Abstract
Nodulation outer protein M (NopM) is an IpaH family type three (T3) effector secreted by the nitrogen-fixing nodule bacterium Sinorhizobium sp. strain NGR234. Previous work indicated that NopM is an E3 ubiquitin ligase required for an optimal symbiosis between NGR234 and the host legume Lablab purpureus Here, we continued to analyze the function of NopM. Recombinant NopM was biochemically characterized using an in vitro ubiquitination system with Arabidopsis thaliana proteins. In this assay, NopM forms unanchored polyubiquitin chains and possesses auto-ubiquitination activity. In a NopM variant lacking any lysine residues, auto-ubiquitination was not completely abolished, indicating noncanonical auto-ubiquitination of the protein. In addition, we could show intermolecular ubiquitin transfer from NopM to C338A (enzymatically inactive NopM form) in vitro Bimolecular fluorescence complementation analysis provided clues about NopM-NopM interactions at plasma membranes in planta NopM, but not C338A, expressed in tobacco cells induced cell death, suggesting that E3 ubiquitin ligase activity of NopM induced effector-triggered immunity responses. Likewise, expression of NopM in Lotus japonicus caused reduced nodule formation, whereas expression of C338A showed no obvious effects on symbiosis. Further experiments indicated that serine residue 26 of NopM is phosphorylated in planta and that NopM can be phosphorylated in vitro by salicylic acid-induced protein kinase (NtSIPK), a mitogen-activated protein kinase (MAPK) of tobacco. Hence, NopM is a phosphorylated T3 effector that can interact with itself, with ubiquitin, and with MAPKs.
Highlights
Nodulation outer protein M (NopM) is an IpaH family type three (T3) effector secreted by the nitrogen-fixing nodule bacterium Sinorhizobium sp. strain NGR234
Previous work indicated that NopM is an E3 ubiquitin ligase required for an optimal symbiosis between NGR234 and the host legume Lablab purpureus
At least some rhizobial T3 effectors interfere with host defense signaling, and successful symbiosis may depend on the rhizobial ability to suppress plant defense reactions (8 –12)
Summary
T3SS, type III protein secretion system; APase, alkaline phosphatase; E1, enzyme 1 of the ubiquitination system (ubiquitin-activating enzyme); E2, enzyme 2 of the ubiquitination system (ubiquitin-conjugating enzyme); E3, enzyme 3 of the ubiquitination system (ubiquitin ligase); ETI, effector-triggered immunity; GST, glutathione S-transferase, LRR, leucine-rich repeats; MAPK, mitogen-activated protein kinase; NEL domain, novel E3 ubiquitin ligase domain; Nop, nodulation outer protein; RFP, red fluorescent protein; ROS, reactive oxygen species; YFP, yellow fluorescent protein; T3, type III; Ub, ubiquitin; BiFC, bimolecular fluorescence complementation; CaMV, cauliflower mosaic virus. NopM possesses E3 ubiquitin ligase activity in an in vitro system with commercially available mammalian proteins [11]. Ubiquitination in eukaryotic cells can affect proteins in many ways It can function as a signal for their degradation via the 26S proteasome, alter their subcellular localization, influence their activity, and affect protein–protein interactions. A ubiquitin ligase (E3) transfers the activated ubiquitin from a ubiquitin-loaded E2 to the ⑀-amino group of a given lysine residue in the protein substrate, forming a glycine–lysine isopeptide bond [26]. IpaH family effectors can form free (unanchored) polyubiquitin chains that contain glycine–lysine isopeptide bonds between two ubiquitin molecules. We found that NopM preferentially catalyzes unanchored Lys-48 – dependent polyubiquitination reactions and possesses auto-ubiquitination activity. We found that a serine residue of NopM is phosphorylated in vivo and in vitro and that NopM is a mitogen-activated protein kinase (MAPK) substrate
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