Johannes Fütterer

Posttranscriptional trans-activation in cauliflower mosaic virus

The ability of plant cells to translate dicistronic mRNAs that mimic a segment of the polycistronic 35S RNA from cauliflower mosaic virus has been tested. The chloramphenicol acetyltransferase and beta-glucuronidase open reading frames (ORFs) were fused in-frame to the second viral cistron (ORF I). Efficient reporter expression from the corresponding plasmids in plant protoplasts was observed only upon cotransfection with viral DNA. The trans-activating gene maps at ORF VI, which is expressed from a separate, monocistronic messenger (19S RNA). Deletion analysis shows that trans-activation selectively enhances downstream gene expression; the high expression of the upstream ORF is not further increased. The major reporter transcript remained bicistronic upon trans-activation, and its abundance varied only to a limited extent. Results indicate that trans-activation enhances the translation of downstream ORFs on polycistronic mRNAs derived from cauliflower mosaic virus.

Translation in plants — rules and exceptions

Translation processes in plants are very similar to those in other eukaryotic organisms and can in general be explained with the scanning model. Particularly among plant viruses, unconventional mRNAs are frequent, which use modulated translation processes for their expression: leaky scanning, translational stop codon readthrough or frameshifting, and transactivation by virus-encoded proteins are used to translate polycistronic mRNAs; leader and trailer sequences confer (cap-independent) efficient ribosome binding, usually in an end-dependent mechanism, but true internal ribosome entry may occur as well; in a ribosome shunt, sequences within an RNA can be bypassed by scanning ribosomes. Translation in plant cells is regulated under conditions of stress and during development, but the underlying molecular mechanisms have not yet been determined. Only a small number of plant mRNAs, whose structure suggests that they might require some unusual translation mechanisms, have been described.

Arabidopsis RETINOBLASTOMA-RELATED Is Required for Stem Cell Maintenance, Cell Differentiation, and Lateral Organ Production

Plant development depends on the balance of stem cell renewal and differentiation in several stem cell niches that is achieved by yet unknown regulatory mechanisms. Using inducible RNA interference-mediated downregulation, Arabidopsis RETINOBLASTOMA-RELATED protein is shown to regulate the proliferation, maintenance, and differentiation of stem cells and organ production from stem cell niches. Several genes involved in the regulation of postembryonic organ initiation and growth have been identified. However, it remains largely unclear how developmental cues connect to the cell cycle. RETINOBLASTOMA RELATED (RBR) is a plant homolog of the tumor suppressor Retinoblastoma (pRb), which is a key regulator of the cell cycle. Using inducible RNA interference (RNAi) against Arabidopsis thaliana RBR (RBRi), we reduced RBR expression levels at different stages of plant development. Conditional reduction or loss of RBR function disrupted cell division patterns, promoted context-dependent cell proliferation, and negatively influenced establishment of cell differentiation. Several lineages of toti- and pluripotent cells, including shoot apical meristem stem cells, meristemoid mother cells, and procambial cells, failed to produce appropriately differentiated cells. Meristem activity was altered, leading to a disruption of the CLAVATA-WUSCHEL feedback loop and inhibition of lateral organ formation. Release of RBR from RNAi downregulation restored meristem activity. Gene profiling analyses soon after RBRi induction revealed that a change in RBR homeostasis is perceived as a stress, even before genes regulated by RBR-E2F become deregulated. The results establish RBR as a key cell cycle regulator required for coordination of cell division, differentiation, and cell homeostasis.

Effects of combined expression of antifungal barley seed proteins in transgenic wheat on powdery mildew infection

Transgenicwheat plants (variety Frisal) constitutively expressing a number of potentialantifungal proteins alone or in combinations were generated and tested forincreased resistance to Blumeria graminis f.sp. tritici(powdery mildew) in a detached leaf infection assay. The most significativerateof protection was obtained with an apoplastic ribosome-inactivation proteinfrombarley seed. Apoplastic Barnase was less efficient and individual plant linesharbouring a barley seed chitinase and β-1,3-glucanase showed linespecificphenotypes from increased resistance to increased susceptibility. Combinationbycrossing of three barley seed proteins did not lead to significant improvementof protection.

Expression of active barley seed ribosome-inactivating protein in transgenic wheat

Abstract Phenotypically normal, transgenic wheat (Triticum aestivum, var. Frisal) plants expressing a barley seed ribosome-inactivating protein (RIP) were produced. Expression was controlled by an intron-enhanced cauliflower mosaic virus 35S promoter and has been completely stable over four generations so far, possibly due to matrix-associated regions (MARs) that flank the transgenes. An engineered fusion to a signal peptide derived from the barley seed ß-1,3-glucanase caused the transport of RIP to the apoplast. Activity of the accumulated protein could be shown by significant inhibition of a rabbit reticulocyte transcription/translation system. Plants expressing high levels of RIP were protected only moderately or not at all against infection by the fungal pathogen Erysiphe graminis.

Role of a Short Open Reading Frame in Ribosome Shunt on the Cauliflower Mosaic Virus RNA Leader

The pregenomic 35 S RNA of cauliflower mosaic virus (CaMV) belongs to the growing number of mRNAs known to have a complex leader sequence. The 612-nucleotide leader contains several short open reading frames (sORFs) and forms an extended hairpin structure. Downstream translation of 35 S RNA is nevertheless possible due to the ribosome shunt mechanism, by which ribosomes are directly transferred from a take-off site near the capped 5′ end of the leader to a landing site near its 3′ end. There they resume scanning and reach the first long open reading frame. We investigated in detail how the multiple sORFs influence ribosome migration either via shunting or linear scanning along the CaMV leader. The sORFs together constituted a major barrier for the linear ribosome migration, whereas the most 5′-proximal sORF, sORF A, in combination with sORFs B and C, played a positive role in translation downstream of the leader by diverting scanning ribosomes to the shunt route. A simplified, shunt-competent leader was constructed with the most part of the hairpin including all the sORFs except sORF A replaced by a scanning-inhibiting structure. In this leader as well as in the wild type leader, proper translation and termination of sORF A was required for efficient shunt and also for the level of shunt enhancement by a CaMV-encoded translation transactivator. sORF A could be replaced by heterologous sORFs, but a one-codon (start/stop) sORF was not functional. The results implicate that in CaMV, shunt-mediated translation requires reinitiation. The efficiency of the shunt process is influenced by translational properties of the sORF.

RETINOBLASTOMA-RELATED PROTEIN controls the transition to autotrophic plant development.

Seedling establishment is a crucial phase during plant development when the germinating heterotrophic embryo switches to autotrophic growth and development. Positive regulators of embryonic development need to be turned off, while the cell cycle machinery is activated to allow cell cycle entry and organ primordia initiation. However, it is not yet understood how the molecular mechanisms responsible for the onset of cell division, metabolism changes and cell differentiation are coordinated during this transition. Here, we demonstrate that the Arabidopsis thaliana RETINOBLASTOMA-RELATED protein (RBR) ortholog of the animal tumor suppressor retinoblastoma (pRB) not only controls the expression of cell cycle-related genes, but is also required for persistent shut-down of late embryonic genes by increasing their histone H3K27 trimethylation. Seedlings with reduced RBR function arrest development after germination, and stimulation with low amounts of sucrose induces transcription of late embryonic genes and causes ectopic cell division. Our results suggest a model in which RBR acts antagonistically to sucrose by negatively regulating the cell cycle and repressing embryonic genes. Thus, RBR is a positive regulator of the developmental switch from embryonic heterotrophic growth to autotrophic growth. This establishes RBR as a new integrator of metabolic and developmental decisions.

Upstream and downstream sequence elements determine the specificity of the rice tungro bacilliform virus promoter and influence RNA production after transcription initiation.

The contribution of sequences upstream and downstream of the transcription start site to the strength and specificity of the promoter of rice tungro bacilliform virus was analysed in transgenic rice plants. The promoter is strongly stimulated by downstream sequences which include an intron and is active in all vascular and epidermal cells. Expression in the vascular tissue requires a promoter element located between −100 and −164 to which protein(s) from rice nuclear extracts bind. Elimination of this region leads to specificity for the epidermis. Due to the presence of a polyadenylation signal in the intron, short-stop RNA is produced from the promoter in addition to full-length primary transcript and its spliced derivatives. The ratio between short-stop RNA and full-length or spliced RNA is determined by upstream promoter sequences, suggesting the assembly of RNA polymerase complexes with different processivity on this promoter.

Simultaneous analysis of the bidirectional African cassava mosaic virus promoter activity using two different luciferase genes.

The expression of geminivirus genes is controlled by bidirectional promoters which are located in the large intergenic region of the circular DNA genomes and specifically regulated by virus encoded proteins. In order to study the simultaneous regulation of both orientations of the DNA A and DNA B promoters of African cassava mosaic virus (ACMV), they were cloned between two different luciferase genes with the firefly luciferase gene in complementary-sense and the Renilla luciferase gene in virion-sense orientation. The regulation of the ACMV promoters by proteins encoded by the complete DNA A, as well as by the individually expressed transactivator (TrAP) or replication-associated (Rep) proteins was assessed in tobacco and cassava protoplasts using dual luciferase assays. In addition, the regulation of the DNA A promoter integrated into tobacco genome was also assessed. The results show that TrAP activates virion-sense expression strongly both in cassava and tobacco protoplasts, but not in transgenic tobacco plants. In contrast to this, DNA A encoded proteins activate virion-sense expression both in protoplasts and in transgenic plants. At the same time they reduce the expression of the complementary-sense Rep gene on DNA A but activate the expression of the complementary-sense movement protein (MPB) gene on DNA B. The degree of MBP activation is higher in cassava than in tobacco protoplasts, indicating that the plant host also influences the promoter strength. Transient transformation experiments using linearized DNA indicate that the different regulation of the ACMV DNA A promoter in protoplasts and transgenic plants could be due to different DNA curvature in free plasmids and in genes integrated in plant genomic DNA.

Differential inhibition of downstream gene expression by the cauliflower mosaic virus 35S RNA leader

The effect of the 600 nucleotide-long CaMV 35S RNA 5′ leader sequence on the expression of downstream genes was analyzed both in plant protoplasts and in vitro. For transient expression studies in protoplasts derived from host and nonhost plants, the bacterial chloramphenicol acetyl transferase (CAT) gene was fused to the initiation codon of ORF VII. The leader sequence reduced CAT expression two- to four-fold in protoplasts derived from three host species, but 10- to 50-fold in protoplasts derived from three different nonhost species. For in-vitro studies the 35S promoter was replaced by the SP6 promoter. The leader reduced in-vitro translation of SP6 transcripts approximately six-fold, indicating that at least part of the inhibition observed in protoplasts is directly due to the interference of the leader sequence with translation. Other steps in gene expression that may also be affected are discussed.