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Rhizobium bacteria live in soil and plant environments, are capable of inducing symbiotic nodules on legumes, invade these nodules, and develop into bacteroids that fix atmospheric nitrogen into ammonia. Rhizobial lipopolysaccharide (LPS) is anchored in the bacterial outer membrane through a specialized lipid A containing a very long-chain fatty acid (VLCFA). VLCFA function for rhizobial growth in soil and plant environments is not well understood. Two genes, acpXL and lpxXL, encoding acyl carrier protein and acyltransferase, are among the six genes required for biosynthesis and transfer of VLCFA to lipid A. Rhizobium leguminosarum mutant strains acpXL, acpXL - /lpxXL - , and lpxXL - were examined for LPS structure, viability, and symbiosis. Mutations in acpXL and lpxXL abolished VLCFA attachment to lipid A. The acpXL mutant transferred a shorter acyl chain instead of VLCFA. Strains without lpxXL neither added VLCFA nor a shorter acyl chain. In all strains isolated from nodule bacteria, lipid A had longer acyl chains compared with laboratory-cultured bacteria, whereas mutant strains displayed altered membrane properties, modified cationic peptide sensitivity, and diminished levels of cyclic β-glucans. In pea nodules, mutant bacteroids were atypically formed and nitrogen fixation and senescence were affected. The role of VLCFA for rhizobial environmental fitness is discussed.

Recent work has shown that the rhizospheric and phyllospheric microbiomes of plants are composed of highly diverse microbial species. Though the information pertaining to the diversity of the aboveground and belowground microbes associated with plants is known, an understanding of the mechanisms by which these diverse microbes function is still in its infancy. Plants are sessile organisms, that depend upon chemical signals to interact with the microbiota. Of late, the studies related to the impact of microbes on plants have gained much traction in the research literature, supporting diverse functional roles of microbes on plant health. However, how these microbes interact as a community to confer beneficial traits to plants is still poorly understood. Recent advances in the use of "biologicals" as bio-fertilizers and biocontrol agents for sustainable agricultural practices is promising, and a fundamental understanding of how microbes in community work on plants could help this approach be more successful. This review attempts to highlight the importance of different signaling events that mediate a beneficial plant microbe interaction. Fundamental research is needed to understand how plants react to different benign microbes and how these microbes are interacting with each other. This review highlights the importance of chemical signaling, and biochemical and genetic events which determine the efficacy of benign microbes to promote the development of beneficial traits in plants.

Three soybean [Glycine max (L) Merr.] small RNA libraries were generated and sequenced using the Illumina platform to examine the role of miRNAs during soybean nodulation. The small RNA libraries were derived from root hairs inoculated with Bradyrhizobium japonicum (In_RH) or mock-inoculated with water (Un_RH), as well as from the comparable inoculated stripped root samples (i.e. inoculated roots with the root hairs removed). Sequencing of these libraries identified a total of 114 miRNAs, including 22 novel miRNAs. A comparison of miRNA abundance among the 114 miRNAs identified 66 miRNAs that were differentially expressed between root hairs and stripped roots, and 48 miRNAs that were differentially regulated in infected root hairs in response to B. japonicum when compared to uninfected root hairs (P ≤ 0.05). A parallel analysis of RNA ends (PARE) library was constructed and sequenced to reveal a total of 405 soybean miRNA targets, with most predicted to encode transcription factors or proteins involved in protein modification, protein degradation and hormone pathways. The roles of gma-miR4416 and gma-miR2606b during nodulation were further analysed. Ectopic expression of these two miRNAs in soybean roots resulted in significant changes in nodule numbers. miRNA target information suggested that gma-miR2606b regulates a Mannosyl-oligosaccharide 1, 2-alpha-mannosidase gene, while gma-miR4416 regulates the expression of a rhizobium-induced peroxidase 1 (RIP1)-like peroxidase gene, GmRIP1, during nodulation., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Bacillus subtilis, which belongs to the phylum Firmicutes, is the most widely studied Gram-positive model organism. It is found in a wide variety of environments and is particularly abundant in soils and in the gastrointestinal tracts of ruminants and humans. Here, we present the complete genome sequence of the newly described B. subtilis strain UD1022. The UD1022 genome consists of a 4.025-Mbp chromosome, and other major findings from our analysis will provide insights into the genomic basis of it being a plant growth-promoting rhizobacterium (PGPR) with biocontrol potential., (Copyright © 2015 Bishnoi et al.)

We report on a nondestructive clearing technique that enhances transmission of light through specimens from diverse plant species, opening unique opportunities for microscope-enabled plant research. After clearing, plant organs and thick tissue sections are amenable to deep imaging. The clearing method is compatible with immunocytochemistry techniques and can be used in concert with common fluorescent probes, including widely adopted protein tags such as GFP, which has fluorescence that is preserved during the clearing process., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

The impact of nano-TiO₂ on Rhizobium-legume symbiosis was studied using garden peas and the compatible bacterial partner Rhizobium leguminosarum bv. viciae 3841. Exposure to nano-TiO₂ did not affect the germination of peas grown aseptically, nor did it impact the gross root structure. However, nano-TiO₂ exposure did impact plant development by decreasing the number of secondary lateral roots. Cultured R. leguminosarum bv. viciae 3841 was also impacted by exposure to nano-TiO₂, resulting in morphological changes to the bacterial cells. Moreover, the interaction between these two organisms was disrupted by nano-TiO₂ exposure, such that root nodule development and the subsequent onset of nitrogen fixation were delayed. Further, the polysaccharide composition of the walls of infected cells of nodules was altered, suggesting that the exposure induced a systemic response in host plants. Therefore, nano-TiO₂ contamination in the environment is potentially hazardous to the Rhizobium-legume symbiosis system., (© 2013 Elsevier B.V. All rights reserved.)

Medicago truncatula NIP/LATD gene, required for symbiotic nitrogen fixing nodule and root architecture development, encodes a member of the NRT1(PTR) family that demonstrates high-affinity nitrate transport in Xenopus laevis oocytes. Of three Mtnip/latd mutant proteins, one retains high-affinity nitrate transport in oocytes, while the other two are nitrate-transport defective. To further examine the mutant proteins' transport properties, the missense Mtnip/latd alleles were expressed in Arabidopsis thaliana chl1-5, resistant to the herbicide chlorate because of a deletion spanning the nitrate transporter AtNRT1.1(CHL1) gene. Mtnip-3 expression restored chlorate sensitivity in the Atchl1-5 mutant, similar to wild type MtNIP/LATD, while Mtnip-1 expression did not. The high-affinity nitrate transporter AtNRT2.1 gene was expressed in Mtnip-1 mutant roots; it did not complement, which could be caused by several factors. Together, these findings support the hypothesis that MtNIP/LATD may have another biochemical activity.

The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are low-affinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 μm) concentrations than at higher (5 mm) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1.1 gene in mutant Mtnip-1 roots partially rescued Mtnip-1 for root architecture defects but not for nodulation defects. This suggests that the spectrum of activities inherent in AtNRT1.1 is different from that possessed by MtNIP/LATD, but it could also reflect stability differences of each protein in M. truncatula. Collectively, the data show that MtNIP/LATD is a high-affinity nitrate transporter and suggest that it could have another function.

Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those that are deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide (nt) "phased" intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs (phasiRNAs) found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt microRNA (miRNA) families that target conserved domains in these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of small RNAs were matched to >60% of all ∼540 encoded Medicago NB-LRRs; in the potato, a model for mycorrhizal interactions, phasiRNAs were also produced from NB-LRRs. DCL2 and SGS3 transcripts were also cleaved by these 22-nt miRNAs, generating phasiRNAs, suggesting synchronization between silencing and pathogen defense pathways. In addition, a new example of apparent "two-hit" phasiRNA processing was identified. Our data reveal complex tasiRNA-based regulation of NB-LRRs that potentially evolved to facilitate symbiotic interactions and demonstrate miRNAs as master regulators of a large gene family via the targeting of highly conserved, protein-coding motifs, a new paradigm for miRNA function.

Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species. Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing ∼94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa's genomic toolbox.

Rhizobium leguminosarum is a Gram-negative bacterium that forms nitrogen-fixing symbioses with compatible leguminous plants via intracellular invasion and establishes a persistent infection within host membrane-derived subcellular compartments. Notably, an unusual very-long-chain fatty acid (VLCFA) is found in the lipid A of R. leguminosarum as well as in the lipid A of the medically relevant pathogens Brucella abortus, Brucella melitensis, Bartonella henselae, and Legionella pneumophila, which are also able to persist within intracellular host-derived membranes. These bacterial symbionts and pathogens each contain a homologous gene region necessary for the synthesis and transfer of the VLCFA to the lipid A. Within this region lies a gene that encodes the specialized acyl carrier protein AcpXL, on which the VLCFA is built. This study describes the biochemical and infection phenotypes of an acpXL mutant which lacks the VLCFA. The mutation was created in R. leguminosarum bv. phaseoli strain 8002, which forms symbiosis with Phaseolus vulgaris, a determinate nodulating legume. Structural analysis using gas chromatography and mass spectrometry revealed that the mutant lipid A lacked the VLCFA. Compared to the parent strain, the mutant was more sensitive to the detergents deoxycholate and dodecyl sulfate and the antimicrobial peptide polymyxin B, suggesting a compromise to membrane stability. In addition, the mutant was more sensitive to higher salt concentrations. Passage through the plant restored salt tolerance. Electron microscopic examination showed that the mutant was developmentally delayed during symbiotic infection of the host plant Phaseolus vulgaris and produced abnormal symbiosome structures., (Copyright © 2011, American Society for Microbiology. All Rights Reserved.)

Legume root architecture involves not only elaboration of the root system by the formation of lateral roots but also the formation of symbiotic root nodules in association with nitrogen-fixing soil rhizobia. The Medicago truncatula LATD/NIP gene plays an essential role in the development of both primary and lateral roots as well as nodule development. We have cloned the LATD/NIP gene and show that it encodes a member of the NRT1(PTR) transporter family. LATD/NIP is expressed throughout the plant. pLATD/NIP-GFP promoter-reporter fusions in transgenic roots establish the spatial expression of LATD/NIP in primary root, lateral root and nodule meristems and the surrounding cells. Expression of LATD/NIP is regulated by hormones, in particular by abscisic acid which has been previously shown to rescue the primary and lateral root meristem arrest of latd mutants. latd mutants respond normally to ammonium but have defects in responses of the root architecture to nitrate. Taken together, these results suggest that LATD/NIP may encode a nitrate transporter or transporter of another compound.

Background: Small non-coding RNAs (21 to 24 nucleotides) regulate a number of developmental processes in plants and animals by silencing genes using multiple mechanisms. Among these, the most conserved classes are microRNAs (miRNAs) and small interfering RNAs (siRNAs), both of which are produced by RNase III-like enzymes called Dicers. Many plant miRNAs play critical roles in nutrient homeostasis, developmental processes, abiotic stress and pathogen responses. Currently, only 70 miRNA have been identified in soybean., Methods: We utilized Illumina's SBS sequencing technology to generate high-quality small RNA (sRNA) data from four soybean (Glycine max) tissues, including root, seed, flower, and nodules, to expand the collection of currently known soybean miRNAs. We developed a bioinformatics pipeline using in-house scripts and publicly available structure prediction tools to differentiate the authentic mature miRNA sequences from other sRNAs and short RNA fragments represented in the public sequencing data., Results: The combined sequencing and bioinformatics analyses identified 129 miRNAs based on hairpin secondary structure features in the predicted precursors. Out of these, 42 miRNAs matched known miRNAs in soybean or other species, while 87 novel miRNAs were identified. We also predicted the putative target genes of all identified miRNAs with computational methods and verified the predicted cleavage sites in vivo for a subset of these targets using the 5' RACE method. Finally, we also studied the relationship between the abundance of miRNA and that of the respective target genes by comparison to Solexa cDNA sequencing data., Conclusion: Our study significantly increased the number of miRNAs known to be expressed in soybean. The bioinformatics analysis provided insight on regulation patterns between the miRNAs and their predicted target genes expression. We also deposited the data in a soybean genome browser based on the UCSC Genome Browser architecture. Using the browser, we annotated the soybean data with miRNA sequences from four tissues and cDNA sequencing data. Overlaying these two datasets in the browser allows researchers to analyze the miRNA expression levels relative to that of the associated target genes. The browser can be accessed at http://digbio.missouri.edu/soybean_mirna/.

In Medicago truncatula nodules, the soil bacterium Sinorhizobium meliloti reduces atmospheric dinitrogen into nitrogenous compounds that the legume uses for its own growth. In nitrogen-fixing nodules, each infected cell contains symbiosomes, which include the rhizobial cell, the symbiosome membrane surrounding it, and the matrix between the bacterium and the symbiosome membrane, termed the symbiosome space. Here, we describe the localization of ENOD8, a nodule-specific esterase. The onset of ENOD8 expression occurs at 4 to 5 days postinoculation, before the genes that support the nitrogen fixation capabilities of the nodule. Expression of an ENOD8 promoter-gusA fusion in nodulated hairy roots of composite transformed M. truncatula plants indicated that ENOD8 is expressed from the proximal end of interzone II to III to the proximal end of the nodules. Confocal immunomicroscopy using an ENOD8-specific antibody showed that the ENOD8 protein was detected in the same zones. ENOD8 protein was localized in the symbiosome membrane or symbiosome space around the bacteroids in the infected nodule cells. Immunoblot analysis of fractionated symbiosomes strongly suggested that ENOD8 protein was found in the symbiosome membrane and symbiosome space, but not in the bacteroid. Determining the localization of ENOD8 protein in the symbiosome is a first step in understanding its role in symbiosome membrane and space during nodule formation and function.

Symbiotic association of legume plants with rhizobia bacteria culminates in organogenesis of nitrogen-fixing root nodules. In indeterminate nodules, plant cells accommodate rhizobial infection by enclosing each bacterium in a membrane-bound, organelle-like compartment called the symbiosome. Numerous symbiosomes occupy each nodule cell; therefore an enormous amount of membrane material must be delivered to the symbiosome membrane for its development and maintenance. Protein delivery to the symbiosome is thought to rely on the plant secretory system; however, the targeting mechanisms are not well understood. In this study, we report the first in-depth analysis of a syntaxin localized on symbiosome membranes. Syntaxins help define a biochemical identity to each compartment in the plant secretory system and facilitate vesicle docking and fusion. Here, we present biochemical and cytological evidence that the SNARE MtSYP132, a Medicago truncatula homologue of Arabidopsis thaliana Syntaxin of Plants 132, localizes to the symbiosome membrane. Using a specific anti-MtSYP132 peptide antibody, we also show that MtSYP132 localizes to the plasma membrane surrounding infection threads and is most abundant on the infection droplet membrane. These results indicate that MtSYP132 may function in infection thread development or growth and the early stages of symbiosome formation.

Legume rhizobia symbiotic nitrogen (N2) fixation plays a critical role in sustainable nitrogen management in agriculture and in the Earth's nitrogen cycle. Signaling between rhizobia and legumes initiates development of a unique plant organ, the root nodule, where bacteria undergo endocytosis and become surrounded by a plant membrane to form a symbiosome. Between this membrane and the encased bacteria exists a matrix-filled space (the symbiosome space) that is thought to contain a mixture of plant- and bacteria-derived proteins. Maintenance of the symbiosis state requires continuous communication between the plant and bacterial partners. Here, we show in the model legume Medicago truncatula that a novel family of six calmodulin-like proteins (CaMLs), expressed specifically in root nodules, are localized within the symbiosome space. All six nodule-specific CaML genes are clustered in the M. truncatula genome, along with two other nodule-specific genes, nodulin-22 and nodulin-25. Sequence comparisons and phylogenetic analysis suggest that an unequal recombination event occurred between nodulin-25 and a nearby calmodulin, which gave rise to the first CaML, and the gene family evolved by tandem duplication and divergence. The data provide striking evidence for the recruitment of a ubiquitous Ca(2+)-binding gene for symbiotic purposes.

Members of the Rhizobiaceae contain 27-hydroxyoctacosanoic acid (27OHC(28:0)) in their lipid A. A Rhizobium leguminosarum 3841 acpXL mutant (named here Rlv22) lacking a functional specialized acyl carrier lacked 27OHC(28:0) in its lipid A, had altered growth and physiological properties (e.g., it was unable to grow in the presence of an elevated salt concentration [0.5% NaCl]), and formed irregularly shaped bacteroids, and the synchronous division of this mutant and the host plant-derived symbiosome membrane was disrupted. In spite of these defects, the mutant was able to persist within the root nodule cells and eventually form, albeit inefficiently, nitrogen-fixing bacteroids. This result suggested that while it is in a host root nodule, the mutant may have some mechanism by which it adapts to the loss of 27OHC(28:0) from its lipid A. In order to further define the function of this fatty acyl residue, it was necessary to examine the lipid A isolated from mutant bacteroids. In this report we show that addition of 27OHC(28:0) to the lipid A of Rlv22 lipopolysaccharides is partially restored in Rlv22 acpXL mutant bacteroids. We hypothesize that R. leguminosarum bv. viciae 3841 contains an alternate mechanism (e.g., another acp gene) for the synthesis of 27OHC(28:0), which is activated when the bacteria are in the nodule environment, and that it is this alternative mechanism which functionally replaces acpXL and is responsible for the synthesis of 27OHC(28:0)-containing lipid A in the Rlv22 acpXL bacteroids.

Nodulins encoding repetitive proline-rich cell wall proteins (PRPs) are induced during early interactions with rhizobia, suggesting a massive restructuring of the plant extracellular matrix during infection and nodulation. However, the proteins corresponding to these gene products have not been isolated or characterized, nor have cell wall localizations been confirmed. Posttranslational modifications, conformation, and interactions with other wall polymers are difficult to predict on the basis of only the deduced amino acid sequence of PRPs. PsENOD2 is expressed in nodule parenchyma tissue during nodule organogenesis and encodes a protein with distinctive PRP motifs that are rich in glutamate and basic amino acids. A database search for the ENOD2 signature motifs indicates that similar proteins may have a limited phylogenetic distribution, as they are presently only known from legumes. To determine the ultrastructural location of the proteins, antibodies were raised against unique motifs from the predicted ENOD2 sequence. The antibodies recognized nodule-specific proteins in pea (Pisum sativum), with a major band detected at 110 kDa, representing a subset of PRPs from nodules. The protein was detected specifically in organelles of the secretory pathway and intercellular spaces in the nodule parenchyma, but it was not abundant in primary walls. Similar proteins with an analogous distribution were detected in soybean (Glycine max). The use of polyclonal antibodies raised against signature motifs of extracellular matrix proteins thus appears to be an effective strategy to identify and isolate specific structural proteins for functional analysis.

The trafficking and function of cell surface proteins in eukaryotic cells may require association with detergent-resistant sphingolipid- and sterol-rich membrane domains. The aim of this work was to obtain evidence for lipid domain phenomena in plant membranes. A protocol to prepare Triton X-100 detergent-resistant membranes (DRMs) was developed using Arabidopsis (Arabidopsis thaliana) callus membranes. A comparative proteomics approach using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry revealed that the DRMs were highly enriched in specific proteins. They included eight glycosylphosphatidylinositol-anchored proteins, several plasma membrane (PM) ATPases, multidrug resistance proteins, and proteins of the stomatin/prohibitin/hypersensitive response family, suggesting that the DRMs originated from PM domains. We also identified a plant homolog of flotillin, a major mammalian DRM protein, suggesting a conserved role for this protein in lipid domain phenomena in eukaryotic cells. Lipid analysis by gas chromatography-mass spectrometry showed that the DRMs had a 4-fold higher sterol-to-protein content than the average for Arabidopsis membranes. The DRMs were also 5-fold increased in sphingolipid-to-protein ratio. Our results indicate that the preparation of DRMs can yield a very specific set of membrane proteins and suggest that the PM contains phytosterol and sphingolipid-rich lipid domains with a specialized protein composition. Our results also suggest a conserved role of lipid modification in targeting proteins to both the intracellular and extracellular leaflet of these domains. The proteins associated with these domains provide important new experimental avenues into understanding plant cell polarity and cell surface processes.

To investigate the legume-Rhizobium symbiosis, we isolated and studied a novel symbiotic mutant of the model legume Medicago truncatula, designated nip (numerous infections and polyphenolics). When grown on nitrogen-free media in the presence of the compatible bacterium Sinorhizobium meliloti, the nip mutant showed nitrogen deficiency symptoms. The mutant failed to form pink nitrogen-fixing nodules that occur in the wild-type symbiosis, but instead developed small bump-like nodules on its roots that were blocked at an early stage of development. Examination of the nip nodules by light microscopy after staining with X-Gal for S. meliloti expressing a constitutive GUS gene, by confocal microscopy following staining with SYTO-13, and by electron microscopy revealed that nip initiated symbiotic interactions and formed nodule primordia and infection threads. The infection threads in nip proliferated abnormally and very rarely deposited rhizobia into plant host cells; rhizobia failed to differentiate further in these cases. nip nodules contained autofluorescent cells and accumulated a brown pigment. Histochemical staining of nip nodules revealed this pigment to be polyphenolic accumulation. RNA blot analyses demonstrated that nip nodules expressed only a subset of genes associated with nodule organogenesis, as well as elevated expression of a host defense-associated phenylalanine ammonia lyase gene. nip plants were observed to have abnormal lateral roots. nip plant root growth and nodulation responded normally to ethylene inhibitors and precursors. Allelism tests showed that nip complements 14 other M. truncatula nodulation mutants but not latd, a mutant with a more severe nodulation phenotype as well as primary and lateral root defects. Thus, the nip mutant defines a new locus, NIP, required for appropriate infection thread development during invasion of the nascent nodule by rhizobia, normal lateral root elongation, and normal regulation of host defense-like responses during symbiotic interactions.

•  A rapid method for detailed analysis of nodule formation has been developed. •  Inoculated root tissues were stained with SYTO 13, a cell-permeant fluorescent nucleic acid-binding dye, and visualized using confocal laser scanning microscopy (CLSM). Structures with high concentrations of DNA and RNA, such as plant cell nuclei and bacteria, labeled strongly. The autofluorescent properties of cell walls made it possible to use CLSM to visualize both plant and rhizobial structures and generate a three-dimensional reconstruction of the root and invading bacteria. •  This method allowed clear observation of stages and structures important in nodule formation, such as rhizobial attachment to root hairs, hair deformation, infection thread ramification, nodule primordium development and nodule cell invasion. Bacteroid structures were easily assessed without the need for fixation that might alter cellular integrity. Plant nodulation mutants with phenotypic differences in thread growth, cellular invasion and plant defense response were also documented. •  Multiple samples can be assessed using detailed microscopy without the need for extensive preparative work, labor-intensive analysis, or the generation of genetically modified samples.

Lipopolysaccharides from pea-nodulating strain Rhizobium leguminosarum by. viciae 3841, as all other members of the family Rhizobiaceae with the possible exception of Azorhizobium caulinodans, contains a very long chain fatty acid; 27-hydroxyoctacosanoic acid (27OHC28:0) in its lipid A region. The exact function and importance of this residue, however, is not known. In this work, a previously constructed mutant, Rhizobium leguminosarum by. viciae 22, deficient in the fatty acid residue, was analyzed for its symbiotic phenotype. While the mutant was able to form nitrogen-fixing nodules, a detailed study of the timing and efficiency of nodulation using light and electron microscopy showed that there was a delay in the onset of nodulation and nodule tissue invasion. Further, microscopy showed that the mutant was unable to differentiate normally forming numerous irregularly shaped bacteroids, that the resultant mature bacteroids were unusually large, and that several bacteroids were frequently enclosed in a single symbiosome membrane, a feature not observed with parent bacteroids. In addition, the mutant nodules were delayed in the onset of nitrogenase production and showed reduced nitrogenase throughout the testing period. These results imply that the lack of 27OHC28:0 in the lipid A in mutant bacteroids results in altered membrane properties that are essential for the development of normal bacteroids.

The symbiosome membrane represents a specialized plant membrane that forms both a structural and a functional interface between the legume plant and its bacterial counterpart. In this study, the symbiosome membrane protein profile from the model system Medicago truncatula and the corresponding bacterium Sinorhizobium meliloti was examined using two-dimensional electrophoresis and microcapillary high-performance liquid chromatography (HPLC) tandem mass spectrometry. The identities of 51 proteins were obtained and these proteins were categorized into functional classes to indicate biochemical roles. Symbiosome membrane proteins include an H(+)-ATPase, ENOD16, ENOD8, nodulin-25, BiP, HSP70, PDI, multifunctional aquaporin, a putative syntaxin, and other proteins of known and unknown identity and function. The majority of the proteins identified were involved with protein destination and storage. These results allow us to understand better the biochemical composition of the symbiosome membrane and thus provide a basis to hypothesize mechanisms of symbiosome membrane formation and function.

The structure of the lipid A from Rhizobium etli and Rhizobium leguminosarum lipopolysaccharides (LPSs) lacks phosphate and contains a galacturonosyl residue at its 4' position, an acylated 2-aminogluconate in place of the proximal glucosamine, and a very long chain omega-1 hydroxy fatty acid, 27-hydroxyoctacosanoic acid (27OHC28:0). The 27OHC28:0 moiety is common in lipid A's among members of the Rhizobiaceae and also among a number of the facultative intracellular pathogens that form chronic infections, e.g., Brucella abortus, Bartonella henselae, and Legionella pneumophila. In this paper, a mutant of R. leguminosarum was created by placing a kanamycin resistance cassette within acpXL, the gene which encodes the acyl carrier protein for 27OHC28:0. The result was an LPS containing a tetraacylated lipid A lacking 27OHC28:0. A small amount of the mutant lipid A may contain an added palmitic acid residue. The mutant is sensitive to changes in osmolarity and an increase in acidity, growth conditions that likely occur in the nodule microenvironment. In spite of the probably hostile microenvironment of the nodule, the acpXL mutant is still able to form nitrogen-fixing root nodules even though the appearance and development of nodules are delayed. Therefore, it is possible that the acpXL mutant has a host-inducible mechanism which enables it to adapt to these physiological changes.

Glycosylphosphatidylinositol (GPI) anchoring of proteins provides a potential mechanism for targeting to the plant plasma membrane and cell wall. However, relatively few such proteins have been identified. Here, we develop a procedure for database analysis to identify GPI-anchored proteins (GAP) based on their possession of common features. In a comprehensive search of the annotated Arabidopsis genome, we identified 167 novel putative GAP in addition to the 43 previously described candidates. Many of these 210 proteins show similarity to characterized cell surface proteins. The predicted GAP include homologs of beta-1,3-glucanases (16), metallo- and aspartyl proteases (13), glycerophosphodiesterases (6), phytocyanins (25), multi-copper oxidases (2), extensins (6), plasma membrane receptors (19), and lipid-transfer-proteins (18). Classical arabinogalactan (AG) proteins (13), AG peptides (9), fasciclin-like proteins (20), COBRA and 10 homologs, and novel potential signaling peptides that we name GAPEPs (8) were also identified. A further 34 proteins of unknown function were predicted to be GPI anchored. A surprising finding was that over 40% of the proteins identified here have probable AG glycosylation modules, suggesting that AG glycosylation of cell surface proteins is widespread. This analysis shows that GPI anchoring is likely to be a major modification in plants that is used to target a specific subset of proteins to the cell surface for extracellular matrix remodeling and signaling.

We introduce the use of Arabidopsis thaliana callus culture as a system for proteomic analysis of plant organelles using liquid-grown callus. This callus is relatively homogeneous, reproducible and cytoplasmically rich, and provides organelles in sufficient quantities for proteomic studies. A database was generated of mitochondrial, endoplasmic reticulum (ER), Golgi/prevacuolar compartment and plasma membrane (PM) markers using two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) and peptide sequencing or mass spectrometric methods. The major callus membrane-associated proteins were characterised as being integral or peripheral by Triton X-114 phase partitioning. The database was used to define specific proteins at the Arabidopsis callus plasma membrane. This database of organelle proteins provides the basis for future characterisation of the expression and localisation of novel plant proteins.

Remodeling of the plant cell surface occurs during the establishment of cell polarity, cellular differentiation, and organ development. This report demonstrates the existence of multiple glycosylphosphatidylinositol (GPI)-anchored proteins in the model plant Arabidopsis. Using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we also show that GPI-anchored proteins are a relatively abundant class of protein and that they are present at the plant plasma membrane. Furthermore, some of these proteins are released into the extracellular matrix. At least one of these is an arabinogalactan protein (AGP), a class of proteins known to be associated with cellular differentiation. Analysis of the amino acid sequences of two novel AGP-like proteins from Arabidopsis predicts that these proteins contain consensus signals for GPI-anchor addition. These findings support a model where GPI-anchored proteins are involved in the generation of specialized cell surfaces and extracellular signaling molecules.

Glycosyltransferases in the Golgi apparatus synthesize cell wall polysaccharides and elaborate the complex glycans of glycoproteins. To investigate the targeting of this type of enzyme to plant Golgi compartments, we generated transgenic Arabidopsis plants expressing alpha-2,6-sialyltransferase, a glycosyltransferase of the mammalian trans-Golgi cisternae and the trans-Golgi network. Biochemical analysis as well as immunolight and immunoelectron microscopy of these plants indicate that the protein is targeted specifically to the Golgi apparatus. Moreover, the protein is predominantly localized to the cisternae and membranes of the trans side of the organelle. When supplied with the appropriate substrates, the enzyme has significant alpha-2,6-sialyltransferase activity. These results indicate a conservation of glycosyltransferase targeting mechanisms between plant and mammalian cells and also demonstrate that glycosyltransferases can be subcompartmentalized to specific cisternae of the plant Golgi apparatus.

The plant Golgi apparatus has an important role in protein glycosylation and sorting, but is also a major biosynthetic organelle that synthesises large quantities of cell wall polysaccharides. This is reflected in the organisation of the Golgi apparatus as numerous individual stacks of cisternae that are dispersed through the cell. Each stack is polarised: the shape of the cisternae and the staining of the membranes change in a cis to trans direction, and the cisternae on the trans side contain more polysaccharides. Numerous glycosyltransferases are required for the synthesis of the complex cell wall polysaccharides. Microscopy and biochemical fractionation studies suggest that these enzymes are compartmentalised within the stack. Although there is no obvious cis Golgi network, the trans-most cisterna or trans Golgi network often buds clathrin-coated and sometimes smooth dense vesicles as well. Here, vacuolar proteins are sorted from the secreted proteins and polysaccharides. This review highlights unique aspects of the organisation and function of the plant Golgi apparatus. Fundamentally similar processes probably underlie Golgi organisation in all organisms, and consideration of the plant Golgi specialisations can therefore be generally informative, as well as being of central importance to plant cell biology.

A 27-kD glycoprotein antigen recognized by monoclonal antibody MAC266 was purified from isolated symbiosomes derived from pea (Pisum sativum) root nodules containing Rhizobium. The N-terminal amino acid sequence was obtained, and the corresponding cDNA clone was isolated by a polymerase chain reaction-based strategy. The clone contained a single open reading frame, and the gene was termed PsNlec1. Phylogenetic analysis of 31 legume sequences showed that the PsNlec1 protein is related to the legume lectin family but belongs to a subgroup that is very different from pea seed lectin. Expression of the PsNlec1 transcript was much stronger in nodules than in other parts of the plant. It was found in both infected and uninfected cells in the central tissue of the nodule and in the stele of the root near the attachment point of the nodule. When uninfected pea seedlings were grown on medium containing nitrate, weak transcription of PsNlec1 was observed in the root system. The identification of PsNlec1 inside the symbiosome is consistent with the observation that legume lectins are generally vacuolar proteins that may serve as transient storage components.

Root nodules on peanut (Arachis hypogaea L.) accumulate a galactose/lactose-binding lectin that is similar, but not identical, to the major seed lectin in peanut. The function of the peanut nodule lectin (PNL) is not known. In the current study, we have investigated the location of lectin in the nodule using immunogold labeling and enzyme-linked immunosorbant assays (ELISA). Lectin was most abundant in the nodule parenchyma, where it accumulated in vacuoles, suggesting a possible role as a vegetative storage protein. Lectin was also detected in the extracellular matrix in the nodule parenchyma, a location that corresponds to the tissue layer forming a barrier to oxygen diffusion. The potential for interactions between PNL and other cell wall components, including a previously described high-molecular weight glycoprotein that co-localizes with PNL, is discussed. Within infected cells, lectin was not detectable by immunogold labeling within the cytoplasm, but light labeling was suggestive of lectin localization within the symbiosome lumen. Analysis of fractionated symbiosomes by the more sensitive ELISA technique confirmed that lectin was present within the symbiosome, but was not bound to bacteroids. Our results indicate that PNL probably plays several roles in this nitrogen-fixing symbiosis.