Early Nodulation Research Articles

The Sinorhizobium (Ensifer) fredii HH103 Type 3 Secretion System Suppresses Early Defense Responses to Effectively Nodulate Soybean.

Plants that interact with pathogenic bacteria in their natural environments have developed barriers to block or contain the infection. Phytopathogenic bacteria have evolved mechanisms to subvert these defenses and promote infection. Thus, the type 3 secretion system (T3SS) delivers bacterial effectors directly into the plant cells to alter host signaling and suppress defenses, providing an appropriate environment for bacterial multiplication. Some rhizobial strains possess a symbiotic T3SS that seems to be involved in the suppression of host defenses to promote nodulation and determine the host range. In this work, we show that the inactivation of the Sinorhizobium (Ensifer) fredii HH103 T3SS negatively affects soybean nodulation in the early stages of the symbiotic process, which is associated with a reduction of the expression of early nodulation genes. This symbiotic phenotype could be the consequence of the bacterial triggering of soybean defense responses associated with the production of salicylic acid (SA) and the impairment of the T3SS mutant to suppress these responses. Interestingly, the early induction of the transcription of GmMPK4, which negatively regulates SA accumulation and defense responses in soybean via WRKY33, could be associated with the differential defense responses induced by the parental and the T3SS mutant strain.

Genome-wide identification of CAMTA gene family members in Medicago truncatula and their expression during root nodule symbiosis and hormone treatments.

Calmodulin-binding transcription activators (CAMTAs) are well-characterized calmodulin-binding transcription factors in the plant kingdom. Previous work shows that CAMTAs play important roles in various biological processes including disease resistance, herbivore attack response, and abiotic stress tolerance. However, studies that address the function of CAMTAs during the establishment of symbiosis between legumes and rhizobia are still lacking. This study undertook comprehensive identification and analysis of CAMTA genes using the latest updated M. truncatula genome. All the MtCAMTA genes were expressed in a tissues-specific manner and were responsive to environmental stress-related hormones. The expression profiling of MtCAMTA genes during the early phase of Sinorhizobium meliloti infection was also analyzed. Our data showed that the expression of most MtCAMTA genes was suppressed in roots by S. meliloti infection. The responsiveness of MtCAMTAs to S. meliloti infection indicated that they may function as calcium-regulated transcription factors in the early nodulation signaling pathway. In addition, bioinformatics analysis showed that CAMTA binding sites existed in the promoter regions of various early rhizobial infection response genes, suggesting possible MtCAMTAs-regulated downstream candidate genes during the early phase of S. meliloti infection. Taken together, these results provide basic information about MtCAMTAs in the model legume M. truncatula, and the involvement of MtCAMTAs in nodule organogenesis. This information furthers our understanding of MtCAMTA protein functions in M. truncatula and opens new avenues for continued research.

The micro-RNA72c-APETALA2-1 node as a key regulator of the common bean-Rhizobium etli nitrogen fixation symbiosis.

Micro-RNAs are recognized as important posttranscriptional regulators in plants. The relevance of micro-RNAs as regulators of the legume-rhizobia nitrogen-fixing symbiosis is emerging. The objective of this work was to functionally characterize the role of micro-RNA172 (miR172) and its conserved target APETALA2 (AP2) transcription factor in the common bean (Phaseolus vulgaris)-Rhizobium etli symbiosis. Our expression analysis revealed that mature miR172c increased upon rhizobial infection and continued increasing during nodule development, reaching its maximum in mature nodules and decaying in senescent nodules. The expression of AP2-1 target showed a negative correlation with miR172c expression. A drastic decrease in miR172c and high AP2-1 mRNA levels were observed in ineffective nodules. Phenotypic analysis of composite bean plants with transgenic roots overexpressing miR172c or a mutated AP2-1 insensitive to miR172c cleavage demonstrated the pivotal regulatory role of the miR172 node in the common bean-rhizobia symbiosis. Increased miR172 resulted in improved root growth, increased rhizobial infection, increased expression of early nodulation and autoregulation of nodulation genes, and improved nodulation and nitrogen fixation. In addition, these plants showed decreased sensitivity to nitrate inhibition of nodulation. Through transcriptome analysis, we identified 114 common bean genes that coexpressed with AP2-1 and proposed these as being targets for transcriptional activation by AP2-1. Several of these genes are related to nodule senescence, and we propose that they have to be silenced, through miR172c-induced AP2-1 cleavage, in active mature nodules. Our work sets the basis for exploring the miR172-mediated improvement of symbiotic nitrogen fixation in common bean, the most important grain legume for human consumption.

Lotus japonicus clathrin heavy Chain1 is associated with Rho-Like GTPase ROP6 and involved in nodule formation.

Mechanisms underlying nodulation factor signaling downstream of the nodulation factor receptors (NFRs) have not been fully characterized. In this study, clathrin heavy chain1 (CHC1) was shown to interact with the Rho-Like GTPase ROP6, an interaction partner of NFR5 in Lotus japonicus. The CHC1 gene was found to be expressed constitutively in all plant tissues and induced in Mesorhizobium loti-infected root hairs and nodule primordia. When expressed in leaves of Nicotiana benthamiana, CHC1 and ROP6 were colocalized at the cell circumference and within cytoplasmic punctate structures. In M. loti-infected root hairs, the CHC protein was detected in cytoplasmic punctate structures near the infection pocket along the infection thread membrane and the plasma membrane of the host cells. Transgenic plants expressing the CHC1-Hub domain, a dominant negative effector of clathrin-mediated endocytosis, were found to suppress early nodulation gene expression and impair M. loti infection, resulting in reduced nodulation. Treatment with tyrphostin A23, an inhibitor of clathrin-mediated endocytosis of plasma membrane cargoes, had a similar effect on down-regulation of early nodulation genes. These findings show an important role of clathrin in the leguminous symbiosis with rhizobia.

Co-Inoculation of Soybean with <i>Bradyrhizobium</i> and <i>Azospirillum</i> Promotes Early Nodulation

Soybean inoculation with elite strains of Bradyrhizobium to improve nodulation, N2 fixation, and grain yield is well established worldwide. However, when grown in soils where N is deficient, soy- bean undergoes an initial phase of N starvation that may last up to 20 days after seedling germina- tion due to the lack of synchronism between the phase when seed N reserves are exhausted and the moment when plants begin to benefit from the nitrogen fixed by the bacteria. Practices that promote early nodulation may play a key role in reducing the N starvation period. Azospirillum is a plant growth promoting rhizobacteria (PGPR) that can stimulate root hair formation and root growth, creating more sites for early root infection and nodule formation by N2-fixing Bradyrhizo- bium spp. In this study, the effects of co-inoculating soybeans with Bradyrhizobium spp. and Azos- pirillum brasilense on nodulation precocity and N2 fixation were evaluated under greenhouse and field conditions. Nodule number and dry weight, as well as plant and root dry weight and N accu- mulated in shoots at 15, 18, 21, 24 and 30 days after emergence (DAE) were evaluated in response to inoculation with Bradyrhizobium spp. alone or when co-inoculated with Azospirillum sp. In the greenhouse, co-inoculated plants nodulated precociously as indicated by a significant increase (p < 0.05) in nodule biomass observed at (include) 21 DAE. More pronounced effects of co-inocula- tion were observed in the field as early as 18 DAE, suggesting that the presence of Azospirillum helps plants to overcome environmental stresses. * Corresponding author.

Exopolysaccharide production in response to medium acidification is correlated with an increase in competition for nodule occupancy.

Sinorhizobium meliloti strains unable to utilize galactose as a sole carbon source, due to mutations in the De-Ley Doudoroff pathway (dgoK), were previously shown to be more competitive for nodule occupancy. In this work, we show that strains carrying this mutation have galactose-dependent exopolysaccharide (EPS) phenotypes that were manifested as aberrant Calcofluor staining as well as decreased mucoidy when in an expR(+) genetic background. The aberrant Calcofluor staining was correlated with changes in the pH of the growth medium. Strains carrying dgoK mutations were subsequently demonstrated to show earlier acidification of their growth medium that was correlated with an increase expression of genes associated with succinoglycan biosynthesis as well as increased accumulation of high and low molecular weight EPS in the medium. In addition, it was shown that the acidification of the medium was dependent on the inability of S. meliloti strains to initiate the catabolism of galactose. To more fully understand why strains carrying the dgoK allele were more competitive for nodule occupancy, early nodulation phenotypes were investigated. It was found that strains carrying the dgoK allele had a faster rate of nodulation. In addition, nodule competition experiments using genetic backgrounds unable to synthesize either succinoglycan or EPSII were consistent with the hypothesis that the increased competition phenotype was dependent upon the synthesis of succinoglycan. Fluorescent microscopy experiments on infected root-hair cells, using the acidotropic dye Lysotracker Red DND-99, provide evidence that the colonized curled root hair is an acidic compartment.

Endoreduplication-mediated initiation of symbiotic organ development in Lotus japonicus.

Many leguminous plants have a unique ability to reset and alter the fate of differentiated root cortical cells to form new organs of nitrogen-fixing root nodules during legume-Rhizobium symbiosis. Recent genetic studies on the role of cytokinin signaling reveal that activation of cytokinin signaling is crucial to the nodule organogenesis process. However, the genetic mechanism underlying the initiation of nodule organogenesis is poorly understood due to the low number of genes that have been identified. Here, we have identified a novel nodulation-deficient mutant named vagrant infection thread 1 (vag1) after suppressor mutant screening of spontaneous nodule formation 2, a cytokinin receptor gain-of-function mutant in Lotus japonicus. The VAG1 gene encodes a protein that is putatively orthologous to Arabidopsis ROOT HAIRLESS 1/HYPOCOTYL 7, a component of the plant DNA topoisomerase VI that is involved in the control of endoreduplication. Nodule phenotype of the vag1 mutant shows that VAG1 is required for the ploidy-dependent cell growth of rhizobial-infected cells. Furthermore, VAG1 mediates the onset of endoreduplication in cortical cells during early nodule development, which may be essential for the initiation of cortical cell proliferation that leads to nodule primordium formation. In addition, cortical infection is severely impaired in the vag1 mutants, whereas the epidermal infection threads formation is normal. This suggests that the VAG1-mediated endoreduplication of cortical cells may be required for the guidance of symbiotic bacteria to host meristematic cells.

The CCAAT box-binding transcription factor NF-YA1 controls rhizobial infection

Symbiosis between legume plants and soil rhizobia culminates in the formation of a novel root organ, the ‘nodule’, containing bacteria differentiated as facultative nitrogen-fixing organelles. MtNF-YA1 is a Medicago truncatula CCAAT box-binding transcription factor (TF), formerly called HAP2-1, highly expressed in mature nodules and required for nodule meristem function and persistence. Here a role for MtNF-YA1 during early nodule development is demonstrated. Detailed expression analysis based on RNA sequencing, quantitiative real-time PCR (qRT-PCR), as well as promoter–β-glucuronidase (GUS) fusions reveal that MtNF-YA1 is first induced at the onset of symbiotic development during preparation for, and initiation and progression of, symbiotic infection. Moreover, using a new knock-out mutant, Mtnf-ya1-1, it is shown that MtNF-YA1 controls infection thread (IT) progression from initial root infection through colonization of nodule tissues. Extensive confocal and electronic microscopic observations suggest that the bulbous and erratic IT growth phenotypes observed in Mtnf-ya1-1 could be a consequence of the fact that walls of ITs in this mutant are thinner and less coherent than in the wild type. It is proposed that MtNF-YA1 controls rhizobial infection progression by regulating the formation and the wall of ITs.

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant &lt;em&gt;Medicago truncatula&lt;/em&gt; with the Rhizobial Symbiont &lt;em&gt;Sinorhizobium meliloti &lt;/em&gt;

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.

A novel GRAS protein gene MtSymSCL1 plays a role in regulating nodule number in Medicago truncatula

Legume-rhizobium symbiosis requires the expression of a series of plant genes playing roles in nodulation signaling that leads to the formation of functional nodules. A few plant-specific GRAS proteins have been shown to be involved in early nodulation signaling. Here, we isolated a novel SCARECROW-like GRAS protein gene, MtSymSCL1, from the model legume Medicago truncatula. MtSymSCL1 has unique gene and protein domain structures, and its homologs are identified from various plant species. Phylogenetic analysis showed that MtSymSCL1 could be classified into a novel subclass in the GRAS protein family. MtSymSCL1 transcript accumulated highly in nodulated roots at 14 days after inoculation of Sinorhizobium meliloti. Translationally fused MtSymSCL1-GFP expressed from a viral or the native promoter was co-localized with nuclear localization signal-fused RFP in leaf mesophyll protoplasts, Agrobacterium rhizogenes-transformed hairy root cells, and epidermal cells of nodules that developed on the transformed roots. Unlike SCARECROW of Arabidopsis, however, RNA silencing of MtsymSCL1 did not affect root growth, nor root hair curling and infection thread growth. Nevertheless, MtSymSCL1-silenced roots developed significantly fewer nodules than GUS-silenced roots, as shown by replicated nodulation assay with pot- or agar plate-grown plants. The structure of MtSymSCL1-silenced root nodules appeared similar to that of the wild type, as shown by sectioned nodules stained with green fluorescent dye. Together, these results suggest that MtSymSCL1 plays a role in regulating nodule number during legume-rhizobium symbiosis.

Rhizobial and mycorrhizal symbioses in Lotus japonicus require lectin nucleotide phosphohydrolase, which acts upstream of calcium signaling.

Nodulation in legumes requires the recognition of rhizobially made Nod factors. Genetic studies have revealed that the perception of Nod factors involves LysM domain receptor-like kinases, while biochemical approaches have identified LECTIN NUCLEOTIDE PHOSPHOHYDROLASE (LNP) as a Nod factor-binding protein. Here, we show that antisense inhibition of LNP blocks nodulation in Lotus japonicus. This absence of nodulation was due to a defect in Nod factor signaling based on the observations that the early nodulation gene NODULE INCEPTION was not induced and that both Nod factor-induced perinuclear calcium spiking and calcium influx at the root hair tip were blocked. However, Nod factor did induce root hair deformation in the LNP antisense lines. LNP is also required for infection by the mycorrhizal fungus Glomus intraradices, suggesting that LNP plays a role in the common signaling pathway shared by the rhizobial and mycorrhizal symbioses. Taken together, these observations indicate that LNP acts at a novel position in the early stages of symbiosis signaling. We propose that LNP functions at the earliest stage of the common nodulation and mycorrhization symbiosis signaling pathway downstream of the Nod factor receptors; it may act either by influencing signaling via changes in external nucleotides or in conjunction with the LysM receptor-like kinases for recognition of Nod factor.

Positive and negative regulation of cortical cell division during root nodule development in Lotus japonicus is accompanied by auxin response

Nodulation is a form of de novo organogenesis that occurs mainly in legumes. During early nodule development, the host plant root is infected by rhizobia that induce dedifferentiation of some cortical cells, which then proliferate to form the symbiotic root nodule primordium. Two classic phytohormones, cytokinin and auxin, play essential roles in diverse aspects of cell proliferation and differentiation. Although recent genetic studies have established how activation of cytokinin signaling is crucial to the control of cortical cell differentiation, the physiological pathways through which auxin might act in nodule development are poorly characterized. Here, we report the detailed patterns of auxin accumulation during nodule development in Lotus japonicus. Our analyses showed that auxin predominantly accumulates in dividing cortical cells and that NODULE INCEPTION, a key transcription factor in nodule development, positively regulates this accumulation. Additionally, we found that auxin accumulation is inhibited by a systemic negative regulatory mechanism termed autoregulation of nodulation (AON). Analysis of the constitutive activation of LjCLE-RS genes, which encode putative root-derived signals that function in AON, in combination with the determination of auxin accumulation patterns in proliferating cortical cells, indicated that activation of LjCLE-RS genes blocks the progress of further cortical cell division, probably through controlling auxin accumulation. Our data provide evidence for the existence of a novel fine-tuning mechanism that controls nodule development in a cortical cell stage-dependent manner.

Systemic Regulation of Soybean Nodulation by Acidic Growth Conditions

Mechanisms inhibiting legume nodulation by low soil pH, although highly prevalent and economically significant, are poorly understood. We addressed this in soybean (Glycine max) using a combination of physiological and genetic approaches. Split-root and grafting studies using an autoregulation-of-nodulation-deficient mutant line, altered in the autoregulation-of-nodulation receptor kinase GmNARK, determined that a systemic, shoot-controlled, and GmNARK-dependent mechanism was critical for facilitating the inhibitory effect. Acid inhibition was independent of aluminum ion concentration and occurred early in nodule development, between 12 and 96 h post inoculation with Bradyrhizobium japonicum. Biological effects were confirmed by measuring transcript numbers of known early nodulation genes. Transcripts decreased on both sides of split-root systems, where only one side was subjected to low-pH conditions. Our findings enhance the present understanding of the innate mechanisms regulating legume nodulation control under acidic conditions, which could benefit future attempts in agriculture to improve nodule development and biological nitrogen fixation in acid-stressed soils.

Nitric oxide (NO): a key player in the senescence of Medicago truncatula root nodules

Nitric oxide (NO) is a signalling and defence molecule involved in diverse plant developmental processes, as well as in the plant response to pathogens. NO has also been detected at different steps of the symbiosis between legumes and rhizobia. NO is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction, but little is known about the role of NO in mature nodules. Here, we investigate the role of NO in the late steps of symbiosis. Genetic and pharmacological approaches were conducted to modulate the NO level inside root nodules, and their effects on nitrogen fixation and root nodule senescence were monitored. An increase in endogenous NO levels led to a decrease in nitrogen fixation and early nodule senescence, characterized by cytological modifications of the nodule structure and the early expression of a specific senescence marker. By contrast, a decrease in NO levels led to a delay in nodule senescence. Together, our results strongly suggest that NO is a signal in developmental as well as stress-induced nodule senescence. In addition, this work demonstrates the pivotal role of the bacterial NO detoxification response in the prevention of early nodule senescence, and hence the maintenance of efficient symbiosis.

Transient Nod factor‐dependent gene expression in the nodulation‐competent zone of soybean (Glycine max [L.] Merr.) roots

All lateral organ development in plants, such as nodulation in legumes, requires the temporal and spatial regulation of genes and gene networks. A total mRNA profiling approach using RNA-seq to target the specific soybean (Glycine max) root tissues responding to compatible rhizobia [i.e. the Zone Of Nodulation (ZON)] revealed a large number of novel, often transient, mRNA changes occurring during the early stages of nodulation. Focusing on the ZON enabled us to discard the majority of root tissues and their developmentally diverse gene transcripts, thereby highlighting the lowly and transiently expressed nodulation-specific genes. It also enabled us to concentrate on a precise moment in early nodule development at each sampling time. We focused on discovering genes regulated specifically by the Bradyrhizobium-produced Nod factor signal, by inoculating roots with either a competent wild-type or incompetent mutant (nodC(-) ) strain of Bradyrhizobium japonicum. Collectively, 2915 genes were identified as being differentially expressed, including many known soybean nodulation genes. A number of unknown nodulation gene candidates and soybean orthologues of nodulation genes previously reported in other legume species were also identified. The differential expression of several candidates was confirmed and further characterized via inoculation time-course studies and qRT-PCR. The expression of many genes, including an endo-1,4-β-glucanase, a cytochrome P450 and a TIR-LRR-NBS receptor kinase, was transient, peaking quickly during the initiation of nodule ontogeny. Additional genes were found to be down-regulated. Significantly, a set of differentially regulated genes acting in the gibberellic acid (GA) biosynthesis pathway was discovered, suggesting a novel role of GAs in nodulation.

Renal Histopathology of a Baboon Model with Type 2 Diabetes

Naturally occurring type 2 diabetes has been found in a colony of baboons. Ongoing characterization of the baboon colony maintained at the Southwest National Primate Research Center has revealed a significant range of glucose sensitivity with some animals clearly diabetic. Seven baboons, four with diabetes and three without diabetes, underwent histopathological investigation. Three diabetic animals were diagnosed using fasting blood glucose, hemoglobin A1C, and intravenous glucose tolerance test, and a fourth one was known to have hyperglycemia. One control baboon and three baboons with diabetes had microalbuminuria. On kidney biopsy, diabetic baboons had thickening of the glomerular basement membrane and mesangial matrix expansion compared to controls. Immunohistochemistry showed the diabetic animals had increased mesangial expression of cellular fibronectin ED-A. Two diabetic animals with microalbuminuria had evidence of mesangiolysis with the formation of an early nodule. One diabetic animal had a Kimmestiel-Wilson nodule. We conclude that the baboon represents a useful primate model of diabetes and nephropathy that resembles the nephropathy associated with type 2 diabetes in humans.

Evaluation of selected growth factor expression in canine spirocercosis (Spirocerca lupi)-associated non-neoplastic nodules and sarcomas

The study aims to assess the expression of vascular endothelial growth factor (VEGF)-A, fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) in the progression of the spirocercosis-induced oesophageal nodule in the dog from early, non-neoplastic, inflammatory nodule to sarcomatous neoplasia.Triplicate 4μm-thick sections from 62 paraffin blocks containing Spirocerca-induced nodules were treated according to the steptavidin-biotin (LSAB) immunohistochemical method using polyclonal goat-anti-canine VEGF and anti-human FGF and PDGF antibodies. The nodules were classified as early inflammatory (n=15), pre-neoplastic (n=27) and neoplastic (n=20). Additionally, 10 sections of normal distal third of the oesophagus and 21 non-spirocercosis-related sarcomas were evaluated and compared with the Spirocerca-induced nodules. Five non-overlapping high power fields per case were evaluated under the light microscope and the fibroblasts were evaluated for percentage of labelled cells. The intensity of labelling was further classified as weak (score 1) or strong (score 2). The intensity score was multiplied by the percentage of labelled fibroblasts to yield a field score and the final score was obtained by calculating an average of the five fields. Antigen labelling was compared between the different histological grades and the controls using the Kruskal–Wallis test followed by the Mann–Whitney test for comparison between specific groups. The level of significance was set at 0.05.There were significant differences between the groups’ score in all the growth factors that were examined. The normal oesophagus showed no labelling for any of the growth factors. FGF scored highest in the non-spirocercosis-related sarcoma group (median 118, 3–194) followed by the spirocercosis-induced sarcoma (34.5, 0–138), pre-neoplastic nodule (8, 0–99) and early nodule (0, 0–30) groups. All the differences among the groups were significant. VEGF scored highest in the non-spirocercosis-related sarcoma group (median 47, 1–110) followed by the spirocercosis-induced sarcoma (26, 0–136), pre-neoplastic nodule (0, 0–62) and early nodule (0, 0–35) groups. PDGF scored highest in the non-spirocercosis-related sarcoma group (median 29.2, 0–70) followed by the pre-neoplastic nodule (23.4, 0–95), early nodule (13.6, 0–132) and spirocercosis-induced sarcoma (0, 0–47) groups.The expression of VEGF and FGF increased as the nodule progressed from early inflammation to sarcoma, but it was not limited to spirocercosis-induced sarcomas. The expression of PDGF in spirocercosis was restricted to the early stages of nodule progression. Further investigation is warranted to establish whether FGF, VEGF or PDGF play a role in the pathogenesis of the malignant transformation in canine spirocercosis or are they simply integral to angiogenesis induction?

Adaptation of Medicago truncatula to nitrogen limitation is modulated via local and systemic nodule developmental responses

Adaptation of Medicago truncatula to local nitrogen (N) limitation was investigated to provide new insights into local and systemic N signaling. The split-root technique allowed a characterization of the local and systemic responses of NO(3)(-) or N(2)-fed plants to localized N limitation. (15)N and (13)C labeling were used to monitor plant nutrition. Plants expressing pMtENOD11-GUS and the sunn-2 hypernodulating mutant were used to unravel mechanisms involved in these responses. Unlike NO(3)(-)-fed plants, N(2)-fixing plants lacked the ability to compensate rapidly for a localized N limitation by up-regulating the N(2)-fixation activity of roots supplied elsewhere with N. However they displayed a long-term response via a growth stimulation of pre-existing nodules, and the generation of new nodules, likely through a decreased abortion rate of early nodulation events. Both these responses involve systemic signaling. The latter response is abolished in the sunn mutant, but the mutation does not prevent the first response. Local but also systemic regulatory mechanisms related to plant N status regulate de novo nodule development in Mt, and SUNN is required for this systemic regulation. By contrast, the stimulation of nodule growth triggered by systemic N signaling does not involve SUNN, indicating SUNN-independent signaling.

A Small GTPase of the Rab Family Is Required for Root Hair Formation and Preinfection Stages of the Common Bean–RhizobiumSymbiotic Association

Legume plants are able to establish a symbiotic relationship with soil bacteria from the genus Rhizobium, leading to the formation of nitrogen-fixing root nodules. Successful nodulation requires both the formation of infection threads (ITs) in the root epidermis and the activation of cell division in the cortex to form the nodule primordium. This study describes the characterization of RabA2, a common bean (Phaseolus vulgaris) cDNA previously isolated as differentially expressed in root hairs infected with Rhizobium etli, which encodes a protein highly similar to small GTPases of the RabA2 subfamily. This gene is expressed in roots, particularly in root hairs, where the protein was found to be associated with vesicles that move along the cell. The role of this gene during nodulation has been studied in common bean transgenic roots using a reverse genetic approach. Examination of root morphology in RabA2 RNA interference (RNAi) plants revealed that the number and length of the root hairs were severely reduced in these plants. Upon inoculation with R. etli, nodulation was completely impaired and no induction of early nodulation genes (ENODs), such as ERN1, ENOD40, and Hap5, was detected in silenced hairy roots. Moreover, RabA2 RNAi plants failed to induce root hair deformation and to initiate ITs, indicating that morphological changes that precede bacterial infection are compromised in these plants. We propose that RabA2 acts in polar growth of root hairs and is required for reorientation of the root hair growth axis during bacterial infection.

A mechanistic molecular test of the plant-sanction hypothesis in legume–rhizobia mutualism

The origin and persistence of mutualism is difficult to explain because of the widespread occurrence of exploitative, ‘cheating’ partners. As a policing strategy stabilising intraspecific cooperation, host sanctions against non-N 2 fixing, cheating symbionts have been proposed to stabilise mutualism in legume-rhizobium symbiosis. Mechanism of penalisations would include decreased nodular rhizobial viability and/or early nodule senescence. We tested these potential mechanisms of penalisations in split-root experiments using two soybean varieties and two rhizobial strains, a cooperative, normal N 2-fixing strain and an isogenic non-fixing derivative. We found no differences in the number of viable rhizobia recovered from nodules and no differential expression of a nodular senescence molecular marker. Thus, our results do not support the hypothesis of plant sanctions acting against cheating rhizobia in our experimental conditions.