454 Genome sequencing of Pseudoperonospora cubensis reveals effector proteins with a QXLR translocation motif

Pseudoperonospora cubensis is a biotrophic oomycete pathogen that causes downy mildew of cucurbits, a devastating foliar disease threatening cucurbit production worldwide. We sequenced P. cubensis genomic DNA using 454 pyrosequencing and obtained random genomic sequences covering approximately 14% of the genome, thus providing the first set of useful genomic sequence information for P. cubensis. Using bioinformatics approaches, we identified 32 putative RXLR effector proteins. Interestingly, we also identified 29 secreted peptides with high similarity to RXLR effectors at the N-terminal translocation domain, yet containing an R-to-Q substitution in the first residue of the translocation motif. Among these, a family of QXLR-containing proteins, designated as PcQNE, was confirmed to have a functional signal peptide and was further characterized as being localized in the plant nucleus. Internalization of secreted PcQNE into plant cells requires the QXLR-EER motif. This family has a large number of near-identical copies within the P. cubensis genome, is under diversifying selection at the C-terminal domain, and is upregulated during infection of plants, all of which are common characteristics of characterized oomycete effectors. Taken together, the data suggest that PcQNE are bona fide effector proteins with a QXLR translocation motif, and QXLR effectors are prevalent in P. cubensis. Furthermore, the massive duplication of PcQNE suggests that they might play pivotal roles in pathogen fitness and pathogenicity.     

Tricyclic aminopyrimidine histamine H4 receptor antagonists

This report discloses the development of a series of tricyclic histamine H(4) receptor antagonists. Starting with a low nanomolar benzofuranopyrimidine HTS hit devoid of pharmaceutically acceptable properties, we navigated issues with metabolism and solubility to furnish a potent, stable and water soluble tricyclic histamine H(4) receptor antagonist with desirable physiochemical parameters which demonstrated efficacy a mouse ova model.     

N5-phosphonoacetyl-L-ornithine (PALO): a convenient synthesis and investigation of influence on regulation of amino acid biosynthetic genes in Saccharomyces cerevisiae

A scalable four-step synthesis of the ornithine transcarbamylase inhibitor N⁵-phosphonoacetyl-l-ornithine (PALO) is achieved through boroxazolidinone protection of ornithine. Investigations in the model organism Saccharomyces cerevisiae found that, in contrast to a previous report, PALO did not influence growth rate or expression of genes involved in arginine metabolism.     

Identification of 5,6-substituted 4-aminothieno[2,3-d]pyrimidines as LIMK1 inhibitors

4-Aminobenzothieno[3,2-d]pyrimidines were previously identified in a high throughput screening campaign as LIMK1 inhibitors. Scaffold reversal led to the identification of a series of simple 5,6-substituted 4-aminothieno[2,3-d]pyrimidines with low micromolar inhibition of LIMK1.     

Pyrazolopyridine inhibitors of B-RafV600E. Part 2: structure-activity relationships

Structure-activity relationships around a novel series of B-Raf(V600E) inhibitors are reported. The enzymatic and cellular potencies of inhibitors derived from two related hinge-binding groups were compared and3-methoxypyrazolopyridine proved to be superior. The 3-alkoxy group of lead B-Raf(V600E) inhibitor 1 was extended and minimally affected potency. The propyl sulfonamide tail of compound 1, which occupies the small lipophilic pocket formed by an outward shift of the αC-helix, was expanded to a series of arylsulfonamides. X-ray crystallography revealed that this lipophilic pocket unexpectedly enlarges to accommodate the bulkier aryl group.     

The role of NDR1 in pathogen perception and plant defense signaling

The biochemical and cellular function of NDR1 in plant immunity and defense signaling has long remained elusive. Herein, we describe a novel role for NDR1 in both pathogen perception and plant defense signaling, elucidated by exploring a broader, physiological role for NDR1 in general stress responses and cell wall adhesion. Based on our predictive homology modeling, coupled with a structure-function approach, we found that NDR1 shares a striking similarity to mammalian integrins, well-characterized for their role in mediating the interaction between the extracellular matrix and stress signaling. ndr1-1 mutant plants exhibit higher electrolyte leakage following pathogen infection, compared to wild type Col-0. In addition, we observed an altered plasmolysis phenotype, supporting a role for NDR1 in maintaining cell wall-plasma membrane adhesions through mediating fluid loss under stress.Key words: NDR1, integrin, RGD, plant defenseNON-RACE SPECIFIC DISEASE RESISTANCE-1(NDR1) was first identified as playing an essential role in plant defense activation following the perception of the bacterial phytopathogen Pseudomonas syringae pv tomato DC3000.¹ Over the past decade, the role of NDR1 in plant defense signaling has emerged through the elucidation of the genetic interactions in which NDR1 participates. This includes the activation of the coiled-coil nucleotide binding site leucine rich repeat (CC-NB-LRR) family of resistance (R) proteins. In parallel, the function of ENHANCED DISEASE SUCEPTIBILITY-1 (EDS1 ²) has been extensively described through its genetic and biochemical relationship with the activation of Toll-Interleukin Receptor (TIR)-NB-LRR R-proteins.³ As central activators required for defense signaling, NDR1 and EDS1 represent critical nodes required for the activation of host resistance.     

A physical map for the Amborella trichopoda genome sheds light on the evolution of angiosperm genome structure

Background

Recent phylogenetic analyses have identified Amborella trichopoda, an understory tree species endemic to the forests of New Caledonia, as sister to a clade including all other known flowering plant species. The Amborella genome is a unique reference for understanding the evolution of angiosperm genomes because it can serve as an outgroup to root comparative analyses. A physical map, BAC end sequences and sample shotgun sequences provide a first view of the 870 Mbp Amborella genome.

Results

Analysis of Amborella BAC ends sequenced from each contig suggests that the density of long terminal repeat retrotransposons is negatively correlated with that of protein coding genes. Syntenic, presumably ancestral, gene blocks were identified in comparisons of the Amborella BAC contigs and the sequenced Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa genomes. Parsimony mapping of the loss of synteny corroborates previous analyses suggesting that the rate of structural change has been more rapid on lineages leading to Arabidopsis and Oryza compared with lineages leading to Populus and Vitis. The gamma paleohexiploidy event identified in the Arabidopsis, Populus and Vitis genomes is shown to have occurred after the divergence of all other known angiosperms from the lineage leading to Amborella.

Conclusions

When placed in the context of a physical map, BAC end sequences representing just 5.4% of the Amborella genome have facilitated reconstruction of gene blocks that existed in the last common ancestor of all flowering plants. The Amborella genome is an invaluable reference for inferences concerning the ancestral angiosperm and subsequent genome evolution.     

Secreted amyloid precursor protein β and secreted amyloid precursor protein α induce axon outgrowth in vitro through Egr1 signaling pathway

Background

sAPPα released after α secretase cleavage of Amyloid Precursor Protein (APP) has several functions including the stimulation of neurite outgrowth although detailed morphometric analysis has not been done. Two domains involved in this function have been described and are present in sAPPβ released at the first step of amyloid peptide cleavage, raising the possibility that sAPPβ could also stimulate neurite outgrowth. We investigated the morphological effects of sAPPα and sAPPβ on primary neurons and identified a key signaling event required for the changes observed.

Methodology/Principal Findings

Final concentrations of 50 to 150 nM bacterial recombinant sAPPα or sAPPβ added to primary neuronal cultures after 1 day in vitro decreased cell adhesion 24 hours later and primary dendrite length 96 hours later. 150 nM sAPPα and sAPPβ induced a similar increase of axon outgrowth, although this increase was already significant at 100 nM sAPPα. These morphological changes induced by sAPPs were also observed when added to differentiated neurons at 5 days in vitro. Real time PCR and immunocytochemistry showed that sAPPα and sAPPβ stimulated Egr1 expression downstream of MAPK/ERK activation. Furthermore, in primary neurons from Egr1 −/− mice, sAPPs affected dendritic length but did not induce any increase of axon length.

Conclusion/Significance

sAPPα and sAPPβ decrease cell adhesion and increase axon elongation. These morphological changes are similar to what has been observed in response to heparan sulfate. The sAPPα/sAPPβ stimulated increase in axon growth requires Egr1 signaling. These data suggest that sAPPβ is not deleterious per se. Since sAPPβ and sAPPα are present in the embryonic brain, these two APP metabolites might play a role in axon outgrowth during development and in response to brain damage.