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 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.

[1]  Xiaojing Liu,et al.  Effects of sgRNA length and number on gene editing efficiency and predicted mutations generated in rice , 2021, The Crop Journal.

[2]  B. Staskawicz,et al.  Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance , 2021, Proceedings of the National Academy of Sciences.

[3]  Yulin Cheng,et al.  Advances in application of genome editing in tomato and recent development of genome editing technology , 2021, Theoretical and Applied Genetics.

[4]  D. Halterman,et al.  Phytophthora infestans (Late blight) Infection Assay in a Detached Leaf of Potato. , 2021, Bio-protocol.

[5]  A. Moglia,et al.  Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L. , 2020, Frontiers in Plant Science.

[6]  R. Visser,et al.  CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew , 2020, BMC Plant Biology.

[7]  Yuriko Osakabe,et al.  Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering , 2020, BMC Plant Biology.

[8]  Keith Lindsey,et al.  CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects , 2020, Advanced science.

[9]  R. Visser,et al.  Breeding Has Increased the Diversity of Cultivated Tomato in The Netherlands , 2019, Front. Plant Sci..

[10]  F. Badeck,et al.  Changes in yield components, morphological, physiological and fruit quality traits in processing tomato cultivated in Italy since the 1930’s , 2019, Scientia Horticulturae.

[11]  Hongliang Zhu,et al.  CRISPR technology is revolutionizing the improvement of tomato and other fruit crops , 2019, Horticulture Research.

[12]  R. Deshmukh,et al.  Mutation Breeding in Tomato: Advances, Applicability and Challenges , 2019, Plants.

[13]  V. Nekrasov,et al.  CRISPR/Cas precision: do we need to worry about off-targeting in plants? , 2018, Plant Cell Reports.

[14]  M. S. Mukhtar,et al.  Genome Editing: Targeting Susceptibility Genes for Plant Disease Resistance. , 2018, Trends in biotechnology.

[15]  Anob M. Chakrabarti,et al.  Target-Specific Precision of CRISPR-Mediated Genome Editing , 2018, bioRxiv.

[16]  Z. Nimchuk,et al.  Cutting Edge Genetics: CRISPR/Cas9 Editing of Plant Genomes. , 2018, Plant & cell physiology.

[17]  G. Venkataraman,et al.  CRISPR for Crop Improvement: An Update Review , 2018, Front. Plant Sci..

[18]  Yuriko Osakabe,et al.  Efficient Multiplex Genome Editing Induces Precise, and Self-Ligated Type Mutations in Tomato Plants , 2018, Front. Plant Sci..

[19]  Reza Ehsani,et al.  Detection of multi-tomato leaf diseases (late blight, target and bacterial spots) in different stages by using a spectral-based sensor , 2018, Scientific Reports.

[20]  B. van Steensel,et al.  Easy quantification of template-directed CRISPR/Cas9 editing , 2017, bioRxiv.

[21]  R. Panstruga,et al.  The powdery mildew-resistant Arabidopsis mlo2 mlo6 mlo12 triple mutant displays altered infection phenotypes with diverse types of phytopathogens , 2017, Scientific Reports.

[22]  Congmao Wang,et al.  Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion , 2017, Scientific Reports.

[23]  M. C. Baracat-Pereira,et al.  Comparative analysis of constitutive proteome between resistant and susceptible tomato genotypes regarding to late blight , 2017, Functional & Integrative Genomics.

[24]  A. May,et al.  DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks. , 2016, Molecular cell.

[25]  Michael Lynch,et al.  Evolution of the Insertion-Deletion Mutation Rate Across the Tree of Life , 2016, G3: Genes, Genomes, Genetics.

[26]  R. Visser,et al.  Silencing of six susceptibility genes results in potato late blight resistance , 2016, Transgenic Research.

[27]  G. Lu,et al.  CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations , 2016, Scientific Reports.

[28]  T. Takumi,et al.  Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system , 2016, Scientific Reports.

[29]  E. Zimnoch-Guzowska,et al.  Virulence and aggressiveness of Phytophthora infestans isolates collected in Poland from potato and tomato plants identified no strong specificity , 2016, European Journal of Plant Pathology.

[30]  Charles E. Vejnar,et al.  CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo , 2015, Nature Methods.

[31]  Christine B. Peterson,et al.  TreeQTL: hierarchical error control for eQTL findings , 2015, bioRxiv.

[32]  H. Ashrafi,et al.  Detached-Leaflet Evaluation of Tomato Germplasm for Late Blight Resistance and Its Correspondence to Field and Greenhouse Screenings. , 2015, Plant disease.

[33]  Z. Lippman,et al.  Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System1 , 2014, Plant Physiology.

[34]  F. Takken,et al.  Susceptibility genes 101: how to be a good host. , 2014, Annual review of phytopathology.

[35]  M. Foolad,et al.  Response of accessions within tomato wild species, Solanum pimpinellifolium to late blight , 2014 .

[36]  Feng Zhang,et al.  Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA , 2014, Cell.

[37]  V. Bergougnoux.  The history of tomato: from domestication to biopharming. , 2014, Biotechnology advances.

[38]  R. Visser,et al.  Powdery Mildew Resistance in Tomato by Impairment of SlPMR4 and SlDMR1 , 2013, PloS one.

[39]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[40]  Marzena Nowakowska,et al.  Potato and Tomato Late Blight Caused by Phytophthora infestans: An Overview of Pathology and Resistance Breeding. , 2012, Plant disease.

[41]  H. Kanamori,et al.  Molecular spectrum of somaclonal variation in regenerated rice revealed by whole-genome sequencing. , 2012, Plant & cell physiology.

[42]  Konstantin Severinov,et al.  Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence , 2011, Proceedings of the National Academy of Sciences.

[43]  Richard M. Clark,et al.  The Rate and Molecular Spectrum of Spontaneous Mutations in Arabidopsis thaliana , 2010, Science.

[44]  R. Visser,et al.  Loss of susceptibility as a novel breeding strategy for durable and broad-spectrum resistance , 2009, Molecular Breeding.

[45]  W. Fry,et al.  Phytophthora infestans: the plant (and R gene) destroyer. , 2008, Molecular plant pathology.

[46]  J. Ton,et al.  Interplay between JA, SA and ABA signalling during basal and induced resistance against Pseudomonas syringae and Alternaria brassicicola. , 2007, The Plant journal : for cell and molecular biology.

[47]  Jörg Durner,et al.  Conserved requirement for a plant host cell protein in powdery mildew pathogenesis , 2006, Nature Genetics.

[48]  S. Simpson.  How to Be a Good Host , 2004, Science.

[49]  D. S. St. Clair,et al.  Fine mapping of three quantitative trait loci for late blight resistance in tomato using near isogenic lines (NILs) and sub-NILs , 2004, Theoretical and Applied Genetics.

[50]  R. Horsch,et al.  Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens , 1986, Plant Cell Reports.

[51]  Nicolai Strizhov,et al.  An Arabidopsis Callose Synthase, GSL5, Is Required for Wound and Papillary Callose Formation Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.016097. , 2003, The Plant Cell Online.

[52]  Monica Stein,et al.  Loss of a Callose Synthase Results in Salicylic Acid-Dependent Disease Resistance , 2003, Science.

[53]  N. Eckardt.  Plant Disease Susceptibility Genes? , 2002, The Plant Cell Online.

[54]  S. Somerville,et al.  Isolation and characterization of powdery mildew-resistant Arabidopsis mutants. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[55]  V. Henriquez.  Breeding for resistance. , 2000 .

[56]  M. Wolter,et al.  The Barley Mlo Gene: A Novel Control Element of Plant Pathogen Resistance , 1997, Cell.

[57]  J. Jinks,et al.  Spontaneous variability of single isolates of Phytophthora infestans , 1970 .

[58]  J. Jinks,et al.  Spontaneous variability of single isolates of Phytophthora infestans. I. Cultural variation , 1968 .