Genomic asymmetry of the Brassica napus seed: epigenetic contributions of DNA methylation and small RNAs to subgenome bias.

Polyploidy is a persistent phenomenon in angiosperm genome evolution that is hypothesized to have contributed to the diversity of extant flowering plants. Brassica napus, one of the world's most important angiosperm oilseed species, originated from the interspecific hybridization of Brassica rapa (An ) and Brassica oleracea (Cn ). While the trends of genome dominance in transcriptomics are beginning to emerge, less is known about the epigenetic and small RNA landscapes in polyploids during reproductive development. The seed is the pivotal developmental transition into the new sporophytic generation, and experiences substantial epigenetic modifications over time. Here, we investigated the prevalence of bias in the contexts of DNA methylation and small interfering (si)RNA profiles in both subgenomes (An and Cn ), as well as the ancestral fractionated genomes across B. napus seed development. We report ubiquitous Cn subgenome bias of siRNA expression and cytosine methylation, with DNA methylation being particularly abundant on gene promoters in the Cn subgenome. Further, we provide evidence that siRNA transcriptional patterns were conserved within the ancestral triplicated subgenomes of B. napus, but not across the An and Cn subgenomes. We discuss how methylation patterns in the B. napus seed relate to genes, promoter regions, siRNA loci and transposable elements through the lens of genome fractionation and polyploidization. Taken together we provide evidence for epigenetic regulation selectively silencing the Cn subgenome during seed development, and explore the impact of genome fractionation on the epigenetic components of the B. napus seed.

Gene expression profiling reveals transcription factor networks and subgenome bias during Brassica napus seed development.

We profiled the global gene expression landscape across the reproductive lifecycle of Brassica napus. Comparative analysis of this nascent amphidiploid revealed the contribution of each subgenome to plant reproduction. Whole-genome transcription factor networks identified BZIP11 as a transcriptional regulator of early B. napus seed development. Knockdown of BZIP11 using RNA interference resulted in a similar reduction in gene activity of predicted gene targets, and a reproductive-lethal phenotype. Global mRNA profiling revealed lower accumulation of Cn subgenome transcripts relative to the An subgenome. Subgenome-specific transcription factor networks identified distinct transcription factor families enriched in each of the An and Cn subgenomes early in seed development. Analysis of laser-microdissected seed subregions further reveal subgenome expression dynamics in the embryo, endosperm and seed coat of early stage seeds. Transcription factors predicted to be regulators encoded by the An subgenome are expressed primarily in the seed coat, whereas regulators encoded by the Cn subgenome were expressed primarily in the embryo. Data suggest subgenome bias are characteristic features of the B. napus seed throughout development, and that such bias might not be universal across the embryo, endosperm and seed coat of the developing seed. Transcriptional networks spanning both the An and Cn genomes of the whole B. napus seed can identify valuable targets for seed development research and that -omics level approaches to studying gene regulation in B. napus can benefit from both broad and high-resolution analyses.

Transcriptome landscape of the early Brassica napus seed.

Brassica napus L. (canola) is one of the world's most economically important oilseeds. Despite our growing knowledge of Brassica genetics, we still know little about the genes and gene regulatory networks underlying early seed development. In this work, we use laser microdissection coupled with RNA sequencing to profile gene activity of both the maternal and filial subregions of the globular seed. We find subregions of the chalazal end including the chalazal endosperm, chalazal proliferating tissue, and chalazal seed coat, have unique transcriptome profiles associated with hormone biosynthesis and polysaccharide metabolism. We confirm that the chalazal seed coat is uniquely enriched for sucrose biosynthesis and transport, and that the chalazal endosperm may function as an important regulator of the maternal region through brassinosteroid synthesis. The chalazal proliferating tissue, a poorly understood subregion, was specifically enriched in transcripts associated with megasporogenesis and trehalose biosynthesis, suggesting this ephemeral structure plays an important role in both sporophytic development and carbon nutrient balance, respectively. Finally, compartmentalization of transcription factors and their regulatory circuits has uncovered previously unknown roles for the chalazal pole in early seed development.

Control of white mold (Sclerotinia sclerotiorum) through plant-mediated RNA interference.

The causative agent of white mold, Sclerotinia sclerotiorum, is capable of infecting over 600 plant species and is responsible for significant crop losses across the globe. Control is currently dependent on broad-spectrum chemical agents that can negatively impact the agroecological environment, presenting a need to develop alternative control measures. In this study, we developed transgenic Arabidopsis thaliana (AT1703) expressing hairpin (hp)RNA to silence S. sclerotiorum ABHYDROLASE-3 and slow infection through host induced gene silencing (HIGS). Leaf infection assays show reduced S. sclerotiorum lesion size, fungal load, and ABHYDROLASE-3 transcript abundance in AT1703 compared to wild-type Col-0. To better understand how HIGS influences host-pathogen interactions, we performed global RNA sequencing on AT1703 and wild-type Col-0 directly at the site of S. sclerotiorum infection. RNA sequencing data reveals enrichment of the salicylic acid (SA)-mediated systemic acquired resistance (SAR) pathway, as well as transcription factors predicted to regulate plant immunity. Using RT-qPCR, we identified predicted interacting partners of ABHYDROLASE-3 in the polyamine synthesis pathway of S. sclerotiorum that demonstrate co-reduction with ABHYDROLASE-3 transcript levels during infection. Together, these results demonstrate the utility of HIGS technology in slowing S. sclerotiorum infection and provide insight into the role of ABHYDROLASE-3 in the A. thaliana-S. sclerotiorum pathosystem.

Host induced gene silencing of the Sclerotinia sclerotiorum ABHYDROLASE-3 gene reduces disease severity in Brassica napus.

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.