A member of the ALOG gene family has a novel role in regulating nodulation in Lotus japonicus

Legumes can control the number of symbiotic nodules that form on their roots, thus balancing nitrogen assimilation and energy consumption. Two major pathways participate in nodulation: the Nod factor(NF)signaling pathway which involves recognition of rhizobial bacteria by root cells and promotion of nodulation, and the autoregulation of nodulation(AON) pathway which involves long-distance negative feedback between roots and shoots. Although a handful of genes have a clear role in the maintenance of nodule number, additional unknown factors may also be involved in this process. Here, we identify a novel function for a Lotus japonicus ALOG(Arabidopsis LSH1 and Oryza G1) family member, LjALOG1,involved in positively regulating nodulation. LjALOG1 expression increased substantially after inoculation with rhizobia, with high levels of expression in whole nodule primordia and in the base of developing nodules. The ljalog1 mutants, which have an insertion of the LORE1 retroelement in LjALOG1, had significantly fewer nodules compared with wild type, along with increased expression of LjCLE-RS1(L. japonicus CLE Root Signal 1), which encodes a nodulation suppressor in the AON pathway. In summary,our findings identified a novel factor that participates in controlling nodulation, possibly by suppressing the AON pathway.     

Nodulation gene regulation and quorum sensing control density-dependent suppression and restriction of nodulation in the Bradyrhizobium japonicum-soybean symbiosis

The nodulation of Glycine max cv. Lambert and the nodulation-restricting plant introduction (PI) genotype PI 417566 by wild-type Bradyrhizobium japonicum USDA110 is regulated in a population-density-dependent manner. Nodulation on both plant genotypes was suppressed (inhibited) when plants received a high-density inoculum (10(9) cells/ml) of strain USDA110 grown in complex medium, and more nodules were produced on plants receiving a low-cell-density inoculum (10(5) cells/ml). Since cell-free supernatants from strain USDA110 grown to high cell density in complex medium decreased the expression of an nodY-lacZ fusion, this phenomenon was attributed to bradyoxetin-induced repression of nod gene expression. Inoculation of either the permissive soybean genotype (cv. Lambert) or PI 417566 with 10(9) cells/ml of the nodD2, nolA, nodW, and nwsB mutants of USDA110 enhanced nodulation (up to 24%) relative to that seen with inoculations done with 10(5) cells/ml of the mutants or the wild-type strain, indicating that these genes are involved in population-density-dependent nodulation of soybeans. In contrast, the number of nodules produced by an nodD1 mutant on either soybean genotype was less than those seen with the wild-type strain inoculated at a low inoculum density. The nodD2 mutant outcompeted B. japonicum strain USDA123 for nodulation of G. max cv. Lambert at a high or low inoculum density, and the results of root-tip-marking and time-to-nodulate studies indicated that the nolA and nodD2 mutants nodulated this soybean genotype faster than wild-type USDA110. Taken together, the results from these studies indicate that the nodD2 mutant of B. japonicum may be useful to enhance soybean nodulation at high inoculum densities and that NodD2 is a key repressor influencing host-controlled restriction of nodulation, density-dependent suppression of nodulation, perception of bradyoxetin, and competitiveness in the soybean-B. japonicum symbiosis.     

Positive and negative control of nod gene expression in Rhizobium meliloti is required for optimal nodulation

We show that expression of common nodulation genes in Rhizobium meliloti is under positive as well as negative control. A repressor protein was found to be involved in the negative control of nod gene expression. Whereas the activator NodD protein binds to the conserved cis-regulatory element (nod-box) required for coordinated regulation of nod genes, the repressor binds to the overlapping nodD1 and nodA promoters, at the RNA polymerase binding site. A model depicting the possible interaction of the plant-derived nod gene inducer (luteolin), the NodD and the repressor with the nod promoter elements is presented. Mutants lacking the repressor exhibited delayed nodulation phenotype, indicating that fine tuning of nod gene expression is required for optimal nodulation of the plant host.     

Bradyrhizobium (Arachis) sp. strain NC92 contains two nodD genes involved in the repression of nodA and a nolA gene required for the efficient nodulation of host plants.

The common nodulation locus and closely linked nodulation genes of Bradyrhizobium (Arachis) sp. strain NC92 have been isolated on an 11.0-kb EcoRI restriction fragment. The nucleotide sequence of a 7.0-kb EcoRV-EcoRI subclone was determined and found to contain open reading frames (ORFs) homologous to the nodA, nodB, nodD1, nodD2, and nolA genes of Bradyrhizobium japonicum and Bradyrhizobium elkanii. Nodulation assays of nodD1, nodD2, or nolA deletion mutants on the host plants Macroptilium atropurpureum (siratro) and Vigna unguiculata (cowpea) indicate that nolA is required for efficient nodulation, as nolA mutants exhibit a 6-day nodulation delay and reduced nodule numbers. The nolA phenotype was complemented by providing the nolA ORF in trans, indicating that the phenotype is due to the lack of the nolA ORF. nodD1 mutants displayed a 2-day nodulation delay, whereas nodD2 strains were indistinguishable from the wild type. Translational nodA-lacZ, nodD1-lacZ, nodD2-lacZ, and nolA-lacZ fusions were created. Expression of the nodA-lacZ fusion was induced by the addition of peanut, cowpea, and siratro seed exudates and by the addition of the isoflavonoids genistein and daidzein. In a nodD1 or nodD2 background, basal expression of the nodA-lacZ fusion increased two- to threefold. The level of expression of the nodD2-lacZ and nolA-lacZ fusions was low in the wild type but increased in nodD1, nodD2, and nodD1 nodD2 backgrounds independently of the addition of the inducer genistein. nolA was required for increased expression of the nodD2-lacZ fusion. These data suggest that a common factor is involved in the regulation of nodD2 and nolA, and they are also consistent with a model of nod gene expression in Bradyrhizobium (Arachis) sp. strain NC92 in which negative regulation is mediated by the products of the nodD1 and nodD2 genes.     

Stimulation of nodulation in Medicago truncatula by low concentrations of ammonium: quantitative reverse transcription PCR analysis of selected genes

Although mineral nitrogen generally has negative effects on nodulation in legume–rhizobia symbioses, low concentrations of ammonium stimulate nodulation in some legumes. In this study, the effects of ammonium and nitrate on growth, nodulation and expression of 2 nitrogen transport and 12 putative nodulation-related genes of the model symbiosis of Medicago truncatula – Sinorhizobium meliloti are investigated. After 3 weeks of hydroponic growth, whole-plant nodulation was enhanced in all the ammonium treatments and up to three-fold in the 0.5 m M treatment compared with the zero-nitrogen control. Specific nodulation (nodules g⁻¹ root dry weight) was greatly stimulated in the 0.1 and 0.5 m M     treatments, to a lower extent in the 0.1 m M     treatment, and inhibited in all other treatments. Expression of the 14 selected genes was observed at 0, 6, 12 and 24 h after exposure to rhizobia and nitrogen. Expression of nitrogen transporter genes increased significantly, but responses of the three genes putatively associated with symbiosis signaling/nodule initiation were mixed. There were infrequent responses of genes coding for an ABA-activated protein kinase or a gibberellin-regulated protein, but an ethylene-responsive element-binding factor showed increased expression in various treatments and sampling times. Three auxin-responsive genes and three cytokinin-responsive genes showed varied responses to ammonium and nitrate. This study indicates that low concentrations of ammonium stimulate nodulation in M. truncatula , but the data were inconclusive in verifying the hypothesis that a relatively high ratio of cytokinin to auxin in roots may be an underlying mechanism in this stimulation of nodulation.     

nodZ, a unique host-specific nodulation gene, is involved in the fucosylation of the lipooligosaccharide nodulation signal of Bradyrhizobium japonicum.

The nodulation genes of rhizobia are regulated by the nodD gene product in response to host-produced flavonoids and appear to encode enzymes involved in the production of a lipo-chitose signal molecule required for infection and nodule formation. We have identified the nodZ gene of Bradyrhizobium japonicum, whose product is required for the addition of a 2-O-methylfucose residue to the terminal reducing N-acetylglucosamine of the nodulation signal. This substitution is essential for the biological activity of this molecule. Mutations in nodZ result in defective nodulation of siratro. Surprisingly, although nodZ clearly codes for nodulation function, it is not regulated by NodD and, indeed, shows elevated expression in planta. Therefore, nodZ represents a unique nodulation gene that is not under the control of NodD and yet is essential for the synthesis of an active nodulation signal.     

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.     

Chemotaxis,induced gene expression and competitiveness in the rhizosphere

Rhizobia are soil bacteria which symbiotically infect legume roots and generate nodules in which they fix atmospheric nitrogen for the plant in exchange for photosynthetically fixed carbon. A crucial aspect of signal exchange between these symbionts is the secretion of phenolic compounds by the host root which induce nodulation gene expression in the bacteria. Stimulation of nod gene expression by host phenolics is required for nodule formation, is biochemically specific at 10^-6 M, and is mediated by nodD. We and others have shown that rhizobia display chemotaxis to 10^-9 M of the same phenolic compounds. Chemotaxis to inducer phenolics is selectively reduced or abolished by mutations in certain nod genes governing nodulation efficiency or host specificity. Conversely, mutations in rhizobia that affect general motility or chemotaxis have substantial effects on nodulation efficiency and competitiveness. These findings suggest that microbes entering the rhizosphere environment may utilize minor, non-nutrient components in root exudates as signals to guide their movement towards the root surface and elicit changes in gene expression appropriate to this environment.     

Cloning and sequence analysis of the arginine deiminase gene from Mycoplasma arginini

Summary A deletion mutant of Rhizobium leguminosarum biovar viciae lacking the host-specific nodulation (nod) gene region (nodFEL nodMNT and nodO) but retaining the other nod genes (nodD nodABCIJ) was unable to nodulate peas or Vicia hirsuta, although it did induce root hair deformation. The mutant appeared to be blocked in its ability to induce infection threads and could be rescued for nodulation of V. hirsuta in mixed inoculation experiments with an exopolysaccharide deficient mutant (which is also Nod^–). The nodulation deficiency of the deletion mutant strain could be partially restored by plasmids carrying the nodFE, nodFEL or nodFELMNT genes but not by nodLMN. Surprisingly, the mutant strain could also be complemented with a plasmid that did not carry any of the nodFELMNT genes but which did carry the nodO gene on a 30 kb cloned region of DNA. Using appropriate mutations it was established that nodO is essential for nodulation in the absence of nodFE. Thus, either of two independent nod gene regions can complement the deletion mutant for nodulation of V. hirsuta. Similar observations were made for pea nodulation except that nodL was required in addition to nodO for nodulation in the absence of the nodFE genes. These observations show that nodulation can occur via either of two pathways encoded by non-homologous genes.Dedicated to the memory of the late Dr. David Goodchild