The MAP4 kinase NbM4K3 regulates immune responses in Nicotiana benthamiana

Abstract The mitogen-activated protein kinase kinase kinase kinase (M4K) family is evolutionarily conserved across plants and animals. In Arabidopsis, the protein kinase SIK1, an M4K member, is known to positively modulate reactive oxygen species (ROS) production during pattern-triggered immunity (PTI) by stabilizing BIK1, a key receptor-like cytoplasmic kinase (RLCK). While homologs of SIK1 exhibit conserved protein domain architectures across a range of land plants, their functional conservation remains incompletely understood. This study investigates the functional conservation and divergence of SIK1 homologs, focusing particularly on NbM4K3 in Nicotiana benthamiana . Silencing NbM4K3 resulted in an impairment of the flg22-induced ROS burst and expression of PTI marker genes. Additionally, silencing NbM4K3 led to diminished protein accumulation of RLCKs, while overexpression of the RLCKs prominently enhanced the flg22-induced ROS burst in NbM4K3 -silenced plants. Furthermore, NbM4K3 -silenced plants exhibited a compromised hypersensitive response (HR), reduced ROS accumulation, and diminished expression of effector-triggered immunity (ETI) marker genes when challenged with the avirulent strains Ralstonia solanacearum GMI1000 and Pseudomonas syringae DC3000, suggesting that NbM4K3 is a positive regulator of ETI. The attenuated HR phenotype observed in NbM4K3 -silenced plants upon expression of RipP1 or RipE1, two avirulent type III effectors of GMI1000, further supports the affirmative role of NbM4K3 in ETI. In summary, our data indicate that the M4K NbM4K3 positively regulates both PTI and ETI in N. benthamiana , potentially by stabilizing RLCKs. These findings not only strengthen the role of M4K family in plant immunity but also suggest its potential in improving disease resistance in plants.