Downy Mildew Resistance in Arabidopsis by Mutation of HOMOSERINE KINASE

Plant disease resistance is commonly triggered by early pathogen recognition and activation of immunity. An alternative form of resistance is mediated by recessive downy mildew resistant 1 (dmr1) alleles in Arabidopsis thaliana. Map-based cloning revealed that DMR1 encodes homoserine kinase (HSK). Six independent dmr1 mutants each carry a different amino acid substitution in the HSK protein. Amino acid analysis revealed that dmr1 mutants contain high levels of homoserine that is undetectable in wild-type plants. Surprisingly, the level of amino acids downstream in the aspartate (Asp) pathway was not reduced in dmr1 mutants. Exogenous homoserine does not directly affect pathogen growth but induces resistance when infiltrated in Arabidopsis. We provide evidence that homoserine accumulation in the chloroplast triggers a novel form of downy mildew resistance that is independent of known immune responses.     

Balance between myogenic,flow-dependent,and metabolic flow control in coronary arterial tree: a model study

Myogenic response, flow-dependent dilation, and direct metabolic control are important mechanisms controlling coronary flow. A model was developed to study how these control mechanisms interact at different locations in the arteriolar tree and to evaluate their contribution to autoregulatory and metabolic flow control. The model consists of 10 resistance compartments in series, each representing parallel vessel units, with their diameters determined by tone depending on either flow and pressure [flow-dependent tone reduction factor (TRF(flow)) x Tone(myo)] or directly on metabolic factors (Tone(meta)). The pressure-Tone(myo) and flow-TRF(flow) relations depend on the vessel size obtained from interpolation of data on isolated vessels. Flow-dependent dilation diminishes autoregulatory properties compared with pressure-flow lines obtained from vessels solely influenced by Tone(myo). By applying Tone(meta) to the four distal compartments, the autoregulatory properties are restored and tone is equally distributed over the compartments. Also, metabolic control and blockage of nitric oxide are simulated. We conclude that a balance is required between the flow-dependent properties upstream and the constrictive metabolic properties downstream. Myogenic response contributes significantly to flow regulation.     

Role of endogenous nitric oxide in endotoxin-induced alteration of hypoxic pulmonary vasoconstriction in mice

Pulmonary vasoconstriction in response to alveolar hypoxia (HPV) is frequently impaired in patients with sepsis or acute respiratory distress syndrome or in animal models of endotoxemia. Pulmonary vasodilation due to overproduction of nitric oxide (NO) by NO synthase 2 (NOS2) may be responsible for this impaired HPV after administration of endotoxin (LPS). We investigated the effects of acute nonspecific (N(G)-nitro-L-arginine methyl ester, L-NAME) and NOS2-specific [L-N6-(1-iminoethyl)lysine, L-NIL] NOS inhibition and congenital deficiency of NOS2 on impaired HPV during endotoxemia. The pulmonary vasoconstrictor response and pulmonary vascular pressure-flow (P-Q) relationship during normoxia and hypoxia were studied in isolated, perfused, and ventilated lungs from LPS-pretreated and untreated wild-type and NOS2-deficient mice with and without L-NAME or L-NIL added to the perfusate. Compared with lungs from untreated mice, lungs from LPS-challenged wild-type mice constricted less in response to hypoxia (69 +/- 17 vs. 3 +/- 7%, respectively, P < 0.001). Perfusion with L-NAME or L-NIL restored this blunted HPV response only in part. In contrast, LPS administration did not impair the vasoconstrictor response to hypoxia in NOS2-deficient mice. Analysis of the pulmonary vascular P-Q relationship suggested that the HPV response may consist of different components that are specifically NOS isoform modulated in untreated and LPS-treated mice. These results demonstrate in a murine model of endotoxemia that NOS2-derived NO production is critical for LPS-mediated development of impaired HPV. Furthermore, impaired HPV during endotoxemia may be at least in part mediated by mechanisms other than simply pulmonary vasodilation by NOS2-derived NO overproduction.     

Identification of arabidopsis loci required for susceptibility to the downy mildew pathogen Hyaloperonospora parasitica

Plants are susceptible to a limited number of pathogens. Most infections fail due to active defense or absence of compatibility. Many components of the plant's surveillance system and defense arsenal have been identified in the last decades. However, knowledge is limited on compatibility; in particular, the role of plant factors in the infection process. To gain insight into these processes, we have initiated an Arabidopsis thaliana mutant screen for reduced susceptibility to the downy mildew pathogen Hyaloperonospora parasitica. Ethyl methane sulfonate (EMS) mutants were generated in the highly susceptible Arabidopsis line Ler eds1-2. Eight downy mildew-resistant (dmr) mutants were analyzed in detail, corresponding to six different loci. Microscopic analysis showed that, in all mutants, H. parasitica growth was severely reduced. Resistance of dmr3, dmr4, and dmr5 was associated with constitutive expression of PR-1. Furthermore, dmr3 and dmr4, but not dmr5, also were resistant to Pseudomonas syringae and Golovinomyces orontii, respectively. However, enhanced activation of plant defense was not observed in dmr1, dmr2, and dmr6. We postulate that, in these susceptibility mutants, cellular processes are disrupted which are required for H. parasitica infection. This interesting new set of mutants provides a basis to elucidate the molecular processes underlying susceptibility to downy mildew in Arabidopsis.     

Microsatellite typing of the rhesus macaque MHC region

To improve the results gained by serotyping rhesus macaque major histocompatibility complex (MHC) antigens, molecular typing techniques have been established for class I and II genes. Like the rhesus macaque Mamu-DRB loci, the Mamu-A and -B are not only polymorphic but also polygenic. As a consequence, sequence-based typing of these genes is time-consuming. Therefore, eight MHC-linked microsatellites, or short tandem repeats (STRs), were evaluated for their use in haplotype characterization. Polymorphism analyses in rhesus macaques of Indian and Chinese origin showed high STR allelic diversity in both populations but different patterns of allele frequency distribution between the groups. Pedigree data for class I and II loci and the eight STRs allowed us to determine extended MHC haplotypes in rhesus macaque breeding groups. STR sequencing and comparisons with the complete rhesus macaque MHC genomic map allowed the exact positioning of the markers. Strong linkage disequilibria were observed between Mamu-DR and -DQ loci and adjacent STRs. Microsatellite typing provides an efficient, robust, and quick method of genotyping and deriving MHC haplotypes for rhesus macaques regardless of their geographical origin. The incorporation of MHC-linked STRs into routine genetic tests will contribute to efforts to improve the genetic characterization of the rhesus macaque for biomedical research and can provide comparative information about the evolution of the MHC region.     

Flow cytometric analysis of polysomaty and in vitro genetic instability in potato

The analysis of nuclear DNA contents in various tissues of potato genotypes showed that flow cytometry is a rapid method to characterize large populations of cells for polysomaty, that is, the occurrence of cells with normal DNA levels together with cells containing endoreduplicated nuclei. The proportion of endoreduplicated nuclei varied in different tissues and genotypes of potato. The analysis of callus and cell cultures showed that the temporal changes in nuclear DNA contents during in vitro growth can be followed and the degree of polyploidization quantified. It is concluded that flow cytometry is a highly suitable method to detect ploidy changes in differentiated plant tissues and calli which are often not amenable for chromosome number determination.     

Efficient induction by amiprophos-methyl and flow-cytometric sorting of micronuclei in Nicotiana plumbaginifolia

Amiprophos-methyl (APM) is a potential herbicide which acts at the level of microtubules. By exposure of suspension cells of Nicotiana plumbaginifolia to this agent, a high degree of metaphase arrest was observed and single as well as groups of chromosomes were scattered throughout the cell, offering good prospects for application in cytology and chromosome isolation. After prolonged exposure to the drug, the chromosomes decondensed and micronuclei were formed. Based on their DNA content, the micronuclei were sorted by flow cytometry. Prospects for application of isolated micronuclei for partial genome transfer and gene mapping are discussed.Abbreviation APM amiprophos-methyl     

Application of enzymehistochemical methods to isolated subcellular fractions and to sucrose-ficoll density gradients

Summary To compare histochemical and biochemical determinations of enzyme activities, enzymehistochemical procedures are applied to sections of pellets of subcellular fractions. These investigations are of value to determine the subcellular localization of histochemically demonstrable enzyme activities and to test the homogeneity of an isolated fraction. In homogenating duckling liver a great part of the endothelial cells is not destructed and consequently is found in the nuclear fraction. Kupffer cell lysosomes land in the heavy mitochondrial fraction, whereas hepatocyte lysosomes are chiefly found in the light mitochondrial fraction. -Glucuronidase activity shows a preferentially microsomal localization. Application of enzymehistochemical staining reactions to discontinuous gradients and comparison with biochemical data provides additional information about the validity of an enzymehistochemical reaction. In rat liver the tetrazolium reductases show a distinctly dual localization: activity in the mitochondrial band and in microsomal bands. As to their localization in different bands of the gradients non-specific esterases demonstrate a clear pH-dependency.     

Mentor pollen effects on gametophytic incompatibility in Nicotiana,Oenothera and Lycopersicum

Summary Attempts were made, through mentor pollen techniques, to overcome self-incompatibility in species belonging to the genera Nicotiana and Oenothera and in a hybrid of Lycopersicum, which are characterized by a gametophytic system of incompatibility. While radiation-killed incompatible pollen did not generate mentor effects in any of the material tested, radiation-killed compatible pollen was found to promote a high level of illegitimate fertilizations by incompatible pollen in N. alata. No evidence was obtained that radiation-killed compatible pollen could induce mentor effects in strictly self-incompatible clones of O. organensis and of the interspecific hybrid L. esculentum x L. peruvianum.This publication is contribution no 1563 from the Biology Radio-protection and Medical Research programme of the Directorate General XII of the Commission of the European Communities