David Tepfer

Transformation of several species of higher plants by agrobacterium rhizogenes: Sexual transmission of the transformed genotype and phenotype

The T-DNA of the Ri plasmid from Agrobacterium rhizogenes is compatible with the regeneration of whole plants from genetically transformed roots and is transmitted through meiosis to the progeny of genetically transformed plants in carrot, tobacco, and morning glory (Convolvulus arvensis). The presence of Ri T-DNA is correlated with a phenotype that in some respects is invariable from species to species and in other respects varies as a function of species, organ clone within species, or individual. The transformed phenotype concerns a variety of morphological and physiological traits, is dominantly inherited in tobacco, but does not in general appear to be deleterious. The Ri T-DNA may provide a molecular starting point for studying a number of basic phenomena in plant morphology and physiology.

Tropane derivatives from Calystegia sepium.

Abstract A family of novel polyhydroxy- nor -tropanes is described. Three of their structures were determined by mass spectrometry and 1 H and 13 C NMR spectroscopy. These calystegins occur in two species of the Convolvulaceae and in Atropa belladonna . Their potential ecological significance is discussed.

Common evolutionary origin of the central portions of the Ri TL-DNA of Agrobacterium rhizogenes and the Ti T-DNAs of Agrobacterium tumefaciens

Analysis of published sequences for Ri TL-DNA (root-inducing left-hand transferred DNA) of Agrobacterium rhizogenes revealed several unsuspected structural features. First, Ri TL-DNA genes are redundant. Using redundancy as a criterion, three regions (left, middle and right) were discerned. The left one, ORFs (open reading frames) 1–7, contains no detectable redundancy. In the middle region a highly diverged gene family was detected in ORFs 8, 11, 12, 13 and 14. The right region contains an apparently recent duplication (ORF 15 =18+17). We interpret the phenomenon of redundancy, particularly in the central region that encodes the transformed phenotype, to be an adaptation that ensures function in a variety of host species. Comparison of Ri TL-DNA and Ti T-DNAs from Agrobacterium tumefaciens revealed common structures, unpredicted by previous nucleic acid hybridization studies. Ri TL-DNA ORF 8 is a diverged Ti T-DNA tms1. Both Agrobacterium genes consist of a member of the diverged gene family detected in the central part of the Ri TL-DNA, but fused to a sequence similar to iaaM of Pseudomonas savastonoi. Other members of this gene family were found scattered throughout Ti T-DNA. We argue that the central region of Ri and the part of Ti T-DNA including ORFs 5–10 evolved from a common ancestor. We present the hypothesis that the gene family encodes functions that alter developmental plasticity in higher plants.

Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of Withania somnifera

Developmental variability was introduced into Withania somnifera using genetic transformation by Agrobacterium rhizogenes, with the aim of changing withasteroid production. Inoculation of W. somnifera with A. rhizogenes strains LBA 9402 and A4 produced typical transformed root lines, transformed callus lines, and rooty callus lines with simultaneous root dedifferentiation and redifferentiation. These morphologically distinct transformed lines varied in T-DNA content, growth rates, and withasteroid accumulation. All of the lines with the typical transformed root morphology contained the TL T-DNA, and 90% of them carried the TR T-DNA, irrespective of the strain used for infection. Accumulation of withaferin A was maximum (0.44% dry weight) in the transformed root line WSKHRL-1. This is the first detection of withaferin A in the roots of W. somnifera. All of the rooty callus lines induced by strain A4 contained both the TL and the TR-DNAs. In contrast, 50% of the rooty-callus lines obtained with strain LBA 9402 contained only the TR T-DNA. All the rooty callus lines accumulated both withaferin A and withanolide D. The callusing lines induced by LBA 9402 lacked the TL T-DNA genes, while all the callusing lines induced by strain A4 contained the TL DNA. Four of these callus lines produced both withaferin A (0.15–0.21% dry weight) and withanolide D (0.08–0.11% dry weight), and they grew faster than the transformed root lines. This is the first report of the presence of withasteroids in undifferentiated callus cultures of W. somnifera.

Changes in flowering and the accumulation of polyamines and hydroxycinnamic acid-polyamine conjugates in tobacco plants transformed by the rolA locus from the Ri TL-DNA of Agrobacterium rhizogenes

Abstract Tobacco plants containing rolA (root locus A) from the Ri TL-DNA (root-inducing, left-hand, transferred DNA) from Agrobacterium rhizogenes have an altered phenotype, including male sterility, abnormal flower morphology, wrinkled leaves and shortened internodes. This phenotype is unstable, reverting to an attenuated form in lateral branches. We show that the pleiotropic effects of rolA are correlated with inhibition of the accumulation of conjugates between polyamines and the hydroxycinnamic acids. The compounds associated with male fertility are primarily affected, particularly the feruloyl derivatives.

Chemical synthesis, expression and mutagenesis of a gene encoding β-cryptogein, an elicitin produced by Phytophthora cryptogea

Elicitins are 10 kDa holoproteins secreted by Phytophthora fungi, that elicit an incompatible hypersensitive reaction, leading to resistance against fungal and bacterial plant pathogens. Comparison of primary sequences of α-elicitins and β-elicitins indicated several potential necrotic activity-determining residues. All of the highly necrotic β-elicitins have a hydrophilic residue (usually lysine) at position 13, whereas in the less necrotic α-elicitins this residue is replaced by a valine. Here, we report the synthesis and expression of a gene encoding a highly necrotic elicitin, β-cryptogein, and we show that the substitution of Lys-13 of this recombinant protein by a valine leads to a drastic alteration to the necrotic activity of the recombinant protein.

Use of Agrobacterium rhizogenes to create transgenic apple trees having an altered organogenic response to hormones

SummaryThe apple rootstock, M26, was genetically and phenotypically transformed using the Agrobacterium wild-type strain, A4. First, chimeric plants were obtained having transformed roots and normal aerial parts. Transformed plants were then produced through regeneration from transformed roots. Transformation was demonstrated by molecular hybridization and opine analysis. The effects of hormones on organogenesis was altered in transformants: cytokinins were required to form roots, whereas auxin was toxic at the concentration used to induce rooting in the control.

Homology-dependent DNA transfer from plants to a soil bacterium under laboratory conditions: implications in evolution and horizontal gene transfer.

DNA transfer was demonstrated from six species of donor plants to the soil bacterium, Acinetobacter spp. BD413, using neomycin phosphotransferase (nptII) as a marker for homologous recombination. These laboratory results are compatible with, but do not prove, DNA transfer in nature. In tobacco carrying a plastid insertion of nptII, transfer was detected with 0.1 g of disrupted leaves and in oilseed rape carrying a nuclear insertion with a similar quantity of roots. Transfer from disrupted leaves occurred in sterile soil and water, without the addition of nutrients. It was detected using intact tobacco leaves and intact tobacco and Arabidopsis plants in vitro. Transfer was dose-dependent and sensitive to DNase, and mutations in the plant nptII were recovered in receptor bacteria. DNA transfer using intact roots and plants in vitro was easily demonstrated, but with greater variability. Transfer varied with plant genome size and the number of repeats of the marker DNA in the donor plant. Transfer was not detected in the absence of a homologous nptII in the receptor bacteria. We discuss these results with reference to non-coding DNA in plant genomes (e.g., introns, transposons and junk DNA) and the possibility that DNA transfer could occur in nature.

Pisum sativum mutants insensitive to nodulation are also insensitive to invasion in vitro by the mycorrhizal fungus, Gigaspora margarita

Abstract Transformed root cultures were established using Agrobacterium rhizigenes inoculation of pea mutants, altered in their interaction with Rhizobium . They were tested in an in vitro model for sensitivity to the vesicular-arbuscular mycorrhizal (VAM) fungus, gigaspora margarita . VAM development was assessed using light and electron microscopy. Two non-nodulating, non-nitrogen fixing (Nod − , Fix − ) pea mutants were resistant to VAM colonization in vitro: Mycelium developed on the root surface but failed to colonize the interior. A nodulating (Nod + ) genotype, which was unable to fix nitrogen (Fix − ) in association with Rhizobium and a parental line, Lincoln (Nod + , Fix + ), interacted normally with the fungus, showing extensive internal colonization. These results confirm, under axenic conditions, previous reports showing that defective nodulation is correlated with defective mycorrhization. We propose using this in vitro model to identify factors necessary to initiating and maintaining the VAM/plant symbiosis.

Isolation and identification of TL-DNA/plant junctions in Convolvulus arvensis transformed by Agrobacterium rhizogenes strain A4.

We have constructed a Charon 4A phage library containing insert DNA isolated from a morning glory (Convolvulus arvensis) plant (clone 7) regenerated from a root organ culture incited by Agrobacterium rhizogenes, strain A4. Using a subcloned region of the Ri plasmid as 32P‐labeled probe, two lambda clones containing most of the ‘left’ T‐DNA (TL) region were isolated. One of these lambda clones contains the left TL‐DNA/plant junction, which was located by comparing nucleotide sequences from the appropriate regions of the Ri plasmid and this lambda clone. A 25‐bp sequence found near this left TL‐DNA/plant junction matches the 25‐bp terminal sequence found at or near T‐DNA/plant junctions of both nopaline‐ and octopine‐type A. tumefaciens Ti plasmids. A possible location for the right Ri TL‐DNA/plant junction in C. arvensis clone 7 was found by obtaining the nucleotide sequence surrounding its mapped location. Hybridization of plant DNA found adjacent to the left TL‐DNA/plant junction against total C. arvensis DNA shows that this T‐DNA integration occurred in a plant DNA region that does not contain highly repetitive DNA sequences. Nucleotide sequence analysis of 1004 bp of this plant DNA revealed no complete or partial open reading frames, but this plant DNA does have the potential to form various secondary structures which might play a role in the T‐DNA integration event.