AlPlanta Institute for Plant Research

Germany

AlPlanta Institute for Plant Research

Germany

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Lamprecht R.L.,Stellenbosch University | Spaltman M.,Stellenbosch University | Stephan D.,Stellenbosch University | Wetzel T.,AlPlanta Institute for Plant Research | Burger J.T.,Stellenbosch University
Viruses | Year: 2013

The complete sequences of RNA1, RNA2 and satellite RNA have been determined for a South African isolate of Grapevine fanleaf virus (GFLV-SACH44). The two RNAs of GFLV-SACH44 are 7,341 nucleotides (nt) and 3,816 nt in length, respectively, and its satellite RNA (satRNA) is 1,104 nt in length, all excluding the poly(A) tail. Multiple sequence alignment of these sequences showed that GFLV-SACH44 RNA1 and RNA2 were the closest to the South African isolate, GFLV-SAPCS3 (98.2% and 98.6% nt identity, respectively), followed by the French isolate, GFLV-F13 (87.3% and 90.1% nt identity, respectively). Interestingly, the GFLV-SACH44 satRNA is more similar to three Arabis mosaic virus satRNAs (85%-87.4% nt identity) than to the satRNA of GFLV-F13 (81.8% nt identity) and was most distantly related to the satRNA of GFLV-R2 (71.0% nt identity). Full-length infectious clones of GFLV-SACH44 satRNA were constructed. The infectivity of the clones was tested with three nepovirus isolates, GFLV-NW, Arabis mosaic virus (ArMV)-NW and GFLV-SAPCS3. The clones were mechanically inoculated in Chenopodium quinoa and were infectious when co-inoculated with the two GFLV helper viruses, but not when co-inoculated with ArMV-NW. © 2013 by the authors; licensee MDPI, Basel, Switzerland.


Marcone C.,University of Salerno | Jarausch B.,AlPlanta Institute for Plant Research | Jarausch W.,AlPlanta Institute for Plant Research
Journal of Plant Pathology | Year: 2010

'Candidatus Phytoplasma prunorum' is an important prokaryotic pathogen that infects stone fruits in Europe. It is known to cause several economically relevant disorders of Prunus spp. which are collectively referred to as European stone fruit yellows (ESFY). This organism is phylogenetically closely related to other important fruit trees pathogens such as apple proliferation (AP), pear decline (PD) and peach yellow leaf roll (PYLR) agents. Together they form a distinct phylogenetic cluster, the AP or 16SrX group. Like the other fruit tree phytoplasmas of the AP group, 'Ca. P. prunorum' exhibits a high host specificity. In nature, this pathogen has been reported to infect only plants in the genus Prunus and to be transmitted by one insect vector species, the psyllid (Psyllidae) Cacopsylla pruni. Also, 'Ca. P. prunorum' includes strains which greatly differ in virulence. This review summarizes the current knowledge of 'Ca. P. prunorum' with emphasis on advances that have been made during the last two decades in understanding molecular and epidemiological aspects. Prospects for disease management and future research which could provide insights into the largely unknown mechanisms involved in pathogenicity of 'Ca. P. prunorum', are also critically discussed.


Guan X.,Karlsruhe Institute of Technology | Buchholz G.,AlPlanta Institute for Plant Research | Nick P.,Karlsruhe Institute of Technology
Journal of Experimental Botany | Year: 2013

Plant innate immunity is composed of two layers. Basal immunity is triggered by pathogen-associated molecular patterns (PAMPs) such as the fagellin-peptide flg22 and is termed PAMP-triggered immunity (PTI). In addition, effector-triggered immunity (ETI) linked with programmed cell death and cytoskeletal reorganization can be induced by pathogen-derived factors, such as the Harpin proteins originating from phytopathogenic bacteria. To get insight into the link between cytoskeleton and PTI or ETI, this study followed the responses of actin flaments and microtubules to flg22 and HrpZ in vivo by spinning-disc confocal microscopy in GFP-tagged marker lines of tobacco BY-2. At a con-centration that clearly impairs mitosis, flg22 can induce only subtle cytoskeletal responses. In contrast, HrpZ causes a rapid and massive bundling of actin microflaments (completed in ∼20 min, i.e. almost simultaneously with extracellular alkalinization), which is followed by progressive disintegration of actin cables and cytoplasmic microtubules, a loss of cytoplasmic structure, and vacuolar disintegration. Cytoskeletal disruption is proposed as an early event that discriminates HrpZ-triggered ETI-like defence from flg22-triggered PTI. © The Author(s) [2013].


Dadami E.,AlPlanta Institute for Plant Research | Moser M.,Research and Innovation Center | Zwiebel M.,AlPlanta Institute for Plant Research | Krczal G.,AlPlanta Institute for Plant Research | And 3 more authors.
FEBS Letters | Year: 2013

In plants, transgenes are generally more sensitive against RNA silencing than endogenes are. In this study, we generated a transgene that structurally mimicks an endogene. It is composed of endogenous promoter, 5′-UTR, introns, 3′-UTR and terminator elements. Our data revealed that, in contrast to a conventional transgene, an endogene-resembling transgene was more stably expressed and poorly processed into small RNAs. In addition, although both constructs triggered methylation of homologous DNA sequences at similar levels, the endogene-resembling transgene exhibited significantly delayed onset of local and systemic silencing. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.


Dadami E.,AlPlanta Institute for Plant Research | Dalakouras A.,AlPlanta Institute for Plant Research | Zwiebel M.,AlPlanta Institute for Plant Research | Krczal G.,AlPlanta Institute for Plant Research | And 2 more authors.
RNA Biology | Year: 2014

In plants, endogenes are less prone to RNA silencing than transgenes. While both can be efficiently targeted by small RNAs for post-transcriptional gene silencing (PTGS), generally only transgene PTGS is accompanied by transitivity, RNA-directed DNA methylation (RdDM) and systemic silencing. In order to investigate whether a transgene could mimick an endogene and thus be less susceptible to RNA silencing, we generated an intron-containing, endogene-resembling GREEN FLUORESCENT PROTEIN (GFP) transgene (GFPendo). Upon agroinfiltration of a hairpin GFP (hpF) construct, transgenic Nicotiana benthamiana plants harboring GFPendo(Nb-GFPendo) were susceptible to local PTGS. Yet, in the local area, PTGS was not accompanied by RdDM of the GFPendo coding region. Importantly, hpF-agroinfiltrated Nb-GFPendo plants were resistant to systemic silencing. For reasons of comparison, transgenic N. benthamiana plants (Nb-GFPcDNA) carrying a GFP cDNA transgene (GFPcDNA) were included in the analysis. HpF-agroinfiltrated Nb-GFPcDNA plants exhibited local PTGS and RdDM. In addition, systemic silencing was established in Nb-GFPcDNA plants. In agreement with previous reports using grafted scions, in systemically silenced tissue, siRNAs mapping to the 3′ of GFP were predominantly detectable by Northern blot analysis. Yet, in contrast to other reports, in systemically silenced leaves, PTGS was also accompanied by dense RdDM comprising the entire GFPcDNA coding region. Overall, our analysis indicated that cDNA transgenes are prone to systemic PTGS and RdDM, while endogene-resembling ones are resistant to RNA silencing. © 2014 Landes Bioscience.


Dalakouras A.,AlPlanta Institute for Plant Research | Dadami E.,AlPlanta Institute for Plant Research | Zwiebel M.,AlPlanta Institute for Plant Research | Krczal G.,AlPlanta Institute for Plant Research | And 2 more authors.
Epigenetics | Year: 2012

In plants, RNa-directed DNa methylation (RdDM) can target both transgene promoters and coding regions/gene bodies. RdDM leads to methylation of cytosines in all sequence contexts: cG, chG and chh. Upon segregation of the RdDM trigger, at least cG methylation can be maintained at promoter regions in the progeny. So far, it is not clear whether coding region methylation can be also maintained. We showed that the body of potato spindle tuber viroid (pSTVd) transgene constructs became densely de novo methylated at cG, chG and chh sites upon pSTVd infection. In this study, we demonstrate that in viroid-free progeny plants, asymmetric chh and chG methylation was completely lost. however, symmetric cG methylation was stably maintained for at least two generations. Importantly, the presence of transgene body methylation did not lead to an increase of dimethylation of histone h3 lysine 9 or a decrease of acetylation of h3. Our data supports the view that cG methylation can be maintained not only in promoters but also in the body of transgenes. They further suggest that maintenance of methylation may occur independently of tested chromatin modifications. © 2012 Landes Bioscience.


Dalakouras A.,AlPlanta Institute for Plant Research | Wassenegger M.,AlPlanta Institute for Plant Research | Wassenegger M.,Center for Organismal Studies Heidelberg
RNA Biology | Year: 2013

RNA-directed DNA methylation (RdDM) involves sequence-specific guiding of the de novo methylation machinery to complementary genomic DNA by RNA molecules. It is still elusive whether guide RNAs bind directly to DNA or to nascent transcripts produced from it. Even the nature of the guide RNAs is not elucidated. RNA interference (RNAi) studies provided a link between RNAi and RdDM indicating that small interfering RNAs (siRNAs) trigger and guide cytosine methylation. The "siRNA hypothesis" is generally accepted. However, recent data demonstrated that RdDM is not always associated with the accumulation of corresponding siRNAs. RdDM triggers may differ from guide RNAs and further studies are needed to clarify if guide RNAs are small or long RNAs, if they are single or double stranded and if they target DNA or nascent transcript. © 2013 Landes Bioscience.


Boonrod K.,AlPlanta Institute for Plant Research | Munteanu B.,AlPlanta Institute for Plant Research | Jarausch B.,AlPlanta Institute for Plant Research | Jarausch W.,AlPlanta Institute for Plant Research | Krczal G.,AlPlanta Institute for Plant Research
Molecular Plant-Microbe Interactions | Year: 2012

The phytopathogenic, cell-wall-less phytoplasmas exhibit a dual life cycle: they multiply in the phloem of their host plant and in the body of their insect vector. Their membrane proteins are in direct contact with both hosts and are supposed to play a crucial role in the phytoplasma spread within the plant as well as by the insect vector. Three types of nonhomologous but highly abundant and immunodominant membrane proteins (IDP) have been identified within the phytoplasmas: Amp, IdpA, and Imp. Although recent results indicate that Amp is involved in vector specificity interacting with insect proteins such as actin, little is known about the interaction of IDP with the plant. We could demonstrate that transiently expressed Imp of 'Candidatus Phytoplasma mali' as well as the Imp without transmembrane domain (ImpΔ™) bind with plant actins in vivo. Moreover, in vitro co-sediment and binding assays showed that Escherichia coli-expressed recombinant ImpΔ™- His binds to both G- and F-actins isolated from rabbit muscle. Transgenic plants expressing Imp- or ImpΔ™-green fluorescent protein did not exhibit any remarkable change of phenotype compared with the wildtype plant. These results indicate that Imp specifically binds to plant actin and a role of Imp-actin binding in phytoplasma motility is hypothesized. © 2012 The American Phytopathological Society.


Jarausch B.,AlPlanta Institute for Plant Research | Jarausch W.,AlPlanta Institute for Plant Research
Journal of Pest Science | Year: 2014

The univoltine psyllid Cacopsylla picta (Foerster) is the main vector of 'Ca. Phytoplasma mali' in Germany, the causal agent of apple proliferation, an economically important disease of apple in Europe. Its population dynamics on its host plant apple (Malus spp.) and migration to coniferous shelter plants for overwintering have been described for Southwest Germany. Temporary greenhouse rearing under controlled conditions, starting from field-collected overwintered adults, was set up to produce homogeneous offspring. New generation adults were transferred to conifers in the greenhouse at the beginning of the natural migration phase to obtain a continuous rearing spanning the aestivation and hibernation period. However, efforts to directly overwinter C. picta on different conifer species failed. The natural overwintering sites were therefore identified and their climatic conditions, mainly moderate summer temperatures with increased humidity and natural cold winter conditions including frost were reproduced. Teneral individuals of the new generation did not switch instantaneously from host plants to conifers. Successful overwintering under experimental conditions was only achieved using a gradual host plant transfer. By incorporating these key factors two continuous rearings of C. picta were maintained over 3 years with both, spruce and pine, as suitable shelter plants. This is the first report of a permanent rearing of C. picta under experimental conditions. Our data show experimentally that C. picta is univoltine and that conifers act only as shelter plants for overwintering and not as hosts for reproduction. © 2014 Springer-Verlag Berlin Heidelberg.


Munteanu B.,AlPlanta Institute for Plant Research | Braun M.,AlPlanta Institute for Plant Research | Boonrod K.,AlPlanta Institute for Plant Research
Journal of Zhejiang University: Science B | Year: 2012

QuickChange mutagenesis is the method of choice for site-directed mutagenesis (SDM) of target sequences in a plasmid. It can be applied successfully to small plasmids (up to 10 kb). However, this method cannot efficiently mutate bigger plasmids. Using KOD Hot Start polymerase in combination with high performance liquid chromatography (HPLC) purified primers, we were able to achieve SDM in big plasmids (up to 16 kb) involving not only a single base change but also multiple base changes. Moreover, only six polymerase chain reaction (PCR) cycles and 0.5 μl of polymerase (instead of 18 PCR cycles and 1.0 μl of enzyme in the standard protocol) were sufficient for the reaction.

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