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.

Drought and heat are major abiotic stresses frequently coinciding to threaten rice production. Despite hundreds of stress-related genes being identified, only a few have been confirmed to confer resistance to multiple stresses in crops. Here we report ONAC023, a hub stress regulator that integrates the regulations of both drought and heat tolerance in rice. ONAC023 positively regulates drought and heat tolerance at both seedling and reproductive stages. Notably, the functioning of ONAC023 is obliterated without stress treatment and can be triggered by drought and heat stresses at two layers. The expression of ONAC023 is induced in response to stress stimuli. We show that overexpressed ONAC23 is translocated to the nucleus under stress and evidence from protoplasts suggests that the dephosphorylation of the remorin protein OSREM1.5 can promote this translocation. Under drought or heat stress, the nuclear ONAC023 can target and promote the expression of diverse genes, such as OsPIP2;7, PGL3, OsFKBP20-1b, and OsSF3B1, which are involved in various processes including water transport, reactive oxygen species homeostasis, and alternative splicing. These results manifest that ONAC023 is fine-tuned to positively regulate drought and heat tolerance through the integration of multiple stress-responsive processes. Our findings provide not only an underlying connection between drought and heat responses, but also a promising candidate for engineering multi-stress-resilient rice.

Myeloblastosis (MYB)-related transcription factors comprise a large subfamily of the MYB family. They play significant roles in plant development and in stress responses. However, MYB-related proteins have not been comprehensively investigated in rapeseed (Brassica napus L.). In the present study, a genome-wide analysis of MYB-related transcription factors was performed in rapeseed. We identified 251 Brassica napus MYB (BnMYB)-related members, which were divided phylogenetically into five clades. Evolutionary analysis suggested that whole genome duplication and segmental duplication events have played a significant role in the expansion of BnMYB-related gene family. Selective pressure of BnMYB-related genes was estimated using the Ka/Ks ratio, which indicated that BnMYB-related genes underwent strong purifying selection during evolution. In silico analysis showed that various development-associated, phytohormone-responsive, and stress-related cis-acting regulatory elements were enriched in the promoter regions of BnMYB-related genes. Furthermore, MYB-related genes with tissue or organ-specific, stress-responsive expression patterns were identified in B. napus based on temporospatial and abiotic stress expression profiles. Among the stress-responsive MYB-related genes, BnMRD107 was strongly induced by drought stress, and was therefore selected for functional study. Rapeseed seedlings overexpressing BnMRD107 showed improved resistance to osmotic stress. Our findings not only lay a foundation for further functional characterization of BnMYB-related genes, but also provide valuable clues to determine candidate genes for future genetic improvement of B. napus.