Type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS) are not only virulence factors of pathogenic bacteria, but also influence symbiotic interactions between nitrogen-fixing nodule bacteria (rhizobia) and leguminous host plants. In this study, we characterized NopM (nodulation outer protein M) of Rhizobium sp. strain NGR234, which shows sequence similarities with novel E3 ubiquitin ligase (NEL) domain effectors from the human pathogens Shigella flexneri and Salomonella enterica. NopM expressed in Escherichia coli, but not the non-functional mutant protein NopM-C338A, showed E3 ubiquitin ligase activity in vitro. In vivo, NopM, but not inactive NopM-C338A, promoted nodulation of the host plant Lablab purpureus by NGR234. When NopM was expressed in yeast, it inhibited mating pheromone signaling, a mitogen-activated protein (MAP) kinase pathway. When expressed in the plant Nicotiana benthamiana, NopM inhibited one part of the plant's defense response, as shown by a reduced production of reactive oxygen species (ROS) in response to the flagellin peptide flg22, whereas it stimulated another part, namely the induction of defense genes. In summary, our data indicate the potential for NopM as a functional NEL domain E3 ubiquitin ligase. Our findings that NopM dampened the flg22-induced ROS burst in N. benthamiana but promoted defense gene induction are consistent with the concept that pattern-triggered immunity is split in two separate signaling branches, one leading to ROS production and the other to defense gene induction.
Many Gram-negative bacterial pathogens possess type 3 secretion systems, which deliver effector proteins into eukaryotic host cells through needle-like structures. Effectors manipulate the host cell and many of them suppress host defense responses. Interestingly, certain symbiotic strains of rhizobia also possess such secretion systems. Rhizobia infect legume roots and induce root nodules, where the bacteria convert atmospheric nitrogen into ammonia. Here, we characterize the effector NopM of Rhizobium sp. strain NGR234. We demonstrate that NopM possesses E3 ubiquitin ligase activity, indicating that NopM can “tag" proteins with ubiquitin, and thus target them for proteasome-dependent degradation. Using a mutant approach, we demonstrate that enzymatically active NopM promotes establishment of symbiosis with Lablab purpureus, the host plant from which NGR234 was originally isolated. We further examine effects of NopM when directly expressed in eukaryotic cells and show that NopM interferes with specific signaling pathways. NopM expressed in the model plant Nicotiana benthamiana dampened generation of reactive oxygen species (ROS), which are formed in response to the bacterial flagellin peptide flg22. We suggest that NopM promotes nodule initiation by reducing the levels of harmful ROS during the infection process.