CRISPR/Cas9-Targeted Knockout of Rice Susceptibility Genes OsDjA2 and OsERF104 Reveals Alternative Sources of Resistance to Pyricularia oryzae

doi:10.1016/j.rsci.2022.04.001

Abstract

Abstract:

Rice genes OsDjA2 and OsERF104, encoding a chaperone protein and an APETELA2/ ethylene-responsive factor, respectively, are strongly induced in a compatible interaction with blast fungus, and also have function in plant susceptibility validated through gene silencing. Here, we reported the CRISPR/Cas9 knockout of OsDjA2 and OsERF104 genes resulting in considerable improvement of blast resistance. A total of 15 OsDjA2 (62.5%) and 17 OsERF104 (70.8%) T₀ transformed lines were identified from 24 regenerated plants for each target and used in downstream experiments. Phenotyping of homozygous T₁ mutant lines revealed not only a significant decrease in the number of blast lesions but also a reduction in the percentage of diseased leaf area, compared with the infected control plants. Our results supported CRISPR/Cas9-mediated target mutation in rice susceptibility genes as a potential and alternative breeding strategy for building resistance to blast disease.

Key words: gene editing, plant-pathogen interaction, Magnaporthe pathosystem, plant immunity, blast resistance, S-gene, rice

Fabiano T. P. K. Távora, Anne Cécile Meunier, Aurore Vernet, Murielle Portefaix, Joëlle Milazzo, Henri Adreit, Didier Tharreau, Octávio L. Franco, Angela Mehta. CRISPR/Cas9-Targeted Knockout of Rice Susceptibility Genes OsDjA2 and OsERF104 Reveals Alternative Sources of Resistance to Pyricularia oryzae[J]. Rice Science, 2022, 29(6): 535-544.

Figures/Tables 4

Fig. 1. CRISPR/Cas9 design and T7EI assay for sgRNA gene-editing activity. A, Schematic map of gRNA target sites on genomic regions of OsDjA2 and OsERF104. Exons are indicated as blue boxes, interspaced by introns shown as lines. Promoter and transcription termination sites are represented by green and yellow boxes, respectively. Protospacer adjacent motif is underlined and represented as white boxes. ATG and TGA represent start codon and stop codon, respectively. B, Simplified schematic representation of CRISPR/Cas9 T-DNA structure. LB and RB, T-DNA left and right borders, respectively; HPT, Hygromycin resistance gene; CaMV35S, Cauliflower mosaic virus 35S promoter; ZmpUbi, Maize ubiquitin promoter; SpCas9, Streptococcus pyogenes Cas9 gene; OsU3, Oryza sativa PolII U3 promoter sequence. C, Assessment of gRNA cleavage activity of rice protoplast genomic DNA via T7EI assay. ‘-’ means non-cleaved PCR product derived from wild type protoplast transformed with a control plasmid; ‘+’ means cleaved PCR product derived from protoplasts transformed with CRISPR/Cas9 final vector. M, Marker.

Table 1. Efficiency of CRISPR/Cas9-mediated genome editing of target genes and ratios of mutant genotypes in T0 plants.

Parameter	OsDjA2 (LOC_Os02g56040)	OsERF104 (LOC_Os08g36920)
No. of total T₀ transgenic plants	24	24
No. of total T₀ transgenic plants with T-DNA PCR positive	23	23
No. of total T₀ transgenic plants harboring 1-2 copies of T-DNA	15	17
No. of total T₀ transgenic plants harboring site-mutations (Proportion, %)	14 (93.3%)	12 (70.6%)
Biallelic mutation (Proportion, %)	8 (57.1%)	5 (41.6%)
Homozygous mutation (Proportion, %)	5 (35.7%)	6 (50.0%)
Heterozygous mutation (Proportion, %)	1 (7.1%)	1 (8.3%)

Fig. 2. PCR-based screening for presence of T-DNA in rice mutant plants. A, T0 homozygous primary transformants L1 (OsDjA2_20.1), L2 (OsDjA2_24.1), L3 (OsERF104_1.1), L4 (OsERF104_5.1) and L5 (OsERF104_6.1). B, T1 progeny plants (n = 6 of each independent mutant line as #1, #2, #3, #4, #5 and #6), using specific Cas9 primer pair. M, DNA molecular ladder; C+, CRISPR plasmid; C-, Genomic DNA of wild type Nipponbare; ‘×’ indicates PCR negative for T-DNA.

Fig. 3. Identification of blast resistance in CRISPR/Cas9-edited rice mutant plants. A, Phenotypes upon blast infection of wild type Nipponbare, and T0 homozygous mutant plants (OsDjA2_20.1 and OsDjA2_24.1; OsERF104_1.1, OsERF104_5.1 and OsERF104_6.1) of each target gene. The fourth leaves of each line were detached at 7 d post-infection, scanned and analyzed for the number of blast lesions (A-I) and the percentage of the lesioned foliar area (A-II) using the software Quant®. B and C, Boxplot merged with swarmplot data representation for the number of blast lesions (B) and the percentage of foliar lesioned area (C), respectively, observed on each of the 20 leaves. The numbers above the boxplot indicate statistical significance (P < 0.05, two-sample t-test).

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