Natural variation in BnaA07.MKK9 confers resistance to Sclerotinia stem rot in oilseed rape.

Sclerotinia stem rot (SSR), caused by the necrotrophic fungus Sclerotinia sclerotiorum, is one of the most devastating diseases for several major oil-producing crops. Despite its impact, the genetic basis of SSR resistance in plants remains poorly understood. Here, through a genome-wide association study, we identify a key gene, BnaA07. MKK9, that encodes a mitogen-activated protein kinase kinase that confers SSR resistance in oilseed rape. Our functional analyses reveal that BnaA07.MKK9 interacts with BnaC03.MPK3 and BnaC03.MPK6 and phosphorylates them at the TEY activation motif, triggering a signaling cascade that initiates biosynthesis of ethylene, camalexin, and indole glucosinolates, and promotes accumulation of H2O2 and the hypersensitive response, ultimately conferring resistance. Furthermore, variations in the coding sequence of BnaA07.MKK9 alter its kinase activity and improve SSR resistance by ~30% in cultivars carrying the advantageous haplotype. These findings enhance our understanding of SSR resistance and may help engineer novel diversity for future breeding of oilseed rape.

Genome-wide analysis of the G-box regulating factors protein family reveals its roles in response to Sclerotinia sclerotiorum infection in rapeseed (Brassica napus L.).

The G-box regulating factors (GRFs) are involved in a wide array of signal transduction pathway and play important roles in plant physiological and developmental processes and stress responses. The GRF proteins have previously been described in several plant species, but not in rapeseed (Brassica napus L.). In this study, we carried out genome-wide analysis of GRFs in B. napus based on the available genome sequence information, and analyzed their expression in different tissues under different hormone treatments and after inoculation with Sclerotinia sclerotiorum. We identified 46 putative BnaGRF genes in rapeseed, unevenly distributed on 18 chromosomes. Like the 14-3-3 proteins in other plant species, the 46 putative BnaGRFs could be classified into two major evolutionary branches: epsilon (ε) group and non-epsilon (non-ε) group. Evolutionary analysis indicated that the BnaGRF gene family expanded in both groups much before speciation. We discovered an expansion of the 14-3-3 gene family that likely occurred during a recent gene duplication event. Collinearity analysis revealed that most of the BnaGRF genes shared syntenic relationships. Global gene expression profiling of BnaGRFs by RNA-seq analysis showed 41.3% (19/46) response to S. sclerotiorum infection, and this response was probably mediated through jasmonic acid (JA) and salicylic acid (SA) signaling pathways. These results provide key insights into the role of 14-3-3s in the biotic stress response and enhance our understanding of their multiple functions in B. napus.

Overexpression of Cinnamoyl-CoA Reductase 2 in Brassica napus Increases Resistance to Sclerotinia sclerotiorum by Affecting Lignin Biosynthesis.

Sclerotinia sclerotiorum causes severe yield and economic losses for many crop and vegetable species, especially Brassica napus. To date, no immune B. napus germplasm has been identified, giving rise to a major challenge in the breeding of Sclerotinia resistance. In the present study, we found that, compared with a Sclerotinia-susceptible line (J902), a Sclerotinia-resistant line (J964) exhibited better xylem development and a higher lignin content in the stems, which may limit the invasion and spread of S. sclerotiorum during the early infection period. In addition, genes involved in lignin biosynthesis were induced under S. sclerotiorum infection in both lines, indicating that lignin was deposited proactively in infected tissues. We then overexpressed BnaC.CCR2.b, which encodes the first rate-limiting enzyme (cinnamoyl-CoA reductase) that catalyzes the reaction of lignin-specific pathways, and found that overexpression of BnaC.CCR2.b increased the lignin content in the stems of B. napus by 2.28-2.76% under normal growth conditions. We further evaluated the Sclerotinia resistance of BnaC.CCR2.b overexpression lines at the flower-termination stage and found that the disease lesions on the stems of plants in the T2 and T3 generations decreased by 12.2-33.7% and 32.5-37.3% compared to non-transgenic control plants, respectively, at 7days post-inoculation (dpi). The above results indicate that overexpression of BnaC.CCR2.b leads to an increase in lignin content in the stems, which subsequently leads to increased resistance to S. sclerotiorum. Our findings demonstrate that increasing the lignin content in the stems of B. napus is an important strategy for controlling Sclerotinia.

Targeted Knockout of BnTT2 Homologues for Yellow-Seeded Brassica napus with Reduced Flavonoids and Improved Fatty Acid Composition.

Brassica napus is one of the important oil crops grown worldwide, and oil quality improvement is a major goal in rapeseed breeding. Yellow seed is an excellent trait, which has great potential in improving seed quality and economic value. In this study, we created stable yellow seed mutants using a CRISPR/Cas9 system and obtained the yellow seed phenotype only when the four alleles of two BnTT2 homologues were knocked out, indicating that the two BnTT2 homologues had conserved but redundant functions in regulating seed color. Histochemical staining and flavonoid metabolic analysis proved that the BnTT2 mutation hindered the synthesis and accumulation of proanthocyanidins. Transcriptome analysis also showed that the BnTT2 mutation inhibited the expression of genes in the phenylpropanoid and flavonoid biosynthetic pathway, which might be regulated by the complex of BnTT2, BnTT8 and BnTTG1. In addition, the homozygous mutants of BnTT2 homologues increased oil content and improved fatty acid composition with higher linoleic acid (C18:2) and linolenic acid (C18:3), which could be used for the genetic improvement of rapeseed. Overall, this research showed that the BnTT2 mutation can be used for yellow seed breeding and oil improvement, which is of great significance in improving the economic value of rapeseeds.

CRISPR/Cas9-Mediated Multiplex Genome Editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L.

Targeted genome editing is a desirable means of basic science and crop improvement. The clustered, regularly interspaced, palindromic repeat (CRISPR)/Cas9 (CRISPR-associated 9) system is currently the simplest and most commonly used system in targeted genomic editing in plants. Single and multiplex genome editing in plants can be achieved under this system. In Arabidopsis, AtWRKY11 and AtWRKY70 genes were involved in JA- and SA-induced resistance to pathogens, in rapeseed (Brassica napus L.), BnWRKY11 and BnWRKY70 genes were found to be differently expressed after inoculated with the pathogenic fungus, Sclerotinia sclerotiorum (Lib.) de Bary. In this study, two Cas9/sgRNA constructs targeting two copies of BnWRKY11 and four copies of BnWRKY70 were designed to generate BnWRKY11 and BnWRKY70 mutants respectively. As a result, twenty-two BnWRKY11 and eight BnWRKY70 independent transformants (T0) were obtained, with the mutation ratios of 54.5% (12/22) and 50% (4/8) in BnWRKY11 and BnWRKY70 transformants respectively. Eight and two plants with two copies of mutated BnWRKY11 and BnWRKY70 were obtained respectively. In T1 generation of each plant examined, new mutations on target genes were detected with high efficiency. The vast majority of BnWRKY70 mutants showed editing in three copies of BnWRKY70 in examined T1 plants. BnWRKY70 mutants exhibited enhanced resistance to Sclerotinia, while BnWRKY11 mutants showed no significant difference in Sclerotinia resistance when compared to non-transgenic plants. In addition, plants that overexpressed BnWRKY70 showed increased sensitivity when compared to non-transgenic plants. Altogether, our results demonstrated that BnWRKY70 may function as a regulating factor to negatively control the Sclerotinia resistance and CRISPR/Cas9 system could be used to generate germplasm in B. napus with high resistance against Sclerotinia.

Homoeolog expression bias and expression level dominance in resynthesized allopolyploid Brassica napus.

BACKGROUND: Allopolyploids require rapid genetic and epigenetic modifications to reconcile two or more sets of divergent genomes. To better understand the fate of duplicate genes following genomic mergers and doubling during allopolyploid formation, in this study, we explored the global gene expression patterns in resynthesized allotetraploid Brassica napus (AACC) and its diploid parents B. rapa (AA) and B. oleracea (CC) using RNA sequencing of leaf transcriptomes. RESULTS: We found that allopolyploid B. napus formation was accompanied by extensive changes (approximately one-third of the expressed genes) in the parental gene expression patterns ('transcriptome shock'). Interestingly, the majority (85%) of differentially expressed genes (DEGs) were downregulated in the allotetraploid. Moreover, the homoeolog expression bias (relative contribution of homoeologs to the transcriptome) and expression level dominance (total expression level of both homoeologs) were thoroughly investigated by monitoring the expression of 23,766 B. oleracea-B. rapa orthologous gene pairs. Approximately 36.5% of the expressed gene pairs displayed expression bias with a slight preference toward the A-genome. In addition, 39.6, 4.9 and 9.0% of the expressed gene pairs exhibited expression level dominance (ELD), additivity expression and transgressive expression, respectively. The genome-wide ELD was also biased toward the A-genome in the resynthesized B. napus. To explain the ELD phenomenon, we compared the individual homoeolog expression levels relative to those of the diploid parents and found that ELD in the direction of the higher-expression parent can be explained by the downregulation of homoeologs from the dominant parent or upregulation of homoeologs from the nondominant parent; however, ELD in the direction of the lower-expression parent can be explained only by the downregulation of the nondominant parent or both homoeologs. Furthermore, Gene Ontology (GO) enrichment analysis suggested that the alteration in the gene expression patterns could be a prominent cause of the phenotypic variation between the newly formed B. napus and its parental species. CONCLUSIONS: Collectively, our data provide insight into the rapid repatterning of gene expression at the beginning of Brassica allopolyploidization and enhance our knowledge of allopolyploidization processes.