Arbuscular-Mycorrhizal Symbiosis in Medicago Regulated by the Transcription Factor MtbHLHm1;1 and the Ammonium Facilitator Protein MtAMF1;3.

Root systems of most land plants are colonised by arbuscular mycorrhiza fungi. The symbiosis supports nutrient acquisition strategies predominantly associated with plant access to inorganic phosphate. The nutrient acquisition is enhanced through an extensive network of external fungal hyphae that extends out into the soil, together with the development of fungal structures forming specialised interfaces with root cortical cells. Orthologs of the bHLHm1;1 transcription factor, previously described in soybean nodules (GmbHLHm1) and linked to the ammonium facilitator protein GmAMF1;3, have been identified in Medicago (Medicago truncatula) roots colonised by AM fungi. Expression studies indicate that transcripts of both genes are also present in arbuscular containing root cortical cells and that the MtbHLHm1;1 shows affinity to the promoter of MtAMF1;3. Both genes are induced by AM colonisation. Loss of Mtbhlhm1;1 expression disrupts AM arbuscule abundance and the expression of the ammonium transporter MtAMF1;3. Disruption of Mtamf1;3 expression reduces both AM colonisation and arbuscule development. The respective activities of MtbHLHm1;1 and MtAMF1;3 highlight the conservation of putative ammonium regulators supporting both the rhizobial and AM fungal symbiosis in legumes.

Balancing nitrate acquisition strategies in symbiotic legumes.

MAIN CONCLUSION: Legumes manage both symbiotic (indirect) and non-symbiotic (direct) nitrogen acquisition pathways. Understanding and optimising the direct pathway for nitrate uptake will support greater legume growth and seed yields. Legumes have multiple pathways to acquire reduced nitrogen to grow and set seed. Apart from the symbiotic N2-fixation pathway involving soil-borne rhizobia bacteria, the acquisition of nitrate and ammonia from the soil can also be an important secondary nitrogen source to meet plant N demand. The balance in N delivery between symbiotic N (indirect) and inorganic N uptake (direct) remains less clear over the growing cycle and with the type of legume under cultivation. In fertile, pH balanced agricultural soils, NO3- is often the predominant form of reduced N available to crop plants and will be a major contributor to whole plant N supply if provided at sufficient levels. The transport processes for NO3- uptake into legume root cells and its transport between root and shoot tissues involves both high and low-affinity transport systems called HATS and LATS, respectively. These proteins are regulated by external NO3- availability and by the N status of the cell. Other proteins also play a role in NO3- transport, including the voltage dependent chloride/nitrate channel family (CLC) and the S-type anion channels of the SLAC/SLAH family. CLC's are linked to NO3- transport across the tonoplast of vacuoles and the SLAC/SLAH's with NO3- efflux across the plasma membrane and out of the cell. An important step in managing the N requirements of a plant are the mechanisms involved in root N uptake and the subsequent cellular distribution within the plant. In this review, we will present the current knowledge of these proteins and what is understood on how they function in key model legumes (Lotus japonicus, Medicago truncatula and Glycine sp.). The review will examine their regulation and role in N signalling, discuss how post-translational modification affects NO3- transport in roots and aerial tissues and its translocation to vegetative tissues and storage/remobilization in reproductive tissues. Lastly, we will present how NO3-influences the autoregulation of nodulation and nitrogen fixation and its role in mitigating salt and other abiotic stresses.

Expression of the GFP-mammalian pleckstrin homology (PH) domain of the phospholipase C δ1 in Saccharomyces cerevisiae BY4741.

BACKGROUND: Pleckstrin homology (PH) domains are common modules of ∼120 amino acids found in proteins involved in signalling, cytoskeletal organization, membrane transport, and modification of phospholipids. Previous live cell studies have involved the use of the green-fluorescent protein (GFP) labelling of PH-domain of phospholipase C δ1 (PLC δ1) to study the interactions of molecules at the membrane interface. METHODS AND RESULTS: For this study, the aim was to construct and express the GFP-PH domain of PLC δ1 in the Saccharomyces cerevisiae BY4741. The transformants expressing GFP-PH domain of PLC δ1 displayed localised fluorescence to the cell periphery (plasma membrane) while the negative control expressed GFP within the cytoplasm only. No GFP was observed in the non-transformed yeast cells. CONCLUSIONS: Thus, this technique could be useful in future molecular interactions studies targeted specifically at the yeast cell membrane interface in live yeast cells.

Caspofungin resistance in Candida albicans: genetic factors and synergistic compounds for combination therapies.

Caspofungin and other echinocandins have been used for the treatment of human infections by the opportunistic yeast pathogen, Candida albicans. There has been an increase in infections by non-albicans Candida species such as Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, and Candida auris in clinical or hospital settings. This is problematic to public health due to the increasing prevalence of echinocandin resistant species/strains. This review will present a summary on various studies that investigated the inhibitory action of caspofungin on 1,3-ß-D-glucan synthesis, on cell wall structure, and biofilm formation of C. albicans. It will highlight some of the issues linked to caspofungin resistance or reduced caspofungin sensitivity in various Candida species and the potential benefits of antimicrobial peptides and other compounds in synergy with caspofungin.

Rapid screening method of Saccharomyces cerevisiae mutants using calcofluor white and aniline blue.

Fungal cell walls are composed of polysaccharide scaffold that changes in response to environment. The structure and biosynthesis of the wall are unique to fungi, with plant and mammalian immune systems evolved to recognize wall components. Additionally, the enzymes that assemble fungal cell wall components are excellent targets for antifungal chemotherapies and fungicides. Understanding changes in the cell wall are important for fundamental understanding of cell wall dynamics and for drug development. Here we describe a screening technique to monitor the gross morphological changes of two key cell wall polysaccharides of chitin and ß-1,3-glucan combined with polymerase chain reaction (PCR) genotyping. Changes in chitin and ß-1,3-glucan were detected microscopically by using the dyes calcofluor white and aniline blue. Combining PCR and fluorescence microscopy, as a quick and easy screening technique, confirmed both the phenotype and genotype of the wild-type, h chitin synthase mutants (chs1Δ and chs3Δ) and one ß-1,3-glucan synthase mutant fks2Δ from Saccharomyces cerevisiae knockout library. This combined screening method highlighted that the fks1Δ strain obtained commercially was in fact not FKS1 deletion strain, and instead had both wild-type genotype and phenotype. A new ß-1,3-glucan synthase knockout fks1::URA3 strain was created. Fluorescence microscopy confirmed its phenotype revealing that the chitin and the new ß-1,3-glucan profiles were elevated in the mother cells and in the emerging buds respectively in the fks1Δ cell walls. This combination of PCR with fluorescence microscopy is a quick and easy screening method to determine and verify morphological changes in the S. cerevisiae cell wall.

Propidium iodide enabled live imaging of Pasteuria sp.-Pratylenchus zeae infection studies under fluorescence microscopy.

Live imaging allows observations of cell structures and processes in real time, to monitor dynamic changes within living organisms compared to fixed organisms. Fluorescence microscopy was used to monitor the dynamic infection process of the nematode parasitic bacterium Pasteuria sp. and the sugarcane root-lesion nematode, Pratylenchus zeae. Under fluorescence microscopy, green-autofluorescent globules were observed in live control and Pasteuria sp.-infected nematodes. Only nematodes killed by Pasteuria sp. or heat treated displayed a diffuse pattern of autofluorescence. Propidium iodide (PI), used as a cell membrane integrity indicator, confirmed that the nematode's cuticle acts as an impermeable barrier. PI stained cells/DNA of heat-treated control and Pasteuria sp.-infected P. zeae. PI as a counterstain facilitated the location of Pasteuria endospores on the cuticle surface of P. zeae. No PI staining was observed in sporangia and in endospores within the nematode body. However, PI specifically stained endospores on the cuticle surface and within the cuticle carcass showing, in mature propagules, a ring-like pattern. Live imaging, combined with fluorescence microscopy and fluorescent dyes such as PI, appears useful in live studies on plant nematode interactions with nematophagous bacteria.

Phytophthora palmivora-Cocoa Interaction.

Phytophthora palmivora (Butler) is an hemibiotrophic oomycete capable of infecting over 200 plant species including one of the most economically important crops, Theobroma cacao L. commonly known as cocoa. It infects many parts of the cocoa plant including the pods, causing black pod rot disease. This review will focus on P. palmivora's ability to infect a plant host to cause disease. We highlight some current findings in other Phytophthora sp. plant model systems demonstrating how the germ tube, the appressorium and the haustorium enable the plant pathogen to penetrate a plant cell and how they contribute to the disease development in planta. This review explores the molecular exchange between the oomycete and the plant host, and the role of plant immunity during the development of such structures, to understand the infection of cocoa pods by P. palmivora isolates from Papua New Guinea.

The Arabidopsis NRT1.1 transceptor coordinately controls auxin biosynthesis and transport to regulate root branching in response to nitrate.

In agricultural systems, nitrate is the main source of nitrogen available for plants. Besides its role as a nutrient, nitrate has been shown to act as a signal molecule in plant growth, development, and stress responses. In Arabidopsis, the NRT1.1 nitrate transceptor represses lateral root (LR) development at low nitrate availability by promoting auxin basipetal transport out of the LR primordia (LRPs). Here we show that NRT1.1 acts as a negative regulator of the TAR2 auxin biosynthetic gene in the root stele. This is expected to repress local auxin biosynthesis and thus to reduce acropetal auxin supply to the LRPs. Moreover, NRT1.1 also negatively affects expression of the LAX3 auxin influx carrier, thus preventing the cell wall remodeling required for overlying tissue separation during LRP emergence. NRT1.1-mediated repression of both TAR2 and LAX3 is suppressed at high nitrate availability, resulting in nitrate induction of the TAR2 and LAX3 expression that is required for optimal stimulation of LR development by nitrate. Altogether, our results indicate that the NRT1.1 transceptor coordinately controls several crucial auxin-associated processes required for LRP development, and as a consequence that NRT1.1 plays a much more integrated role than previously expected in regulating the nitrate response of root system architecture.

Interactions of endoparasitic and ectoparasitic nematodes within the plant root system.

Root-knot and cyst nematodes have sophisticated mechanisms to invade their plant hosts to reprogram the plant developmental program to induce feeding structures essential for nematode survival and reproduction. This has a detrimental effect on the plant as this sedentary endoparasitic interaction affects the growth and yields of many crop plants. However, other migratory endoparasitic nematodes that do not establish root feeding sites are as aggressive on many crop plants. With new information gained from the genome and transcriptomes of the migratory endoparasitic nematode, Pratylenchus spp., this review compares the different lifestyles and the pathogenic interactions these nematodes have with their plant host. Pratylenchus spp. utilises a common arsenal of effectors involved in plant cell wall degradation and the manipulation of plant host innate immunity. The absence of specific cell reprogramming effector genes may explain its migratory endoparasitic lifestyle, making it relevant to pest management approaches in Australia.

Nitrate Controls Root Development through Posttranscriptional Regulation of the NRT1.1/NPF6.3 Transporter/Sensor.

Plants are able to modulate root growth and development to optimize their nitrogen nutrition. In Arabidopsis (Arabidopsis thaliana), the adaptive root response to nitrate (NO3-) depends on the NRT1.1/NPF6.3 transporter/sensor. NRT1.1 represses emergence of lateral root primordia (LRPs) at low concentration or absence of NO3- through its auxin transport activity that lowers auxin accumulation in LR. However, these functional data strongly contrast with the known transcriptional regulation of NRT1.1, which is markedly repressed in LRPs in the absence of NO3- To explain this discrepancy, we investigated in detail the spatiotemporal expression pattern of the NRT1.1 protein during LRP development and combined local transcript analysis with the use of transgenic lines expressing tagged NRT1.1 proteins. Our results show that although NO3- stimulates NRT1.1 transcription and probably mRNA stability both in primary root tissues and in LRPs, it acts differentially on protein accumulation, depending on the tissues considered with stimulation in cortex and epidermis of the primary root and a strong repression in LRPs and to a lower extent at the primary root tip. This demonstrates that NRT1.1 is strongly regulated at the posttranscriptional level by tissue-specific mechanisms. These mechanisms are crucial for controlling the large palette of adaptive responses to NO3- mediated by NRT1.1 as they ensure that the protein is present in the proper tissue under the specific conditions where it plays a signaling role in this particular tissue.

The Control of Auxin Transport in Parasitic and Symbiotic Root-Microbe Interactions.

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.

Role of auxin during intercellular infection of Discaria trinervis by Frankia.

Nitrogen-fixing nodules induced by Frankia in the actinorhizal plant Discaria trinervis result from a primitive intercellular root invasion pathway that does not involve root hair deformation and infection threads. Here, we analyzed the role of auxin in this intercellular infection pathway at the molecular level and compared it with our previous work in the intracellular infected actinorhizal plant Casuarina glauca. Immunolocalisation experiments showed that auxin accumulated in Frankia-infected cells in both systems. We then characterized the expression of auxin transporters in D. trinervis nodules. No activation of the heterologous CgAUX1 promoter was detected in infected cells in D. trinervis. These results were confirmed with the endogenous D. trinervis gene, DtAUX1. However, DtAUX1 was expressed in the nodule meristem. Consistently, transgenic D. trinervis plants containing the auxin response marker DR5:VENUS showed expression of the reporter gene in the meristem. Immunolocalisation experiments using an antibody against the auxin efflux carrier PIN1, revealed the presence of this transporter in the plasma membrane of infected cells. Finally, we used in silico cellular models to analyse auxin fluxes in D. trinervis nodules. Our results point to the existence of divergent roles of auxin in intercellularly- and intracellularly-infected actinorhizal plants, an ancestral infection pathways leading to root nodule symbioses.

Agp2p, the plasma membrane transregulator of polyamine uptake, regulates the antifungal activities of the plant defensin NaD1 and other cationic peptides.

Cationic antifungal peptides (AFPs) act through a variety of mechanisms but share the common feature of interacting with the fungal cell surface. NaD1, a defensin from Nicotiana alata, has potent antifungal activity against a variety of fungi of both hyphal and yeast morphologies. The mechanism of action of NaD1 occurs via three steps: binding to the fungal cell surface, permeabilization of the plasma membrane, and internalization and interaction with intracellular targets to induce fungal cell death. The targets at each of these three stages have yet to be defined. In this study, the screening of a Saccharomyces cerevisiae deletion collection led to the identification of Agp2p as a regulator of the potency of NaD1. Agp2p is a plasma membrane protein that regulates the transport of polyamines and other molecules, many of which carry a positive charge. Cells lacking the agp2 gene were more resistant to NaD1, and this resistance was accompanied by a decreased uptake of defensin. Agp2p senses and regulates the uptake of the polyamine spermidine, and competitive inhibition of the antifungal activity of NaD1 by spermidine was observed in both S. cerevisiae and the plant pathogen Fusarium oxysporum. The resistance of agp2Δ cells to other cationic antifungal peptides and decreased binding of the cationic protein cytochrome c to agp2Δ cells compared to that of wild-type cells have led to a proposed mechanism of resistance whereby the deletion of agp2 leads to an increase in positively charged molecules at the cell surface that repels cationic antifungal peptides.

Silencing of the chalcone synthase gene in Casuarina glauca highlights the important role of flavonoids during nodulation.

Nitrogen-fixing root nodulation is confined to four plant orders, including > 14,000 Leguminosae, one nonlegume genus Parasponia and c. 200 actinorhizal species that form symbioses with rhizobia and Frankia bacterial species, respectively. Flavonoids have been identified as plant signals and developmental regulators for nodulation in legumes and have long been hypothesized to play a critical role during actinorhizal nodulation. However, direct evidence of their involvement in actinorhizal symbiosis is lacking. Here, we used RNA interference to silence chalcone synthase, which is involved in the first committed step of the flavonoid biosynthetic pathway, in the actinorhizal tropical tree Casuarina glauca. Transformed flavonoid-deficient hairy roots were generated and used to study flavonoid accumulation and further nodulation. Knockdown of chalcone synthase expression reduced the level of specific flavonoids and resulted in severely impaired nodulation. Nodule formation was rescued by supplementing the plants with naringenin, which is an upstream intermediate in flavonoid biosynthesis. Our results provide, for the first time, direct evidence of an important role for flavonoids during the early stages of actinorhizal nodulation.

Symbiotic signaling in actinorhizal symbioses.

Actinorhizal symbioses are mutualistic associations between plants belonging to eight angiosperm families and soil bacteria of the genus Frankia. These interactions lead to the formation of new root organs, actinorhizal nodules, where the bacteria are hosted and fix atmospheric nitrogen thus providing the plant with an almost unlimited source of nitrogen for its nutrition. It involves an elaborate signaling between both partners of the symbiosis. In recent years, our knowledge of this signaling pathway has increased tremendously thanks to a series of technical breakthroughs including the sequencing of three Frankia genomes [1] and the implementation of RNA silencing technology for two actinorhizal species. In this review, we describe all these recent advances, current researches on symbiotic signaling in actinorhizal symbioses and give some potential future research directions.

Nitrate transceptor(s) in plants.

The availability of mineral nutrients in the soil dramatically fluctuates in both time and space. In order to optimize their nutrition, plants need efficient sensing systems that rapidly signal the local external concentrations of the individual nutrients. Until recently, the most upstream actors of the nutrient signalling pathways, i.e. the sensors/receptors that perceive the extracellular nutrients, were unknown. In Arabidopsis, increasing evidence suggests that, for nitrate, the main nitrogen source for most plant species, a major sensor is the NRT1.1 nitrate transporter, also contributing to nitrate uptake by the roots. Membrane proteins that fulfil a dual nutrient transport/signalling function have been described in yeast and animals, and are called 'transceptors'. This review aims to illustrate the nutrient transceptor concept in plants by presenting the current evidence indicating that NRT1.1 is a representative of this class of protein. The various facets, as well as the mechanisms of nitrate sensing by NRT1.1 are considered, and the possible occurrence of other nitrate transceptors is discussed.

Auxin carriers localization drives auxin accumulation in plant cells infected by Frankia in Casuarina glauca actinorhizal nodules.

Actinorhizal symbioses are mutualistic interactions between plants and the soil bacteria Frankia that lead to the formation of nitrogen-fixing root nodules. Little is known about the signaling mechanisms controlling the different steps of the establishment of the symbiosis. The plant hormone auxin has been suggested to play a role. Here we report that auxin accumulates within Frankia-infected cells in actinorhizal nodules of Casuarina glauca. Using a combination of computational modeling and experimental approaches, we establish that this localized auxin accumulation is driven by the cell-specific expression of auxin transporters and by Frankia auxin biosynthesis in planta. Our results indicate that the plant actively restricts auxin accumulation to Frankia-infected cells during the symbiotic interaction.

Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants.

Nitrate is both a nitrogen source for higher plants and a signal molecule regulating their development. In Arabidopsis, the NRT1.1 nitrate transporter is crucial for nitrate signaling governing root growth, and has been proposed to act as a nitrate sensor. However, the sensing mechanism is unknown. Herein we show that NRT1.1 not only transports nitrate but also facilitates uptake of the phytohormone auxin. Moreover, nitrate inhibits NRT1.1-dependent auxin uptake, suggesting that transduction of nitrate signal by NRT1.1 is associated with a modification of auxin transport. Among other effects, auxin stimulates lateral root development. Mutation of NRT1.1 enhances both auxin accumulation in lateral roots and growth of these roots at low, but not high, nitrate concentration. Thus, we propose that NRT1.1 represses lateral root growth at low nitrate availability by promoting basipetal auxin transport out of these roots. This defines a mechanism connecting nutrient and hormone signaling during organ development.

Strain competition and agar affect the interaction of rhizobia with rice.

Competition assays with Sinorhizobium meliloti 1021 and its GFP-labelled pSymA cured and deleted derivatives, SmA818 and SmA146, demonstrated that Sm1021 could still inhibit rice seedling growth even when outnumbered by a large excess of the noninhibitory cured or deleted strain. The wild-type strain Sm1021 also inhibited the growth of its noninhibitory pSymA-cured strain SmA818(gfp) and its pSymA-deleted strain SmA146(gfp) in a manner suggesting that Sm1021 produced a bacteriocin-like substance. The production of, and resistance to, this substance seemed to be pSymA-associated, but it was not the cause of killing in competition experiments on rice, suggesting that the killing of SmA818(gfp) and SmA146(gfp) was medium dependent. The addition of agar in liquid F10 medium at concentrations < or = 0.4% (m/v) abolished the rice growth inhibition of strain Sm1021 and Sm1021(gfp). The increased medium viscosity at higher agar concentrations decreased the diffusion of gases and small molecules through the media. Thus, the low agar concentrations may mimic waterlogged soil conditions leading to the production of inhibitory compounds by the bacterial strains under microaerobic conditions.

Infection process and the interaction of rice roots with rhizobia.

Most rhizobial strains inhibit rice root growth in the presence of calcium or potassium nitrates, but not ammonium nitrate. Certain rhizobial strains, however, such as strain R4, do not inhibit rice growth and can enter rice roots and multiply in the intercellular spaces. By using the green fluorescent protein (GFP) as a visual marker, it was found that Rhizobium became intimately associated with rice seedling roots within 24-48 h. During this initial period it was observed that strain R4 could cause structural changes resembling infection threads within the rice root hairs. Generally, the sites of the emerging lateral roots provide a temporary entry point for rhizobia, either by root hair entry or crack entry. All tested GFP-labelled Rhizobium strains infected the root hairs near the base of growing lateral roots. This study suggests that some strains may have the ability to infect rice root tissues via root hairs located at the emerging lateral roots and to spread extensively throughout the rice root.