Abstract
Sweet basil (Ocimum basilicum) is an economically important allotetraploid (2n = 4x = 48) herb whose global production is threatened by downy mildew disease caused by the obligate biotrophic oomycete, Peronospora belbahrii. Generation of disease resistant cultivars by mutagenesis of susceptibility (S) genes via CRISPR/Cas9 is currently one of the most promising strategies to maintain favored traits while improving disease resistance. Previous studies have identified Arabidopsis DMR6 (Downy Mildew Resistance 6) as an S gene required for pathogenesis of the downy mildew-causing oomycete pathogen Hyaloperonospora arabidopsidis. In this study, a sweet basil homolog of DMR6, designated ObDMR6, was identified in the popular sweet basil cultivar Genoveser and found to exist with a high copy number in the genome with polymorphisms among the variants. Two CRISPR/Cas9 constructs expressing one or two single guide RNAs (sgRNAs) targeting the conserved regions of ObDMR6 variants were generated and used to transform Genoveser via Agrobacterium-mediated transformation. 56 T0 lines were generated, and mutations of ObDMR6 were detected by analyzing the Sanger sequencing chromatograms of an ObDMR6 fragment using the Interference of CRISPR Edits (ICE) software. Among 54 lines containing mutations in the targeted sites, 13 had an indel percentage greater than 96% suggesting a near-complete knockout (KO) of ObDMR6. Three representative transgene-free lines with near-complete KO of ObDMR6 determined by ICE were identified in the T1 segregating populations derived from three independent T0 lines. The mutations were further confirmed using amplicon deep sequencing. Disease assays conducted on T2 seedlings of the above T1 lines showed a reduction in production of sporangia by 61–68% compared to the wild-type plants and 69–93% reduction in relative pathogen biomass determined by quantitative PCR (qPCR). This study not only has generated transgene-free sweet basil varieties with improved downy mildew resistance, but also contributed to our understanding of the molecular interactions of sweet basil-P. belbahrii.
Highlights
Sweet basil (Ocimum basilicum) is an economically important crop that is cultivated for its favorable biochemical properties used in the culinary, pharmaceutical, cosmetic, and biodiesel industries [1,2,3]
Three transcripts with significant homology to AtDMR6 were identified with E-values of 2e-150, 4e-124 and 2e123, respectively. comp38697_c0_seq1 contained a full open reading frame (ORF) of 1011 bp, which was translated to a protein of 336 amino acids
Using the amino acid sequence of comp38697_c0_seq1, we performed BLASTP against Arabidopsis protein database Araport11 using The Arabidopsis Information Resource (TAIR) and the best hit was AtDMR6, suggesting that the transcript comp38697_c0_seq1 encodes a DMR6 ortholog in O. basilicum, thereby designated ObDMR6. comp38697_c1_seq3 and comp38697_c1_seq2 contained partial ORF sequences, which were highly similar to ORF comp38697_c0_seq1 with a few single nucleotide polymorphisms (SNPs) among them, suggesting that they are the variants of ObDMR6 (S3 Appendix)
Summary
Sweet basil (Ocimum basilicum) is an economically important crop that is cultivated for its favorable biochemical properties used in the culinary, pharmaceutical, cosmetic, and biodiesel industries [1,2,3]. Basil downy mildew (BDM) caused by the obligate biotrophic oomycete Peronospora belbahrii threatens the global production of sweet basil [5]. This devastating foliar disease can cause damage at most growth stages of sweet basil [6]. BDM is characterized as a dark-hued mat observed on the abaxial surface of infected leaves caused by the emergence of sporangiophores and sporangia [5, 7]. On the adaxial surface, infected leaves develop chlorotic lesions that lead to necrosis and abscission of the leaves [7], resulting in unmarketable products
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