A single transcript CRISPR/Cas9 mediated mutagenesis of CaERF28 confers anthracnose resistance in chilli pepper (Capsicum annuum L.)

T-DNA-free homozygous mutant lines developed through a single transcript CRISPR/Cas9 system harboring the desired modification in the CaERF28 locus exhibited significantly enhanced resistance to the anthracnose pathogen Colletotrichum truncatum coupled with the improved expression of defense responsive genes. Anthracnose, caused by Colletotrichum species, is a major disease of chilli (Capsicum annuum) accounting for significant pre- and post-harvest yield losses across the tropical and subtropical regions of the world. Management of chilli anthracnose using traditional methods have not met with noticeable success. In the present study, we have demonstrated an enhanced anthracnose resistance through a single transcript unit CRISPR/Cas9 mediated alteration of the susceptibility gene CaERF28 in C. annuum. A construct with a single Pol II promoter-driven expression of Cas9, sgRNA and a hammerhead ribozyme (RZ) was designed to modify the CaERF28 gene in the susceptible chilli genotype Arka Lohit. Fourty-five C-ERF28-induced mutant lines (72.5%) were identified from 62 T0 transgenic plants. Further, simultaneously targeted multiple sites within CaERF28 showed increased mutation (85.7%) efficiency. DNA sequence analysis showed that these plants harboured multiple InDels at the target site. The allelic mutants of C-ERF28 were transferred to the following generations by simple Mendelian inheritance. Segregation in the T1 and T2 generations resulted in the identification of T-DNA free and marker-free C-ERF28 mutant lines. Five homozygous mutants demonstrated enhanced resistance to anthracnose compared to wild type as demonstrated by reduced spore count and fungal growth as well as induced expression of defense-related genes. Our results demonstrated that the STU-CRISPR/Cas9 mediated editing of the CaERF28 gene is a rapid, safe and versatile approach for enhancing anthracnose resistance in chilli pepper and pave way for its utilization in the improvement of other solanaceous crops.