Analysis of Nod-factor-induced calcium signaling in root hairs of symbiotically defective mutants of Lotus japonicus

Nodulation (Nod)-factor signaling molecules are essential for rhizobia to initiate the nitrogen-fixing symbiotic interaction with legumes. Using a dual dye ratiometric calcium imaging technique, we have shown that 10 nM Nod factor added to roots of Lotus japonicus seedlings induces an intracellular calcium increase (calcium flux) that precedes oscillations in intracellular calcium (calcium spiking). The calcium flux was not observed with 1 or 0.1 nM Nod factor, which did induce calcium spiking. The calcium flux was variable in timing of initiation and duration and was observed in approximately half of the root hairs examined. Representatives from 11 complementation groups of symbiotically defective mutants were analyzed for the calcium flux. Mutants from four groups (sym6, ccamk, sym35, and nin) which retained calcium spiking all showed a normal calcium flux. Two classes of mutants (nfr1 and nfr5) lacked both calcium influx and calcium spiking, whereas five classes of mutants (symRK, castor, pollux, nup133, and sym24) defective for calcium spiking retained a calcium flux. There was no correlation between calcium spiking and induction of root hair deformation by Nod factor. We propose that increased bacterial numbers within infection foci in root hairs leads to accumulation of Nod factor to sufficient levels to activate the calcium flux, and this may drive infection thread growth.     

New nodulation mutants responsible for infection thread development in Lotus japonicus

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

Characterization of Tn5-induced symbiotically defective mutants of cowpea rhizobia

Symbiotically defective mutants of cowpea rhizobia strain IRC256 were isolated by random Tn5 mutagenesis and characterized. One auxotroph (MS1) requiring adenine and thiamine was a non-nodulating mutant (Nod⁻) and three prototrophic mutants were Nod⁺ Fix⁻ which formed small and ineffective nodules on cowpeas (Vigna unguiculata). Acetylene reduction activity of the Nod⁺ Fix⁻ mutants was reduced to 80–94% of that of the wild-type strain. The non-nodulating mutant (MS1) induced root-hair curling but did not show any nodule initiation or nodule development. Ultrastructural examination of nodules formed by Fix⁻ mutants showed that these contained few bacteroids, indicating either early senescence or a reduction in bacterial release into the cytoplasm of the host cell. DNA hybridization of total DNAs from a representative number of Tn5 mutants showed that each of them had one copy of the transposon Tn5 which was randomly inserted into the genome of cowpea rhizobia.     

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

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

ERN1 and CYCLOPS coordinately activate NIN signaling to promote infection thread formation in Lotus japonicus

Journal of Plant Research - Legumes engage in symbiosis with nitrogen-fixing soil bacteria, collectively called rhizobia, under nitrogen-limited conditions. In many legumes, the root invasion of...     

The Mesorhizobium loti purB gene is involved in infection thread formation and nodule development in Lotus japonicus

The purB and purH mutants of Mesorhizobium loti exhibited purine auxotrophy and nodulation deficiency on Lotus japonicus. In the presence of adenine, only the purH mutant induced nodule formation and the purB mutant produced few infection threads, suggesting that 5-aminoimidazole-4-carboxamide ribonucleotide biosynthesis catalyzed by PurB is required for the establishment of symbiosis.     

Pollen development and tube growth are affected in the symbiotic mutant of Lotus japonicus, crinkle

The symbiotic mutant of Lotus japonicus, crinkle (crk), exhibits abnormal nodulation and other alterations in the root hairs, trichomes, and seedpods. Defective nodulation in crk mutant is due to the arrested infection thread growth from the epidermis into the cortex. Here, we describe that crk is also affected in male fertility that causes the production of small pods with few seeds. Under in vitro conditions, pollen germination and tube growth were markedly reduced in the crk mutant. A swollen tip phenotype with disorganized filamentous actin (F-actin) was observed in the mutant pollen tubes after prolonged in vitro culture. During pollen development, the striking difference noted in the mutant was the small size of the microspores that remained spherical. Histological examination of ovule development, as well as outcrosses of the mutant as female to wild type as male, showed no evidence of abnormality in the female gametophyte development. Based on these findings, the Crk gene, aside from its role in the infection process during nodulation, is also involved in male gametophyte development and function. Therefore, this gene represents a connection between nodule symbiosis, polar tip growth, and other plant developmental processes.     

Isolation of photorespiratory mutants from Lotus japonicus deficient in glutamine synthetase

A mutagenesis programme using ethyl methanesulphonate (EMS) was carried out on Lotus japonicus (Regel) Larsen cv. Gifu in order to isolate photorespiratory mutants in this model legume. These mutants were able to grow in a CO₂-enriched atmosphere [0.7% (v/v) CO₂] but showed stress symptoms when transferred to air. Among them, three mutants displayed low levels of glutamine synthetase (GS; EC 6.3.1.2) activity in leaves. The mutants accumulated ammonium in leaves upon transfer from 0.7% (v/v) CO₂ to air. F₁ plants of back crosses to wild type were viable in air and F₂ populations segregated 3 : 1 (viable in air : air-sensitive) indicative of a single Mendelian recessive trait. Complementation tests showed that the three mutants obtained were allelic. Chromatography on DEAE-Sephacel used to separate the cytosolic and plastidic GS isoenzymes together with immunological data showed that: (1) mutants were specifically affected in the plastidic GS isoform, and (2) in L. japonicus the plastidic GS isoform eluted at lower ionic strength than the cytosolic isoform, contrary to what happens in most plants. The plastidic GS isoform present in roots of wild type L. japonicus was also absent in roots of the mutants, indicating that this plastidic isoform from roots was encoded by the same gene than the GS isoform expressed in leaf tissue. Viability of mutant plants in high-CO₂ conditions indicates that plastidic GS is not essentially required for primary ammonium assimilation. Nevertheless, mutant plants did not grow as well as wild type plants in high-CO₂ conditions.     

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.     

Reactions of Lotus japonicus ecotypes and mutants to root parasitic plants

Witchweeds (Striga spp.) and broomrapes (Orobanche spp.) are obligate root parasitic plants on economically important field and horticultural crops. The parasites' seeds are induced to germinate by root-derived chemical signals. The radicular end is transformed into a haustorium which attaches, penetrates the host root and establishes connection with the vascular system of the host. Reactions of Lotus japonicus, a model legume for functional genomics, were studied for furthering the understanding of host-parasite interactions. Lotus japonicus was compatible with Orobanche aegyptiaca, but not with Orobanche minor, Striga hermonthica and Striga gesnerioides. Orobanche minor successfully penetrated Lotus japonicus roots, but failed to establish connections with the vascular system. Haustoria in Striga hermonthica attached to the roots, but penetration and subsequent growth of the endophyte in the cortex were restricted. Striga gesnerioides did not parasitize Lotus japonicus. Among seven mutants of Lotus japonicus (castor-5, har1-5, alb1-1, ccamk-3, nup85-3, nfr1-3 and nsp2-1) with altered characteristics in relation to rhizobial nodulation and mycorrhizal colonization, castor-5 and har1-5 were parasitized by Orobanche aegyptiaca with higher frequency than the wild type. In contrast, Orobanche aegyptiaca tubercle development was delayed on the mutants nup85-3, nfr1-3 and nsp2-1. These results suggest that nodulation, mycorrhizal colonization and infection by root parasitic plants in Lotus japonicus may be modulated by similar mechanisms and that Lotus japonicus is a potential model legume for studying plant-plant parasitism.     

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

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

Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus

Nitrogen-fixing root nodules develop on legumes as a result of an interaction between host plants and soil bacteria collectively referred to as rhizobia. The organogenic process resulting in nodule development is triggered by the bacterial microsymbiont, but genetically controlled by the host plant genome. Using T-DNA insertion as a tool to identify novel plant genes that regulate nodule ontogeny, we have identified two putatively tagged symbiotic loci, Ljsym8 and Ljsym13, in the diploid legume Lotus japonicus. The sym8 mutants are arrested during infection by the bacteria early in the developmental process. The sym13 mutants are arrested in the final stages of infection, and ineffective nodules are formed. These two plant mutant lines were identified in progeny from 1112 primary transformants obtained after Agrobacterium tumefaciens T-DNA-mediated transformation of L. japonicus and subsequent screening for defects in the symbiosis with Mesorhizobium loti. Additional nontagged mutants arrested at different developmental stages were also identified and genetic complementation tests assigned all the mutations to 16 monogenic symbiotic loci segregating recessive mutant alleles. In the screen reported here independent symbiotic loci thus appeared with a frequency of ～1.5%, suggesting that a relatively large set of genes is required for the symbiotic interaction.     

Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development

Arbuscular mycorrhiza (AM) fungi form nutrient‐acquiring symbioses with the majority of higher plants. Nutrient exchange occurs via arbuscules, highly branched hyphal structures that are formed within root cortical cells. With a view to identifying host genes involved in AM development, we isolated Lotus japonicus AM‐defective mutants via a microscopic screen of an ethyl methanesulfonate‐mutagenized population. A standardized mapping procedure was developed that facilitated positioning of the defective loci on the genetic map of L. japonicus, and, in five cases, allowed identification of mutants of known symbiotic genes. Two additional mutants representing independent loci did not form mature arbuscules during symbiosis with two divergent AM fungal species, but exhibited signs of premature arbuscule arrest or senescence. Marker gene expression patterns indicated that the two mutants are affected in distinct steps of arbuscule development. Both mutants formed wild‐type‐like root nodules upon inoculation with Mesorhizobium loti, indicating that the mutated loci are essential during AM but not during root nodule symbiosis.     

Identification and characterization of NBS-encoding disease resistance genes in Lotus japonicus

Nucleotide-binding site (NBS) disease resistance genes play an important role in defending plants from a range of pathogens and insect pests. Consequently, NBS-encoding genes have been the focus of a number of recent studies in molecular disease resistance breeding programs. However, little is known about NBS-encoding genes in Lotus japonicus. In this study, a full set of disease resistance (R) candidate genes encoding NBS from the complete genome of L. japonicus was identified and characterized using structural diversity, chromosomal locations, conserved protein motifs, gene duplications, and phylogenetic relationships. Distinguished by N-terminal motifs and leucine-rich repeat motifs (LRRs), 92 regular NBS genes of 158 NBS-coding sequences were classified into seven types: CC-NBS-LRR, TIR-NBS-LRR, NBS-LRR, CC-NBS, TIR-NBS, NBS, and NBS-TIR. Phylogenetic reconstruction of NBS-coding sequences revealed many NBS gene lineages, dissimilar from results for Arabidopsis but similar to results from research on rice. Conserved motif structures were also analyzed to clarify their distribution in NBS-encoding gene sequences. Moreover, analysis of the physical locations and duplications of NBS genes showed that gene duplication events of disease resistance genes were lower in L. japonicus than in rice and Arabidopsis, which may contribute to the relatively fewer NBS genes in L. japonicus. Sixty-three NBS-encoding genes with clear conserved domain character were selected to check their gene expression levels by semi-quantitative RT-PCR. The results indicated that 53 of the genes were most likely to be acting as the active genes, and exogenous application of salicylic acid improved expression of most of the R genes.     

Floral patterning in Lotus japonicus

Floral patterning in Papilionoideae plants, such as pea (Pisum sativum) and Medicago truncatula, is unique in terms of floral organ number, arrangement, and initiation timing as compared to other well-studied eudicots. To investigate the molecular mechanisms involved in the floral patterning in legumes, we have analyzed two mutants, proliferating floral meristem and proliferating floral organ-2 (pfo-2), obtained by ethyl methanesulfonate mutagenesis of Lotus japonicus. These two mutants showed similar phenotypes, with indeterminate floral structures and altered floral organ identities. We have demonstrated that loss of function of LjLFY and LjUFO/Pfo is likely to be responsible for these mutant phenotypes, respectively. To dissect the regulatory network controlling the floral patterning, we cloned homologs of the ABC function genes, which control floral organ identity in Arabidopsis (Arabidopsis thaliana). We found that some of the B and C function genes were duplicated. RNA in situ hybridization showed that the C function genes were expressed transiently in the carpel, continuously in stamens, and showed complementarity with the A function genes in the heterogeneous whorl. In proliferating floral meristem and pfo-2 mutants, all B function genes were down-regulated and the expression patterns of the A and C function genes were drastically altered. We conclude that LjLFY and LjUFO/Pfo are required for the activation of B function genes and function together in the recruitment and determination of petals and stamens. Our findings suggest that gene duplication, change in expression pattern, gain or loss of functional domains, and alteration of key gene functions all contribute to the divergence of floral patterning in L. japonicus.     

Petal Development in Lotus japonicus

Previous studies have demonstrated that petal shape and size in legume flowers are determined by two separate mechanisms, dorsoventral (DV) and organ internal (IN) asymmetric mechanisms, respectively. However, little is known about the molecular mechanisms controlling petal development in legumes. To address this question, we investigated petal development along the floral DV axis in Lotus japonicus with respect to cell and developmental biology by comparing wild‐type legumes to mutants. Based on morphological markers, the entire course of petal development, from initiation to maturity, was grouped to define 3 phases or 13 stages. In terms of epidermal micromorphology from adaxial surface, mature petals were divided into several distinct domains, and characteristic epidermal cells of each petal differentiated at stage 9, while epidermal cells of all domains were observed until stage 12. TCP and MIXTA‐like genes were found to be differentially expressed in various domains of petals at stages 9 and 12. Our results suggest that DV and IN mechanisms interplay at different stages of petal development, and their interaction at the cellular and molecular level guides the elaboration of domains within petals to achieve their ideal shape, and further suggest that TCP genes determine petal identity along the DV axis by regulating MIXTA‐like gene expression.     

Nodule Organogenesis in Lotus japonicus

Lotus japonicus. Received 25 September 2000/ Accepted in revised form 9 October 2000