Selective removal of seedling root hairs for studies of the Rhizobium-legume symbiosis

A freeze-fracture method has been developed for the selective removal of root hairs from white clover (Trifolium repens L.) and alfalfa (Medicago sativa L.) seedling. This procedure yields sufficient material for analysis of root hair proteins by polyacrylamide gel electrophoresis and can be adapted to study in vivo protein synthesis in these differentiated epiderman cells. Clover root hairs which have been injected by the nitrogen-fixing symbiont, Rhizobium trifolii 0403, are also detached from roots by this process, yielding appropriate material to study root responses to the bacterial symbiont during the infection process.     

丛枝菌根真菌和根瘤菌对白三叶氮同化的影响

为揭示丛枝菌根真菌(AMF)和根瘤菌在白三叶氮(N)同化中的作用,该研究对白三叶进行单一或联合接种隐类球囊霉(Paraglomus occultum)和三叶草根瘤菌(Rhizobium trifolii),分析其对白三叶的生长、光合作用、叶片N和氨基酸含量以及N同化相关酶活性的影响。结果表明:(1)单一接种AMF或根瘤菌以及联合接种AMF和根瘤菌均显著增加了白三叶的株高、匍匐茎长度、叶片数、地上部生物量、总生物量、叶绿素b和总叶绿素含量、稳态光量子效率和叶片N含量,这种增强效应是联合接种>单一AMF>单一根瘤菌>未接种处理。(2)联合接种AMF和根瘤菌显著增加了白三叶叶片中丙氨酸、精氨酸、天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸和组氨酸的含量,显著提升了叶片N同化相关酶如硝酸还原酶、亚硝酸还原酶、谷氨酰胺合成酶、谷氨酸合成酶、谷氨酸脱氢酶、天冬酰胺合成酶和天冬氨酸转氨酶的活性,显著促进AMF对白三叶根系的侵染。综上认为,联合接种AMF和根瘤菌通过激活N同化相关酶活性有效促进N同化,产生更多氨基酸,进一步促进白三叶植株生长; 联合接种AMF和根瘤菌具有协同作用,有效促进了白三叶的N同化。     

Transient susceptibility of root cells in four common legumes to nodulation by rhizobia

Root cells of four common legumes were found to remain susceptible to nodulation by rhizobia for only a short period of time. Delayed inoculation experiments conducted with these legume hosts indicated that the initially susceptible region of the root became progressively less susceptible if inoculations were delayed by a few hours. Profiles of the frequency of nodule formation relative to marks indicating the regions of root and root hair development at the time of inoculation indicated that nodulation of Vigna sinensis (L.) Endl. cv California Black Eye and Medicago sativa L. cvs Moapa and Vernal roots was inhibited just below the region that was most susceptible at the time of inoculation. This result suggests the existence of a fast-acting regulatory mechanism in these hosts that prevents overnodulation. Nodulation in white clover may occur in two distinct phases. In addition to the transient susceptibility of preemergent and developing root hair cells, there appeared to be an induced susceptibility of mature clover root hair cells. A cell-free bacterial exudate preparation from Rhizobium trifolii cells was found to render mature root hair cells of white clover more rapidly susceptible to nodulation.     

Calcium localization pattern in clover root hair cells associated with infection process: Studies with aureomycin

Summary Localized regions of calcium were shown to change in clover root hair cells after infection withRhizobium trifolii as shown with the Ca⁺⁺ binding antibiotic, chlorotetracycline. Some root hairs from the immature zone showed increased calcium fluorescence distributed through most of the cell. These root hair had a morphology with a notched side wall, typical of cells infected by bacteria. The maturing root hairs that show a growing infection thread or start of an infection thread, have a similar increased calcium fluorescence, especially around the area of origin of infection thread and tip of the root hair cell.Kettering contribution no. 722.     

Root Hair Deformations Associated with Fractionated Extracts from Rhizobium trifolii

Components from culture fluid and whole cells of Rhizobium trifolii were examined for effects on root hair morphology of white clover seedlings (Trifolium repens var. Ladino). Cell-free culture fluid, exopolysaccharides, supernatant fluid from the precipitation of the exopolysaccharides, capsular polysaccharides, lipopolysaccharides, and a protein fraction from culture fluids were assayed for morphogenetic effects on the root hairs of axenically grown clover seedlings. Crude fractions were chromatographed on Bio Gel A-5m (Bio-Rad Laboratories), and fractions collected were similarly assayed. Hexose, uronic acid, and protein concentrations were determined for all fractions assayed. Gel chromatography indicated the materials with deforming ability to be of high molecular weight (>10,000). For all fractions except exopolysaccharide, deforming ability was associated with a protein component. This suggested that two components were associated with deformation; both contained polysaccharides and one contained protein. Crude fractions differed in their ability to cause deformations and indicated the following relative ability (in decreasing order) to deform root hairs: cell-free culture fluid, capsular polysaccharides, protein from culture fluids, exopolysaccharide, and cell envelope. Lipopolysaccharides had no effect.     

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.     

Regulation by fixed nitrogen of host-symbiont recognition in the Rhizobium-clover symbiosis

Either NO₃⁻ (16 millimolar) or NH₄⁺ (1 millimolar) completely inhibited infection and nodulation of white clover seedlings (Trifoliin repens) inoculated with Rhizobium trifolii. The binding of R. trifolii to root hairs and the immunologically detectable levels of the plant lectin, trifoliin, on the root hair surface had parallel declining slopes as the concentration of either NO₃⁻ or NH₄⁺ was increased in the rooting medium. This supports the role of trifoliin in binding R. trifolii to clover root hairs. Agglutination of R. trifolii by trifoliin from seeds was not inhibited by these levels of NO₃⁻ or NH₄⁺. The results suggest that these fixed N ions may play important roles in regulating an early recognition process in the Rhizobium-clover symbiosis, namely the accumulation of high numbers of infective R. trifolii cells on clover root hairs.     

Receptor Site on Clover and Alfalfa Roots for Rhizobium

Sites on white clover and alfalfa roots that bind Rhizobium trifolii and R. meliloti capsular polysaccharides, respectively, were examined by fluorescence microscopy. Fluorescein isothiocyanate-labeled capsular material from R. trifolii bound specifically to root hairs of clover but not alfalfa. Binding was most intense at the root hair tips. Treatment of clover roots with 2-deoxyglucose (2-dG) prevented binding of R. trifolii capsular material to the roots. The sugar 2-dG enhanced the elution of clover root protein, which could bind to and specifically agglutinate R. trifolii but not R. meliloti or R. japonicum. The mild elution procedure left the roots intact. Agglutination of R. trifolii and passive hemagglutination of rabbit erythrocytes coated with the capsular material of R. trifolii were specifically inhibited by 2-dG. These results suggest that clover roots contain proteins that cross-link complementary polysaccharides on the surface of clover root hairs and infective R. trifolii through 2-dG-sensitive binding sites. Alfalfa root hairs were shown to specifically bind to a surface polysaccharide from R. meliloti.     

The effect of gum arabic on infection and nodulation of cluster clover by Rhizobium

Summary Gum arabic is an adhesive used in pelleting legume seeds with Rhizobium. The present study shows that the gum generally enhances the number of infected root hairs, promotes early nodulation and significantly increases the number of leaves during initial nodulation of cluster clover (Trifolium glomeratum) inoculated with Rhizobium trifolii under bacteriologically controlled conditions.     

Nodulation of specific legumes is controlled by several distinct loci in Rhizobium trifolii

Summary Three distinct loci (designated regions III, IV and V) were identified in the 14 kb Nod region of Rhizobium trifolii strain ANU843 and were found to determine the host range characteristics of this strain. Deletion of region III or region V only from the 14 kb Nod region affected clover nodulation capacity. The introduction to R. Leguminosarum of DNA fragments on multicopy vectors carrying regions III, IV and V (but not smaller fragments) extended the host range of R. leguminosarum so that infection threads and nodules occurred on white clover plants. The same DNA fragments were introduced to the Sym plasmid-cured strain (ANU845) carrying the R. meliloti recombinant nodulation plasmid pRmSL26. Plasmid pRmSL26 alone does not confer root hair curling or nodulation on clover plants. However, the introduction to ANU845 (pRmSL26) of a 1.4 kb fragment carrying R. trifolii region IV only, resulted in the phenotypic activation of marked root hair curling ability to this strain on clovers but no infection events or nodules resulted. Only the transfer of regions III, IV and V to strain ANU845 (pRmSL26) conferred normal nodulation and host range ability of the original wild type R. trifolii strain. These results indicate that the host range genes determine the outcome of early plant-bacterial interactions primarily at the stage of root hair curling and infection.     

Tn5 mutagenesis of Rhizobium trifolii host-specific nodulation genes result in mutants with altered host-range ability

Summary A 14 kb DNA fragment from the Sym plasmid of the Rhizobium trifolii strain ANU843, known to carry common nodulation nod and host specific nodulation hsn genes, was extensively mutagenised with transposon Tn5. A correlation between the site of Tn5 insertion and the induced nodulation defect led to the identification of three specific regions (designated I, II, III) which affected nodulation ability. Twenty-three Tn5 insertions into region I (ca. 3.5 kb) affected normal root hair curling ability and abolished infection thread formation. The resulting mutants were unable to nodulate all tested plant species. Tn5 insertions in regions II and III resulted in mutants which showed an exaggerated root hair curling (Hac⁺⁺) response on clover plants. Ten region II mutants which occurred over a 1.1 kb area showed a greatly reduced nodulation ability on clovers and produced aborted, truncated infection threads. Tn5 insertions into region III (ca. 1.5 kb) altered the outcome of crucial early plant recognition and infection steps by R. trifolii. Seven region III mutants displayed host-range properties which differed from the original parent strain. Region III mutants were able to induce marked root hair distortions, infection threads, and nodules on Pisum sativum including the recalcitrant Afghanistan variety. In addition region III mutants showed a poor nodulation ability on Trifolium repens even though the ability to induce infection threads was retained on this host. The altered host-range properties of region III mutants could only be revealed by mutation and the mutant phenotype was shown to be recessive.     

Microscopic analysis of the effect ofRhizobium leguminosarum biovartrifolii host specific nodulation genes in the infection of white clovers

Summary A microscopic assessment is presented of the comparative infection capacity of wild-type and hybrid strains ofRhizobium leguminosarum bv.viciae withR. l. bv.trifolii strain ANU 843 on white clover seedlings. TheR. l. bv.viciae hybrid strains contained defined DNA segments coding for different combinations ofR. l. bv.trifolii host-specific nodulation genes. White clover plants were examined over a 72 h period to assessRhizobium infectivity, the morphological changes in root hair growth; colonisation ability of rhizobia; infection thread initiation and the ability to induce cortical cell division.R. l. bv.viciae strain 300 induced root hair curling more slowly than strain ANU 843 or any of the hybrid strain 300 bacteria, and when curling had taken place, there was poorer colonization by strain 300 within the folded hair cell, no evidence of infection thread formation and only limited cortical cell division 72 h after inoculation. The addition of the host-specific nodulation genes ofR. l. bv.trifolii to strain 300 was necessary to induce infection threads and establish a normal pattern of nodulation of the roots of white clovers.     

Influence of Soil and Nonsoil Environments on Nodulation by Rhizobium trifolii

Indigenous serotypes 1-01 and 2-02 of Rhizobium trifolii occupied similar percentages (18 to 23%) of root nodules on soil-grown subclover (Trifolium subterraneum L.) and were virtually absent (4.5%) from nodules of soil-grown white clover (Trifolium repens L.). In contrast (with the exception of one dilution [10⁻⁴]), serotype 1-01 occupied a substantial portion of nodules (16 to 40%) on white clover seedlings grown on mineral salts agar and exposed to samples of the same soil in the form of a 10-fold dilution series (10⁻¹ to 10⁻⁵). Under the latter conditions, occupancy of subclover nodules by 1-01 and of nodules of both plant species by 2-02 was consistent with the results obtained with soil-grown plants.     

Soil acidity factors and nodulation ofTrifolium repens

Summary Effects of factors associated with soil acidity (low pH, low calcium, high aluminium and high manganese) on theTrifolium repens-Rhizobium trifolii symbiosis were investigted under laboratory conditions using an axenic solution-culture technique. 200 μM manganese increased root elongation in the range pH 4.3–5.5, but had no effect on root hair formation, the number of Rhizobium in the rhizosphere, or nodule formation. Root elongation and root hair formation were unaffected at pH 4.3 when 500 or 1000μM calcium was supplied, whereas multiplication of Rhizobium in the rhizosphere and nodulation were inhibited at pH 4.3 and 4.7.50–1000μM calcium had no effect either on the multiplication of Rhizobium in the range pH 4.3–5.5, or on nodule formation in the absence of aluminium. 50 μM aluminium inhibited, root elongation and root hair formation at pH 4.3 and 4.7; the effect on root elongation was reduced by increasing the calcium concentration from 50 to 1000μM. 50μM aluminium also inhibited Rhizobium multiplication in the rhizosphere and reduced nodule formation at pH 5.5 (at which aluminium precipitated out of solution), but root elongation and root hair formation were unaffected. These, effects of aluminium at pH 5.5 may explain the poor response to inoculation by white clover in acid mineral soils after liming.     

Interaction of Azospirillum and Rhizobium Strains Leading to Inhibition of Nodulation

Rhizobium-Azospirillum interactions during establishment of Rhizobium-clover symbiosis were studied. When mixed cultures of Azospirillum and Rhizobium trifolii strains were simultaneously inoculated onto clover plants, no nodulation by R. trifolii was observed. R. trifolii ANU1030, which nodulated clover plants without attacking root hairs, i.e., does not cause root hair curling (Hac⁻), did not show inhibition of nodulation when inoculated together with Azospirillum strains. Isolation of bacteria from surface-sterilized roots showed that azospirilla could be isolated both from within root segments and from nodules. Inhibition of nodulation could be mimicked by the addition of auxins to the plant growth medium.     

Interaction of yeasts and some nitrogen fixing bacteria on nodulation of legumes

Summary Combined inoculation ofRhizobium trifolii withSaccharomyces cerevisiae and other yeasts generally enhanced the number of nodules, length of plants and dry weight of Egyptian clover (Trifolium alexandrinum) seedlings grown on agar slopes. Similar effects were observed when seedlings were inoculated withR. trifolii in the presence of dialyzed culture filtrate ofS. cerevisiae.     

Subnanomolar concentrations of membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii are fully capable of eliciting symbiosis-related responses on white clover

Axenic seedling bioassays were performed on white clover, vetch, and alfalfa to assess the variety and dose responses of biological activities exhibited by membrane chitolipooligosaccharides (CLOSs) from wild type Rhizobium leguminosarum bv. trifolii ANU843. Subnanomolar concentrations of CLOSs induced deformation of root hairs (Had) and increased the number of foci of cortical cell divisions (Ccd) in white clover, some of which developed into nodule meristems. In contrast, ANU843 CLOSs were unable to induce Had in alfalfa and required a 10⁴-fold higher threshold concentration to induce this response in vetch. Also, ANU843 CLOSs were not mitogenic on either of these non-host legumes. In addition, CLOS action also increased chitinase activity in white clover root exudate. Thus, the membrane CLOSs from wild type R. leguminosarum bv. trifolii are fully capable of eliciting various symbiosis-related responses in white clover in the same concentration range as extracellular CLOSs of other rhizobia on their respective legume hosts. These results and our earlier studies indicate that membrane CLOSs represent one of many different classes of bioactive metabolites made by R. leguminosarum bv. trifolii which elicit more intense symbiosis-related responses in white clover than in other legumes. Therefore, CLOSs evidently play an important role in symbiotic development, but they may not be the sole determinant of host-range in the Rhizobium-clover symbiosis.Abbreviations Ccd cortical cell division - CLOS chitolipooligosaccharide - Had root hair deformation     

Lack of a direct nitrate-trifoliin A interaction in the Rhizobium-clover symbiosis

Summary Nitrate added at critical concentrations to plant growth medium inhibits the infection of legume roots by Rhizobium. The direct interaction, of nitrate and trifoliin A, a Rhizobium-recognition lection from white clover (Trifolium repens L.), was examined as a possible basis for this regulation. Selective molecular ultrafiltration studies to detect ligand-protein interactions showed that radioactive¹³NO₃ ⁻ did not bind directly to trifoliin A when incubated at two molar ratios. Immunoprecipitation of trifoliin A by its homologous antibody was unaffected by 15 mM NO₃ ⁻. In addition, there was no apparent reduction in attachment ofR. trifolii 0403 to root hairs of clover seedings during 1 h of incubation in the presence of 15 mM NO₃ ⁻. These results show that nitrate inhibition of these early steps of the infection process is not due to a direct interaction of nitrate with trifoliin A or its glycosylated receptors.     

Trifolin: A Rhizobium recognition protein from white clover

A protein agglutinin, trifoliin, was purified from white clover seeds and seedling roots. Trifoliin specifically agglutinates the symbiont of clover, Rhizobium trifolii, at concentrations as low as 0.2 μg protein/ml, and binds to the surface of encapsulated R. trifolii 0403. This clover protein has a subunit with M_r ≈ 50 000, an isoelectric point of 7.3, and contains carbohydrate. Antibody to purified trifoliin binds to the root hair region of 24-h-old clover seedlings, but does not bind to alfalfa, birdsfoot trefoil or joint vetch. The highest concentration of trifoliin on a clover root is present at sites where material in the capsule of R. trifolii binds. 2-Deoxy-d-glucose elutes trifoliin from intact clover-seedling roots, suggesting that this protein is anchored to root cell walls through its carbohydrate binding sites. We propose that trifoliin on the root hair surface plays an important role in the recognition of R. trifolii by clover.     

Scanning Electron Microscopy of Rhizobium trifolii Infection Sites on Root Hairs of White Clover

White clover root hairs which were inoculated with Rhizobium trifolii 4S (infectious strain) contained infection threads which were observed by light microscopy and scanning electron microscopy. Three morphological types of root hairs retaining infection threads were recognized. The bacteria were strongly attached between the surfaces of two plant cell walls as follows: between surfaces of a root hair tip curled back on itself, between a protuberance from a root hair and its cell surface, or between two root hair tips clinging together. An anatomical analysis documented the attachment site of the infection thread sheath from the inside of the root hair cell.