Expression of DNA Coding for Diphtheria Toxin Chain A Is Toxic to Plant Cells

Abstract

DNA coding for the enzymatically active subunit A of diphtheria toxin was placed under the control of the cauliflower mosaic virus 35S promoter and the Agrobacterium left transfer-DNA gene 7 polyadenylation signal. Agrobacteria carrying a binary plant vector with the chimeric diphtheria toxin A gene had very low transforming activity in tobacco (Nicotiana tabacum L.), and greatly diminished the recovery of stable transformants when mixed together with agrobacteria which alone transformed plant cells well. As reporter genes, the bacterial cat2 and fl-glucuronidase genes have been used most widely (14,19). Both gene activities can be quantified, and the latter can be detected in situ; however, the assays are still tedious and destruct the material. In addition, they are sometimes complicated by endogenous inhibitors or background activity (10,15,19 (8), and the active site is distinct from that for the ADP-ribosylation (6). The structural gene for DTx carried by corynebacteriophages codes for a protein with a leader peptide which is removed during secretion from Corynebacterium diphtheriae. The mature toxin is composed of two domains: the NH2-terminal portion (A chain) which carries the active sites of the toxin and the COOH-terminal portion (B chain) which is required for binding to specific receptors, transport of the toxin into target cells, and is dispensable for intracellular toxicity. The toxin protein is unmasked and activated by reduction of the disulfide linkages and by hydrolysis of a peptide bond located in the loop between the sulfur atoms of the disulfide bridge located in the NH2-terminal portion of the intact toxin.The two fragments remain firmly held together by weak interactions (27).Because polypeptide fragments of microbial and plant toxins (including DTx) that are devoid of cell receptor binding and transfer domains are not cytotoxic, it has become attractive to use these polypeptides in yeast (29) and mammalian (21, 26) systems in the assembly of chimeric genes. Here we present evidence that expression of a chimeric DTx-A gene construct in tobacco is toxic. This finding provides a potent new negative selection gene for probing the function of plant promoters. MATERIALS AND METHODS Strains and Molecular CloningEscherichia coli strain MC 1000 (7) was used as a recipient for plasmids constructed by standard procedures. The Agrobacterium tumefaciens strain LBA4404 (17) carrying the pAL4404 disarmed helper Ti plasmid in a Ach5 chromosomal background was used for plant transformation. The DNA coding for the DTx fragment A was obtained from pTH 1 (21). The binary plant expression vectors pGA642 and pGA643, carrying a polylinker site between the nopaline synthase promoter and CaMV 35S promoter, respectively, and the pTiA6 octopine-type Ti-plasmid TL-DNA 7 gene terminator were described previously (4). The binary vectors were transferred into Agrobacterium by the direct transformation method (2).

Meristem-Localized Inducible Expression of a UDP-Glycosyltransferase Gene Is Essential for Growth and Development in Pea and Alfalfa

“…We conclude that this result represents lethality of the expressed gene. This conclusion is based on control experiments in which a known cytolytic gene encoding diphtheria toxin A, whose expression in a given cell results in cell death (Czako and An, 1991), was placed into A. rhizogenes under the control of the 35S promoter. When used to inoculate pea, the resulting lethal phenotype was indistinguishable from that which occurs in response to expression of the PsUGT1 promoter-PsUGT1 antisense cDNA construct (data not shown).…”

Antisense Expression of the Peptide Transport Gene AtPTR2-B Delays Flowering and Arrests Seed Development in Transgenic Arabidopsis Plants

“…The pGA941 plasmids containing the antisense or sense AtPTR2-B construct were transformed into Escherichia cozi MClOOO (Casadaban and Cohen, 1980; this strain gives a higher efficiency of transformation than other strains tested), and transformants were selected on Luria broth medium containing kanamycin (10 p g / mL) and tetracycline (3 kg/mL) (Czako and An, 1991). The plasmids were then isolated and transformed into A. tumefaciens strain EHA105 (Hood et al, 1993) by electroporation (Sambrook et al, 1989).…”

“…To clone AtPTR2-B into the XbaI site of the plant expression vector pGA941 (with the 35s RNA promoter sequence and kanamycin resistance gene nptll Czako and An, 1991]), AtPTR2-B was subcloned sequentially into pUC19 and pBluescript SK. The SacI-XbaI fragment of AtPTX2-B from pBluescript SK-ZB, which has a 100-bp deletion of the coding region and lacks 230 bp from the 3' noncoding region of the AtPTR2-B cDNA, was inserted between the Sacl and XbnI restriction sites of pUC19.…”

The Anti-nptII Gene (A Potential Negative Selectable Marker for Plants)

“…A chimeric RAS2 gene from yeast also had a "killer" effect in N. plumbaginifolia (Hilson et al, 1990). The expression of diphtheria toxin chain A gene in tobacco was demonstrated to be toxic (Czako and An, 1991). These negative Plant Physiol.…”

“…Therefore, the anti-nptll gene has potential as a negative selectable marker for gene targeting in higher plants. Other negative selection genes have been reported (Depiker et al, 1988;Hilson et al, 1990;Czako and An, 1991). The amido hydrolase gene 2 of Agrobacterium T-DNA was proposed for the elimination of a specific group of cells or tissues and for fundamental studies on gene inactivation (Depiker et al, 1988).…”