Plant Molecular Biology

Section for Plant Molecular Biology

Description of research

Understanding interactions between cells and organisms by investigating the role of polysaccharides exposed on cell surfaces and secreted polysaccharide signal molecules

Jens Stougaard

The aim and perspective are to determine structural requirements for recognition of complex polysaccharides and the role of ligand-receptor interactions in the relationships between different cells and organisms. Characterisation of such cellular communication systems is important for understanding 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.

The centre aims at understanding the interactions between cells and organisms by investigating the role of cell wall glycans and polysaccharides exposed on cell surfaces, and polysaccharide signal molecules secreted as part of a complex interaction between organisms. Our centre focuses on interdisciplinary approaches for understanding biological recognition and response processes at the molecular level.

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 and the development of their specialised organs and tissues.

The Centre will undertake an integrated functional characterisation of receptor-ligand mechanisms mediating recognition of surface-exposed polysaccharides and subsequent signal amplification. Experimental approaches from chemical biology, bioinformatics, structural biology, functional genomics, proteomics, bioorganic chemistry, and nanobioscience tools will be used in an interdisciplinary environment combining experience from laboratories with complementary research expertise.

The Centre activities will focus on two model organisms: zebrafish and the legume plant Lotus japonicus, and their interactions with pathogenic and symbiotic microorganisms.

Project description

We aim at understanding the interactions between cells and organisms by investigating the role of cell wall glycans and polysaccharides exposed on cell surfaces, and polysaccharide signal molecules secreted as part of a complex interaction between organisms.

The perspective is to determine structural requirements for recognition of complex polysaccharides and the role of ligand-receptor interactions in the relationships between different cells and organisms. Characterisation of such cellular communication systems is important for understanding 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.

Identification of a new class of LysM lipochitin-oligosaccharide receptors in the rhizobium-legume interaction - coupled with the ability to manipulate both the ligand and the individual domains of the receptor experimentally - has provided new opportunities for functional analysis of polysaccharide receptors. LysM domains are widespread and appear to possess an unusual flexibility in ligand-binding specificity combined with a possible multi-domain mode of ligand binding. Structural and functional characterisation of human, zebrafish and plant LysM domains, their ligand-binding properties and their mechanisms for converting recognition into signalling and cellular responses are therefore of broad scientific interest and a central theme in the Centre's activities.

CARB's approach to unravel cell-to-cell communication will focus on studies of polysaccharide signalling in the legume and zebrafish systems where in vivo activity and in vitro binding can be compared directly. Effective plant and bacterial genetic methods will be used to identify components recognising exo- and lipopolysaccharides exposed on cell surfaces.

To accomplish the Centre's aims an international team will apply interdisciplinary approaches building on their expertise ranging from molecular genetics, carbohydrate chemistry to nanobioscience and bioinformatics. Interactions between synthesised oligosaccharide ligands and receptor binding sites will be determined. Selected recombinant domains including LysM domains from uncharacterised human and zebrafish proteins will be purified and binding of oligosaccharide ligands characterised biochemically and structurally to determine the nature of ligand-binding site interactions. Biochemical binding assays, biophysical methodology, NMR and crystallographic methods will be employed.

The mechanisms of single-membrane-pass receptors translating ligand-binding at extracellular domains into intracellular kinase activation will be characterised in terms of conformational and structural changes. By combining an assortment of genome information and technologies available in the two model organisms, the Centre aims at taking the analysis of signalling processes in multicellular organisms to a new level, distinguishing events in tissues, cells and nuclei. It is the Centre's ambition to establish a unique understanding of fundamental life-processes in animals, humans and plants and provide a scientific environment allowing young scientists to pioneer new developments and do cutting-edge research at a level beyond what could be accomplished by individual participants.

Understanding interactions between cells and organisms by investigating the role of polysaccharides exposed on cell surfaces and secreted polysaccharide signal molecules

Research description

Aim and perspective

The aim and perspective are to determine structural requirements for recognition of complex polysaccharides and the role of ligand-receptor interactions in the relationships between different cells and organisms. Characterisation of such cellular communication systems is important for understanding 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.

The centre aims at understanding the interactions between cells and organisms by investigating the role of cell wall glycans and polysaccharides exposed on cell surfaces, and polysaccharide signal molecules secreted as part of a complex interaction between organisms. Our centre focuses on interdisciplinary approaches for understanding biological recognition and response processes at the molecular level.

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 and the development of their specialised organs and tissues.

The Centre will undertake an integrated functional characterisation of receptor-ligand mechanisms mediating recognition of surface-exposed polysaccharides and subsequent signal amplification. Experimental approaches from chemical biology, bioinformatics, structural biology, functional genomics, proteomics, bioorganic chemistry, and nanobioscience tools will be used in an interdisciplinary environment combining experience from laboratories with complementary research expertise.

The Centre activities will focus on two model organisms: zebrafish and the legume plant Lotus japonicus, and their interactions with pathogenic and symbiotic microorganisms.

Project description

We aim at understanding the interactions between cells and organisms by investigating the role of cell wall glycans and polysaccharides exposed on cell surfaces, and polysaccharide signal molecules secreted as part of a complex interaction between organisms.

The perspective is to determine structural requirements for recognition of complex polysaccharides and the role of ligand-receptor interactions in the relationships between different cells and organisms. Characterisation of such cellular communication systems is important for understanding 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.

Identification of a new class of LysM lipochitin-oligosaccharide receptors in the rhizobium-legume interaction - coupled with the ability to manipulate both the ligand and the individual domains of the receptor experimentally - has provided new opportunities for functional analysis of polysaccharide receptors. LysM domains are widespread and appear to possess an unusual flexibility in ligand-binding specificity combined with a possible multi-domain mode of ligand binding. Structural and functional characterisation of human, zebrafish and plant LysM domains, their ligand-binding properties and their mechanisms for converting recognition into signalling and cellular responses are therefore of broad scientific interest and a central theme in the Centre's activities.

CARB's approach to unravel cell-to-cell communication will focus on studies of polysaccharide signalling in the legume and zebrafish systems where in vivo activity and in vitro binding can be compared directly. Effective plant and bacterial genetic methods will be used to identify components recognising exo- and lipopolysaccharides exposed on cell surfaces.

To accomplish the Centre's aims an international team will apply interdisciplinary approaches building on their expertise ranging from molecular genetics, carbohydrate chemistry to nanobioscience and bioinformatics. Interactions between synthesised oligosaccharide ligands and receptor binding sites will be determined. Selected recombinant domains including LysM domains from uncharacterised human and zebrafish proteins will be purified and binding of oligosaccharide ligands characterised biochemically and structurally to determine the nature of ligand-binding site interactions. Biochemical binding assays, biophysical methodology, NMR and crystallographic methods will be employed.

The mechanisms of single-membrane-pass receptors translating ligand-binding at extracellular domains into intracellular kinase activation will be characterised in terms of conformational and structural changes. By combining an assortment of genome information and technologies available in the two model organisms, the Centre aims at taking the analysis of signalling processes in multicellular organisms to a new level, distinguishing events in tissues, cells and nuclei. It is the Centre's ambition to establish a unique understanding of fundamental life-processes in animals, humans and plants and provide a scientific environment allowing young scientists to pioneer new developments and do cutting-edge research at a level beyond what could be accomplished by individual participants.


Simona Radutoiu

Plant-endophyte interaction

Simona Radutoiu’s research deals with the studies of plant-endophyte interaction. Endophytes are bacteria or fungi that live inside the plant colonising various tissues, sometimes inducing local plant cell death for its own proliferation, while under certain conditions or in certain hosts become parasites. Most plants, and especially agronomically important crops like rice, wheat, barley, maize and sugarcane largely benefit from this less refined plant-microbe association which leads to growth promotion, increased biomass, enhanced resistance to salt, limited soil nutrients, water or pathogenic stress.

The molecular mechanisms used by the plant to recognise and adapt to surrounding beneficial or pathogenic microbes are crucial for survival, growth and productivity. Despite its ubiquity and great influence that plant-endophyte interaction has on crops and natural ecosystems, the area has got limited attention in molecular research. Little is known about the genetic and molecular components controlling the fine-tuned balance between various hosts and endophytes, which leads to beneficial rather than pathogenic interaction. The research focuses on identifying plant genetic factors required for increased benefit from the endophyte interaction, like microbe colonization, increased host biomass and ability to grow on nutrient-limited conditions.

The research builds on findings and resources from studies of the symbiotic interactions between the model plant Lotus japonicus, and nitrogen-fixing bacteria and mycorrhizae fungi, and uses the latest techniques and methods in genetics and genomic analysis to elucidate the molecular dialogue between host plants and microbes.


Projects

Identification and analysis of novel symbiotic mutants (with Niels Sandal)

To characterize the function of  a specific gene, it is important to have access to plant lines carrying a loss-of-function allele of the gene of interest. At the CARB centre, we are using the mobile element Lotus retrotransposon 1 (LORE1) to establish a large mutant population. We have annotated all the LORE1 insertions in this population using high-throughput Illumina sequencing, and we are now screening the population for aberrant symbiotic phenotypes. Once a mutant with an interesting phenotype has been identified, we can quickly identify the causal gene because of the well-characterized mutant population. The project is aimed at the identification and detailed characterization of novel symbiotic mutants to improve our understanding of symbiotic nitrogen fixation.

Natural variation in plant symbiotic and pathogen responses

Plants continuously interact with countless microorganisms of both pathogenic and symbiotic nature. Because the handling of these interactions is vital to the survival of the plant, there is a strong selective pressure on the genetic components regulating them. Based on a collection of Lotus japonicus ecotypes, we use association and QTL analysis to identify the genetic components responsible for the variation in plant-microorganism interactions in natural populations. The project involves bioinformatic data analysis and quantitative phenotyping based on image analysis.

Regulation of plant defense responses during symbiosis

To allow a symbiotic infection, microorganism invasion has to be coordinated with plant pathogen defense systems. So far, little is known about the mechanisms behind this cross-talk. Using the LORE1 resource, we are now establishing a collection of Lotus japonicus defense mutants. By investigating the effects of defense gene mutations on the symbiotic process, we can identify the primary defense signaling pathways influencing symbiosis.


Bjarne Jochimsen - emeritus

Research project: degradation of phosphonate compounds

Phosphonates are organic compounds containing an extremely stable chemical bond between carbon and phosphorus, a property exploited by the pharmaceutical, chemical and agricultural industries for synthesising drugs, detergents and herbicides. Researchers have now found that the bacteria's ability to degrade phosphonates takes place via a complicated complex of five different proteins.

It is estimated that 20,000 tons of the above phosphonate compounds are released into the environment every year in the western hemisphere alone, and these persistent compounds are suspected to be harmful to the environment. The research results therefore represent an important contribution to finding solutions to reduce the presence of these unwanted phosphonates in the environment.

An example of a phosphonate compound is glyphosate, which is the active ingredient in the herbicide Roundup®. This is considered a safe herbicide as it relatively quickly disappears from the soil, where it is converted into the compound aminomethylphosphonate (abbreviated AMPA). AMPA is highly stable in the soil, from where it constitutes a potential environmental problem, especially for our groundwater.

Certain bacteria that occur in different environments are able to degrade phosphonates. One example is the well-known intestinal bacterium E. coli. To cope with the degradation, E. coli needs 14 different proteins encoded by 14 genes that are arranged together in an operon: phnCDEFGHIJKLMNOP. Three of these genes (phnCDE) code for proteins carrying phosphonates into the bacterium, while a single gene (phnF) codes for a regulatory protein. The remaining 10 genes are believed to code for enzymes that are necessary for the decomposition of phosphonates.

The enzymatic function of the three proteins coded by the genesphnNOP has been solved. By studying the expression of the genesphnGHIJKLM, it was shown that five proteins deriving from thephnGHIJK remain together in a complex during the application of various protein purification principles.

The function of this protein complex is not yet known in detail, but the results suggest that we are dealing with the enzyme that cleaves the carbon-phosphorus bond (CP lyase).

The researchers are now focusing on expanding their studies with a detailed characterisation of the complex’s enzymatic function and mechanism, using X-ray crystallography to clarify the structure of the protein complex.


Projects

Small RNAs inovlved in Lotus japonicus root symbiosis

Most genes in both plants and animals encode proteins. If such a gene is active, the cell first produces a corresponding RNA transcript, which then is translated into a protein – and it is in this form that the gene exerts its biological role. But an increasing number of RNAs are being discovered that never get translated. Certain groups of such noncoding transcripts are processed into small RNAs only 19-24 nt in length. These can regulate the activity of protein coding genes in diverse developmental and physiological processes. We are investigating the roles of regulatory small RNAs, in particular micro RNAs, in Lotus japonicus interactions with symbiotic bacteria and fungi. The project involves high throughput detection of small RNA populations using next generation sequencing, bioinformatic data analysis, as well as plant experiments to validate small RNA functions.

The Lotus japonicus small RNA machinery

To understand the role of regulatory small RNAs in root symbiosis, we have identified loss-of-function mutants in a number of genes that are potentially necessary for small RNA production and activity in the cell. We are examining these mutants and evaluate their ability to interact with nitrogen fixing rhizobial bacteria as well as mycorrhizal fungi. Defects or differences in symbiosis development in these mutants compared to genetically healthy wild type plants where small RNA regulation is intact help us identify the roles the respective genes, and their associated small RNAs, play in these processes.

Cell type specific transcript and small RNA responses in Lotus japonicus roots

Although we begin to understand more about the plant genes as well as small RNAs that respond to an encounter with root symbiotic microorganisms, we know little about where in the plant root these genes are active. Most previous studies rely on pooling different cell types of the root organ such that we can no longer pinoint precisely where changes in gene activity occur during symbiosis initiation. Based on promoters expressed in particular cell types only, we have therefore developed a system that allows the analysis of both coding and small noncoding RNA transcripts in defined root cell types. We are using this system for cell type specific detection as well as manipulation of transcript abundances in a quest to gain further insights into plant responses during root symbiosis.

Peer-reviewed publications

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Patents

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Section coordinator

Jens Stougaard

Professor
M
H bldg. 3134, 204
P +4587155504
P +4560202649

Associate Professors/Senior Researchers


Simona Radutoiu

Associate professor


Team Leaders

Stig Uggerhøj Andersen

Associate professor

The Plant Molecular Biology Group

29 August 2013

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