Killers or protectors: Contrasting roles of alfalfa SIMKK and PRKK in Nicotiana benthamiana cell death

Mitogen-activated protein kinases (MAPKs) play important roles in immune signalling pathways following pathogen attack or elicitor treatment. A key plant defence strategy is a localised cell death (CD) mechanism that restricts pathogen spread. This study investigated the potential role of alfalfa ( Medicago sativa ) MAPK kinases (MAPKKs) and MAPK pairs in regulating CD. In vitro kinase assays confirmed that STRESS-INDUCED MAPKK (SIMKK) and PATHOGEN-RESPONSIVE MAPKK (PRKK) could activate downstream targets, including STRESS-INDUCED MAPK (SIMK), STRESS-ACTIVATED MAPK (SAMK), MEDICAGO MAPK 2 (MMK2), and MEDICAGO MAPK 3 (MMK3). SIMKK was more active than PRKK, with the highest activation observed in SIMKK combinations with SIMK and SAMK, perhaps associated with CD. To study this, SIMKK and PRKK were co-transformed pairwise with SIMK, SAMK, MMK2 , and MMK3 in Nicotiana benthamiana , representing a reliable in planta expression system. Microscopy revealed co-localisation of fluorescently-tagged MAPKKs with MAPKs in nuclei and cytoplasm. In the case of the SIMKK–SAMK combination, stress granule (SG)-like structures appeared in the cytoplasm, and phenotypic observations showed CD symptoms including increased level of reactive oxygen species (ROS) two days and death tissues six days post-transformation. Further phenotype analysis indicated that SIMKK pairwise overexpression with SAMK or SIMK induced ROS production and CD, suggesting SIMKK promotes immune signalling that may restrict pathogen invasion. Conversely, PRKK overexpression with the same MAPKs suppressed ROS production and CD, suggesting a protective role against excessive CD. These findings highlight a differential function between SIMKK and PRKK, offering a basis for enhancing alfalfa disease resistance through genetic approaches.