Lowell D. Owens

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.

Activity of cecropin SB37 against protoplasts from several plant species and their bacterial pathogens

Abstract The lethal concentration of cecropin SB37 was determined for protoplasts from 11 plant cultivars or experimental lines representing 7 plant species and for 9 bacterial species that are pathogens of these plants. Lethal concentrations ranged from 4.5 μM for tomato ( Lycopersicon esculentum cv Red Cherry) to 41 μM for sugarbeet ( Beta vulgaris REL 1). Cecropin SB37 exhibited potent antibacterial activity against all of the bacterial pathogens tested. Lethal concentrations ranged from 0.1–4.5 μM. The difference between the lethal concentrations of cecropin SB37 for protoplasts from certain plants and their respective bacterial pathogens suggests it may be feasible to protect against these pathogens by introducing a modified cecropin gene into the plant.

Measurement and effects of gel matric potential and expressibility on production of morphogenic callus by cultured sugarbeet leaf discs

During studies to optimize production of morphogenic callus from cultured leaf discs of sugarbeet (Beta vulgaris L.) large differences were observed associated with the gelling agent employed. Water availability, as determined mainly by gel matric potential, was found to be the dominant factor. A simple method was devised to measure the relative matric potential of different gels. A precisely moistened filter-paper disc was placed on the gel surface, allowed to equilibrate, removed and weighed. The relative gain or loss of water from the paper disc was a measure of the matric potential of the gel and varied with both gel type and concentration. Leaf disc expansion and production of callus-derived embryos and shoots were shown to be directly proportional to gel matric potential. Water availability may also be affected by the ease with which liquid is expressed from gels in response to localized pressure caused by explant expansion and contortion. This property, called gel expressibility, was easily measured with a weight and capillary pipette and shown also to vary with gel type and concentration. Validity of the technique for measuring relative matric potential was verified physiologically by culturing leaf discs on filter-paper overlays to eliminate expressibility differences among gels. Additionally, comparison of leaf disc growth on uncovered gel surfaces versus filter-paper overlays demonstrated the contribution of liquid expression to overall water availability. Expression of liquid by explants on uncovered gel surfaces greatly enhanced the production of morphogenic callus.

Selenocysteyl-tRNAs recognize UGA in Betavulgaris, 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.

Introduction of pathogen defense genes and a cytokinin biosynthesis gene into sugarbeet (Beta vulgaris L.) by Agrobacterium or particle bombardment

Abstract Two different methods for sugarbeet (Beta vulgaris L.) transformation were developed, one using Agrobacterium with excised cotyledons, the other, particle bombardment of embryogenic hypocotyl callus. Transformation efficiencies averaged 0.7% for the Agrobacterium method (number of transgenic plants obtained per treated cotyledon) and about 8% for the bombardment method (number of transgenic plants obtained per plate of embryogenic callus treated). Transgenic sugarbeet plants were produced carrying genes encoding either pathogen-defense-related proteins or the reporter enzyme β-glucuronidase (GUS) under transcriptional control of stress- or wound-inducible promoters. In addition, two plants were regenerated carrying a gene associated with enhanced insect resistance, the cytokinin biosynthesis gene, fused to a patatin gene promoter from potato. Expression of the GUS gene (gusA) under the control of the tobacco osmotin promoter was wound inducible with detectable activity at 8 h and maximal activity at 72 h post-wounding.

Rhizobium-synthesized phytotoxin: An inhibitor of β-cystathionase in Salmonella typhimurium

Abstract Rhizobitoxine, a phytotoxin synthesized by Rhizobium japonicum, inhibited the growth of Salmonella typhimurium wild-type and methionine-requiring mutant me-B on a medium supplemented with cystathionine. Neither strain was inhibited by the toxin when the medium was supplemented with homocysteine or methionine. Salmonella β-cystathionase was inhibited by very low concentrations of rhizobitoxine. Cystathionine γ-synthase and Neurospora γ-cystathionase were also inhibited, but at toxin concentrations 1000 times higher. These results indicate that growth inhibition is caused by a methionine deficiency induced by rhizobitoxine inhibition of β-cystathionase.

Evidence for the breakdown of cecropin B by proteinases in the intercellular fluid of peach leaves

Abstract Peach ( Prunus persica (L.) Batsch) leaves were found to tolerate relatively high levels of the bacteriocidal polypeptide cecropin B as determined by infiltration tests. Detached leaves showed no symptoms when infiltrated with 25 μM cecropin B, and only infrequent necrotic symptoms with 50 μM. Incubation of cecropin B with intercellular fluid (ICF) extracted from peach leaves reduced its biotoxicity toward the peach pathogen Pseudomonas syringae pv. syringae . The reduction in toxicity was lessened by adding proteinase inhibitors to ICF — and prevented by boiling ICF prior to incubation with cecropin B. Electrophoretic evidence suggested that ICF constituents caused endopeptidase cleavage of cecropin during the first hour of incubation followed by complete digestion of the cleavage products during the following 8 h. Incubations of various levels of cecropin B and ICF indicated that as much as 90% of the activity could be destroyed in 10 min, but that levels adequate for lethality against P. syringae pv. syringae would remain. We conclude that peach trees transgenic for cecropin B could feasibly produce a wide range of cecropin levels sufficient to control pathogenic bacteria without damaging the leaf tissue.

Kanamycin promotes morphogenesis of plant tissues

Abstract Low levels of the aminoglycoside antibiotic kanamycin enhanced shoot differentiation of protoplast-derived microcalli of tobacco, leaf sections of two tobacco cultivars and callus cultures of several carrot cultivars. Morphogenesis by one carrot cultivar occurred only in the presence of kanamycin. The aminoglycoside antibiotic streptomycin also enhanced morphogenesis, but chloramphenicol, sparsomycin or cycloheximide did not.

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

SummaryChimeric 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.

Sugarbeet leaf disc culture: an improved procedure for inducing morphogenesis

In preparation for gene transfer experiments we investigated factors that might affect the production of shoots and somatic embryos from the wound callus of cultured sugarbeet leaf discs. A complex interaction was found between the leaf disc plating density, the disc culture medium, the source-shoot culture medium and the frequency of disc transfer to fresh medium. The most productive protocol utilized: source shoots maintained on MS medium containing 0.25 mg 1-1 BA; multiple leaf discs (ten 4-mm discs/plate) plated onto an enriched modification of MS medium (RV) containing 1.0 mg 1-1 BA and solidified with 0.3% Gelrite (not permitted to dry during hardening); and transfer of the discs to fresh medium every two weeks during the first month. This standard protocol produced more callus per plate and higher rates of morphogenesis per unit dry weight of callus than did the one-step method of Saunders and Doley. Water availability considerations were found to be critical to obtaining high morphogenic rates. Root induction frequency and quality was superior on shoots transplanted to MS medium containing 1 mg 1-1 NAA as the sole growth regulator compared to IAA at the same concentration.