TECHNICAL SESSION: Fungal and oomycete pathogens: New approaches to disease resistance
Targeted mutagenesis of basil candidate susceptibility gene DMR1 using CRISPR/Cas9
Natasha Navet - University of Hawaii at Manoa. Miaoying Tian- University of Hawaii at Manoa
Production of sweet basil is in jeopardy due to obligate-biotrophic oomycete Peronospora belbahrii causing basil downy mildew (BDM). Emerging biotechnology tools promises to broaden disease resistance and accelerate molecular understanding of basil-P. belbahrii interactions. The CRISPR/Cas9-mediated gene-editing has revolutionized crop improvement and functional genomics. The efficacy of this robust genomic tool in tetraploid sweet basil is being tested to knockout a potential susceptibility (S) gene. Genes exploited by pathogens in facilitating infection in plants are considered as plant S genes. ObDMR1, basil homolog of Arabidopsis DMR1 (Downy Mildew Resistance 1) is a S gene that upon knockout confers nearly complete resistance against Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis, was chosen for mutagenesis. Two binary constructs having one sgRNA and two sgRNAs respectively were generated targeting DMR1 sequence encoding the N-terminal half of the protein. A total of 41 transgenic lines were obtained via Agrobacterium-mediated stable transformation exhibiting no apparent phenotypic aberration. Mutation analysis of 24 first-generation transgenic lines carrying one sgRNA verified 29.16% chimeric plants and 20.83% with indels. Also, a mutation frequency of 11.76% was obtained from 17 transgenic plants carrying two sgRNAs. Mutants with all four homeoalleles mutated in second-generation will be subjected to pathogen inoculation assay. This study demonstrates the feasibility of CRISPR/Cas9 in basil functional genomics and to develop BDM resistant varieties.