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PubMed2005

Topolins and hydroxylated thidiazuron derivatives are substrates of cytokinin O-glucosyltransferase with position specificity related to receptor recognition.

Glucosides of trans-zeatin occur widely in plant tissues, formed either by O-glucosylation of the hydroxylated side chain or N-glucosylation of the purine ring structure. O-Glucosylation is stereo-specific: the O-glucosyltransferase encoded by the Phaseolus lunatus ZOG1 gene has high affinity for trans-zeatin as the substrate, whereas the enzyme encoded by the maize (Zea mays) cisZOG1 gene prefers cis-zeatin. Here we show that hydroxylated derivatives of benzyladenine (topolins) are also substrates of ZOG1 and cisZOG1. The m-OH and o-OH derivatives are the preferred substrate of ZOG1 and cisZOG1, respectively. Among the hydroxylated derivatives of thidiazuron tested, the only enzyme/substrate combination resulting in conversion was cisZOG1/(o-OH) thidiazuron. The abilities of these cytokinins to serve as substrates to the glucosyltransferases were in a large part correlated with their biological activities in the P. lunatus callus bioassay, indicating that there may be similarities between cytokinin-binding sites on the enzymes and cytokinin receptors. Further support for this interpretation is provided by cytokinin recognition studies involving the Arabidopsis (Arabidopsis thaliana) CRE1/WOL/AHK4 and maize ZmHK1 receptors. The AHK4 receptor responded to trans-zeatin and m-topolin, while the ZmHK1 receptor responded also to cis-zeatin and o-topolin. Three-dimensional molecular models of the substrates were applied to explain the results.

Adenine↗
PubMed2005

Regulation of photosynthetic GAPDH dissected by mutants.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants catalyzes an NADPH-consuming reaction, which is part of the Calvin cycle. This reaction is regulated by light via thioredoxins and metabolites, while a minor NADH-dependent activity is constant and constitutive. The major native isozyme is formed by A- and B-subunits in stoichiometric ratio (A2B2, A8B8), but tetramers of recombinant B-subunits (GapB) display similar regulatory features to A2B2-GAPDH. The C-terminal extension (CTE) of B-subunits is essential for thioredoxin-mediated regulation and NAD-induced aggregation to partially inactive oligomers (A8B8, B8). Deletion mutant B(minCTE) is redox insensitive and invariably tetrameric, and chimeric mutant A(plusCTE) acquired redox sensitivity and capacity to aggregate to very large oligomers in presence of NAD. Redox regulation principally affects the turnover number, without significantly changing the affinity for either 1,3-bisphosphoglycerate or NADPH. Mutant R77A of GapB, B(R77A), is down-regulated and mimics the behavior of oxidized GapB under any redox condition, whereas mutant B(E362Q) is constantly up-regulated, resembling reduced GapB. Despite their redox insensitivity, both B(R77A) and B(E362Q) mutants are notably prone to aggregate in presence of NAD. Based on structural data and current functional analysis, a model of GAPDH redox regulation is presented. Formation of a disulfide in the CTE induces a conformational change of the GAPDH with repositioning of the terminal amino acid Glu-362 in the proximity of Arg-77. The latter residue is thus distracted from binding the 2'-phosphate of NADP, with the final effect that the enzyme relaxes to a conformation leading to a slower NADPH-dependent catalytic activity.

Amino Acid Sequence↗
PubMed2002

A major light-harvesting polypeptide of photosystem II functions in thermal dissipation.

Under high-light conditions, photoprotective mechanisms minimize the damaging effects of excess light. A primary photoprotective mechanism is thermal dissipation of excess excitation energy within the light-harvesting complex of photosystem II (LHCII). Although roles for both carotenoids and specific polypeptides in thermal dissipation have been reported, neither the site nor the mechanism of this process has been defined precisely. Here, we describe the physiological and molecular characteristics of the Chlamydomonas reinhardtii npq5 mutant, a strain that exhibits little thermal dissipation. This strain is normal for state transition, high light-induced violaxanthin deepoxidation, and low light growth, but it is more sensitive to photoinhibition than the wild type. Furthermore, both pigment data and measurements of photosynthesis suggest that the photosystem II antenna in the npq5 mutant has one-third fewer light-harvesting trimers than do wild-type cells. The npq5 mutant is null for a gene designated Lhcbm1, which encodes a light-harvesting polypeptide present in the trimers of the photosystem II antennae. Based on sequence data, the Lhcbm1 gene is 1 of 10 genes that encode the major LHCII polypeptides in Chlamydomonas. Amino acid alignments demonstrate that these predicted polypeptides display a high degree of sequence identity but maintain specific differences in their N-terminal regions. Both physiological and molecular characterization of the npq5 mutant suggest that most thermal dissipation within LHCII of Chlamydomonas is dependent on the peripherally associated trimeric LHC polypeptides.

Algal Proteins↗
PubMed2002

Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae.

Pro has been shown to play an important role in ameliorating environmental stress in plants and microorganisms, including heavy metal stress. Here, we describe the effects of the expression of a mothbean delta(1)-pyrroline-5-carboxylate synthetase (P5CS) gene in the green microalga Chlamydomonas reinhardtii. We show that transgenic algae expressing the mothbean P5CS gene have 80% higher free-Pro levels than wild-type cells, grow more rapidly in toxic Cd concentrations (100 microM), and bind fourfold more Cd than wild-type cells. In addition, Cd-K edge extended x-ray absorption fine structure studies indicated that Cd does not bind to free Pro in transgenic algae with increased Pro levels but is coordinated tetrahedrally by sulfur of phytochelatin. In contrast to P5CS-expressing cells, Cd is coordinated tetrahedrally by two oxygen and two sulfur atoms in wild-type cells. Measurements of reduced/oxidized GSH ratios and analyses of levels of malondialdehyde, a product of the free radical damage of lipids, indicate that free Pro levels are correlated with the GSH redox state and malondialdehyde levels in heavy metal-treated algae. These results suggest that the free Pro likely acts as an antioxidant in Cd-stressed cells. The resulting increased GSH levels facilitate increased phytochelatin synthesis and sequestration of Cd, because GSH-heavy metal adducts are the substrates for phytochelatin synthase.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed2001

Identification of a mannitol transporter, AgMaT1, in celery phloem.

A celery petiole phloem cDNA library was constructed and used to identify a cDNA that gives Saccharomyces cerevisiae cells the ability to grow on mannitol and transport radiolabeled mannitol in a manner consistent with a proton symport mechanism. This cDNA was named AgMaT1 (Apium graveolens mannitol transporter 1). The expression profile in source leaves and phloem was in agreement with a role for mannitol in phloem loading in celery. The identification in eukaryotes of a mannitol transporter is important because mannitol is not only a primary photosynthetic product in species such as celery but is also considered a compatible solute and antioxidant implicated in resistance to biotic and abiotic stress.

Amino Acid Sequence↗
PubMed2005

Preservation of a transgenic strain of the sawfly, Athalia rosae (Hymenoptera) by artificial fertilization using cryopreserved sperm.

Germline transformation using a piggyBac-derived vector is feasible in the sawfly, Athalia rosae. A previously generated transgenic line carrying green fluorescence protein (GFP) genes as reporters was successfully maintained and preserved without consecutive rearing. Sperm taken from males that were frozen directly in liquid nitrogen and stored at -80 degrees C for a year were microinjected into mature unfertilized eggs dissected from female ovaries. A fraction of the sperm-injected eggs was fertilized and developed into diploid females, and all of them expressed GFP. Haploid male progeny from these females segregated into GFP-positive and GFP-negative individuals in a ratio of 1:1 indicating heterozygosity of the parental females. The GFP genes were stably inherited staying at the location where they were originally integrated.

Animals↗
PubMed2005

Transposable element insertion location bias and the dynamics of gene drive in mosquito populations.

Some vector-borne disease control strategies using transgenic mosquitoes require transgene spread to high frequency in populations. Transposable elements (TEs) are DNA sequences that replicate and transpose within the genomes of other organisms and may therefore be represented in the next generation in higher frequencies than predicted by Mendelian segregation. This over-representation has allowed some TEs to spread through natural populations. Transgenes incorporated within a TE sequence are expected to be driven into populations as long as there is a positive balance between fitness costs and over-representation. Models have been used to examine parameters that affect this balance but did not take into account biased insertion of TEs to linked sites in the genome. A simulation model was created to examine the impact of insertion bias on TE spread in mosquito populations. TEs that induce no fitness costs are predicted to increase in frequency over a wide range of parameter values but spread is slower for lower levels of transposition and non-local movement. If TEs are costly, high proportions of local movement can slow or halt spread. To function as a robust transgene drive mechanism a TE should replicate and transpose > 10%/insert/generation, induce < 1% fitness cost/insert, and move preferentially to unlinked sites in the genome.

Animals↗
PubMed2005

Development of food-grade cloning and expression vectors for Lactococcus lactis.

AIMS: To develop food-grade cloning and expression vectors for use in genetic modification of Lactococcus lactis. METHODS AND RESULTS: Two plasmid replicons and three dominant selection markers were isolated from L. lactis and used to construct five food-grade cloning vectors. These vectors were composed of DNA only from L. lactis and contained no antibiotic resistance markers. Three of the vectors (pND632, pND648 and pND969) were based on the same plasmid replicon and carried, either alone or in combination, the three different selectable markers encoding resistance to nisin, cadmium and/or copper. The other two (pND965DJ and pND965RS) were derived from a cadmium resistance plasmid, and carried a constitutive promoter and a copper-inducible promoter, respectively, immediately upstream of a multicloning site. All vectors were stable in L. lactis LM0230 for at least 40 generations without selection pressure. The two groups of vectors were compatible in L. lactis LM0230. The vectors pND648 and pND965RS, as representatives of the two groups, were transferred successfully by electroporation into and maintained in an industrial strain of L. lactis. The usefulness of the vectors was further demonstrated by expressing a phage resistance gene (abiI) in another industrial strain of L. lactis. CONCLUSIONS: The five food-grade vectors constructed are potentially useful for industrial strains of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: These vectors represent a new set of molecular tools useful for food-grade modifications of L. lactis.

Base Sequence↗
PubMed2005

Phenylacetic acid-producing Rhizoctonia solani represses the biosynthesis of nematicidal compounds in vitro and influences biocontrol of Meloidogyne incognita in tomato by Pseudomonas fluorescens strain CHA0 and its GM derivatives.

AIMS: The aim of the present investigation was to determine the influence of Rhizoctonia solani and its pathogenicity factor on the production of nematicidal agent(s) by Pseudomonas fluorescens strain CHA0 and its GM derivatives in vitro and nematode biocontrol potential by bacterial inoculants in tomato. METHODS AND RESULTS: One (Rs7) of the nine R. solani isolates from infected tomato roots inhibited seedling emergence and caused root rot in tomato. Thin layer chromatography revealed that culture filtrates of two isolates (Rs3 and Rs7) produced brown spots at Rf-values closely similar to synthetic phenylacetic acid (PAA), a phytotoxic factor. Filtrates from isolate Rs7, amended with the growth medium of P. fluorescens, markedly repressed nematicidal activity and PhlA'-'LacZ reporter gene expression of the bacteria in vitro. On the contrary, isolate Rs4 enhanced nematicidal potential of a 2,4-diacetylphloroglucinol overproducing mutant, CHA0/pME3424, of P. fluorescens strain CHA0 in vitro. Therefore, R. solani isolates Rs4 and Rs7 were tested more rigorously for their potential to influence biocontrol effectiveness of the bacterial agents. Methanol extract of the culture filtrates of PAA-producing isolate Rs7 resulting from medium amended with phenylalanine enhanced fungal repression of the production of nematicidal agents by bacteria, while amendments with zinc or molybdenum eliminated such fungal repression, thereby restoring bacterial potential to cause nematode mortality in vitro. A pot experiment was carried out, 3-week-old tomato seedlings were infested with R. solani isolates Rs4 or Rs7 and/or inoculated with Meloidogyne incognita, the root-knot nematode. The infested soil was treated with aqueous cell suspensions (10(8) CFU) of P. fluorescens strain CHA0 or its GM derivatives or left untreated (as a control). Observations taken 45 days after nematode inoculation revealed that, irrespective of the bacterial treatments, galling intensity per gram of fresh tomato roots was markedly higher in soil amended with isolate Rs4 than in Rs7-amended soils. Soil amendments with R. solani and the bacterial antagonists resulted in substantial reductions of the number of galls per gram of root. These results are contradictory to those obtained under in vitro conditions where culture filtrates of PAA-positive Rs7 repressed the production of nematicidal compounds. Plants grown in Rs7-amended soils, with or without bacterial inoculants, had lesser shoot and root weights than plants grown in nonamended or Rs4-amended soils. Moreover, amendments with Rs7 substantially retarded root growth and produced necrotic lesions that reduced the number of entry sites for invasion and subsequent infection by nematodes. Populations of P. fluorescens in the tomato rhizosphere were markedly higher in Rs7-amended soils. CONCLUSIONS: PAA-producing virulent R. solani drastically affects the potential of P. fluorescens to cause death of M. incognita juveniles in vitro and influences bacterial effectiveness to suppress nematodes in tomato roots. SIGNIFICANCE AND IMPACT OF THE STUDY: As most agricultural soils are infested with root-infecting fungi, including R. solani, it is likely that some PAA-producing isolates of the fungus may also be isolated from such soils. The inhibitory effect of PAA-producing R. solani on the biosynthesis of nematicidal agent(s) critical in biocontrol may reduce or even eliminate the effectiveness of fluorescent pseudomonads against root-knot nematodes, both in nursery beds and in field conditions. Introduction of bacterial inoculants, for the control of any plant pathogen, should be avoided in soils infested with PAA-producing R. solani. Alternatively, the agents could be applied together with an appropriate quantity of fungicide or chemicals such as zinc to create an environment more favourable for bacterial biocontrol action.

Animals↗
PubMed2005

Construction of transgenic Bacillus mucilaginosus strain with improved phytase secretion.

AIMS: To construct a transgenic Bacillus mucilaginosus strain to increase the secretion capability of a wild-type isolate of B. mucilaginosus D4B1 to hydrolyse phytate phosphorus, which can be used as a microbial fertilizer in field application. METHODS AND RESULTS: We constructed a phytase secreting expression vector pSP43 with a mini-Tn5 transposon and a Aspergillus fumigatus phytase expression cassette. The vector pSP43 was successfully transferred into the wild-type B. mucilaginosus using the particle bombardment method, and three transgenic strains with a stable copy of phytase expression cassette integrated into the chromosome of the B. mucilaginosus by Tn5 transposition were selected. The phytase activity of the engineered strains increased 36-46-fold when compared with the wild-type strain of D4B1. CONCLUSIONS: The A. fumigatus phytase gene can be expressed under the direction of p43 promoter in B. mucilaginosus. The expression protein is secreted extracellularly and newly constructed strains showed a high phytase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: A transgenic Bacillus strain by the particle bombardment method was constructed.

6-Phytase↗
PubMed2004

piggyBac transformation of the New World screwworm, Cochliomyia hominivorax, produces multiple distinct mutant strains.

Sterile insect technique (SIT) programs are designed to eradicate pest species by releasing mass-reared, sterile insects into an infested area. The first major implementation of SIT was the New World Screwworm Eradication Program, which successfully eliminated the New World screwworm (NWS), Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), from the Continental US, Mexico and much of Central America. Ionizing radiation is currently used for sterilization, but transgenic insect techniques could replace this method, providing a safer, more cost-effective alternative. Genetic transformation methods have been demonstrated in NWS, and verified by Southern blot hybridization, PCR and sequencing of element insertion junctions. A lethal insertional mutation and enhancer detection-like phenotypic expression variations are presented and discussed. In addition to supporting the eradication efforts, transformation methods offer potential means to identify genes and examine gene function in NWS.

Animals↗
PubMed2004

Allelic polymorphisms in apical membrane antigen-1 are responsible for evasion of antibody-mediated inhibition in Plasmodium falciparum.

Apical membrane antigen-1 (AMA-1) is a target of antibodies that inhibit invasion of Plasmodium falciparum into human erythrocytes and is a candidate for inclusion in a malaria vaccine. We have identified a line of P. falciparum (W2mef) less susceptible to anti-AMA1 antibodies raised to the protein from a heterologous parasite line (3D7). We have constructed transgenic P. falciparum expressing heterologous AMA-1 alleles. In vitro invasion assays show that these transgenic parasites differ from parental lines in susceptibility to inhibitory antibodies, providing direct evidence that sequence polymorphisms within AMA-1 are responsible for evasion of immune responses that inhibit parasite invasion. We also generated a parasite line that would express a chimeric AMA-1 protein, in which highly polymorphic residues within domain 1 were exchanged. Inhibition assays suggest that these residues are not sufficient for inhibition by invasion-blocking antibodies. This study is the first to use P. falciparum allelic exchange to examine the relationship between genetic diversity and susceptibility to protective antibodies. The findings have important implications for the development of an AMA-1-based malaria vaccine.

Alleles↗
PubMed2004

AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis.

For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K(0.5) = 416 microm) for Ala-Asp compared with Ala-Ala (K(0.5) = 54 microm) and Ala-Lys (K(0.5) = 112 microm). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides.

Arabidopsis↗
PubMed2004

Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas.

RNA interference (RNAi), the double-stranded RNA (dsRNA) triggered post-transcriptional gene silencing, is becoming a powerful tool for reverse genetics studies. Stable RNAi, induced by the expression of inverted repeat (IR) transgenes, has been achieved in protozoa, algae, fungi, plants, and metazoans. However, the level of gene silencing is often quite variable, depending on the type of construct, transgene copy number, site of integration, and target gene. This is a hindrance in functional genomics studies, where it is desirable to suppress target genes reliably to analyze unknown phenotypes. Consequently, we explored strategies for direct selection of effective transgenic RNAi lines in Chlamydomonas reinhardtii. We initially attempted to suppress expression of the Rubisco small subunit multigene family by placing an IR, homologous to the conserved coding sequence, in the 3'UTR of a transgene conferring resistance to bleomycin. However, this approach was fairly inefficient at inducing RNAi as many strains displayed defective transgene integration, resulting in partial or complete deletion of the IR, or low levels of dsRNA expression, presumably due to transcriptional silencing of the integrated IR transgenes. To overcome these problems we designed a system consisting of tandem IR transgenes that consistently triggered co-silencing of a gene with a selectable RNAi-induced phenotype (encoding tryptophan synthase beta subunit) and another gene of interest (encoding either Ku80, an RNA-binding protein, or a thioredoxin isoform). We anticipate that this approach will be useful for generating stable hypomorphic epi-mutants in high-throughput phenotypic screens.

Animals↗
PubMed2004

Potassium carrier TRH1 is required for auxin transport in Arabidopsis roots.

Disruption of the TRH1 potassium transporter impairs root hair development in Arabidopsis, and also affects root gravitropic behaviour. Rescue of these morphological defects by exogenous auxin indicates a link between TRH1 activity and auxin transport. In agreement with this hypothesis, the rate of auxin translocation from shoots to roots and efflux of [3H]IAA in isolated root segments were reduced in the trh1 mutant, but efflux of radiolabelled auxin was accelerated in yeast cells transformed with the TRH1 gene. In roots, Pro(TRH1):GUS expression was localized to the root cap cells which are known to be the sites of gravity perception and are central for the redistribution of auxin fluxes. Consistent with these findings, auxin-dependent DR5:GUS promoter-reporter construct was misexpressed in the trh1 mutant indicating that partial block of auxin transport through the root cap is associated with upstream accumulation of the phytohormone in protoxylem cells. When [K+] in the medium was reduced from 20 to 0.1 mm, wild type roots showed mild agravitropic phenotype and DR5:GUS misexpression in stelar cells. This pattern of response to low external [K+] was also affected by trh1 mutation. We conclude that the TRH1 carrier is an important part of auxin transport system in Arabidopsis roots.

Arabidopsis↗