SPIKE1 Activates the GTPase ROP6 to Guide the Polarized Growth of Infection Threads in Lotus japonicus

In legumes, rhizobia attach to root hair tips and secrete nodulation factor to activate rhizobial infection and nodule organogenesis. Endosymbiotic rhizobia enter nodule primordia via a specialized transcellular compartment known as the infection thread (IT). The IT elongates by polar tip growth, following the path of the migrating nucleus along and within the root hair cell. Rho-family ROP GTPases are known to regulate the polarized growth of cells, but their role in regulating polarized IT growth is poorly understood. Here, we show that LjSPK1, a DOCK family guanine nucleotide exchange factor (GEF), interacts with three type I ROP GTPases. Genetic analyses showed that these three ROP GTPases are involved in root hair development, but only LjROP6 is required for IT formation after rhizobia inoculation. Misdirected ITs formed in the root hairs of Ljspk1 and Ljrop6 mutants. We show that LjSPK1 functions as a GEF that activates LjROP6. LjROP6 enhanced the plasma membrane localization LjSPK1 in Nicotiana benthamiana leaf cells and Lotus japonicus root hairs, and LjSPK1 and LjROP6 interact at the plasma membrane. Taken together, these results shed light on how the LjROP6-LjSPK1 module mediates the polarized growth of ITs in L. japonicus.     

Early cytological events in the infection of the root hairs ofTrifolium repens byRhizobium leguminosarum biovartrifolii observed by glass slide culture in living seedlings

This report describes the early cytological events in the infection byRhizobium leguminosarum biovartrifolii of the root hairs ofTrifolium repens seedlings kept alive on agar medium in glass slide culture experiment. The infection threads bearing rhizobia were formed as soon as the epidermal cells began to emerge as root hairs. On the top of some of these infected emerging root hairs, there were smoky, cell-debris-like bodies, which appeared to be derived from the cell wall dug by rhizobia. Similar bodies were also observed in longer root hairs. None of the root hair cells along the length of the roots which contained infection threads were curled or distorted. A substantial number of pink-colored nodules were later formed on the roots with non-curled infected root hairs.     

Early development ofRhizobium-induced root nodules ofParasponia rigida. I. Infection and early nodule initiation

Summary The first of two major steps in the infection process in roots ofParasponia rigida (Ulmaceae) following inoculation byRhizobium strain RP501 involves the invasion ofRhizobium into the intercellular space system of the root cortex. The earliest sign of root nodule initiation is the presence of clumps of multicellular root hairs (MCRH), a response apparently unique amongRhizobium-root associations. At the same time or shortly after MCRH are first visible, cell divisions are initiated in the outer root cortex of the host plant, always subjacent to the MCRH. No infection threads were observed in root hairs or cortical cells in early stages. Rhizobial entry through the epidermis and into the root cortex was shown to occur via intercellular invasion at the bases of MCRH. The second major step in the infection process is the actual infectionper se of host cells by the rhizobia and formation of typical intracellular infection threads with host cell accommodation. This infection step is probably the beginning of the truly symbiotic relationship in these nodules. Rhizobial invasion and infection are accompanied by host cortical cell divisions which result in a callus-like mass of cortical cells. In addition to infection thread formation in some of these host cortical cells, another type of rhizobial proliferation was observed in which large accumulations of rhizobia in intercellular spaces are associated with host cell wall distortion, deposition of electron-dense material in the walls, and occasional deleterious effects on host cell cytoplasm.     

Phosphorylation of MtRopGEF2 by LYK3 mediates MtROP activity to regulate rhizobial infection in Medicago truncatula

The formation of nitrogen-fixing no dules on legume roots requires the coordination of infection by rhizobia at the root epidermis with the initiation of cell divisions in the root cortex. During infection, rhizobia attach to the tip of elongating root hairs which then curl to entrap the rhizobia. However, the mechanism of root hair deformation and curling in response to symbiotic signals is still elusive. Here, we found that small GTPases (MtRac1/MtROP9 and its homologs) are required for root hair development and rhizobial infection in Medicago truncatula. Our results show that the Nod factor receptor LYK3 phosphorylates the guanine nucleotide exchange factor MtRopGEF2 at S73 which is critical for the polar growth of root hairs. In turn, phosphorylated MtRopGEF2 can activate MtRac1. Activated MtRac1 was found to localize at the tips of root hairs and to strongly interact with LYK3 and NFP. Taken together, our results support the hypothesis that MtRac1, LYK3, and NFP form a polarly localized receptor complex that regulates root hair deformation during rhizobial infection.     

The legume Rhizosphere

Summary Examination of the root surfaces of Medicago tribuloides Desr. with phase contrast microscopy or electron microscopy using thin sections revealed the presence of a layer of material outside the root surface. In thin sections of KMnO₄ fixed roots this layer was composed of a thin electron dense layer, an electron dense granular matrix of varying width and an enclosing electron dense membrane. After inoculation with an effective Rhizobium strain, rhizobia were found aggregated in a definite zone adjacent to the root surface when either living roots were examined by phase microscopy or thin sections by electron microscopy. This layer was also found in inoculated and uninoculated roots of Trifolium fragiferum and T. pratense. The bacteria were packed with inclusion granules and lay enclosed by a membrane layer adjacent to the granular matrix seen in uninoculated roots. The ultrastructural organisation of root hairs is essentially similar to that of other differentiated root cells. The replicated surface of the uninoculated root hair wall is largely amorphous with a few sculptured portions resembling a cuticle layer. The inoculated root hair wall often shows areas of exposed, open microfibrillar meshwork with rhizobia sitting on the microfibrils. The rhizobia resemble a flagellated, coccoid swarmer form of Rhizobium which is found in the barrel medic rhizosphere.     

Aperiplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins

A search was performed for a periplasmic molecular chaperone which may assist outer membrane proteins of Escherichia coli on their way from the cytoplasmic to the outer membrane. Proteins of the periplasmic space were fractionated on an affinity column with sepharose-bound outer membrane porin OmpF. A 17kDa polypeptide was the predominant protein retained by this column. The corresponding gene was found in a gene bank; it encodes the periplasmic protein Skp. The protein was isolated and it could be demonstrated that it bound outer membrane proteins, following SDS-PAGE, with high selectivity. Among these were OmpA, OmpC, OmpF and the maltoporin LamB. The chromosomal skp gene was inactivated by a deletion causing removal of most of the signal peptide plus 107 residues of the 141-residue mature protein. The mutant was viable but possessed much-reduced concentrations of outer membrane proteins. This defect was fully restored by a plasmid-borne skp gene which may serve as a periplasmic chaperone.     

Exposed and hidden lectin-binding epitopes at the surface of<Emphasis Type="Italic">Borrelia burgdorferi</Emphasis>

The presence of surface- and subsurface-located lectin-binding epitopes of Borrelia burgdorferi was examined by electron microscopy using a variety of gold-labeled lectins. Concanavalin A reacted predominantly with extracellular material adjacent to the spirochetes. Wheat germ agglutinin bound weakly to the surface of borreliae; however, alterations of the outer membrane by preincubation in 100 ppm Triton X-100 or boiling uncovered numerous periplasmic sites recognized by the lectin. The periplasmic flagella liberated by some cells after detergent treatment were labeled with concanavalin A, wheat germ agglutinin and Ulex europaeus agglutinin UEA-I. No surface-exposed or periplasmic epitopes for the lectins from Glycine max, Dolichos biflorus or Helix pomatia were detected.     

Ultrastructure of infection-thread development during the infection of soybean by Rhizobium japonicum

The location and topography of infection sites in soybean (Glycine max (L.) Merr.) root hairs spot-inoculated with Rhizobium japonicum have been studied at the ultrastructural level. Infections commonly developed at sites created when the induced deformation of an emerging root hair caused a portion of the root-hair cell wall to press against an adjacent epidermal cell, entrapping rhizobia within the pocket between the two host cells. Infections were initiated by bacteria which became embedded in the mucigel in the enclosed groove. Infection-thread formation in soybean appears to involve degradation of mucigel material and localized disruption of the outer layer of the folded hair cell wall by one or more entrapped rhizobia. Rhizobia at the site of penetration are separated from the host cytoplasm by the host plasmalemma and by a layer of wall material that appears similar or identical to the normal inner layer of the hair cell wall. Proliferation of the bacteria results in an irregular, wall-bound sac near the site of penetration. Tubular infection threads, bounded by wall material of the same appearance as that surrounding the sac, emerge from the sac to carry rhizobia roughly single-file into the hair cell. Growing regions of the infection sac or thread are surrounded by host cytoplasm with high concentrations of organelles associated with synthesis and deposition of membrane and cell-wall material. The threads follow a highly irregular path toward the base of the hair cell. Threads commonly run along the base of the hair cell for some distance, and may branch and penetrate into subjacent cortical cells at several points in a manner analagous to the initial penetration of the root hair.     

Nod factors alter the microtubule cytoskeleton in Medicago truncatula root hairs to allow root hair reorientation

The microtubule (MT) cytoskeleton is an important part of the tip-growth machinery in legume root hairs. Here we report the effect of Nod factor (NF) on MTs in root hairs of Medicago truncatula. In tip-growing hairs, the ones that typically curl around rhizobia, NF caused a subtle shortening of the endoplasmic MT array, which recovered within 10 min, whereas cortical MTs were not visibly affected. In growth-arresting root hairs, endoplasmic MTs disappeared shortly after NF application, but reformed within 20 min, whereas cortical MTs remained present in a high density. After NF treatment, growth-arresting hairs were swelling at their tips, after which a new outgrowth formed that deviated with a certain angle from the former growth axis. MT depolymerization with oryzalin caused a growth deviation similar to the NF; whereas, combined with NF, oryzalin increased and the MT-stabilizing drug taxol suppressed NF-induced growth deviation. The NF-induced disappearance of the endoplasmic MTs correlated with a loss of polar cytoarchitecture and straight growth directionality, whereas the reappearance of endoplasmic MTs correlated with the new set up of polar cytoarchitecture. Drug studies showed that MTs are involved in determining root hair elongation in a new direction after NF treatment.     

Endophytic colonisation of Bacillus subtilis in the roots of Robinia pseudoacacia L

The endophytic colonisation of Bacillus subtilis strain GXJM08, isolated from roots of Podocarpus imbricatus B1. Enum. P1. Jav., in roots of the leguminous plant Robinia pseudoacacia L. was investigated. Ultrastructure observations showed that B. subtilis caused morphological changes in the root hair and colonised the plant through infected root hairs. The structure of the infection thread was similar to that of rhizobia, but the structure of infected cells was different. B. subtilis is also different from rhizobia and plant pathogens in terms of the formation of a peribacteroid membrane and the mode of penetration through the host cell wall. Our results provide a basis for studying development of the mutualistic symbiotic relationship between B. subtilis and plants, and a basis for studying the mechanism of the B. subtilis–plant interaction.     

Populations ofRhizobium japonicum associated with the surfaces of soil-grown roots

Summary Populations of nativeRhizobium japonicum 123 in the rhizospheres of field and pot grown plants as determined by immunofluorescence were calculated on the basis of root surface area. The density ofR. japonicum 123 on the root fluctuated between a few hundred to over a thousand per square centimeter of root surface. As root volume expanded rapidly, the Rhizobium density fell to less than one hundred per unit area. There was no appreciable effect due to different plant, nitrogen amendment, or addition of another strain ofR. japonicum, on the surface density of the nativeR. japonicum population on roots. Nor did the native population influence the added strain. Direct examination of root surface segments revealed that naturalized rhizobia existed sparsely on root surfaces in the form of short rods. They were observed to be attached sideways or in a polar manner on root hairs, epidermal cells, and at junctions of tap and lateral roots. There was no evidence of specific stimulation of the homologous Rhizobium by the host plant as a prelude to nodulation.     

Effects of sodium chloride and polyethylene glycol on root-hair infection and nodulation of Vicia faba L. plants by Rhizobium leguminosarum

The effects of sodium chloride and polyethylene glycol (PEG) on the interaction between Rhizobium leguminosarum strain 29d and root hairs of field bean (Vicia faba L. cv. Maris Bead) plants were investigated. Two levels each of NaCl (50 and 100 mol·m^–3) and PEG (100 and 200 mol·m^–3) were given at the time of root-hair formation. Scanning electron microscopy showed rhizobial attachment and colonization on root-hair tips. Adhesion of rhizobia in both lateral and polar orientation, sometimes associated with microfibrils, occurred mainly in crooks at the root-hair tips; most of the infections also occurred here. Bacterial colonization and root-hair curling were both reduced by stress treatments. Polyethylene glycol but not NaCl significantly reduced root-hair diameter. The proportion of root hairs containing infection threads was reduced by 30% under NaCl and by 52% under PEG. The structure of some of the root hairs, epidermal and hypodermal cells, as seen by light microscopy in ultrasections, was distorted as a result of NaCl and PEG treatments; cells showed plasmolysis and folded membranes. After three weeks of treatment, both NaCl and PEG inhibited nodule number by about 50% and nodule weight by more than 60%. It is concluded that the root-hair infection process in Vicia faba is impaired by NaCl and PEG treatments and this in turn results in fewer nodules being produced.Abbreviation PEG polyethylene glycol     

Isolation of root hairs from seedlings of Pisum sativum. Identification of root hair specific proteins by in situ labeling

A procedure was developed which allows the large-scale isolation of root hairs from seedlings of Pisum sativum . L. cvs. Kleine Rheinländerin and Rosa Krone. The method may yield up to 50 g fresh weight of root hairs per 3.10⁴ seedlings. In a modified form considerable amounts of root hair material may be harvested, even after incubation of the roots in aqueous solutions. Thus, detailed biochemical studies on the root hair system have become feasible.
The occurrence of specific proteins in membrane fractions of P. sativum root hairs was demonstrated as follows: Incubation of root hairs in situ with 3-azidonaphthalene-2,7-disulfonate – a strongly anionic, photoactivated fluorescent marker – followed by gel electrophoresis of membrane fractions showed the presence of root-hair specific proteins which, since the system was intact, suggests that they are on the outer surface of the cells.     

Cell polarity and PIN protein positioning in Arabidopsis require STEROL METHYLTRANSFERASE1 function

Plants have many polarized cell types, but relatively little is known about the mechanisms that establish polarity. The orc mutant was identified originally by defects in root patterning, and positional cloning revealed that the affected gene encodes STEROL METHYLTRANSFERASE1, which is required for the appropriate synthesis and composition of major membrane sterols. smt1(orc) mutants displayed several conspicuous cell polarity defects. Columella root cap cells revealed perturbed polar positioning of different organelles, and in the smt1(orc) root epidermis, polar initiation of root hairs was more randomized. Polar auxin transport and expression of the auxin reporter DR5-beta-glucuronidase were aberrant in smt1(orc). Patterning defects in smt1(orc) resembled those observed in mutants of the PIN gene family of putative auxin efflux transporters. Consistently, the membrane localization of the PIN1 and PIN3 proteins was disturbed in smt1(orc), whereas polar positioning of the influx carrier AUX1 appeared normal. Our results suggest that balanced sterol composition is a major requirement for cell polarity and auxin efflux in Arabidopsis.     

Nod factors of Rhizobium are a key to the legume door

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lopo-oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis-specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR-factors to signal-production-deficient mutants of the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium japorticum strain USDA110. Between 10 ⁻⁷ M and 10⁻⁶ M NodNGR factors permitted these NodABC mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a non-host. Detailed cytological investigations of V. unguiculata showed that the NodABC mutant UGR AnodABC, in the presence of NodNGR factors, entered roots in the same way as the wild-type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.     

Some Observations on Infection of Arachis hypogaea L. by Rhizobium

The infection process in Arachis hypogaea by rhizobia differsfrom that normally found in Trifolium spp. in that no infectionthreads are formed. The root hairs, which are long (up to 4mm), septate, and often with large basal cells, occur only atthe sites of emerging lateral roots. Infection occurs only wherethe root hairs have large basal cells. Rhizobia cause curlingand deformation of the root hairs (as in Trifolium spp.) butenter the root at the junction of the root hair and the epidermaland cortical cells. The bacteria are distributed intercellularlyvia the middle lamellae and enter the cortical cells throughthe structurally altered cell wall, often close to the hostcell nucleus. The root hairs and large basal cells become infectedin the same way. Within the cortical cells of the emerging lateralroot the rhizobia multiply rapidly and the invaded cells dividerepeatedly to form the nodule tissue. Bacteriod formation occursonly when the host cell ceases to divide.     

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.