Pectin modifications promote haustoria development in the parasitic plant Phtheirospermum japonicum.

Parasitic plants are globally prevalent pathogens with important ecological functions but also potentially devastating agricultural consequences. Common to all parasites is the formation of the haustorium which requires parasite organ development and tissue invasion into the host. Both processes involve cell wall modifications. Here, we investigated a role for pectins during haustorium development in the facultative parasitic plant Phtheirospermum japonicum. Using transcriptomics data from infected Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), we identified genes for multiple P. japonicum pectin methylesterases (PMEs) and their inhibitors (PMEIs) whose expression was upregulated by haustoria formation. Changes in PME and PMEI expression were associated with tissue-specific modifications in pectin methylesterification. While de-methylesterified pectins were present in outer haustorial cells, highly methylesterified pectins were present in inner vascular tissues, including the xylem bridge that connects parasite to host. Specifically blocking xylem bridge formation in the haustoria inhibited several PME and PMEI genes from activating. Similarly, inhibiting PME activity using chemicals or by overexpressing PMEI genes delayed haustoria development. Our results suggest a dynamic and tissue-specific regulation of pectin contributes to haustoria initiation and to the establishment of xylem connections between parasite and host.

Nitrogen represses haustoria formation through abscisic acid in the parasitic plant Phtheirospermum japonicum.

Parasitic plants are globally prevalent pathogens that withdraw nutrients from their host plants using an organ known as the haustorium. The external environment including nutrient availability affects the extent of parasitism and to understand this phenomenon, we investigated the role of nutrients and found that nitrogen is sufficient to repress haustoria formation in the root parasite Phtheirospermum japonicum. Nitrogen increases levels of abscisic acid (ABA) in P. japonicum and prevents the activation of hundreds of genes including cell cycle and xylem development genes. Blocking ABA signaling overcomes nitrogen's inhibitory effects indicating that nitrogen represses haustoria formation by increasing ABA. The effect of nitrogen appears more widespread since nitrogen also inhibits haustoria in the obligate root parasite Striga hermonthica. Together, our data show that nitrogen acts as a haustoria repressing factor and suggests a mechanism whereby parasitic plants use nitrogen availability in the external environment to regulate the extent of parasitism.

Oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida.

BACKGROUND: Spironucleus salmonicida is an anaerobic parasite that can cause systemic infections in Atlantic salmon. Unlike other diplomonad parasites, such as the human pathogen Giardia intestinalis, Spironucleus species can infiltrate the blood stream of their hosts eventually colonizing organs, skin and gills. How this presumed anaerobe can persist and invade oxygenated tissues, despite having a strictly anaerobic metabolism, remains elusive. RESULTS: To investigate how S. salmonicida response to oxygen stress, we performed RNAseq transcriptomic analyses of cells grown in the presence of oxygen or antioxidant-free medium. We found that over 20% of the transcriptome is differentially regulated in oxygen (1705 genes) and antioxidant-depleted (2280 genes) conditions. These differentially regulated transcripts encode proteins related to anaerobic metabolism, cysteine and Fe-S cluster biosynthesis, as well as a large number of proteins of unknown function. S. salmonicida does not encode genes involved in the classical elements of oxygen metabolism (e.g., catalases, superoxide dismutase, glutathione biosynthesis, oxidative phosphorylation). Instead, we found that genes encoding bacterial-like oxidoreductases were upregulated in response to oxygen stress. Phylogenetic analysis revealed some of these oxygen-responsive genes (e.g., nadh oxidase, rubrerythrin, superoxide reductase) are rare in eukaryotes and likely derived from lateral gene transfer (LGT) events into diplomonads from prokaryotes. Unexpectedly, we observed that many host evasion- and invasion-related genes were also upregulated under oxidative stress suggesting that oxygen might be an important signal for pathogenesis. CONCLUSION: While oxygen is toxic for related organisms, such as G. intestinalis, we find that oxygen is likely a gene induction signal for host invasion- and evasion-related pathways in S. salmonicida. These data provide the first molecular evidence for how S. salmonicida could tolerate oxic host environments and demonstrate how LGT can have a profound impact on the biology of anaerobic parasites.

Developing a thief: Haustoria formation in parasitic plants.

Parasitic plants are widespread pathogens that infect numerous plant species and cause devastating agricultural losses. They efficiently withdraw water, nutrients and sugars from their hosts by fusing tissues and connecting their vasculature to the host vasculature. This ability to parasitize is found in a wide range of species and has evolved at least eleven independent times, suggesting a recurring and flexible developmental strategy. Despite multiple independent origins, a common feature to parasitism is the formation of an invasive organ termed the haustorium. Parasitic plants form haustoria in their stems or roots and use this structure to penetrate host tissues and form vascular connections, often with distantly related species. This ability to join to an unrelated species is remarkable, and together with the economic importance of parasitism, there is a strong need to further understand how parasitic plants infect their hosts. Here, we discuss the developmental basis for plant parasitism, focusing on haustorial initiation, penetration and vascular formation. We also discuss future directions and outstanding questions in this emerging field.

Visualization of the membrane engineering concept: evidence for the specific orientation of electroinserted antibodies and selective binding of target analytes.

Membrane engineering is a generic methodology for increasing the selectivity of a cell biosensor against a target molecule, by electroinserting target-specific receptor-like molecules on the cell surface. Previous studies have elucidated the biochemical aspects of the interaction between various analytes (including viruses) and their homologous membrane-engineered cells. In the present study, purified anti-biotin antibodies from a rabbit antiserum along with in-house prepared biotinylated bovine serum albumin (BSA) were used as a model antibody-antigen pair of molecules for facilitating membrane engineering experiments. It was proven, with the aid of fluorescence microscopy, that (i) membrane-engineered cells incorporated the specific antibodies in the correct orientation and that (ii) the inserted antibodies are selectively interacting with the homologous target molecules. This is the first time the actual working concept of membrane engineering has been visualized, thus providing a final proof of the concept behind this innovative process. In addition, the fluorescence microscopy measurements were highly correlated with bioelectric measurements done with the aid of a bioelectric recognition assay.

Anti-MuSK- and anti-AChR-positive myasthenia gravis induced by d-penicillamine.

BACKGROUND: Myasthenia gravis (MG) is an autoimmune disorder of the neuromuscular junction usually caused by antibodies to the nicotinic acetylcholine receptor (AChR) and occasionally to muscle-specific kinase (MuSK). D-penicillamine is a therapeutic agent for several diseases, but can also induce a number of immune-mediated disorders, including MG, as a side-effect. In most patients with D-penicillamine-induced MG, anti-AChR antibodies are detected, but the presence of anti-MuSK antibodies has not been reported previously. CASE: The case reported was a female patient who presented with myasthenic symptoms after D-penicillamine administration for scleroderma. RESULTS: Both anti-AChR and anti-MuSK antibodies were identified in the patient's serum. The anti-MuSK antibodies were of the IgG4 subclass, as in idiopathic MG. Both types of antibody gradually disappeared after discontinuation of D-penicillamine. A significant improvement in symptoms was observed and the patient gradually became free of MG symptoms, without requiring any treatment for MG. Another four double-positive (anti-AChR and anti-MuSK antibodies) patients were identified during a retrospective study, but none had been treated with D-penicillamine. CONCLUSION: D-penicillamine can cause anti-AChR and anti-MuSK antibody-positive MG, a rare phenomenon which is reversed after discontinuation of D-penicillamine treatment.

Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci.

The isolation of human antibodies against muscle acetylcholine receptor (AChR), the autoantigen involved in myasthenia gravis (MG), is important for the development of therapeutically useful reagents. Monovalent antibody fragments from monoclonal antibodies against the main immunogenic region (MIR) of AChR protect the receptor from the destructive activity of MG autoantibodies. Human anti-AChR alpha-subunit antibody fragments with therapeutic potential have been isolated using phage display antibody libraries. An alternative approach for obtaining human mAb has been provided by the development of humanized mice. In this report, we show that immunization of transgenic mouse strains with the extracellular domain of the human AChR alpha-subunit results in antibody responses and isolation of hybridomas producing human mAb. Four specific IgM mAb were isolated and analyzed. mAb170 recognized the native receptor the best and was capable of inducing AChR antigenic modulation, suggesting its specificity for a pathogenic epitope. Moreover, the recombinant antigen-binding (Fab) fragment of this mAb competed with an anti-MIR mAb, revealing that its antigenic determinant lies in or near the MIR. Finally, Fab170 was able to compete with MG autoantibodies and protect the AChR against antigenic modulation induced by MG sera. This approach will be useful for isolating additional mAb with therapeutic potential against the other AChR subunits.