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

Abstract

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.

Genomic Analysis Highlights Putative Defective Susceptibility Genes in Tomato Germplasm

“…Although we did not delve into these effects in detail, they are worth monitoring in order to gain a deeper understanding of altered S-genes. Among the 119 SNPs, 10 were validated in 10 genotypes readily available within the research group facilities ( , accessed on 1 April 2022) through Sanger sequencing with a 90% validation rate ( Table S3 ); indeed, some non-validated SNPs were mutations detected in a heterozygous condition or possessed the same allelic profile as the reference; the emergence of such heterozygous/reference-like SNPs during the validation step can be explained by the high genetic diversity existing within the analyzed germplasm set ( Figure 3 ), as observed by Li et al [ 15 ].…”

“…The incidence of deleterious SNPs in S-genes in a heterozygous condition was comparatively lesser than that of homozygous ones, as observed in both the complete germplasm collection (ALL) and the BIG tomato varieties ( Table 2 , Figure 4 ). This frequency may be due to the genetic structure of tomato as an inbred species, which tends to have a low number of heterozygous mutations [ 15 ]. However, the number appears relatively high because such mutations, although harmful, can be maintained in the genome if the normal allelic copy continues to function.…”

“…Mutations in the PMR4 (powdery-mildew-resistant) gene have also been found to induce not only resistance to powdery mildew but also late blight [ 14 ]. Its CRISPR/Cas9-based disabling reduced susceptibility to both pathogens in tomato [ 15 , 16 ] and in potato to late blight as well as several diseases [ 17 ]. PMR5 , a pectin acetyltransferase, and PMR6, a pectate-lyase-like gene, increased powdery mildew resistance in their Arabidopsis mutants, despite the mutants showing higher susceptibility to multiple strains of Botrytis cinerea [ 18 , 19 , 20 ].…”

Recent Advances in Tomato Gene Editing

“…The biochemical characterization of DMR6 enzymes suggests that they play a role in converting salicylic acid to its inactive form [ 157 ]. In other work, the CRISPR/Cas9 system has been used to mutate the susceptibility gene POWDERY MILDEW RESISTANCE 4 ( PMR4 ) Solyc07g053980 , with the product functioning as a callose synthase conferring resistance to O. neolycopersici and Phytophtora infestans in tomatoes [ 158 , 159 ]. These results demonstrate that the CRISPR/Cas9 system is suitable for facilitating broad resistance to bacterial and fungal pathogens by precisely targeting susceptibility genes and negative regulators involved in the plant defense mechanism.…”

New insights on the role ofSlDMR6-1in drought avoidance in tomato

“…A classic example of a S-gene is Mildew resistance locus O (Mlo1) (Bü et al, 1997), of which loss-of-function natural mutants have been exploited for over 70 years in barley breeding programs (Piffanelli et al, 2002). In tomato, successful examples of CRISPR/Cas9 genome editing aimed at disabling S-genes have been reported to confer resistance against distinct classes of pathogens like viruses (Atarashi et al, 2020;Yoon et al, 2020;Kuroiwa et al, 2022), bacteria (Ortigosa et al, 2019;De Toledo Thomazella et al, 2021), fungi (Nekrasov er al, 2017;Santillán Martínez et al, 2020) and oomycetes (De Toledo Thomazella et al, 2021;Li et al, 2022). Among S-genes, DOWNY MILDEW RESISTANCE 6 (DMR6) is particularly interesting.…”