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

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...

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Autores principales: Li, Ruiling, Maioli, Alex, Yan, Zhe, Bai, Yuling, Valentino, Danila, Milani, Anna Maria, Pompili, Valerio, Comino, Cinzia, Lanteri, Sergio, Moglia, Andrea, Acquadro, Alberto
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9735651/
https://www.ncbi.nlm.nih.gov/pubmed/36498869
http://dx.doi.org/10.3390/ijms232314542
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author Li, Ruiling
Maioli, Alex
Yan, Zhe
Bai, Yuling
Valentino, Danila
Milani, Anna Maria
Pompili, Valerio
Comino, Cinzia
Lanteri, Sergio
Moglia, Andrea
Acquadro, Alberto
author_facet Li, Ruiling
Maioli, Alex
Yan, Zhe
Bai, Yuling
Valentino, Danila
Milani, Anna Maria
Pompili, Valerio
Comino, Cinzia
Lanteri, Sergio
Moglia, Andrea
Acquadro, Alberto
author_sort Li, Ruiling
collection PubMed
description 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 knocked out at the PMR4 locus, showed reduced disease symptoms (reduction in susceptibility from 55 to 80%) compared to control plants. 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. 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.
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spelling pubmed-97356512022-12-11 CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars Li, Ruiling Maioli, Alex Yan, Zhe Bai, Yuling Valentino, Danila Milani, Anna Maria Pompili, Valerio Comino, Cinzia Lanteri, Sergio Moglia, Andrea Acquadro, Alberto Int J Mol Sci Article 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 knocked out at the PMR4 locus, showed reduced disease symptoms (reduction in susceptibility from 55 to 80%) compared to control plants. 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. 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. MDPI 2022-11-22 /pmc/articles/PMC9735651/ /pubmed/36498869 http://dx.doi.org/10.3390/ijms232314542 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Li, Ruiling
Maioli, Alex
Yan, Zhe
Bai, Yuling
Valentino, Danila
Milani, Anna Maria
Pompili, Valerio
Comino, Cinzia
Lanteri, Sergio
Moglia, Andrea
Acquadro, Alberto
CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars
title CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars
title_full CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars
title_fullStr CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars
title_full_unstemmed CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars
title_short CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars
title_sort crispr/cas9-based knock-out of the pmr4 gene reduces susceptibility to late blight in two tomato cultivars
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9735651/
https://www.ncbi.nlm.nih.gov/pubmed/36498869
http://dx.doi.org/10.3390/ijms232314542
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