Reviewer #2 (Public Review): The Rhizobial effector NopT targets Nod factor receptors to regulate symbiosis in Lotus japonicus

It is well-studied that type-III effectors are essential required by gram-negative bacteria to directly target different cellular pathways from hosts, thus enhancing their infection. However, in the unique context of legume-rhizobium symbiosis, the role of rhizobial effectors in regulating plant symbiotic pathways remains a subject of hypothesized understanding. Here, we show that NopT, a YopT-type cysteine protease from Sinorhizobium fredii NGR234 that could directly target symbiotic signaling pathway by associating with two Nod Factor Receptors (NFR1 and NFR5) from Lotus japonicus. NopT is the only effector out of 15 effectors from S. fredii NGR234 that inhibits cell death triggered by co-overexpression of NFR1 and NFR5 in Nicotiana benthamiana. NopT interacts with both NFR1 and NFR5. NopT proteolytically cleaves NFR5 in both in vitro and in vivo assays, but is later inactivated via phosphorylation by NFR1. NopT plays an essential role in mediating rhizobial infection in L. japonicus. The truncated NopT, lacking 50 N–terminal amino acids post-autocleavage, retains the ability to cleave NFR5 but no longer interacts with NFR1. Intriguingly, certain Sinorhizobium species exclusivley harbor this truncated NopT in their genomes. These findings unveil an intricate regulatory interplay between rhizobia and legumes, wherein rhizobial NopT actively cleaves NFR5, and this cleavage process is subsequently suppressed following NopT phosphorylation by NFR1. This discovery presents a compelling narrative of a nuanced regulatory interplay between rhizobia and legumes, highlighting rhizobial effector as a direct mediator targeting symbiotic pathway from plants.The rhizobial effector NopT forms a mutual interaction with plant’s two Nod factor receptors, NFR1 and NFR5, to precisely regulate rhizobial symbiosis in Lout japonicus