An Alternative Mode of Early Land Plant Colonization by Putative Endomycorrhizal Fungi

Rhizomatous axes of Nothia aphylla, a land plant from the 400-myr-old Rhynie chert, host a fungus that closely resembles Glomites rhyniensis (Glomeromycota), the endomycorrhizal fungus of the Rhynie chert plant Aglaophyton major. However, G. rhyniensis is an intercellular endophyte that becomes intracellular exclusively within a well-defined region of the cortex, while the fungus in N. aphylla initially is intracellular but later becomes intercellular in the cortex. We hypothesize that N. aphylla displays an alternative mode of colonization by endomycorrhizal fungi, perhaps related to the peculiar internal anatomy of the lower portion of the rhizomatous axis, in which the radial arrangement of cells, along with the virtual absence of intercellular spaces, provides no intercellular infection pathway into the cortex.Key Words: Aglaophyton major, endomycorrhiza, Glomeromycota, Nothia aphylla, Early Devonian, Rhynie chertThe Early Devonian (c. 400 Ma) Rhynie chert is an in situ silicified hot springs environment that has become significant in our understanding of the complexity of life in early terrestrial ecosystems because of the extraordinary preservation of plants, animals, and microorganisms.¹ Moreover, various associations and interactions between different organisms can be directly examined,² including the earliest fossil examples of arbuscular endomycorrhizae.^3,4 The Rhynie chert land plant Aglaophyton major is characterized by arching, stomatiferous prostrate axes that grow along the substrate surface, and form rhizoid-bearing bulges, usually around stomata, upon contact with the substrate. Extramatrical hyphae of the mycorrhizal fungal enter the axes through these stomata, and spread out through the intercellular system of the hypodermis and cortex, subsequently penetrating individual cells within a well-defined region of the cortex (i.e., the mycorrhizal arbuscule-zone) to form arbuscules.⁴A recently published study⁵ reports on three fungal endophytes that (co-)occur in the Rhynie chert plant Nothia aphylla. This plant consists of upright aerial axes arising from a system of non-stomatiferous, subterranean rhizomatous axes characterized by a prominent ventral rhizoidal ridge.^6,7 The rhizoidal ridge, which is unique among Rhynie chert land plants, consists of a rhizoid-bearing epidermis, a multi-layered hypodermis, files of parenchyma cells that connect to the stele, and extra-stelar conducting elements (Fig. 1A); intercellular spaces are virtually absent.Open in a separate windowFigure 1Nothia aphylla from the Lower Devonian Rhynie chert. (A) Ventral portion of a rhizomatous axis with rhizoidal ridge (cross section); bar = 250 µm. (B) Fungal hypha [arrows] entering the axis through a rhizoid; bar = 30 µm. (C) Sheathed intracellular hyphae [arrows] in hypodermal cells (transverse section); bar = 20 µm. (D) Intercellular hyphae and vesicles in the cortex (longitudinal section); bar scale = 50 µm. (E) Hyphae, vesicles and a thick-walled spore in the cortex (longitudinal section); bar = 100 µm. All images from the original paper; reproduced with permission.One of the fungal endophytes in N. aphylla closely resembles Glomites rhyniensis (Glomeromycota), the endomycorrhizal fungus of A. major.⁴ In N. aphylla, this fungus occurs as an intracellular endophyte in rhizoids and tissues of the rhizoidal ridge. Moreover, it is abundant in the intercellular system of the cortex of both prostrate and proximal portions of aerial axes. The fungus enters the axes through rhizoids (Fig. 1B). Once in the hypodermis, hyphae become sheathed by cell wall material (Fig. 1C). In the cortex, the fungus produces intercellular vesicles (Fig. 1D) and thick-walled spores (Fig. 1E). Based on the presence of vesicles that are similar to those of G. rhyniensis, and spores like those in extant Glomeromycota, we hypothesize that this fungus is an endomycorrhizal member of the Glomeromycota; however, arbuscules have not been observed to date.If this interpretation is accurate, N. aphylla displays an alternative pattern of colonization by endomycorrhizal fungi. Although the morphology of the fungus and distribution in N. aphylla correspond to that of G. rhyniensis in A. major, the infection pathway is distinctly different. While G. rhyniensis is an intercellular endophyte that penetrates individual cells exclusively within the mycorrhizal arbuscule-zone,⁴ the fungus of N. aphylla enters the plant as an intracellular endophyte, and remains intracellular until it reaches the cortex. The host plant apparently does not respond to the invading fungus because infected rhizoids are not altered morphologically. Once in the hypodermis, however, hyphae become separated from the host cell protoplast. This feature suggests a shift from (i) uncontrolled intracellular occurrence of the fungus in the rhizoids, to (ii) controlled intracellular occurrence in the rhizoidal ridge, to (iii) intercellular occurrence in the cortex.The fact that the rhizomatous axes of N. aphylla are subterranean, along with the peculiar internal anatomy of the rhizoidal ridge, may have provided the selective pressure for an alternative mode of colonization by endomycorrhizal fungi. The fungus probably enters the plant through rhizoids because the axes are non-stomatiferous. Moreover, the morphology and radial arrangement of cells in the rhizoidal ridge, along with the virtual absence of intercellular spaces, perhaps does not provide an intercellular infection pathway into the cortex. We speculate that N. aphylla tolerated intracellular penetration in the rhizoids and within the tissues of the rhizoidal ridge in order to become inoculated. Tolerating (or even facilitating) intracellular penetration within a limited area of the axis may simultaneously have provided the plant with a means of recognizing and subsequently distinguishing the endomycorrhizal fungus from potentially harmful parasites (e.g., by surface features of the hyphae or chemical signals). Once recognized, the endomycorrhizal fungi become sheathed and “guided” through the ridge without being able to extract nutrients from the host, and into the cortex where intracellular penetration is not longer possible. The parasites, once recognized, are confined in the tissues of the rhizoidal ridge by specific or unspecific host responses, e.g., secondarily thickened cell walls.⁵ Conversely, if the endomycorrhizal fungus entered the plant through surface openings, and spread out exclusively through the intercellular system, the mechanisms that might confine simultaneous parasite infections were probably much more limited.Endomycorrhizal relationships are believed to have evolved from parasitic interactions.⁸ It has been postulated that modern enodomycorrhizal fungi in some way control parasites because both compete for the same resources.⁹ It may be that, during the evolution of fungal endophytism, the initial benefits of mycorrhizae included protection of the host from pathogenic fungi.¹⁰ Nothia aphylla from the Lower Devonian Rhynie chert adds support to this hypothesis, and may demonstrate that more than a single pattern of colonization by endomycorrhizal fungi occurred during the early evolution of land plants.     

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

Background

Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

Results

As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K_d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.

Conclusion

Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.     

Inhibiting alternative pathway complement activation by targeting the factor D exosite

By virtue of its amplifying property, the alternative complement pathway has been implicated in a number of inflammatory diseases and constitutes an attractive therapeutic target. An anti-factor D Fab fragment (AFD) was generated to inhibit the alternative complement pathway in advanced dry age-related macular degeneration. AFD potently prevented factor D (FD)-mediated proteolytic activation of its macromolecular substrate C3bB, but not proteolysis of a small synthetic substrate, indicating that AFD did not block access of the substrate to the catalytic site. The crystal structures of AFD in complex with human and cynomolgus FD (at 2.4 and 2.3 Å, respectively) revealed the molecular details of the inhibitory mechanism. The structures show that the AFD-binding site includes surface loops of FD that form part of the FD exosite. Thus, AFD inhibits FD proteolytic function by interfering with macromolecular substrate access rather than by inhibiting FD catalysis, providing the molecular basis of AFD-mediated inhibition of a rate-limiting step in the alternative complement pathway.     

Evolutionarily conserved paired immunoglobulin-like receptor α (PILRα) domain mediates its interaction with diverse sialylated ligands

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ～22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.     

Endophytic cyanobacteria in a 400-million-yr-old land plant: A scenario for the origin of a symbiosis?

Direct evidence for the origin and evolution of land plant/cyanobacterial symbioses is virtually absent from the fossil record. Here we report on rare occurrences of prostrate mycorrhizal axes of the Early Devonian land plant Aglaophyton major that host a filamentous cyanobacterium, which enters the plant through the stomata and colonizes the substomatal chambers and intercellular spaces in the outer cortex. In dead ends of the intercellular system, the filaments form loops and continue growth in reverse direction. Some filaments penetrate parenchyma cells close to and within the mycorrhizal arbuscule-zone and form intracellular coils. This discovery represents the earliest direct evidence for cyanobacteria growing inside land plants, and offers a model for the types of associations that may have preceded the evolution of mutualistic land plant/cyanobacterial symbioses.     

The small regulatory RNA molecule MicA is involved in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium biofilm formation

Background  

LuxS is the synthase enzyme of the quorum sensing signal AI-2. In Salmonella Typhimurium, it was previously shown that a luxS deletion mutant is impaired in biofilm formation. However, this phenotype could not be complemented by extracellular addition of quorum sensing signal molecules.     

Functional characterisation of the regulation of CAAT enhancer binding protein alpha by GSK-3 phosphorylation of Threonines 222/226

Background  

Glycogen Synthase Kinase-3 (GSK3) activity is repressed following insulin treatment of cells. Pharmacological inhibition of GSK3 mimics the effect of insulin on Phosphoenolpyruvate Carboxykinase (PEPCK), Glucose-6 Phosphatase (G6Pase) and IGF binding protein-1 (IGFBP1) gene expression. CAAT/enhancer binding protein alpha (C/EBPα) regulates these gene promoters in liver and is phosphorylated on two residues (T222/T226) by GSK3, although the functional outcome of the phosphorylation has not been established. We aimed to establish whether CEBPα is a link between GSK3 and these gene promoters.     

Signaling Defect in the Activation of Caspase-3 and PKCδ in Human TUR Leukemia Cells Is Associated with Resistance to Apoptosis

Exposure of the two related human leukemic cell lines U937 and TUR to chemotherapeutic compounds resulted in opposite effects on induction and resistance to apoptosis. Incubation of U937 cells with 1-β- -arabinofuranosylcytosine or the etoposide VP-16 was accompanied by growth arrest in G₀/G₁of the cell cycle and an accumulation of a population in the sub-G₁phase which exhibited characteristics typical for the apoptotic pathway. In contrast, human TUR leukemia cells demonstrated no significant effects after a similar treatment with Ara-C and VP-16. Thus, TUR cells continued to proliferate in the presence of these anti-cancer drugs and the number of apoptotic cells as evaluated by propidium iodide staining and the detection of internucleosomal DNA fragmentation was significantly reduced when compared to the parental U937 cells. Similar effects were observed upon serum-starvation demonstrating resistance to apoptosis in TUR cells. Whereas induction of apoptosis is regulated by a network of distinct factors including the activation of proteolytically active caspases, we investigated these pathways in both cell lines. U937 cells demonstrated activation of the 32-kDa caspase-3 upon drug treatment by cleavage into the 20-kDa activated form. However, there was no 20-kDa caspase-3 fragment detectable in TUR cells. Simultaneously, the enzymatic activity of caspase-3 was significantly increased in drug-treated U937 cells as measuredin vitroby enhanced metabolization of a fluorescence substrate andin vivoby cleavage of an appropriate substrate for caspase-3, namely, protein kinase Cδ. In contrast, there was little if any caspase-3 activation detectable in drug-treated TUR cells. Taken together, these data suggest a signaling defect in the activation of the caspase-3 proteolytic system in TUR cells upon treatment with chemotherapeutic compounds which is associated with resistance to apoptosis in these human leukemia cells.     

Polarized distribution of Na+, K(+)-ATPase alpha-subunit isoforms in electrocyte membranes

We have previously demonstrated that Na+, K(+)-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the alpha subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of alpha isoforms (alpha1 and alpha2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na+, K(+)-ATPase activity 2.6-fold more sensitive to ouabain (I50=1.0+/-0.1 microM) than the activity of innervated membranes (I50=2.6+/-0.2 microM). As depicted in [3H]ouabain binding experiments, when the [3H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na+, K(+)-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of alpha1 and alpha2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na+, K(+)-ATPase alpha-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.     

Identification of skunk species submitted for rabies testing in the desert southwest

Skunks usually are identified by their common name (skunk) when submitted for rabies testing. In the desert southwest (Arizona, New Mexico, Texas, USA; and northern Mexico), there are five species of skunks; four of which can occur in sympatry. To better understand the ecology of skunk rabies in these areas, it is imperative that species be properly identified. We used the displacement loop (d-loop) of the mitochondrial genome to identify to species 24 skunk brain samples submitted for rabies testing in New Mexico from 2001 to 2002. Most were identified as striped skunks (Mephitis mephitis), but hooded (Mephitis macroura) and hog-nosed (Conepatus leuconotus) skunks were also found.