Transposon tagging of the sulfur gene of tobacco using engineered maize Ac/Ds elements.

The Sulfur gene of tobacco is nuclearly encoded. A Su allele at this locus acts as a dominant semilethal mutation and causes reduced accumulation of chlorophyll, resulting in a yellow color in the plant. An engineered transposon tagging system, based upon the maize element Ac/Ds, was used to mutate the gene. High frequency of transposon excision from the Su locus produced variegated sectors. Plants regenerated from the variegated sector exhibited a similar variegated phenotype. Genetic analyses showed that the variegation was always associated with the transposase construct and the transposon was linked to the Su locus. Sequences surrounding the transposon were isolated, and five revertant sectors possessed typical direct repeats following Ds excisions. These genetic and molecular data are consistent with the tagging of the Su allele by the transposon.

Gene silencing from plant DNA carried by a Geminivirus.

The geminivirus tomato golden mosaic virus (TGMV) replicates in nuclei and expresses genes from high copy number DNA episomes. The authors used TGMV as a vector to determine whether episomal DNA can cause silencing of homologous, chromosomal genes. Two markers were used to asses silencing: (1) the sulfur allele (su) of magnesium chelatase, an enzyme required for chlorophyll formation; and (2) the firefly luciferase gene (luc). Various portions of both marker genes were inserted into TGMV in place of the coat protein open-reading frame and the constructs were introduced into intact plants using particle bombardment. When TGMV vectors carrying fragments of su (TGMV::su) were introduced into leaves of wild type Nicotiana benthamiana, circular, yellow spots with an area of several hundred cells formed after 3-5 days. Systemic movement of TGMV::su subsequently produced varigated leaf and stem tissue. Fragments that caused silencing included a 786 bp 5' fragment of the 1392 bp su cDNA in sense and anti-sense orientation, and a 403 bp 3' fragment. TGMV::su-induced silencing was propogated through tissue culture, along with the viral episome, but was not retained through meiosis. Systemic downregulation of a constitutively expresse luciferase transgene in plants was achieved following infection with TGMV vectors carrying a 623 bp portion of luc in sense or anti-sense orientation. These results establish that homologous DNA sequences localized in nuclear episomes can modulate the expression of active chromosomal genes.

A triggered-suicide system designed as a defense against bacteriophages.

A novel bacteriophage protection system for Lactococcus lactis based on a genetic trap, in which a strictly phage-inducible promoter isolated from the lytic phage phi31 is used to activate a bacterial suicide system after infection, was developed. The lethal gene of the suicide system consists of the three-gene restriction cassette LlaIR+, which is lethal across a wide range of gram-positive bacteria. The phage-inducible trigger promoter (phi31P) and the LlaIR+ restriction cassette were cloned in Escherichia coli on a high-copy-number replicon to generate pTRK414H. Restriction activity was not apparent in E. coli or L. lactis prior to phage infection. In phage challenges of L. lactis(pTRK414H) with phi31, the efficiency of plaquing was lowered to 10(-4) and accompanied by a fourfold reduction in burst size. Center-of-infection assays revealed that only 15% of infected cells released progeny phage. In addition to phage phi31, the phi31P/LlaIR+ suicide cassette also inhibited four phi31-derived recombinant phages at levels at least 10-fold greater than that of phi31. The phi31P/LlaIR+-based suicide system is a genetically engineered form of abortive infection that traps and eliminates phages potentially evolving in fermentation environments by destroying the phage genome and killing the propagation host. This type of phage-triggered suicide system could be designed for any bacterium-phage combination, given a universal lethal gene and an inducible promoter which is triggered by the infecting bacteriophage.

Sugar Repression of Mannitol Dehydrogenase Activity in Celery Cells.

We present evidence that the activity of the mannitol-catabolizing enzyme mannitol dehydrogenase (MTD) is repressed by sugars in cultured celery (Apium graveolens L.) cells. Furthermore, this sugar repression appears to be mediated by hexokinases (HKs) in a manner comparable to the reported sugar repression of photosynthetic genes. Glucose (Glc)-grown cell cultures expressed little MTD activity during active growth, but underwent a marked increase in MTD activity, protein, and RNA upon Glc starvation. Replenishment of Glc in the medium resulted in decreased MTD activity, protein, and RNA within 12 h. Addition of mannoheptulose, a competitive inhibitor of HK, derepressed MTD activity in Glc-grown cultures. In contrast, the addition of the sugar analog 2-deoxyglucose, which is phosphorylated by HK but not further metabolized, repressed MTD activity in mannitol-grown cultures. Collectively, these data suggest that HK and sugar phosphorylation are involved in signaling MTD repression. In vivo repression of MTD activity by galactose (Gal), which is not a substrate of HK, appeared to be an exception to this hypothesis. Further analyses, however, showed that the products of Gal catabolism, Glc and fructose, rather than Gal itself, were correlated with MTD repression.

Subcellular localization of celery mannitol dehydrogenase. A cytosolic metabolic enzyme in nuclei.

Mannitol dehydrogenase (MTD) is the first enzyme in mannitol catabolism in celery (Apium graveolens L. var dulce [Mill] Pers. cv Florida 638). Mannitol is an important photoassimilate, as well as providing plants with resistance to salt and osmotic stress. Previous work has shown that expression of the celery Mtd gene is regulated by many factors, such as hexose sugars, salt and osmotic stress, and salicylic acid. Furthermore, MTD is present in cells of sink organs, phloem cells, and mannitol-grown suspension cultures. Immunogold localization and biochemical analyses presented here demonstrate that celery MTD is localized in the cytosol and nuclei. Although the cellular density of MTD varies among different cell types, densities of nuclear and cytosolic MTD in a given cell are approximately equal. Biochemical analyses of nuclear extracts from mannitol-grown cultured cells confirmed that the nuclear-localized MTD is enzymatically active. The function(s) of nuclear-localized MTD is unknown.

Immunolocalization of mannitol dehydrogenase in celery plants and cells.

Immunolocalization of mannitol dehydrogenase (MTD) in celery (Apium graveolens L.) suspension cells and plants showed that MTD is a cytoplasmic enzyme. MTD was found in the meristems of celery root apices, in young expanding leaves, in the vascular cambium, and in the phloem, including sieve-element/companion cell complexes, parenchyma, and in the exuding phloem sap of cut petioles. Suspension cells that were grown in medium with mannitol as the sole carbon source showed a high anti-MTD cross-reaction in the cytoplasm, whereas cells that were grown in sucrose-containing medium showed little or no cross-reaction. Gel-blot analysis of proteins from vascular and nonvascular tissues of mature celery petioles showed a strong anti-MTD sera cross-reactive band, corresponding to the 40-kD molecular mass of MTD in vascular extracts, but no cross-reactive bands in nonvascular extracts. The distribution pattern of MTD within celery plants and in cell cultures that were grown on different carbon sources is consistent with the hypothesis that the Mtd gene may be regulated by sugar repression. Additionally, a developmental component may regulate the distribution of MTD within celery plants.

Sequence analysis of a mannitol dehydrogenase cDNA from plants reveals a function for the pathogenesis-related protein ELI3.

Mannitol is the most abundant sugar alcohol in nature, occurring in bacteria, fungi, lichens, and many species of vascular plants. Celery (Apium graveolens L.), a plant that forms mannitol photosynthetically, has high photosynthetic rates thought to results from intrinsic differences in the biosynthesis of hexitols vs. sugars. Celery also exhibits high salt tolerance due to the function of mannitol as an osmoprotectant. A mannitol catabolic enzyme that oxidizes mannitol to mannose (mannitol dehydrogenase, MTD) has been identified. In celery plants, MTD activity and tissue mannitol concentration are inversely related. MTD provides the initial step by which translocated mannitol is committed to central metabolism and, by regulating mannitol pool size, is important in regulating salt tolerance at the cellular level. We have now isolated, sequenced, and characterized a Mtd cDNA from celery. Analyses showed that Mtd RNA was more abundant in cells grown on mannitol and less abundant in salt-stressed cells. A protein database search revealed that the previously described ELI3 pathogenesis-related proteins from parsley and Arabidopsis are MTDs. Treatment of celery cells with salicylic acid resulted in increased MTD activity and RNA. Increased MTD activity results in an increased ability to utilize mannitol. Among other effects, this may provide an additional source of carbon and energy for response to pathogen attack. These responses of the primary enzyme controlling mannitol pool size reflect the importance of mannitol metabolism in plant responses to divergent types of environmental stress.

Purification of NAD-dependent mannitol dehydrogenase from celery suspension cultures.

Mannitol dehydrogenase, a mannitol:mannose 1-oxidoreductase, constitutes the first enzymatic step in the catabolism of mannitol in nonphotosynthetic tissues of celery (Apium graveolens L.). Endogenous regulation on the enzyme activity in response to environmental cues is critical in modulating tissue concentration of mannitol, which, importantly, contribute to stress tolerance of celery. The enzyme was purified to homogeneity from celery suspension cultures grown on D-mannitol as the carbon source. Mannitol dehydrogenase was purified 589-fold to a specific activity of 365 mumol h-1 mg-1 protein with a 37% yield of enzyme activity present in the crude extract. A highly efficient and simple purification protocol was developed involving polyethylene glycol fractionation, diethylaminoethyl-anion-exchange chromatography, and NAD-agarose affinity chromatography using NAD gradient elution. Sodium dodecylsulfate gel electrophoresis of the final preparation revealed a single 40-kD protein. The molecular mass of the native protein was determined to be approximately 43 kD, indicating that the enzyme is a monomer. Polyclonal antibodies raised against the enzyme inhibited enzymatic activity of purified mannitol dehydrogenase. Immunoblots of crude protein extracts from mannitol-grown celery cells and sink tissues of celery, celeriac, and parsley subjected to sodium dodecyl sulfate gel electrophoresis showed a single major immuno-reactive 40-kD protein.

Root-knot nematode--directed expression of a plant root--specific gene.

Root-knot nematodes are obligate plant parasites that induce development of an elaborate feeding site during root infection. Feeding-site formation results from a complex interaction between the pathogen and the host plant in which the nematode alters patterns of plant gene expression within the cells destined to become the feeding site. Expression of TobRB7, a gene expressed only in tobacco roots, is induced during feeding site development. The cis-acting sequences that mediate induction by the nematode are separate from those that control normal root-specific expression. Reporter transgenes driven by the nematode-responsive promoter sequences exhibit expression exclusively in the developing feeding site.

Development and characterization of a generalized gene tagging system for higher plants using an engineered maize transposon Ac.

This report describes a series of transposon tagging vectors for dicotyledonous plants based on the maize transposable element Ac. This binary system includes the transposase (Ts) and the tagging element (Ds) on separate T-DNA vectors. Ts elements include versions in which transcription is driven either by the endogenous Ac promoter or by the cauliflower mosaic virus (CaMV) 35S promoter. Ds tagging element includes a gene conferring methotrexate (Mtx) resistance for selection and a supF gene to facilitate cloning of tagged sequences. The Ds element is flanked by a CaMV 35S promoter and the beta-glucuronidase (GUS) coding sequence so that GUS expression occurs upon excision of the element. We have transformed these Ts and Ds elements into tobacco and demonstrated that the Ts is functional with either promoter, and that the artificial Ds elements are capable of transposition. The amount of excision was found to depend upon both the individual Ts and Ds primary transformants used. Somatic excision of Ds was seen in up to 100% of progeny seedlings containing Ts and Ds. Germinal excision was detected in up to 48% of the progeny of plants containing both elements. Hence, this system can generate a sufficient number of events to be useful in gene tagging.

Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription.

Nitrate reductase, the first enzyme in nitrate assimilation, is located at the crossroad of two energy-consuming pathways: nitrate assimilation and carbon fixation. Light, which regulates the expression of many higher-plant carbon fixation genes, also regulates nitrate reductase gene expression. Located in the cytosol, nitrate reductase obtains its reductant not from photosynthesis but from carbohydrate catabolism. This relationship prompted us to investigate the indirect role that light might play, via photosynthesis, in the regulation of nitrate reductase gene expression. We show that sucrose can replace light in eliciting an increase of nitrate reductase mRNA accumulation in dark-adapted green Arabidopsis plants. We show further that sucrose alone is sufficient for the full expression of nitrate reductase genes in etiolated Arabidopsis plants. Finally, using a reporter gene, we show that a 2.7-kilobase region of 5' flanking sequence of the nitrate reductase gene is sufficient to confer the light or the sucrose response.

Comparison of the capsid protein cistron from serologically distinct strains of sweetpotato feathery mottle virus (SPFMV).

Complementary DNA clones corresponding to the 3' terminus of sweetpotato feathery mottle virus (SPFMV) strains RC and C were synthesized and sequenced. An open reading frame followed by a 3' terminal non-coding region of 222 nucleotides and a terminal polyadenylation track was present in clones from both strains. Putative N-terminal capsid protein cleavage sites were identified for both strains 945 nucleotides 5' of the first stop codon. Sequence comparisons of these strains show 98% nucleic acid identity in the last 351 nucleotides of the capsid protein cistron and 100% in the corresponding amino acids. This relatively short homologous sequence element near the C terminus is responsible for the wide spectrum hybridization among SPFMV strains using in vitro transcribed antiviral RNA probes (riboprobes). The sequence similarity in the remaining N terminal 645 nucleotides is only 62% and 65% for their predicted amino acids. A tendency of decreasing nucleotide mismatches in the alignment from 5' to 3' end of both capsid protein cistrons was detected. Although the alignment of the predicted amino acid sequence of the SPFMV-RC capsid protein with those of other potyviruses showed significant homology, hybridization with riboprobes from both the 5' and 3' regions of the capsid protein cistron of SPFMV was virus-specific.

Differential expression of the two Arabidopsis nitrate reductase genes.

The differential regulation of the two nitrate reductase (NR, EC 1.6.6.1) genes of Arabidopsis thaliana L. Heynh was examined. cDNAs corresponding to each of the NR genes (NR1 and NR2) were used to measure changes in the steady-state levels of NR mRNA in response to nitrate, light, circadian rhythm, and tissue specificity. Although nitrate-induction kinetics of the two genes are very similar, NR1 is expressed in the absence of nitrate at a higher basal level than NR2. Nitrate induction is transient both in the roots and leaves, however the kinetics are different: the induction and decline in the roots precede that in the leaves. Light induces the expression of each of the genes with significantly different kinetics: NR2 reached saturation more rapidly than did NR1. Both genes showed similar diurnal patterns of circadian rhythm, with NR2 mRNA accumulating earlier in the morning.

Characterization of cis-acting sequences regulating root-specific gene expression in tobacco.

The expression of the tobacco root-specific gene TobRB7 was characterized. Gel blot hybridizations to RNA isolated from various tobacco tissues demonstrated that steady-state TobRB7 mRNA is not detected in expanded leaf, stem, or shoot apex tissue. To determine the spatial pattern of expression, in situ hybridization to root sections revealed that TobRB7 expression is localized to root meristem and immature central cylinder regions. The 5' flanking region of the gene was studied with respect to its ability to direct root-specific expression. Deletions of 5' flanking sequence were fused to the beta-glucuronidase (GUS) reporter gene and transformed into tobacco. Our data demonstrated that sequences 636 base pairs from the site of transcription initiation are sufficient to direct the root-specific GUS expression in transgenic tobacco, whereas sequences 299 base pairs from the site of transcription initiation fail to direct root-specific expression. A negative regulatory element was apparent between 813 base pairs and 636 base pairs 5' of the transcription initiation site. Histochemical localization of GUS activity in transgenic plants was consistent with in situ hybridization results: GUS activity was localized to the root meristem and central cylinder regions. GUS activity appeared 2 days post-germination in the primary root meristem. In lateral roots, GUS activity was detected from the time of initiation.

Isolation of transcriptionally regulated root-specific genes from tobacco.

Four root-specific cDNA clones and their corresponding genomic clones have been isolated from tobacco (Nicotiana tabacum) by a novel differential hybridization procedure. The genes are expressed at high levels in roots and are not detectable in leaves. The cDNAs are encoded by small gene families of two to four members. Transcription experiments with isolated nuclei demonstrate that the genes are, at least in part, transcriptionally regulated. Constructions in which 1.4 kilobase pairs of 5' flanking region of one of the root-specific genes was fused to a reporter gene (beta-glucuronidase) were transformed into tobacco. beta-Glucuronidase activity in transgenic plants was localized in the roots, demonstrating the cis-acting sequences regulating root-specific expression are present on the 5' flanking region.

Isolation and characterization of conditional alleles of bacteriophage T4 genes uvsX and uvsY.

The bacteriophage T4 uvsW, uvsX and uvsY gene functions are required for wild-type levels of recombination and for normal survival and mutagenesis after treatments with ultraviolet (UV) and ionizing radiations. The ability of uvsX and uvsY mutations to suppress the lethality of gene 49 mutations was used to select temperature-sensitive and amber alleles of these two genes. (uvsW mutations do not suppress gene 49 mutations.) A simple and powerful complementation test was developed to assist in assigning uvs mutations to genes. The amber alleles of uvsX and uvsY behave as simple null alleles, fully suppressing a gene 49 defect, enhancing UV killing and abolishing UV mutagenesis. However, the properties of the ts alleles of uvsX and uvsY demonstrated that suppression of a gene 49 defect, sensitivity to UV-induced inactivation and UV mutability can be partially uncoupled. These results prompt the hypothesis that radiation mutagenesis occurs during DNA chain elongation past template damage within a recombinational intermediate rather than within a conventional replication fork.

Thermal rescue of UV-irradiated bacteriophage T4 and biphasic mode of action of the WXY system.

When ultraviolet-irradiated bacteriophage T4 is assayed at plating temperatures ranging from 20 degrees to 40 degrees, its survival increases at the higher temperatures. This "thermal rescue" requires an intact WXY system but not the denV pyrimidine dimer excision system. Mutation rates decrease with increasing temperature, indicating that some lesions processed in a mutagenic manner at lower temperatures are accurately repaired or circumvented at high temperatures. When both the cold sensitivity of UV survival in the wild type and the temperature sensitivity of newly isolated ts mutants of uvsX and uvsY were used, expression of the WXY system was monitored in temperature shift UV survival experiments and was found to be biphasic: the uvsX and uvsY functions increase UV survival in two increments, one at an early and another at a late stage of infection. The uvsW function, however, increases UV survival only early in infection.