Martin D. Watson

Chemotaxis of Rhizobium leguminosarum biovar phaseoli towards Flavonoid Inducers of the Symbiotic Nodulation Genes

Summary: Chemotaxis of Rhizobium leguminosarum biovar phaseoli RP8002 towards a range of carbohydrates, phenolic compounds and flavonoids was assayed. Xylose (peak response 10−4 M), sucrose (peak response 10−6 M) and raffinose (peak response 10−5 M) were strong chemoattractants amongst the carbohydrates, whilst glucose, fructose, galactose and maltose produced little or no detectable response. Of the monocyclic phenolic compounds, vanillyl alcohol, p-hydroxybenzoic acid (both peak responses 10−6 M) and 3,4-dihydroxybenzoic acid (peak response 10−4 M) all evoked strong chemotactic responses. Amongst the nod-inducing flavonoids, apigenin and luteolin were both strong chemoattractants (peaks at 10−5 M) while naringenin produced a very low response. Competition experiments suggest that apigenin and luteolin are recognized by a common receptor, but that there exists a separate receptor for luteolin alone. The inhibitors of nod-induction, umbelliferone and acetosyringone, both produced strong chemotactic responses, with peaks at 10−3 M and 10−2 M respectively. This evidence is indicative of a role for chemotaxis towards nod-inducing flavonoids in the initiation of root nodule formation by rhizobia, and also suggests that chemotaxis may influence the host range of the interaction.

Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases

We selected cDNA plasmid clones that corrected the temperature-sensitive phenotype of Escherichia coli strain JC201, which is deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity. A plasmid-based maize endosperm cDNA library was used for complementation and a plasmid that enabled the cells to grow at 44°C on ampicillin was isolated. Addition of this plasmid (pMAT1) to JC201 restored 1-acyl-sn-glycerol-3-phosphate acyltransferase activity to the cells. Total phospholipid labelling showed that the substrate for the enzyme, lysophosphatidic acid, accumulated in JC201 and was further metabolised to phosphatidylethanolamine in complemented cells. Membranes isolated from such cells were able to convert lysophosphatidic acid to phosphatidic acid in acyltransferase assays. The cDNA insert of pMAT1 contains one long open reading frame of 374 amino acids which encodes a protein of relative molecular weight 42 543. The sequence of this protein is most similar to SLC1, which is thought to be able to acylate glycerol at the sn-2 position during synthesis of inositol-containing lipids. Homologies between the SLC1 protein, the 1-acyl-sn-glycerol-3-phosphate acyltransferase of E. coli (PlsC) and the maize ORF were found with blocks of conserved amino acids, whose spacing was conserved between the three proteins, identifiable.

The Pisum sativum MAP kinase homologue (PsMAPK) rescues the Saccharomyces cerevisiae hog1 deletion mutant under conditions of high osmotic stress.

Previous analysis of the MAP kinase homologue from Pisum sativum (PsMAPK) revealed a potential MAP kinase motif homologous to that found in eukaryotic cdc2 kinases. Sequence comparison showed a 47% identity on amino acid sequence basis to the Saccharomyces cerevisiae Hog 1p MAP kinase involved in the osmoregulatory pathway. Under conditions of salt-stress aberrant morphology of a hog1 deletion mutant was completely restored and growth was partially restored by expression of the PsMAPK. This shows that PsMAPK is functionally active as a MAP kinase in S. cerevisiae. Comparison of PsMAPK with other kinases involved in osmosensitivity, showed a high degree of homology and implicates a possible role for PsMAPK in a P. sativum osmosensing signal transduction pathway.

Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.)

Oleosins of Brassica napus L. (oilseed rape) synthesized by in-vitro translation were found to be very efficiently targeted to microsomal membranes but only poorly translocated to oil bodies or emulsified oil. The use of other bilayer membranes as controls showed that this interaction was specific. The rate of oleosin synthesis in the presence of microsomes was enhanced about threefold, indicative of the involvement of the signal-recognition particle in the targeting process. There is no evidence for the cleavage of the protein during targeting and the protein sequence reveals no consensus cleavage site for the signal peptide. Protection experiments using Proteinase K revealed that about 6 kDa of the protein is exposed on the cytoplasmic side of the ER but the remainder is protected. Carbonate (pH 11) washing of microsomal membranes after in-vitro translation confirmed that oleosins have a domain which remains inserted in the ER rather than the protein being transported completely into the lumen of the ER. These results indicate that oleosins are transported via the ER prior to their accumulation on oil bodies.

Differential, temporal and spatial expression of genes involved in storage oil and oleosin accumulation in developing rapeseed embryos: implications for the role of oleosins and the mechanisms of oil-body formation

The temporal and spatial expression of oleosin and Δ9-stearoyl-ACP desaturase genes and their products has been examined in developing embryos of rapeseed, Brassica napus L. var. Topas. Expression of oleosin and stearate desaturase genes was measured by in situ hybridisation at five different stages of development ranging from the torpedo stage to a mature-desiccating embryo. The temporal pattern of gene expression varied dramatically between the two classes of gene. Stearate desaturase gene expression was relatively high, even at the torpedo stage, whereas oleosin gene expression was barely detectable at this stage. By the stage of maximum embryo fresh weight, stearate desaturase gene expression had declined considerably while oleosin gene expression was at its height.In contrast to their differential temporal expression, the in situ labelling of both classes of embryo-specific gene showed similar, relatively uniform patterns of spatial expression throughout the embryo sections. Immunogold labelling of ultra-thin sections from radicle tissue with anti-oleosin antibodies showed similar patterns to sections from cotyledon tissue. However, whereas at least three oleosin isoforms were detectable on western blots of homogenates from cotyledons, only one isoform was found in radicles. This suggests that some of the oleosin isoforms may be expressed differentially in the various types of embryo tissue. The differential timing of stearate desaturase and oleosin gene expression was mirrored by similar differences in the timing of the accumulation of their ultimate products, i.e. storage oil and oleosin proteins. Oil-body fractions prepared from young (2.5 mg) embryos contained very little oleosin protein, as examined by SDS-PAGE and western blotting, whereas identically prepared fractions from dry seeds contained over 10% (w/w) oleosin. Dehydration of oil bodies from young embryos resulted in their breakdown and coalescence into large clumps of oil which could not be re-emulsified, even after rehydration. In contrast, the oleosin-rich oil bodies from mature embryos were stable to dehydration and subsequent rehydration. It is suggested that, in developing rapeseed embryos, the accumulation of storage oil and oleosins is not concomitant but that the eventual deposition of oleosins onto the surfaces of storage oil bodies is essential for their stability during seed desiccation.

Xenopus NK cells identified by novel monoclonal antibodies

Early‐thymectomized (Tx) Xenopus frogs, which are permanently deficient in T cells, are used as a model sytem for the characterization of novel monoclonal antibodies (mAb) which identify candidate NK cells at the amphibian level of evolution. Hybridomas, generated from mice immunized with splenocytes from Tx Xenopus following B cell and thrombocyte depletion, were screened by flow cytometry. Three mAb (1F8, 4D4 and 1G5) were identified that stained increased proportions of splenocytes from Tx compared with control frogs. These mAb identified lymphoid populations from Xenopus spleen, liver and gut which, after 48 h culture in growth factor‐rich medium, exhibited spontanous killing of MHC‐deficient allotumor targets. mAb‐defined splenocytes also rapidly induced apoptosis of such tumor targets. Dual color analysis confirmed that NK cells are neither T nor B cells. Cytospins of splenocytes isolated with anti‐NK mAb revealed large lymphoid cells with distinct pseudopodia. Immunohistology indicated each anti‐NK mAb routinely labeled cells within the gut epithelium but NK cells were difficult to visualize in spleen sections. Western blotting of spleen, liver and intestinal lysates subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that 1G5 reacted strongly with protein bands of ∼ 70 – 85 kDa, whereas mAb 1F8 and 4D4 stained less intensely, but identified similar protein bands.

T-cell and natural killer cell development in thymectomized Xenopus

Summary: The Xenopus early‐thymectomy model system is used to investigate the extent to which the thymus controls T‐cell development and to probe the evolution of natural killer (NK) ceils. Loss of T‐cell function following thymectomy, together with the paucity of cells expressing monoclonal antibody‐defined T‐cell surface markers, and greatly reduced expression of T‐cell receptor β transcripts in spleen, ever and intestine, indicate that T‐cell development is minimal in the absence of the thymus. Our findings therefore mitigate against the idea that a substantial extrathymic pathway of T‐cell development exists in early vertebrate evolution. Rather, they suggest that in this amphibian representative T cells are predominately thymus dependent. In vitro studies with control and thymectomized Xenopus splenocytes reveal that a non‐T/non‐B population and also two T‐cell subsets all display natural cytotoxicity towards allogeneic thymus lymphoid tumour cells (which are deficient in MHC antigen expression). Since Xenopus thymectomized early in larval development are permanently deficient in T cells, they may provide a useful phylogenetic model for the study of NK cells.

NK-like activity against allogeneic tumour cells demonstrated in the spleen of control and thymectomized Xenopus

This paper addresses the issue of natural killer (NK) cell evolution by searching for NK‐like activity in an amphibian representative, the immunologically well‐characterized clawed frog. Xenopus laevis. Using in vitro6 h 51 chromium release assays, we have shown that splenocyte effectors from early thymectomized (Tx) year‐old frogs, but not from control siblings, are able to spontaneously lyse allogeneic thymus tumour cell lines that lack MHC antigen expression. Such lytic capacity can be readily induced in control Xenopus and elevated in Tx frogs by a single injection of tumour cells, with maximal splenocyte cytotoxicity occurring 3 days postinjection, the amount of 51 Cr‐release correlating directly with effector: target ratios. Splenocytes, even those from tumour‐injected frogs, are unable to lyse allogeneic splenic lymphoblasts or erythrocyte targets, even when the latter are coated with IgY (the Xenopus IgG equivalent); moreover, we were unable to demonstrate any splenocyte‐induced lysis of the human NK cell target K562. Lymphokine‐activated killing (LAK) in Xenopus is suggested, since Tx splenocytes cultured in cytokine‐rich medium (from concanavalin A‐stimulated control splenocytes) display significantly elevated killing of allogeneic tumour targets. Flow cytometric analysis highlights the loss of T cell markers from the spleen of Tx frogs and reveals a variable staining pattern of both control and Tx splenocytes when treated with a mAb that binds to both fish non‐specific cytotoxic cells and human NK cells. Prospects for identifying the cellular basis of NK‐like activity in Xenopus are discussed in the light of these experiments.

Ontogeny of Xenopus NK cells in the absence of MHC class I antigens

This paper explores the ontogeny of NK cells in control and early-thymectomized (Tx) Xenopus laevis through phenotypic analysis of cells expressing the NK cell antigen 1F8 and by performing in vitro cytotoxic assays. Dual color flow cytometry reveals that a few 1F8positive splenocytes first emerge in late larval life, at approximately 7-weeks post-fertilization. This is about 2-weeks after the time when surface MHC class Ia expression can first be detected. The proportion of splenocytes expressing 1F8 remains very low in 3-4 month-old froglets, but by 1 year there is a sizeable 1F8positive population, which is proportionally elevated in Tx frogs. The ontogeny of NK cell function is monitored by a 5 h DNA fragmentation (JAM) assay. Control and Tx larval splenocytes (from either 5- or 7-week-old tadpoles) fail to kill MHC-deficient thymus-derived tumor cell targets. Such in vitro killing is still relatively poor in 3-4 month froglets, compared with high levels of tumor cell cytotoxicity mediated by splenocytes from older frogs. Immunoprecipitation studies identify that the major ligand for the 1F8 mAb is a 55 kDa polypeptide. Finally, further evidence is provided that 1F8positive lymphocytes are indeed bona fide NK cells, distinct from T cells, since purified 1F8positive splenocytes from Tx Xenopus fail to express fully rearranged TCRbeta V region transcripts. We conclude that NK cells fail to develop prior to MHC class I protein expression and, therefore, do not contribute to the larval immune system, whereas they do provide an important backup for T cells in the adult frog by contributing to anti-tumor immunity.

Natural cytotoxicity towards allogeneic tumour targets in Xenopus mediated by diverse splenocyte populations

We have recently demonstrated NK-like activity in the spleen of the clawed frog, Xenopus laevis. This paper investigates the cellular basis of this natural cytotoxicity. Significant levels of cytotoxicity towards B3B7 allogeneic thymus tumour targets, that express neither class Ia nor class II MHC proteins, occurred after splenocytes from either control or early-thymectomized (Tx) year-old Xenopus were cultured for 48 hours. Killing by Tx cells required their culture in growth factor-rich medium (GFM) obtained from concanavalin A-stimulated cells. Immunomagnetic cell sorting revealed that cytotoxic effectors in both control and Tx frogs were found in the B cell-depleted population, but never in the B cell-enriched fraction. Splenocytes from control Xenopus, depleted of T cells by magnetic sorting and following culture in GFM, also developed natural cytotoxicity towards allotumour cells. Magnetic cell sorting also revealed that purified (CD5+) T cells cultured for 48 hours in GFM also became able to lyse the allogeneic tumour targets. Cytotoxicity mediated by T cells resided not only in the CD5+, CD8+ population, but also in the CD5+, CD8- (putative CD4+) T cell subset. Ontogenetic studies revealed that splenocytes from 6-7 week-old (stage 56-57) control larvae, even after 48 hr culture in GFM, were unable to spontaneously lyse the allotumour targets, whereas cultured splenocytes from 6 month old froglets were effective killers. Thymocytes from larvae or adults routinely failed to kill tumour cells. The work highlights the need to use Tx Xenopus to further explore non-T-cell-mediated, NK-like cytotoxicity at the amphibian level of evolution.