Microtubule array formation during root hair infection thread initiation and elongation in the Mesorhizobium-Lotus symbiosis

Nuclear migration during infection thread (IT) development in root hairs is essential for legume-Rhizobium symbiosis. However, little is known about the relationships between IT formation, nuclear migration, and microtubule dynamics. To this aim, we used transgenic Lotus japonicus expressing a fusion of the green fluorescent protein and tubulin-α6 from Arabidopsis thaliana to visualize in vivo dynamics of cortical microtubules (CMT) and endoplasmic microtubules (EMTs) in root hairs in the presence or absence of Mesorhizobium loti inoculation. We also examined the effect of microtubule-depolymerizing herbicide, cremart, on IT initiation and growth, since cremart is known to inhibit nuclear migration. In live imaging studies of M. loti-treated L. japonicus root hairs, EMTs were found in deformed, curled, and infected root hairs. The continuous reorganization of the EMT array linked to the nucleus appeared to be essential for the reorientation, curling, and IT initiation and the growth of zone II root hairs which are susceptible to rhizobial infection. During IT initiation, the EMTs appeared to be linked to the root hair surface surrounding the M. loti microcolonies. During IT growth, EMTs dissociated from the curled root hair tip, remained linked to the nucleus, and appeared to surround the IT tip. Lack or disorganized EMT arrays that were no longer linked to the nucleus were observed only in infection-aborted root hairs. Cremart affected IT formation and nodulation in a concentration-dependent manner, suggesting that the microtubule (MT) organization and successive nuclear migration are essential for successful nodulation in L. japonicus by M. loti.     

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.     

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     

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.     

3-Phosphoglycerate dehydrogenase in Mesorhizobioum loti is essential for maintaining symbiotic nitrogen fixation of Lotus japonicus root nodules

Mesorhizobium loti is a Gram negative bacterium that induces N₂-fixing root nodules on the model legume Lotus japonicus. Proteomic analysis in M. loti indicated that 3-phosphoglycerate dehydrogenase (EC. 1.1.1.95, PHGDH) protein content was 2.2 times higher in bacteroids than in cultured bacteria. A M. loti mutant (STM5) with a transposon insertion in the PHGDH gene, mll3875, showed an absolute dependence on serine or glycine in minimal medium for growth. When L. japonicus plants were infected with STM5, the roots formed nodules in numbers comparable to those formed by wild type M. loti; however, the nodules showed very low acetylene reduction activity, and significant starch granule accumulation was observed in the uninfected cells. In such nodules, vast necrosis occurred in the central tissue of the nodules, although bacteroids were detected in the infected cell of the nodules. These data indicate that serine or glycine biosynthesis by PHGDH is important for maintaining symbiosis and nitrogen fixation in L. japonicus nodules.     

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 hairs are the most important root trait for rhizosheath formation of barley (Hordeum vulgare), maize (Zea mays) and Lotus japonicus (Gifu)

Background and AimsRhizosheaths are defined as the soil adhering to the root system after it is extracted from the ground. Root hairs and mucilage (root exudates) are key root traits involved in rhizosheath formation, but to better understand the mechanisms involved their relative contributions should be distinguished.MethodsThe ability of three species [barley (Hordeum vulgare), maize (Zea mays) and Lotus japonicus (Gifu)] to form a rhizosheath in a sandy loam soil was compared with that of their root-hairless mutants [bald root barley (brb), maize root hairless 3 (rth3) and root hairless 1 (Ljrhl1)]. Root hair traits (length and density) of wild-type (WT) barley and maize were compared along with exudate adhesiveness of both barley and maize genotypes. Furthermore, root hair traits and exudate adhesiveness from different root types (axile versus lateral) were compared within the cereal species.Key ResultsPer unit root length, rhizosheath size diminished in the order of barley > L. japonicus > maize in WT plants. Root hairs significantly increased rhizosheath formation of all species (3.9-, 3.2- and 1.8-fold for barley, L. japonicus and maize, respectively) but there was no consistent genotypic effect on exudate adhesiveness in the cereals. While brb exudates were more and rth3 exudates were less adhesive than their respective WTs, maize rth3 bound more soil than barley brb. Although both maize genotypes produced significantly more adhesive exudate than the barley genotypes, root hair development of WT barley was more extensive than that of WT maize. Thus, the greater density of longer root hairs in WT barley bound more soil than WT maize. Root type did not seem to affect rhizosheath formation, unless these types differed in root length.ConclusionsWhen root hairs were present, greater root hair development better facilitated rhizosheath formation than root exudate adhesiveness. However, when root hairs were absent root exudate adhesiveness was a more dominant trait.     

Rearrangement of Actin Cytoskeleton Mediates Invasion of Lotus japonicus Roots by Mesorhizobium loti

Infection thread–dependent invasion of legume roots by rhizobia leads to internalization of bacteria into the plant cells, which is one of the salient features of root nodule symbiosis. We found that two genes, Nap1 (for Nck-associated protein 1) and Pir1 (for 121F-specific p53 inducible RNA), involved in actin rearrangements were essential for infection thread formation and colonization of Lotus japonicus roots by its natural microsymbiont, Mesorhizobium loti. nap1 and pir1 mutants developed an excess of uncolonized nodule primordia, indicating that these two genes were not essential for the initiation of nodule organogenesis per se. However, both the formation and subsequent progression of infection threads into the root cortex were significantly impaired in these mutants. We demonstrate that these infection defects were due to disturbed actin cytoskeleton organization. Short root hairs of the mutants had mostly transverse or web-like actin filaments, while bundles of actin filaments in wild-type root hairs were predominantly longitudinal. Corroborating these observations, temporal and spatial differences in actin filament organization between wild-type and mutant root hairs were also observed after Nod factor treatment, while calcium influx and spiking appeared unperturbed. Together with various effects on plant growth and seed formation, the nap1 and pir1 alleles also conferred a characteristic distorted trichome phenotype, suggesting a more general role for Nap1 and Pir1 in processes establishing cell polarity or polar growth in L. japonicus.     

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.     

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.     

Gene expression and localization of a β-1,3-glucanase of <Emphasis Type="Italic">Lotus japonicus</Emphasis>

Phytohormone abscisic acid (ABA) inhibits root nodule formation of leguminous plants. LjGlu1, a β-1,3-glucanase gene of Lotus japonicus, has been identified as an ABA responsive gene. RNA interference of LjGlu1 increased nodule number. This suggests that LjGlu1 is involved in the regulation of nodule formation. Host legumes control nodule number by autoregulation of nodulation (AON), in which the presence of existing root nodules inhibits further nodulation. For further characterization of LjGlu1, we focused on the expression of LjGlu1 in relation to AON. In a split-root system, LjGlu1 expression peaked when AON was fully induced. Hairy roots transformed with LjCLE-RS1, a gene that induces AON, were generated. Expression of LjGlu1 was greater in the transgenic roots than in untransformed roots. LjGlu1 was not induced in a hypernodulating mutant inoculated with Mesorhizobium loti. These results suggest that the expression of LjGlu1 is involved in the system of AON. However, neither hypernodulation nor enlarged nodulation zone was observed on the transgenic hairy roots carrying LjGlu1-RNAi, suggesting that LjGlu1 is not a key player of AON. Recombinant LjGlu1 showed endo-β-1,3-glucanase activity. LjGlu1-mOrange fusion protein suggested that LjGlu1 associated with M. loti on the root hairs. Exogenous β-1,3-glucanase inhibited infection thread formation by both the wild type and the mutant, and nodule numbers were reduced. These results suggest that LjGlu1 is expressed in response to M. loti infection and functions outside root tissues, resulting in the inhibition of infection.     

Lipochito-oligosaccharide nodulation factors stimulate cytoplasmic polarity with longitudinal endoplasmic reticulum and vesicles at the tip in vetch root hairs

Vetch root hair development has four stages: bulge, growing, growth terminating, and full-grown hair. In the assay we used, the nodulation factor induced swellings and outgrowths in growth-terminating hairs. Bulges, swellings, and full-grown hairs have transverse endoplasmic reticulum (ER) and no tip-accumulated vesicles. Growing hairs and outgrowths show vesicle accumulation in the tip and longitudinal subapical ER. Bulge walls and walls of swellings appear mottled.     

Glutamine synthetase I-deficiency in Mesorhizobium loti differentially affects nodule development and activity in Lotus japonicus

In this study, we focused on the effect of glutamine synthetase (GSI) activity in Mesorhizobium loti on the symbiosis between the host plant, Lotus japonicus, and the bacteroids. We used a signature-tagged mutant of M. loti (STM30) with a transposon inserted into the GSI (mll0343) gene. The L. japonicus plants inoculated with STM30 had significantly more nodules, and the occurrence of senesced nodules was much higher than in plants inoculated with the wild-type. The acetylene reduction activity (ARA) per nodule inoculated with STM30 was lowered compared to the control. Also, the concentration of chlorophyll, glutamine, and asparagine in leaves of STM30-infected plants was found to be reduced. Taken together, these data demonstrate that a GSI deficiency in M. loti differentially affects legume–rhizobia symbiosis by modifying nodule development and metabolic processes.     

Intragenic complementation at the Lotus japonicus CELLULOSE SYNTHASE-LIKE D1 locus rescues root hair defects

Root hair cells form the primary interface of plants with the soil environment, playing key roles in nutrient uptake and plant defense. In legumes, they are typically the first cells to become infected by nitrogen-fixing soil bacteria during root nodule symbiosis. Here, we report a role for the CELLULOSE SYNTHASE-LIKE D1 (CSLD1) gene in root hair development in the legume species Lotus japonicus. CSLD1 belongs to the cellulose synthase protein family that includes cellulose synthases and cellulose synthase-like proteins, the latter thought to be involved in the biosynthesis of hemicellulose. We describe 11 Ljcsld1 mutant alleles that impose either short (Ljcsld1-1) or variable (Ljcsld1-2 to 11) root hair length phenotypes. Examination of Ljcsld1-1 and one variable-length root hair mutant, Ljcsld1-6, revealed increased root hair cell wall thickness, which in Ljcsld1-1 was significantly more pronounced and also associated with a strong defect in root nodule symbiosis. Lotus japonicus plants heterozygous for Ljcsld1-1 exhibited intermediate root hair lengths, suggesting incomplete dominance. Intragenic complementation was observed between alleles with mutations in different CSLD1 domains, suggesting CSLD1 function is modular and that the protein may operate as a homodimer or multimer during root hair development.

Intragenic complementation reveals that Lotus japonicus CELLULOSE SYNTHASE-LIKE D1 multimers facilitate root hair development.     

Some Observations on Root-hair Infection by Nodule Bacteria

The infection by nodule bacteria of the root hairs of Viciahirsuta and of twelve species of Trifolium described. The proportion of root hairs deformed by bacterial secretionsvaries between host species and form point to point along theroot but is unaffected by bacterial strain. Infection is morefrequent in regions of maximum curling. Infection threads differin their place of origin in the hair (apical or lateral), theirmode of development and detailed morphology; some of these differencesare characteristic of host species. The growth of infectionthreads is often arrested in the root hair. Least arrested growthoccurs on 2-week-old seedlings infected at about twenty sites.The proportion of lateral infections increases with seedlingage. Root-hair curling, infection-thread initiation and growth areassociated with the near presence of the host cell nucleus. The observational data support the hypothesis of formation ofthe infection thread by invagination of the root-hair wall. No infections were observed in the root hairs of nodulated plantsof Lotus hispida, Lotus angustissimus, and Anthyllis vulneraria.     

Arbuscular mycorrhizal colonization outcompetes root hairs in maize under low phosphorus availability

Background and AimsAn increase in root hair length and density and the development of arbuscular mycorrhiza symbiosis are two alternative strategies of most plants to increase the root–soil surface area under phosphorus (P) deficiency. Across many plant species, root hair length and mycorrhization density are inversely correlated. Root architecture, rooting density and physiology also differ between species. This study aims to understand the relationship among root hairs, arbuscular mycorrhizal fungi (AMF) colonization, plant growth, P acquisition and mycorrhizal-specific P_i transporter gene expression in maize.MethodsUsing nearly isogenic maize lines, the B73 wild type and the rth3 root hairless mutant, we quantified the effect of root hairs and AMF infection in a calcareous soil under P deficiency through a combined analysis of morphological, physiological and molecular factors.Key ResultsWild-type root hairs extended the rhizosphere for acid phosphatase activity by 0.5 mm compared with the rth3 hairless mutant, as measured by in situ zymography. Total root length of the wild type was longer than that of rth3 under P deficiency. Higher AMF colonization and mycorrhiza-induced phosphate transporter gene expression were identified in the mutant under P deficiency, but plant growth and P acquisition were similar between mutant and the wild type. The mycorrhizal dependency of maize was 33 % higher than the root hair dependency.ConclusionsThe results identified larger mycorrhizal dependency than root hair dependency under P deficiency in maize. Root hairs and AMF inoculation are two alternative ways to increase P_i acquisition under P deficiency, but these two strategies compete with each other.     

Infection threads in the root hairs of soybean (Glycine max) plants inoculated withRhizobium japonicum

Summary The mode of infection leading to nodulation was studied in soybean (Glycine max) plants inoculated withRhizobium japonicum strains 61A76, 3I1b83, 3Ilb142, and 3Ilb143 and a commercial inoculum. Infection threads were noticed in the root hairs of plants grown in small field plots, Leonard bottle-jar assemblies and on agar slants. Two infection threads per root hair were commonly observed. Root hairs with infection threads were persistent on the nodules. The maximum number of infection threads per plant was observed in Leonard bottle-jar assemblies.Contribution No. 603 from Charles F. Kettering Research Laboratory.     

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.