CRISPR/Cas9-mediated resurrection of tobacco NB-LRR class virus resistance gene from a susceptible allele with partial duplication

It is still difficult to manipulate the nucleotide-binding site-leucine-rich repeat (NB-LRR) class disease resistance genes because of their large multigenic family. Here, we report the successful application of CRISPR/Cas9 to resurrect a functional allele from a susceptible allele of the N′ tobamovirus resistance gene. The susceptible alleles of N′ from some Nicotiana tabacum cultivars (Nt-n′) have a partial duplication of the N′ coding sequence upstream of a complete coding sequence of the N′ gene, which likely abolished the N′-mediated resistance. We first established a transgenic tobacco line expressing Cas9 under the control of a chemically inducible promoter. The plant line was retransformed with a construct expressing a guide RNA targeting the sequences common to the duplicated partial sequence in the upstream and the complete sequence in the downstream. The T₀ transformants had different ratios of the sequences devoid of the duplicated partial sequence. Sequencing proved that some of them had sequences identical to that of the functional N′ gene, suggesting the successful resurrection of the functional N′ gene. The resurrected allele, N′^-R, was inherited by a few T₁ progenies and subsequent generations with the least mutation at the target site under Cas9-uninduced conditions. The plants homozygous for N′^-R showed resistance to a tobamovirus, indicating that the resurrected N′^-R allele is functional.