Transgenic production of cytokinin suppresses bacterially induced hypersensitive response symptoms and increases antioxidative enzyme levels in Nicotiana spp.

Responses of cytokinin overproducing transgenic Nicotiana plants to infections with compatible and incompatible Pseudomonas syringae pathovars were compared. Plants used were transformed with the ipt(isopentenyl transferase) gene that catalyzes the synthesis of cytokinin. In cytokinin overproducing lines that carry the ipt gene fused to the CaMV 35S (Nt+ipt), the wound-inducible proteinase inhibitor II (Ntx+ipt), or the light-inducible Rubisco small subunit protein (Npl+ipt) promoter, development of the hypersensitive response (HR) after infection with incompatible bacteria (P. syringae pv. tomato) was significantly inhibited as compared to the untransformed (Nt) controls. Over a 12 h period following inoculation, P. syrinage pv. tomato populations were slightly reduced in leaves of the cytokinin-overproducing Nt-ipt line compared with the Nt control. When the compatible P. syringae. pv. tabaci was used to infect the ipt transformed lines, slight or no significant differences in necrosis development were observed. Following infection, the titer of P. syringae pv. tabaci increased rapidly in both the transgenic and control lines but was higher in Nt+ipt plants. Leaf superoxide dismutase and catalase enzyme activities were about 60% higher in ipt leaf extracts than in the controls. This augmented antioxidant capacity likely decreased the amount of H(2)O(2) that may be associated with the higher tolerance of plants to pathogen-induced necrosis. In addition, the Nt+ipt lines had a significantly lower molar ratio of free sterols to phospholipids. The more stable membrane lipid composition and the higher antioxidant capacity likely contributed to the suppressed HR symptoms in the cytokinin overproducing Nt+ipt plants. In conclusion, the overproduction of cytokinins in tobacco appears to suppress the HR symptoms induced by incompatible bacteria.

Effect of insertions, deletions, and double-strand breaks on homologous recombination in mouse L cells.

We have used DNA-mediated gene transfer to study homologous recombination in cultured mammalian cells. A family of plasmids with insertion and deletion mutations in the coding region of the herpes simplex type 1 thymidine kinase (tk) gene served as substrates for DNA-mediated gene transfer into mouse Ltk- cells by the calcium phosphate technique. Intermolecular recombination events were scored by the number of colonies in hypoxanthine-aminopterin-thymidine selective medium. We used supercoiled plasmids containing tk gene fragments to demonstrate that an overlap of 62 base pairs (bp) of homologous DNA was sufficient for intermolecular recombination. Addition of 598 bp of flanking homology separated from the region of recombination by a double-strand gap, deletion, or insertion of heterologous DNA increased the frequency of recombination by 300-, 20-, or 40-fold, respectively. Linearizing one of the mutant plasmids in a pair before cotransfer by cutting in the area of homology flanking a deletion of 104 bp or an insertion of less than 24 bp increased the frequency of recombination relative to that with uncut plasmids. However, cutting an insertion mutant of greater than or equal to 24 bp in the same manner did not increase the frequency. We show how our data are consistent with models that postulate at least two phases in the recombination process: homologous pairing and heteroduplex formation.

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors.

Proteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiol-activators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or L-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM L-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN(2) and Z-Phe-Ala-CHN(2), suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.

Cytokinin content and tissue distribution in plants transformed by a reconstructed isopentenyl transferase gene.

The cytokinin gene, isopentenyl transferase (ipt), was placed under the control of a heat-inducible promoter from the Drosophila melanogaster hsp70 gene and introduced into Nicotiana plumbaginifolia by cocultivation with Agrobacterium tumefaciens. Transformants were analyzed for organ-specific expression, cytokinin levels and effects on plant development before and after the heat induction. The ipt gene transcripts were detected in leaves and stems but not roots of transgenic plants following a 2 hour, 45 degrees C treatment. Maximum mRNA levels observed occurred 2 hours after heat treatment and 46 hours later were detected only in leaves. Zeatin and zeatinriboside concentrations 2 hours after heat shock ranged from over 900 to 2000 pmol/g, representing a greater than 140- to 200-fold increase over uninduced levels. After 46 hours, approximately 50% of the cytokinins are still present in the leaves as opposed to much reduced levels in the stems. Transgenic plants were greener, shorter, had an underdeveloped root system, reduced leaf width, and increased growth of axillary buds. After a single heat treatment, plants exhibited a darker green pigment and continued growth of lateral buds. Transient accumulations of endogenous cytokinins following thermal induction did not appear to alter the plant's preprogrammed pattern of differentiation.

Cytokinin gene fused with a strong promoter enhances shoot organogenesis and zeatin levels in transformed plant cells.

The isopentenyltransferase (ipt) gene associated with cytokinin biosynthesis in plants was cloned from a tumor-inducing plasmid carried by Agrobacterium tumefaciens and placed under the control of promoters of differing activities, the cauliflower mosaic virus 35S promoter and the nopaline synthase promoter. These promoter-gene constructs were introduced into wounded Nicotiana stems, leaf pieces, and cucumber seedlings by A. tumefaciens infection. Shoots were observed in the infection site on all responding genotypes of Nicotiana plants infected with the 35S promoter construct (35S-ipt), whereas only 41% responded similarly to infection with the unmodified gene. Furthermore, shoots were observed 19 days after infection with the 35S-ipt gene but not until 28 to 45 days with the unaltered ipt gene. Shoots were more numerous (>40) on galls incited by 35S-ipt and were up to 6 times taller than shoots induced by the native gene. On Cucumis (cucumber), shoots were observed only on galls incited by the 35S-ipt construct. These galls were on the average 7.5 times larger than those incited by the nopaline synthase promoter construct (NOS-ipt) or the unmodified ipt gene. Zeatin and zeatinriboside concentrations averaged 23 times greater in the 35S-ipt transformed shoots than in ones transformed with the native ipt gene. These results suggest that a more active promoter on the ipt gene can enhance or change the morphogenic potential of transformed plant cells by increasing their endogenous cytokinin levels.

Transformation of Soybean Cells Using Mixed Strains of Agrobacterium tumefaciens and Phenolic Compounds.

Cotyledon explants from germinated 1-day-old soybean seedling were inoculated with single or mixed strains of Agrobacterium tumefaciens. Mixed-strain infections with the supervirulent L,L-succinamopine type strain A281 (pTiBo542) and strain LBA4404 carrying an octopine type virulence (vir) region and a binary vector (pBin6) with a chimeric gene for kanamycin detoxification gave rise to tumors of which 25% were both kanamycin resistant and capable of hormone-independent growth. Singlestrain inoculations with LBA4404 (pBin6) failed to give rise to kanamycin-resistant callus. Syringaldehyde, a compound which induces vir genes carried on the Ti plasmid, increased the number of galls incited on excised cotyledons by the weakly virulent octopine type strain A348 (pTiA6). Similar results were obtained with whole plants treated with this strain in the presence of the vir-inducing compound acetosyringone. Our results indicate that the recovery of transformed soybean cells can be enabled in some instances by coinfecting with a supervirulent strain or in other instances promoted by adding a phenolic compound to the inoculum.

Cytokinin-to-Auxin Ratios and Morphology of Shoots and Tissues Transformed by a Chimeric Isopentenyl Transferase Gene.

Tissues transformed with the isopentenyl transferase (ipt) gene cloned from the T-DNA region of the Ti plasmid or with the ipt gene placed under the control of the cauliflower mosaic virus 35S promoter (35S-ipt) were analyzed for auxin and cytokinin. Free and total indole-3-acetic acid (IAA) levels in 35S-ipt-transformed Nicotiana tabacum and cucumber cells were reduced by 12 to 78% in comparison to untransformed tissues. In contrast, free IAA concentrations in 35S-ipt-transformed Nicotiana plumbaginifolia were almost three times those of untransformed tissues, while total IAA levels were not significantly affected. Cytokinin levels in these antibodies were elevated an average of 300-fold resulting in a 24- to over 2000-fold increase in the cytokinin-to-auxin ratios. High cytokinin-to-auxin ratios correlated with the shooty phenotype of transformed tissues propagated in vitro in the absence of added growth hormones. We conclude that increased endogenous cytokinin-to-auxin ratios can induce cells to undergo morphogenesis and that elevated cytokinin levels can also induce auxin-autonomous growth of transformed tissues without causing a corresponding increase in endogenous IAA levels.

Novel transmissible factors in a non-O1 Vibrio cholerae and a Vibrio sp.

Transmissible factors encoding production of lacunae (L factors) were demonstrated in a non-O1 Vibrio cholerae and a Vibrio sp. of recent environmental origin. Lacunae were produced in lawns of non-O1 V. cholerae indicator strains under the same assay conditions as those where lacunae were produced by the well characterized P fertility plasmid of V. cholerae O1 and the V fertility factor found in a non-cholera vibrio strain. The origin of the lacunae produced by strains harbouring the V and L factors was examined. No vibriocin or phage activity was found in culture supernates or in lacunae produced by the strains, suggesting that, as in the case of the P plasmid, the lacunae probably represent sites of active mating. Unlike the P plasmid, neither the Vn or L factor could be detected or isolated by conventional plasmid techniques.

Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco.

Cytokinins are plant growth regulators that induce shoot formation, inhibit senescence and root growth. Experiments with hydroponically grown tobacco plants, however, indicated that exogenously applied cytokinin led to the accumulation of proline and osmotin. These responses were also associated with environmental stress reactions, such as salt stress, in many plant species. To test whether increased endogenous cytokinin accumulation led to NaCl stress symptoms, the gene ipt from Agrobacterium tumefaciens, encoding isopentenyl transferase, was transformed into Nicotiana tabacum cv. SR-1 under the control of the light-inducible rbcS-3A promoter from pea. In high light (300 mumol PPFD m-2 s-1), ipt mRNA was detected and zeatin/zeatin glucoside levels were 10-fold higher than in control plants or when transformants were grown in low light (30 mumol PPFD m-2 s-1). High light treatment was accompanied by increased levels of proline and osmotin when compared to low light grown transformed and untransformed control plants. Elevated in planta cytokinin levels induced responses also stimulated by salt stress, suggesting either common or overlapping signaling pathways are initiated independently by cytokinin and NaCl, setting in motion gene expression normally elicited by developmental processes such as flowering or environmental stress.

Double-strand gap repair results in homologous recombination in mouse L cells.

Previous studies have demonstrated that the presence of double-strand breaks or double-strand gaps increases the frequency of homologous recombination between two cotransferred DNAs when they are introduced into cultured mammalian cells. Here we demonstrate that the repair of these double-strand gaps is a major mechanism for homologous recombination between exogenous DNAs. In particular, when a plasmid DNA containing a 104-base-pair (bp) gap in its tk gene (herpes simplex virus gene for thymidine kinase) undergoes recombination in mouse L cells to generate an intact gene, the majority of events result from direct repair of the double-strand gap using a cotransferred DNA as the template. We analyzed the recombination events by comparing the frequency of tk+ colonies, Southern blotting of cloned tk+ cell lines, and cloning recombined functional tk genes by plasmid rescue. In addition, by creating double-strand breaks within or adjacent to heterologous insertions in a mutant tk gene, we estimate that the L cell can generate a double-strand gap of between 152 and 248 bp and then can repair the gap to create a functional tk gene. We conclude that double-strand breaks and double-strand gaps are recombinogenic in transferred plasmid DNAs because they serve as intermediates in homologous recombination by double-strand gap repair, a nonreciprocal exchange of DNA or gene conversion event.