The symbiotic association of legumes with rhizobia involves bacterially derived Nod factor, which is sufficient to activate the formation of nodules on the roots of the host plant. Perception of Nod factor by root hair cells induces calcium oscillations that are a component of the Nod factor signal transduction pathway. Perception of the calcium oscillations is a function of a calcium-and calmodulin-dependent protein kinase, and this activates nodulation gene expression via two GRAS domain transcriptional regulators, Nodulation Signaling Pathway1 (NSP1) and NSP2, and an ERF transcription factor required for nodulation. Here, we show that NSP1 and NSP2 form a complex that is associated with the promoters of early nodulin genes. We show that NSP1 binds directly to ENOD promoters through the novel cis-element AATTT. While NSP1 shows direct binding to the ENOD11 promoter in vitro, this association in vivo requires NSP2. The NSP1-NSP2 association with the ENOD11 promoter is enhanced following Nod factor elicitation. Mutations in the domain of NSP2 responsible for its interaction with NSP1 highlight the significance of the NSP1-NSP2 heteropolymer for nodulation signaling. Our work reveals direct binding of a GRAS protein complex to DNA and highlights the importance of the NSP1-NSP2 complex for efficient nodulation in the model legume Medicago truncatula.

Bacterial infection of interior tissues of legume root nodules is controlled at the epidermal cell layer and is closely coordinated with progressing organ development. Using spontaneous nodulating Lotus japonicus plant mutants to uncouple nodule organogenesis from infection, we have determined the role of 16 genes in these two developmental processes. We show that host-encoded mechanisms control three alternative entry processes operating in the epidermis, the root cortex and at the single cell level. Single cell infection did not involve the formation of trans-cellular infection threads and was independent of host Nod-factor receptors and bacterial Nod-factor signals. In contrast, Nod-factor perception was required for epidermal root hair infection threads, whereas primary signal transduction genes preceding the secondary Ca2+ oscillations have an indirect role. We provide support for the origin of rhizobial infection through direct intercellular epidermal invasion and subsequent evolution of crack entry and root hair invasions observed in most extant legumes.

A new nodulation-defective mutant of Lotus japonicus does not initiate nodule cortical cell division in response to Mesorhizobium loti, but induces root hair deformation, Nod factor-induced calcium spiking, and mycorrhization. This phenotype, together with mapping data, suggested that the mutation could be in the ortholog of the Medicago truncatula NSP1 gene (MtNSP1). The sequence of the orthologous gene (LjNSP1) in the L. japonicus mutant (Ljnsp1-1) revealed a mutation causing a premature stop resulting in loss of the C-terminal 23 amino acids. We also sequenced the NSP2 gene from L. japonicus (LjNSP2). A mutant (Ljnsp2-3) with a premature stop codon was identified by TILLING showing a similar phenotype to Ljnsp1-1. Both LjNSP1 and LjNSP2 are predicted GRAS (GAI, RGA, SCR) domain transcriptional regulators. Transcript steady-state levels of LjNSP1 and LjNSP2 initially decreased and then increased following infection by M. loti. In hairy root transformations, LjNSP1 and MtNSP1 complemented both Mtnsp1-1 and Ljnsp1-1 mutants, demonstrating that these orthologous proteins have a conserved biochemical function. A Nicotiana benthamiana NSP1-like gene (NbNSP1) was shown to restore nodule formation in both Ljnsp1-1 and Mtnsp1-1 mutants, indicating that NSP1 regulators from legumes and non-legumes can propagate the Nod factor-induced signal, activating appropriate downstream targets. The L. japonicus nodules complemented with NbNSP1 contained some cells with abnormal bacteroids and could fix nitrogen. However, the NbNSP1-complemented M. truncatula nodules did not fix nitrogen and contained very few bacteria released from infection threads. These observations suggest that NSP1 is also involved in infection, bacterial release, and normal bacteroid formation in nodule cells.Legumes produce root nodules in response to Nod factors secreted by rhizobia. These Nod factor signals are essential for root hair deformation, induction of early nodulation genes, formation of nodule primordia, and infection by rhizobia. The earliest plant responses to Nod factors include an influx of calcium, plasmamembrane depolarizations, and then induction of cytosolic calcium spiking around the nucleus of epidermal root cells (Oldroyd and Downie, 2004). Purified Nod factors are sufficient to cause a range of early responses involved in the host developmental program (Hirsch and Fang, 1994;Schultze and Kondorosi, 1998;Downie and Walker, 1999). The Nod factors are the principle determinants by which legumes can be nodulated by specific rhizobia; the basis for this host specificity is the structure of the Nod factor, suggesting that highly specific plant receptors perceive Nod factor signals, thereby initiating the plant developmental response.Nod factor-induced root hair deformation is associated with reorganization of actin filaments in preparation for infection (Cardenas et al., 1998;Sieberer et al., 2005). The root hairs bend back, entrapping bacteria and thereby allowing infection foci to form as entrapped microcolonies. The infection thread, i...

To allow rhizobial infection of legume roots, plant cell walls must be locally degraded for plant-made infection threads (ITs) to be formed. Here we identify a Lotus japonicus nodulation pectate lyase gene (LjNPL), which is induced in roots and root hairs by rhizobial nodulation (Nod) factors via activation of the nodulation signaling pathway and the NIN transcription factor. Two Ljnpl mutants produced uninfected nodules and most infections arrested as infection foci in root hairs or roots. The few partially infected nodules that did form contained large abnormal infections. The purified LjNPL protein had pectate lyase activity, demonstrating that this activity is required for rhizobia to penetrate the cell wall and initiate formation of plant-made infection threads. Therefore, we conclude that legume-determined degradation of plant cell walls is required for root infection during initiation of the symbiotic interaction between rhizobia and legumes.Medicago truncatula | Mesorhizobium | pectin | polygalacturonase T he infection of legumes by nitrogen-fixing rhizobia occurs via plant-made infection threads (ITs). These tube-like structures, lined with a plant cell wall and membrane, are usually initiated in curled root hairs and grow down through the root hair and continue growing through epidermal and cortical cells (1). When the growing IT reaches the dividing root cells that make up the nodule primordium, the plant cell wall of the IT is lost and the bacteria are budded off into the plant cytoplasm surrounded by a plant-derived membrane. The bacteria then differentiate into nitrogen-fixing forms called bacteroids and in the mature nodule, they fix N 2 , producing ammonia that is translocated to the plant.The initiation and growth of ITs require signaling between rhizobia and legumes. Rhizobial nodulation (Nod) factors activate nuclear-associated calcium spiking via a signaling cascade that requires LysM-receptor kinases and a leucine-rich repeat receptor-like kinase in the plasma membrane and nucleoporins and ion channels in the nuclear membrane. The subsequent activation of a calcium and calmodulin-dependent kinase then activates transcription factors required for the induction of nodulation and infection genes (2). Nod factors also induce a calcium influx that is associated with depolarization of the plasma membrane; this calcium influx has been proposed to be important for initiation of infection (3). Oligosaccharides derived from the synthesis of the rhizobial exopolysaccharide also play a crucial role in initiation of infection, possibly by suppressing plant defense responses (4, 5). Membrane-associated remorins and flotillins that promote protein interactions and alter membrane dynamics are also important for infection (6, 7).Initiation of infection in root hairs requires localized degradation of the root-hair cell wall and the initiation of inward growth of the cell wall and membrane. Genes that play a role in remodeling the cytoskeleton are required for infection initiation (8, 9). However, although oth...