Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); 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 1 and 3 helices of the PrP(C) 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 PrP(C), was exacerbated by PrP(C) 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 PrP(C) 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 PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-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. (Fig. 1b). None of three high-affinity antibodies to the octapeptide repeats (OR, residues 50-90 embedded within the FT) were neurotoxic (Fig. 1b). Antibodies POM3 and D13, which bind the "charged cluster-2" 11 (CC2, residues 95-110), were innocuous at 67 nM but neurotoxic at 200 nM (Fig. 1b). None of the tested antibodies were toxic to Prnp o/o COCS ( Supplementary Fig. 2a). The identity of the targeted epitopes appeared to be a better predictor of PrP C antibody toxicity than their affinity to PrP C , suggesting that neurotoxicity resulted from the interaction of antibodies with specific PrP C domains (Supplementary Table 2).The mechanisms of neurotoxicity were further explored using POM1, a highly toxic antibody targeting the GD. Wild-type (wt) and tga20 COCS lost most granule cells (CGC) within 28 and 14 days post-exposure (dpe) to POM1, respectively (Fig. 2a-c). Controls included POM1-treated Prnp o/o COCS 12 , t...
Background: Members 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.
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.
Sclerotinia stem rot caused by Sclerotinia sclerotiorum affects canola production worldwide. Emerging evidence suggests that long non-coding RNAs (lncRNAs) play important roles in the regulation of gene expression in plants, in response to both abiotic and biotic stress. So far, identification of lncRNAs has been limited to a few model plant species, and their roles in mediating responses to biotic stresses are yet to be characterized in Brassica napus. The present study reports the identification of novel lncRNAs responsive to S. sclerotiorum infection in B. napus at two time points after infection (24 hpi and 48 hpi) using a stranded RNA-Sequencing technique and a detection pipeline for lncRNAs. Of the total 3,181 lncRNA candidates, 2,821 lncRNAs were intergenic, 111 were natural antisense transcripts, 76 possessed exonic overlap with the reference coding transcripts while the remaining 173 represented novel lnc- isoforms. Forty one lncRNAs were identified as the precursors for microRNAs (miRNAs) including miR156, miR169 and miR394, with significant roles in mediating plant responses to fungal phytopathogens. A total of 931 differentially expressed lncRNAs were identified in response to S. sclerotiorum infection and the expression of 12 such lncRNAs was further validated using qRT-PCR. B. napus antisense lncRNA, TCONS_00000966, having 90% overlap with a plant defensin gene, showed significant induction at both infection stages, suggesting its involvement in the transcriptional regulation of defense responsive genes under S. sclerotiorum infection. Additionally, nine lncRNAs showed overlap with cis-regulatory regions of differentially expressed genes of B. napus. Quantitative RT-PCR verification of a set of S. sclerotiorum responsive sense/antisense transcript pairs revealed contrasting expression patterns, supporting the hypothesis that steric clashes of transcriptional machinery may lead to inactivation of sense promoter. Our findings highlight the potential contributions of lncRNAs in regulating expression of plant genes that respond to biotic stress.
A database of lightning-related deaths and injuries in Bangladesh was developed from 1990 to mid-2016 from a variety of sources that contains a total of 5468 casualties, composed of 3086 fatalities and 2382 injuries. Spatial, temporal, and demographic aspects of these lightning casualties are evaluated in order to aid relevant entities in effective management of lightning-related meteorological hazards. The annual averages for Bangladesh are 114 fatalities and 89 injuries over the entire period. Weighting by population reveals a fatality rate of 0.92 per million people per year and an injury rate of 0.71. In contrast, the latest 6 years have a fatality rate of 1.6 and injury rate of 1.4. The rural portion of lightning fatalities is 93%. Most fatalities occurred between early morning (0600 LST) and early evening (2000). Through the year, more fatalities occur during the premonsoon season of March through May than during the monsoon season (June–September). The interannual time series of fatalities indicates an increase since the late 2000s, which is a result of greatly improved communications leading to better media reporting of lightning casualties. Bangladesh has also become much more populous in recent years. As a result, the most recent 6 years have 251 fatalities per year, which may be considered as the current estimate. The majority of lightning-related deaths occurred to males. Farming is the major activity at the time of lightning fatalities followed by being inside a dwelling and returning home or walking around homesteads/courtyards.
Sclerotinia stem rot, caused by the necrotrophic fungal pathogen Sclerotinia sclerotiorum, is a serious disease of canola (Brassica napus L.). To increase the understanding of the B. napus- S. sclerotiorum interaction, proteins potentially involved in mediating this interaction were identified and characterized. Upon infection of canola leaves by S. sclerotiorum, necrosis of host leaves was observed by 12 h and rapidly progressed during the later time points. These morphological observations were supported by microscopic study performed at different time points after pathogen challenge. Leaf proteins were extracted and analyzed by 2-DE, which revealed the modulation of 32 proteins (12 down- and 20 up-regulated). The identities of these proteins were established by ESI-q-TOF MS/MS and included proteins involved in photosynthesis and metabolic pathways, protein folding and modifications, hormone signaling, and antioxidant defense. Gene expression analysis of selected genes was performed by qRT-PCR, whereas the elevated levels of the antioxidant enzymes peroxidase and superoxide dismutase were validated by enzyme assays. To the authors' best knowledge, this is the first proteomics-based investigation of B. napus-S. sclerotiorum interaction, and the roles of many of the proteins identified are discussed within the context of this pathosystem.
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.
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