Nuclear-associated oscillations in calcium act as a secondary messenger in the symbiotic signaling pathway of legumes. These are decoded by a nuclear-localized calcium and calmodulin-dependent protein kinase, the activation of which is sufficient to drive downstream responses. This implies that the calcium oscillations within the nucleus are the predominant signals for legume symbiosis. However, the mechanisms that allow targeted release of calcium in the nuclear region have not been defined. Here we show that symbiosis-induced calcium changes occur in both the nucleoplasm and the perinuclear cytoplasm and seem to originate from the nuclear membranes. Reaction diffusion simulations suggest that spike generation within the nucleoplasm is not possible through transmission of a calcium wave from the cytoplasm alone and that calcium is likely to be released across the inner nuclear membrane to allow nuclear calcium changes. In agreement with this, we found that the cation channel DMI1, which is essential for symbiotic calcium oscillations, is preferentially located on the inner nuclear membrane, implying an essential function for the inner nuclear membrane in symbiotic calcium signaling. Furthermore, a sarco/endoplasmic reticulum calcium ATPase (SERCA) essential for symbiotic calcium oscillations is targeted to the inner nuclear membrane, as well as the outer nuclear membrane and endoplasmic reticulum (ER). We propose that release of calcium across the inner nuclear membrane allows targeted release of the ER calcium store, and efficient reloading of this calcium store necessitates the capture of calcium from the nucleoplasm and nuclear-associated cytoplasm.L egumes form mutualistic symbiotic interactions with mycorrhizal fungi and with rhizobial bacteria that aid in the uptake of nutrients (1, 2). Establishment of both symbioses requires the common symbiosis (Sym) signaling pathway (1, 2) that involves calcium oscillations after perception of diffusible signals from the symbionts (3, 4): Nod factors from rhizobia and Myc factors from mycorrhizal fungi (4-7). The calcium oscillations are concentrated in the perinuclear region (3), and a nuclear-targeted calcium reporter showed that part of these oscillations occurs in the nucleoplasm (8). The decoder of the calcium oscillations, a calcium and calmodulin-dependent protein kinase (CCaMK), is localized to the nucleoplasm (9, 10), implying that intranuclear calcium changes are paramount. Furthermore, some of the components of the Sym pathway required for the induction of calcium oscillations are localized to the nuclear envelope: two cation channels and three components of the nuclear pore (11-14). All of this points to the nuclear membrane as playing a central role in symbiotic calcium oscillations.The question of whether calcium changes can derive from the nucleus has been a contentious point for many years (15, 16). In animals, it is widely accepted that calcium events in and around the nucleus have significant effects on signaling pathways in the nucleus (15). Depend...

Lateral root base nodulation on the tropical, semiaquatic legume Sesbania rostrata results from two coordinated, Nod factor-dependent processes: formation of intercellular infection pockets and induction of cell division. Infection pocket formation is associated with cell death and production of hydrogen peroxide. Pharmacological experiments showed that ethylene and reactive oxygen species mediate Nod factor responses and are required for nodule initiation, whereby induction of division and infection could not be uncoupled. Application of purified Nod factors triggered cell division, and both Nod factors and ethylene induced cavities and cell death features in the root cortex. Thus, in S. rostrata, ethylene and reactive oxygen species act downstream from the Nod factors in pathways that lead to formation of infection pockets and initiation of nodule primordia.

Upon submergence, Azorhizobium caulinodans infects the semiaquatic legume Sesbania rostrata via the intercellular crack entry process, resulting in lateral root-based nodules. A gene encoding a gibberellin (GA) 20-oxidase, SrGA20ox1, involved in GA biosynthesis, was transiently up-regulated during lateral root base nodulation. Two SrGA20ox1 expression patterns were identified, one related to intercellular infection and a second observed in nodule meristem descendants. The infection-related expression pattern depended on bacterially produced nodulation (Nod) factors. Pharmacological studies demonstrated that GAs were involved in infection pocket and infection thread formation, two Nod factor-dependent events that initiate lateral root base nodulation, and that they were also needed for nodule primordium development. Moreover, GAs inhibited the root hair curling process. These results show that GAs are Nod factor downstream signals for nodulation in hydroponic growth.Legume plants develop a symbiotic interaction with rhizobia by forming root nodules in which the bacteria fix atmospheric nitrogen. Nodule formation integrates several developmental processes, such as induction of cortical and pericycle cell division and rhizobial invasion, which are coordinated in time and space. The onset of the symbiosis is marked by a complex exchange of signals, involving plant flavonoids and bacterial nodulation (Nod) factors. Recognition of specific Nod factors will switch on the nodulation program in the legume host.The best known mode of invasion is the root hair curling (RHC) mechanism that is used by most crop legumes and the model legumes barrel medic (Medicago truncatula) and Lotus japonicus. Rhizobia induce growing root hairs to curl in the root zone I, just above the root meristem, whereby a rhizobial microcolony is entrapped. Local cell wall degradation and subsequent inward growth of the root hair plasma membrane result in the formation of an infection thread (IT) that guides the bacteria to the cortical cells. RHC is Nod factor dependent, and purified compatible Nod factors trigger several nodulation-related effects within the root hair, such as deformation, gene expression, Ca 21 spiking, membrane depolarization, and ion effluxes (Oldroyd and Downie, 2004). Several compo-