Salicylic acid and its location in response to biotic and abiotic stress

Salicylic acid (SA) is an important signal involved in the activation of plant defence responses against abiotic and biotic stress. SA may derive from the phenylpropanoid pathway or via isochorismate synthase as demonstrated in Nicotiana benthamiana, tomato and Arabidopsis thaliana. The phenylpropanoid pathway as well as isochorismate synthase are localized in the chloroplasts but it remains unknown if the end product SA is in the same organelle. We have studied the localization of SA in A. thaliana using the salicylate hydroxylase (NahG) gene expressed with a chloroplast targeting sequence. Plants expressing NahG in the chloroplasts are unable to accumulate SA induced after pathogen or UV exposure. Our data infer that SA is initially located in the chloroplasts.     

Guided cobamide biosynthesis for heterologous production of reductive dehalogenases

Cobamides (Cbas) are essential cofactors of reductive dehalogenases (RDases) in organohalide-respiring bacteria (OHRB). Changes in the Cba structure can influence RDase function. Here, we report on the cofactor versatility or selectivity of Desulfitobacterium RDases produced either in the native organism or heterologously. The susceptibility of Desulfitobacterium hafniense strain DCB-2 to guided Cba biosynthesis (i.e. incorporation of exogenous Cba lower ligand base precursors) was analysed. Exogenous benzimidazoles, azabenzimidazoles and 4,5-dimethylimidazole were incorporated by the organism into Cbas. When the type of Cba changed, no effect on the turnover rate of the 3-chloro-4-hydroxy-phenylacetate-converting enzyme RdhA6 and the 3,5-dichlorophenol-dehalogenating enzyme RdhA3 was observed. The impact of the amendment of Cba lower ligand precursors on RDase function was also investigated in Shimwellia blattae, the Cba producer used for the heterologous production of Desulfitobacterium RDases. The recombinant tetrachloroethene RDase (PceA_Y51) appeared to be non-selective towards different Cbas. However, the functional production of the 1,2-dichloroethane-dihaloeliminating enzyme (DcaA) of Desulfitobacterium dichloroeliminans was completely prevented in cells producing 5,6-dimethylbenzimidazolyl-Cba, but substantially enhanced in cells that incorporated 5-methoxybenzimidazole into the Cba cofactor. The results of the study indicate the utilization of a range of different Cbas by Desulfitobacterium RDases with selected representatives apparently preferring distinct Cbas.     

Planar Cell Polarity Pathway Regulates Nephrin Endocytosis in Developing Podocytes

The noncanonical Wnt/planar cell polarity (PCP) pathway controls a variety of cell behaviors such as polarized protrusive cell activity, directional cell movement, and oriented cell division and is crucial for the normal development of many tissues. Mutations in the PCP genes cause malformation in multiple organs. Recently, the PCP pathway was shown to control endocytosis of PCP and non-PCP proteins necessary for cell shape remodeling and formation of specific junctional protein complexes. During formation of the renal glomerulus, the glomerular capillary becomes enveloped by highly specialized epithelial cells, podocytes, that display unique architecture and are connected via specialized cell-cell junctions (slit diaphragms) that restrict passage of protein into the urine; podocyte differentiation requires active remodeling of cytoskeleton and junctional protein complexes. We report here that in cultured human podocytes, activation of the PCP pathway significantly stimulates endocytosis of the core slit diaphragm protein, nephrin, via a clathrin/β-arrestin-dependent endocytic route. In contrast, depletion of the PCP protein Vangl2 leads to an increase of nephrin at the cell surface; loss of Vangl2 functions in Looptail mice results in disturbed glomerular maturation. We propose that the PCP pathway contributes to podocyte development by regulating nephrin turnover during junctional remodeling as the cells differentiate.     

Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice

Phospholemman (FXYD1), a 72-amino acid transmembrane protein abundantly expressed in the heart and skeletal muscle, is a major substrate for phosphorylation in the cardiomyocyte sarcolemma. Biochemical, cellular, and electrophysiological studies have suggested a number of possible roles for this protein, including ion channel modulator, taurine-release channel, Na(+)/Ca(2+) exchanger modulator, and Na-K-ATPase-associated subunit. We have generated a phospholemman-deficient mouse. The adult null mice exhibited increased cardiac mass, larger cardiomyocytes, and ejection fractions that were 9% higher by magnetic resonance imaging compared with wild-type animals. Notably, this occurred in the absence of hypertension. Total Na-K-ATPase activity was 50% lower in the phospholemman-deficient hearts. Expression (per unit of membrane protein) of total Na-K-ATPase was only slightly diminished, but expression of the minor alpha(2)-isoform, which has been specifically implicated in the control of contractility, was reduced by 60%. The absence of phospholemman thus results in a complex response, including a surprisingly large reduction in intrinsic Na-K-ATPase activity, changes in Na-K-ATPase isoform expression, increase in ejection fraction, and increase in cardiac mass. We hypothesize that a primary effect of phospholemman is to modulate the Na-K-ATPase and that its reduced activity initiates compensatory responses.     

Bioprecipitation: a feedback cycle linking Earth history,ecosystem dynamics and land use through biological ice nucleators in the atmosphere

Landscapes influence precipitation via the water vapor and energy fluxes they generate. Biologically active landscapes also generate aerosols containing microorganisms, some being capable of catalyzing ice formation and crystal growth in clouds at temperatures near 0 °C. The resulting precipitation is beneficial for the growth of plants and microorganisms. Mounting evidence from observations and numerical simulations support the plausibility of a bioprecipitation feedback cycle involving vegetated landscapes and the microorganisms they host. Furthermore, the evolutionary history of ice nucleation‐active bacteria such as Pseudomonas syringae supports that they have been part of this process on geological time scales since the emergence of land plants. Elucidation of bioprecipitation feedbacks involving landscapes and their microflora could contribute to appraising the impact that modified landscapes have on regional weather and biodiversity, and to avoiding inadvertent, negative consequences of landscape management.     

Selecting an ornamental pepper banker plant for Amblyseius swirskii in floriculture crops

Preference of phytoseiid mite, Amblyseius swirskii (Athias-Henriot) was assessed on four cultivars of ornamental pepper banker plant candidates; Red Missile (RM), Masquerade (MA), Explosive Ember (EE) and Black Pearl (BP) for potential control of pestiferous insects in floriculture. Significant differences in cultivar preference by A. swirskii was observed in choice experiments whether the test was pre- (with pollen) or during bloom. Overall, female mites laid more eggs when pollen was provided as a food source. The number of tuft domatia per cultivar leaf appeared to positively influence host preference in the choice plant tests pre-bloom. In addition, cultivar RM had the highest mean number ± SEM of tuft domatia per leaf (5.1 ± 0.3) and motiles per plant (4.0 ± 1.2), followed by MA, EE and BP. In choice tests on blooming plants, A. swirskii showed preference for both cultivars RM and MA compared to EE. These experiments indicated that the number of tuft domatia and availability of pollen can influence the host preference of A. swirskii for an ornamental pepper banker plant cultivar. Results from this study will help growers, researchers, educators and extension personnel in understanding the plant phenology promoting adoption of suitable banker plants for managing greenhouse and landscape insect pests.     

A residue in MutY important for catalysis identified by photocross-linking and mass spectrometry

MutY is an adenine glycosylase in the base excision repair (BER) superfamily that is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA. MutY contains a [4Fe-4S]2+ cluster that is part of a novel DNA binding motif, referred to as the iron-sulfur cluster loop (FCL) motif. This motif is found in a subset of members of the BER glycosylase superfamily, defining the endonuclease III-like subfamily. Site-specific cross-linking was successfully employed to investigate the DNA-protein interface of MutY. The photoreactive nucleotide 4-thiothymidine (4ST) incorporated adjacent to the OG:A mismatch formed a specific cross-link between the substrate DNA and MutY. The amino acid participating in the cross-linking reaction was characterized by positive ion electrospray ionization (ESI) tandem mass spectrometry. This analysis revealed Arg 143 as the site of modification in MutY. Arg 143 and nearby Arg 147 are conserved throughout the endo III-like subfamily. Replacement of Arg 143 and Arg 147 with alanine by site-directed mutagenesis reduces adenine glycosylase activity of MutY toward OG:A and G:A mispairs. In addition, the R143A and R147A enzymes exhibit a reduced affinity for duplexes containing the substrate analogue 2'-deoxy-2'-fluoroadenosine opposite OG and G. Modeling of MutY bound to DNA using an endonuclease III-DNA complex structure shows that these two conserved arginines are located within close proximity to the DNA backbone. The insight from mass spectrometry experiments combined with functional mutagenesis results indicate that these two amino acids in the [4Fe-4S]2+ cluster-containing subfamily play an important role in recognition of the damaged DNA substrate.     

Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis

Background

Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species.

Methods and Findings

We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity.

Conclusions

The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.