Project description:Brassica napus leaves(18 days old) were inoculated by Sclerotinia sclerotiorum with leaves harvested after 12, 24 and 48 h. Arabidopsis thaliana full-genome 70mer microarray representing at least 23,686 genes were used.
Project description:RNA-seq of Sclerotinia sclerotiorum-infected B. napus leaves during a timecourse after inoculation. This is a time course experiment. Sclerotinia hyphae were place on detached B. napus leaves, then the combined tissues were harvested at 1, 3, 6, 12, 24, and 48 hours post-innoculation. 3 biological replicates were collected for each time point, and sclerotinia from liquid medium was used as a control.
Project description:Global transcriptome profiling of suceptible and tolerant lines of Brassica napus infected with Sclerotinia sclerotiorum using a petal inoculation method that mimics field conditions.
Project description:Sclerotinia sclerotiorum, the causal agent of white mould, is a necrotrophic fungal pathogen responsible for extensive crop loss. Current control options rely heavily on the application of chemical fungicides that are becoming less effective and may lead to the development of fungal resistance. In the current study, we used a foliar spray application of boron to protect Brassica napus (canola) from S. sclerotiorum infection using whole plant infection assays. Application of boron to aerial surfaces of the canola plant reduced the number of S. sclerotiorum forming lesions by 87% compared to an untreated control. We used dual RNA sequencing to profile the effect of boron on both the host plant and fungal pathogen during the infection process. Differential gene expression analysis and gene ontology term enrichment further revealed the mode of action of a foliar boron spray at the mRNA level. A single foliar application of boron primed the plant defense response through the induction of genes associated with systemic acquired resistance while an application of boron followed by S. sclerotiorum infection induced genes associated with defense-response-related cellular signalling cascades. Additionally, in S. sclerotiorum inoculated on boron-treated B. napus, we uncovered gene activity in response to salicylic acid breakdown, consistent with salicylic-acid-dependent systemic acquired resistance induction within the host plant. Taken together, this study demonstrates that a foliar application of boron results in priming of the B. napus plant defense response, likely through systemic acquired resistance, thereby contributing to increased tolerance to S. sclerotiorum infection.
Project description:Sclerotinia sclerotiorum is a pathogenic fungus that infects hundreds of crop species, causing extensive yield loss every year. Chemical fungicides are used to control this phytopathogen, but with concerns about increasing resistance and impacts on non-target species, there is a need to develop alternative control measures. In the present study, we engineered Brassica napus to constitutively express a hairpin (hp)RNA molecule to silence ABHYRDOLASE-3 in S. sclerotiorum. We demonstrate the potential for Host Induced Gene Silencing (HIGS) to protect B. napus from S. sclerotiorum using leaf, stem and whole plant infection assays. The interaction between the transgenic host plant and invading pathogen was further characterized at the molecular level using dual-RNA sequencing and at the anatomical level through microscopy to understand the processes and possible mechanisms leading to increased tolerance to this damaging necrotroph. We observed significant shifts in the expression of genes relating to plant defense as well as cellular differences in the form of structural barriers around the site of infection in the HIGS-protected plants. Our results provide proof-of-concept that HIGS is an effective means of limiting damage caused by S. sclerotiorum to the plant and demonstrates the utility of this biotechnology in the development of resistance against fungal pathogens.
Project description:Sclerotinia sclerotiorum is a pathogenic fungus that infects hundreds of crop species, causing extensive yield loss every year. Chemical fungicides are used to control this phytopathogen, but with concerns about increasing resistance and impacts on non-target species, there is a need to develop alternative control measures. In the present study, we engineered Brassica napus to constitutively express a hairpin (hp)RNA molecule to silence ABHYRDOLASE-3 in S. sclerotiorum. We demonstrate the potential for Host Induced Gene Silencing (HIGS) to protect B. napus from S. sclerotiorum using leaf, stem and whole plant infection assays. The interaction between the transgenic host plant and invading pathogen was further characterized at the molecular level using dual-RNA sequencing and at the anatomical level through microscopy to understand the processes and possible mechanisms leading to increased tolerance to this damaging necrotroph. We observed significant shifts in the expression of genes relating to plant defense as well as cellular differences in the form of structural barriers around the site of infection in the HIGS-protected plants. Our results provide proof-of-concept that HIGS is an effective means of limiting damage caused by S. sclerotiorum to the plant and demonstrates the utility of this biotechnology in the development of resistance against fungal pathogens.
Project description:MicroRNAs are multifunctional non-coding short nucleotide molecules. Nevertheless, the role of miRNAs in the interactions between plants and necrotrophic pathogens is largely unknown. Here, we report the identification of the miRNA repertoire of the economically important oil crop oilseed rape (Brassica napus) and those involved in interacting with its most devastating necrotrophic pathogen Sclerotinia sclerotiorum. We identified 280 B. napus miRNA candidates, including 53 novel candidates and 227 canonical members or variants of known miRNA families, by high-throughput deep sequencing of small RNAs from both normal and S. sclerotiorum-inoculated leaves. Target genes of 15 novel candidates and 222 known miRNAs were further identified by sequencing of degradomes from the two types of samples. MicroRNA microarray analysis revealed that 68 miRNAs were differentially expressed between S. sclerotiorum-inoculated and uninoculated leaves. A set of these miRNAs target genes involved in plant defense to S. sclerotiorum and/or other pathogens such as NBS-LRR R genes and nitric oxygen and reactive oxygen species related genes. Additionally, three miRNAs target AGO1 and AGO2, key components of post-transcriptional gene silencing (PTGS). Expression of several viral PTGS suppressors reduced resistance to S. sclerotiorum. Arabidopsis mutants of AGO1 and AGO2 exhibited reduced resistance while transgenic lines over-expressing AGO1 displayed increased resistance to S. sclerotiorum in an AGO1 expression level-dependent manner. Moreover, transient over-expression of miRNAs targeting AGO1 and AGO2 decreased resistance to S. sclerotiorum in oilseed rape. Our results demonstrate that the interactions between B. napus and S. sclerotiorum are tightly regulated at miRNA level and probably involve PTGS.
Project description:MicroRNAs are multifunctional non-coding short nucleotide molecules. Nevertheless, the role of miRNAs in the interactions between plants and necrotrophic pathogens is largely unknown. Here, we report the identification of the miRNA repertoire of the economically important oil crop oilseed rape (Brassica napus) and those involved in interacting with its most devastating necrotrophic pathogen Sclerotinia sclerotiorum. We identified 280 B. napus miRNA candidates, including 53 novel candidates and 227 canonical members or variants of known miRNA families, by high-throughput deep sequencing of small RNAs from both normal and S. sclerotiorum-inoculated leaves. Target genes of 15 novel candidates and 222 known miRNAs were further identified by sequencing of degradomes from the two types of samples. MicroRNA microarray analysis revealed that 68 miRNAs were differentially expressed between S. sclerotiorum-inoculated and uninoculated leaves. A set of these miRNAs target genes involved in plant defense to S. sclerotiorum and/or other pathogens such as NBS-LRR R genes and nitric oxygen and reactive oxygen species related genes. Additionally, three miRNAs target AGO1 and AGO2, key components of post-transcriptional gene silencing (PTGS). Expression of several viral PTGS suppressors reduced resistance to S. sclerotiorum. Arabidopsis mutants of AGO1 and AGO2 exhibited reduced resistance while transgenic lines over-expressing AGO1 displayed increased resistance to S. sclerotiorum in an AGO1 expression level-dependent manner. Moreover, transient over-expression of miRNAs targeting AGO1 and AGO2 decreased resistance to S. sclerotiorum in oilseed rape. Our results demonstrate that the interactions between B. napus and S. sclerotiorum are tightly regulated at miRNA level and probably involve PTGS.