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

Abstract

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.

Keywords: Brassica napus; CRISPR/Cas9; Mutation pattern; Sclerotinia sclerotiorum; WRKY.

Figure 1

Phylogenetic tree of WRKY11 and WRKY70 and the expression level of BnWRKY11 and BnWRKY70 in response to S. sclerotiorum inoculation. (A) Phylogenetic tree of BnWRKY11 and the homologs from Arabidopsis; (B) Phylogenetic tree of BnWRKY70 and the homologs from Arabidopsis; (C,D) The expression level of BnWRKY11 and BnWRKY70 in response to S. sclerotiorum inoculation [51]. The tree was generated using the DNAMAN program by maximum likelihood (ML) methods. Bootstrap values are displayed with red numbers. hpi, hours post-inoculation.

Figure 2

Position of target sites and primers on BnWRKY11 and BnWRKY70 and physical maps of the T-DNA regions of Cas9/sgRNA constructs. (A,B) the target sites for BnWRKY11 and BnWRKY70 respectively and the primers for the amplification were shown as well. Tgt1-Tgt3 means the chosen target sites, the locations of target sites are marked with black arrows; primers are shown in red arrows. (C) Physical maps of the T-DNA regions of Cas9/sgRNA constructs. LB/RB, left/right border of T-DNA; P_35S:Cas9, Cas9 gene which driven by CMV35S promoter; P_35S:Kan^R, NTP gene which driven by CMV35S promoter. AtU3/AtU6, Arabidopsis U3/U6 promoter.

Figure 3

Multiplex mutagenesis of B. napus genome in T₀ generation. The protospace adjacent motif (PAM) is shown in bold blue letters; red dashes mark the deletions; the inserted nucleotide is marked by a green letter. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. Tgt1-Tgt3 means the target sequence used to generate sgRNA expression cassette.

Figure 4

Mutation types and frequency in transgenic plants. Mutation types and frequency from combined data of four different targets at T₀ generation. Left insert, occurrence of insertion (I), deletion (D) and substitution (S) mutation types. Right insert, counts of different mutation length. In x-axis: I^#, ^# of bp inserted at target site; D^#, ^# of bp deleted from target site.

Figure 5

Lesion area on leaves of BnWRKY70 knockout B. napus lines inoculated with S. sclerotiorum. (A) Representatives of disease symptom on the Non-Transgenic (Non-Trans), BnWRKY70 knockout lines. Leaves of 6-week-old plants were inoculated with S. sclerotiorum. Photos were taken 48 h post-inoculation. (B) Lesion area on leaves of BnWRKY70 overexpression lines. ** indicate that the means are statistically different (p < 0.01).

Figure 6

Expression analysis and lesion area on leaves of BnWRKY70 overexpression B. napus lines inoculated with S. sclerotiorum. (A) RT-qPCR analysis of BnWRKY70 expression in overexpression plants. BnActin7 was used as reference gene. The data shown are the mean of three independent experiments ± standard error (SE). (B) Representatives of disease symptom on the Non-Transgenic (Non-Trans), BnWRKY70 knockout lines. Leaves of 6-week-old plants were inoculated with S. sclerotiorum. Photos were taken 48 h post-inoculation. (C) Lesion area on leaves of BnWRKY70 overexpression lines. ** indicate that the means are statistically different (p < 0.01).