Abstract
Legumes are plants essential to human nutrition, because of their seeds that
include beans, lentils and peas. In academia, legumes are especially studied
due to their auxiliary ability to fix nitrogen, which is derived from a symbiosis
with rhizobial bacteria. This thesis seeks to expand the current knowledge on
the molecular signals regulating this symbiosis.
In the legume-rhizobium symbiosis, carbohydrate molecular signals such as
bacterial exopolysaccharides and nodulation (Nod) factors are pivotal for development
of root nodules in which nitrogen fixation take place. However, as
several carbohydrates are released by both symbiotic and pathogenic bacteria,
recognition of these signals not only ensures plant growth but also survival of
the plant.
This thesis showed how purification and characterization of rhizobial Nod
factors were performed. It also described two novel methods for immobilizing
carbohydrates onto surfaces. Novel chemistry was used to synthesize a range of
biotinylated rhizobial carbohydrate signal compounds. Biolayer interferometry
(forteBio) direct binding assays were introduced, and carbohydrate conjugates
were immobilized on streptavidin-coated biosensors. When allowed to interact
with tentative receptors, lysine motif receptor like kinases (LysM-RLKs), it
was possible to obtain binding data that can determine the specificity legumes
exhibit towards rhizobia. Using this approach, the project seeked to build a
’host-specificity study’. In this study, it could potentially be determined if
LysM-RLKs from the model legumes L. japonicus and M. truncatula were able
to bind Nod factors from rhizobia that they do not naturally form symbiosis
with. These data could be useful in exploring the exact nature of the symbiosis.
For this experiment proof of concept was obtained, but further work is required
to determine the optimal parameters for biolayer interferometry experiments.
This work is ongoing, and will hopefully result in high quality binding data.
The thesis also described how M. loti exopolysaccharide octaose was purified
and for the first time fully characterized, by fragmentation analysis using
Fourier transform ion cyclotron mass spectrometry. As for Nod factors, biotinylated
exopolysaccharide octaose was also used in direct binding assays
with the suspected exopolysaccharide receptor LYS3. These assays lead to the
determination of a KD value of 2.7 ± 0.2 μM, which fully supported genetic
studies performed by Kawaharada et al. at Aarhus University. As LYS3 was
confirmed to be a receptor for exopolysaccharides this led to the protein being
renamed ExoPolysaccharide Receptor 3 (EPR3
include beans, lentils and peas. In academia, legumes are especially studied
due to their auxiliary ability to fix nitrogen, which is derived from a symbiosis
with rhizobial bacteria. This thesis seeks to expand the current knowledge on
the molecular signals regulating this symbiosis.
In the legume-rhizobium symbiosis, carbohydrate molecular signals such as
bacterial exopolysaccharides and nodulation (Nod) factors are pivotal for development
of root nodules in which nitrogen fixation take place. However, as
several carbohydrates are released by both symbiotic and pathogenic bacteria,
recognition of these signals not only ensures plant growth but also survival of
the plant.
This thesis showed how purification and characterization of rhizobial Nod
factors were performed. It also described two novel methods for immobilizing
carbohydrates onto surfaces. Novel chemistry was used to synthesize a range of
biotinylated rhizobial carbohydrate signal compounds. Biolayer interferometry
(forteBio) direct binding assays were introduced, and carbohydrate conjugates
were immobilized on streptavidin-coated biosensors. When allowed to interact
with tentative receptors, lysine motif receptor like kinases (LysM-RLKs), it
was possible to obtain binding data that can determine the specificity legumes
exhibit towards rhizobia. Using this approach, the project seeked to build a
’host-specificity study’. In this study, it could potentially be determined if
LysM-RLKs from the model legumes L. japonicus and M. truncatula were able
to bind Nod factors from rhizobia that they do not naturally form symbiosis
with. These data could be useful in exploring the exact nature of the symbiosis.
For this experiment proof of concept was obtained, but further work is required
to determine the optimal parameters for biolayer interferometry experiments.
This work is ongoing, and will hopefully result in high quality binding data.
The thesis also described how M. loti exopolysaccharide octaose was purified
and for the first time fully characterized, by fragmentation analysis using
Fourier transform ion cyclotron mass spectrometry. As for Nod factors, biotinylated
exopolysaccharide octaose was also used in direct binding assays
with the suspected exopolysaccharide receptor LYS3. These assays lead to the
determination of a KD value of 2.7 ± 0.2 μM, which fully supported genetic
studies performed by Kawaharada et al. at Aarhus University. As LYS3 was
confirmed to be a receptor for exopolysaccharides this led to the protein being
renamed ExoPolysaccharide Receptor 3 (EPR3
| Original language | English |
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| Publisher | Department of Chemistry, Faculty of Science, University of Copenhagen |
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| Publication status | Published - 2015 |