CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance.

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 (...

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Main Authors: Jeremieh Abram R Hasley, Natasha Navet, Miaoying Tian
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2021-01-01
Series:PLoS ONE
Online Access:https://doi.org/10.1371/journal.pone.0253245
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author Jeremieh Abram R Hasley
Natasha Navet
Miaoying Tian
author_facet Jeremieh Abram R Hasley
Natasha Navet
Miaoying Tian
author_sort Jeremieh Abram R Hasley
collection DOAJ
description 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.
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spelling doaj.art-2fdec371349d427aafce1dbfebcdb4c92022-12-21T21:27:12ZengPublic Library of Science (PLoS)PLoS ONE1932-62032021-01-01166e025324510.1371/journal.pone.0253245CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance.Jeremieh Abram R HasleyNatasha NavetMiaoying TianSweet 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.https://doi.org/10.1371/journal.pone.0253245
spellingShingle Jeremieh Abram R Hasley
Natasha Navet
Miaoying Tian
CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance.
PLoS ONE
title CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance.
title_full CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance.
title_fullStr CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance.
title_full_unstemmed CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance.
title_short CRISPR/Cas9-mediated mutagenesis of sweet basil candidate susceptibility gene ObDMR6 enhances downy mildew resistance.
title_sort crispr cas9 mediated mutagenesis of sweet basil candidate susceptibility gene obdmr6 enhances downy mildew resistance
url https://doi.org/10.1371/journal.pone.0253245
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AT miaoyingtian crisprcas9mediatedmutagenesisofsweetbasilcandidatesusceptibilitygeneobdmr6enhancesdownymildewresistance