Hans-Peter Mühlbach

Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.)

Abstract A gene derived from grapevine (Vitis vinifera) coding for stilbene synthase has been transferred into protoplasts of the commercially important japonica rice cultivar Nipponbare using PEG-mediated direct gene transfer. Transgenic plants were regenerated from calli selected on kanamycin. Southern blot analysis of genomic DNA isolated from regenerants and progeny plants demonstrated that the stilbene synthase gene is stably integrated in the genome of transgenic rice plants and inherited in the offspring. The transient formation of stilbene-synthase-specific mRNA shortly after inoculation with the fungus of the rice blast Pyricularia oryzae has demonstrated that the grapevine stilbene synthase promoter is also active in monocotyledonous plants. Preliminary results indicate an enhanced resistance of transgenic rice to P. oryzae.

Emaravirus: a novel genus of multipartite, negative strand RNA plant viruses.

Ringspot symptoms in European mountain ash (Sorbus aucuparia L.), fig mosaic, rose rosette, raspberry leaf blotch, pigeonpea sterility mosaic (Cajanus cajan) and High Plains disease of maize and wheat were found to be associated with viruses that share several characteristics. They all have single-stranded multipartite RNA genomes of negative orientation. In some cases, double membrane-bound virus-like particles of 80 to 200 nm in diameter were found in infected tissue. Furthermore, at least five of these viruses were shown to be vectored by eriophyid mites. Sequences of European mountain ash ringspot-associated virus (EMARaV), Fig mosaic virus (FMV), rose rosette virus (RRV), raspberry leaf blotch virus (RLBV), pigeonpea sterility mosaic virus and High Plains virus strongly support their potential phylogenetic relationship. Therefore, after characterization of EMARaV, the novel genus Emaravirus was established, and FMV was the second virus species assigned to this genus. The recently sequenced RRV and RLBV are supposed to be additional members of this new group of plant RNA viruses.

Double-stranded RNA pattern and partial sequence data indicate plant virus infection associated with the ringspot disease of European mountain ash (Sorbus aucuparia L.)

Summary.Double-stranded RNA (dsRNA) has been extracted from tissue of European mountain ash trees (Sorbus aucuparia L.) showing typical ringspot and mottling symptoms on leaves and a gradual decay in general. A characteristic dsRNA pattern was found in leaf samples of symptomatic mountain ash trees from various stands in Germany. Bands of dsRNA molecules of approximately 7 kb, 2.3 kb, 1.5 kb, and 1.3 kb, respectively, were repeatedly detected. By random primed reverse transcription cDNA was synthesised from dsRNA and amplified by degenerate oligonucleotide primed PCR. After TA cloning, the cDNA clones obtained were screened with an enhanced-chemiluminescence-labelled dsRNA probe. Positive clones were further analysed by using them as hybridisation probes in Northern blots of total plant RNA and in Southern hybridisation with genomic DNA from Sorbus aucuparia leaves. From cDNA clones that were found to be specific for dsRNA in Northern analysis, primers were deduced for 5′-RACE analyses and further cloning. Finally, a cDNA fragment of 3,737 bp was obtained, which showed homology to viral proteins, particularly to the RNA-dependent RNA polymerase of members of the family Bunyaviridae, but without high similarity to a known genus. The dsRNA pattern and the sequence information strongly indicate a virus associated with the mountain ash ringspot disease. The putative virus remains still unidentified.

Detection and tissue distribution of potato spindle tuberviroid in infected tomato plants by tissue print hybridization

SummaryPotato spindle tuber viroid (PSTVd) was detected in two cultivars of tomato (Lycopersicon esculentum Mill.) by tissue print hybridization of cross-sections of stem and rhachis, using a 35S-labeled PSTVd RNA probe. PSTVd was detectable in the viroid-sensitive and symptom-developping cv “Rutgers” 2 weeks p.i., and in the viroid-tolerant and practically symptomless cv “Goldkugel” 3 weeks p.i. In both tomato cultivars, PSTVd accumulated in the upper parts of the plants newly grown after inoculation. It was predominantly found in association with the ring formed by the vascular tissue. The final accumulation of PSTVd as well as its spatial distribution were similar in the sensitive and in the tolerant tomato cultivar, as estimated from the tissue print autoradiographs. Thus, tissue print hybridization provides a rapid and sensitive means for viroid diagnosis and for the assessment of tissue-specific localization of the viroid RNA.

Use of plant cell cultures in biotechnology

Plant cell cultures are being widely used in scientific studies on the physiology, biochemistry and molecular biology of primary and secondary metabolism, developmental regulation and cellular responses to pathogens and stress. In this chapter the significance of plant cell cultures in biotechnology is discussed with special emphasis on commercial production of secondary metabolites and pharmaceuticals, the potential of genetically transformed cell cultures, photosynthetically active cell cultures, production of somatic embryos, and novel assay systems based on the use of plant cells. Future aspects of biotechnical applications with respect to the potentials and limitations of these approaches are assessed, particularly in comparison with the productivity of lower eucaryotes.

Photosynthetically active suspension cultures of potato spindle tuber viroid infected tomato cells as tools for studying viroid — host cell interaction

Photosynthetically active callus and cell suspension cultures were established from uninfected Lycopersicon peruvianum plants and from uninfected and potato spindle tuber viroid (PSTVd) infected plants of Lycopersicon esculentum cv. Rutgers. Viroid infection was maintained in photoheterotrophic culture on media containing 3% sucrose, but during continuous photo-mixotrophic culture in low sucrose media (1% sucrose), the level of PSTVd accumulation decreased. Photoautotrophic cell suspensions could be established with uninfected, but not with viroid infected tomato cells. As compared to uninfected cells, PSTVd infected cells grew slowly, were morphologically different in size and shape, and formed tight cell aggregates. Electronmicroscopy showed that starch accumulation in chloroplasts, deformation of the chloroplast envelope and irregular plasmalemmasomes at the cell membrane were associated with PSTVd infection.

Detection of European mountain ash ringspot-associated virus-specific RNA and protein P3 in the pear leaf blister mite Phytoptus pyri (Eriophyidae)

The means by which European mountain ash ringspot-associated virus (EMARaV), a minus-strand ssRNA virus and the type member of the genus Emaravirus, is naturally spread, is unknown. In attempts to identify an EMARaV vector, galls induced by the eriophyid mite Phytoptus pyri were frequently found on infected leaves. By immunofluorescence microscopy, the presence of EMARaV nucleocapsid protein P3 was demonstrated in P. pyri individuals collected from diseased plants. Furthermore, RT-PCR analysis of entire P. pyri individuals revealed the presence of both viral genomic ss(−)RNAs and antigenomic ss(+)RNAs, suggesting that P. pyri might be a candidate vector of EMARaV.

Taxonomy of the family Arenaviridae and the order Bunyavirales: update 2018

In 2018, the family Arenaviridae was expanded by inclusion of 1 new genus and 5 novel species. At the same time, the recently established order Bunyavirales was expanded by 3 species. This article presents the updated taxonomy of the family Arenaviridae and the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

Detection of potato spindle tuber viroid (PSTVd) in minute amounts of potato (Solanum tuberosum L.) leaf tissue by hybridization techniques and, together with potato viruses, by multiplex RT-PCR

Potato spindle tuber viroid (PSTVd) is one of the major constraints for the improvement of potato production worldwide. Sensitive and sophisticated diagnostic procedures such as multiplex RT-PCR will help to control the spread of this and other pathogens, but are technically demanding and expensive. We therefore analyzed the usefulness of hybridization techniques for detection of PSTVd in minute amounts of tissue from potato plants raised through in vitro culture, which is the routine way of potato production. Tissue print, dot blot and Northern blot hybridization with a digoxigenin-labeled PSTVd specific riboprobe allowed detection of PSTVd on a nylon membrane at a minimum quantity of 10 pg per spot or, correspondingly, in RNA extracted from microgram quantities of potato leaf tissue. These findings underlined the usefulness of hybridization techniques in PSTVd diagnosis. The application of multiplex RT-PCR for PSTVd detection in the presence of five potato viruses clearly demonstrated the advantages of this technique, but its potential drawbacks, when used under field conditions in developing countries, must not be underestimated.ZusammenfassungDas Kartoffelspindelknollen-Viroid (PSTVd) stellt eines der größten Probleme in der weltweiten Erhöhung der Kartoffel-produktion dar. Zwar gibt es hochempfindliche Diagnoseverfahren wie die RT-PCR oder ”Multiplex RT-PCR“ zur simultanen Entdeckung mehrere Pathogene, doch sind diese Verfahren gerade in den Entwicklungsländern schwer einsetzbar, weil sie eine aufwendige Laborausstattung und gut ausgebildetes Personal erfordern. Wir haben daher an jungen Kartoffelpflanzen, die nach dem heutigen Standard durch in vitro-Vermehrung produziert wurden, geprüft, inwieweit die technisch leichter zu etablierenden Hybridisierungsmethoden wie Gewebeabdruck-, Dot-Blot- und Northern-Blot-Hybridisierung mit nichtradioaktiv markierten RNA-Sonden ein vergleichbar sensitives diagnostisches Instrument darstellen. Mit diesen Verfahren konnten noch 10 pg PSTVd-RNA auf einer Nylonmembran nachgewiesen werden; im Dot-Blot ließ sich PSTVd in einer RNA-Menge nachweisen, die aus wenigen Mikrogramm Blattmaterial isoliert worden war. Dies unterstützt unsere Annahme der Nutzbarkeit von Hybridierungs-methoden in der Diagnostik von PSTVd. Der Vorteil der Multiplex RT-PCR, PSTVd auch in Gegenwart von häufig vorkommenden Kartoffelviren eindeutig nachzuweisen, wurde in Vergleichsuntersuchungen bestätigt, jedoch müssen hier die Probleme der Anwendung unter Feldbedingungen in Entwicklungsländern berücksichtigt werden.

Circadian oscillations of Lhc mRNAs in a photoautotrophic cell culture of Lycopersicon peruvianum.

Fourteen genes encoding proteins of the light harvesting complex (Lhc) are expressed in a photoautotrophic cell culture from the wild species of tomato (Lycopersicon peruvianum). For two genes, Lhca2 (cab7) and Lhcb2*1 (cab4), a rhythmic oscillation of the transcript accumulation is observed under light/dark and constant dark conditions indicating that gene expression is controlled by a circadian clock in the tomato cell culture. The circadian expression of the Lhc genes remains present after application of 2,2′-dipyridyl. However, the amplitude of Lhc mRNA oscillations and the photosynthetic capacity (Fmax/Fo) decrease significantly. The transcript accumulations of psbA, rbcS and rbcL are less or not at all affected by 2,2′-dipyridyl.