Identification of lncRNAs in response to infection by Plasmodiophora brassicae in Brassica napus and development of lncRNA-based SSR markers

Clubroot resistance in spring canola has been introgressed from different Brassica sources; however, molecular mechanism underlying this resistance, especially the involvement of long non-coding RNAs (lncRNAs), yet to be understood. We identified 464 differentially expressed (DE) lncRNAs from the roots of clubroot resistant canola, carrying resistance on chromosome BnaA03, and susceptible canola lines challenged with Plasmodiophora brassicae pathotype 3. Pathway enrichment analysis showed that most of the target genes regulated by these DE lncRNAs belonged to plant-pathogen interaction and hormone signaling, as well as primary and secondary metabolic pathways. Comparative analysis of these lncRNAs with the previously reported 530 DE lncRNAs, identified using resistance located on BnaA08, detected 12 lncRNAs which showed a similar trend of upregulation in both types of resistant lines; these lncRNAs probably play a fundamental role in clubroot resistance. We identified SSR markers within 196 DE lncRNAs. Genotyping of two DH populations carrying resistance on BnaA03 identified a marker capable of detecting the resistance in 98% of the DH lines. To our knowledge, this is the first report of the identification of SSRs within the lncRNAs responsive to P. brassicae infection demonstrating the potential use of the lncRNAs in the breeding of Brassica crops.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

The crystal structure of an octapeptide repeat of the Prion protein in complex with a fab fragment of the POM2 antibody

Prion diseases are progressive, infectious neurodegenerative disorders caused primarily by the misfolding of the cellular prion protein, PrP(c) , into an insoluble, protease-resistant, aggregated isoform termed PrP(sc) . In native conditions, PrP(c) has a structured C-terminal domain and a highly flexible N-terminal domain. A part of this N-terminal domain consists of 4-5 repeats of an unusual glycine rich, eight amino acids long peptide known as the octapeptide repeat (OR) domain. In this paper, we successfully report the first crystal structure of an octapeptide repeat of PrP(c) bound to the POM2 Fab antibody fragment. The structure was solved at a resolution of 2.3 Å by molecular replacement. While several studies have previously predicted a β-turn like structure of the unbound octapeptide repeats, our structure shows an extended conformation of the octapeptide repeat when bound to a molecule of the POM2 Fab indicating that the bound Fab disrupts any putative native β turn conformation of the octapeptide repeats. Encouraging results from several recent studies have shown that administering small molecule ligands or antibodies targeting the OR domain of PrP result in arresting the progress of peripheral prion infections both in ex vivo and in in vivo models. This makes the structural study of the interactions of POM2 Fab with the OR domain very important as it would help us to design smaller and tighter binding OR ligands

Crystallization and preliminary X-ray diffraction analysis of prion protein bound to the Fab fragment of the POM1 antibody

Prion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein PrP(c) to the pathogenic isoform PrP(sc). Several antibodies are known to interact with the cellular prion protein and to inhibit this transition. An antibody Fab fragment, Fab POM1, was produced that recognizes a structural motif of the C-terminal domain of mouse prion protein. To study the mechanism by which Fab POM1 recognizes and binds the prion molecule, the complex between Fab POM1 and the C-terminal domain of mouse prion (residues 120-232) was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group C2, with unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°

qRT-PCR validation of pathogen induced fold changes in 12 selected <i>B</i>. <i>napu</i>s lncRNAs detected by RNA-seq.

<p>At each time point, the expression level in <i>S</i>. <i>sclerotiorum</i> infected sample was normalized to that in its corresponding mock sample. Standard error bars for the fold changes determined by qRT-PCR are shown.</p

Differential Expression of miRNAs in <i>Brassica napus</i> Root following Infection with <i>Plasmodiophora brassicae</i>

<div><p>Canola (oilseed rape, <i>Brassica napus</i> L.) is susceptible to infection by the biotrophic protist <i>Plasmodiophora brassicae</i>, the causal agent of clubroot. To understand the roles of microRNAs (miRNAs) during the post-transcriptional regulation of disease initiation and progression, we have characterized the changes in miRNA expression profiles in canola roots during clubroot disease development and have compared these to uninfected roots. Two different stages of clubroot development were targeted in this miRNA profiling study: an early time of 10-dpi for disease initiation and a later 20-dpi, by which time the pathogen had colonized the roots (as evident by visible gall formation and histological observations). <i>P. brassicae</i> responsive miRNAs were identified and validated by qRT-PCR of miRNAs and the subsequent validation of the target mRNAs through starBase degradome analysis, and through 5′ RLM-RACE. This study identifies putative miRNA-regulated genes with roles during clubroot disease initiation and development. Putative target genes identified in this study included: transcription factors (TFs), hormone-related genes, as well as genes associated with plant stress response regulation such as cytokinin, auxin/ethylene response elements. The results of our study may assist in elucidating the role of miRNAs in post-transcriptional regulation of target genes during disease development and may contribute to the development of strategies to engineer durable resistance to this important phytopathogen.</p></div

Categories of initial assemblies and classification of novel lncRNAs from <i>B</i>. <i>napus</i> post infection with <i>S</i>. <i>sclerotiorum</i>.

<p>Categories of initial assemblies and classification of novel lncRNAs from <i>B</i>. <i>napus</i> post infection with <i>S</i>. <i>sclerotiorum</i>.</p