Fig 1.
NF perception and the common symbiosis signalling pathway.
Flavonoid exudates from legume roots act as signals to relevant rhizobia in the soil, activating production of NF. A receptor complex at the root surface allows NF recognition, through binding to LysM receptor kinases LYK3 (also known as NFR1) and NFP (also known as NFR5), coupled to the LRR-containing receptor kinase SYMRK (also known as DMI2) that activates downstream signalling. A number of RIPs have been identified that may facilitate downstream signal transduction including ROP-GTPases and GEFs, which are particularly associated with rhizobial infection; a group of receptor-like cytoplasmic kinases, which includes SYMRK INTERACTING PROTEINS and the NFR5-INTERACTING CYTOPLASMIC KINASE4 that can activate phosphorylation cascades; and a HMGR, which interacts with SYMRK and induces the production of mevalonate. The action of one or many of these RIPs may produce a secondary messenger that links receptor activation at the plasma membrane to induction of cation channels on the nuclear envelope. The cyclic nucleotide-gated channels CNGC15s release calcium from the nuclear envelope lumen into the nucleus, while other cation channels are required for symbiotic calcium spiking, CASTOR and POLLUX (also known as DMI1): DMI1 interacts with CNGC15 and appears to coordinate the release of calcium from this channel. The calcium ATPase MCA8 pumps calcium back into the nuclear envelope to maintain nuclear calcium homeostasis. Components of the nuclear pore complex, like NUP85, NUP133, and NENA, are also required for symbiosis signalling, and these are thought to direct the necessary channels onto the inner nuclear envelope. The coordinated action of the channels and pumps creates nuclear calcium oscillations that promote the CCaMK (also known as DMI3). CCamK/DMI3 phosphorylates CYCLOPS/IPD3, which, in turn, promotes symbiotic transcription, such as the induction of NIN. Gain-of-functions in NFR1, NFR5, SYMRK, DMI1, CCaMK, and CYCLOPs, all autoactivate nodulation, demonstrating that activation at any point in this pathway is necessary and sufficient for nodule initiation. Created with BioRender.com. CaM, calmodulin; CCaMK, calcium and CaM-dependent serine/threonine protein kinase; DMI2, DOES NOT MAKE INFECTIONS 2; GEF, Guanine-nucleotide Exchange Factor; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; IPD3, INTERACTING PROTEIN OF DMI3; Lj, Lotus japonicus; LysM, lysine motif; Mt, Medicago truncatula; NF, Nod factor; NIN, NODULE INCEPTION; NUP, NUCLEAR PORE COM-PLEX PROTEIN; RIP, receptor-interacting protein; ROP-GTPase, Rho of plants–guanosine triphosphatase.
Fig 2.
Rhizobia can enter the root immediately through a process of intracellular infection (A) or through differing levels of intercellular infection (B). Whichever route is taken, bacteria always end up intracellularly accommodated. (C) Initiation of intracellular accommodation starts with receptor activation through the stringent perception of NFs or exopolysaccharides. ROPs, which interact with the NF receptors, activate RBOHs, which regulate reactive oxygen species, which can coordinate multiple aspects of cell functionality and signalling. The SCAR/WAVE complex, which governs the ARP2/3 complex coordinating actin dynamics for infection thread initiation. Actin dynamics alongside two scaffold proteins, FLOT4 and the remorin protein SYMREM1, facilitate the formation of a nanodomain and reduce the mobility of the NF receptors, a process vital for rhizobial infection. Coordinating the microtubule organisation with cell wall and plasma membrane dynamics is in part fulfilled by SYFO1. NPL, which plays a critical role in cell wall remodelling for infection thread development, is stimulated and accumulates in the infection pocket in response to NFs. The infectosome, which is made up of VPY, the E3 ligase LIN/CERBERUS, RPG, and the exocyst complex EXOCYST subunit H4 (EXO70 H4), is located at the tip of infection threads and regulates polar development by controlling vesicle membrane fusion. Perfect synchronisation of infection and nodule production is required for effective nodulation, which is regulated by NIN and ERN1. NIN regulates rhizobial infection in epidermal cells by up-regulating NPL. On the other hand, in epidermal cells, NIN competes with ERN1 to limit the production of ENOD11 and probably other genes. At the same time, NIN promotes the transfer of an unknown mobile signal, perhaps cytokinin, from the epidermis to the cortex to initiate cell divisions in cortical cells, leading to the formation of the nodule meristem. Created with BioRender.com. ARP2/3, actin-related protein 2/3; ENOD11, EARLY NODULIN 11; ERN1, Ethylene Response Factor Required for Nodulation 1; FLOT4, FLOTILLIN 4; LIN, LUMPY INFECTION; NF, Nod factor; NIN, NODULE INCEPTION; NPL, NODULE PECTATE LYASE; RBOH, Respiratory Burst Oxidase Homolog; ROP, Rho of plants; RPG, RHIZOBIUM-DIRECTED POLAR GROWTH; SCAR/WAVE, Suppressor of cAMP receptor defect/WASP family verpolin homologous protein; SYFO1, SYMBIOTIC FORMIN 1; VPY, VAPYRIN.
Fig 3.
(A) The regulatory network underpinning nodule development. In cortical cells, activation of the cytokinin receptor CRE1/LHK1 induces NIN expression. In return, NIN promotes the transcription of CRE1, creating a feedforward loop that can increase cytokinin signalling and NIN accumulation. NIN controls the expression of LBD16, an auxin-responsive transcription factor that activates the auxin symbiosis pathway via SHI/STY transcription factors, which, in turn, promote expression of YUCCAs, rate-limiting enzymes in auxin biosynthesis. The expression and distribution of auxin transporters, PINs and LAXs, are precisely regulated during different stages of nodule development to control the dynamics of the accumulating auxin maximum. NIN also induces NF-YA1 expression, which further enhances STY/SHI expression, as well as likely contributing to other aspects of the nodule meristem and bacterial infection. We propose the existence of an unknown component that dictates nodule identity, which, in turn, likely affects expression of NOOT genes that are necessary to maintain nodule identity. NIN also controls the nodule maturation process to transition into the nitrogen-fixing state. The DNF1 signal peptidase complex cleaves the NIN protein and generates a C-terminal NIN fragment, which activates genes involved in bacteroid differentiation and nitrogen fixation, including NCRs, GRPs, and leghemoglobin. (B, C) The developmental patterns of indeterminate and determinate legume nodules at initiation and maturation stages. (B) For indeterminate nodules, the initial cell divisions forming the nodule primordia occur in inner cortical cells, while in determinate nodules, this occurs in the outer cortical cells (C). Despite their anatomical differences, cell divisions in the pericycle have been observed in both nodule types. (B) Mature indeterminate nodules contain a persistent meristem at the tip of the nodule, which is commonly observed in M. truncatula and Pisum sativum. (C) Mature determinate nodules form without having a persistent meristem, which is often seen in L. japonicus, Phaseolus vulgaris, and Glycine max. Created with BioRender.com. DNF1, DEFECTIVE IN NITROGEN FIXATION1; GRP, glycine-rich peptide; LBD16, LATERAL ORGAN BOUNDARIES DOMAIN 16; NCR, nodule-specific cysteine-rich peptides; NIN, NODULE INCEPTION; NOOT, NODULE ROOT; SHI/STY, SHORT-INTERNODES/STYLISH.
Fig 4.
Creating an environment for nitrogen fixation and nitrogen delivery.
Once the infection thread reaches the nodule primordium, infection threads release droplets into the cell containing bacteria, always surrounded by a plant-derived membrane. These structures, the so-called symbiosomes, are organelle-like. Symbiosomes can continue to divide, and the bacteria can differentiate into bacteroids. The DNF1-cleaved N-NCR is delivered into the symbiosome via membrane vesicle trafficking and induces bacteroid differentiation. DNF1 also proteolytically cleaves the NIN protein and generates a C-NIN, activating genes involved in terminal differentiation and nitrogen fixation. The nitrogenase enzyme complex in rhizobia converts N2 into ammonia that is released from the bacteroid via diffusion or unknown channels. The H+-ATPases on the symbiosome membrane create an acidic peribacteroid space, which traps ammonium by protonating ammonia and producing ammonium cations. Ammonium cations are exported into the cytoplasm of plant cells and then assimilated into Gln and Glu by GS and by GOGAT, transferring the amide group from Gln to AKG. Depending on legume species and nodule types, Gln and Glu are converted into Asn or ureides for long-distance transport through the xylem. In the end, the nodule undergoes senescence and bacteroids lyse. Different CPs are highly expressed in nodules at the senescence stage, especially a papain peptidase (CP6) and a VPE, which are controlled by a transcription factor NAC969. Created with BioRender.com. AKG, α-ketoglutarate; Asn, Asparagine; C-NIN, C-terminal NIN peptide fragment; CP, cysteine proteinase; Gln, glutamine; Glu, glutamate; GOGAT, glutamate synthase; GS, glutamine synthetase; NAC, NAM/ATAF/CUC; NAC969, NAC-encoding 969; NIN, NODULE INCEPTION; N-NCR, N-terminal signal peptide of NCRs; UPS, Ureide Permease; VPE, vacuolar processing enzyme.