Mitogen activated protein kinase (MAPK) cascades are key players in plant immune signalling pathways. MAP kinase singling consists of a number of associated proteins where MAPKKKs are located upstream of MAPK cascade activating multiple MAPKs. MAPKKKε, a positive regulator of cell death, is associated with plant immunity. Phytophthora infestans is a pathogenic oomycete that inflicts potato late blight disease, delivering RXLR effector proteins to plant cells to modulate host immune signalling and colonization incident. The RXLR effector PexRD2 interacts with the kinase domain of MAPKKKε. Using a CRISPR/Cas9 system, a construct was made to introduce four guide RNAs (gRNAs) in potato cultivar, Agria. Thirty-five mutant lines were regenerated, in which three and two transgenic lines with deletions and insertions were analysed, respectively. A mutant line with a three-allelic knock down of MAPKKKε was obtained, showing ~70% reduction in MAPKKKε expression level resulting in enhanced susceptibility to P. infestans. The results of this study provide evidences that editing MAPKKKε to modulate PexRD2 interaction, enhances susceptibility to P. infestans, suggesting that MAPKKKε plays a key role in resistance to P. infestans in potato. Therefore, it can be anticipated that MAPKKKε gene can be used in future potato breeding programs to generate biotic stress resistance plants.