Host range and molecular and ultrastructural analyses of Asparagus virus 1 pathotypes isolated from garden asparagus Asparagus officinalis L.

Asparagus samples were examined from growing areas of Germany and selected European as well as North, Central and South American countries. Overall, 474 samples were analyzed for Asparagus virus 1 (AV1) using DAS-ELISA. In our survey, 19 AV1 isolates were further characterized. Experimental transmission to 11 species belonging to Aizoaceae, Amarantaceae, Asparagaceae, and Solanaceae succeeded. The ultrastructure of AV1 infection in asparagus has been revealed and has been compared with the one in indicator plants. The cylindrical inclusion (CI) protein, a core factor in viral replication, localized within the cytoplasm and in systemic infections adjacent to the plasmodesmata. The majority of isolates referred to pathotype I (PI). These triggered a hypersensitive resistance in inoculated leaves of Chenopodium spp. and were incapable of infecting Nicotiana spp. Only pathotype II (PII) and pathotype III (PIII) infected Nicotiana benthamiana systemically but differed in their virulence when transmitted to Chenopodium spp. The newly identified PIII generated amorphous inclusion bodies and degraded chloroplasts during systemic infection but not in local lesions of infected Chenopodium spp. PIII probably evolved via recombination in asparagus carrying a mixed infection by PI and PII. Phylogeny of the coat protein region recognized two clusters, which did not overlap with the CI-associated grouping of pathotypes. These results provide evidence for ongoing modular evolution of AV1.

Csf1r mediates enhancement of intestinal tumorigenesis caused by inactivation of Mir34a.

The CSF1 receptor (CSF1R) encoding mRNA represents a direct target of miR-34a. However, the in vivo relevance of the suppression of CSF1R by miR-34a for intestinal tumor suppression mediated by the p53/miR-34a pathway has remained unknown. Here, Apc Min/+ mice with intestinal-epithelial cell (IEC)-specific deletions of Mir34a showed increased formation of adenomas and decreased survival, whereas deletion of Csf1r decreased adenoma formation and increased survival. In adenomas deletion of Mir34a enhanced proliferation, STAT3 signaling, infiltration with fibroblasts, immune cells and microbes, and tumor stem cell abundance and decreased apoptosis. Deletion of Csf1r had the opposite effects. In addition, homeostasis of intestinal secretory and stem cells, and tumoroid formation were affected in opposite directions by deletion of Mir34a and CSF1R. Concomitant deletion of Csf1r and Mir34a neutralized the effects of the single deletions. mRNAs containing Mir34a seed-matching sites, which encode proteins related to EMT (epithelial-mesenchymal transition), stemness and Wnt signaling, were enriched after Mir34a inactivation in adenomas and derived tumoroids. Netrin-1/Ntn1 and Transgelin/Tagln were characterized as direct targets of Mir34a and Csf1r signaling. Mir34a-inactivation related expression signatures were associated with CMS4/CRISB+D, stage 4 CRCs and poor patient survival. In tumoroids the loss of Mir34a conferred resistance to 5-FU which was mediated by Csf1r. This study provides genetic evidence for a requirement of Mir34a-mediated Csf1r suppression for intestinal stem/secretory cell homeostasis and tumor suppression, and suggests that therapeutic targeting of CSF1R may be effective for the treatment of CRCs with defects in the p53/miR-34a pathway.

Transfer of the Dominant Virus Resistance Gene AV-1 pro From Asparagus prostratus to Chromosome 2 of Garden Asparagus A. officinalis L.

An introgression breeding programme was carried out to transfer the virus resistance gene AV-1 pro from the wild species Asparagus prostratus to the garden asparagus Asparagus officinalis. Serious crossing barriers caused by genetic distance and different ploidy levels of the crossing parents have been overcome using embryo rescue for the F1, BC1, and BC2 generations. The male and female fertility was widely restored in BC2 and was shown to be comparable to the cultivated asparagus. Five AV-1 resistant diploid (2n = 2x = 20) BC2 plants were selected and reciprocally backcrossed with asparagus cultivars. Segregation analyses of fourteen seedborne BC3 progenies suggested a monogenic dominant inheritance of the AV-1 resistance. Genotyping by sequencing analysis gave a strong hint for location of the resistance gene on asparagus Chromosome 2. Using an Axiom single nucleotide polymorphism (SNP) genotyping array for the analysis of three BC3 families with 10 AV-1 resistant and 10 AV-1 susceptible plants each, as well as 25 asparagus cultivars, the AV-1 pro locus on Chromosome 2 was further narrowed down. The SNP with the highest LOD score was converted to a kompetitive allele specific PCR (KASP) marker, shown to be useful for the further backcross programme and serving as the starting point for the development of a diagnostic marker.

Introgression of Two Broad-Spectrum Late Blight Resistance Genes, Rpi-Blb1 and Rpi-Blb3, From Solanum bulbocastanum Dun Plus Race-Specific R Genes Into Potato Pre-breeding Lines.

There is a wealth of resistance genes in the Mexican wild relative of cultivated Solanum, but very few of these species are sexually compatible with cultivated Solanum tuberosum. The most devastating disease of potato is late blight caused by the oomycete Phytophthora infestans (Pi). The wild hexaploid species S. demissum, which it is able to cross with potato, was used to transfer eleven race-specific genes by introgressive hybridization that were subsequently widely used in potato breeding. However, there are now more virulent races of Pi that can overcome all of these genes. The most sustainable strategy for protecting potatoes from late blight is to pyramid or stack broad-spectrum resistance genes into the cultivars. Recently four broad-spectrum genes (Rpi) conferring resistance to Pi were identified and cloned from the sexually incompatible species S. bulbocastanum: Rpi-blb1 (RB), Rpi-blb2, Rpi-blb3, and Rpi-bt1. For this research, a resistant S. bulbocastanum accession was selected carrying the genes Rpi-blb1 and Rpi-blb3 together with race-specific R3a and R3b genes. This accession was previously used to produce a large number of somatic hybrids (SHs) with five commercial potato cultivars using protoplast electrofusion. In this study, three SHs with cv. 'Delikat' were selected and backcross generations (i.e., BC1 and BC2) were obtained using cvs. 'Baltica', 'Quarta', 'Romanze', and 'Sarpo Mira'. Their assessment using gene-specific markers demonstrates that these genes are present in the SHs and their BC progenies. We identified plants carrying all four genes that were resistant to foliage blight in greenhouse and field trials. Functionality of the genes was shown by using agro-infiltration with the effectors of corresponding Avr genes. For a number of hybrids and BC clones yield and tuber number were not significantly different from that of the parent cultivar 'Delikat' in field trials. The evaluation of agronomic traits of selected BC2 clones and of their processing qualities revealed valuable material for breeding late blight durable resistant potato. We show that the combination of somatic hybridization with the additional use of gene specific markers and corresponding Avr effectors is an efficient approach for the successful identification and introgression of late blight resistance genes into the potato gene pool.

The conformational bases for the two functionalities of 2-cysteine peroxiredoxins as peroxidase and chaperone.

2-Cysteine peroxiredoxins (2-CysPrxs) are ubiquitous and highly abundant proteins that serve multiple functions as peroxidases, chaperones, and thiol oxidases and in redox-dependent cell signalling. The chloroplast protein plays a role in seedling development and protection of the photosynthetic apparatus. This study aimed to unequivocally link conformation and function. To this end, a set of non-tagged site-directed mutagenized At2-CysPrx variants was engineered, which mimicked the conformational states and their specific functions: hyperoxidized form (C54D), reduced form (C54S, C176S), oxidized form (C54DC176K), phosphorylated form (T92D), reduced ability for oligomerization by interfering with the dimer-dimer interface (F84R) and a C-terminally truncated form [ΔC (-20 aa)]. These variants were fully or partly fixed in their quaternary structure and function, respectively, and were analysed for their conformational state and peroxidase and chaperone activity, as well as for their sensitivity to hyperoxidation. The presence of a His6-tag strongly influenced the properties of the protein. The ΔC variant became insensitive to hyperoxidation, while T92D and F84R became more sensitive. The C54D variant revealed the highest chaperone activity. The highest peroxidase activity was observed for the F84R and ΔC variants. Efficient interaction with NADP-dependent thioredoxin reductase C depended on the presence of Cys residues and the C-terminal tail. The results suggest that the structural flexibility is important for the switch between peroxidase and chaperone function and that evolution has conserved the functional switch instead of maximizing a single function. These variants are ideal tools for future conformation-specific studies in vivo and in vitro.

Redox-mediated mechanisms regulate DNA binding activity of the G-group of basic region leucine zipper (bZIP) transcription factors in Arabidopsis.

Plant genes that contain the G-box in their promoters are responsive to a variety of environmental stimuli. Bioinformatics analysis of transcriptome data revealed that the G-box element is significantly enriched in promoters of high light-responsive genes. From nuclear extracts of high light-treated Arabidopsis plants, we identified the AtbZIP16 transcription factor as a component binding to the G-box-containing promoter fragment of light-harvesting chlorophyll a/b-binding protein2.4 (LHCB2.4). AtbZIP16 belongs to the G-group of Arabidopsis basic region leucine zipper (bZIP) type transcription factors. Although AtbZIP16 and its close homologues AtbZIP68 and AtGBF1 bind the G-box, they do not bind the mutated half-sites of the G-box palindrome. In addition, AtbZIP16 interacts with AtbZIP68 and AtGBF1 in the yeast two-hybrid system. A conserved Cys residue was shown to be necessary for redox regulation and enhancement of DNA binding activity in all three proteins. Furthermore, transgenic Arabidopsis lines overexpressing the wild type version of bZIP16 and T-DNA insertion mutants for bZIP68 and GBF1 demonstrated impaired regulation of LHCB2.4 expression. Finally, overexpression lines for the mutated Cys variant of bZIP16 provided support for the biological significance of Cys(330) in redox regulation of gene expression. Thus, our results suggest that environmentally induced changes in the redox state regulate the activity of members of the G-group of bZIP transcription factors.

Mechanisms and dynamics in the thiol/disulfide redox regulatory network: transmitters, sensors and targets.

Plant cells sense, weigh and integrate various endogenous and exogenous cues in order to optimize acclimation and resource allocation. The thiol/disulfide redox network appears to be in the core of this versatile integration process. In plant cells its complexity exceeds by far that of other organisms. Recent research has elucidated the multiplicity of the diversified input elements, transmitters (thioredoxin, glutaredoxins), targets and sensors (peroxiredoxins and other peroxidases), controlled processes and final acceptors (reactive oxygen species). An additional level of thiol/disulfide regulation is achieved by introducing dynamics in time and subcompartment and complex association.

Antitumor quinol PMX464 is a cytocidal anti-trypanosomal inhibitor targeting trypanothione metabolism.

Better drugs are urgently needed for the treatment of African sleeping sickness. We tested a series of promising anticancer agents belonging to the 4-substituted 4-hydroxycyclohexa-2,5-dienones class ("quinols") and identified several with potent trypanocidal activity (EC(50) < 100 nM). In mammalian cells, quinols are proposed to inhibit the thioredoxin/thioredoxin reductase system, which is absent from trypanosomes. Studies with the prototypical 4-benzothiazole-substituted quinol, PMX464, established that PMX464 is rapidly cytocidal, similar to the arsenical drug, melarsen oxide. Cell lysis by PMX464 was accelerated by addition of sublethal concentrations of glucose oxidase implicating oxidant defenses in the mechanism of action. Whole cells treated with PMX464 showed a loss of trypanothione (T(SH)(2)), a unique dithiol in trypanosomes, and tryparedoxin peroxidase (TryP), a 2-Cys peroxiredoxin similar to mammalian thioredoxin peroxidase. Enzyme assays revealed that T(SH)(2), TryP, and a glutathione peroxidase-like tryparedoxin-dependent peroxidase were inhibited in time- and concentration-dependent manners. The inhibitory activities of various quinol analogues against these targets showed a good correlation with growth inhibition of Trypanosoma brucei. The monothiols glutathione and L-cysteine bound in a 2:1 ratio with PMX464 with K(d) values of 6 and 27 µM, respectively, whereas T(SH)(2) bound more tightly in a 1:1 ratio with a K(d) value of 430 nM. Overexpression of trypanothione synthetase in T. brucei decreased sensitivity to PMX464 indicating that the key metabolite T(SH)(2) is a target for quinols. Thus, the quinol pharmacophore represents a novel lead structure for the development of a new drug against African sleeping sickness.

Dynamic translocation of ligand-complexed DNA through solid-state nanopores with optical tweezers.

We investigated the threading and controlled translocation of individual lambda-DNA (λ-DNA) molecules through solid-state nanopores with piconewton force sensitivity, millisecond time resolution and picoampere ionic current sensitivity with a set-up combining quantitative 3D optical tweezers (OT) with electrophysiology. With our virtually interference-free OT set-up the binding of RecA and single peroxiredoxin protein molecules to λ-DNA was quantitatively investigated during dynamic translocation experiments where effective forces and respective ionic currents of the threaded DNA molecule through the nanopore were measured during inward and outward sliding. Membrane voltage-dependent experiments of reversible single protein/DNA translocation scans yield hysteresis-free, asymmetric single-molecule fingerprints in the measured force and conductance signals that can be attributed to the interplay of optical trap and electrostatic nanopore potentials. These experiments allow an exact localization of the bound protein along the DNA strand and open fascinating applications for label-free detection of DNA-binding ligands, where structural and positional binding phenomena can be investigated at a single-molecule level.

Structural and mechanistic insights into type II trypanosomatid tryparedoxin-dependent peroxidases.

TbTDPX (Trypanosoma brucei tryparedoxin-dependent peroxidase) is a genetically validated drug target in the fight against African sleeping sickness. Despite its similarity to members of the GPX (glutathione peroxidase) family, TbTDPX2 is functional as a monomer, lacks a selenocysteine residue and relies instead on peroxidatic and resolving cysteine residues for catalysis and uses tryparedoxin rather than glutathione as electron donor. Kinetic studies indicate a saturable Ping Pong mechanism, unlike selenium-dependent GPXs, which display infinite K(m) and V(max) values. The structure of the reduced enzyme at 2.1 A (0.21 nm) resolution reveals that the catalytic thiol groups are widely separated [19 A (0.19 nm)] and thus unable to form a disulphide bond without a large conformational change in the secondary-structure architecture, as reported for certain plant GPXs. A model of the oxidized enzyme structure is presented and the implications for small-molecule inhibition are discussed.

A comparative study of type I and type II tryparedoxin peroxidases in Leishmania major.

The genome of Leishmania major, the causative agent of cutaneous leishmaniasis, contains three almost identical genes encoding putative glutathione peroxidases, which differ only at their N- and C-termini. Because the gene homologues are essential in trypanosomes, they may also represent potential drug targets in Leishmania. Recombinant protein for the shortest of these showed negligible peroxidase activity with glutathione as the electron donor indicating that it is not a bone fide glutathione peroxidase. By contrast, high peroxidase activity was obtained with tryparedoxin, indicating that these proteins belong to a new class of monomeric tryparedoxin-dependent peroxidases (TDPX) distinct from the classical decameric 2-Cys peroxiredoxins (TryP). Mass spectrometry studies revealed that oxidation of TDPX1 with peroxides results in the formation of an intramolecular disulfide bridge between Cys35 and Cys83. Site-directed mutagenesis and kinetic studies showed that Cys35 is essential for peroxidase activity, whereas Cys83 is essential for reduction by tryparedoxin. Detailed kinetic studies comparing TDPX1 and TryP1 showed that both enzymes obey saturation ping-pong kinetics with respect to tryparedoxin and peroxide. Both enzymes show high affinity for tryparedoxin and broad substrate specificity for hydroperoxides. TDPX1 shows higher affinity towards hydrogen peroxide and cumene hydroperoxide than towards t-butyl hydroperoxide, whereas no specific substrate preference could be detected for TryP1. TDPX1 exhibits rate constants up to 8 x 10(4) m(-1).s(-1), whereas TryP1 exhibits higher rate constants approximately 10(6) m(-1).s(-1). All three TDPX proteins together constitute approximately 0.05% of the L. major promastigote protein content, whereas the TryPs are approximately 40 times more abundant. Possible specific functions of TDPXs are discussed.

Role of the cysteine residues in Arabidopsis thaliana cyclophilin CYP20-3 in peptidyl-prolyl cis-trans isomerase and redox-related functions.

Cyps (cyclophilins) are ubiquitous proteins of the immunophilin superfamily with proposed functions in protein folding, protein degradation, stress response and signal transduction. Conserved cysteine residues further suggest a role in redox regulation. In order to get insight into the conformational change mechanism and functional properties of the chloroplast-located CYP20-3, site-directed mutagenized cysteine-->serine variants were generated and analysed for enzymatic and conformational properties under reducing and oxidizing conditions. Compared with the wild-type form, elimination of three out of the four cysteine residues decreased the catalytic efficiency of PPI (peptidyl-prolyl cis-trans isomerase) activity of the reduced CYP20-3, indicating a regulatory role of dithiol-disulfide transitions in protein function. Oxidation was accompanied by conformational changes with a predominant role in the structural rearrangement of the disulfide bridge formed between Cys(54) and Cys(171). The rather negative E(m) (midpoint redox potential) of -319 mV places CYP20-3 into the redox hierarchy of the chloroplast, suggesting the activation of CYP20-3 in the light under conditions of limited acceptor availability for photosynthesis as realized under environmental stress. Chloroplast Prx (peroxiredoxins) were identified as interacting partners of CYP20-3 in a DNA-protection assay. A catalytic role in the reduction of 2-Cys PrxA and 2-Cys PrxB was assigned to Cys(129) and Cys(171). In addition, it was shown that the isomerization and disulfide-reduction activities are two independent functions of CYP20-3 that both are regulated by the redox state of its active centre.

Reaction mechanism of plant 2-Cys peroxiredoxin. Role of the C terminus and the quaternary structure.

Barley 2-cysteine peroxiredoxin (2-Cys Prx) was analyzed for peroxide reduction, quaternary structure, thylakoid attachment, and function as well as in vivo occurrence of the inactivated form, with emphasis on the role of specific amino acid residues. Data presented show the following. 1) 2-Cys Prx has a broad substrate specificity and reduces even complex lipid peroxides such as phosphatidylcholine dilineoyl hydroperoxide, although at low rates. 2) 2-Cys Prx partly becomes irreversibly oxidized by peroxide substrates during the catalytic cycle in a concentration-dependent manner, particularly by bulky hydroperoxides. 3) Using dithiothreitol and thioredoxin (Trx) as reductants, amino acids were identified that are important for peroxide reduction (Cys64, Arg140, and Arg163), regeneration by Trx (Cys185), and conformation changes from dimer to oligomer (Thr66, Trp99, and Trp189). 4) Oligomerization decreased the rate of Trx-dependent peroxide detoxification. 5) Comparison of PrxWT, W99L, and W189L using static and time-resolved LIF techniques demonstrated the contributions of the tryptophan residues and yielded information about their local environment. Data indicated protein dynamics in the catalytic site and the carboxyl terminus during the reduction-oxidation cycle. 6) Reduced and inactivated barley 2-Cys Prx oligomerized and attached to the thylakoid membrane in isolated chloroplasts. The in vivo relevance of inactivation was shown in leaves subjected to cold and wilting stress and during senescence. Based on these results, it is hypothesized that in addition to its function in peroxide detoxification, 2-Cys Prx may play a role as a structural redox sensor in chloroplasts.

Divergent light-, ascorbate-, and oxidative stress-dependent regulation of expression of the peroxiredoxin gene family in Arabidopsis.

Peroxiredoxins (prxs) are peroxidases with broad substrate specificity. The seven prx genes expressed in Arabidopsis shoots were analyzed for their expressional response to changing photon fluence rates, oxidative stress, and ascorbate application. The results reveal a highly variable and gene-specific response to reducing and oxidizing conditions. The steady-state transcript amounts of the chloroplast-targeted prxs, namely the two-cysteine (2-Cys) prxs, prx Q and prx II E, decreased upon application of ascorbate. prx Q also responded to peroxides and diamide treatment. prx II B was induced by tertiary butylhydroperoxide, but rather unaffected by ascorbate. The strongest responses were observed for prx II C, which was induced with all treatments. The two Arabidopsis 2-Cys Prxs and four Prx II proteins were expressed heterologously in Escherichia coli. In an in vitro test system, they all showed peroxidase activity, but could be distinguished by their ability to accept dithiothreitol and thioredoxin as electron donor in the regeneration reaction. The midpoint redox potentials (E(m)') of Prx II B, Prx II C, and Prx II E were around -290 mV and, thus, less negative than E(m)' of Prx II F, 2-Cys Prx A, and 2-Cys Prx B (-307 to -322 mV). The data characterize expression and function of the mitochondrial Prx II F and the chloroplast Prx II E for the first time, to our knowledge. Antibodies directed against 2-Cys Prx and Prx II C showed a slight up-regulation of Prx II protein in strong light and of 2-Cys Prx upon transfer both to high and low light. The results are discussed in context with the subcellular localization of the Prx gene products.

The plant-specific function of 2-Cys peroxiredoxin-mediated detoxification of peroxides in the redox-hierarchy of photosynthetic electron flux.

The 2-cysteine peroxiredoxins (2-Cys Prx) constitute an ancient family of peroxide detoxifying enzymes and have acquired a plant-specific function in the oxygenic environment of the chloroplast. Immunocytochemical analysis and work with isolated intact chloroplasts revealed a reversible binding of the oligomeric form of 2-Cys Prx to the thylakoid membrane. The oligomeric form of the enzyme was enhanced under stress. The 2-Cys Prx has a broad substrate specificity with activity toward hydrogen peroxides and complex alkyl hydroperoxides. During the peroxide reduction reaction, 2-Cys Prx is alternatively oxidized and reduced as it catalyzes an electron flow from an electron donor to peroxide. Escherichia coli thioredoxin, but also spinach thioredoxin f and m were able to reduce oxidized 2-Cys Prx. The midpoint redox potential of -315 mV places 2-Cys Prx reduction after Calvin cycle activation and before switching the malate valve for export of excess reduction equivalents to the cytosol. Thus the 2-Cys Prx has a defined and preferential place in the hierarchy of photosynthetic electron transport. The activity of 2-Cys Prx also is linked to chloroplastic NAD(P)H metabolism as indicated by the presence of the reduced form of the enzyme after feeding dihydroxyacetone phosphate to intact chloroplasts. The function of the 2-Cys Prx is therefore not confined to its role in the water-water cycle pathway for energy dissipation in photosynthesis but also mediates peroxide detoxification in the plastids during the dark phase.