Abstract Our current understanding of pathogen-associated molecular pattern (PAMP)-triggered immunity signaling pathways in plants is limited due to the redundancy of several components or the lethality of mutants in Arabidopsis (Arabidopsis thaliana). To overcome this, we used a virus-induced gene silencing-based approach in combination with pharmacological studies to decipher links between early PAMP-triggered immunity events and their roles in immunity following PAMP perception in Nicotiana benthamiana. Two different calcium influx inhibitors suppressed the reactive oxygen species (ROS) burst: activation of the mitogen-activated protein kinases (MAPKs) and PAMP-induced gene expression. The calcium burst was unaffected in plants specifically silenced for components involved in ROS generation or for MAPKs activated by PAMP treatment. Importantly, the ROS burst still occurred in plants silenced for the two major defense-associated MAPK genes NbSIPK (for salicylic acid-induced protein kinase) and NbWIPK (for wound-induced protein kinase) or for both genes simultaneously, demonstrating that these MAPKs are dispensable for ROS production. We further show that NbSIPK silencing is sufficient to prevent PAMP-induced gene expression but that both MAPKs are required for bacterial immunity against two virulent strains of Pseudomonas syringae and their respective nonpathogenic mutants. These results suggest that the PAMP-triggered calcium burst is upstream of separate signaling branches, one leading to MAPK activation and then gene expression and the other to ROS production. In addition, this study highlights the essential roles of NbSIPK and NbWIPK in antibacterial immunity. Unexpectedly, negative regulatory mechanisms controlling the intensity of the PAMP-triggered calcium and ROS bursts were also revealed by this work.