Expression of an Engineered Cecropin Gene Cassette in Transgenic Tobacco Plants Confers Disease Resistance to Pseudomonas syringae pv. tabaci.

ABSTRACT A chimeric gene fusion cassette, consisting of a secretory sequence from barley alpha-amylase joined to a modified cecropin (MB39) coding sequence and placed under control of the promoter and terminator from the potato proteinase inhibitor II (PiII) gene, was introduced into tobacco by Agrobacterium-mediated transformation. Transgenic and control plants reacted differently when inoculated with tobacco wildfire pathogen Pseudomonas syringae pv. tabaci at various cell concentrations. With control plants (transformed with a PiII-GUS [beta-D-glucuronidase] gene fusion), necrosis was clearly visible in leaf tissue infiltrated with bacterial inoculum levels of 10(2), 10(3), 10(4), 10(5), and 10(6) CFU/ml. With MB39-transgenic plants, however, necrosis was observed only in the areas infiltrated with the two highest levels (10(5) and 10(6) CFU/ml). No necrosis was evident in areas infiltrated with bacterial concentrations of 10(4) CFU/ml or less. Bacterial multiplication in leaves of MB39-transgenic plants was suppressed more than 10-fold compared to control plants, and absence of disease symptom development was associated with this growth suppression. We conclude that the pathogen-induced promoter and the secretory sequence were competent elements for transforming a cecropin gene into an effective disease-control gene for plants.

A single amino acid substitution in the antimicrobial defense protein cecropin B is associated with diminished degradation by leaf intercellular fluid.

Degradation is one of several factors that may affect the level of accumulation of transgene products in plants. In plants engineered to secrete antimicrobial proteins to the intercellular compartment of leaves, the degenerative activity of proteases residing in leaf intercellular fluid (IF) could be critical to achieving the expected transgene function. We synthesized a structural analogue (MB39) of the antibacterial protein cecropin B and compared the susceptibility of both proteins to degradation in vitro by IF extracted from leaves of various crops. The half-life of the two proteins in the various IF extracts ranged from 3 min to 25.5 h, with the analogue MB39 displaying the longer half-life in IF from nine of 10 species. Overall, the half-life of MB39 averaged 2.9 times greater than that of cecropin B. Analysis of the peptides produced by endopeptidase activity in potato iF indicated that the 5.7-fold lower degradation rate of MB39 was associated with the substitution of valine for methionine at residue 11 of cecropin B. These findings point to the possibility of tailoring antimicrobial protein genes to reduce the rate of protein degradation in a particular target crop.

Effect of promoter-leader sequences on transient expression of reporter gene chimeras biolistically transferred into sugarbeet (Beta vulgaris) suspension cells.

Chimeric constructs consisting of the gus coding region fused downstream of promoterun-translated leader sequences from the tobacco osmotin and PR-S genes, the potato proteinase inhibitor 2 gene (pin2), and the cauliflower mosaic virus (CaMV) 35S promoter were biolistically transferred into sugarbeet suspension cells. Each construct was expressed in recipient cells at 6 h after bombardment with maximum levels observed between 12 and 48 h. Expression of the PR-S construct mimicked the time-course expression of the constitutively expressed 35S construct but reached levels almost 50% higher. The pin2-promoter construct was ultimately expressed at levels similar to that of PR-S. Expression of the osmotin promoter-leader construct was highest, reaching levels approximately 2.5-fold higher than those of the 35S construct.

Selenocysteyl-tRNAs recognize UGA in Beta vulgaris, a higher plant, and in Gliocladium virens, a filamentous fungus.

Selenocysteyl-tRNAs that decode UGA were previously identified in representatives of three of the five life kingdoms which were the monera, animal and protist kingdoms. In the present study, we show that these tRNAs also occur in representatives of the two remaining kingdoms, plants and fungi; i.e., selenocysteyl-tRNAs which code for UGA occur in Beta vulgaris, a higher plant, and in Gliocladium virens, a filamentous fungus. The fact that selenocysteyl-tRNAs are present in all five life kingdoms strongly suggests that UGA, in addition to dictating the cessation of protein synthesis, also codes for selenocysteine in the universal genetic code.

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.

Somaclonal variation in soybean plants regenerated from tissue culture.

Callus cultures of soybean (Glycine max (L.) Merr.) genotypes PI 88788, PI 438489B, and cultivar Bedford were initiated in vitro from seedling explants consisting of the cotyledonary node plus epicotyl from germinated mature seed. Plants were regenerated from these callus cultures and subsequently evaluated for qualitative variation in three to four subsequent generations. Variant phenotypes observed that have not been previously reported from tissue culture include lanceolate leaves, leaf variegation (chimeral variegated plants), pod variegation on otherwise normal plants, and change in growth habit from indeterminate to determinate. The lanceolate leaf, chimeral variegated plant, and change from indeterminate to determinate growth habit characters were inherited through at least three generations (R0-R2), and segregation occurred in each generation. Pod variegation was inherited through the two generations tested thus far and segregation occurred in each generation. No variation was observed in control plants derived from normal seed. Variants appeared more frequently in regenerants from PI 88788 and PI 438489B than from Bedford. These results confirm and extend the finding that certain tissue culture techniques may be used to induce novel plant formation from somatic tissue of soybean.

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 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.

An improved medium for adventitious shoot formation and callus induction in Beta vulgaris L. in vitro.

Six sugarbeet (Beta vulgaris L.) lines (GWI-248, SPB-11, MonoHy 55, SMS-1, EL45 and FC607) were tested for regeneration. Shoot cultures were initiated in vitro from naked, sterilized embryos obtained from mature seed. Excised petioles from cultured shoots were plated on Gamborg's B5 medium and four modified Murashige and Skoog (MS) media. A medium containing MS inorganic salts supplemented with 0.4 mg/1 N(6)-benzyladenine, 0.1 mg/1 indole-3-butyric acid, ten vitamins and six amino acids, termed RV, was superior for both adventitious shoot and callus formation. Callus was observed only on RV medium and only on petioles that did not develop adventitious buds directly. Rooting of regenerated shoots and development of complete plants was accomplished by transfer to Gamborg's B5 medium with 5 mg/l indole-3-butyric acid as the sole phytohormone. The complete process of regeneration through adventitious shoot production took from 4 to 6 weeks from explants to rooted plants. The callus that formed on nonorganogenic petioles was regenerative when transferred to fresh RV medium. Regeneration from callus occurred mainly by shoot organogenesis but also by somatic embryogenesis at a low frequency.

Behavior and viability of tobacco protoplasts in response to electrofusion parameters.

This investigation examines responses of protoplasts in a systematic and quantitative way to the various electrical treatments used to achieve electrofusion and their individual and cumulative effect on protoplast viability. Mesophyll and cell suspension protoplasts from two species of the same genera, Nicotiana tabacum and N. rustica var brasilia were used in these experiments. Optimal frequencies for alignment of tobacco protoplasts were between 500 kilohertz and 2 megahertz at 100 volts per centimeter. Variations in frequency and voltage of the alternating current (AC) field caused predictable movements of protoplasts within an electrofusion chamber. AC frequencies below 10 hertz or above 5 megahertz significantly decreased the viability of protoplasts in the fusion chamber as estimated by fluorescein diacetate staining 1 hour after treatment. Although the direct current (DC) pulse appeared to have a slight detrimental effect on protoplast viability, this effect was not significantly different from untreated control preparations.Protoplasts from both leaf mesophyll cells and suspension cells were induced to fuse with one or more 10 to 30 microseconds DC square wave pulses of approximately 1 kilovolt per centimeter after the protoplasts had been closely appressed with an AC field.

Genotypic variability of soybean response to agrobacterium strains harboring the ti or ri plasmids.

Twenty four diverse cultivars of soybean (Glycine max [L.] Merrill) and three lines of its annual wild progenitor Glycine soja Sieb and Zucc. were tested for their response to Agrobacterium strains harboring either the Ti (tumor-inducing) plasmid (pTi) from Agrobacterium tumefaciens or the Ri (root-inducing) plasmid (pRi) from Agrobacterium rhizogenes following uniform wounding and inoculation. Based upon gall weight at 8 weeks postinfection, three G. max cultivars (Biloxi, Jupiter, and Peking) and one G. soja line, Plant Introduction (PI) 398.693B, were judged highly susceptible to A. tumefaciens strain A348 (pTiA6), ten genotypes moderately susceptible, 11 weakly susceptible, and two nonsusceptible. Of 26 genotypes inoculated with strain R1000 (pRiA4b), only seven responded in a clearly susceptible fashion by forming small, fleshy roots at internodal infection sites. Cotyledons excised from 1- or 3-day old seedlings of Peking and Biloxi cultivars also formed galls when infected in vitro with agrobacteria carrying either the Ti or Ri plasmid. Tumor lines established from cotyledon and stem galls induced by A. tumefaciens A348 (pTiA6) exhibited the T-DNA borne traits of phytohormone-independent growth and octopine synthesis. Additionally, DNA isolated from cultured tumors hybridized with labeled T-DNA probe.

Characteristics of teratomas regenerated in vitro from octopine-type crown gall.

Crown galls induced by infection of tobacco plants with Agrobacterium tumefaciens strain C58-Cl(pTiB6S3) were excised and cultured in vitro. After about one year of culture on medium-lacking phytohormones, two noncloned lines spontaneously formed shoots. Leaf explants from shoots of tumor-line T5 were capable of growing on hormone-free medium, and the resulting mixture of organized and unorganized tissue synthesized octopine. Detached leaves from T5 shoots also synthesized octopine. These results establish that shoots from this octopine-type tumor contain transformed cells and are true crown-gall teratomas.

Characterization of Kanamycin-Resistant Cell Lines of Nicotiana tabacum.

Two kanamycin-resistant variants of Nicotiana tabacum were derived by culturing seedling leaf sections on a shoot-inducing medium containing kanamycin. The variants displayed a higher resistance to the structurally related antibiotic streptomycin than to kanamycin. The resistance phenotype was expressed when the tissue was cultured either as callus or as a differentiating tissue and was stably maintained in the absence of kanamycin. Plants regenerated from the variant lines had morphologically abnormal flowers and produced few viable seeds. These resistant lines are potentially useful for protoplast fusion or genetic modification experiments requiring selectable phenotypic markers.

Ethylene production by callus and suspension cells from cortex tissue of postclimacteric apples.

Cortex tissue from postclimacteric ;Golden Delicious' apples (Malus domestica, Borkh.) stored at 0 C for 9 months after harvest were induced to form callus in vitro. Cell suspension cultures were subsequently formed from calli. Of five media tested, only the medium of Schenk and Hildebrandt (Can J Bot 1972, 50: 192) and that of Uchimiya and Murashige (Plant Physiol 1974, 54: 936) allowed callus formation. During growth both the callus and cell cultures produced ethylene in a pattern which showed a rapid rise and then a fall as the culture grew. (14)C-Labeled methionine was converted to labeled ethylene by the cell suspension cultures, which also could be inhibited from producing ethylene by a rhizobitoxine analog or free radical scavengers. Ethylene production in these cultures, like that in intact fruit tissue slices, could be stimulated by IAA or suppressed by N(6)-(gamma,gamma-dimethylallyl) adenosine and GA(3).

Binding of ColE1-kan Plasmid DNA by Tobacco Protoplasts: Nonexpression of Plasmid Gene.

Protoplasts prepared from cultured tobacco cells were treated with ColE1-kan plasmid DNA, a hybrid of ColE1 and pSC105 plasmids bearing a gene for kanamycin resistance. The conditions employed permitted the uptake or irreversible binding of 2.9% of the added DNA in acid-insoluble form. Upon commencement of division, the treated cells were plated in agar medium containing kanamycin and differentiating hormones. Plantlets or shoots obtained as presumptive transformants were further tested on kanamycin medium by subculturing small leaf pieces. No evidence was obtained for expression of the kanamycin resistance gene of ColE1-kan in tobacco tissue.

Control of Free Methionine Production in Wild Type and Ethionine-resistant Mutants of Chlorella sorokiniana.

Mutants of Chlorella sorokiniana selected for resistance to the methionine analogue ethionine took up ethionine at the same rate as did the wild type strain. Cells of two ethionine-resistant mutants produced severalfold higher levels of free methionine and cysteine than did wild type cells.Exogenous methionine had no apparent effect on free methionine production in a mutant that produces excessive levels of free methionine. Under the same conditions, production of free methionine was relatively inhibited in wild type cells and in a mutant that produces wild type levels of free methionine.The results suggest that free methionine production in the wild type strain is subject to endproduct control, and that this control is lacking in one class of ethionine-resistant mutants.

beta-Cystathionase In Vivo Inactivation by Rhizobitoxine and Role of the Enzyme in Methionine Biosynthesis in Corn Seedlings.

Rhizobitoxine has previously been shown to inactivate irreversibly beta-cystathionase isolated from spinach. In the present studies, rhizobitoxine was shown to inhibit partially beta-cystathionase of spinach and corn seedlings in vivo. An activity of 30 to 40% of normal remained in toxin-treated seedlings of both spinach and corn. Possible reasons for the partial inhibition are discussed.Rhizobitoxine-treated and control corn seedlings were allowed to assimilate (35)SO(4) (2-) for 3 or 6 hours, and the radioactivity incorporated into sulfur amino acids at these times was studied. The most striking effect of rhizobitoxine was an increase (up to 22-fold) in radioactive cystathionine. Accumulation of radioactivity in methionine was only slightly inhibited by rhizobitoxine treatment. The results strongly suggest that the transsulfuration pathway contributes to methionine biosynthesis, and that metabolism via this pathway is impaired, but not entirely eliminated, by rhizobitoxine treatment.The present data do not permit decisions about the relative contributions of the transsulfuration and the direct sulfhydration pathways to methionine biosynthesis, or whether the pathological effects of rhizobitoxine are due chiefly to inhibition of beta-cystathionase.

Inhibition of ethylene production by rhizobitoxine.

Rhizobitoxine, an inhibitor of methionine biosynthesis in Salmonella typhimurium, inhibited ethylene production about 75% in light-grown sorghum seedlings and in senescent apple tissue. Ethylene production stimulated by indoleacetic acid and kinetin in sorghum was similarly inhibited. With both apple and sorghum, the inhibition could only be partially relieved by additions of methionine. A methionine analogue, alpha-keto-gamma-methylthiobutyric acid, which has been suggested as an intermediate between methionine and ethylene, had no effect on the inhibition.Incorporation of (14)C from added methionine-(14)C into ethylene was curtailed by rhizobitoxine to about the same extent as was ethylene production. These results suggest that rhizobitoxine interferes with ethylene biosynthesis by blocking the conversion of methionine to ethylene and not indirectly by inhibiting the biosynthesis of methionine. Ethylene production by Penicillium digitatum, a fungus which produces ethylene via pathways not utilizing methionine as a precursor, was not affected by rhizobitoxine.