CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars

作者：Ruiling Li, Alex Maioli, Zhe Yan, Yuling Bai, Danila Valentino, A. Milani, Valerio Pompili, Cinzia Comino, Sergio Lanteri, Andrea Moglia, Alberto Acquadro · 发表于：International Journal of Molecular Sciences · 年份：2022 · DOI：10.3390/ijms232314542 · 被引用次数：26 · 研究领域：CRISPR and Genetic Engineering、Plant Virus Research Studies、Insect-Plant Interactions and Control

Phytophthora infestans, the causal agent of late blight (LB) in tomato (Solanum lycopersicum L.), is a devastating disease and a serious concern for plant productivity. The presence of susceptibility (S) genes in plants facilitates pathogen proliferation; thus, disabling these genes may help provide a broad-spectrum and durable type of tolerance/resistance. Previous studies on Arabidopsis and tomato have highlighted that knock-out mutants of the PMR4 susceptibility gene are tolerant to powdery mildew. Moreover, PMR4 knock-down in potato has been shown to confer tolerance to LB. To verify the same effect in tomato in the present study, a CRISPR–Cas9 vector containing four single guide RNAs (sgRNAs: sgRNA1, sgRNA6, sgRNA7, and sgRNA8), targeting as many SlPMR4 regions, was introduced via Agrobacterium-tumefaciens-mediated transformation into two widely grown Italian tomato cultivars: ‘San Marzano’ (SM) and ‘Oxheart’ (OX). Thirty-five plants (twenty-six SM and nine OX) were selected and screened to identify the CRISPR/Cas9-induced mutations. The different sgRNAs caused mutation frequencies ranging from 22.1 to 100% and alternatively precise insertions (sgRNA6) or deletions (sgRNA7, sgRNA1, and sgRNA8). Notably, sgRNA7 induced in seven SM genotypes a −7 bp deletion in the homozygous status, whereas sgRNA8 led to the production of fifteen SM genotypes with a biallelic mutation (−7 bp and −2 bp). Selected edited lines were inoculated with P. infestans, and four of them, fully knock...