Fabiano T.P.K.Távora, Anne Cécile Meunier, Aurore Vernet, Murielle Portefaix, Joëlle Milazzo, Henri Adreit, Didier Tharreau, Octávio L. Franco, Angela Mehta

Rice Science; Volume 29, Issue 6, November 2022, Pages 535-544

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.

See https://www.sciencedirect.com/science/article/pii/S1672630822000762

Fig. 1. CRISPR/Cas9 design and T7EI assay for sgRNAgene-editing activity.

A,Schematic map of gRNA target sites on genomic regions of OsDjA2and 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-DNAleft and rightborders, respectively;HPT, Hygromycin resistance gene; CaMV35S, Cauliflower mosaic virus35S promoter;ZmpUbi, Maize ubiquitinpromoter; SpCas9, Streptococcus pyogenesCas9gene; OsU3, Oryza sativaPolII U3 promoter sequence.

C, Assessment of gRNAcleavage activity of rice protoplast genomic DNA via T7EI assay. ‘–’ means non-cleaved PCR product derived from wild typeprotoplast transformed with a control plasmid; ‘+’ means cleaved PCR product derived from protoplasts transformed with CRISPR/Cas9 final vector.M, Marker.