from 6 days after inoculation on. The alhl mutant, impaired in the cross talk between ethylene and auxins, is resistant to P. brassicae. A differential protein analysis of infected versus noninfected roots and hypocotyls was performed using two-dimensional gel electrophoresis and quantitative image analysis, coupled to matrix-assisted laser desorption ionization time of flight-time of flight mass spectrometry-based protein identification. Of the visualized proteins, 12% show altered abundance compared with the noninfected plants, including proteins involved in metabolism, cell defense, cell differentiation, and detoxification. Combining the hormone and proteome data, we postulate that, at the very first stages of Plasmodiophora infection, plasmodial-produced cytokinins trigger a local re-initiation of cell division in the root cortex. Consequently, a de novo meristematic area is established that acts as a sink for host-derived indole-3-acetic acid, carbohydrates, nitrogen, and energy to maintain the pathogen and to trigger gall development.     

Crystal structure at 1.45-A resolution of the major allergen endo-beta-1,3-glucanase of banana as a molecular basis for the latex-fruit syndrome

Resolution of the crystal structure of the banana fruit endo-beta-1,3-glucanase by synchrotron X-ray diffraction at 1.45-A resolution revealed that the enzyme possesses the eightfold beta/alpha architecture typical for family 17 glycoside hydrolases. The electronegatively charged catalytic central cleft harbors the two glutamate residues (Glu94 and Glu236) acting as hydrogen donor and nucleophile residue, respectively. Modeling using a beta-1,3 linked glucan trisaccharide as a substrate confirmed that the enzyme readily accommodates a beta-1,3-glycosidic linkage in the slightly curved catalytic groove between the glucose units in positions -2 and -1 because of the particular orientation of residue Tyr33 delimiting subsite -2. The location of Phe177 in the proximity of subsite +1 suggested that the banana glucanase might also cleave beta-1,6-branched glucans. Enzymatic assays using pustulan as a substrate demonstrated that the banana glucanase can also cleave beta-1,6-glucans as was predicted from docking experiments. Similar to many other plant endo-beta-1,3-glucanases, the banana glucanase exhibits allergenic properties because of the occurrence of well-conserved IgE-binding epitopes on the surface of the enzyme. These epitopes might trigger some cross-reactions toward IgE antibodies and thus account for the IgE-binding cross-reactivity frequently reported in patients with the latex-fruit syndrome.     

Swelling-activated K+ efflux and regulatory volume decrease efficiency in human bronchial epithelial cells

This study describes the correlation between cell swelling-induced K+ efflux and volume regulation efficiency evaluated with agents known to modulate ion channel activity and/or intracellular signaling processes in a human bronchial epithelial cell line, 16HBE14o(-1). Cells on permeable filter supports, differentiated into polarized monolayers, were monitored continuously at room temperature for changes in cell height (T(c)), as an index of cell volume, whereas (86)Rb efflux was assessed for K+ channel activity. The sudden reduction in osmolality of both the apical and basolateral perfusates (from 290 to 170 mosmol/kg H(2)O) evoked a rapid increase in cell volume by 35%. Subsequently, the regulatory volume decrease (RVD) restored cell volume almost completely (to 94% of the isosmotic value). The basolateral (86)Rb efflux markedly increased during the hyposmotic shock, from 0.50 +/- 0.03 min(-1) to a peak value of 6.32 +/- 0.07 min(-1), while apical (86)Rb efflux was negligible. Channel blockers, such as GdCl(3) (0.5 mM), quinine (0.5 mM) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, 100 microM), abolished the RVD. The protein tyrosine kinase inhibitors tyrphostin 23 (100 microM) and genistein (150 microM) attenuated the RVD. All agents decreased variably the hyposmosis-induced elevation in (86)Rb efflux, whereas NPPB induced a complete block, suggesting a link between basolateral K(+) and Cl(-1) efflux. Forskolin-mediated activation of adenylyl cyclase stimulated the RVD with a concomitant increase in basolateral (86)Rb efflux. These data suggest that the basolateral extrusion of K+ and Cl(-1) from 16HBE14o(-1) cells in response to cell swelling determines RVD efficiency.     

Localization and in vitro binding studies suggest that the cytoplasmic/nuclear tobacco lectin can interact in situ with high-mannose and complex N-glycans

The possible in vivo interaction of the Nicotiana tabacum agglutinin (Nictaba) with endogenous glycoproteins was corroborated using a combination of confocal/electron microscopy of an EGFP-Nictaba fusion protein expressed in tobacco Bright Yellow-2 (BY-2) cells and biochemical analyses. In vitro binding studies demonstrated that the expressed EGFP-Nictaba possesses carbohydrate-binding activity. Microscopic analyses confirmed the previously reported cytoplasmic/nuclear location of Nictaba in jasmonate-treated tobacco leaves and provided evidence for the involvement of a nuclear localization signal-dependent transport mechanism. In addition, it became evident that the lectin is not uniformly distributed over the nucleus and the cytoplasm of BY-2 cells. Far Western blot analysis of extracts from whole BY-2 cells and purified nuclei revealed that Nictaba interacts in a glycan inhibitable way with numerous proteins including many nuclear proteins. Enzymatic deglycosylation with PNGase F indicated that the observed interaction depends on the presence of N-glycans. Glycan array screening, which showed that Nictaba exhibits a strong affinity for high-mannose and complex N-glycans, provided a reasonable explanation for this observation. The cytoplasmic/nuclear localization of a plant lectin that has a high affinity for high-mannose and complex N-glycans and specifically interacts with conspecific glycoproteins suggests that N-glycosylated proteins might be more important in the cytoplasm and nucleus than is currently believed.     

RNAi in Haemonchus contortus: a potential method for target validation

RNA interference (RNAi) is a method for the functional analysis of specific genes, and is particularly well developed in the free-living nematode Caenorhabditis elegans. There have been several attempts to apply this method to parasitic nematodes. In a recent study undertaken in Haemonchus contortus, Geldhof and colleagues concluded that, although a mechanism for RNAi existed, the methods developed for RNAi in C. elegans had variable efficacy in this parasitic nematode. The potential benefits of RNAi are clear; however, further studies are required to characterize the mechanism present in parasitic nematodes, and to improve culture systems for these nematodes to monitor the long-term effects of RNAi. Only then could RNAi become a reliable assay of gene function.     

Correlation between cell survival,clonogenic activity and micronuclei induction in DMBA-OC-1R cells treated with immunospecific albumin microspheres containing cisplatin and 5-fluorouracil

An ideal chemotherapeutic strategy would be to deliver a high concentration of drug that would be released in sustained small amounts from targeted microspheres to effectively kill only the tumour cells and thus reduce toxicity to normal tissue. Clonogenic and cell survival growth curve assays, as well as the micronucleus assay, were used to determine the feasibility of employing targeted immunomicrospheres in the treatment of cancer. Cells of a rodent ovarian carcinoma cell line, were exposed to cisplatin and 5-fluorouracil, either as free drug or encapsulated in albumin microspheres that were either conjugated to monoclonal antibodies or not. In cell survival growth curve assays, cell survival was reduced to 1.2% of the control when cells were treated with drug-containing immunomicrospheres. 3.2-fold more micronuclei were found in those cells that had been exposed to the drugs in immunomicrospheres than in those subjected to untargeted microspheres. All three assays demonstrated that the targeted immunomicrospheres were more effective in delivering cisplatin and 5-fluorouracil directly to the cells than the unconjugated microspheres, thus suggesting that targeted chemotherapy might be a more effective option in the treatment of cancer.     

Crystal structure of a heterodimer of editosome interaction proteins in complex with two copies of a cross-reacting nanobody

The parasite Trypanosoma brucei, the causative agent of sleeping sickness across sub-Saharan Africa, depends on a remarkable U-insertion/deletion RNA editing process in its mitochondrion. A approximately 20 S multi-protein complex, called the editosome, is an essential machinery for editing pre-mRNA molecules encoding the majority of mitochondrial proteins. Editosomes contain a common core of twelve proteins where six OB-fold interaction proteins, called A1-A6, play a crucial role. Here, we report the structure of two single-strand nucleic acid-binding OB-folds from interaction proteins A3 and A6 that surprisingly, form a heterodimer. Crystal growth required the assistance of an anti-A3 nanobody as a crystallization chaperone. Unexpectedly, this anti-A3 nanobody binds to both A3(OB) and A6, despite only ~40% amino acid sequence identity between the OB-folds of A3 and A6. The A3(OB)-A6 heterodimer buries 35% more surface area than the A6 homodimer. This is attributed mainly to the presence of a conserved Pro-rich loop in A3(OB). The implications of the A3(OB)-A6 heterodimer, and of a dimer of heterodimers observed in the crystals, for the architecture of the editosome are profound, resulting in a proposal of a 'five OB-fold center' in the core of the editosome.     

Understanding the development of roots exposed to contaminants and the potential of plant-associated bacteria for optimization of growth

Background and Scope

Plant responses to the toxic effects of soil contaminants, such as excess metals or organic substances, have been studied mainly at physiological, biochemical and molecular levels, but the influence on root system architecture has received little attention. Nevertheless, the precise position, morphology and extent of roots can influence contaminant uptake. Here, data are discussed that aim to increase the molecular and ecological understanding of the influence of contaminants on root system architecture. Furthermore, the potential of plant-associated bacteria to influence root growth by their growth-promoting and stress-relieving capacities is explored.

Methods

Root growth parameters of Arabidopsis thaliana seedlings grown in vertical agar plates are quantified. Mutants are used in a reverse genetics approach to identify molecular components underlying quantitative changes in root architecture after exposure to excess cadmium, copper or zinc. Plant-associated bacteria are isolated from contaminated environments, genotypically and phenotypically characterized, and used to test plant root growth improvement in the presence of contaminants.

Key Results

The molecular determinants of primary root growth inhibition and effects on lateral root density by cadmium were identified. A vertical split-root system revealed local effects of cadmium and copper on root development. However, systemic effects of zinc exposure on root growth reduced both the avoidance of contaminated areas and colonization of non-contaminated areas. The potential for growth promotion and contaminant degradation of plant-associated bacteria was demonstrated by improved root growth of inoculated plants exposed to 2,4-di-nitro-toluene (DNT) or cadmium.

Conclusions

Knowledge concerning the specific influence of different contaminants on root system architecture and the molecular mechanisms by which this is achieved can be combined with the exploitation of plant-associated bacteria to influence root development and increase plant stress tolerance, which should lead to more optimal root systems for application in phytoremediation or safer biomass production.     

Mechanism of entomotoxicity of the plant lectin from Hippeastrum hybrid (Amaryllis) in Spodoptera littoralis larvae

Plant lectins have received a lot of attention because of their insecticidal properties. When orally administered in artificial diet or in transgenic plants, lectins provoke a wide range of detrimental effects, including alteration of the digestive enzyme machinery, fecundity drop, reduced feeding, changes in oviposition behavior, growth and development inhibition and mortality. Although many studies reported the entomotoxicity of lectins, only a few of them investigated the mode of action by which lectins exert toxicity. In the present paper we have studied for the first time the insecticidal potential of the plant lectin from Hippeastrum hybrid (Amaryllis) (HHA) bulbs against the larvae of the cotton leafworm (Spodoptera littoralis). Bioassays on neonate larvae showed that this mannose-specific lectin affected larval growth, causing a development retardation and larval weight decrease. Using primary cell cultures from S. littoralis midguts and confocal microscopy we have elucidated FITC-HHA binding and internalization mechanisms. We found that HHA did not exert a toxic effect on S. littoralis midgut cells, but HHA interaction with the brush border of midgut cells interfered with normal nutrient absorption in the S. littoralis midgut, thereby affecting normal larval growth in vivo. This study thus confirms the potential of mannose-specific lectins as pest control agents and sheds light on the mechanism underlying lectin entomotoxicity.