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Abstract Background Cytokinins are a class of phytohormone that play a crucial role in the development of plants. They are involved in the regulation of nearly every aspect of plant growth, from germination to senescence. The role of cytokinins in many developmental programs is complex and varies both spatially and temporally. Current techniques used to investigate the functions of cytokinins in plant development lack this spatial and temporal resolution required to observe cell-type specific effects. Results To this end, we present a method of activating a caged cytokinin in single cells. A caged benzyladenine was synthesized, along with caged adenine as a negative control. In vitro testing confirmed ultraviolet light-mediated uncaging, and subsequent root growth assays demonstrated that uncaging produced a cytokinin phenotype. This uncaging was confined to single cells using multiphoton confocal microscopy. Using an Arabidopsis thaliana cytokinin reporter line expressing TCSn::GFP, the resulting GFP expression was confined to the uncaging region, including in single cells. This study presents a novel cell-targeted method of cytokinin delivery, which has the potential to elucidate a broad range of processes in plant development. Conclusions We combined multiphoton confocal microscopy and a caged cytokinin treatment, allowing cell type-specific uncaging of a cytokinin in Arabidopsis roots.

Legumes form two types of root organs in response to signals from microbes, namely, nodules and root galls. In the field, these interactions occur concurrently and often interact with each other. The outcomes of these interactions vary and can depend on natural variation in rhizobia and nematode populations in the soil as well as abiotic conditions. While rhizobia are symbionts that contribute fixed nitrogen to their hosts, parasitic root-knot nematodes (RKN) cause galls as feeding structures that consume plant resources without a contribution to the plant. Yet, the two interactions share similarities, including rhizosphere signaling, repression of host defense responses, activation of host cell division, and differentiation, nutrient exchange, and alteration of root architecture. Rhizobia activate changes in defense and development through Nod factor signaling, with additional functions of effector proteins and exopolysaccharides. RKN inject large numbers of protein effectors into plant cells that directly suppress immune signaling and manipulate developmental pathways. This review examines the molecular control of legume interactions with rhizobia and RKN to elucidate shared and distinct mechanisms of these root-microbe interactions. Many of the molecular pathways targeted by both organisms overlap, yet recent discoveries have singled out differences in the spatial control of expression of developmental regulators that may have enabled activation of cortical cell division during nodulation in legumes. The interaction of legumes with symbionts and parasites highlights the importance of a comprehensive view of root-microbe interactions for future crop management and breeding strategies.

Abstract Background The formation of infection threads in the symbiotic infection of rhizobacteria in legumes is a unique, fascinating, and poorly understood process. Infection threads are tubes of cell wall material that transport rhizobacteria from root hair cells to developing nodules in host roots. They form in a type of reverse tip-growth from an inversion of the root hair cell wall, but the mechanism driving this growth is unknown, and the composition of the thread wall remains unclear. High resolution, 3-dimensional imaging of infection threads, and cell wall component specific labelling, would greatly aid in our understanding of the nature and development of these structures. To date, such imaging has not been done, with infection threads typically imaged by GFP-tagged rhizobia within them, or histochemically in thin sections. Results We have developed new methods of imaging infection threads using novel and traditional cell wall fluorescent labels, and laser confocal scanning microscopy. We applied a new Periodic Acid Schiff (PAS) stain using rhodamine-123 to the labelling of whole cleared infected roots of Medicago truncatula; which allowed for imaging of infection threads in greater 3D detail than had previously been achieved. By the combination of the above method and a calcofluor-white counter-stain, we also succeeded in labelling infection threads and plant cell walls separately, and have potentially discovered a way in which the infection thread matrix can be visualized. Conclusions Our methods have made the imaging and study of infection threads more effective and informative, and present exciting new opportunities for future research in the area.

Legumes are generally considered to be more responsive to elevated CO2 (eCO2) conditions due to the benefits provided by symbiotic nitrogen fixation. In response to high carbohydrate demand from nodules, legumes display autoregulation of nodulation (AON) to restrict nodules to the minimum number necessary to sustain nitrogen supply under current photosynthetic levels. AON mutants super-nodulate and typically grow smaller than wild-type plants under ambient CO2. Here, we show that AON super-nodulating mutants have substantially higher biomass under eCO2 conditions, which is sustained through increased photosynthetic investment. We examined photosynthetic and physiological traits across super-nodulating rdn1-1 (Root Determined Nodulation) and sunn4 (Super Numeric Nodules) and non-nodulating nfp1 (Nod Factor Perception) Medicago truncatula mutants. Under eCO2 conditions, super-nodulating plants exhibited increased rates of carboxylation (Vcmax) and electron transport (J) relative to wild-type and non-nodulating counterparts. The substantially higher rate of CO2 assimilation in eCO2-grown sunn4 super-nodulating plants was sustained through increased production of key photosynthetic enzymes, including Rieske FeS. We hypothesize that AON mutants are carbon-limited and can perform better at eCO2 through improved photosynthesis. Nodulating legumes, especially those with higher nitrogen fixation capability, are likely to out-perform non-nodulating plants under future CO2 conditions and will be important tools for understanding carbon and nitrogen partitioning under eCO2 conditions and future crop improvements.

Because of the developmental similarities between root nodules induced by symbiotic rhizobia and root galls formed by parasitic nematodes, we investigated the involvement of nodulation genes in the infection of Medicago truncatula by the root knot nematode (RKN), Meloidogyne javanica. We found that gall formation, including giant cell formation, pericycle and cortical cell division, as well as egg laying, occurred successfully in the non-nodulating mutants nfp1 (nod factor perception1), nin1 (nodule inception1) and nsp2 (nodulation signaling pathway2) and the cytokinin perception mutant cre1 (cytokinin receptor1). Gall and egg formation were significantly reduced in the ethylene insensitive, hypernodulating mutant skl (sickle), and to a lesser extent, in the low nodulation, abscisic acid insensitive mutant latd/nip (lateral root-organ defective/numerous infections and polyphenolics). Despite its supernodulation phenotype, the sunn4 (super numeric nodules4) mutant, which has lost the ability to autoregulate nodule numbers, did not form excessive numbers of galls. Co-inoculation of roots with nematodes and rhizobia significantly reduced nodule numbers compared to rhizobia-only inoculated roots, but only in the hypernodulation mutant skl. Thus, this effect is likely to be influenced by ethylene signaling, but is not likely explained by resource competition between galls and nodules. Co-inoculation with rhizobia also reduced gall numbers compared to nematode-only infected roots, but only in the wild type. Therefore, the protective effect of rhizobia on nematode infection does not clearly depend on nodule number or on Nod factor signaling. Our study demonstrates that early nodulation genes that are essential for successful nodule development are not necessary for nematode-induced gall formation, that gall formation is not under autoregulation of nodulation control, and that ethylene signaling plays a positive role in successful RKN parasitism in M. truncatula.

Abstract Background Plant roots release a variety of organic compounds into the soil which alter the physical, chemical and biological properties of the rhizosphere. Root exudates are technically challenging to measure in soil because roots are difficult to access and exudates can be bound by minerals or consumed by microorganisms. Exudates are easier to measure with hydroponically-grown plants but, even here, simple compounds such as sugars and organic acids can be rapidly assimilated by microorganisms. Sterile hydroponic systems avoid this shortcoming but it is very difficult to maintain sterility for long periods especially for larger crop species. As a consequence, studies often use small model species such as Arabidopsis to measure exudates or use seedlings of crop plants which only have immature roots systems. Results We developed a simple hydroponic system for cultivating large crop plants in sterile conditions for more than 30 days. Using this system wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) plants were grown in sterile conditions for 30 days by which time they had reached the six-leaf stage and developed mature root systems with seminal, nodal and lateral roots. To demonstrate the utility of this system we characterized the aluminium-activated exudation of malate from the major types of wheat roots for the first time. We found that all root types measured released malate but the amounts were two-fold greater from the seminal and nodal axile roots compared with the lateral roots. Additionally, we showed that this sterile growth system could be used to collect exudates from intact whole root systems of barley. Conclusions We developed a simple hydroponic system that enables cereal plants to be grown in sterile conditions for longer periods than previously recorded. Using this system we measured, for the first time, the aluminium-activated efflux of malate from the major types of wheat roots. We showed the system can also be used for collecting exudates from intact root systems of 30-day-old barley plants. This hydroponic system can be modified for various purposes. Importantly it enables the study of exudates from crop species with mature root systems.

The root-infecting necrotrophic fungal pathogen Rhizoctoniasolani causes significant disease to all the world’s major food crops. As a model for pathogenesis of legumes, we have examined the interaction of R. solani AG8 with Medicago truncatula. RNAseq analysis of the moderately resistant M. truncatula accession A17 and highly susceptible sickle (skl) mutant (defective in ethylene sensing) identified major early transcriptional reprogramming in A17. Responses specific to A17 included components of ethylene signaling, reactive oxygen species metabolism, and consistent upregulation of the isoflavonoid biosynthesis pathway. Mass spectrometry revealed accumulation of the isoflavonoid-related compounds liquiritigenin, formononetin, medicarpin, and biochanin A in A17. Overexpression of an isoflavone synthase in M. truncatula roots increased isoflavonoid accumulation and resistance to R. solani. Addition of exogenous medicarpin suggested this phytoalexin may be one of several isoflavonoids required to contribute to resistance to R. solani. Together, these results provide evidence for the role of ethylene-mediated accumulation of isoflavonoids during defense against root pathogens in legumes. The involvement of ethylene signaling and isoflavonoids in the regulation of both symbiont-legume and pathogen-legume interactions in the same tissue may suggest tight regulation of these responses are required in the root tissue.

Actinorhizal nodules are structurally different from legume nodules and show a greater similarity to lateral roots. Because of the important role of auxins in lateral root and nodule formation, auxin profiles were examined in roots and nodules of the actinorhizal species Datisca glomerata and the model legume Medicago truncatula. The auxin response in roots and nodules of both species was analyzed in transgenic root systems expressing a beta-glucuronidase gene under control of the synthetic auxin-responsive promoter DR5. The effects of two different auxin on root development were compared for both species. The auxin present in nodules at the highest levels was phenylacetic acid (PAA). No differences were found between the concentrations of active auxins of roots vs. nodules, while levels of the auxin conjugate indole-3-acetic acid-alanine were increased in nodules compared to roots of both species. Because auxins typically act in concert with cytokinins, cytokinins were also quantified. Concentrations of cis-zeatin and some glycosylated cytokinins were dramatically increased in nodules compared to roots of D. glomerata, but not of M. truncatula. The ratio of active auxins to cytokinins remained similar in nodules compared to roots in both species. The auxin response, as shown by the activation of the DR5 promoter, seemed significantly reduced in nodules compared to roots of both species, suggesting the accumulation of auxins in cell types that do not express the signal transduction pathway leading to DR5 activation. Effects on root development were analyzed for the synthetic auxin naphthaleneacetic acid (NAA) and PAA, the dominant auxin in nodules. Both auxins had similar effects, except that the sensitivity of roots to PAA was lower than to NAA. However, while the effects of both auxins on primary root growth were similar for both species, effects on root branching were different: both auxins had the classical positive effect on root branching in M. truncatula, but a negative effect in D. glomerata. Such a negative effect of exogenous auxin on root branching has previously been found for a cucurbit that forms lateral root primordia in the meristem of the parental root; however, root branching in D. glomerata does not follow that pattern.

Bacteria use quorum sensing signaling for cell-to-cell communication, which is also important for their interactions with plant hosts. Quorum sensing via N-acyl-homoserine lactones (AHLs) is important for successful symbioses between legumes and nitrogen-fixing rhizobia. Previous studies have shown that plant hosts can recognize and respond to AHLs. Here, we tested whether the response of the model legume Medicago truncatula to AHLs from its symbiont and other bacteria could be modulated by the abundance and composition of plant-associated microbial communities. Temporary antibiotic treatment of the seeds removed the majority of bacterial taxa associated with M. truncatula roots and significantly altered the effect of AHLs on nodule numbers, but lateral root density, biomass, and root length responses were much less affected. The AHL 3-oxo-C14-HSL (homoserine lactone) specifically increased nodule numbers but only after the treatment of seeds with antibiotics. This increase was associated with increased expression of the early nodulation genes RIP1 and ENOD11 at 24 h after infection. A 454 pyrosequencing analysis of the plant-associated bacteria showed that antibiotic treatment had the biggest effect on bacterial community composition. However, we also found distinct effects of 3-oxo-C14-HSL on the abundance of specific bacterial taxa. Our results revealed a complex interaction between plants and their associated microbiome that could modify plant responses to AHLs.

Root nodule symbiosis (RNS) is a feature confined to a single clade of plants, the Fabids. Among Fabids capable of RNS, legumes form root cortex-based nodules in symbioses with rhizobia, while actinorhizal species form lateral root-based nodules with actinomycetes. Cytokinin has previously been shown to be sufficient for “pseudonodule” initiation in model legumes. Here, we tested whether this response correlates with the ability to nodulate across a range of plant species. We analyzed the formation of pseudonodules in 17 nodulating and non-nodulating legume species, and 11 non-legumes, including nodulating actinorhizal species, using light and fluorescence microscopy. Cytokinin-induced pseudonodules arising from cortical cell divisions occurred in all nodulating legume species, but not in any of the other species, including non-nodulating legumes. Pseudonodule formation was dependent on the CRE1 cytokinin receptor in Medicago truncatula. Inhibition of root growth by cytokinin occurred across plant groups, indicating that pseudonodule development is the result of a specific cortical cytokinin response unique to nodulating legumes. Lack of a cortical cytokinin response from the Arabidopsis thaliana cytokinin reporter TCSn::GFP supported this hypothesis. Our results suggest that the ability to form cortical cell-derived nodules was gained in nodulating legumes, and likely lost in non-nodulating legumes, due to a specific root cortical response to cytokinin.

Legumes enter into a symbiotic relationship with nitrogen-fixing rhizobia, leading to nodule development. Two main types of nodules have been widely studied, indeterminate and determinate, which differ in the location of the first cell division in the root cortex, and persistency of the nodule meristem. Here, we compared the control of auxin transport, content, and response during the early stages of indeterminate and determinate nodule development in the model legumes Medicago truncatula and Lotus japonicus, respectively, to investigate whether differences in auxin transport control could explain the differences in the location of cortical cell divisions. While auxin responses were activated in dividing cortical cells during nodulation of both nodule types, auxin (indole-3-acetic acid) content at the nodule initiation site was transiently increased in M. truncatula, but transiently reduced in L. japonicus. Root acropetal auxin transport was reduced in M. truncatula at the very start of nodule initiation, in contrast to a prolonged increase in acropetal auxin transport in L. japonicus. The auxin transport inhibitors 2,3,5-triiodobenzoic acid and 1-N-naphthylphthalamic acid (NPA) only induced pseudonodules in legume species forming indeterminate nodules, but failed to elicit such structures in a range of species forming determinate nodules. The development of these pseudonodules in M. truncatula exhibited increased auxin responses in a small primordium formed from the pericycle, endodermis, and inner cortex, similar to rhizobia-induced nodule primordia. In contrast, a diffuse cortical auxin response and no associated cortical cell divisions were found in L. japonicus. Collectively, we hypothesize that a step of acropetal auxin transport inhibition is unique to the process of indeterminate nodule development, leading to auxin responses in pericycle, endodermis, and inner cortex cells, while increased auxin responses in outer cortex cells likely require a different mechanism during the formation of determinate nodules.

Most field-grown plants are surrounded by microbes, especially from the soil. Some of these, including bacteria, fungi and nematodes, specifically manipulate the growth and development of their plant hosts, primarily for the formation of structures housing the microbes in roots. These developmental processes require the correct localization of the phytohormone auxin, which is involved in the control of cell division, cell enlargement, organ development and defense, and is thus a likely target for microbes that infect and invade plants. Some microbes have the ability to directly synthesize auxin. Others produce specific signals that indirectly alter the accumulation of auxin in the plant by altering auxin transport. This review highlights root–microbe interactions in which auxin transport is known to be targeted by symbionts and parasites to manipulate the development of their host root system. We include case studies for parasitic root–nematode interactions, mycorrhizal symbioses as well as nitrogen fixing symbioses in actinorhizal and legume hosts. The mechanisms to achieve auxin transport control that have been studied in model organisms include the induction of plant flavonoids that indirectly alter auxin transport and the direct targeting of auxin transporters by nematode effectors. In most cases, detailed mechanisms of auxin transport control remain unknown.

Nitrogen deficiency limits crop performance under elevated CO2 (eCO2), depending on the ability of plant N uptake. However, the dynamics and redistribution of N2 fixation, and fertilizer and soil N use in legumes under eCO2 have been little studied. Such an investigation is essential to improve the adaptability of legumes to climate change. We took advantage of genotype-specific responses of soybean to increased CO2 to test which N-uptake phenotypes are most strongly related to enhanced yield. Eight soybean cultivars were grown in open-top chambers with either 390 ppm (aCO2) or 550 ppm CO2 (eCO2). The plants were supplied with 100 mg N kg−1 soil as 15N-labeled calcium nitrate, and harvested at the initial seed-filling (R5) and full-mature (R8) stages. Increased yield in response to eCO2 correlated highly (r = 0.95) with an increase in symbiotically fixed N during the R5 to R8 stage. In contrast, eCO2 only led to small increases in the uptake of fertilizer-derived and soil-derived N during R5 to R8, and these increases did not correlate with enhanced yield. Elevated CO2 also decreased the proportion of seed N redistributed from shoot to seeds, and this decrease strongly correlated with increased yield. Moreover, the total N uptake was associated with increases in fixed-N per nodule in response to eCO2, but not with changes in nodule biomass, nodule density, or root length.

Subtilisin-like serine protease (MtSBT), serine carboxypeptidase (MtSCP), MtN5, non-specific lipid transfer protein (MtnsLTP), early nodulin2-like protein (MtENOD2-like), FAD-binding domain containing protein (MtFAD-BP1), and rhicadhesin receptor protein (MtRHRE1) were among 34 proteins found in the supernatant of M. truncatula 2HA and sickle cell suspension cultures. This study investigated the expression of genes encoding those proteins in roots and developing nodules. Two methods were used: quantitative real time RT-PCR and gene expression analysis (with promoter:GUS fusion) in roots. Those proteins are predicted as secreted proteins which is indirectly supported by the findings that promoter:GUS fusions of six of the seven genes encoding secreted proteins were strongly expressed in the vascular bundle of transgenic hairy roots. All six genes have expressed in 14-day old nodule. The expression levels of the selected seven genes were quantified in Sinorhizobium-inoculated and control plants using quantitative real time RT-PCR. In conclusion, among seven genes encoding secreted proteins analyzed, the expression level of only one gene, MtN5, was up-regulated significantly in inoculated root segments compared to controls. The expression of MtSBT1, MtSCP1, MtnsLTP, MtFAD-BP1, MtRHRE1 and MtN5 were higher in root tip than in other tissues examined.

Measurement of auxin transport capacity provides quantitative data on the physiological machinery involved in auxin transport within plants. This technique is easy to perform and gives quick results. Radiolabelled auxin (indole-3-acetic-acid) is fed into the roots of Medicago truncatula via an agar block. The resulting radioactivity from radiolabelled auxin uptake in the roots is measured with a liquid scintillation counter. Here, we describe the measurement of auxin transport capacity around the nodulation susceptible zone in young seedling roots of M. truncatula in response to rhizobia inoculation. Similar assays could be adapted in other plant species and to answer other biological questions.

Auxins represent a major group of phytohormones controlling plant development. The spatio-temporal regulation of auxin gradients is essential for the initiation, growth and correct development of plant organs. Because auxins and their metabolites occur at trace levels in plant tissue, experiments requiring identification plus their selective and specific quantification can be most conveniently achieved using mass spectrometry (MS) and the associated chromatographic methods. With the advent of appropriate liquid-based ionisation techniques, emphasis has moved from the use of gas chromatography as the sample interface to the MS (GC/MS), with its concomitant need for derivatisation, to the more sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS). We describe an optimized liquid chromatography electrospray-ionisation quadrupole time-of-flight (LC-ESI-QTOF) methodology for the quantification of auxins. While the solvent extraction of young Medicago truncatula (M. truncatula) roots, as described herein, is relatively straightforward, older, woody or oily plant tissues may also be analyzed with appropriate modification to remove interferences and/or enhance extraction efficiency. In our hands, the analytical assay has proved sufficiently sensitive for the quantification of auxins to investigate their roles in various organogenic events, such as root nodulation in M. truncatula. Further increases in sensitivity can be expected with the use of the latest generation of instruments.

Most land plants can become infected by plant parasitic nematodes in the field. Plant parasitic nematodes can be free-living or endoparasitic, and they usually infect plant roots. Most damaging are endoparasites, which form feeding sites inside plant roots that damage the root system and redirect nutrients towards the parasite. This process involves developmental changes to the root in parallel with the induction of defense responses. Plant flavonoids are secondary metabolites that have roles in both root development and plant defense responses against a range of microorganisms. Here, we review our current knowledge of the roles of flavonoids in the interactions between plants and plant parasitic nematodes. Flavonoids are induced during nematode infection in plant roots, and more highly so in resistant compared with susceptible plant cultivars, but many of their functions remain unclear. Flavonoids have been shown to alter feeding site development to some extent, but so far have not been found to be essential for root–parasite interactions. However, they likely contribute to chemotactic attraction or repulsion of nematodes towards or away from roots and might help in the general plant defense against nematodes. Certain flavonoids have also been associated with functions in nematode reproduction, although the mechanism remains unknown. Much remains to be examined in this area, especially under field conditions.

Flavonoids are structurally diverse secondary metabolites in plants, with a multitude of functions. These span from functions in regulating plant development, pigmentation, and UV protection, to an array of roles in defence and signalling between plants and microorganisms. Because of their prevalence in the human diet, many flavonoids constitute important components of medicinal plants and are used in the control of inflammation and cancer prevention. Advances in the elucidation of flavonoid biosynthesis and its regulation have led to an increasing number of studies aimed at engineering the flavonoid pathway for enhancing nutritional value and plant defences against pathogens and herbivores, as well as modifying the feeding value of pastures. Many future opportunities await for the exploitation of this colourful pathway in crops, pastures, and medicinal plants.

Nodule formation in Rhizobium-legume symbiosis is negatively regulated by ethylene. Ethylene inhibitors such as L-α-(2-amino-ethoxyvinyl)-glycine (AVG) and silver ions (Ag+), the ethylene-insensitive sickle mutant, and transgenic plants were used to study ethylene-mediated responses in nodulation. The mode of action of ethylene inhibitors AVG and Ag+, and the skl mutation occur at different steps in ethylene biosynthesis and perception. Their effects on root growth and nodulation phenotypes, in particular nodule distribution along the primary root, were compared in this study. Ag+ and AVG treatments showed similar root growth responses to skl mutant. However, nodule distribution in the hypernodulating skl mutant is different from that of wild-type plants grown on either AVG or Ag+. AVG increased nodule numbers and widened the nodulation zone, while the skl mutant had an increased number of nodules within the susceptible zone of nodulation. Ag+ reduced nodule numbers, restricted the nodulation zone, and restored the nodulation phenotype of skl to that of the wild type.

Nodule formation in Rhizobium-legume symbiosis is negatively regulated by ethylene. Ethylene inhibitors such as L--(2-amino-ethoxyvinyl)-glycine (AVG) and silver ions (Ag+), the ethylene-insensitive sickle mutant, and transgenic plants were used to study ethylene-mediated responses in nodulation. The mode of action of ethylene inhibitors AVG and Ag+, and the skl mutation occur at different steps in ethylene biosynthesis and perception. Their effects on root growth and nodulation phenotypes, in particular nodule distribution along the primary root, were compared in this study. Ag+ and AVG treatments showed similar root growth responses to skl mutant. However, nodule distribution in the hypernodulating skl mutant is different from that of wild-type plants grown on either AVG or Ag+. AVG increased nodule numbers and widened the nodulation zone, while the skl mutant had an increased number of nodules within the susceptible zone of nodulation. Ag+ reduced nodule numbers, restricted the nodulation zone, and restored the nodulation phenotype of skl to that of the wild type.

The rhizosphere microbiome is regulated by plant genotype, root exudates and environment. There is substantial interest in breeding and managing crops that host root microbial communities that increase productivity. The eudicot model species Arabidopsis has been used to investigate these processes, however a model for monocotyledons is also required. We characterized the rhizosphere microbiome and root exudates of Brachypodium distachyon, to develop it as a rhizosphere model for cereal species like wheat. The Brachypodium rhizosphere microbial community was dominated by Burkholderiales. However, these communities were also dependent on how tightly they were bound to roots, the root type they were associated with (nodal or seminal roots), and their location along the roots. Moreover, the functional gene categories detected in microorganisms isolated from around root tips differed from those isolated from bases of roots. The Brachypodium rhizosphere microbiota and root exudate profiles were similar to those reported for wheat rhizospheres, and different to Arabidopsis. The differences in root system development and cell wall chemistry between monocotyledons and eudicots may also influence the microorganism composition of these major plant types. Brachypodium is a promising model for investigating the microbiome of wheat.

We examined the timing and location of several early root responses to Rhizobium leguminosarum bv. trifolii infection, compared with a localized addition of cytokinin in white clover, to study the role of cytokinin in early signaling during nodule initiation. Induction of ENOD40 expression by either rhizobia or cytokinin was similar in timing and location and occurred in nodule progenitor cells in the inner cortex. Inoculation of rhizobia in the mature root failed to induce ENOD40 expression and cortical cell divisions (ccd). Nitrate addition at levels repressing nodule formation inhibited ENOD40 induction by rhizobia but not by cytokinin. ENOD40 expression was not induced by auxin, an auxin transport inhibitor, or an ethylene precursor. In contrast to rhizobia, cytokinin addition was not sufficient to induce a modulation of the auxin flow, the induction of specific chalcone synthase genes, and the accumulation of fluorescent compounds associated with nodule initiation. However, cytokinin addition was sufficient for the localized induction of auxin-induced GH3 gene expression and the initiation of ccd. Our results suggest that rhizobia induce cytokinin-mediated events in parallel to changes in auxin-related responses during nodule initiation and support a role of ENOD40 in regulating ccd. We propose a model for the interactions of cytokinin with auxin, ENOD40, flavonoids, and nitrate during nodulation.

We examined a range of responses of root cortical cells to Rhizobium sp. inoculation to investigate why rhizobia preferentially nodulate legume roots in the zone of emerging root hairs, but generally fail to nodulate the mature root. We tested whether the inability to form nodules in the mature root is due to a lack of plant flavonoids to induce the bacterial genes required for nodulation or a failure of mature cortical cells to respond to Rhizobium spp. When rhizobia were inoculated in the zone of emerging root hairs, changes in β-glucuronidase (GUS) expression from an auxin-responsive promoter (GH3), expression from three chalcone synthase promoters, and the accumulation of specific flavonoid compounds occurred in cortical cells prior to nodule formation. Rhizobia failed to induce these responses when inoculated in the mature root, even when co-inoculated with nod gene-inducing flavonoids. However, mature root hairs remained responsive to rhizobia and could support infection thread formation. This suggests that a deficiency in signal transduction is the reason for nodulation failure in the mature root. However, nodules could be initiated in the mature root at sites of lateral root emergence. A comparison between lateral root and nodule formation showed that similar patterns of GH3:gusA expression, chalcone synthase gene expression, and accumulation of a particular flavonoid compound occurred in the cortical cells involved in both processes. The results suggest that rhizobia can“ hijack” cortical cells next to lateral root emergence sites because some of the early responses required for nodule formation have already been activated by the plant in those cells.

We examined the site-specific induction of the flavonoid pathway before and during nodule initiation in white clover with transgenic plants, fluorescence microscopy, and microspectrofluorometry to test if flavonoids play a role in nodule organogenesis. A chalcone synthase regulated β-glucuronidase (GUS) transgene (CHS3:gusA) was up-regulated from 3 h post inoculation (p.i.) until cell division (around 40 h p.i.) in inner cortex cells underlying the inoculation site. Intracellular fluorescence occurred in vacuoles of those inner cortex cells from 13 h p.i. until the fluorescent cells divided. Fluorescence emission spectra of contents of individual fluorescing cortex cells were measured in situ and compared with emission spectra of compounds purified from root extracts. The fluorescing compound located in cells of the inner cortex after Rhizobium leguminosarum bv. trifolii infection was identified as a water-soluble derivative of 7,4′-dihydroxyflavone. Nodule primordium cells contained a different fluorescent compound, identified as the isoflavonoid formononetin. CHS3:gusA expression and flavonoid accumulation were only induced in inner cortex cells by a nodulating Rhizo-bium strain and by clover-specific lipo-chitinoligosac-charides, but not by non-nodulating rhizobia. Fluorescence was also induced by compatible rhizobia in other legumes such as alfalfa, pea, and siratro in the cells that participate in nodule initiation. Our results show that fluorescent flavonoids are useful markers in nodule or-ganogenesis in clover and may have direct roles in nodule formation.

S. flexneri strains, most frequently linked with endemic outbreaks of shigellosis, invade the colonic and rectal epithelium of their host and cause severe tissue damage. Here we have attempted to elucidate the contribution of the periplasmic enzyme, L-asparaginase (AnsB) to the pathogenesis of S. flexneri. Using a reverse genetic approach we found that ansB mutants showed reduced adherence to epithelial cells in vitro and attenuation in two in vivo models of shigellosis, the Caenorhabditis elegans and the murine pulmonary model. To investigate how AnsB affects bacterial adherence, we compared the proteomes of the ansB mutant with its wild type parental strain using two dimensional differential in-gel electrophoresis and identified the outer membrane protein, OmpA as up-regulated in ansB mutant cells. Bacterial OmpA, is a prominent outer membrane protein whose activity has been found to be required for bacterial pathogenesis. Overexpression of OmpA in wild type S. flexneri serotype 3b resulted in decreasing the adherence of this virulent strain, suggesting that the up-regulation of OmpA in ansB mutants contributes to the reduced adherence of this mutant strain. The data presented here is the first report that links the metabolic enzyme AnsB to S. flexneri pathogenesis.

The Gram-negative bacterium Shigella flexneri is the causative agent of shigellosis, a diarrhoeal disease also known as bacillary dysentery. S. flexneri infects the colonic and rectal epithelia of its primate host and induces a cascade of inflammatory responses that culminates in the destruction of the host intestinal lining. Molecular characterization of host-pathogen interactions in this infection has been challenging due to the host specificity of S. flexneri strains, as it strictly infects humans and non-human primates. Recent studies have shown that S. flexneri infects the soil dwelling nematode Caenorhabditis elegans, however, the interactions between S. flexneri and C. elegans at the cellular level and the cause of nematode death are unknown. Here we attempt to gain insight into the complex host-pathogen interactions between S. flexneri and C. elegans. Using transmission electron microscopy, we show that live S. flexneri cells accumulate in the nematode intestinal lumen, produce outer membrane vesicles and invade nematode intestinal cells. Using two-dimensional differential in-gel electrophoresis we identified host proteins that are differentially expressed in response to S. flexneri infection. Four of the identified genes, aco-1, cct-2, daf-19 and hsp-60, were knocked down using RNAi and ACO-1, CCT-2 and DAF-19, which were identified as up-regulated in response to S. flexneri infection, were found to be involved in the infection process. aco-1 RNAi worms were more resistant to S. flexneri infection, suggesting S. flexneri-mediated disruption of host iron homeostasis. cct-2 and daf-19 RNAi worms were more susceptible to infection, suggesting that these genes are induced as a protective mechanism by C. elegans. These observations further our understanding of the processes involved in S. flexneri infection of C. elegans, which is immensely beneficial to the routine use of this new in vivo model to study S. flexneri pathogenesis.

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin-regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.

The incidence of major diseases such as cardiovascular disease, thrombosis and cancer increases with age and is the major cause of mortality world-wide, with neonates and children somehow protected from such diseases of ageing. We hypothesized that there are major developmental differences in plasma proteins and that these contribute to age-related changes in the incidence of major diseases. We evaluated the human plasma proteome in healthy neonates, children and adults using the 2D-DIGE approach. We demonstrate significant changes in number and abundance of up to 100 protein spots that have marked differences in during the transition of the plasma proteome from neonate and child through to adult. These proteins are known to be involved in numerous physiological processes such as iron transport and homeostasis, immune response, haemostasis and apoptosis, amongst others. Importantly, we determined that the proteins that are differentially expressed with age are not the same proteins that are differentially expressed with gender and that the degree of phosphorylation of plasma proteins also changes with age. Given the multi-functionality of these proteins in human physiology, understanding the differences in the plasma proteome in neonates and children compared to adults will make a major contribution to our understanding of developmental biology in humans.