NDLI: CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew

Content Provider	Springer Nature : BioMed Central
Author	Santillán Martínez, Miguel I. Bracuto, Valentina Koseoglou, Eleni Appiano, Michela Jacobsen, Evert Visser, Richard G. F. Wolters, Anne-Marie A. Bai, Yuling
Abstract	Background The development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) gene PowderyMildewResistance 4 (PMR4) in tomato has been shown to enhance resistance against the powdery mildew pathogen Oidium neolycopersici (On). Results To study whether full knock-out of the tomato PMR4 gene would result in a higher level of resistance than in the RNAi-silenced transgenic plants we generated tomato PMR4 CRISPR mutants. We used a CRISPR/Cas9 construct containing four single-guide RNAs (sgRNAs) targeting the tomato PMR4 gene to increase the possibility of large deletions in the mutants. After PCR-based selection and sequencing of transformants, we identified five different mutation events, including deletions from 4 to 900-bp, a 1-bp insertion and a 892-bp inversion. These mutants all showed reduced susceptibility to On based on visual scoring of disease symptoms and quantification of relative fungal biomass. Histological observations revealed a significantly higher occurrence of hypersensitive response-like cell death at sites of fungal infection in the pmr4 mutants compared to wild-type plants. Both haustorial formation and hyphal growth were diminished but not completely inhibited in the mutants. Conclusion CRISPR/Cas-9 targeted mutagenesis of the tomato PMR4 gene resulted in mutants with reduced but not complete loss of susceptibility to the PM pathogen On. Our study demonstrates the efficiency and versatility of the CRISPR/Cas9 system as a powerful tool to study and characterize S-genes by generating different types of mutations.
Related Links	https://bmcplantbiol.biomedcentral.com/counter/pdf/10.1186/s12870-020-02497-y.pdf
Ending Page	13
Page Count	13
Starting Page	1
File Format	HTM / HTML
ISSN	14712229
DOI	10.1186/s12870-020-02497-y
Journal	BMC Plant Biology
Issue Number	1
Volume Number	20
Language	English
Publisher	BioMed Central
Publisher Date	2020-06-19
Access Restriction	Open
Subject Keyword	Plant Sciences Agriculture Tree Biology PMR4 CRISPR/Cas9 Targeted mutagenesis Powdery mildew Susceptibility gene
Content Type	Text
Resource Type	Article
Subject	Plant Science
Journal Impact Factor	4.3/2023
5-Year Journal Impact Factor	5.2/2023

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