Mutation introduced in DDTFR10/A gene of ethylene response element-binding protein (EREBP) family through CRISPR/Cas9 genome editing confers increased Fusarium wilt tolerance in tomato

Abstract

We investigated the role of the DDTFR10/A gene of the ethylene response element-binding protein (EREBP) family through the CRISPR/Cas9 genome editing approach. The associated role of this gene in tomato fruit ripening was known. The involvement of ripening-regulatory proteins in plant defense has been documented; therefore, to find the involvement of the DDTFR10/A gene in host susceptibility, we introduced the mutation in DDTFR10/A gene through CRISPR/cas9 in the genome of the tomato plant. The 50% biallelic and 50% homozygous mutations were observed in the T₀ generation. The CRISPR/Cas9 edited plants showed 40% reduced symptoms of Fusarium wilt compared to control plants (non-edited). The DDTFR10/A gene expression in tomato plants was evaluated against biotic (Fusarium wilt) and abiotic (salinity) stresses, and the upregulated expression of this gene was found under both challenges. However, a comparative increase in DDTFR10/A gene expression was observed in tomato plants upon inoculation with Fusarium oxysporum f. sp. lycopersici. The phenotypic assay performed on edited tomato plants demonstrated the role of the DDTFR10/A gene in contributing toward susceptibility against Fusarium wilt.

Supplementary information: The online version contains supplementary material available at 10.1007/s12298-022-01273-6.

Keywords: CRISPR/Cas9 genome editing; DDTFR10/A gene; Negative regulator; Salinity; Tomato wilt.

Fig. 1

Expression level of the DDTFR10/A gene using RT-qPCR analysis was analyzed under wilt and salinity challenges. a The DDTFR10/A gene expression under salinity stress on tomato cv. Sandal was shown to be upregulated compared to control plants. The expression level of the DDTFR10/A gene was increased with an increase in treatment level, 8dS/m and10dS/m, respectively. b Upon inoculation, the expression level of the DDTFR10/A gene in wilt-susceptible tomato cv. Sandal was high compared to the control plants (non-inoculated). However, the expression of the DDTFR10/A gene in control plants and inoculated plants of wilt-tolerant tomato cv. Sahel was similar

Fig. 2

Detected mutation in targeted DDTFR10/A gene and disease assay of T₀ tomato plants a Ten tomato plants, after basta selection, were screened for Indel detection with EcoRI digestion. The targeted plants, TP1, TP3, TP4, TP6, TP9, and TP10, appeared to be un-edited and produced double bands (~ 210 bp and ~ 110 bp), while TP2, TP5, TP7, and TP8 produced single bands (~ 326), indicates fully edited alleles. b Direct sequencing of PCR product containing the targeted site of fully edited tomato T₀ plants. The overlapping traces of the sequencing chromatogram were decoded by the DSD method. Arrows indicate the site of mutation