Abstract
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 eIF4G was designed and cloned into a binary vector, pHUE401. This recombinant vector was used to generate mutations in the rice cultivar Nipponbare via Agrobacterium-mediated transformation. This approach produced heritable homozygous mutations in the transgene-free T1 generation. Sequence analysis of the eIF4G 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 eIF4G may plays an important role in rice normal development, as there were no eIF4G 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 eif4g 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 eIF4G transcript level was reduced twofold in the mutant plants. These results indicate that site-specific mutation of rice eIF4G successfully conferred partial resistance specific to RBSDV associated with less transcription of eIF4G in mutants. Therefore, this study demonstrates that the novel eIF4G alleles generated by CRISPR/Cas9 represent valuable disease-resistant resources that can be used to develop RBSDV-resistant varieties.
Highlights
Rice black-streaked dwarf virus (RBSDV), transmitted by the small brown planthopper (SBPH: Laodelphax striatellus Fallén), mainly causes rice black-streaked dwarf disease and maize rough dwarf diseases, which lead to serious cereal crop yield losses in China [1,2].Rice plants infected by RBSDV typically exhibit acute growth abnormalities, such as severe dwarfism, dark green and stiff leaves, and tumors on leaves and stems [3,4,5]
To design sgRNAs, we identified eukaryotic translation initiation factor 4G (eIF4G) target sgRNA sequences using clustered regularly interspersed palindromic repeats (CRISPR) PLANT tools. sgRNAs targeting the first exon with the Bsg I restriction site were used for genome editing (Figure 1A)
We cloned this sgRNA into a binary vector [26], and this pHUE401-eIF4G CRISPR/Cas9 construct was introduced into rice plants (Nipponbare) by Agrobacterium-mediated transformation
Summary
Rice black-streaked dwarf virus (RBSDV), transmitted by the small brown planthopper (SBPH: Laodelphax striatellus Fallén), mainly causes rice black-streaked dwarf disease and maize rough dwarf diseases, which lead to serious cereal crop yield losses in China [1,2]. Rice plants infected by RBSDV typically exhibit acute growth abnormalities, such as severe dwarfism, dark green and stiff leaves, and tumors on leaves and stems [3,4,5]. RBSDV is a double-stranded RNA virus and belongs to the genus Fijivirus within the family Reoviridae [7]. RBSDV virus particles are icosahedral, with double-layer shells containing spikes, and contain 10 double-stranded RNAs, S1–S10 [8,9]. SP8 encoded by SRBSDV, a closely related Fijivirus, can interact with rice auxin response factor 17
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
