Peter H. Schreier

Size, Location and Polarity of T-DNA-Encoded Transcripts in Nopaline Crown Gall Tumors - Common Transcripts in Octopine and Nopaline Tumors

Up to thirteen T-DNA-encoded, polyadenylated transcripts of different relative abundance were detected by Northern blot hybridization in the tobacco nopaline BT37 crown gall teratoma tissue. Their sizes range from 900 to 2,700 bases. The polarity of eight of the thirteen transcripts was assigned by hybridization of labeled RNA to single-stranded DNA fragments of the T-region obtained by cloning in an M13 vector. Both strands of the T-DNA are transcribed. Our data indicate that most, if not all, transcripts are generated via independent promoter and poly(A)-addition sites on the T-DNA. Comparison of T-DNA-encoded transcripts present in crown gall tumors showing teratoma-like growth (BT37) with those from an unorganized tumor line (W38C58) reveals that this difference in phenotype is accompanied by a difference in the expression of the T-DNA. T-DNA sequences common to both octopine and nopaline tumors encode at least five, and probably six, cross-hybridizing transcripts of the same size, location, polarity and function. These transcripts are involved in the process of plant tumor formation and maintenance.

Peptide-mediated broad-spectrum plant resistance to tospoviruses

Plant viruses have a significant impact on agronomic losses worldwide. A new strategy for engineering virus-resistant plants by transgenic expression of a dominant interfering peptide is presented here. This peptide of 29 aa strongly interacts with the nucleocapsid proteins (N) of different tospoviruses. Transgenic Nicotiana benthamiana lines expressing the peptide fused to a carrier protein were challenged with five different tospoviruses that have a nucleocapsid protein interacting with the peptide. In the transgenic plants, strong resistance to tomato spotted wilt virus, tomato chlorotic spot virus, groundnut ring spot virus, and chrysanthemum stem necrosis virus was observed. This therefore demonstrates the feasibility of using peptide “aptamers” as an in vivo tool to control viral infection in higher plants.

A functional analysis of T-DNA gene 6b: the fine tuning of cytokinin effects on shoot development.

SummaryThe physiological function in planta of T-DNA gene 6b was studied under various experimental conditions. For this purpose the coding region of gene 6b was cloned behind the 1′-promoter of the TR-DNA to enhance expression of the gene product in transformed plant cells. Expression of the recombinant gene in leaf discs of Nicotiana tabacum altered the capacity for shoot formation of the discs, induced by exogenous (i.e. BAP in the growth medium or agrobacterial trans-zeatin produced under control of gene tzs) or endogenous cytokinins (i.e. isopentenyladenosine produced under control of T-DNA gene 4). The data obtained indicate a reduction of cytokinin activity within the plant cells by the product of T-DNA gene 6b.

Novel P-Type ATPases Mediate High-Affinity Potassium or Sodium Uptake in Fungi

ABSTRACT Fungi have an absolute requirement for K+, but K+ may be partially replaced by Na+. Na+ uptake in Ustilago maydis and Pichia sorbitophila was found to exhibit a fast rate, low Km, and apparent independence of the membrane potential. Searches of sequences with similarity to P-type ATPases in databases allowed us to identify three genes in these species, Umacu1, Umacu2, and PsACU1, that could encode P-type ATPases of a novel type. Deletion of the acu1 and acu2 genes proved that they encoded the transporters that mediated the high-affinity Na+ uptake of U. maydis. Heterologous expressions of the Umacu2 gene in K+ transport mutants of Saccharomyces cerevisiae and transport studies in the single and double Δacu1 and Δacu2 mutants of U. maydis revealed that the acu1 and acu2 genes encode transporters that mediated high-affinity K+ uptake in addition to Na+ uptake. Other fungi also have genes or pseudogenes whose translated sequences show high similarity to the ACU proteins of U. maydis and P. sorbitophila. In the phylogenetic tree of P-type ATPases all the identified ACU ATPases define a new cluster, which shows the lowest divergence with type IIC, animal Na+,K+-ATPases. The fungal high-affinity Na+ uptake mediated by ACU ATPases is functionally identical to the uptake that is mediated by some plant HKT transporters.

Characterization of two class II chitinase genes from peanut and expression studies in transgenic tobacco plants.

Two different genes encoding class II chitinases from peanut (Arachis hypogaea L. cv. NC4), A.h.Chi2;1 and A.h.Chi2;2, have been cloned. In peanut cell suspension cultures, mRNA levels of A.h.Chi2;2 increased after ethylene or salicylate treatment and in the presence of conidia from Botrytis cinerea. The second gene, A.h.Chi2;1, was only expressed after treatment with the fungal spores. Transgenic tobacco plants containing the complete peanut A.h.Chi2;1 gene exhibited essentially the same expression pattern in leaves as observed in peanut cell cultures. Expression characteristics of transgenic tobacco carrying a promoter-GUS fusion of A.h.Chi2;1 are described.

Elicitor-specific induction of one member of the chitinase gene family in Arachis hypogaea

SummaryChitinases are believed to play an important role in plant defence against bacterial and fungal attack. In peanut (Arachis hypogaea) chitinase genes form a small multigene family. Four chitinase cDNAs (chit 1–4) were isolated from cultured peanut cells. Expression of individual chit genes was assayed by the polymerase chain reaction (PCR) followed by analysis of restriction fragment length polymorphisms (RFLP). UV irradiation, dilution of cell cultures and treatment with Phytophthora megasperma (Pmg) elicitor or yeast extract were used to induce expression of chit genes. The chit 3 gene is constitutively expressed at a low level in untreated as well as in treated cultures; the expression of chit 4 gene is induced by each of the stimuli tested, whereas the chit 1 gene is activated by cell culture dilution and by yeast extract treatment. The chit 2 gene is strongly activated by treatment with cell wall components from the fungus Phytophthora megasperma but not by the other stimuli. These results indicate that chit 2 gene expression may be controlled by pathogen-specific regulatory elements.

Targeting of protein to chloroplasts in transgenic tobacco by fusion to mutated transit peptide

SummaryTransport of foreign proteins into chloroplasts was studied in a transgenic plant expressing two different fusion proteins, the transit peptide (TP) of ribulose-bisphosphate carboxylase small subunit (SS) fused to neomycin phosphotransferase (TP-NPT II) and, the same transit peptide plus the amino-terminal 23 amino acids of mature SS linked to NPT II. The second fusion protein (TP-SS-NPT II) was found in isolated chloroplasts but accumulated to a lesser degree than the first (TP-NPT II). This finding does not support the hypothesis that the highly conserved amino acid sequence surrounding the cleavage site between the transit peptide (TP) and mature SS is required for efficient transport. This cleavage region shows a markedly higher conservation than either the mature protein or the TP sequences in SS genes from different plant species. Evidence is presented indicating that the transport of the TP-SS-NPT II precursor is diminished as a result of competition between the rate of its uptake and the rate of its degradation by cytosolic proteases. In an attempt to identify further regions in the TP involved in transport and processing, we designed derivatives of both the TP-SS-NPT II and TP-NPT II precursors. A derivative of TP-SS-NPT II lacking the amino acids at the processing site was expressed in plants and was shown to be transported and processed. A derivative of TP-NPT II comprising the first 41 amino acids (out of 57) of the transit peptide linked to NPT II was also expressed in plants. This protein was not imported into the organelles; however a significant amount of partially processed fusion protein was found to be attached to the outer membrane of the chloroplast.

Plastid Transformation: A Progress Report

Reproducible methods, allowing the introduction of defined DNA fragments in the plant nuclear genome, are presently available (see previous chapters in this volume) and are contributing significantly to the experimental analysis of the gene structure-function relationships governing regulated gene-expression in plants.