Dataset Information

CRISPR/Cas9-mediated VvPR4b editing decreases downy mildew resistance in grapevine (Vitis vinifera L.).

ABSTRACT: Downy mildew of grapevine (Vitis vinifera L.), caused by the oomycete pathogen Plasmopara viticola, is one of the most serious concerns for grape production worldwide. It has been widely reported that the pathogenesis-related 4 (PR4) protein plays important roles in plant resistance to diseases. However, little is known about the role of PR4 in the defense of grapevine against P. viticola. In this study, we engineered loss-of-function mutations in the VvPR4b gene from the cultivar "Thompson Seedless" using the CRISPR/Cas9 system and evaluated the consequences for downy mildew resistance. Sequencing results showed that deletions were the main type of mutation introduced and that no off-target events occurred. Infection assays using leaf discs showed that, compared to wild-type plants, the VvPR4b knockout lines had increased susceptibility to P. viticola. This was accompanied by reduced accumulation of reactive oxygen species around stomata. Measurement of the relative genomic abundance of P. viticola in VvPR4b knockout lines also demonstrated that the mutants had increased susceptibility to the pathogen. Our results confirm that VvPR4b plays an active role in the defense of grapevine against downy mildew.

SUBMITTER: Li MY

PROVIDER: S-EPMC7458914 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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CRISPR/Cas9-mediated VvPR4b editing decreases downy mildew resistance in grapevine (Vitis vinifera L.).

Li Meng-Yuan MY Jiao Yun-Tong YT Wang Yu-Ting YT Zhang Na N Wang Bian-Bian BB Liu Rui-Qi RQ Yin Xiao X Xu Yan Y Liu Guo-Tian GT

Horticulture research 20200901

Downy mildew of grapevine (Vitis vinifera L.), caused by the oomycete pathogen Plasmopara viticola, is one of the most serious concerns for grape production worldwide. It has been widely reported that the pathogenesis-related 4 (PR4) protein plays important roles in plant resistance to diseases. However, little is known about the role of PR4 in the defense of grapevine against P. viticola. In this study, we engineered loss-of-function mutations in the VvPR4b gene from ...[more]

PMID: 32922821

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Project 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.

Dataset Information

CRISPR/Cas9-mediated VvPR4b editing decreases downy mildew resistance in grapevine (Vitis vinifera L.).

Publications

CRISPR/Cas9-mediated <i>VvPR4b</i> editing decreases downy mildew resistance in grapevine (<i>Vitis vinifera</i> L.).

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