Rice black-streaked dwarf disease, caused by rice black-streaked dwarf virus (RBSDV), is a serious constraint in Chinese rice production. Breeding disease-resistant varieties through multigene aggregation is considered an effective way to control diseases, but few disease-resistant resources have been characterized thus far. To develop novel resources for resistance to RBSDV through CRISPR/Cas9-mediated genome editing, a guide RNA sequence targeting exon 1 of was designed and cloned into a binary vector, pHUE401. This recombinant vector was used to generate mutations in the rice cultivar Nipponbare via -mediated transformation. This approach produced heritable homozygous mutations in the transgene-free T1 gene... More
Rice black-streaked dwarf disease, caused by rice black-streaked dwarf virus (RBSDV), is a serious constraint in Chinese rice production. Breeding disease-resistant varieties through multigene aggregation is considered an effective way to control diseases, but few disease-resistant resources have been characterized thus far. To develop novel resources for resistance to RBSDV through CRISPR/Cas9-mediated genome editing, a guide RNA sequence targeting exon 1 of was designed and cloned into a binary vector, pHUE401. This recombinant vector was used to generate mutations in the rice cultivar Nipponbare via -mediated transformation. This approach produced heritable homozygous mutations in the transgene-free T1 generation. Sequence analysis of the target region from T1 transgenic plants identified 3 bp deletion mutants, and analysis of the predicted amino acid sequence identified one amino acid deletion in mutants that possess near full-length eIF4G. Furthermore, our data suggest that may plays an important role in rice normal development, as there were no knock-out homozygous mutants in T1 generation plants. When homozygous mutant lines were inoculated with RBSDV, they exhibited enhanced tolerance to virus infection, without visibly affecting plant growth and development. However, the mutant plants showed the same sensitivity to rice stripe virus (RSV) infection as wild-type plants. Notably, the wild-type and mutant N-termini of eIF4G interacted directly with RBSDV P8 in yeast and in planta. Additionally, compared to wild-type plants, the transcript level was reduced twofold in the mutant plants. These results indicate that site-specific mutation of rice successfully conferred partial resistance specific to RBSDV associated with less transcription of in mutants. Therefore, this study demonstrates that the novel alleles generated by CRISPR/Cas9 represent valuable disease-resistant resources that can be used to develop RBSDV-resistant varieties.
