CRISPR/Cas9-Mediated Mutation in XSP10 and SlSAMT Genes Impart Genetic Tolerance to Fusarium Wilt Disease of Tomato (Solanum lycopersicum L.)

Debbarma, J and Saikia, B and Singha, D L and Das, D and Keot, A J and Maharana, J and Velmurugan, N and Arunkumar, K P and Reddy, P S and Chikkaputtaiah, C (2023) CRISPR/Cas9-Mediated Mutation in XSP10 and SlSAMT Genes Impart Genetic Tolerance to Fusarium Wilt Disease of Tomato (Solanum lycopersicum L.). Genes (TSI), 14. 01-22. ISSN 2073-4425

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Abstract

Fusarium wilt is a major devastating fungal disease of tomato (Solanum lycopersicum L.) caused by Fusarium oxysporum f. sp. lycopersici (Fol) which reduces the yield and production. Xylem sap protein 10 (XSP10) and Salicylic acid methyl transferase (SlSAMT) are two putative negative regulatory genes associated with Fusarium wilt of tomato. Fusarium wilt tolerance in tomato can be developed by targeting these susceptible (S) genes. Due to its efficiency, high target specificity, and versatility, CRISPR/Cas9 has emerged as one of the most promising techniques for knocking out disease susceptibility genes in a variety of model and agricultural plants to increase tolerance/resistance to various plant diseases in recent years. Though alternative methods, like RNAi, have been attempted to knock down these two S genes in order to confer resistance in tomato against Fusarium wilt, there has been no report of employing the CRISPR/Cas9 system for this specific intent. In this study, we provide a comprehensive downstream analysis of the two S genes via CRISPR/Cas9-mediated editing of single (XSP10 and SlSAMT individually) and dual-gene (XSP10 and SlSAMT simultaneously). Prior to directly advancing on to the generation of stable lines, the editing efficacy of the sgRNA-Cas9 complex was first validated using single cell (protoplast) transformation. In the transient leaf disc assay, the dual-gene editing showed strong phenotypic tolerance to Fusarium wilt disease with INDEL mutations than single-gene editing. In stable genetic transformation of tomato at the GE1 generation, dual-gene CRISPR transformants of XSP10 and SlSAMT primarily exhibited INDEL mutations than single-gene-edited lines. The dual-gene CRISPR-edited lines (CRELs) of XSP10 and SlSAMT at GE1 generation conferred a strong phenotypic tolerance to Fusarium wilt disease compared to single-gene-edited lines. Taken together, the reverse genetic studies in transient and stable lines of tomato revealed that, XSP10 and SlSAMT function together as negative regulators in conferring genetic tolerance to Fusariumwilt disease.

Item Type:	Article
Divisions:	Global Research Program - Accelerated Crop Improvement
CRP:	UNSPECIFIED
Uncontrolled Keywords:	CRISPR/Cas9, dual-gene editing, INDEL, Fusarium wilt, genetic tolerance, tomato
Subjects:	Others > Genetic Engineering Others > Genetics and Genomics
Depositing User:	Mr Nagaraju T
Date Deposited:	22 Jun 2023 10:49
Last Modified:	22 Jun 2023 10:49
URI:	http://oar.icrisat.org/id/eprint/12118
Official URL:	https://www.mdpi.com/2073-4425/14/2/488
Projects:	Early Career Research Grant, Focused Basic Research Network Project on Genome Editing
Funders:	Science and Engineering Research Board (SERB), Council of Scientific and Industrial Research (CSIR)
Acknowledgement:	Authors would like to acknowledge Director, CSIR-NEIST, Jorhat for providing necessary facilities to conduct this study.
Links:	Google Scholar

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