CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera)

Dong-Yan Wan,Yang Hu,Shunyuan Xiao,Ying-Qiang Wen,Yuan Cheng,Yuejin Wang,Ye Guo

doi:10.1038/s41438-020-0339-8

Abstract

Grapevine (Vitis vinifera), one of the most economically important fruit crops in the world, suffers significant yield losses from powdery mildew, a major fungal disease caused by Erysiphe necator. In addition to suppressing host immunity, phytopathogens modulate host proteins termed susceptibility (S) factors to promote their proliferation in plants. In this study, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) technology was used to enable the targeted mutagenesis of MLO (mildew resistance Locus O) family genes that are thought to serve as S factors for powdery mildew fungi. Small deletions or insertions were induced in one or both alleles of two grapevine MLO genes, VvMLO3 and VvMLO4, in the transgenic plantlets of the powdery mildew-susceptible cultivar Thompson Seedless. The editing efficiency achieved with different CRISPR/Cas9 constructs varied from 0 to 38.5%. Among the 20 VvMLO3/4-edited lines obtained, one was homozygous for a single mutation, three harbored biallelic mutations, seven were heterozygous for the mutations, and nine were chimeric, as indicated by the presence of more than two mutated alleles in each line. Six of the 20 VvMLO3/4-edited grapevine lines showed normal growth, while the remaining lines exhibited senescence-like chlorosis and necrosis. Importantly, four VvMLO3-edited lines showed enhanced resistance to powdery mildew, which was associated with host cell death, cell wall apposition (CWA) and H2O2 accumulation. Taken together, our results demonstrate that CRISPR/Cas9 genome-editing technology can be successfully used to induce targeted mutations in genes of interest to improve traits of economic importance, such as disease resistance in grapevines.

Highlights

Grapevine is one of the most extensively cultivated and economically valuable horticultural crops in the world, with 7.6 million hectares in production and an annual value of ~$3.6 billion in wine export markets alone (Organisation Internationale de la Vigne et du Vin, 2017)
Grape production is limited by various diseases, including powdery mildew caused by the fungus Erysiphe necator
Our work suggests that CRISPR/Cas9-targeted mutagenesis is a valuable tool for generating disease-resistant grapevines

Summary

Introduction

Grapevine is one of the most extensively cultivated and economically valuable horticultural crops in the world, with 7.6 million hectares in production and an annual value of ~$3.6 billion in wine export markets alone (Organisation Internationale de la Vigne et du Vin, 2017). Wan et al Horticulture Research (2020)7:116 based on the phenotypes caused by mutations of a target gene)[2]. The introduction of functional mutations through targeted genome editing is a powerful reverse genetics approach for understanding gene function. In 2013, three research groups simultaneously reported the use of the CRISPRCas[9] system for targeted genome modification in Arabidopsis thaliana (Arabidopsis), tobacco, rice and wheat for the first time[4,5,6]. This system has been successfully applied in maize[7], cotton[8], and other plants. There have been only a few reports on the use of CRISPR-Cas[9] for genome editing in grapevine[5,9,10,11]

Methods

Results

Conclusion