Background: Members of plant WRKY transcription factor families are widely implicated in defense responses and various other physiological processes. For canola (Brassica napusL.), no WRKY genes have been described in detail. Because of the economic importance of this crop, and its evolutionary relationship toArabidopsis thaliana, we sought to characterize a subset of canolaWRKYgenes in the context of pathogen and hormone responses.Results: In this study, we identified 46WRKYgenes from canola by mining the expressed sequence tag (EST) database and cloned cDNA sequences of 38BnWRKYs. 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 comparedBnWRKYsto the 72WRKYgenes fromArabidopsisand 91WRKYfrom rice, and we identified 46 presumptive orthologs ofAtWRKYgenes. 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 selectedBnWRKYgenes to two fungal pathogens,Sclerotinia sclerotiorumandAlternaria brassicae, were analyzed by quantitative real time-PCR (qRT-PCR). Transcript abundance of 13BnWRKYgenes changed significantly following pathogen challenge: transcripts of 10WRKYs increased in abundance, twoWRKYtranscripts decreased after infection, and one decreased at 12 h post-infection but increased later on (72 h). We also observed that transcript abundance of 13/16BnWRKYgenes 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 inArabidopsisand rice, and observed both similarities and differences in expression patterns.Conclusion: We identified a set of 13BnWRKYgenes from among 16BnWRKYgenes 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.

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.