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.     

Effect of Root Temperature on the Infection Processes and Nodulation in Lotus and Stylosanthes

Infection threads were observed abundantly in the root hairsof Lotus corniculatus L., but very rarely in L. hispidus, Desf.,in response to infection by Rhizobium strains 3001 and 3002.Numbers of infections differed between species and strains andwere also affected by temperature. In L. corniculatus all thenodules originated from infection threads, but in L. hispidusmost nodules appeared to originate by direct bacterial penetrationthrough the epidermis, and infected root hairs were very rarelyseen. Both species of Lotus were tolerant to cold temperatures,the minimum temperature for nodulation being 10 ?C. The optimumtemperature for nodulation of L. corniculatus was 20 ?C with3001 and between 27 and 30 ?C with 3002, a few nodules beingformed with both strains at 35 ?C. L. hispidus formed more nodulesthan L. corniculatus and the optimum temperature for both thestrains was between 25 and 27 ?C. No infection threads were seen in root hairs or nodules of Stylosanthesguyanensis (Aubl.) S. W. and S. humilis H.B.K. infected withRhizobium strain CB1552, and all the nodules were formed inthe axils of lateral roots. Optimum temperature for nodulationin S. guyanensis and S. humilis was around 27 ?C; nodulationwas completely inhibited at 15 ?C and very few nodules wereformed at 35 ?C. Both in Lotus and Stylosanthes the transfer of plants from suboptimalto optimal and supraoptimal temperatures increased nodulation.Delayed inoculation and excision of root tips increased nodulation.     

Simulation of marked root hair curling in Rhizobium-legume symbiosis

The soil bacterium Rhizobium infects its leguminous host plants in temperate regions of the world mostly by way of the growing root hairs. Root hair curling is a prerequisite for root hair infection, although sidelong root hair infections occasionally have been observed. The processes underlying Rhizobium -induced root hair curling are unknown.
Computer simulation of root hair growth indicates that one-sided tip growth inhibition by Rhizobium can result in root hair curling when three conditions are simultaneously fulfilled: 1) rhizobial growth inhibition is strong enough to prevent removal out of the tip growth range: 2) root hair surface growth between the attached Rhizobium and the root hair top is inhibited; 3) rhizobial growth inhibition is limited to one side of the root hair.
The results predict that root hair curling by stimulation of tip growth is improbable. This study accentuates the need for information about the growth processes contributing to tip growth in leguminous root hairs.     

The Influence of Temperature on Root Hair Infection of Trifolium parviflorum and T. glomeratum by Root Nodule Bacteria: I. THE EFFECTS OF CONSTANT ROOT TEMPERATURE ON INFECTION AND RELATED ASPECTS OF PLANT DEVELOPMENT

The onset and rate of infection in root hairs of T. parviflorumand T. glomeratum inoculated with Rhizobium trifolli strain5 varied much with root temperature. At moderate root temperature(18, 24, and 30 °C) infections were initiated earlier andin larger numbers than at low (6 and 12 °C) or moderatelyhigh (36 °C) temperatures. Both species showed a broad temperatureoptimum between 18 and 30 °C. The site of thread initiation(apically or laterally in a hair) was independent of temperature,as was also the proportion of successful threads penetratingthe root cortex, which increased with seedling age. Threadsgrew more slowly at low temperatures. The size of hair nucleinear infection threads remained unaffected by temperature, butnuclei associated with laterally originating threads were largerthan those associated with apical threads. Infection was non-randomly distributed along the main root atall temperatures. More zones of infection were found at moderatetemperatures than at temperature extremes (6–12 or 36°C). Probit plots of numbers of infections for individualplants were steplike, the linear sloping parts correspondingto normal distributions of infection within zones. Between 18 and 30 °C numbers of infections increased exponentiallyin two phases, the first more rapid phase ending at about thetime nodules appeared. A model devised for the infection processand fitted to the data suggested the existence of two kindsof infections: primary ones occuring randomly at a slow rateand probably not affected by temperature and secondary infectionsthat appeared to increase with rising temperatures in the range12 to 30 °C. Nodule numbers were relatively more sensitive to high and lowtemperatures than infection. The numbers of infections and nodulesand the root lengths of T. parviflorum were twice those of T.glomeratum except at the temperature extremes. Numbers of infectionswere otherwise unrelated to root length or cotyledon or leafareas. The development of lateral organs (primordia, lateralroots, and nodules) was reduced at temperatures below 18 °Cand above 30 °C.     

Time Course of Cell Biological Events Evoked in Legume Root Hairs by Rhizobium Nod Factors: State of the Art

In many common legumes, when host-specific nodule bacteria meettheir legume root they attach to it and enter through root hairs.The bacteria can intrude these cells because they instigatein the hairs the formation of an inward growing tube, the infectionthread, which consists of wall material. Prior to infectionthread formation, the bacteria exploit the cell machinery forwall deposition by inducing the hairs to form a curl, in whichthe dividing bacteria become entrapped. In most species, Nodfactor alone (a lipochito-oligosaccharide excreted by bacteria)induces root hair deformation, though without curling, thusmost aspects of the initial effects of Nod factor can be elucidatedby studying root hair deformation. In this review we discussthe cellular events that host-specific Nod factors induce intheir host legume root hairs. The first event, detectable onlya few seconds after Nod factor application, is a Ca²⁺influxat the root hair tip, followed by a transient depolarizationof the plasma membrane potential, causing an increase in cytosolic[Ca²⁺] at the root hair tip. Also within minutes, Nod factorschange the cell organization by acting on the actin cytoskeleton,enhancing tip cell wall deposition so that root hairs becomelonger than normal for their species. Since the remodellingof the actin cytoskeleton precedes the second calcium event,Ca²⁺spiking, which is observed in the perinuclear area, we proposethat the initial cytoskeleton events taking place at the hairtip are related to Ca²⁺influx in the hair tip and that Ca²⁺spikingserves later events involving gene expression. Copyright 2001Annals of Botany Company Review, Nod factor, tip growth, root hair, Rhizobium, legume, cytoskeleton, calcium, symbiosis     

Endoplasmic reticulum-targeted GFP reveals ER remodeling in Mesorhizobium-treated Lotus japonicus root hairs during root hair curling and infection thread formation

The endoplasmic reticulum (ER) of the model legume Lotus japonicus was visualized using green fluorescent protein (GFP) fused with the KDEL sequence to investigate the changes in the root hair cortical ER in the presence or absence of Mesorhizobium loti using live fluorescence imaging. Uninoculated root hairs displayed dynamic forms of ER, ranging from a highly condensed form to an open reticulum. In the presence of M. loti, a highly dynamic condensed form of the ER linked with the nucleus was found in deformed, curled, and infected root hairs, similar to that in uninoculated and inoculated growing zone I and II root hairs. An open reticulum was primarily found in mature inoculated zone III root hairs, similar to that found in inactive deformed/curled root hairs and infected root hairs with aborted infection threads. Co-imaging of GFP-labeled ER with light transmission demonstrated a correlation between the mobility of the ER and other organelles and the directionality of the cytoplasmic streaming in root hairs in the early stages of infection thread formation and growth. ER remodeling in root hair cells is discussed in terms of possible biological significance during root hair growth, deformation/curling, and infection in the Mesorhizobium–L. japonicus symbiosis.     

Root hair deformation in the white clover/Rhizobium trifolii symbiosis

Rhizobium trifolii most frequently infects its host white clover (Trifolium repens L.) by means of infection threads formed in markedly curled root hairs. Rhizobium infections are classified as either lateral or apical based on whether they originate in the branches or at the apex of the root hairs. A quantitative estimate of lateral and apical infection in the region of the host root (Trifolium repens L. cv. Regal Ladino) that possessed mature and immature root hairs at the time of inoculation with Rhizobium trifolii TAI (CSIRO, Canberra City, Australia) indicated that lateral infection occurred more frequently in the mature root hair region of the root. Apical infections were more common in the immature root hair region. Cell free filtrates collected from R. trifolii cultured in association with the host roots induced branching in white clover root hairs. A partially purified preparation of the branching factor was obtained from freeze-dried filtrates by ethanol extraction and ion exchange chromatography. Preliminary studies on the characteristics of these substances suggest that some are dialyzable and heat stable white others are non-dialyzable and heat labile. The dialyzable, heat-stable compounds contain neutral sugars and range between 1200 to 10000 daltons in size. In roots that were exposed to low concentrations (6–25 μg-ml^?1) of these partially purified deformation factors before inoculation, the developmentally mature root hairs were deformed at the time of inoculation. Nodules appeared in the mature and immature root hair region of these plants at the same time. In plants exposed to water, nodules were observed in the immature root hair region and mature root hair regions 3 and 5 days after inoculation, respectively. Based on these results, we conclude that the nodule development was hastened in the plants exposed to the root hair-deforming substances because the mature root hairs of these plants were made infectible at the time of inoculation by this exposure.     

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.     

Hair Curling Induced in Heterologous Legumes and Monocots by Flavonoid-Treated Rhizobia

The curling of root hairs and the deformation response wereobserved when white clover was infected with homologous strainsof Rhizobium leguminosarum biovar trifolii 4S and 0403. In thecase of Rhizobium meliloti NZ and Rhizobium leguminosarum biovarviciae 128C53, however, curling was only induced when thesebacteria were pretreated with flavonoids: luteolin in the caseof R. meliloti and naringenin for R.I. viciae. The same resultswere obtained with oat, a monocotyledonous non-leguminous plant.The two flavonoids mentioned are secreted from the host plantsand induce the expression of genes for root hair curling (Hac)on Sym plasmid in homologous rhizobia, therefore, the curlingresponse in both white clover and oat appears to be correlatedwith the activation of the Hac genes. These results suggestthat a factor(s) that activates the Hac genes, such as 7,4'-dihydroxyflavonewhich is known as the factor required by R. I. trifolii, issecreted from the oat roots. (Received June 12, 1989; Accepted November 9, 1989)     

Signaling and Gene Expression for Water-Tolerant Legume Nodulation

In the symbiotic interaction with rhizobia, legumes develop nodules in which nitrogen fixation takes place. Upon submersion, most temperate legumes are incapable of nodulation, but tropical legumes that grow in waterlogged soils have acquired water stress tolerance for growth and nodulation. One well-studied model plant, the tropical, semi-aquatic Sesbania rostrata, develops stem-located adventitious root primordia that grow out into adventitious roots upon submergence and develop into stem nodules after inoculation with the microsymbiont, Azorhizobium caulinodans. Sesbania rostrata also has a nodulated underground root system. On well-aerated roots, nodules form via root hair curling infection in the zone, just above the root tip, where root hairs develop; on hydroponic roots, an alternative process is used, recruiting a cortical intercellular invasion program at the lateral root bases that skips the epidermal responses. This intercellular cortical invasion entails infection pocket formation, a process that involves cell death features and reactive oxygen species. The plant hormones ethylene and gibberellin are the major signals that act downstream from the bacterial nodulation factors in the nodulation and invasion program. Both hormones block root hair curling infection, but cooperate to stimulate lateral root base invasion and play a role in infection thread formation, meristem establishment, and differentiation of meristem descendants.     

Medicago LYK3, an entry receptor in rhizobial nodulation factor signaling

Rhizobia secrete nodulation (Nod) factors, which set in motion the formation of nitrogen-fixing root nodules on legume host plants. Nod factors induce several cellular responses in root hair cells within minutes, but also are essential for the formation of infection threads by which rhizobia enter the root. Based on studies using bacterial mutants, a two-receptor model was proposed, a signaling receptor that induces early responses with low requirements toward Nod factor structure and an entry receptor that controls infection with more stringent demands. Recently, putative Nod factor receptors were shown to be LysM domain receptor kinases. However, mutants in these receptors, in both Lotus japonicus (nfr1 and nfr5) and Medicago truncatula (Medicago; nfp), do not support the two-receptor model because they lack all Nod factor-induced responses. LYK3, the putative Medicago ortholog of NFR1, has only been studied by RNA interference, showing a role in infection thread formation. Medicago hair curling (hcl) mutants are unable to form curled root hairs, a step preceding infection thread formation. We identified the weak hcl-4 allele that is blocked during infection thread growth. We show that HCL encodes LYK3 and, thus, that this receptor, besides infection, also controls root hair curling. By using rhizobial mutants, we also show that HCL controls infection thread formation in a Nod factor structure-dependent manner. Therefore, LYK3 functions as the proposed entry receptor, specifically controlling infection. Finally, we show that LYK3, which regulates a subset of Nod factor-induced genes, is not required for the induction of NODULE INCEPTION.     

The Infection of Trifolium subterraneum Root Hairs by Rhizobium trifolii

Both host cultivar and Rhizobium strain influence the numberof infected root hairs of Trifolium subl-errctneum, seedlings;Yarloop had fewer infections than Cranmore, Mount Barker, orTallarook and Rhizobium trifolii strain 5 infected fewer hairsthan strain TA1. Hybrid lines bred for sparse or abundant nodulationhad similar numbers of infected hairs, but. as in the cultivars,these always greatly exceeded the number of nodules formed.More infection threads aborted early during growth in the roothairs of Cranmore than in other hosts and early abortion wasmore common with strain 5 than strain TA1 In all hosts and with both Rhizobium strains, infection beganon day 3 and was initially restricted to one or two zones alongthe root with later infections extending these zones or initiatingnew ones. The exponential rate of infection (least for Yarloop)slows sharply when nodules appear. Early nodules and lateral roots formed at different places indifferent hosts, and in most cultivars and hybrid lines nodulesand laterals occurred in mutually exclusive zones. Primordiaarising above the first nodule failed to develop.     

Infection and Nodule Development in Aotus ericoides (Vent.) G. Don, A Woody Native Australian Legume

Infection and nodule development were studied by light and electronmicroscopy in Aotus ericoides, a woody native Australian legume,inoculated with a slow-growing field isolate of Rhizobium. Rhizobiabound to straight, but not deformed, root hairs, as detectedby immunofluorescence. Neither markedly curled root hairs norroot hairs with infection threads were seen. Nodules were indeterminate(astragaloid), with a peripheral meristematic layer, few vasculartraces and both infected and uninfected cells in the centralinfected zone. Infection threads containing contorted bacteriawere present throughout the nodule. Swollen, rod-shaped bacteriain infected cells were in groups in vesicles bounded by plasmalemma-derivedperibacteroid membranes. Senescence in infected cells was associatedwith accumulation of a fibrillar matrix inside peribacteroidmembranes, distortion of bacteria and destruction of most cytoplasmiccontents of the bacteria and host cells; however, most bacterialand plant membranes and plant cell walls remained intact. Ineffectivenesswas associated with relatively little, short-lived infectedtissue. Events in infection and nodule development were similarto those in most herbaceous legumes but showed characters ofboth determinate and indeterminate nodules. Key words: Bacteroids, Legume, Nitrogen-fixing, Nodule, Rhizobium     

Interaction between rhizobia and potato tissues10

Several cultivars of Solanum tuberosum L., the potato, weregrown on tissue culture media and their roots inoculated withstrains of rhizobia known to infect legumes at root junctionsor between epidermal cells. Infection incidence and severityshowed considerable cultivar/bacterial strain interaction. Bacteriaspread through intercellular spaces and invaded cells in a non-structuredway: some infections penetrated to the root xylem. There wasno evidence that potato root cells produced nod-inducing factorsand nitrogenase activity was not detected. In some host/rhizobialcombinations outgrowths were formed on roots. These varied fromloose infected callus tissue at the junctions of lateral roots,to modified lateral roots of limited growth which showed somecolonization by rhizobia. Key words: Solarium tuberosum L, potato, glycoprotein, immunogold labelling, rhizobia, flavonoids     

Rhizobium-stimulated Callose Formation in Clover Root Hairs and its Relation to Infection

Callose was detected in the cell walls of the tips of growingroot hairs of Trifolium species and the non-legume Phleum pratenseusing u.v. fluorescence of fresh material stained with 0·005%aniline blue. Inoculation of the roots with Rhizobium trifolii,R. leguminosarum, R. meliloti, and R. japonicum, or additionof 10^–7 and 10^–8 M indole-3-acetic acid (IAA) increasedtip callose formation. Most tip callose was formed at 12 °C, and amounts declinedprogressively at 18, 24, and 30 °C, with very little formedat 36 °C. Tip calloso usually became less and disappearedin individual root hairs as they aged. Callose which appeared prominently in the host cell walls atthe points of initiation of infection threads did not usuallydisappear as the hairs matured. There was little or no extensionof callose along the infection thread and none in the threadtip or in the cell nucleus. Presumptive regions of callose hadsimilar structure and electron density as root hair wall materialand were sometimes related to arrays of vesicles in the hostcytoplasm. The external surface of the hair wall bore smallpegs or papillae (0·1–0·2 µm) continuouswith the outer layer of the wall and possibly associated withattachment of bacteria. Bacteria were usually umboriate at thepoint of attachment and their polyphosphate granules were muchlarger near the root hair than at the distal end.     

Studies on the Physiology of Nodule Formation: VIII. The Influence of the Size of the Rhizosphere Population of Nodule Bacteria on Root Hair Infection in Clover

With an adequate inoculum the number of infected root hairsin three species of clover (Trifolium parviflorum, T. patensand T. glomeratum) increased exponentially with time in twophases; the increase was rapid during the first 8-10 days beforenodulation begins, but slower afterwards. T. parviflorum hadmost infections and T. glomeratum the fewest. Experiments on varying inoculum size, using an avirulent mutanstrain of Rhizobhtm trifolii as diluent, showed that root-hairinfection was differentially limited by inoculum size duringthe two phases. Infection in all three species was about doubledby doubling the density of the virulent bacteria in the rhizospherebefore nodulation begins. After nodulation bacterial densityhad to be increased much more than twice to double the numberof infections. This increase in the infecting population wasinversely related to the numbers of infections formed on thethree host species. Early infection and nodulation were promoted by high bacterialdensity in the rhizosphere.     

Studies on the Physiology of Nodule Formation: IX. The Influence of Combined Nitrogen, Glucose, Light Intensity and Day Length on Root-hair Infection in Clover

Small amounts of nitrate or nitrite salts (10 µg N/plant)in the root medium of Trifolium glomeratum or T. repens delayednodulation, prolonged the initial rapid phase of root infectionand slightly stimulated lateral root formation, whereas equivalentquantities of ammonium sulphate or urea did not. Growth of rootsand root hairs was unaffected by any of these substances at10 µg N/plant. Altering the carbohydrate status of the clover seedlings byadding glucose to the root medium, or by changing day lengthor light intensity, influenced neither the stimulation of root-hairinfection nor the delay in nodulation induced by nitrate at10 fig N/plant, except that plants grown in total darkness hadfewer hairs infected when the root medium contained small amountsof nitrate. The nitrogenous compounds at 100 µg to 1,000 µg N/plant generally delayed and decreased nodulation,increased lateral root formation, slowed hair infection, andincreased root growth.     

豇豆根瘤菌NGR234和紫云英根瘤菌109感染紫云英的比较研究

利用光学和电子显微镜对紫云英根瘤菌菌株109和广宿主的快生型根瘤菌菌株NGR234感染温带型豆科植物紫云英进行了研究,结果表明根瘤菌感染紫云英是通过在根毛中形成侵染线的途径。电子显微镜研究揭示了固氮根瘤中细胞内侵染线的存在。接种二天后,首先可观察到根毛的卷曲或分枝。接种四至五天后,在每株植物卷曲的根毛中可看到侵染线。接种八至十天后的植株出现肉眼可见的根瘤。菌株NGR234能够在紫云英上诱导根毛的卷曲,侵染线和根瘤的形成,但所形成的根瘤却未能固氮,根瘤中无明显的类菌体区,但有少数包有细菌的侵染线。NGR234抗抗菌素的衍生菌均未能使紫云英结瘤。将NGR234的共生质粒转移至三叶草、苜蓿、豌豆、快生型大豆根瘤菌和农杆菌,亦未能使这些细菌获得紫云英上结瘤的能力。     

Infection and Invasion of Roots by Symbiotic, Nitrogen-Fixing Rhizobia during Nodulation of Temperate Legumes

Bacteria belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Azorhizobium (collectively referred to as rhizobia) grow in the soil as free-living organisms but can also live as nitrogen-fixing symbionts inside root nodule cells of legume plants. The interactions between several rhizobial species and their host plants have become models for this type of nitrogen-fixing symbiosis. Temperate legumes such as alfalfa, pea, and vetch form indeterminate nodules that arise from root inner and middle cortical cells and grow out from the root via a persistent meristem. During the formation of functional indeterminate nodules, symbiotic bacteria must gain access to the interior of the host root. To get from the outside to the inside, rhizobia grow and divide in tubules called infection threads, which are composite structures derived from the two symbiotic partners. This review focuses on symbiotic infection and invasion during the formation of indeterminate nodules. It summarizes root hair growth, how root hair growth is influenced by rhizobial signaling molecules, infection of root hairs, infection thread extension down root hairs, infection thread growth into root tissue, and the plant and bacterial contributions necessary for infection thread formation and growth. The review also summarizes recent advances concerning the growth dynamics of rhizobial populations in infection threads.     

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.