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ABSTRACT
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SUMMARYIn plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)‐inducing molecules produced by the endonucleolytic cleavage of double‐stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs... more
SUMMARYIn plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)‐inducing molecules produced by the endonucleolytic cleavage of double‐stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA‐DIRECTED RNA POLYMERASE 6 (RDR6)‐mediated dsRNA synthesis using siRNA‐targeted RNA. The newly synthesized dsRNA is subsequently cleaved into secondary siRNAs by DICER‐LIKE (DCL) endonucleases. Just like primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA‐induced silencing complex reinforcing the cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the green fluorescent protein (GFP)‐expressing Nicotiana benthamiana 16C line, high‐pressure spraying protocol, and synthetic 22‐nucleotide (nt) long siRNAs. We found that the 22‐nt siRNA targeting the 3ʹ of the GFP transgene was less efficient in inducing silencing when compared with the siRNAs targeting the 5ʹ and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs is shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6‐mediated dsRNA synthesis is not primed by primary siRNAs and that dsRNA synthesis appears to be generally initiated at the 3ʹ‐end of the target RNA.
Research Interests: RNA, Plant Biology, Biology, RNA silencing, Medicine, and 4 moreGene Silencing, RNA interference, Argonaute-2, and Plant
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Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA)... more
Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (-)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses.
Research Interests: Microbiology, RNA, Plant Biology, Biology, RNA silencing, and 15 moreInnate immunity, Plant Molecular Biology, Medicine, Potato, RNA interference, Plant diseases, Molecular plant pathology, Temperature, Tomato, Plant viruses, Viroids, Chromosome segregation, Nucleic Acid Conformation, Lycopersicon esculentum, and reverse transcriptase polymerase chain reaction
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In plants, transgenes frequently become spontaneously silenced for unknown reasons. Typically, transgene silencing involves the generation of small interfering RNAs (siRNAs) that directly or indirectly target cognate DNA and mRNA... more
In plants, transgenes frequently become spontaneously silenced for unknown reasons. Typically, transgene silencing involves the generation of small interfering RNAs (siRNAs) that directly or indirectly target cognate DNA and mRNA sequences for methylation and degradation, respectively. In this report, we compared spontaneous silencing of a transgene in Nicotiana benthamiana and Nicotiana tabacum. In both species, abundant siRNAs were produced. In N. benthamiana, the self-silencing process involved mRNA degradation and dense DNA methylation of the homologous coding region. In N. tabacum, self-silencing occurred without complete mRNA degradation and with low methylation of the cognate coding region. Our data indicated that in plants, siRNA-mediated spontaneous silencing is, in addition to mRNA degradation, based on translational inhibition. Differences in the initiation and establishment of self-silencing together with marked differences in the degree of de novo DNA methylation showed that the mechanistic details of RNA silencing, although largely conserved, may vary also in genetically close plant species.
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Research Interests: RNA, Biology, RNA silencing, Medicine, Gene Silencing, and 3 moreRNA interference, Argonaute-2, and Plant
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The satellite RNA of the grapevine isolate NW of Arabis mosaic virus (ArMV) was cloned and sequenced, and showed 75% identity at the nucleotide level to the satellite RNA of the lilac isolate of ArMV. In order to survey ArMV isolates from... more
The satellite RNA of the grapevine isolate NW of Arabis mosaic virus (ArMV) was cloned and sequenced, and showed 75% identity at the nucleotide level to the satellite RNA of the lilac isolate of ArMV. In order to survey ArMV isolates from various geographical origins and natural hosts for the presence of large satellite RNAs and analyse their degree of variability, a RT/PCR-partial restriction enzymatic mapping (PREM) method was developed. The method is based on the incorporation of 5-methyl-dCTP in the RT/PCR reaction, and the subsequent digestion of the RT/PCR products by methyl-sensitive restriction enzymes. Satellites RNAs were detected by RT/PCR in eight isolates out of 47, six of them originating from grapevine, one from hop and one from lilac. The partial restriction digestion patterns allowed to distinguish six different types of satellites. Cloning and sequencing of the different satellites confirmed these results, the PREM proving able to discriminate sequences with 96% identity. The sizes of the different satellites varied between 1092 and 1139 nucleotides, their encoded proteins between 338 and 360 amino acids. Conserved domains were found in the amino and carboxy-termini between the sequences of the proteins encoded by the satellites of the different isolates of ArMV.
Research Interests: Microbiology, Medical Microbiology, Biology, Medicine, DNA, and 15 moreDNA fingerprinting, Satellite, Molecular cloning, Amino Acid Profile, Vitis, Amino Acid Sequence, Restriction Site, Nucleotides, Virus isolation, Molecular Sequence Data, restriction enzyme, Restriction Mapping, reverse transcriptase polymerase chain reaction, conserved sequence , and DNA Restriction Enzymes
HC-Pro is a helper component-proteinase which acts as a multifunctional protein in the potyviral life cycle. Apart from its proteolytic activity, HC-Pro has the capacity to bind duplex small RNAs (sRNAs). To investigate HC-Pro-mediated... more
HC-Pro is a helper component-proteinase which acts as a multifunctional protein in the potyviral life cycle. Apart from its proteolytic activity, HC-Pro has the capacity to bind duplex small RNAs (sRNAs). To investigate HC-Pro-mediated sRNA binding in vitro, high amounts of purified protein are required. For this purpose, the Zucchini yellow mosaic virus (ZYMV) HC-Pro was expressed as a fusion with hexa-histidine (6xHis) or maltose-binding protein (MBP) in Escherichia coli. The expressed fusion proteins were purified by affinity chromatography. 6xHis:HC-Pro and MBP:HC-Pro were partially soluble. Electrophoretic mobility-shift assays demonstrated that only MBP:HC-Pro exhibits the sRNA binding activity. The recombinant HC-Pro bound 21 bp siRNAs as well as 19 bp and 24 bp siRNAs. A point mutation in the highly conserved FRNK box produced the HC-Pro(FINK) protein, previously shown to be associated with reduced viral symptoms and weak sRNA binding. In this study, sRNA binding of the MBP:HA-HC-Pro(FINK) was not detectable. The high yield of purified HC-Pro offers the possibility to study the biochemistry of the protein in detail.
Research Interests: Biochemistry, Molecular Biology, Biology, Medicine, Gene expression, and 15 moreEscherichia coli, Cucurbita, Life Cycle, Affinity chromatography, Small RNA, Recombinant DNA, Protein expression and purification, Protein Expression, Cysteine endopeptidases, Protein Binding, Fusion Protein, Histidine, Biochemistry and cell biology, Proteolytic activity, and Maltose binding protein
Research Interests: Genetics, RNA, Plant Biology, Biology, Tobacco, and 14 morePlant Molecular Biology, Medicine, Gene Silencing, Recombinant DNA Technology, Potato, RNA interference, DNA methylation, Plant viruses, Secondary Structure, Viroids, Base Sequence, Full Length Movies, Biochemistry and cell biology, and Molecular Sequence Data
The development of 2D and 3D structures on the nanoscale containing viral nanoparticles (VNPs) as interesting nanobuilding blocks has come into focus for a bottom-up approach as an alternative to the top-down approach in... more
The development of 2D and 3D structures on the nanoscale containing viral nanoparticles (VNPs) as interesting nanobuilding blocks has come into focus for a bottom-up approach as an alternative to the top-down approach in nanobiotechnology. Our research has focused on the plant Tomato Bushy Stunt Virus (TBSV). In a previous study, we reported the impact of the pH value on the 2D assembly of viral monolayers. Here, we extend these studies into the third dimension by using specific interactions between the layers in combination with selective side chains on the viral capsid. The virus bilayer structure is prepared by an alternating deposition of His-tagged TBSV (4D6H-TBSV, first layer), Ni-NTA nanogold (second layer) complexes and 4D6H-TBSV, respectively, and 6D-TBSV (6xaspartic acid TBSV) as the third layer, i.e., the second layer of VNPs. The formed layer structures were imaged by using scanning force and scanning electron microscopy. The data show that a virus bilayer structure was ...
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The plant pathogen ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) is the causing agent of apple proliferation that leads to heavy damage in apple production all over Europe. To identify and analyze effector proteins of plant pathogens is... more
The plant pathogen ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) is the causing agent of apple proliferation that leads to heavy damage in apple production all over Europe. To identify and analyze effector proteins of plant pathogens is an important strategy in plant disease research. Here, we report that the SAP11-like protein of ‘Ca. P. mali’ induces crinkled leaves and siliques and witches’ broom symptoms in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to 6 members of class I and all members of class II TCP (TEOSINE BRANCHES/ CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of A. thaliana in yeast two-hybrid assays. Moreover, we demonstrate that the protein localizes actively into the plant nucleus without requiring the nuclear leader sequence (NLS). We also identified a 17 amino acid stretch previously predicted to be a nuclear leader sequence that is important for the binding of some of the TCPs and also responsible for the crinkled leaf and silique ...
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Self‐assembled plant viruses, here tomato bushy stunt virus (TBSV), are interesting building blocks in nanobiotechnology. Especially genetically modified virus types show enhanced self‐assembling properties compared to the wild type (wt)... more
Self‐assembled plant viruses, here tomato bushy stunt virus (TBSV), are interesting building blocks in nanobiotechnology. Especially genetically modified virus types show enhanced self‐assembling properties compared to the wild type (wt) virus. In this article important aspects of the self‐assembly of tetra histidine (4xHis) TBSV (histidine side chains on the capsid surface) are presented in detail. Different virus concentrations show a different wetting behavior of the solid substrate. This leads to different coverages depending on the position within the sample. In the center of the virus covered area, the most stable conditions are found and often also the highest coverage. If these central areas are compared to samples prepared with different virus concentrations, a large variation of the coverage can be detected. For the desired homogeneous monolayer of the virus, the concentration has thus to be optimized. In addition, it could be shown that the drop volume does not influence ...
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It was shown that the SAP11 effector of different Candidatus Phytoplasma can destabilize some TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTORs (TCPs), resulting in plant phenotypes such as witches’ broom and crinkled leaves. Some... more
It was shown that the SAP11 effector of different Candidatus Phytoplasma can destabilize some TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTORs (TCPs), resulting in plant phenotypes such as witches’ broom and crinkled leaves. Some SAP11 exclusively localize in the nucleus, while the others localize in the cytoplasm and the nucleus. The SAP11-like effector of Candidatus Phytoplasma mali strain PM19 (SAP11PM19) localizes in both compartments of plant cells. We show here that SAP11PM19 can destabilize TCPs in both the nucleus and the cytoplasm. However, expression of SAP11PM19 exclusively in the nucleus resulted in the disappearance of leaf phenotypes while still showing the witches’ broom phenotype. Moreover, we show that SAP11PM19 can not only destabilize TCPs but also relocalizes these proteins in the nucleus. Interestingly, three different transgenic Nicotiana species expressing SAP11PM19 show all the same witches’ broom phenotype but different leaf phenotypes. A possible mecha...
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Die pflanzenspezifischen SBP-Box-Gene kodieren für putative Transkriptionsfaktoren, die ubiqutär im Pflanzenreich vorkommen. Charakteristisch für alle Mitglieder dieser GenACHTUNGTRENNUNGfamilie ist die hochkonservierte SBP-Domäne, die... more
Die pflanzenspezifischen SBP-Box-Gene kodieren für putative Transkriptionsfaktoren, die ubiqutär im Pflanzenreich vorkommen. Charakteristisch für alle Mitglieder dieser GenACHTUNGTRENNUNGfamilie ist die hochkonservierte SBP-Domäne, die für die DNAbindende Eigenschaft verantwortlich ist. Außerhalb dieser Domäne besitzen SBP-Box-Gene nur geringe Sequenzähnlichkeiten. Dennoch weisen einige Mitglieder dieser Genfamilie eine höhere Sequenzähnlichkeit sowie eine konservierte Anzahl und Position der Introns auf. Aus Aradopsis thaliana konnten bisher 16 SBP-Box-Gene isoliert werden. Die Funktion der meisten SBP-Box-Gene konnte bisher noch nicht aufgeklärt werden. Bei einer konstitutiven Überexpression zeigte lediglich SPL3 mit deletiertem 3’UTR einen frühblühenden Phänotyp. Am 3’UTR befindet sich das so genannte miRNA responsive element (MRE), an welches die miRNA 156 an die mRNA bindet. Bei den miRNAs handelt es sich um 20 – 24 nt kleine RNAs die komplementär zu ihren Targetsequenzen in der mRNA sind. Die Genregulation über miRNAs verläuft entweder über „Cleavage“ der mRNA oder über Inhibierung der Translation. Brassica napus wurde mit dem Arabidopsis SPL3 Gen mit deletiertem 3’UTR unter Kontrolle des 35S Promotors transformiert. Transgene Regenerate wurden mit Hilfe einer Multiplex PCR identifiziert. In Southern Blot Analysen wurde die Kopienzahl des Transgens bestimmt. 3 Linien, in denen jeweils nur eine Transgenkopie und die Transgenaktivität in Northern Blot Analysen nachgewiesen wurden, wurden im Gewächshaus auf Blühbeginn, Anzahl der vegetativen Seitentriebe, Anzahl der Seitentriebe der Infloreszenz sowie Anzahl der Rosettenblätter bonitiert. 2 der 3 untersuchten Linien zeigten eine Blühverfrühung von ca. 4 Tagen sowie eine um 2 reduzierte Anzahl der Rosettenblätter. Lediglich die transgenen Pflanzen zeigten vegetative Seitentriebe und besaßen im Durchschnitt 2 Infloreszenz-Seitentriebe mehr als die Kontrollpflanzen. Als Kontrollpflanzen wurden Pflanzen der untersuchten 3 Linien ausgewählt, bei denen das Transgen herausgekreuzt wurde. Zur Untersuchung der Struktur der SBPBox Genfamilie in der amphidiploiden Spezies Brassica napus wurden gewebespezifische cDNA Bibliotheken aus Blatt und Blüte angelegt. Mit RACE-PCR Reaktionen und einer anschließenden BLAST Analyse konnten 13 SBP-Box-Gene identifiziert werden. Für SPL3 wurden 2 Gene mit unterschiedlichen UTRs gefunden. Ebenso konnte ein SBPBox-Gen mit bisher unbekannter Sequenz identifiziert werden.
Research Interests: Biology and Food Sciences
Research Interests: Biology and Food Sciences
ABSTRACT
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SAP11 is an effector protein that has been identified in various phytoplasma species. It localizes in the plant nucleus and can bind and destabilize TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors.... more
SAP11 is an effector protein that has been identified in various phytoplasma species. It localizes in the plant nucleus and can bind and destabilize TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors. Although SAP11 of different phytoplasma species share similar activities, their protein sequences differ greatly. Here, we demonstrate that the SAP11-like protein of ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) strain PM19 localizes into the plant nucleus without requiring the anticipated nuclear localization sequence (NLS). We show that the protein induces crinkled leaves and siliques, and witches’ broom symptoms, in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to six members of class I and all members of class II TCP transcription factors of A. thaliana in yeast two-hybrid assays. We also identified a 17 amino acid stretch previously predicted to be a nuclear localization sequence that is important for the binding of some of the TCPs, ...
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Phytoplasmas are the agents associated with numerous diseases in several plant species all over the world, including important food crops. The mode of phytoplasma infection is poorly understood and often based on genomic data. A yeast... more
Phytoplasmas are the agents associated with numerous diseases in several plant species all over the world, including important food crops. The mode of phytoplasma infection is poorly understood and often based on genomic data. A yeast two-hybrid screening was used to find new protein-protein interactions between ‘Candidatus Phytoplasma mali’, the phytoplasma associated with apple proliferation and its host plant. ‘Ca. P. mali’ strain PM19 genome encodes a protein PM19_00185 that interacts with at least five different ubiquitin conjugating enzymes (UBC, E2) of Arabidopsis thaliana. The in vitro ubiquitination assay shows that PM19_00185 is enzymatically active as E3 ligase with A. thaliana E2 UBC09.
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In plants, RNA interference (RNAi) is an effective defense mechanism against pathogens and pests. RNAi mainly involves the micro RNA and the small interfering RNA (siRNA) pathways. The latter pathway is generally based on the processing... more
In plants, RNA interference (RNAi) is an effective defense mechanism against pathogens and pests. RNAi mainly involves the micro RNA and the small interfering RNA (siRNA) pathways. The latter pathway is generally based on the processing of long double stranded RNAs (dsRNA) into siRNAs by DICER-LIKE endonucleases (DCLs). SiRNAs are loaded onto ARGONAUTE proteins to constitute the RNA-induced silencing complex (RISC). Natural dsRNAs derive from transcription of inverted repeats or of specific RNA molecules that are transcribed by RNA-directed RNA polymerase 6 (RDR6). Moreover, replication of infecting viruses/viroids results in the production of dsRNA intermediates that can serve as substrates for DCLs. The high effectiveness of RNAi both locally and systemically implicated that plants could become resistant to pathogens, including viruses, through artificial activation of RNAi by topical exogenous application of dsRNA. The most preferable procedure to exploit RNAi would be to simply ...
Phytoplasmas are the causative agent of numerous diseases of plant species all over the world, including important food crops. The mode by which phytoplasmas multiply and behave in their host is poorly understood and often based on... more
Phytoplasmas are the causative agent of numerous diseases of plant species all over the world, including important food crops. The mode by which phytoplasmas multiply and behave in their host is poorly understood and often based on genomic data. We used yeast two-hybrid screening to find new protein–protein interactions between the causal agent of apple proliferation ‘Candidatus Phytoplasma mali’ and its host plant. Here, we report that the ‘Ca. P. mali’ strain PM19 genome encodes a protein PM19_00185 that interacts with at least six different ubiquitin-conjugating enzymes (UBC; E2) of Arabidopsis thaliana. An in vitro ubiquitination assay showed that PM19_00185 is enzymatically active as E3 ligase with A. thaliana E2 UBC09 and Malus domestica E2 UBC10. We show that a nonhost bacteria (Pseudomonas syringae pv. tabaci) can grow in transgenic A. thaliana plant lines expressing PM19_00185. A connection of phytoplasma effector proteins with the proteasome proteolytic pathway has been re...