![]() Tomato ( Solanum lycopersicum L.) is the most cultivated and consumed vegetable worldwide, with more than 186.8 million tons harvested in 2020. Our results showed, for the first time, a reduced susceptibility to Phytophtora infestans in pmr4 tomato mutants confirming the role of KO PMR4 in providing broad-spectrum protection against pathogens. ![]() The four SM lines were sequenced using Illumina whole-genome sequencing for deeper characterization without exhibiting any evidence of mutations in the candidate off-target regions. infestans, and four of them, fully knocked out at the PMR4 locus, showed reduced disease symptoms (reduction in susceptibility from 55 to 80%) compared to control plants. Selected edited lines were inoculated with P. 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). The different sgRNAs caused mutation frequencies ranging from 22.1 to 100% and alternatively precise insertions (sgRNA6) or deletions (sgRNA7, sgRNA1, and sgRNA8). ![]() Thirty-five plants (twenty-six SM and nine OX) were selected and screened to identify the CRISPR/Cas9-induced mutations. 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). Moreover, PMR4 knock-down in potato has been shown to confer tolerance to LB. Previous studies on Arabidopsis and tomato have highlighted that knock-out mutants of the PMR4 susceptibility gene are tolerant to powdery mildew. 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. 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.
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