Temporal and Spatial Patterns of Gene Expression around Sites of Attempted Fungal Infection in Parsley Leaves.

We analyzed the expression patterns of several pathogen defense-related genes in primary leaf buds of parsley by in situ RNA hybridization. Labeled antisense RNA probes were generated from seven selected cDNAs detecting transcripts from genes that are rapidly and strongly activated in cultured parsley cells upon treatment with fungal elicitor. These genes encode two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, a furanocoumarin-specific bergaptol O-methyltransferase, one pathogenesis-related protein, and three less well characterized proteins, designated as ELI 3, ELI 5, and ELI 7. In uninfected tissue, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase mRNA levels were high in epidermal cells, oil-duct epithelial cells, and cells of the developing xylem; bergaptol O-methyltransferase mRNA was confined to oil-duct epithelial cells; and the pathogenesis-related protein and ELI 3, ELI 5, and ELI 7 mRNAs were undetectable. All seven mRNAs accumulated transiently and locally around infection sites caused by the soybean-pathogenic fungus Phytophthora megasperma f. sp. glycinea, to which parsley is nonhost-resistant. The observed late appearance of bergaptol O-methyltransferase mRNA, as compared with all other mRNAs, is in accord with a similar relative timing of transient gene activation in elicitor-treated cell cultures. Sharp borders were observed between the infection center, where hypersensitive cell death had occurred in response to fungal penetration, the surrounding area of local gene activation, and the remainder of the tissue not showing any apparent response. Some of the genes were also activated, although less sharply localized, upon wounding of parsley leaves.

Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers.

Correlation of Rapid Cell Death with Metabolic Changes in Fungus-Infected, Cultured Parsley Cells.

To study in detail the hypersensitive reaction, one of the major defense responses of plants against microbial infection, we used a model system of reduced complexity with cultured parsley (Petroselinum crispum) cells infected with the phytopathogenic fungus Phytophthora infestans. Experimental conditions were established to maintain maximal viability of the cultured cells during co-cultivation with fungal germlings, and a large proportion of the infected parsley cells responded to fungal infection with rapid cell death, thereby exhibiting major features of the hypersensitive reaction in whole-plant-pathogen interactions. Rapid cell death clearly correlated with termination of further growth and development of the fungal pathogen. Thus, the system fulfilled important prerequisites for investigating cell-death-related metabolic changes in individual infected cells. Using cytochemical methods, we monitored the increase of mitochondrial activity in single infected cells and the intracellular accumulation of reactive oxygen species prior to the occurrence of rapid cell death. We obtained strong correlative evidence for the involvement of these intracellularly accumulating reactive oxygen species in membrane damage and in the resulting abrupt collapse of the cell.

Expression of maize and fungal nitrate reductase genes in arbuscular mycorrhiza.

The role of arbuscular mycorrhizal (AM) fungi in assisting their host plant in nitrate assimilation was studied. With polymerase chain reaction technology, part of the gene coding for the nitrate reductase (NR) apoprotein from either the AM fungus Glomus intraradices or from maize was specifically amplified and subsequently cloned and sequenced. Northern (RNA) blot analysis with these probes indicated that the mRNA level of the maize gene was lower in roots and shoots of mycorrhizal plants than in noncolonized controls, whereas the fungal gene was transcribed in roots of AM plants. The specific NR activity of leaves was significantly lower in AM-colonized maize than in the controls. Nitrite formation catalyzed by NR was mainly NADPH-dependent in roots of AM-colonized plants but not in those of the controls, which is consistent with the fact that NRs of fungi preferentially utilize NADPH as reductant. The fungal NR mRNA was detected in arbuscules but not in vesicles by in situ RNA hybridization experiments. This appears to be the first demonstration of differential formation of transcripts of a gene coding for the same function in both symbiotic partners.

Cyst germination proteins of the potato pathogen Phytophthora infestans share homology with human mucins.

We have cloned genes of Phytophthora infestans, the causal agent of potato late blight, that are activated shortly before the onset of invasion of the host tissue. The three genes isolated appear to be arranged in a genomic cluster and belong to a small polymorphic gene family. A conspicuous feature of the deduced proteins is an internal octapeptide repeat with the consensus sequence TTYAP TEE. Because of this structural motif, these novel P. infestans proteins were named Car (Cyst-germination-specific acidic repeat) proteins. One of the genes, car90, codes for 1,489 amino acids including 120 octapeptide tandem repeats. Car proteins are transiently expressed during germination of cysts and formation of appressoria and are localized at the surface of germlings. The structural motif of tandemly repeated oligopeptides also occurs in a prominent class of proteins, the mucins, from mammals. The P. infestans Car proteins share 51% sequence homology with the tandem repeat region of a subfamily of human mucins. According to the physiological functions ascribed to mucins, we suggest that Car proteins may serve as a mucous cover protecting the germling from desiccation, physical damage, and adverse effects of the plant defense response and may assist in adhesion to the leaf surface.

NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis.

Hterologous complementation of yeast mutants has enabled the isolation of genes encoding several families of amino acid transporters. Among them, NTR1 codes for a membrane protein with weak histidine transport activity. However, at the sequence level, NTR1 is related to rather non-specific oligopeptide transporters from a variety of species including Arabidopsis and to the Arabidopsis nitrate transporter CHL1. A yeast mutant deficient in oligopeptide transport was constructed allowing to show that NTR1 functions as a high affinity, low specificity peptide transporter. In siliques NTR1-expression is restricted to the embryo, implicating a role in the nourishment of the developing seed.

N-terminal amino acid sequences of precursor and mature forms of alpha-1-antitrypsin.

alpha-1-Antitrypsin is found in hepatocytes as a high-mannose glycoprotein (Mr 49 000), extracellularly as a complex-type glycoprotein (Mr 54 000). Deglycosylation of both forms with peptide: N-glycosidase led to proteins of identical app. Mr (41 000). The sequence of 26 N-terminal amino acids of rat alpha 1-antitrypsin was determined. A high content of polar amino acids was found. The partially characterized presequence of in vitro synthesized alpha 1-antitrypsin showed a cluster of hydrophobic amino acids. A pre-peptide of 24 amino acids is proposed. There is no evidence for the existence of a propeptide.

Profilin and Rop GTPases are localized at infection sites of plant cells.

We have found 5 profilin cDNAs in cultured parsley cells, representing a small gene family of about 5 members in parsley. Specific antibodies were produced using heterologously expressed parsley profilin as antigen. Western blot analysis revealed the occurrence of similar amounts of profilin in roots and green parts of parsley plants. Immunocytochemical staining of parsley cells infected with the oomycetous plant pathogen Phytophthora infestans clearly revealed that profilin accumulates at the site on the plasma membrane subtending the oomycetous appressorium, where the actin cables focus. We also observed the accumulation of Rop GTPases around this site, which might point to a potential function in signaling to the cytoskeleton.

Biotransformation of cyclosporin in primary rat, porcine and human liver cell co-cultures.

The purpose of this study was to investigate the species-specific cyclosporin biotransformation in primary rat, human, and porcine liver cell cultures, and to investigate the suitability of a modified sandwich culture technique with non-purified liver cell co-cultures for drug metabolism studies. A sandwich culture was found to enhance hepatocellular metabolic activity and improve cellular morphology and ultrastructure. The cyclosporin metabolites AM9 and AM1 were formed in porcine and human liver cell sandwich co-cultures at levels corresponding to the respective in vivo situations. In contrast, metabolite profiles in rat hepatocytes were at variance with the in vivo situation. However, for all cell types, the overall metabolic activity was positively influenced by sandwich co-culture. The initial levels of albumin synthesis were higher in sandwich cultures than in those without matrix overlay. It is hypothesized that the sandwich culture system provides an improved microenvironment and is, therefore, an advantageous tool for in vitro studies of drug metabolism.

In situ localization of light-induced chalcone synthase mRNA, chalcone synthase, and flavonoid end products in epidermal cells of parsley leaves.

Methods involving in situ RNA hybridization, immunohistochemistry, and microspectrophotometry of individual cells were used to localize the mRNA encoding chalcone synthase (the key enzyme of flavonoid biosynthesis), the enzyme protein, and the biosynthetic end products in cross sections of parsley leaves (Petroselinum crispum). The light-dependent, sequential occurrence of all three components was restricted to epidermal cells. The results are in agreement with the putative function of flavonoids (transcriptionally inducible, UV-protective pigments) and suggest that all biosynthetic steps occur in those cells in which the products accumulate.

Coordinated changes in transcription and translation rates of phenylalanine ammonia-lyase and 4-coumarate: CoA ligase mRNAs in elicitor-treated Petroselinum crispum cells.

Treatment of cultured parsley cells (Petroselinum crispum) with elicitor preparations from the fungus, Phytophthora megasperma f. sp. glycinea, resulted in coordinated, sequential changes in the transcription rates, mRNA amounts and translational activities, and the catalytic activities of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase. In contrast to previous observations (Kuhn et al. 1984, Chappell and Hahlbrock 1984), the coordination included the timing of changes in transcription rates and mRNA amounts if a different elicitor preparation or a different cell culture was used.

The role of chalcone synthase in the regulation of flavonoid biosynthesis in developing oat primary leaves.

The role of chalcone synthase in the regulation of flavonoid biosynthesis during organogenesis of oat primary leaves has been investigated at the level of enzyme activity and mRNA translation in vitro. Chalcone synthase was purified about 500-fold. The apparent Km values were 1.5 and 6.3 microM for 4-coumaroyl-CoA and malonyl-CoA, respectively. The end products of oat flavonoid biosynthesis, three C-glucosylflavones, did not inhibit the reaction at concentrations as measured up to 60 microM each. Apigenin (4',5,7-trihydroxyflavone), a stable structural analog of the reaction product, 2',4,4',6'-tetrahydroxychalcone, was found to be a strong competitive inhibitor of 4-coumaroyl-CoA binding and a strong noncompetitive inhibitor of malonyl-CoA binding. Although apigenin is not supposed to be an intermediate of C-glucosylflavone biosynthesis, this compound might be a valuable tool for future kinetic studies. To date, there is no indication of chalcone synthase regulation by feedback or similar mechanisms which modulate enzyme activity. Mathematical correlation of chalcone synthase activity and flavonoid accumulation during leaf development, however, indicates that chalcone synthase is the rate-limiting enzyme of the pathway. By in vitro translation studies using preparations of total RNA from different leaf stages, we could demonstrate for the first time that the translational activity of chalcone synthase mRNA undergoes marked daily changes. The high values found at the end of the dark phase suggest that light does not exert direct influence on flavonoid biosynthesis but probably functions by controlling the basic diurnal rhythm.

Synthesis of a larger precursor for the subunit IV of rat liver cytochrome c oxidase in a cell-free wheat germ system.

Poly(A)+RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germ. The RNA stimulated the incorporation of [35S]methionine into proteins 20- to 30-fold. The labeled translation products were incubated with an antiserum against cytochrome c oxidase. After binding of the antigen x immunoglobulin complex to and elution from protein A-Sepharose and sodium dodecyl sulfate (SDS)-polyacrylamide step gel electrophoresis, autoradiography was carried out. Mainly one major protein with an apparent molecular weight of 19,500 was visualized. When the unlabeled individual cytochrome c oxidase subunits IV, V, VI, or VII, isolated from preparative SDS-polyacrylamide gels, were added to the translation mixture, it was found that only subunit IV could compete with the in vitro-synthesized protein of 19.5 kilodaltons in respect to the binding to the cytochrome c oxidase antiserum. The in vitro-synthesized product was 3,000 daltons larger than the cytochrome c oxidase subunit polypeptide IV. It is concluded that the subunit IV is synthesized as a precursor. Evidence for the precursor form was obtained from translation experiments with [35S]methionine bound to a specific initiator tRNA which led to a radioactively labeled product of identical electrophoretic mobility as the 19.5 kilodalton protein. Furthermore, two dimensional tryptic fingerprints of subunit IV and its precursor show a high degree of similarity.

Occurrence of phytoalexins and other putative defense-related substances in uninfected parsley plants.

Considerable amounts of the following substances were found in uninfected parsley (Petroselinum crispum) cotyledons: furanocoumarins, the putative phytoalexins of this and some related plant species, two enzymes of the furanocoumarin pathway (S-adenosyl-L-methionine: xanthotoxol and S-adenosyl-L-methionine: bergaptol O-methyltransferases), two hydrolytic enzymes (1,3-ß-glucanase, EC 3.2.1.39, and chitinase, EC 3.2.1.14), and 'pathogenesis-related' proteins. The furanocoumarins and the methyltransferase activities reached their highest levels at the onset of cotyledon senescence as the hydrolytic enzymes increased from low to relatively high activity values. The relative amounts of pathogenesis-related proteins 1 and 2, as well as the corresponding mRNAs, also increased markedly. Two enzymes of general phenylpropanoid metabolism, L-phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, decreased in activity in a biphasic fashion during cotyledon development. At all developmental stages, the levels of these putative defense-related agents in total cotyledon extracts were too high to enable detection of, possibly, additional changes upon infection with zoospores of Phytophthora megasperma f. sp. glycinea, a fungal pathogen to which parsley shows a non-host, hypersensitive resistance response.

Gene structure and in situ transcript localization of pathogenesis-related protein 1 in parsley.

We have analysed three nearly full-length cDNAs complementary to mRNAs encoding two PR1 (pathogenesis-related, class 1) proteins in parsley (Petroselinum crispum). Furthermore, one selected genomic clone containing the PcPR1-1 gene was investigated in detail. The structural organization and possible regulatory elements in the 5' flanking region of this gene are presented. In situ RNA hybridization in fungus-infected parsley leaf tissue demonstrated rapid and massive PR1 mRNA accumulation around infection sites.

Primary structure and expression of acidic (class II) chitinase in potato.

Infection of potato (Solanum tuberosum) leaves by the late blight fungus Phytophthora infestans or treatment with fungal elicitor leads to a strong increase in chitinase activity. We isolated cDNAs encoding acidic (class II) chitinases (ChtA) from potato leaves and determined their structures and expression patterns in healthy and stressed plants. From the total number of cDNAs and the complexity of genomic DNA blots we conclude that acidic chitinase in potato is encoded by a gene family which is considerably smaller than that encoding basic (class I) chitinase (ChtB). The deduced amino acid sequences show 78 to 96% identity to class II chitinases from related plant species tomato, tobacco) whereas the identity to basic chitinases of potato is in the range of 60%. RNA blot analysis revealed that both acidic and basic chitinases were strongly induced by infection or elicitor treatment and that the induction occurred both locally at the site of infection and systemically in upper uninfected leaves. In contrast, a differential response to other types of stress was observed. Acidic chitinase mRNA was strongly induced by salicylic acid, whereas basic chitinase mRNA was induced by ethylene or wounding. In healthy, untreated plants, acidic chitinase mRNA accumulated also in an organ-, cell-type- and development-specific manner as revealed by RNA blot analysis and in situ RNA hybridization. Relatively high transcript levels were observed in old leaves and young internodes as well as in vascular tissue and cells constituting the stomatal complex in leaves and petioles. Lower, but appreciable mRNA levels were also detectable in roots and various flower organs, particularly in sepals and stamens. The possible implications of these findings in pathogen defense, development and growth processes are discussed.

The size and distribution of cytochrome c oxidase subunit IV mRNA between free and membrane-bound polyribosomes.

A length of 990 +/- 80 nucleotides was estimated for the poly(A)-rich mRNA in the higher-molecular-weight precursor of rat liver cytochrome c oxidase subunit IV. Assuming a molecular weight of 19500 for the cytochrome c oxidase subunit IV precursor, a non-coding region of about 350 nucleotides must exist. In experiments on the site of synthesis it is shown that the poly(A)-rich RNA for the higher-molecular-weight precursor of cytochrome c oxidase subunit IV is found in free, loosely and tightly membrane-bound polyribosomes. This finding does not agree with the present picture of free polysomes as the exclusive site of synthesis for mitochondrial proteins.

Proteolytic precursor processing in the biosynthesis of mitochondria.

Essentially all polypeptides synthesized in the cytoplasm and imported into either the matrix or into the inner or outer membrane of mitochondria are made as larger molecular weight precursors. All known examples of in vivo or in vitro synthesized precursors are summarized. Little information on the nature of the proteolytic enzymes involved in the processing of the larger precursor polypeptides exists. The biosynthesis of rat liver cytochrome c oxidase is discussed in detail. In contrast to reported data, the cytoplasmic subunits of rat liver cytochrome c oxidase are synthesized as larger molecular weight precursors and not as a polyprotein. Precursors to subunits IV and V show an extra-peptide sequence of about 3000 daltons. Evidence against the existence of a polyprotein precursor was also obtained, when messenger RNAs for the individual subunits IV and V were isolated and analyzed in respect to their size. A length of 990 +/- 80 and 830 +/- 70 nucleotides was estimated for the poly(A)+-RNA of cytochrome c oxidase subunits IV and V, respectively. In experiments on the site of synthesis, it was found that cytochrome c oxidase subunits IV and V are made on free, loosely and tightly membrane-bound polyribosomes.

Local mechanical stimulation induces components of the pathogen defense response in parsley.

Cell suspension cultures of parsley (Petroselinum crispum) have previously been used as a suitable system for studies of the nonhost resistance response to Phytophthora sojae. In this study, we replaced the penetrating fungus by local mechanical stimulation by using a needle of the same diameter as a fungal hypha, by local application of a structurally defined fungus-derived elicitor, or by a combination of the two stimuli. Similar to the fungal infection hypha, the local mechanical stimulus alone induced the translocation of cytoplasm and nucleus to the site of stimulation, the generation of intracellular reactive oxygen intermediates (ROI), and the expression of some, but not all, elicitor-responsive genes. When the elicitor was applied locally to the cell surface without mechanical stimulation, intracellular ROI also accumulated rapidly, but morphological changes were not detected. A combination of the mechanical stimulus with simultaneous application of low doses of elicitor closely simulated early reactions to fungal infection, including cytoplasmic aggregation, nuclear migration, and ROI accumulation. By contrast, cytoplasmic rearrangements were impaired at high elicitor concentrations. Neither papilla formation nor hypersensitive cell death occurred under the conditions tested. These results suggest that mechanical stimulation by the invading fungus is responsible for the observed intracellular rearrangements and may trigger some of the previously demonstrated changes in the activity of elicitor-responsive genes, whereas chemical stimulation is required for additional biochemical processes. As yet unidentified signals may be involved in papilla formation and hypersensitive cell death.

Development of barley aleurone cells: temporal and spatial patterns of accumulation of cell-specific mRNAs.

The mechanisms underlying the response of the mature barley (Hordeum vulgare L.) aleurone layer to gibberellic acid have received much attention, but little is known about the developmental basis for this response. We have investigated the spatial and temporal accumulation of mRNAs complementary to two barleygrain cDNAs that are differentially expressed in the aleurone layer of the developing endosperm. Messenger RNA complementary to one of these clones (B11E; Jakobsen etal., 1989, Plant Mol. Biol. 12, 285-293) accumulates exclusively in the aleurone layer of developing grains where it is uniformly distributed in all three cell layers. Accumulation of B11E mRNA is first detectable 10 d post an thesis (DPA), increases 200-fold up to 25 DPA, and then declines towards grain maturity. Messenger RNA complementary to the other clone, B22E, shows a more complex pattern of expression. In addition to the aleurone layer, this mRNA accumulates in the vascular tissue of the maternal pericarp and embryo axis, as well as in the parenchyma cells of the embryonic scutellum. In excised immature embryos abscisic acid strongly suppresses accumulation of B22E mRNA. The B22E transcript is absent from mature embryos, but rapidly reappears after germination.