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Publikationen - Molekulare Signalverarbeitung

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Publikation

Gago-Zachert, S.; Schuck, J.; Weinholdt, C.; Knoblich, M.; Pantaleo, V.; Grosse, I.; Gursinsky, T.; Behrens, S.-E.; Highly efficacious antiviral protection of plants by small interfering RNAs identified in vitro Nucleic Acids Res. 47, 9343-9357, (2019) DOI: 10.1093/nar/gkz678

In response to a viral infection, the plant’s RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, a very few of these siRNAs actually interfere with viral replication. A reliable approach to identify these immunologically effective siRNAs (esiRNAs) and to define the characteristics underlying their activity has not been available so far. Here, we develop a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. Tests on the efficacy of such identified esiRNAs of a model virus achieved a virtual full protection of plants against a massive subsequent infection in transient applications. We find that the functionality of esiRNAs depends crucially on two properties: the binding affinity to Argonaute proteins and the ability to access the target RNA. The ability to rapidly identify functional esiRNAs could be of great benefit for all RNA silencing-based plant protection measures against viruses and other pathogens.
Publikation

Rekik, I.; Drira, N.; Grubb, C. D.; Elleuch, A.; Molecular characterization and evolution studies of a SERK like gene transcriptionally induced during somatic embryogenesis in Phoenix Dactylifera L v Deglet Nour Genetika 47, 323-337, (2015) DOI: 10.2298/GENSR1501323R

A somatic embryogenesis receptor kinase like (SERKL) cDNA, designated PhSERKL, was isolated from date palm (Phoenix Dactylifera L) using RACE PCR. PhSERKL protein shared all the characteristic domains of the SERK family, including five leucine-rich repeats, one proline-rich region motif, a transmembrane domain, and kinase domains. Phylogenetic analyses using PHYLIP and Notung 2.7 programs suggest that the SERK proteins of some plant species resulted from relatively ancient duplication events. We predict an ancestor protein of monocots and dicots SERK using FASTML program. Somatic embryogenic cultures of date palm were established following transfer of callus cultures to medium containing 2, 4-dichlorophenoxyacetic acid. The role of PhSERKL gene during establishment of somatic embryogenesis in culture was investigated using quantitative real-time PCR. PhSERKL gene was highly expressed during embryogenic competence acquisition and globular embryo formation in culture. Overall, levels of expression of PhSERKL gene were lower in nonembryogenic tissues and organs than in embryogenic callus.
Publikation

Carbonell, A.; Flores, R.; Gago, S.; Trans-cleaving hammerhead ribozymes with tertiary stabilizing motifs: in vitro and in vivo activity against a structured viroid RNA Nucleic Acids Res. 39, 2432-2444, (2011) DOI: 10.1093/nar/gkq1051

Trans -cleaving hammerheads with discontinuous or extended stem I and with tertiary stabilizing motifs (TSMs) have been tested previously against short RNA substrates in vitro at low Mg 2+ concentration. However, the potential of these ribozymes for targeting longer and structured RNAs in vitro and in vivo has not been examined. Here, we report the in vitro cleavage of short RNAs and of a 464-nt highly structured RNA from potato spindle tuber viroid (PSTVd) by hammerheads with discontinuous and extended formats at submillimolar Mg 2+ . Under these conditions, hammerheads derived from eggplant latent viroid and peach latent mosaic viroid (PLMVd) with discontinuous and extended formats, respectively, where the most active. Furthermore, a PLMVd-derived hammerhead with natural TSMs showed activity in vivo against the same long substrate and interfered with systemic PSTVd infection, thus reinforcing the idea that this class of ribozymes has potential to control pathogenic RNA replicons.
Publikation

Dufour, D.; De la Peña, M.; Gago, S.; Flores, R.; Gallego, J.; Structure–function analysis of the ribozymes of chrysanthemum chlorotic mottle viroid: a loop–loop interaction motif conserved in most natural hammerheads Nucleic Acids Res. 37, 368-381, (2009) DOI: 10.1093/nar/gkn918

Loop–loop tertiary interactions play a key role in the folding and catalytic activity of natural hammerhead ribozymes. Using a combination of NMR spectroscopy, site-directed mutagenesis and kinetic and infectivity analyses, we have examined the structure and function of loops 1 and 2 of the (+) and (–) hammerheads of chrysanthemum chlorotic mottle viroid RNA. In both hammerheads, loop 1 is a heptanucleotide hairpin loop containing an exposed U at its 5′ side and an extrahelical U at its 3′-side critical for the catalytic activity of the ribozyme in vitro and for viroid infectivity in vivo , whereas loop 2 has a key opened A at its 3′-side. These structural features promote a specific loop–loop interaction motif across the major groove. The essential features of this tertiary structure element, base pairing between the 5′ U of loop 1 and the 3′ A of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2, are likely shared by a significant fraction of natural hammerheads.
Publikation

De Nardi, B.; Dreos, R.; Del Terra, L.; Martellossi, C.; Asquini, E.; Tornincasa, P.; Gasperini, D.; Pacchioni, B.; Rathinavelu, R.; Pallavicini, A.; Graziosi, G.; Differential responses of Coffea arabica L. leaves and roots to chemically induced systemic acquired resistance Genome 49, 1594-1605, (2006) DOI: 10.1139/g06-125

Coffea arabica is susceptible to several pests and diseases, some of which affect the leaves and roots. Systemic acquired resistance (SAR) is the main defence mechanism activated in plants in response to pathogen attack. Here, we report the effects of benzo(1,2,3)thiadiazole-7-carbothioic acid-s-methyl ester (BTH), a SAR chemical inducer, on the expression profile of C. arabica. Two cDNA libraries were constructed from the mRNA isolated from leaves and embryonic roots to create 1587 nonredundant expressed sequence tags (ESTs). We developed a cDNA microarray containing 1506 ESTs from the leaves and embryonic roots, and 48 NBS-LRR (nucleotide-binding site leucine-rich repeat) gene fragments derived from 2 specific genomic libraries. Competitive hybridization between untreated and BTH-treated leaves resulted in 55 genes that were significantly overexpressed and 16 genes that were significantly underexpressed. In the roots, 37 and 42 genes were over and underexpressed, respectively. A general shift in metabolism from housekeeping to defence occurred in the leaves and roots after BTH treatment. We observed a systemic increase in pathogenesis-related protein synthesis, in the oxidative burst, and in the cell wall strengthening processes. Moreover, responses in the roots and leaves varied significantly.
Publikation

Carbonell, A.; De la Peña, M.; Flores, R.; Gago, S.; Effects of the trinucleotide preceding the self-cleavage site on eggplant latent viroid hammerheads: differences in co- and post-transcriptional self-cleavage may explain the lack of trinucleotide AUC in most natural hammerheads Nucleic Acids Res. 34, 5613-5622, (2006) DOI: 10.1093/nar/gkl717

Eggplant latent viroid (ELVd) can form stable hammerhead structures in its (+) and (−) strands. These ribozymes have the longest helices I reported in natural hammerheads, with that of the ELVd (+) hammerhead being particularly stable (5/7 bp are G-C). Moreover, the trinucleotide preceding the self-cleavage site of this hammerhead is AUA, which together with GUA also found in some natural hammerheads, deviate from the GUC present in most natural hammerheads including the ELVd (−) hammerhead. When the AUA trinucleotide preceding the self-cleavage site of the ELVd (+) hammerhead was substituted by GUA and GUC, as well as by AUC (essentially absent in natural hammerheads), the values of the self-cleavage rate constants at low magnesium of the purified hammerheads were: ELVd-(+)-AUC≈ELVd-(+)-GUC>ELVd-(+)-GUA> ELVd-(+)-AUA. However, the ELVd-(+)-AUC hammerhead was the catalytically less efficient during in vitro transcription, most likely because of the transient adoption of catalytically-inactive metastable structures. These results suggest that natural hammerheads have been evolutionary selected to function co-transcriptionally, and provide a model explaining the lack of trinucleotide AUC preceding the self-cleavage site of most natural hammerheads. Comparisons with other natural hammerheads showed that the ELVd-(+)-GUC and ELVd-(+)-AUC hammerheads are the catalytically most active in a post-transcriptional context with low magnesium.
Publikation

Quint, M.; Melchinger, A. E.; Dußle, C. M.; Lübberstedt, T.; Breeding for virus resistance in maize Genetika 32, 529-545, (2000)

Sugarcane mosaic virus (SCMV) is an important disease in maize, which is emerging in Germany since 1983. Using this pest as a model for the inheritance of oligogenic traits, we clarified the genetic ba­sis for resistance in early maturing European maize germplasm. Screening of 122 adapted European inbred lines identified three completely resistant lines, which were used for further analyses. The genetics of SCMV resis­tance was investigated by allelism tests in field experiments combined with QTL and bulked segregant analyses (BSA) on the marker level. QTL analyses revealed the presence of two major genes Scm1 and Scm2 plus three minor QTL. Involvement of Scm1 and Scm2 in the inheritance of SCMV resistance could be confirmed by BSA in a second cross. Breeders can make use of tightly linked STS markers for marker-assisted selection (MAS) as well as our SCMV resistant flint lines to improve their elite germplasm. Currently, recurrent backcrossing with phenotypic selection is the most appropriate and cost effective breeding method. With de­creasing costs of DNA chip technology, MAS can be competitive with phenotypic selection in the near future. Further objectives of our research are the isolation and cloning of Scm1 and Scm2. To achieve this goal we follow two different approaches. (1) Positional cloning based on more than 500 AFLP primer combinations resulted in Scm1/Scm2 specific markers with a resolution of approximately 0.2 cM in the respective re­gions. (2) Resistance gene analogues (RGAs), cosegregating with the tar­get genes are used to identify further candidate genes for transformation experiments.
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