CARB: Centre for Carbohydrate Recognition and Signalling

Publiceret April 2012

CARB was established in 2007 and financially supported for an initial five-year period by Danmarks Grundforskningsfond. In 2011, another five-year grant was awarded to allow research to continue in this Centre of Excellence until spring 2017. A team of approximately 50 researchers, PhD/MSc students and technicians contribute to the Centre activities led by Centre Director Professor Jens Stougaard. The central CARB node is at the Department of Molecular Biology and Genetics, Aarhus University. CARB members also include researchers from the University of Copenhagen, under the direction of Professor Knud J. Jensen; the University of Leiden in The Netherlands, led by Professor Herman Spaink; and the University of Otago in New Zealand, directed by Professor Clive Ronson. The Centre aspires to establish a unique understanding of fundamental life-processes in animals, humans and plants and to provide a scientific environment allowing young scientists to pioneer new developments and perform cutting-edge research at a level beyond what could be accomplished by individual participants. A subset of central themes and activities will be presented in this article.

Scientific Themes

Our centre focuses on understanding biological recognition and response processes at the molecular level. In particular, CARB aims to characterise the role of carbohydrate signals, how they are perceived and the responses they trigger, in the interaction of plants or metazoans with microbes. CARB members thus study downstream events of carbohydrate signalling in defence or symbiosis at both cellular and sub-cellular levels. Within this scientific theme, research encompasses several key genetic and genomic topics:

  • Structural analysis of carbohydrate recognition and signalling,
  • Lipochitin-oligosaccharide purification, synthesis and analysis,
  • Comparative analysis of LysM proteins and chitinases,
  • Discovery and analysis of new polysaccharide receptors,
  • Cellular and subcellular signalling events.

Research Approach

CARB research on recognition and signalling exploits two main biological systems; the model legume Lotus japonicus (Lotus) and the zebrafish, and their interactions with symbiotic, pathogenic and endophytic microorganisms. These include symbiotic nitrogen fixing bacteria and mycorrhizal fungi in Lotus, and pathogenic bacteria such as Salmonella and Mycobacterium marinum in zebrafish. The approach to unravel cell-to-cell communication predominantly focuses on studies of signalling by polysaccharides. To accomplish the Centre’s aims, an international team applies interdisciplinary approaches building on their expertise. The research uses a multifaceted approach with a variety of technology and techniques employed, including molecular genetics, genomics and proteomics, advanced microscopy, bioorganic and carbohydrate chemistry, bioinformatics, structural biology, and nanobioscience tools. The advantageous biological features of the model organisms, Lotus and zebrafish, allow in vivo activity and in vitro data to be compared directly.

CARB Research

Carbohydrate signals and extracellular polysaccharides play an important role in cell-to-cell communication processes and are equally important for the organisation of multicellular organisms, including the development of their specialised organs and tissues. Characterisation of such cellular communication systems is necessary to understand the factors determining pathogenesis of microorganisms as well as immune responses, symbiosis and cell-to-cell signalling involved in the development and functioning of multicellular organisms. Interactions between synthesised carbohydrates (oligosaccharide ligands) and receptor binding sites are being characterised biochemically and structurally, to determine the nature of ligand-binding site interactions. Biochemical binding assays, biophysical methodology, NMR and crystallographic methods are all employed. Functional characterisation of these interaction mechanisms, which mediate recognition of polysaccharides that are exposed on cell surfaces or secreted and also subsequent signal amplification, is a key aim for CARB. Accordingly, CARB continues to investigate the structural requirements for recognition of complex polysaccharides and the role of signal-receptor relationships between different cells and organisms.

In studies of Lotus CARB researchers have succeeded in finding a single-cell infection mechanism that can further understanding of how legumes control infection with beneficial bacteria. Significant progress has been made in characterising the receptor proteins that recognise lipochitin-oligosaccharide signal molecules secreted by rhizobia bacteria, which are part of a complex interaction between organisms. Rhizobia trigger a development process in the legume plant that leads to formation of nitrogen-fixing nodules on the plant roots. Hosted within these nodules, rhizobia convert atmospheric nitrogen to ammonium fertiliser that can be absorbed by the plant. The formation of functional root nodules is controlled by at least sixteen genes. Our researchers are investigating how these genes interact, what the specific roles of these genes are and how each one individually contributes to the overall process. Complementing this approach CARB researchers also use zebrafish as a model to examine how carbohydrates influence the development process of animals. Of special interest is the LysM domains found in a number of zebrafish proteins and in the receptor proteins that legumes use to receive lipochitin-oligosacchride signals from rhizobia.

2012-2 Lotus Nodul infected

A 3-week-old Lotus (ecotype Gifu) nodule infected with M. loti bacteria expressing a red fluorescent protein (DsRED).

Using a combination of genetic and biochemical techniques CARB researchers have identified a new class of receptors in the rhizobium-legume interaction, which contain ‘LysM modules’ in their extracellular domains. Coupled with the ability to experimentally manipulate both the ligands and the receptors, this provided new opportunities for functional analysis of polysaccharide receptors, for example, biochemical studies of ligand binding affinities. Structural and functional characterisation of human, zebrafish and plant LysM domains, their binding properties and mechanisms for converting recognition into signalling and cellular responses, are of broad scientific interest and remain a central theme in the Centre’s activities. CARB aims to take the analysis of signalling processes in multicellular organisms to a new level, distinguishing events in tissues, cells and nuclei. In this context small RNAs and miRNAs present in different root cell types has attracted attention. Next-generation sequencing has been used to identify small RNA populations in Lotus tissues. By assaying small RNA pools from different mutant genotypes and symbiotic conditions, a link between specific small RNA species and stages of symbiosis development could be established. Following this global investigation more detailed studies of the role of small RNA-directed gene regulation in symbiosis is now being pursued and the first glimpse of a novel signalling loop has been obtained.

The ability of plants to recognise and permit infections by beneficial bacteria, rather than engage in pathogen defence actions, is also a key theme in CARB. Discovering how a plant knows when, or when not to, defend itself against microorganisms is pivotal to developing plant varieties with improved resistance to pathogens. Plant recognition of surface exposed bacterial exopolysaccharides play a role in distinguishing friend from foe as infection of rhizobial exopolysaccharide mutants is impaired in infection. The complex structures of rhizobial exopolysaccharides are therefore being determined and a combination of bacterial and plant genetics is employed to identify putative plant receptors recognising bacterial surface exopolysaccharides.

Recent Highlight: The LORE1 Insertional Mutagenesis Project.

In 2011 the CARB has invested heavily in establishing the genetic and genomic platforms for the future. Recent availability of the Lotus genome sequence and gene-models based on RNA sequencing allowed development of reverse genetics methods; techniques used to assign biological function to a specific gene. Thus, CARB aimed to and has successfully developed a toolbox of reverse genetic techniques that will accelerate future plant genetic studies and increase our understanding of plant gene function. A cost-effective, high-throughput and comprehensive reverse genetic resource, including an innovative software package, was rigorously tested and its efficacy demonstrated with a set of 3,744 mutant plants. The protocols developed in this CARB project are now the cornerstone of a new LORE1 reverse genetics resource, characterized by efficient mutant line generation and accurate mutation annotation. The project outcome is publicly available and all mutant lines are freely available upon request. The LORE1 mutagenesis initiative was started by CARB and involves collaborators from Japan.

Dissemination of Knowledge and Education

CARB research has been published in prestigious scientific journals and over 60 publications in peer reviewed journals have so far been generated. CARB is also proud of its abundant collaborations, with the high level of international collaboration reflected by 50 co-authored papers.

Presently, 13 CARB students have been awarded Doctor of Philosophy (PhD), 10 their Masters of Science and 14 Bachelor of Science students successfully completed their final year project at CARB. Student education and development will continue to be an integral part of CARBs activities.

2012-2 Lotus Root Hairs infected

Lotus (ecotype Gifu) root hairs infected by symbiotic bacteria expressing GFP or DsRED; The bacterial infection thread is seen fluorescing green or red. The bacteria enter the root hair close to its tip and from there they divide inside the thread, which is surrounded by the plasma membrane and a thin cell wall. When the tread reaches the nodule primordium it starts to branch and bacteria colonize the primordium cells.

Ambitions

CARB will continue to explore the projects described in this article, but also investigate new mechanisms of carbohydrate action to determine the role of carbohydrates in the cell, as well as to understand the developmental biology that is controlled by carbohydrates in both plants and vertebrates. The researchers hope that determining the role of carbohydrates in the cell will continue to provide answers to fundamental questions about the function of carbohydrates as signal molecules, and ultimately contribute to the understanding of diseases in plants and animals. CARB will also continue their investigations of the genetic background of the single-cell infection mechanism to increase understanding about how bacteria cross the cell membrane and establish themselves in the plant cell. This knowledge is expected to reveal how the bacteria avoid being detected by the plant’s defence system. Furthermore, results of these studies may be used to increase understanding of bacterial infection in animals; knowledge that other scientists will be able to use to combat pathogenic infections.

CARB has performed groundbreaking work to reveal how bacteria and plants jointly form root nodules that are able to convert atmospheric nitrogen into fertiliser for the plant. Research has also led to the discovery of the recognition mechanisms in legumes, which ensure that collaboration with the soil dwelling rhizobia can begin. In the long term, results from CARB research projects are expected to contribute to the biotechnological exploitation of the symbiosis between microbes and plants; providing potential opportunities to reduce the use of fertilizers and also to produce plants with improved disease resistance. Importantly, CARB is providing key stepping-stones leading towards enhanced and sustainable agriculture.