Nuclear membranes control symbiotic calcium signaling of legumes.

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.

Sesbania rostrata: a case study of natural variation in legume nodulation.

Legumes acquired the ability to engage in a symbiotic interaction with soil-borne bacteria and establish a nitrogen-fixing symbiosis in a novel root organ, the nodule. Most legume crops and the model legumes Medicago truncatula and Lotus japonicus are infected intracellularly in root hairs via infection threads that lead the bacteria towards a nodule primordium in the root cortex. This infection process, however, does not reflect the great diversity of infection strategies that are used by leguminous plants. An alternative, intercellular invasion occurs in the semiaquatic legume Sesbania rostrata. Bacteria colonize epidermal fissures at lateral root bases and trigger cortical cell death for infection pocket formation and subsequent intercellular and intracellular infection thread progression towards the primordium. This infection mode evolved as an adaptation to waterlogged conditions that inhibit intracellular invasion. In this review, we discuss the molecular basis for this adaptation and how insights into this process contribute to general knowledge of the rhizobial infection process.

Water-tolerant legume nodulation.

Water-tolerant nodulation is an adaptation of legumes that grow in wet or temporarily flooded habitats. This nodulation mode takes place at lateral root bases via intercellular bacterial invasion in cortical infection pockets. The tropical legume Sesbania rostrata has become a model for the study of the molecular basis of crack entry nodulation compared with root hair curl nodulation. For intercellular invasion, Nodulation Factor (NF) signalling recruits an ethylene-dependent, common Sym gene-independent pathway, leading to local cell death. The NF structure requirements are less stringent than for intracellular invasion in root hairs, which is correlated with a very specific NF-induced calcium spiking signature, presumably necessary for correct gene expression to assemble a functional entry complex in the epidermis.

Rhizobium infection: lessons from the versatile nodulation behaviour of water-tolerant legumes.

Water-tolerant legumes provide bacteria with special ways of invading roots to establish N(2)-fixing symbiosis upon flooding. On well-aerated roots, root hair curling (RHC) invasion is used, whereas, under hydroponic conditions, rhizobia enter the cortex through cracks at lateral root bases (LRBs). Here, we compare the physiological and anatomical traits of these invasions. During waterlogging, accumulating ethylene inhibits the epidermal stages of RHC invasion. LRB invasion circumvents this step by direct colonization of the cortical tissue. By avoiding the epidermis for bacterial entry under hydroponic conditions, the stringent nodulation (Nod) factor perception systems that are active within the epidermis are not needed. Consequently, LRB invasion might be useful for analysing the requirement for Nod factor perception and other signal transduction systems downstream of the epidermis.

Calcium spiking patterns and the role of the calcium/calmodulin-dependent kinase CCaMK in lateral root base nodulation of Sesbania rostrata.

Nodulation factor (NF) signal transduction in the legume-rhizobium symbiosis involves calcium oscillations that are instrumental in eliciting nodulation. To date, Ca2+ spiking has been studied exclusively in the intracellular bacterial invasion of growing root hairs in zone I. This mechanism is not the only one by which rhizobia gain entry into their hosts; the tropical legume Sesbania rostrata can be invaded intercellularly by rhizobia at cracks caused by lateral root emergence, and this process is associated with cell death for formation of infection pockets. We show that epidermal cells at lateral root bases respond to NFs with Ca2+ oscillations that are faster and more symmetrical than those observed during root hair invasion. Enhanced jasmonic acid or reduced ethylene levels slowed down the Ca2+ spiking frequency and stimulated intracellular root hair invasion by rhizobia, but prevented nodule formation. Hence, intracellular invasion in root hairs is linked with a very specific Ca2+ signature. In parallel experiments, we found that knockdown of the calcium/calmodulin-dependent protein kinase gene of S. rostrata abolished nodule development but not the formation of infection pockets by intercellular invasion at lateral root bases, suggesting that the colonization of the outer cortex is independent of Ca2+ spiking decoding.

Comparative transcriptome analysis reveals common and specific tags for root hair and crack-entry invasion in Sesbania rostrata.

The tropical legume Sesbania rostrata provides its microsymbiont Azorhizobium caulinodans with versatile invasion strategies to allow nodule formation in temporarily flooded habitats. In aerated soils, the bacteria enter via the root hair curling mechanism. Submergence prevents this epidermal invasion by accumulation of inhibiting concentrations of ethylene and, under these conditions, the bacterial colonization occurs via intercellular cortical infection at lateral root bases. The transcriptome of both invasion ways was compared by cDNA-amplified fragment length polymorphism analysis. Clusters of gene tags were identified that were specific for either epidermal or cortical invasion or were shared by both. The data provide insight into mechanisms that control infection and illustrate that entry via the epidermis adds a layer of complexity to rhizobial invasion.

Gibberellins are involved in nodulation of Sesbania rostrata.

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.

SrSymRK, a plant receptor essential for symbiosome formation.

The symbiosis between legumes and rhizobia is essential for the nitrogen input into the life cycle on our planet. New root organs, the nodules, are established, which house N2-fixing bacteria internalized into the host cell cytoplasm as horizontally acquired organelles, the symbiosomes. The interaction is initiated by bacterial invasion via epidermal root hair curling and cell division in the cortex, both triggered by bacterial nodulation factors. Of the several genes involved in nodule initiation that have been identified, one encodes the leucine-rich repeat-type receptor kinase SymRK. In SymRK mutants of Lotus japonicus or its orthologs in Medicago sp. and Pisum sativum, nodule initiation is arrested at the level of the root hair interaction. Because of the epidermal block, the role of SymRK at later stages of nodule development remained enigmatic. To analyze the role of SymRK downstream of the epidermis, the water-tolerant legume Sesbania rostrata was used that has developed a nodulation strategy to circumvent root hair responses for bacterial invasion. Evidence is provided that SymRK plays an essential role during endosymbiotic uptake in plant cells.

Switch from intracellular to intercellular invasion during water stress-tolerant legume nodulation.

Rhizobia colonize their legume hosts by different modes of entry while initiating symbiotic nitrogen fixation. Most legumes are invaded via growing root hairs by the root hair-curl mechanism, which involves epidermal cell responses. However, invasion of a number of tropical legumes happens through fissures at lateral root bases by cortical, intercellular crack entry. In the semiaquatic Sesbania rostrata, the bacteria entered via root hair curls under nonflooding conditions. Upon flooding, root hair growth was prevented, invasion on accessible root hairs was inhibited, and intercellular invasion was recruited. The plant hormone ethylene was involved in these processes. The occurrence of both invasion pathways on the same host plant enabled a comparison to be made of the structural requirements for the perception of nodulation factors, which were more stringent for the epidermal root hair invasion than for the cortical intercellular invasion at lateral root bases.

Reactive oxygen species and ethylene play a positive role in lateral root base nodulation of a semiaquatic legume.

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.