Arabidopsis RECEPTOR-LIKE PROTEIN30 and Receptor-Like Kinase SUPPRESSOR OF BIR1-1/EVERSHED Mediate Innate Immunity to Necrotrophic Fungi

Effective plant defense strategies rely in part on the perception of non-self determinants, so-called microbe-associated molecular patterns (MAMPs), by transmembrane pattern recognition receptors leading to MAMP-triggered immunity. Plant resistance against necrotrophic pathogens with a broad host range is complex and yet not well understood. Particularly, it is unclear if resistance to necrotrophs involves pattern recognition receptors. Here, we partially purified a novel proteinaceous elicitor called SCLEROTINIA CULTURE FILTRATE ELICITOR1 (SCFE1) from the necrotrophic fungal pathogen Sclerotinia sclerotiorum that induces typical MAMP-triggered immune responses in Arabidopsis thaliana. Analysis of natural genetic variation revealed five Arabidopsis accessions (Mt-0, Lov-1, Lov-5, Br-0, and Sq-1) that are fully insensitive to the SCFE1-containing fraction. We used a forward genetics approach and mapped the locus determining SCFE1 sensitivity to RECEPTOR-LIKE PROTEIN30 (RLP30). We also show that SCFE1-triggered immune responses engage a signaling pathway dependent on the regulatory receptor-like kinases BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1) and SUPPRESSOR OF BIR1-1/EVERSHED (SOBIR1/EVR). Mutants of RLP30, BAK1, and SOBIR1 are more susceptible to S. sclerotiorum and the related fungus Botrytis cinerea. The presence of an elicitor in S. sclerotiorum evoking MAMP-triggered immune responses and sensed by RLP30/SOBIR1/BAK1 demonstrates the relevance of MAMP-triggered immunity in resistance to necrotrophic fungi.     

Strong genetic evidence of DCDC2 as a susceptibility gene for dyslexia

We searched for linkage disequilibrium (LD) in 137 triads with dyslexia, using markers that span the most-replicated dyslexia susceptibility region on 6p21-p22, and found association between the disease and markers within the VMP/DCDC2/KAAG1 locus. Detailed refinement of the LD region, involving sequencing and genotyping of additional markers, showed significant association within DCDC2 in single-marker and haplotype analyses. The association appeared to be strongest in severely affected patients. In a second step, the study was extended to include an independent sample of 239 triads with dyslexia, in which the association--in particular, with the severe phenotype of dyslexia--was confirmed. Our expression data showed that DCDC2, which contains a doublecortin homology domain that is possibly involved in cortical neuron migration, is expressed in the fetal and adult CNS, which--together with the hypothesized protein function--is in accordance with findings in dyslexic patients with abnormal neuronal migration and maturation.     

Immuno-PCR--a new tool for paleomicrobiology: the plague paradigm

Background

The cause of past plague pandemics was controversial but several research teams used PCR techniques and dental pulp as the primary material to reveal that they were caused by Yersinia pestis. However, the degradation of DNA limits the ability to detect ancient infections.

Methods

We used for the first time immuno-PCR to detect Yersinia pestis antigens; it can detect protein concentrations 70 times lower than the standard ELISA. After determining the cut-off value, we tested 34 teeth that were obtained from mass graves of plague, and compared previous PCR results with ELISA and immuno-PCR results.

Results

The immuno-PCR technique was the most sensitive (14 out of 34) followed by the PCR technique (10 out of 34) and ELISA (3 out of 34). The combination of these three methods identified 18 out of 34 (53%) teeth as presumably being from people with the plague.

Conclusion

Immuno-PCR is specific (no false-positive samples were found) and more sensitive than the currently used method to detect antigens of ancient infections in dental pulp. The combination of three methods, ELISA, PCR and immuno-PCR, increased the capacity to identify ancient pathogens in dental pulp.     

Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex

Serum-free conditioned media and cell extracts from cultured human umbilical vein endothelial cells were analyzed for plasminogen activator by SDS-polyacrylamide gel electrophoresis and enzymography on fibrin-indicator gels. Active bands of free and complexed tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) were identified by the incorporation of specific antibodies against, respectively, t-PA or u-PA in the indicator gel. The endothelial cells predominantly released a high-molecular-weight t-PA (95000–135000). This t-PA form was converted to M_r-72000 t-PA by 1.5 M NH₄OH/39 mM SDS. A component with high affinity for both t-PA and u-PA could be demonstrated in serum-free conditioned medium and endothelial cell extract. The complex between this component and M_r-72000 t-PA comigrated with high-molecular-weight t-PA. From the increase in M_r of t-PA or u-PA upon complex formation, the M_r of the endothelial cell component was estimated to be 50000–70000. The reaction between t-PA or u-PA and the plasminogen activator-binding component was blocked by 5 mM p-aminobenzamidine, while the complexes, once formed, could be cleaved by 1.5 M NH₄OH/39 mM SDS. These observations indicated that the active center of plasminogen activator was involed in the complex formation. It was further noted that serum-free conditioned medium of endothelial cell extract inhibited plasminogen activator activity when assayed by the fibrin-plate method. Evidence is provided that the plasminogen activator-binding component was different from a number of the known plasma serine proteinase inhibitors, the placenta inhibitor and the fibroblast surface protein, proteinase-nexin. We conclude that cultured endothelial cells produce a rapid inhibitor of u-PA and t-PA as well as a t-PA-inhibitor complex.     

Selection of Motor Programs for Suppressing Food Intake and Inducing Locomotion in the Drosophila Brain

Central mechanisms by which specific motor programs are selected to achieve meaningful behaviors are not well understood. Using electrophysiological recordings from pharyngeal nerves upon central activation of neurotransmitter-expressing cells, we show that distinct neuronal ensembles can regulate different feeding motor programs. In behavioral and electrophysiological experiments, activation of 20 neurons in the brain expressing the neuropeptide hugin, a homolog of mammalian neuromedin U, simultaneously suppressed the motor program for food intake while inducing the motor program for locomotion. Decreasing hugin neuropeptide levels in the neurons by RNAi prevented this action. Reducing the level of hugin neuronal activity alone did not have any effect on feeding or locomotion motor programs. Furthermore, use of promoter-specific constructs that labeled subsets of hugin neurons demonstrated that initiation of locomotion can be separated from modulation of its motor pattern. These results provide insights into a neural mechanism of how opposing motor programs can be selected in order to coordinate feeding and locomotive behaviors.     

Feline immunodeficiency virus ORF-Ais required for virus particle formation and virus infectivity

The orf-A (orf-2) gene of feline immunodeficiency virus (FIV) is a small open reading frame predicted to encode a 77-amino-acid protein that contains putative domains similar to those of the ungulate lentiviral Tat protein. Orf-A is reported to be critical for efficient viral replication in vitro and in vivo. A series of FIV-pPPR-derived proviruses with in-frame deletions and point mutations within orf-A were constructed and tested for replication in feline lymphoid cells. Orf-A mutant proviruses were also tested for viral gene and protein expression, viral particle formation, and virion infectivity. Deletions within orf-A severely restricted FIV replication in feline peripheral blood mononuclear cells (PBMC) and interleukin-2-dependent T-cell lines. In addition, substitutions of alanines for leucines in the putative leucine-rich domain, for cysteines in the putative cysteine-rich domain, and for a tryptophan at position 43 in Orf-A restricted the replication of FIV mutants. Deletions and point mutations in orf-A imposed a small effect or no effect on FIV long-terminal-repeat-driven viral gene expression and had no effect on viral protein expression. However, release of cell-free, virion-associated viral RNA in supernatants from cells transfected with orf-A mutant proviruses was severely restricted but was rescued by cotransfection with a wild-type Orf-A expression vector. In addition, virions derived from orf-A mutant proviruses expressed reduced infectivity for feline PBMC. Our findings suggest that Orf-A functions involve multiple steps of the FIV life cycle including both virion formation and infectivity. Furthermore, these observations suggest that Orf-A represents an FIV-encoded analog more similar to the accessory gene vpr, vpu, or nef than to the regulatory gene tat encoded by the primate lentiviruses.