Nat N. V. Kav

The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes

PrPSc, a misfolded and aggregated form of the cellular prion protein PrPC, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrPC in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrPC and PrPSc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrPC. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrPC. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrPSc. Other antibodies immunoprecipitate PrPC, but not PrPSc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrPC and PrPSc. Amino-proximal antibodies were found to react with repetitive PrPC epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.

The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein

Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrPC; ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrPC globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrPC, was exacerbated by PrPC overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrPC consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrPC mutant, PrP(Δ94–134), indicating that the flexible tail mediates toxicity in two distinct PrPC-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides.

Functional characterization of four APETALA2-family genes (RAP2.6, RAP2.6L, DREB19 and DREB26) in Arabidopsis

APETALA2 (AP2) transcription factors (TFs) play very important roles in plant growth and development and in defense response. Here, we report functional characterization of four AP2 TF family genes [(RAP2.6 (At1g43160), RAP2.6L (At5g13330), DREB 26 (At1g21910) and DREB19 (At2g38340)] that were identified among NaCl inducible transcripts in abscisic acid responsive 17 (ABR17) transgenic Arabidopsis in our previous microarray analyses. DREB19 and DREB26 function as transactivators and localize in the nucleus. All four genes were abundant in early vegetative and flowering stages, although the magnitude of the expression varied. We observed tissue specific expression patterns for RAP2.6, RAP2.6L, DREB19 and DREB26 in flowers and other organs. RAP2.6 and RAP2.6L were responsive to stress hormones like jasmonic acid, salicylic acid, abscisic acid and ethylene in addition to salt and drought. DREB19 and DREB26 were less responsive to stress hormones, but the former was highly responsive to salt, heat and drought. Overexpression of RAP2.6 in Arabidopsis resulted in a dwarf phenotype with extensive secondary branching and small siliques, and DREB26 overexpression resulted in deformed plants. However, overexpression of RAP2.6L and DREB19 enhanced performance under salt and drought stresses without affecting phenotype. In summary, we have demonstrated that RAP2.6, RAP2.6L, DREB26 and DREB19 are transactivators, they exhibit tissue specific expression, and they participate in plant developmental processes as well as biotic and/or abiotic stress signaling. It is possible that the results from this study on these transcription factors, in particular RAP2.6L and DREB19, can be utilized in developing salt and drought tolerant plants in the future.

Identification and expression analysis of WRKY transcription factor genes in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments

BackgroundMembers of plant WRKY transcription factor families are widely implicated in defense responses and various other physiological processes. For canola (Brassica napus L.), no WRKY genes have been described in detail. Because of the economic importance of this crop, and its evolutionary relationship to Arabidopsis thaliana, we sought to characterize a subset of canola WRKY genes in the context of pathogen and hormone responses.ResultsIn this study, we identified 46 WRKY genes from canola by mining the expressed sequence tag (EST) database and cloned cDNA sequences of 38 BnWRKYs. A phylogenetic tree was constructed using the conserved WRKY domain amino acid sequences, which demonstrated that BnWRKYs can be divided into three major groups. We further compared BnWRKYs to the 72 WRKY genes from Arabidopsis and 91 WRKY from rice, and we identified 46 presumptive orthologs of AtWRKY genes. We examined the subcellular localization of four BnWRKY proteins using green fluorescent protein (GFP) and we observed the fluorescent green signals in the nucleus only.The responses of 16 selected BnWRKY genes to two fungal pathogens, Sclerotinia sclerotiorum and Alternaria brassicae, were analyzed by quantitative real time-PCR (qRT-PCR). Transcript abundance of 13 BnWRKY genes changed significantly following pathogen challenge: transcripts of 10 WRKYs increased in abundance, two WRKY transcripts decreased after infection, and one decreased at 12 h post-infection but increased later on (72 h). We also observed that transcript abundance of 13/16 BnWRKY genes was responsive to one or more hormones, including abscisic acid (ABA), and cytokinin (6-benzylaminopurine, BAP) and the defense signaling molecules jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). We compared these transcript expression patterns to those previously described for presumptive orthologs of these genes in Arabidopsis and rice, and observed both similarities and differences in expression patterns.ConclusionWe identified a set of 13 BnWRKY genes from among 16 BnWRKY genes assayed, that are responsive to both fungal pathogens and hormone treatments, suggesting shared signaling mechanisms for these responses. This study suggests that a large number of BnWRKY proteins are involved in the transcriptional regulation of defense-related genes in response to fungal pathogens and hormone stimuli.

Pea PR 10.1 is a ribonuclease and its transgenic expression elevates cytokinin levels

The constitutive expression of a cDNA encoding a pea (Pisum sativum L.) PR 10 protein in Brassica napus leading to an enhancement of germination under saline conditions has been previously reported. In order to understand the biochemical function of this pea PR 10 protein, its cDNA has been expressed in Escherichia coli and the recombinant protein purified to homogeneity. Ribonuclease activity of the recombinant pea PR 10 protein has been demonstrated for the first time using an in-solution as well as an in-gel RNA degradation assay. Furthermore, in order to characterize the changes brought about as a result of the constitutive expression of the pea PR 10 cDNA in B. napus, we have measured the endogenous concentrations of several phytohormones. Increased cytokinin and, decreased abscisic acid (ABA) were observed in 7-day-old transgenic seedlings whereas no significant changes in the concentrations of gibberellin (GA) or indoleacetic acid (IAA) were observed at this stage of growth and development. The potential role(s) of PR 10 proteins with RNase activity and elevated cytokinins during plant stress responses as well as the possible relationship between PR 10 protein and changes in cytokinin concentrations are discussed.

Application of Proteomics to Investigate Plant-Microbe Interactions

With the completion of genome projects for Arabidopsis thaliana, Oryza sativa and several other plant species, an increasing number of whole genome sequences are now available for plants. In this post-genomic era, a more thorough understanding of gene expression and function can be achieved through the characterization of the products of expression, the proteins, which are essential biological determinants of plant phenotypes. Proteomics offers a continually evolving set of novel techniques to study all facets of protein structure and function. The application of proteomics in plant pathology is becoming more commonplace with techniques such as two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) being used to characterize cellular and extracellular virulence and pathogenicity factors produced by pathogens as well as to identify changes in protein levels in plant hosts upon infection by pathogenic organisms and symbiotic counterparts. This review article summarizes the current status of geland non gel-based proteomic techniques and describes the significant discoveries that have resulted from the various proteome-level investigations into phytopathogenic microorganisms and plant host-microbe interactions.

Proteomic analysis of egg white proteins during storage.

Egg storage causes egg white to lose its viscous nature to form a thin liquid, commonly referred to as egg white thinning. To understand the mechanisms underlying egg white thinning, white‐shell eggs were used in the present study to determine the proteome‐level changes of egg white proteins occurred during storage. Egg white thinning was observed visually after 20 days of storage at ambient temperature (22±2°C) when the maximum number of proteome‐level changes occurred. The proteins that showed significant changes in abundance during storage included ovalbumin, clusterin, ovoinhibitor, ovotransferrin, and prostaglandin D2 synthase. Among these, only the abundance of clusterin was observed to change continuously during the storage period. Hence, it is expected that the increase in the concentrations of clusterin and ovoinhibitor along with the change of ovalbumin content during storage might contribute to egg white thinning. Degradation of ovalbumin/clusterin during egg storage may be due to the combined effect of proteolysis and increase in pH; this may also be partly responsible for egg white thinning phenomenon.

Transcriptional profiling of pea ABR17 mediated changes in gene expression in Arabidopsis thaliana

BackgroundPathogenesis-related proteins belonging to group 10 (PR10) are elevated in response to biotic and abiotic stresses in plants. Previously, we have shown a drastic salinity-induced increase in the levels of ABR17, a member of the PR10 family, in pea. Furthermore, we have also demonstrated that the constitutive expression of pea ABR17 cDNA in Arabidopsis thaliana and Brassica napus enhances their germination and early seedling growth under stress. Although it has been reported that several members of the PR10 family including ABR17 possess RNase activity, the exact mechanism by which the aforementioned characteristics are conferred by ABR17 is unknown at this time. We hypothesized that a study of differences in transcriptome between wild type (WT) and ABR17 transgenic A. thaliana may shed light on this process.ResultsThe molecular changes brought about by the expression of pea ABR17 cDNA in A. thaliana in the presence or absence of salt stress were investigated using microarrays consisting of 70-mer oligonucleotide probes representing 23,686 Arabidopsis genes. Statistical analysis identified number of genes which were over represented among up- or down-regulated transcripts in the transgenic line. Our results highlight the important roles of many abscisic acid (ABA) and cytokinin (CK) responsive genes in ABR17 transgenic lines. Although the transcriptional changes followed a general salt response theme in both WT and transgenic seedlings under salt stress, many genes exhibited differential expression patterns when the transgenic and WT lines were compared. These genes include plant defensins, heat shock proteins, other defense related genes, and several transcriptional factors. Our microarray results for selected genes were validated using quantitative real-time PCR.ConclusionTranscriptional analysis in ABR17 transgenic Arabidopsis plants, both under normal and saline conditions, revealed significant changes in abundance of transcripts for many stress responsive genes, as well as those related to plant growth and development. Our results also suggest that ABR17 may mediate stress tolerance through the modulation of many ABA- and CK-responsive genes and may further our understanding of the role of ABR17 in mediating plant stress responses.

Inhibition of photosynthesis and modification of the wheat leaf proteome by Ptr ToxB: A host‐specific toxin from the fungal pathogen Pyrenophora tritici‐repentis

Tan spot, caused by Pyrenophora tritici‐repentis, is an important foliar disease of wheat. The fungus produces the host‐specific, chlorosis‐inducing toxin Ptr ToxB. To better understand toxin action, we examined the effects of Ptr ToxB on sensitive wheat. Photosynthesis, as measured by infrared gas analysis, declined significantly within 12 h of toxin treatment, prior to the development of chlorosis at 48–72 h. Analysis by 2‐DE revealed a total of 102 protein spots with significantly altered intensities 12–36 h after toxin treatment, of which 66 were more abundant and 36 were less abundant than in the buffer‐treated control. The identities of 47 of these spots were established by MS/MS, and included proteins involved in the light reactions of photosynthesis, the Calvin cycle, and the stress/defense response. Based on the declines in photosynthesis and the identities of the differentially abundant proteins, we hypothesize that Ptr ToxB causes a rapid disruption in the photosynthetic processes of sensitive wheat, leading to the generation of ROS and oxidative stress. Although the photoprotective and repair mechanisms of the host appear to initially still be functional, they are probably overwhelmed by the continued production of ROS, leading to chlorophyll photooxidation and the development of chlorosis.

Oxalic acid-mediated stress responses in Brassica napus L.

Oxalic acid (OA) occurs extensively in nature and plays diverse roles, especially in pathogenic processes involving various plant pathogens. However, proteome changes and modifications of signaling and oxidative network of plants in response to OA are not well understood. In order to investigate the responses of Brassica napus toward OA, a proteome analysis was conducted employing 2‐DE with MS/MS. A total of 37 proteins were identified as responding to OA stress, of which 13 were up‐regulated and 24 were down‐regulated. These proteins were categorized into several functional groups including protein processing, RNA processing, photosynthesis, signal transduction, stress response, and redox homeostasis. Investigation of the effect of OA on phytohormone signaling and oxidative responses revealed that jasmonic acid‐, ethylene‐, and abscisic acid‐mediated signaling pathways appear to increase at later time points, whereas those pathways mediated by salicylic acid appear to be suppressed. Moreover, the activities of the antioxidant enzymes catalase, peroxidase, superoxide dismutase and oxalic acid oxidase, but not NADPH oxidase, were suppressed by OA stress. Our findings are discussed within the context of the proposed role(s) of OA during infection by Sclerotinia sclerotiorum and subsequent disease progression.