Distribution of legume arabinogalactan protein-extensin (AGPE) glycoproteins in symbiotically defective pea mutants with abnormal infection threads

The interface between the host cell and the microsymbiont is an important zone for development and differentiation during consecutive stages of Rhizobium-legume symbiosis. Legume root nodule extensins, otherwise known as arabinogalactan protein-extensins (AGPEs) are abundant components of infection thread matrix. We have characterized the origin and distribution of these glycoproteins at the symbiotic interface of root nodules of symbiotically defective mutants of pea (Pisum sativum L.) by using immunogold localization with MAC265 an anti-AGPE monoclonal antibody. For mutants with defective growth of infection threads, the AGPE epitope was abundant in the extracellular matrix surrounding infected host cells in the central infected tissue of the nodule, as well as in the lumen of Rhizobiuminduced infection threads. This seems to indicate a mistargeting of AGPE as a consequence of abnormal growth of the infection threads. Furthermore, mutants in the gene sym33 showed reduced labeling with MAC265 and, in some infection threads and droplets, the label was completely absent, a phenomenon that is not observed in wild-type nodules. This suggests an alteration in the composition of the infection thread matrix for sym33 mutants, which may be correlated to the absence of endocytosis of rhizobia into the host cytoplasm.     

Development of ectopic roots from abortive nodule primordia

The symbiotic phenotype of five Tn5-induced mutants of Rhizobium etli affected in different anabolic pathways (namely, gluconeogenesis and biosynthesis of lysine, purine, or pyrimidine) was analyzed. These mutants induced, on the root of Phaseolus vulgaris, a normal early sequence of morphogenetics events, including root hair deformation and development of nodule primordia. Later on, however, from the resulting root outgrowths, instead of nodules, one or more ectopic roots (spaced closely related and agravitropic) emerged. Therefore, this group of mutant was collectively called "root inducer" (RIND). It was observed that the RIND-induced infection threads aborted early inside the invaded root hair, and that the resulting abortive nodules lack induction of late nodulin genes. Moreover, experiments performed using a conditional mutant (a methionine-requiring invader) revealed that bacterial invasion plays a key role in the maintenance of the program of nodule development and, in particular, in the differentiation of the most specific symbiotic tissue of globose nodules, the central tissue. These data indicate that, in P. vulgaris, the nodule primordium is a root-specified pro-meristematic tissue.     

Development ofBradyrhizobium infections in supernodulating and non-nodulating mutants of soybean (Glycine max [L.] Merrill)

Summary The early events in the development of nodules induced byBradyrhizobium japonicum were studied in serial sections of a wild type (cv. Bragg), a supernodulating mutant (nts 382) and four non-nodulating mutants (nod49, nod139, nod772, andrj ₁) of soybean (Glycine max [L.] Merrill). Cultivar Bragg responded to inoculation in a similar manner to that described previously for cv. Williams; centres of sub-epidermal cell divisions were observed both with and without associated infection threads and most infection events were blocked before the formation of a nodule meristem. The non-nodulating mutants (nod49, nod772, andrj ₁) had, at most, a few centres of sub-epidermal cell divisions. In general, these were devoid of infection threads and did not develop beyond the very early stages of nodule ontogeny. Sub-epidermal cell divisions or infection threads were never observed on mutant nodl39. This mutant is not allelic to the other non-nodulating mutants and represents a defect in a separate complementation group or gene that is required for nodulation. The supernodulating mutant nts382, which is defective in autoregulation of nodulation, had a similar number of sub-epidermal cell divisions as the wild-type Bragg, but a much greater proportion of these developed to an advanced stage of nodule ontogeny. Mutant nts382, like Bragg, possessed other infection events that were arrested at an early stage of development. The results are discussed in the context of the progression of events in nodule formation and autoregulation of nodulation in soybean.Abbreviations nts nitrate tolerant symbiosis - RT root tip (i.e., position of the tap root tip at the time of inoculation) - SERH shortest emerging root hair (i.e., position of the shortest emerging root hair on the tap root at the time of inoculation) - SCD subepidermal cell divisions     

Dissection of Nodule Development by Supplementation of Rhizobium leguminosarum biovar phaseoli Purine Auxotrophs with 4-Aminoimidazole-5-Carboxamide Riboside

Purine auxotrophs of Rhizobium leguminosarum biovar phaseoli CFN42 elicit uninfected pseudonodules on bean (Phaseolus vulgaris L.). Addition of 4-aminoimidazole-5-carboxamide (AICA) riboside to the root medium during incubation of the plant with these mutants leads to enhanced nodule development, although nitrogenase activity is not detected. Nodules elicited in this manner had infection threads and anatomical features characteristic of normal nodules, such as peripheral vasculature rather than the central vasculature of the pseudonodules that were elicited without AICA riboside supplementation. Although 10⁵ to 10⁶ bacteria could be recovered from these nodules after full development, bacteria were not observed in the interior nodule cells. Instead, large cells with extensive internal membranes were present. Approximately 5% of the normal amount of leghemoglobin and 10% of the normal amount of uricase were detected in these nodules. To promote the development of true nodules rather than pseudonodules, AICA riboside was required no later than the second day through no more than the sixth day following inoculation. After this period, removal of AICA riboside from the root medium did not prevent the formation of true nodules. This observation suggests that there is a critical stage of infection, reached before nodule emergence, at which development becomes committed to forming a true nodule rather than a pseudonodule.     

nip, a symbiotic Medicago truncatula mutant that forms root nodules with aberrant infection threads and plant defense-like response

To investigate the legume-Rhizobium symbiosis, we isolated and studied a novel symbiotic mutant of the model legume Medicago truncatula, designated nip (numerous infections and polyphenolics). When grown on nitrogen-free media in the presence of the compatible bacterium Sinorhizobium meliloti, the nip mutant showed nitrogen deficiency symptoms. The mutant failed to form pink nitrogen-fixing nodules that occur in the wild-type symbiosis, but instead developed small bump-like nodules on its roots that were blocked at an early stage of development. Examination of the nip nodules by light microscopy after staining with X-Gal for S. meliloti expressing a constitutive GUS gene, by confocal microscopy following staining with SYTO-13, and by electron microscopy revealed that nip initiated symbiotic interactions and formed nodule primordia and infection threads. The infection threads in nip proliferated abnormally and very rarely deposited rhizobia into plant host cells; rhizobia failed to differentiate further in these cases. nip nodules contained autofluorescent cells and accumulated a brown pigment. Histochemical staining of nip nodules revealed this pigment to be polyphenolic accumulation. RNA blot analyses demonstrated that nip nodules expressed only a subset of genes associated with nodule organogenesis, as well as elevated expression of a host defense-associated phenylalanine ammonia lyase gene. nip plants were observed to have abnormal lateral roots. nip plant root growth and nodulation responded normally to ethylene inhibitors and precursors. Allelism tests showed that nip complements 14 other M. truncatula nodulation mutants but not latd, a mutant with a more severe nodulation phenotype as well as primary and lateral root defects. Thus, the nip mutant defines a new locus, NIP, required for appropriate infection thread development during invasion of the nascent nodule by rhizobia, normal lateral root elongation, and normal regulation of host defense-like responses during symbiotic interactions.     

Identification of symbiotically defective mutants of Lotus japonicus affected in infection thread growth

During the symbiotic interaction between legumes and rhizobia, the host cell plasma membrane and associated plant cell wall invaginate to form a tunnel-like infection thread, a structure in which bacteria divide to reach the plant root cortex. We isolated four Lotus japonicus mutants that make infection pockets in root hairs but form very few infection threads after inoculation with Mesorhizobium loti. The few infection threads that did initiate in the mutants usually did not progress further than the root hair cell. These infection-thread deficient (itd) mutants were unaffected for early symbiotic responses such as calcium spiking, root hair deformation, and curling, as well as for the induction of cortical cell division and the arbuscular mycorrhizal symbiosis. Complementation tests and genetic mapping indicate that itd2 is allelic to Ljsym7, whereas the itdl, itd3, and itd4 mutations identified novel loci. Bacterial release into host cells did occur occasionally in the itdl, itd2, and itd3 mutants suggesting that some infections may succeed after a long period and that infection of nodule cells could occur normally if the few abnormal infection threads that were formed reached the appropriate nodule cells.     

Entry of Rhizobium leguminosarum bv.viciae into root hairs requires minimal Nod factor specificity, but subsequent infection thread growth requires nodO or nodE

Using various mutant strains of Rhizobium leguminosarum bv. viciae, we have investigated the role of nodO in stimulating infection thread development in vetch and pea. Analysis of R. leguminosarum bv. viciae nodE and nodO mutants revealed no significant difference from the wild-type infection phenotype. Conversely, an R. leguminosarum bv. viciae nodE nodO double mutant was severely impaired in its ability to form normal infection threads. This strain displayed a number of novel infection-related events, including intracellular accumulations of bacteria at the base of root hairs, distended and enlarged infection threads, and reversed threads growing up root hairs. Since normal infection was seen in a nodE mutant, nodO must suppress these abnormal infection phenomena A deletion mutant, retaining only the nodD and nodABCIJ genes, also formed intracellular accumulations at the base of root hairs. Addition of R. leguminosarum bv. viciae nodO could alleviate this phenotype and restore some infection thread formation, although these threads appeared to be abnormal. Exogenous application of R. leguminosarum bv. viciae Nod factors could not alleviate the aberrant infection phenotype. Our results show that the most basic Nod factor structure can allow bacterial entry into the root hair, and that nodO can promote subsequent infection thread development.     

Nodules Initiated by Rhizobium meliloti Exopolysaccharide Mutants Lack a Discrete, Persistent Nodule Meristem

Infection of alfalfa with Rhizobium meliloti exo mutants deficient in exopolysaccharide results in abnormal root nodules that are devoid of bacteria and fail to fix nitrogen. Here we report further characterization of these abnormal nodules. Tightly curled root hairs or shepherd's crooks were found after inoculation with Rm 1021-derived exo mutants, but curling was delayed compared with wild-type Rm 1021. Infection threads were initiated in curled root hairs by mutants as well as by wild-type R. meliloti, but the exo mutant-induced threads aborted within the peripheral cells of the developing nodule. Also, nodules elicited by Rm 1021-derived exo mutants were more likely to develop on secondary roots than on the primary root. In contrast with wild-type R. meliloti-induced nodules, the exo mutant-induced nodules lacked a well defined apical meristem, presumably due to the abortion of the infection threads. The relationship of these findings to the physiology of nodule development is discussed.     

Cloning and characterization of Rhizobium meliloti loci required for symbiotic root nodule invasion

An immunological assay of root nodule polypeptides was used to analyze the nodules induced by 25 symbiotically defective Rhizobium meliloti mutants. Differences in polypeptide accumulation in these nodules were used to divide the mutants into three subsets. One subset, containing two mutant strains, was further analyzed. Nodules induced by these mutant strains lack both infection threads and bacteria. The kinetics of nodule formation by these mutant strains, by an exoB mutant, and by mixed mutant inocula suggest that the gene products required for nodule invasion may also influence nodule meristem induction. One of the two mutants characterized in this study contains a transposon Tn5 insertion in the ndvB locus, which probably results in the loss of beta-glucan synthesis. The second mutant contains a transposon in a previously uncharacterized locus. RNA analysis suggests that the newly identified locus is transcribed in free-living cultures of ndvB and exoB strains, as well as in the parental R. meliloti strain. Southern blot analysis suggests that at least a portion of this locus is duplicated. This duplication may explain the apparently leaky phenotype of the mutant strain.     

crinkle,a novel symbiotic mutant that affects the infection thread growth and alters the root hair,trichome, and seed development in Lotus japonicus

To elucidate the mechanisms involved in Rhizobium-legume symbiosis, we examined a novel symbiotic mutant, crinkle (Ljsym79), from the model legume Lotus japonicus. On nitrogen-starved medium, crinkle mutants inoculated with the symbiont bacterium Mesorhizobium loti MAFF 303099 showed severe nitrogen deficiency symptoms. This mutant was characterized by the production of many bumps and small, white, uninfected nodule-like structures. Few nodules were pale-pink and irregularly shaped with nitrogen-fixing bacteroids and expressing leghemoglobin mRNA. Morphological analysis of infected roots showed that nodulation in crinkle mutants is blocked at the stage of the infection process. Confocal microscopy and histological examination of crinkle nodules revealed that infection threads were arrested upon penetrating the epidermal cells. Starch accumulation in uninfected cells and undeveloped vascular bundles were also noted in crinkle nodules. Results suggest that the Crinkle gene controls the infection process that is crucial during the early stage of nodule organogenesis. Aside from the symbiotic phenotypes, crinkle mutants also developed morphological alterations, such as crinkly or wavy trichomes, short seedpods with aborted embryos, and swollen root hairs. crinkle is therefore required for symbiotic nodule development and for other aspects of plant development.     

Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation

Spontaneous mutants at a new symbiotic locus in Rhizobium meliloti SU47 are resistant to several phages and are conditionally insensitive to a monoclonal antibody to the bacterial surface, apparently because they are deficient in a wild-type exopolysaccharide. On alfalfa, the mutants do not curl root hairs, but penetrate the epidermis directly, forming nodules that contain no visible infection threads or "bacteroids," have a few bacteria in superficial intercellular spaces only and not within the nodule cells, and fail to fix nitrogen (Fix-). Evidently, infection threads are not essential for cell proliferation and nodule formation, which are here induced by a bacterial signal at a distance and uncoupled from the bacterial differentiation that normally goes on as well.     

Rhizobium infection and nodule development in soybean are affected by exposure of the cotyledons to light

The initiation of Rhizobium infections and the development of nodules on the primary root of soybean Glycine max L. Merr cv Williams seedlings are strongly affected by exposure of the cotyledons/hypocotyls to light. Seedlings in plastic growth pouches were inoculated with R. japonicum in dim light and the position of the root tip of each seedling was marked on the face of the pouch. The pouches were covered and kept in the dark for various times before exposing the upper portions of the plants (cotyledons and hypocotyls) to light. Maximum nodulation occurred if the plants were kept in the dark until 1 day after inoculation. The exposure of plants to light 2 days before inoculation reduced the number of nodules by 50% while the number of nodules was reduced by 70% if the plants were kept in the dark until 7 days after inoculation. Anatomical studies revealed that exposure to light prior to inoculation reduced both the number of infection centers with visible infection threads and the number of infections which developed nodule meristems. Plants kept in the dark for 7 days after inoculation formed a normal number of infection threads above the root tip mark, but very few of these infections developed a nodule meristem. It appears that light stimulates soybean to produce substances which can both inhibit the formation of infection threads and enhance the development of nodules from established infection threads. The effects of light on nodulation appear to be expressed independently of the Rhizobium-induced suppression of nodule formation in younger regions of the root.     

Nodulation inhibition by Rhizobium leguminosarum multicopy nodABC genes and analysis of early stages of plant infection.

During analysis of early events in the infection and nodulation of Vicia hirsuta roots inoculated with normal and mutant strains of Rhizobium leguminosarum and strains containing cloned nodulation (nod) genes, a number of novel observations were made. (i) Alternating zones of curled and straight root hairs were seen on roots of V. hirsuta inoculated with the wild-type strain of R. leguminosarum. This phasing of root hair curling was not seen if plants were grown under continuous light or continuous dark conditions. (ii) Reduced nodulation and delayed nodule initiation was observed with a strain carrying a Tn5 mutation in the nodE gene. In addition the phased root hair curling was absent, and root hair curling was observed along the length of the root. (iii) The nodABC genes cloned on a multicopy plasmid in a wild-type strain inhibited nodulation but induced a continuous root hair curling response. Those few nodules that eventually formed were found to contain bacteria which had lost the plasmid carrying the nodABC genes. (iv) With a strain of Rhizobium cured of its indigenous symbiotic plasmid, but containing the cloned nodABCDEF genes, continuous root hair curling on V. hirsuta was observed. However, no infection threads were observed, and surprisingly, it did appear that initial stages of nodule development occurred. Observations of thin sections of these early developing nodules indicated that early nodule meristematic divisions may have occurred but that no bacteria were found within the nodules and no infection threads were observed either within the nodule bumps or within any of the root hairs. It was concluded that for normal infections to occur, precise regulation of the nod genes is required and that overexpression of the root hair curling genes inhibits the normal infection process.     

New nodulation mutants responsible for infection thread development in Lotus japonicus

Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.     

60Ma of legume nodulation. What's new? What's changing?

Current evidence suggests that legumes evolved about 60 million years ago. Genetic material for nodulation was recruited from existing DNA, often following gene duplication. The initial process of infection probably did not involve either root hairs or infection threads. From this initial event, two branched pathways of nodule developmental processes evolved, one involving and one not involving the development of infection threads to 'escort' bacteria to young nodule cells. Extant legumes have a wide range of nodule structures and at least 25% of them do not have infection threads. The latter have uniform infected tissue whereas those that have infection threads have infected cells interspersed with uninfected (interstitial) cells. Each type of nodule may develop indeterminately, with an apical meristem, or show determinate growth. These nodule structures are host determined and are largely congruent with taxonomic position. In addition to variation on the plant side, the last 10 years have seen the recognition of many new types of 'rhizobia', bacteria that can induce nodulation and fix nitrogen. It is not yet possible to fit these into the emerging pattern of nodule evolution.     

Rhizobium fix genes mediate at least two communication steps in symbiotic nodule development

To identify bacterial genes involved in symbiotic nodule development, ineffective nodules of alfalfa (Medicago sativa) induced by 64 different Fix-mutants of Rhizobium meliloti were characterized by assaying for symbiotic gene expression and by morphological studies. The expression of leghemoglobin and nodulin-25 genes from alfalfa and of the nifHD genes from R. meliloti were monitored by hybridizing the appropriate DNA probes to RNA samples prepared from nodules. The mutants were accordingly divided into three groups. In group I none of the genes were expressed, in group II only the plant genes were expressed and in group III all three genes were transcribed. Light and electron microscopical analysis of nodules revealed that nodule development was halted at different stages in nodules induced by different group I mutants. In most cases nodules were empty lacking infection threads and bacteroids or nodules contained infection threads and a few released bacteroids. In nodules induced by a third mutant class bacteria were released into the host cells, however the formation of the peribacteroid membrane was not normal. On this basis we suggest that peribacteroid membrane formation precedes leghemoglobin and nodulin-25 induction, moreover, after induction of nodulation by the nod genes at least two communication steps between the bacteria and the host plants are necessary for the development of the mature nodule. By complementing each mutant of group I with a genomic R. meliloti library made in pLAFRl, four new fix loci were identified, indicating that several bacterial genes are involved in late nodule development.     

Expression of the Medicago truncatula DM12 gene suggests roles of the symbiotic nodulation receptor kinase in nodules and during early nodule development

The Medicago truncatula DMI2 gene encodes a receptorlike kinase required for establishing root endosymbioses. The DMI2 gene was shown to be expressed much more highly in roots and nodules than in leaves and stems. In roots, its expression was not altered by nitrogen starvation or treatment with lipochitooligosaccharidic Nod factors. Moreover, the DMI2 mRNA abundance in roots of the nfp, dmil, dmi3, nsp1, nsp2, and hcl symbiotic mutants was similar to the wild type, whereas lower levels in some dmi2 mutants could be explained by regulation by the nonsense-mediated decay, RNA surveillance mechanism. Using pDMI2::GUS fusions, the expression of DMI2 in roots appeared to be localized primarily in the cortical and epidermal cells of the younger, lateral roots and was not observed in the root apices. Following inoculation with Sinorhizobium meliloti, the DMI2 gene was induced in the nodule primordia, before penetration by the infection threads. No increased expression was seen in lateral-root primordia. In nodules, expression was observed primarily in a few cell layers of the pre-infection zone. These results are consistent with the DMI2 gene mediating Nod factor perception and transduction leading to rhizobial infection, not only in root epidermal cells but also during nodule development.     

Rhizobium meliloti nodulation genes allow Agrobacterium tumefaciens and Escherichia coli to form pseudonodules on alfalfa.

Regions of the Rhizobium meliloti symbiotic plasmid (20 to 40 kilobase pairs long) containing nodulation (nod) genes were transferred to Agrobacterium tumefaciens or Escherichia coli by conjugation. The A. tumefaciens and E. coli transconjugants elicited root hair curling and the formation of ineffective pseudonodules on inoculated alfalfa plants. A tumefaciens elicited pseudonodules formed at a variable frequency, ranging from 15 to 45%, irrespective of the presence of the Ti plasmid. These pseudonodules developed characteristic nodule meristems, and in some nodules, infection threads were found within the interior of nodules. Infrequently, infection threads penetrated deformed root hairs, but these threads were found only in a minority of nodules. There was no evidence of bacterial release from the infection threads. In addition to being found within threads, agrobacteria were also found in intercellular spaces and within nodule cells that had senesced . In the latter case, the bacteria appeared to invade the nodule cells independently of infection threads and degenerated at the same time as the senescing host cells. No peribacteroid membranes enclosed any agrobacteria , and no bacteroid differentiation was observed. In contrast to the A. tumefaciens-induced pseudonodules , the E. coli-induced pseudonodules were completely devoid of bacteria; infection threads were not found to penetrate root hairs or within nodules. Our results suggest that relatively few Rhizobium genes are involved in the earliest stages of nodulation, and that curling of root hairs and penetration of bacteria via root hair infection threads are not prerequisites for nodule meristem formation in alfalfa.     

Morphogenesis of lucerne root nodules incited by Rhizobium meliloti in the presence of combined nitrogen

Combined light and transmission electron microscopy were used to examine the effect of nitrate on the development of root nodules in lucerne (alfalfa, Medicago sativa L.) following induction by the nitrogen-fixing symbiont, Rhizobium meliloti. The timing of NO ₃ ^- addition was varied in order to study its effect on all of the recognized morphogenetic steps of nodule formation. Roots of plants inoculated in the presence of 18 mM NO ₃ ^- had straight root hairs which were devoid of adherent rhizobia and infection threads, and developed no nodules. However, nodules were formed on roots if 18 mM NO ₃ ^- was added 5 d after inoculation. At this time, the initiation of nodule primordia had already commenced in the root cortex. The histology and ultrastructure of young nodules which had developed for 5 d in the absence of NO ₃ ^- and another 5 d in the presence of 18 mM NO ₃ ^- resembled nodules developing under N-free conditions, except that in the infection threads within the infection zone of the nodule 1) some bacteria tended to loose their normal shape and gain more electron density, indicating premature degradation, and 2) the matrix of the infection threads was abnormally enlarged. In the presence of high NO ₃ ^- levels in the medium, lysis and degeneration of the bacteria released from the infection threads were observed in the infection and bacteroid zones of developing nodules, indicative of premature senescence. On the other hand, the nodule meristems continued to proliferate even after 12 d of exposure of 18 mM NO ₃ ^- . This was the only morphogenetic step of root nodulation which was insensitive to levels of combined nitrogen that completely prevented infection if present at the time of inoculation. These data indicate that all of the recognized steps of root nodule morphogenesis in which the bacteria play a key role are sensitive to the inhibitory effect of combined nitrogen.