Functional Analysis of NopM, a Novel E3 Ubiquitin Ligase (NEL) Domain Effector of Rhizobium sp. Strain NGR234

Abstract

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.

Mutualistic Co-evolution of Type III Effector Genes in Sinorhizobium fredii and Bradyrhizobium japonicum

“…Rhizobial T3E genes show genetic patterns indicative of surprising conservation, pointedly contrasting the patterns consistent with the dynamic arms race model of co-evolution dogmatic for T3Es (Figures 3–5). This finding is particularly striking in light of the observations that T3Es of mutualistic rhizobia are similar in regards to those of pathogens in having to maintain sufficiency in engaging and dampening PTI while avoiding ETI [15], [17], [24], [47]. Moreover, we demonstrated that the high conservation of T3Es in rhizobia relative to phytopathogens is not likely driven by differences in host range or phylogenetic diversity among genomes (Figures 1, S2, 4, and 5).…”

Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system

“…However, this and other studies show that B. japonicum is unable to nodulate on either En1282 or Clark-rj 1 , suggesting that B. elkanii possesses a unique T3E(s) responsible for activation of nodulation signaling. Although we have not yet detected any B. elkanii unique T3E candidate genes, B. elkanii encodes several genes for T3E candidates, including nopL, nopM, and nopP, which have been reported to play either a positive or a negative role in symbiosis in other rhizobia (9,25). Intriguingly, CCaMK and LHK1, mentioned above, are protein kinases that play pivotal roles in nodulation and should participate in phosphorylation systems (26).…”

Bacterial E3 Ubiquitin Ligase IpaH4.5 of Shigella flexneri Targets TBK1 To Dampen the Host Antibacterial Response

“…Shigella, which causes bacillary dysentery, produces several closely related invasion plasmid Ag H (IpaH) proteins (16,17). The effectors of the IpaH family produced by mammal, fish, and plant pathogenic bacteria share several structural and functional characteristics (18) and have a highly conserved N-terminal leucine-rich repeat (LRR) domain and a C-terminal region with E3 ubiquitin (Ub) ligase activity found in pathogenic and symbiotic bacteria (19)(20)(21)(22). Recently, IpaH4.5 was found to target NF-kB subunit p65 to downregulate the host immune response (23).…”