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Shahriari M.,Botanical Institute | Richter K.,Botanical Institute | Keshavaiah C.,Botanical Institute | Keshavaiah C.,University College Cork | And 3 more authors.
Plant Molecular Biology | Year: 2011

In yeast, endosomal sorting of monoubiquitylated transmembrane proteins is performed by a subset of the 19 "class E vacuolar protein sorting" proteins. The core machinery consists of 11 proteins that are organised in three complexes termed ESCRT I-III (endosomal sorting complex required for transport I-III) and is conserved in eukaryotic cells. While the pathway is well understood in yeast and animals, the plant ESCRT system is largely unexplored. At least one sequence homolog for each ESCRT component can be found in the Arabidopsis genome. Generally, sequence conservation between yeast/animals and the Arabidopsis proteins is low. To understand details about participating proteins and complex organization we have performed a systematic pairwise yeast two hybrid analysis of all Arabidopsis proteins showing homology to the ESCRT core machinery. Positive interactions were validated using bimolecular fluorescence complementation. In our experiments, most putative ESCRT components exhibited interactions with other ESCRT components that could be shown to occur on endosomes suggesting that despite their low homology to their yeast and animal counterparts they represent functional components of the plant ESCRT pathway. © 2011 Springer Science+Business Media B.V.


Spallek T.,Norwich Research Park | Spallek T.,RIKEN | Beck M.,Norwich Research Park | Ben Khaled S.,Norwich Research Park | And 6 more authors.
PLoS Genetics | Year: 2013

The plant immune receptor FLAGELLIN SENSING 2 (FLS2) is present at the plasma membrane and is internalized following activation of its ligand flagellin (flg22). We show that ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT (ESCRT)-I subunits play roles in FLS2 endocytosis in Arabidopsis. VPS37-1 co-localizes with FLS2 at endosomes and immunoprecipitates with the receptor upon flg22 elicitation. Vps37-1 mutants are reduced in flg22-induced FLS2 endosomes but not in endosomes labeled by Rab5 GTPases suggesting a defect in FLS2 trafficking rather than formation of endosomes. FLS2 localizes to the lumen of multivesicular bodies, but this is altered in vps37-1 mutants indicating compromised endosomal sorting of FLS2 by ESCRT-I loss-of-function. VPS37-1 and VPS28-2 are critical for immunity against bacterial infection through a role in stomatal closure. Our findings identify that VPS37-1, and likewise VPS28-2, regulate late FLS2 endosomal sorting and reveals that ESCRT-I is critical for flg22-activated stomatal defenses involved in plant immunity. © 2013 Spallek et al.


News Article | October 13, 2016
Site: phys.org

Under the microscope (confocal microscopy): The AM fungus (green) reaches the inner root cortex and forms arbuscules (s. arrow; tree-like structure, Latin arbor = tree). Credit: Carolin Heck/KIT Many fungi damage or even kill plants. But there are also plant-friendly fungi: Most land plants live in close community with arbuscular mycorrhiza fungi (AM fungi) that stimulate their growth. Researchers of the "Molecular Phytopathology" Group of Karlsruhe Institute of Technology (KIT) study the development of this symbiosis. The scientists have now identified a gene that is specifically activated by AM fungi and influences the development of the plant root: The GRAS transcription factor MIG1 stimulates growth of more and larger root cortex cells. This is reported by the researchers in Current Biology. Most land plants live in symbiosis with AM fungi. Both sides profit: The AM fungi help the plants extract nutrients, such as nitrogen, phosphate, and water, from the ground, protect them against pests, and stimulate plant growth by influencing root development. In return, the plants supply the AM fungi with carbohydrates produced by photosynthesis. Symbiosis enhances growth and health of the plants even under adverse conditions of nutrient-depleted soil and stress. Controlled cultivation of plants in symbiosis with arbuscular mycorrhiza fungi might help reduce the amount of fertilizers and pesticides needed and, thus, contribute to sustainable agriculture. But how does the plant make friends with fungi? This question is studied by scientists of the "Molecular Phytopathology" Group headed by Professor Natalia Requena of KIT's Botanical Institute. Their fundamental research focuses on molecular processes associated with symbiosis. For the stimulation of plant growth by root development, the scientists have identified a plant gene that is activated specifically by the AM fungi – the GRAS transcription factor MIG1. It determines the size of the root cortex cells. Using Medicago truncatula, a snail clover species, KIT scientists studied the role of MIG1. They report their findings in the Current Biology journal. "Development of symbiosis with arbuscular mycorrhiza fungi requires plants to adapt in an extraordinary and controlled way," Professor Natalia Requena explains. "The plant activates its genetic programs for such a symbiosis even before its first physical contact with the fungus as soon as it receives a signaling substance emitted by the fungus." Then, development of symbiosis is mainly controlled by the plant. Settlement of AM fungi on plant roots is restricted to the epidermal tissue and cortex. Hyphae (cellular threads) of the fungus penetrate deep into the cortex and form widely branched structures, so-called arbuscules. The plant forms a specifically synthesized periarbuscular membrane (PAM) to enclose the arbuscules. Plant-specific proteins of the GRAS protein family take over major functions in the regulation of root colonization and the formation of arbuscules. They act as transcription factors that control, i.e. switch on or off, the activity of other genes. The protein RAM1, for instance, enables branching of arbuscules, RAD1 maintains them, and NSP1, NSP2, and DIP1 control the general colonization process. The researchers working in the group of Professor Natalia Requena identified the transcription factor MIG1 (Mycorrhiza Induced GRAS 1). It is expressed most strongly in cells containing arbuscules. MIG1 significantly modifies root cortex development by stimulating growth of more and larger root cortex cells. The overall diameter of the roots increases considerably. Vice versa, downregulation of MIG1 leads to malformed arbuscules. Explore further: Symbiosis with mycorrhizal fungi provides plants with enhanced access to scarce resources More information: Carolin Heck et al. Symbiotic Fungi Control Plant Root Cortex Development through the Novel GRAS Transcription Factor MIG1, Current Biology (2016). DOI: 10.1016/j.cub.2016.07.059


News Article | April 21, 2016
Site: phys.org

Made by KIT: The microfluidic bioreactor technically reproduces plant tissue. The researchers now start a new project to make the next step. Credit: KIT Plants produce a number of substances that can be used to treat cancer, Alzheimer's or Parkinson's disease. Frequently, however, metabolic pathways to obtain the target substance are so complex that its biotechnological production is hardly effective and very expensive. Scientists of KIT now combine their expertise with the technical know-how of Phyton Biotech GmbH, the biggest producer of pharmaceutical ingredients with plant cells. With the help of a microfluidic bioreactor consisting of coupled modules, the scientists technically reproduce complex plant tissue to produce active substances against cancer or Alzheimer's disease more effectively and at lower costs. According to latest estimates, plants form about a million chemical substances, so-called secondary metabolites. Unlike amino acids or sugar, these secondary metabolites are not of vital importance. However, this vast pool of plant products contains a true treasure of pharmaceutically active substances that inhibit the growth of cancer cells or reduce the formation of Alzheimer-typical plaques in the brain. Many of these valuable ingredients cannot be produced synthetically. Often, they have to be extracted directly from wild plants and processed at high costs. Moreover, many of these plants are rare and endangered: For instance, the discovery of Taxol inhibiting cancer cells brought the Pacific yew to the brink of extermination. "For this reason, biotechnological approaches to producing the respective active substances are of high interest," Peter Nick, Professor for Molecular Cell Biology of KIT's Botanical Institute, says. Often, underlying metabolic pathways are highly complex. In the natural plant, the substance of interest mostly is the product of a long chain of steps with many converted interim products. The chemical processes required for this purpose do not necessarily take place in a single plant cell, but in several specialized cell types found in the plant tissue from the root to the leaf. Many years ago, Phyton demonstrated that plant-based medical substances, such as Taxol, can also be produced with minimum resources and sustainably by the cultivation of plant cells in the lab. "Certain substances, however, can be produced neither in a simple cell culture nor in microorganisms manipulated by genetic engineering, because metabolic pathways are too complex," Peter Nick says. "Within the framework of a new research project, we now want to technically reproduce plant tissue with various cell types using a so-called microfluidic bioreactor. It consists of several modules, in which one cell type each is cultivated. The modules are connected via channels. Metabolic products of one cell type then enter the next module for further processing without the different cell types being mixed. In the end, the target substance can be extracted from the flow and, hence, "harvested". The project is managed by the Jülich Project Management Agency (PtJ) and funded with EUR 750,000 by the Federal Ministry of Education and Research for a period of two years. The project partners are the Botanical Institute and the Institute of Microstructure Technology (both of KIT) and the company Phyton Biotech GmbH. Together, the three partners possess the expertise required for the project. The Botanical Institute contributes its knowledge of molecular cellular biology of plant cell cultures. Professor Andreas Guber and Dr. Ralf Ahrens of the Institute of Microstructure Technology are responsible for the development and fabrication of partial components of microfluidic bioreactors, their microassembly, and interconnection to a functioning system. The industry partner Phyton Biotech GmbH is a worldwide leading company in the area of plant cell fermentation and supplies the expertise and infrastructure needed to analyze potential applications on the industrial scale. "Cooperation with the experts of KIT will allow us to reach a new level of use of plant cells produced by controlled cultivation," Dr. Gilbert Gorr, Research and Development Director of Phyton, says. "Our joint objective is to make further natural substances accessible, which so far have been produced with large difficulties and high costs only." Phyton Biotech produces high-quality active pharmaceutical ingredients by plant cell fermentation (PCF) and is worldwide supplier of Paclitaxel and Docetaxel. The company has been inspected successfully by authorities, such as EDQM, EMA, FDA, KFDA, and TGA. Apart from production, Phyton also offers development services for customers. These cover the development of plant cell lines and fermentation processes for plant ingredients as well as the development of synthesis processes of complex substances. Explore further: Plant growth without light control: Synthetic photoreceptor stimulates germination and development


Hann D.R.,Botanical Institute | Hann D.R.,Ludwig Maximilians University of Munich | Dominguez-Ferreras A.,Botanical Institute | Motyka V.,Academy of Sciences of the Czech Republic | And 7 more authors.
New Phytologist | Year: 2014

We characterized the molecular function of the Pseudomonas syringae pv. tomato DC3000 (Pto) effector HopQ1. In silico studies suggest that HopQ1 might possess nucleoside hydrolase activity based on the presence of a characteristic aspartate motif. Transgenic Arabidopsis lines expressing HopQ1 or HopQ1 aspartate mutant variants were characterized with respect to flagellin triggered immunity, phenotype and changes in phytohormone content by high-performance liquid chromatography-MS (HPLC-MS). We found that HopQ1, but not its aspartate mutants, suppressed all tested immunity marker assays. Suppression of immunity was the result of a lack of the flagellin receptor FLS2, whose gene expression was abolished by HopQ1 in a promoter-dependent manner. Furthermore, HopQ1 induced cytokinin signaling in Arabidopsis and the elevation in cytokinin signaling appears to be responsible for the attenuation of FLS2 expression. We conclude that HopQ1 can activate cytokinin signaling and that moderate activation of cytokinin signaling leads to suppression of FLS2 accumulation and thus defense signaling. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.


Stockinger H.,CNRS Agroecology Lab | Peyret-Guzzon M.,CNRS Agroecology Lab | Koegel S.,Botanical Institute | Bouffaud M.-L.,CNRS Agroecology Lab | Redecker D.,CNRS Agroecology Lab
PLoS ONE | Year: 2014

Due to the potential of arbuscular mycorrhizal fungi (AMF, Glomeromycota) to improve plant growth and soil quality, the influence of agricultural practice on their diversity continues to be an important research question. Up to now studies of community diversity in AMF have exclusively been based on nuclear ribosomal gene regions, which in AMF show high intra-organism polymorphism, seriously complicating interpretation of these data. We designed specific PCR primers for 454 sequencing of a region of the largest subunit of RNA polymerase II gene, and established a new reference dataset comprising all major AMF lineages. This gene is known to be monomorphic within fungal isolates but shows an excellent barcode gap between species. We designed a primer set to amplify all known lineages of AMF and demonstrated its applicability in combination with high-throughput sequencing in a long-term tillage experiment. The PCR primers showed a specificity of 99.94% for glomeromycotan sequences. We found evidence of significant shifts of the AMF communities caused by soil management and showed that tillage effects on different AMF taxa are clearly more complex than previously thought. The high resolving power of high-throughput sequencing highlights the need for quantitative measurements to efficiently detect these effects. © 2014 Stockinger et al.


Bhat A.I.,Indian Institute of Spices Research | Hohn T.,Botanical Institute | Selvarajan R.,National Research Center for Banana
Viruses | Year: 2016

Badnaviruses (Family: Caulimoviridae; Genus: Badnavirus) are non-enveloped bacilliform DNA viruses with a monopartite genome containing about 7.2 to 9.2 kb of dsDNA with three to seven open reading frames. They are transmitted by mealybugs and a few species by aphids in a semi-persistent manner. They are one of the most important plant virus groups and have emerged as serious pathogens affecting the cultivation of several horticultural crops in the tropics, especially banana, black pepper, cocoa, citrus, sugarcane, taro, and yam. Some badnaviruses are also known as endogenous viruses integrated into their host genomes and a few such endogenous viruses can be awakened, e.g., through abiotic stress, giving rise to infective episomal forms. The presence of endogenous badnaviruses poses a new challenge for the fool-proof diagnosis, taxonomy, and management of the diseases. The present review aims to highlight emerging disease problems, virus characteristics, transmission, and diagnosis of badnaviruses. © 2016 by the authors; licensee MDPI, Basel, Switzerland.


Stockinger H.,CNRS Agroecology Lab | Peyret-Guzzon M.,CNRS Agroecology Lab | Koegel S.,Botanical Institute | Bouffaud M.-L.,CNRS Agroecology Lab | Redecker D.,CNRS Agroecology Lab
PloS one | Year: 2014

Due to the potential of arbuscular mycorrhizal fungi (AMF, Glomeromycota) to improve plant growth and soil quality, the influence of agricultural practice on their diversity continues to be an important research question. Up to now studies of community diversity in AMF have exclusively been based on nuclear ribosomal gene regions, which in AMF show high intra-organism polymorphism, seriously complicating interpretation of these data. We designed specific PCR primers for 454 sequencing of a region of the largest subunit of RNA polymerase II gene, and established a new reference dataset comprising all major AMF lineages. This gene is known to be monomorphic within fungal isolates but shows an excellent barcode gap between species. We designed a primer set to amplify all known lineages of AMF and demonstrated its applicability in combination with high-throughput sequencing in a long-term tillage experiment. The PCR primers showed a specificity of 99.94% for glomeromycotan sequences. We found evidence of significant shifts of the AMF communities caused by soil management and showed that tillage effects on different AMF taxa are clearly more complex than previously thought. The high resolving power of high-throughput sequencing highlights the need for quantitative measurements to efficiently detect these effects.


PubMed | University of Tübingen, Australian National University, Botanical Institute, University of Cambridge and Academy of Sciences of the Czech Republic
Type: Journal Article | Journal: The New phytologist | Year: 2015

We characterized the molecular function of the Pseudomonas syringae pv. tomato DC3000 (Pto) effector HopQ1. In silico studies suggest that HopQ1 might possess nucleoside hydrolase activity based on the presence of a characteristic aspartate motif. Transgenic Arabidopsis lines expressing HopQ1 or HopQ1 aspartate mutant variants were characterized with respect to flagellin triggered immunity, phenotype and changes in phytohormone content by high-performance liquid chromatography-MS (HPLC-MS). We found that HopQ1, but not its aspartate mutants, suppressed all tested immunity marker assays. Suppression of immunity was the result of a lack of the flagellin receptor FLS2, whose gene expression was abolished by HopQ1 in a promoter-dependent manner. Furthermore, HopQ1 induced cytokinin signaling in Arabidopsis and the elevation in cytokinin signaling appears to be responsible for the attenuation of FLS2 expression. We conclude that HopQ1 can activate cytokinin signaling and that moderate activation of cytokinin signaling leads to suppression of FLS2 accumulation and thus defense signaling.


News Article | December 8, 2016
Site: phys.org

"To cope with changed requirements on agriculture, development of new plant species is indispensable. For this, we need a better understanding of important crops, such as rice that is considered the most important source of food worldwide," Dr. Michael Riemann of the Molecular Cell Biology Division of KIT's Botanical Institute explains. Together with the startup da-cons, he developed the RiSeGrAn (Rice Seedlings Growth Analysis) system that analyzes the growth of rice seedlings. By comparing genetically different species, conclusions can be drawn with respect to the function of certain genes for resistance against a variety of stress factors. As research concentrates on the first phases of seedling development, gene variations can be classified more quickly. The system uses an infrared camera to take photos of plant seedlings growing in darkness. "At first, the seedlings have to grow in the dark for them to become highly sensitive to light. Then, we can measure the effect of the light on the seedlings," Riemann explains. In the next step, the system evaluates the photos automatically. The system is accommodated in a box of 50 times 50 centimeters in dimension. The interior is illuminated by 20 infrared LEDs. "The seedlings change their appearance depending on whether they grow in the dark or in light. However, the system is to observe the plants and not to influence them. For this reason, the box is designed to prevent visible light from falling on the seedlings," Riemann says. The seedlings are arranged in a sealed plate in water agar, a transparent nutrient medium that supplies the seedlings with water. For a detailed documentation of plant growth, the system takes a picture every hour for a period of ten days in a computer-controlled manner without a person having to look into the box. Algorithms developed by da-cons GmbH are used to determine from the photos the lengths of the shoot, the first leaf, and the root. In addition, the computer transmits the photos automatically to a server, where they can be look at by the researchers. "By means of the system, we can discover unknown properties of known genes. For example, we can precisely measure parameters, such as the time of germination or growth of certain tissues," Riemann explains. "Our measurements can support molecular biology studies by identifying the genes that make plants more resistant to certain stress factors, e.g. saline soils." In the next step, the developers of the RiSeGrAn project plan to establish an online OpenData platform based on the data collected. Scientists can then publish their data on this platform. OpenData means that raw data obtained from experiments are to be made available to other scientists. Researchers are enabled to check initial results or to study the data for certain characteristics. "Based on the data of the RiSeGrAn system, we can now test how to transfer them to the OpenData platform in the best way. In addition, we can estimate the required computer capacity and study various ways of presenting the results," Dr. Michael Kreim, Development Director of da-cons GmbH, says. "In general, technical background processes and the user interface can be developed better with realistic data than with test data." da-cons uses the data sets of the RiSeGrAn project to determine requirements to be met by the platform and to test the latter.

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