Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Data from: Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses
Velzen, R. van; Holmer, R. ; Bu, F. ; Rutten, L.J.J. ; Zeijl, A.L. van; Liu, W. ; Santuari, L. ; Cao, Q. ; Sharma, Trupti ; Shen, D. ; Roswanjaya, Yuda ; Wardhani, T. ; Seifi Kalhor, M. ; Jansen, Joelle ; Hoogen, D.J. van den; Gungor, Berivan ; Hartog, M.V. ; Hontelez, Jan ; Verver, J.W.G. ; Yang, Wei-Cai ; Schijlen, E.G.W.M. ; Repin, Rimi ; Schilthuizen, M. ; Schranz, M.E. ; Heidstra, R. ; Miyata, Kana ; Fedorova, E. ; Kohlen, W. ; Bisseling, A.H.J. ; Smit, S. ; Geurts, R. - \ 2018
comparative genomics - copy number variation - evolution - nitrogen fixation - symbiosis - Parasponia andersonii - Parasponia rigada - Parasponia rugosa - Trema levigata - Trema orientalis - Trema tomentosa
Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION. Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants
Comparative Genomics Highlights Symbiotic Capacities and High Metabolic Flexibility of the Marine Genus Pseudovibrio
Versluis, Dennis ; Nijsse, Bart ; Naim, Mohd Azrul ; Koehorst, Jasper J. ; Wiese, Jutta ; Imhoff, Johannes F. ; Schaap, Peter J. ; Passel, Mark W.J. van; Smidt, Hauke ; Sipkema, Detmer - \ 2018
Genome Biology and Evolution 10 (2018)1. - ISSN 1759-6653 - p. 125 - 142.
domainome - microbiota - phylogeny - secondary metabolites - sponge - symbiosis

Pseudovibrio is a marine bacterial genus members of which are predominantly isolated from sessile marine animals, and particularly sponges. It has been hypothesized that Pseudovibrio spp. form mutualistic relationships with their hosts. Here, we studied Pseudovibrio phylogeny and genetic adaptations that may play a role in host colonization by comparative genomics of 31 Pseudovibrio strains, including 25 sponge isolates. All genomes were highly similar in terms of encoded core metabolic pathways, albeit with substantial differences in overall gene content. Based on gene composition, Pseudovibrio spp. clustered by geographic region, indicating geographic speciation. Furthermore, the fact that isolates from the Mediterranean Sea clustered by sponge species suggested host-specific adaptation or colonization. Genome analyses suggest that Pseudovibrio hongkongensis UST20140214-015BT is only distantly related to other Pseudovibrio spp., thereby challenging its status as typical Pseudovibrio member. All Pseudovibrio genomes were found to encode numerous proteins with SEL1 and tetratricopeptide repeats, which have been suggested to play a role in host colonization. For evasion of the host immune system, Pseudovibrio spp. may depend on type III, IV, and VI secretion systems that can inject effector molecules into eukaryotic cells. Furthermore, Pseudovibrio genomes carry on average seven secondary metabolite biosynthesis clusters, reinforcing the role of Pseudovibrio spp. as potential producers of novel bioactive compounds. Tropodithietic acid, bacteriocin, and terpene biosynthesis clusters were highly conserved within the genus, suggesting an essential role in survival, for example through growth inhibition of bacterial competitors. Taken together, these results support the hypothesis that Pseudovibrio spp. have mutualistic relations with sponges.

Plant immunity and symbiosis signaling mediated by LysM receptors
Desaki, Yoshitake ; Miyata, Kana ; Suzuki, Maruya ; Shibuya, Naoto ; Kaku, Hanae - \ 2018
Innate Immunity 24 (2018)2. - ISSN 1753-4259 - p. 92 - 100.
Arabidopsis - CEBiP - CERK1 - chitin - LysM receptor - Plant immunity - rice - signal transduction - symbiosis
Plants possess the ability to recognize microbe-associated molecular patterns (MAMPs) and PAMPs through the PRRs, and initiate pattern-triggered immunity. MAMPs are derived from cell-envelope components, secreted materials and cytosolic proteins from bacteria, oomycetes or fungi, and some MAMPs play a similar function in the innate immunity in mammals. Chitin is a representative fungal MAMP and triggers defense signaling in a wide range of plant species. The chitin receptors CEBiP and CERK1 on the plasma membrane have LysM (lysin motif) in their ectodomains. These molecules play an important role for the defense responses in rice and Arabidopsis, strictly recognizing the size and acetylated form of chitin oligosaccharides. However, related LysM receptors also play major roles for the signaling in root nodule and arbuscular mycorrhizal symbiosis. This review summarizes current knowledge on the molecular mechanisms of the defense and symbiosis signaling mediated by LysM receptors, including the activation steps of chitin-induced defense signaling downstream of LysM receptors.
Virulence contribution and recognition of homologs of the Verticillium dahliae effector Ave1
Boshoven, Jordi C. - \ 2017
Wageningen University. Promotor(en): Bart Thomma; Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789463436441 - 183
verticillium dahliae - plant pathogenic fungi - plant pathogens - disease resistance - virulence factors - virulence - immunity - host parasite relationships - plant-microbe interactions - symbiosis - mutagenesis - resistance breeding - plantenziekteverwekkende schimmels - plantenziekteverwekkers - ziekteresistentie - virulente factoren - virulentie - immuniteit - gastheer parasiet relaties - plant-microbe interacties - symbiose - mutagenese - resistentieveredeling

Disease resistance in crops is an important aspect of securing global food security. Resistant plants carry immune receptors that sense pathogen invasion often through the recognition of important pathogen virulence factors, known as effectors. Thus, identification and characterization of effectors is important for the fundamental understanding of virulence mechanisms and to aid in resistance breeding. In this thesis the VdAve1 effector of the soil-borne fungal pathogen Verticillium dahliae is studied that is recognized by tomato immune receptor Ve1. Homologs were found in other plant pathogens and the role in virulence in these pathogens was analyzed. Ave1 homologs are differentially recognized by Ve1 and with a combination of domain swaps and truncations a surface exposed patch was identified that contributes to the recognition by Ve1. Knowledge of specific effector-receptor combinations and knowledge of effectors in general can be exploited to aid in breeding for durable resistance in crops.

Dissecting hormonal pathways in nitrogen-fixing rhizobium symbioses
Zeijl, Arjan van - \ 2017
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - Wageningen : Wageningen University - ISBN 9789463436311 - 231
plants - root nodules - rhizobium - symbiosis - cytokinins - plant-microbe interactions - biosynthesis - mutagenesis - genes - nodulation - planten - wortelknolletjes - symbiose - cytokininen - plant-microbe interacties - biosynthese - mutagenese - genen - knobbelvorming

Nitrogen is a key element for plant growth. To meet nitrogen demands, some plants establish an endosymbiotic relationship with nitrogen-fixing rhizobium or Frankia bacteria. This involves formation of specialized root lateral organs, named nodules. These nodules are colonized intracellularly, which creates optimal physiological conditions for the fixation of atmospheric nitrogen by the microbial symbiont. Nitrogen-fixing endosymbioses are found among four related taxonomic orders that together form the nitrogen-fixation clade. Within this clade, nodulation is restricted to ten separate lineages that are scattered among mostly non-nodulating plant species. This limited distribution suggests that genetic adaptations that allowed nodulation to evolve occurred in a common ancestor.

A major aim of the scientific community is to unravel the evolutionary trajectory towards a nitrogen-fixing nodule symbiosis. The formation of nitrogen-fixing root nodules is best studied in legumes (Fabaceae, order Fabales); especially in Lotus japonicus and Medicago truncatula, two species that serve as model. Legumes and Parasponia (Cannabaceae, order Rosales) represent the only two lineages that can form nodules with rhizobium bacteria. Studies on M. truncatula, L. japonicus and Parasponia showed, amongst others, that nodule formation is initiated upon perception of rhizobial secreted lipo-chitooligosaccharide (LCO) signals. These signals are structurally related to the symbiotic signals produced by arbuscular mycorrhizal fungi. These obligate biotropic fungi colonize roots of most land plants and form dense hyphal structures inside existing root cortical cells.

Rhizobial and mycorrhizal LCOs are perceived by LysM-domain-containing receptor-like kinases. These activate a signaling pathway that is largely shared between both symbioses. Symbiotic LCO receptors are closely related to chitin innate immune receptors, and some receptors even function in symbiotic as well as innate immune signaling. In Chapter 2, I review the intertwining of symbiotic LCO perception and chitin-triggered immunity. Furthermore, I discuss how rhizobia and mycorrhiza might employ LCO signaling to modulate plant immunity. In a perspective, I speculate on a role for plant hormones in immune modulation, besides an important function in nodule organogenesis.

In legumes, nodule organogenesis requires activation of cytokinin signaling. Mutants in the orthologous cytokinin receptor genes MtCRE1 and LjLHK1 in M. truncatula and L. japonicus, respectively, are severely affected in nodule formation. However, how cytokinin signaling is activated in response to rhizobium LCO perception and to what extent this contributes to rhizobium LCO-induced signaling remained elusive. In Chapter 3, I show that the majority of transcriptional changes induced in wild-type M. truncatula, upon application of rhizobium LCOs, are dependent on activation of MtCRE1-mediated cytokinin signaling. Among the genes induced in wild type are several involved in cytokinin biosynthesis. Consistently, cytokinin measurements indicate that cytokinins rapidly accumulate in M. truncatula roots upon treatment with rhizobium LCOs. This includes the bioactive cytokinins isopentenyl adenine and trans-zeatin. Therefore, I argue that cytokinin accumulation represents a key step in the pathway leading to legume root nodule organogenesis.

Strigolactones are plant hormones of which biosynthesis is increased in response to nutrient limitation. In rice (Oryza sativa) and M. truncatula, this response requires the GRAS-type transcriptional regulators NSP1 and NSP2. Both proteins regulate expression of DWARF27 (D27), which encodes an enzyme that performs the first committed step in strigolactone biosynthesis. NSP1 and NSP2 are also essential components of the signaling cascade that controls legume root nodule formation. In line with this, I questioned whether the NSP1-NSP2-D27 regulatory module functions in rhizobium symbiosis. In Chapter 4, I show that in M. truncatula MtD27 expression is induced within hours after treatment with rhizobium LCOs. Spatiotemporal expression studies revealed that MtD27 is expressed in the dividing cells of the nodule primordium. At later stages, its expression becomes confined to the meristem and distal infection zone of the mature nodule. Analysis of the expression pattern of MtCCD7 and MtCCD8, two additional strigolactone biosynthesis genes, showed that these genes are co-expressed with MtD27 in nodule primordia and mature nodules. Additionally, I show that symbiotic expression of MtD27 requires MtNSP1 and MtNSP2. This suggests that the NSP1-NSP2-D27 regulatory module is co-opted in rhizobium symbiosis.

Comparative studies between legumes and nodulating non-legumes could identify shared genetic networks required for nodule formation. We recently adopted Parasponia, the only non-legume lineage able to engage in rhizobium symbiosis. However, to perform functional studies, powerful reverse genetic tools for Parasponia are essential. In Chapter 5, I describe the development of a fast and efficient protocol for CRISPR/Cas9-mediated mutagenesis in Agrobacterium tumefaciens-transformed Parasponia andersonii plants. Using this protocol, stable mutants can be obtained in a period of three months. These mutants can be effectively propagated in vitro, which allows phenotypic evaluation already in the T0 generation. As such, phenotypes can be obtained within six months after transformation. As proof-of-principle, we mutated PanHK4, PanEIN2, PanNSP1 and PanNSP2. These genes are putatively involved in cytokinin and ethylene signaling and regulation of strigolactone biosynthesis, respectively. Additionally, orthologues of these genes perform essential symbiotic functions in legumes. Panhk4 and Panein2 knockout mutants display developmental phenotypes associated with reduced cytokinin and ethylene signaling. Analysis of Pannsp1 and Pannsp2 mutants revealed a conserved role for NSP1 and NSP2 in regulation of the strigolactone biosynthesis genes D27 and MAX1 and root nodule organogenesis. In contrast, symbiotic mutant phenotypes of Panhk4 and Panein2 mutants are different from their legume counterparts. This illustrates the value of Parasponia as comparative model - besides legumes - to study the genetics underlying rhizobium symbiosis.

Phylogenetic reconstruction showed that the Parasponia lineage is embedded in the non-nodulating Trema genus. This close relationship suggests that Parasponia and Trema only recently diverged in nodulation ability. In Chapter 6, I exploited this close relationship to question whether the nodulation trait is associated with gene expression differentiation. To this end, I sequenced root transcriptomes of two Parasponia and three Trema species. Principal component analysis separated all Parasponia samples from those of Trema along the first principal component. This component explains more than half of the observed variance, indicating that the root transcriptomes of two Parasponia species are distinct from that of the Trema sister species T. levigata, as well as the outgroup species T. orientalis and T. tomentosa. To determine, whether the transcriptional differences between Parasponia and Trema are relevant in a symbiotic context, I compared the list of differentially expressed genes to a list of genes that show nodule-enhanced expression in P. andersonii. This revealed significant enrichment of nodule-enhanced genes among genes that lower expressed in roots of Parasponia compared to Trema. Among the genes differentially expressed between Parasponia and Trema roots are several involved in mycorrhizal symbiosis as well as jasmonic acid biosynthesis. Measurements of hormone concentrations, showed that Parasponia and Trema roots harbor a difference in jasmonic acid/salicylic acid balance. However, mutants in jasmonic acid biosynthesis are unaffected in nodule development. Therefore, it remains a challenge to determine whether the difference in root transcriptomes between Parasponia and Trema are relevant in a symbiotic context.

In Chapter 7, I review hormone function in nitrogen-fixing nodule symbioses in legumes, Parasponia and actinorhizal species. In this chapter, I question whether different nodulating lineages recruited the same hormonal networks to function in nodule formation. Additionally, I discuss whether nodulating species harbor genetic adaptations in hormonal pathways that correlate with nodulation capacity.

Functional analyses of plant-specific histone deacetylases : Their role in root development, stress responses and symbiotic interactions
Li, Huchen - \ 2017
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Olga Kulikova. - Wageningen : Wageningen University - ISBN 9789463436816 - 188
plants - histones - enzymes - roots - development - symbiosis - gene expression - molecular biology - root nodules - mycorrhizas - planten - histonen - enzymen - wortels - ontwikkeling - symbiose - genexpressie - moleculaire biologie - wortelknolletjes - mycorrhizae

Plants have a sessile lifestyle. To ensure survival, they develop a potential to respond to environmental cues to set up an adaptive growth and development. This adaptation involves transcriptional reprogramming of the genome through chromatin-based mechanisms relying on the dynamic interplay of transcription factors (TFs), post-translational modification of histones, the deposition of histone variants, DNA methylation, and nucleosome remodeling. This thesis is focused on a role of one group of histone post-translational modifiers, plant-specific histone deacetylases (HDTs), in plant development under control condition and variable stresses/symbiotic interactions.

It is well known that HDTs are involved in plant responses to environmental stresses. However, whether they play a role in regulating plant growth and development is elusive. In this thesis it is shown that Arabidopsis thaliana AtHDT1/2 regulate the cell fate switch from division to expansion in the Arabidopsis root. Knock-down of AtHDT1/2 (hdt1,2i) causes that this switch occurs earlier and results in less cells in the root meristem. This process slows down root growth. One target of AtHDT1/2, AtGA2ox2, is identified here. Its overexpression displays the same root phenotype as hdt1/2i , and its knock-out partially rescues hdt1,2i root meristem phenotype. AtGA2ox2 inactivates gibberellin (GA4) whose application increases root meristem cell number in WT, but not in hdt1,2i. Based on these data, we conclude that AtHDT1/2 repress the transcription of AtGA2ox2, and likely fine-tunes GA homeostasis to regulate the switch from cell division to expansion in root tips.

HDTs respond to salt stress in Arabidopsis seedlings. Halotropism is a novel reported tropism allowing roots to avoid a saline environment. Whether the AtHDT1/2-AtGA2ox2 module is operational in halotropism is studied here. We show that hdt1,2i mutants respond more severe in halotropism. AtHDT1/2, as well as AtGA2ox2 display asymmetric localization patterns in halotropism with AtHDT1/2 reduced and AtGA2ox2 induced at high salt side of root tips. Our data indicate that their asymmetric patterns likely results in less GA at high salt side of root tips and this is required for halotropism establishment. In line with this, both constitutive expression of AtHDT2 and exogenous GA application reduce halotropic response. A reduction of GA in root tips causes an earlier switch from cell division to expansion. We discuss that this earlier switch enables roots rapidly to bend away from saline environment.

It has been shown that HDTs play a role under biotic stress in rice and tobacco leaves. We demonstrate that they are also involved in response to biotic stress in Arabidopsis leaves. Arabidopsis hdt2 mutants are more susceptible to virulent Pseudomonas syringae pv. tomato PstDC3000, whereas AtHDT2 overexpression mutants are more resistant. In addition, we detected a translocation of AtHDT2 from nucleolus to nucleoplasm after the perception of flagellin22 in Arabidopsis leaf cells. This translocation is not observed under abiotic stress. A mechanism controlling this translocation is identified. AtMPK3 is activated under biotic stress, it interacts with and phosphorylates AtHDT2. This leads to the accumulation of AtHDT2 in nucleoplasm where it contributes to the repression of defense genes.

During the interaction with symbiotic microorganisms, plants could develop a symbiotic organ/structure. For example, legumes of which Medicago truncatula is a model, can form root nodules or arbuscules by interacting with rhizobia or arbuscular mycorrhiza.

We show that nodule-specific knock-down of MtHDT1/2/3 (MtHDTs RNAi) blocks nodule primordia development and affects the function of nodule meristem. This is consistent with their roles in controlling cell division during root development and suggests that the function of nodule and root meristems is closely related. However, MtHDT2 gains a new sub-nuclear localization pattern in nodule meristem by using a not yet known mechanism, different from that in root meristem. This suggests that these two meristems have different transcriptional landscapes. In the nodule infection zone MtHDTs are also expressed and in MtHDTs RNAi the intracellular release of rhizobia is markedly reduced. Expression of MtHMGR1 and its paralogs, encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductases are down-regulated in MtHDTs RNAi. It has been shown MtHMGR1 interacts with MtDMI2, a component of Nod factor signalling pathway, to control rhizobial infection. Knock-down of MtHMGR1/MtDMI2, as well as inhibiting MtHMGRs enzymatic activity blocks nodule primordia development and rhizobial infection in nodule primordia/mature nodules. This phenotype partially resembles MtHDTs RNAi phenotype. We discuss that MtHDTs regulate expression of MtHMGRs and in this way affect Nod factor signalling and control nodule development.

Similar to nodule symbiosis, during arbuscular mycorrhizal symbiosis cells in the cortex are also intracellularly infected. We show that MtHDT2 is also induced in these arbuscule containing cells. Knock-down of MtHDT2 (MtHDT2i) significantly reduces the intracellular infection of the hyphae on the mycorrhized root segments, indicating that MtHDT2 control mycorrhizal intracellular infection. We discuss whether MtHDTs can regulate mycorrhizal/rhizobial infection in a similar way.

The data obtained in this thesis and the published information related to these subjects are discussed at the end. HDTs are key players in plant responses to environmental cues, whereas they respond to abiotic factors and biotic factors differently. They are also key regulators of plant growth and development that is clearly demonstrated in this thesis on examples of root and nodule development. I also propose a role of AtHDT1/2 in response to salt signal to fine-tune the switch from cell division to expansion in root tips during halotropism.

Interface Symbiotic Membrane Formation in Root Nodules of Medicago truncatula: the Role of Synaptotagmins MtSyt1, MtSyt2 and MtSyt3
Gavrin, A.Y. ; Kulikova, O. ; Bisseling, A.H.J. ; Fedorova, E. - \ 2017
Frontiers in Plant Science 8 (2017). - ISSN 1664-462X - 10 p.
symbiosis - synaptotagmin1 - membrane tension/repair - interface membrane - root nodule - arbuscular
Symbiotic bacteria (rhizobia) are maintained and conditioned to fix atmospheric nitrogen in infected cells of legume root nodules. Rhizobia are confined to the asymmetrical protrusions of plasma membrane (PM): infection threads (IT), cell wall-free unwalled droplets and symbiosomes. These compartments rapidly increase in surface and volume due to the microsymbiont expansion, and remarkably, the membrane resources of the host cells are targeted to interface membrane quite precisely. We hypothesized that the change in the membrane tension around the expanding microsymbionts creates a vector for membrane traffic toward the symbiotic interface. To test this hypothesis, we selected calcium sensors from the group of synaptotagmins: MtSyt1, Medicago truncatula homolog of AtSYT1 from Arabidopsis thaliana known to be involved in membrane repair, and two other homologs expressed in root nodules: MtSyt2 and MtSyt3. Here we show that MtSyt1, MtSyt2, and MtSyt3 are expressed in the expanding cells of the meristem, zone of infection and proximal cell layers of zone of nitrogen fixation (MtSyt1, MtSyt3). All three GFP-tagged proteins delineate the interface membrane of IT and unwalled droplets and create a subcompartments of PM surrounding these structures. The localization of MtSyt1 by EM immunogold labeling has shown the signal on symbiosome membrane and endoplasmic reticulum (ER). To specify the role of synaptotagmins in interface membrane formation, we compared the localization of MtSyt1, MtSyt3 and exocyst subunit EXO70i, involved in the tethering of post-Golgi secretory vesicles and operational in tip growth. The localization of EXO70i in root nodules and arbusculated roots was strictly associated with the tips of IT and the tips of arbuscular fine branches, but the distribution of synaptotagmins on membrane subcompartments was broader and includes lateral parts of IT, the membrane of unwalled droplets as well as the symbiosomes. The double silencing of synaptotagmins caused a delay in rhizobia release and blocks symbiosome maturation confirming the functional role of synaptotagmins. In conclusion: synaptotagmin-dependent membrane fusion along with tip-targeted exocytosis is operational in the formation of symbiotic interface.
Genetic constraints that determine rhizobium-root nodule formation in Parasponia andersonii
Seifi Kalhor, M. - \ 2016
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - Wageningen : Wageningen University - ISBN 9789462579118 - 160 p.
parasponia - rhizobium - root nodules - rhizobium rhizogenes - temperature - nitrates - symbiosis - genetics - wortelknolletjes - temperatuur - nitraten - symbiose - genetica

Bacteria of the genus Rhizobium play a very important role in agriculture by inducing nitrogen-fixing nodules on the roots of legumes. Root nodule symbiosis enables nitrogen‐fixing bacteria (Rhizobium) to convert atmospheric nitrogen into a form that is directly available for plant growth. This symbiosis can relieve the requirements for added nitrogenous fertilizer during the growth of leguminous crops. Research on legume-rhizobium symbioses has emphasized fitness benefits to plants but in our research, we take a different vantage point, focusing on the Parasponia-rhizobium symbiosis. Parasponia is the only non-legume plant capable of establishing mutualistic relation with rhizobia. This study will provide background knowledge for use in applied objectives as well as yielding a wealth of fundamental knowledge with wide implications from rhizobium symbiosis evolution. This thesis describes my research on genetic constrains that determine rhizobium-root nodule formation. To identify these constraints we used Parasponia anadersnii as only non-legume capable to establish nitrogen fixing rhizobium symbiosis. Our main attempt in this thesis was to find the genetic constraints using Parasponia as a key and reconstruct an auto active symbiotic signaling cascade in the non- legume plants. In line with this, a simple and efficient hairy root transformation method was established in this thesis. To determine the genetic elements that underlie the rhizobium symbiosis, we aimed to compare Parasponia with closest non nodulating specious, Trema tomentosa. To do so, we also developed an efficient genetic transformation method for Trema mediated by Agrobacterium tumefaciens. In different attempt we implemented in a physiological study on symbiotic response of Parasponia to nitrate. This research opened a novel view on the Parasponia-rhizobium symbiosis by discovering a different mechanism that control root nodule formation in Parasponia in compare with legumes. We discovered that Parasponai-rhizbium symbiosis is not evolved to regulate the nodule number in presence of the nitrate. According to the fact that Parasponia and legumes are remotely related, it was hypothesized that, Parasponia-rhizobium symbiosis evolved independently. Therefore we put forward our attempt to determine the genes required for nodule formation in Parasponia, by extending our research on symbiotic genes which are available in non nodulating plants with different function, namely NSP1 and NSP2. We showed that NSP1 and NSP2 are involved in both nodulation and mycorrhization. This result highlight the idea that RN and AM symbiosis are conserved in part of the pathway and probably bifurcates into two branches by NSP transcription factor allowing specific activation of nodulation or mycorrhization. Aiming to know the role of hormones in symbiotic behavior, we focused on ethylene as a negative regulator of nodule formation in legumes. We found the negative effect of ethylene on root nodulation of Parasponia. For the first time we reported a hyper nodulation (20 fold nodule number in compare with control plants) phenotype in Parasponia by performing knocked down mutant of EIN2 gene. Finally, the results obtained in this study provide new insight into the fact that rhizobium symbiosis are under tight genetic constraints that guide endosymbiosis in remotely evolved host plants, legumes and Parasponia.

Role of anti-competitor toxins in the origin and maintenance of diversity in Saccharomyces yeast microbial populations
Pieczynska, M.D. - \ 2015
Wageningen University. Promotor(en): Bas Zwaan, co-promotor(en): Arjan de Visser. - Wageningen : Wageningen University - ISBN 9789462573093 - 123
gisten - rna-virussen - toxinen - symbiose - toxiciteit - co-evolutie - fenotypen - fermentatie - yeasts - rna viruses - toxins - symbiosis - toxicity - coevolution - phenotypes - fermentation


Saccharomyces cells occasionally carry cytoplasmic ds-RNA “killer” viruses coding for low-mass proteins, which upon secretion to the environment can kill related cells that do not carry the viral particles. Such killer viruses are not infectious, and can spread only through cell division and during mating. Three principal classes of Saccharomyces viruses (ScV-M1, ScV-M2 and ScV-M28) belonging to the Totiviridae family have been characterised, each capable of forming a specific anti-competitor toxin and corresponding antidote. Presumably, toxic killing provides competitive benefits to the yeast host. However, the ecological and evolutionary significance of toxin production remains poorly understood. For example, it is unknown where yeast killers occur and at what frequency, how evolvable killing ability is, whether it is constrained by possible trade-offs with resource competitive ability and how it is shaped by interactions with toxin-sensitive competitors. Also unknown is how stable yeast-virus symbioses are, and how coevolution between host and virus may affect this stability and the killing phenotype itself. It is believed that killer yeasts are common based on the fact that they have been found among yeasts isolated from different sources over several decades. In chapter 2, we assay two large yeast collections from diverse habitats, including nature and man-made habitats (in total 136 strains with known genome sequences), for killer phenotype and toxin resistance. We find that ~10.3% carry a killer virus, while about 25% are resistant to at least one of the three known killer toxins (12.5% to different combinations of two and ~9% to all three), most likely due to chromosomal mutations. Analyses of their evolutionary relationship indicate that host-virus associations are relatively short lived, whereas the relatively high frequency of resistance suggests that toxins have a substantial impact on yeast evolution.

In order to understand the ecological and evolutionary role of toxin production, it is essential to reliably assess the killing rate of toxin producers by measuring how many toxin-sensitive individuals are killed by a single toxin producer during a given time interval. To identify a convenient method with high sensitivity and reproducibility, in chapter 3 we perform a systematic comparative analysis of four methods, including the conventional “Halo method” and three more quantitative liquid assays. We apply these methods to a set of three known yeast killer strains (K1, K2 and K28) and find that the easy applicable Halo method provides the most sensitive and reproducible killing rate estimates (with best discrimination between killer strains).

Understanding the evolution of the yeast-virus association is crucial for a full understanding of the ecological and evolutionary role of killer strains. In chapter 4, we present experimental tests of the strength of the dependence of yeast host strains on their killer viruses. We cross-infect several viruses among killer strains of the genus Saccharomyces – all expressing the K1-type toxin, and test native and new combinations for the strength of host-virus co-adaptation. We find explicit host-virus co-adaptation, because native yeasts hosts display the highest toxicity and highest stability of killer viruses relative to hosts carrying non-native viruses. Even stronger, we find that curing these wild killer yeasts from their virus reduces their competitive fitness, despite initial fitness costs of viral carriage reported for constructed killer strains. These results demonstrate co-adaptation of host and virus in the natural killer strains resulting in their dependence on the killer virus. To explore the evolutionary costs and benefits of virus carriage and toxin production, and understand whether they are shaped by the coevolution between host and virus and the presence of toxin-sensitive competitors in the environment, we conduct a series of laboratory experiments where we manipulate the opportunity for coevolution (chapter 5). Analyses of killing ability, toxin sensitivity and fitness (i.e. resource competitive ability), show rapid reciprocal changes in killer and sensitive strain when coevolution is allowed, modulated by the rapid invasion of toxin-resistant mutants and subsequent reduction of killing ability. Remarkably, we find that the rapid invasion of toxin-resistant mutants involves two mutational steps, the first being a mutation showing a meiotic drive phenotype as well as a strong fitness benefit in heterozygotes, the second the resistance mutation. Shifts in the competitive fitness of evolved killer isolates with increased killing ability show a clear trade-off between killing rate and resource competitive ability, indicating that resource and interference competitive ability are alternative competitive strategies. Moreover, by cross-infecting the killer virus between the ancestral and an evolved strain, we are able to demonstrate the rapid co-adaptation between host and killer virus, supporting our previous findings of co-adaptive responses in wild yeast killers (chapter 4).

Our analyses are based on screens of natural isolates, laboratory evolution experiments and phenotypic analyses, complemented by classical genetics. To more fully understand the reciprocal nature and molecular mechanisms of adaptive responses, genome analyses are required. The motivation for such analyses and other follow-up studies are proposed in chapter 6. My studies show the usefulness of the killer yeast system to address questions related to interference competition and coevolution, which may proof valuable also given potential applications of killer yeasts in the fermentation industry.

Root and nodule : lateral organ development in N2-fixing plants
Xiao, T.T. - \ 2015
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts; Henk Franssen. - Wageningen : Wageningen University - ISBN 9789462572768 - 198
medicago - wortelknolletjes - endosymbiose - symbiose - mycorrhizae - stikstoffixatie - plantenontwikkeling - moleculaire biologie - root nodules - endosymbiosis - symbiosis - mycorrhizas - nitrogen fixation - plant development - molecular biology

Plants are sessile organisms. This characteristic severely limits their ability of approaching nutrients. To cope with this issue, plants evolved endosymbiotic relationships with soil fungi to extend their interface with surrounding environment. In case of arbuscular mycorrhizae (AM) fungi this occurred about 400 million years ago. The AM fungi can interact with most angiosperms. In this symbiotic relationship, the plant get nutrients, especially phosphate, from the fungi, and plants provide carbohydrates to the fungi in return. About 60 million years ago, a group of plants evolved N2-fixing nodule symbiosis. This includes interactions of legumes plants with rhizobium bacteria and actinorhizal plants with Frankia bacteria. Currently, all plant species that are able to establish a nodule symbiosis belong to the Rosid I clade. In the nodule symbioses the bacteria produce ammonia and the plant provides carbohydrates to the bacteria.

In the root nodule symbiosis, the nitrogen fixing bacteria are hosted in the cell of the root nodule. Although the function and structure of the root nodule are different from the other plant organs, it does share some features with other organs, especially the lateral root. To get further insight into the similarities and differences between root nodule and lateral root, I made use of the model legume (Medicago truncatula) and the non-legume Parasponia (Parasponia andersonii) that is the only genus outside the legumes that forms nodules with rhizobium.

In Chapter 1, I will give a general introduction on the process of root nodule formation in legume plants. I will mainly focus on nodule organogenesis and the plant hormones that are known to be important for this process. Root nodules are supposed to have a close relationship with lateral roots. Therefore a comparison between lateral root and root nodule development will be included in this introduction.

Lateral root development has especially been studied in in Arabidopsis. To be able to compare the root and root nodule developmental process, especially at the early stages, a Medicago lateral root development fate map has been made. This will be described in Chapter 2 and showed that in addition to the pericycle, endodermis and cortex are also mitotically activated during lateral root formation. Pericycle derived cells only form part of the stem cell niche as endodermis derived cells also contribute to this.

In Chapter 3, a Medicago root nodule fate map is presented. In this Chapter, the contribution of different root cell layers to the mature nodule will be described. A set of molecular markers for root tissue, cell cycle and rhizobial infection have been used to facilitate this analysis. The fate map showed that nodule meristem originates from the third cortical layer and many cell layers of the base of the nodule are directly derived from cells of the inner cortical layers, root endodermis and pericycle. The inner cortical cell layers form about 8 cell layers of infected cells while the root endodermis and pericycle derived cells forms the uninfected tissues that are located at the base of the mature nodule. Nodule vascular is formed from the part of the primordium derived from the cortex. The development of primordia was divided in 6 stages. To illustrate the value of this fate map, a few published mutant nodule phenotypes are re-analyzed.

In Chapter 4, the role of auxin at early stages of Medicago nodule formation is studied. In this chapter auxin accumulation is studied during the 6 stages of primordium development. It is studied by using DR5::GUS as an auxin reporter. Auxin accumulation associates with mitotic activity within the primordium. Previously, it has been postulated by theoretical modelling that the accumulation of auxin during nodulation is induced by a local reduction of PIN (auxin efflux carriers) levels. We tested this theory, but this was hampered due to the low level of PIN proteins in the susceptible zone of the root. It is still possible that auxin accumulation is initiated by a decrease of PIN levels. However, the level of 2 PIN already increase before the first divisions are induced. In young primordia they accumulate in all cells. At later stages PINs mainly accumulate at the nodule periphery and the future nodule meristem. The subcellular position of PINs strongly indicates they play a key role in the accumulation of auxin in primordia.

Previous studies showed that a group of root apical meristem regulators is expressed in the nodule meristem. In Chapter 5, we tested whether the Medicago nodule meristem expresses PLETHORA genes that are expressed in the root meristem. These PLETHORAs were functionally analysed, by using RNAi approach using a nodule specific promoter. Knockdown of PLETHORAs expression hampers primordium formation and meristem growth. Hence, we conclude rhizobium recruited key regulators of root development for nodule development.

In Chapter 6, we first introduced the non-legume lateral root and nodule fate maps by using Parasponia. In Parasponia nodules the nodule central vascular bundle is completely derived from the pericycle similar as its lateral roots. The nodule infected cells were shown to be derived from cortex. Together with the data obtained in this thesis, this Chapter further discussed several developmental aspects of the different lateral root organs. Especially, it focused on the vasculature and meristem formation of legume and non-legume nodules.

Exploring microbial diversity of marine sponges by culture-dependent and molecular approaches
Naim, M.A. - \ 2015
Wageningen University. Promotor(en): Hauke Smidt, co-promotor(en): Detmer Sipkema. - Wageningen : Wageningen University - ISBN 9789462572867 - 220
sponsen - microbiële diversiteit - symbiose - gastheerspecificiteit - zeeschimmels - biodiversiteit - sponges - microbial diversity - symbiosis - host specificity - marine fungi - biodiversity

Discovery of sponge-grade metazoans dated 650 million years ago proved that sponges have been around since the Precambrian era. Their resilience to ever-changing environmental conditions and their global distribution is one of the features attributed to the symbionts in sponges, which include Archaea, Bacteria and Eukarya. It is yet unknown how sponges attract and select their bacterial associates but mechanisms to maintain or newly acquire their symbionts have been demonstrated, such as vertical and horizontal transmission.

Discovery of species-specific bacterial communities in the marine sponges H. panicea, H. oculata and H. xena which are dominated by an alpha, beta- and gammaproteobacterium, respectively, confirmed host-specificity of bacterial associates in marine sponges from the North Sea, although their function remains unknown. Detection of Chlamydiae in high relative abundance raised the question as to what is their function in the sponge holobiont as they were only distantly related to other known Chlamydiae.

Little is known about the fungal community in marine sponges. This prompted the study of sponge-associated fungi based on molecular analysis. This was previously a difficult enterprise due the large amount of ‘contaminating’ sponge DNA, which is susceptible to amplification with fungi-specific PCR primers as well. The advent of next generation sequencing technology now for the first time allowed to overcome this hurdle by the sheer numbers of sequences that can be generated. This lead to discovery of novel yeast lineages from the phyla Ascomycota and Basidiomycota in North Sea and Mediterranean marine sponges, indicating a much higher diversity of fungi yet to be explored. For instance, yeasts from the order Malasseziales, which are common pathogens of marine animals, were found as the dominant yeasts in many of the sponges tested that were without apparent disease.

A complementary cultivation-dependent approach provided access to fungal isolates. Fungi belonging to the genus Penicillium were found to be the dominant fungi recovered by isolation from the Mediterranean sponges Aplysina aerophoba, Petrosia ficiformis and Corticium candelabrum. In addition, fungi belonging to the order Alternaria and yeasts affiliated to the genus Rhodotorula were isolated multiple times. No overlap was found with the fungal species observed through the molecular study, which indicates that the great plate anomaly also exists for fungi. Many of the fungal Pencilillium and Alternaria strains isolated were shown to have the genetic capacity for producing polyketide synthases (PKS) or PKS-non ribosomal peptide synthase (PKS-NRPS) hybrids. These enzyme complexes are generally responsible for the production of secondary metabolites with a high biological activity.

Isolation of bacteria from H. panicea in a cultivation experiment with a large diversification of media and growth conditions and subsequent comparison of the retrieved microorganisms to bacteria found in the sponge tissue by a molecular approach revealed the presence of bacterial genera that dominate the cultivation library, but comprise of represent minor components of the sponge microbiome. This includes genera such as Bacillus, Paracoccus and Shewanella. Another genus that was commonly isolated from many marine sponges, but only is found at low relative abundance in the sponge microbiome is Pseudovibrio. Phenotypic characterization based on antibiotic resistance and genotypic differentiation based on bacterial BOX elements and presence of halogenase-encoding genes could discriminate closely related strains that could not be distinguished based on their 16 rRNA gene sequence.

In conclusion, this thesis helps to bridge the gap between cultivation-dependent and cultivation-independent studies of sponge-associated bacteria and fungi by clearly defining the frontiers of the gap. The knowledge derived from this thesis could serve as a scientific foundation and inspiration for future microbial diversity studies and provides perspective for analysing and exploiting sponge symbionts.

The danger of mycorrhizal traps?
Kuyper, T.W. ; Kiers, E.T. - \ 2014
New Phytologist 203 (2014)2. - ISSN 0028-646X - p. 352 - 354.
ectomycorrhizal fungi - nitrogen limitation - boreal forests - mutualism - plants - competition - symbiosis - patterns - partners - markets
Plant-driven genome selection of arbuscular mycorrhizal fungi
Limpens, E.H.M. ; Geurts, R. - \ 2014
Molecular Plant Pathology 15 (2014)6. - ISSN 1464-6722 - p. 531 - 534.
The birth of cooperation
Aanen, D.K. ; Bisseling, T. - \ 2014
Science 345 (2014)6192. - ISSN 0036-8075 - p. 29 - 30.
symbiosis - mutualism - evolution
Mutually beneficial associations between individuals of different species, called mutualistic symbioses, have enabled major ecological innovations and underlie some of the major transitions in evolution (1). For example, the ancestor of plants domesticated endosymbiotic photosynthetic bacteria, today's chloroplasts, for carbon fixation. This association dramatically increased the habitat of these photosynthetic bacteria from the sea to terrestrial ecosystems. However, the colonization of land by plants required an additional symbiotic association, with fungal root symbionts that facilitate nutrient uptake (2). Yet, surprisingly little is known about how mutualistic symbioses evolved and persist. On page 94 of this issue, Hom and Murray show how mutualism may arise without prior coevolution (see the photo) (3)
Fate map of Medicago truncatula root nodules
Xiao, T.T. ; Schilderink, S. ; Moling, S. ; Deinum, E.E. ; Kondorosi, E. ; Franssen, H. ; Kulikova, O. ; Niebel, A. ; Bisseling, T. - \ 2014
Development 141 (2014). - ISSN 0950-1991 - p. 3517 - 3528.
rhizobial infection - cortical-cells - nod factors - arabidopsis - differentiation - protein - expression - initiation - activator - symbiosis
Legume root nodules are induced by N-fixing rhizobium bacteria that are hosted in an intracellular manner. These nodules are formed by reprogramming differentiated root cells. The model legume Medicago truncatula forms indeterminate nodules with a meristem at their apex. This organ grows by the activity of the meristem that adds cells to the different nodule tissues. In Medicago sativa it has been shown that the nodule meristem is derived from the root middle cortex. During nodule initiation, inner cortical cells and pericycle cells are also mitotically activated. However, whether and how these cells contribute to the mature nodule has not been studied. Here, we produce a nodule fate map that precisely describes the origin of the different nodule tissues based on sequential longitudinal sections and on the use of marker genes that allow the distinction of cells originating from different root tissues. We show that nodule meristem originates from the third cortical layer, while several cell layers of the base of the nodule are directly formed from cells of the inner cortical layers, root endodermis and pericycle. The latter two differentiate into the uninfected tissues that are located at the base of the mature nodule, whereas the cells derived from the inner cortical cell layers form about eight cell layers of infected cells. This nodule fate map has then been used to re-analyse several mutant nodule phenotypes. This showed, among other things, that intracellular release of rhizobia in primordium cells and meristem daughter cells are regulated in a different manner.
It’s all about perception : nod factor perception inside nodules of Medicago truncatula
Moling, S.G.J.A. - \ 2014
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Erik Limpens. - Wageningen : Wageningen UR - ISBN 9789462570399 - 190
medicago truncatula - wortelknolletjes - knobbeltjes - rhizobium - symbiose - genexpressie - root nodules - nodules - symbiosis - gene expression

Legumes are unique in that they are able to establish a mutual symbiotic interaction with nitrogen fixing soil bacteria generally referred to as rhizobia. This interaction starts off in the root epidermis where the bacterial signal molecule, the Nod factor, is perceived by the plant (Nod factor signaling). This recognition sets in motion a series of responses leading to the formation of a root nodule. This organ is specifically created to host the bacteria in an intracellular manner. The rhizobia develop into a mature, nitrogen fixing state in these infected cells. The rhizobia are surrounded by a host membrane and this forms a cell wall free symbiotic interface, that allows the exchange of nutrients between the symbionts. The comparison of the mechanism controlling symbiotic interface formation in rhizobium symbiosis and the much more common symbiosis with arbusucal micorrhizal (AM) fungi strongly suggest that rhizobia co-opted parts of the AM symbiosis. The signalling as well as cellular processes controlling symbiotic interface formation in the ancient AM symbiosis have been recruited by the rhizobium nodule symbiosis. In this thesis I present the results of my research on the role of and mechanisms controling Nod factor signalling on symbiotic interface formation in nodules of the model legume Medicago truncatula (Medicago).

To study the role of Nod factor signaling in the formation of a symbiotic interface it was essential to define a fate map for Medicago root nodules. The formation of a nodule starts, after Nod factor perception in the epidermis, with divisions in the cortex, pericycle and endodermis. Devisions in the cortex start from the inner most layer and spread outwards. When cortical layer 4 (C4) and C5 have already divided a few times, mitotic activity is induced in C3 and ultimately C2 divides. After the first periclinal divisions have been induced in C3, cells derived from C4 and C5 stop dividing and form about 8 cell layers. C3 continues to divide and ultimately forms the nodule meristem. The infection thread that developed in the root hairs has to penetrate the primordium before the first periclinal divisions occur in C3. The cells derived from C4 and C5 are then infected by bacteria from the infection thread in the primordium. After the establishment of a meristem this meristem adds new cells to the nodule, which are gradually infected when they leave the meristem and enter the infection zone. The fate map shows that formation of the symbiotic interface occurs in two ways: first in cells of the primordium and later in the infection zone in daughter cells derived from the meristem.

For the infection of daughter cells derived from the meristem the Nod factor receptors (NFP and LYK3 in Medicago) are needed, most likely to perceive the Nod factor. When we knocked down one of the receptors in a nodule specific manner release of bacteria is hampered (NFP) or massive unwalled droplets were observed (LYK3) indicating most likely slow release. This shows that the receptors are needed for the formation of the symbiotic interface in the nodule. In line with this function in the infection zone we found the receptors to accumulate in the nodule apex. The receptors accumulate in a narrow zone of two cell layers that is the border between the merisitem and the infection zone. The receptors accumulate to a zone markedly narrower than the zone where the promoters are activity. In this narrow zone the receptors accumulate at the cell periphery, most likely the plasma membrane. Outside this layer we observed accumulation of the receptors in the vacuoles suggesting degradation. In cells with LYK3 at the plasma membrane LYK3 occurs only in 35% of the cells at the membrane surrounding infection threads. The removal of LYK3 from the plasma membrane appears to be first completed at the membrane surrounding the infection thread. As the receptors are expressed in a broader zone, post-transcriptional mechanisms limit their accumulation at the plasma membrane. Ectopic expression experiments show that broader expression, and most likely accumulation, could induce defence responses (NFP) and reduces infection (LYK3). Therefore the accumulation of the receptors needs to be limited and the receptors are only allowed to accumulate in a narrow zone where Nod factor perception could take place.

As both receptors accumulate in the same cell layers we tested whether they form heteromeric complexes. It was already proposed at their discovery that NFP and LYK3 should form a complex based on their phenotype and the lack of an active kinase domain in NFP. Also most receptors form complexes to modulate their (kinase) activity. We show that a heteromeric complex is also formed in the proper biological context in nodules. The cell death responses that are induced when the receptors are co-expressed in heterologous systems are avoided in Medicago nodules. Also homomeric complexes containing LYK3 were observed in nodules. This homomeric complex is formed either as an addition to the heteromeric complex with NFP or the complex with NFP contains multiple LYK3 molecules.

As receptors often function in larger complexes we performed immunoprecipitation coupled with mass spectrometry to detect possible interactors of LYK3. With this we detected several proteins interactors of LYK3 including a EF1α, Dynamin-2B, PR10, fructose-bisphosphate aldolase and 14-3-3 f2 protein. Some of these proteins have a function in the regulation of vesicle transport and the cytoskeleton. Remarkable, other known or putative interactors of LYK3 (NFP, PUB1, SYMREM1 and FLOT4) were not detected. To test why we could not find these interactors we performed co-localization experiments. These experiments show that the interaction with SYMREM1 and the putative interaction with FLOT4 could take place as both proteins co-localize with LYK3. SYMREM1 accumulates at the membrane surrounding the infection threads and at the symbiosomes. As LYK3 also, although transient, accumulates at the membrane surrounding the infection threads, LYK3 and SYREM1 co-localize there. This shows that these interactors could form a complex with LYK3, although the complex is most likely very transient. This transient nature makes the complexes difficult to detect.

Because co-localization is a prerequisite for two proteins to interact we studied the expression of the Medicago genome. We used laser capture micro-dissection experiment to isolate RNA from different zones of the nodules and measured the differential expression of the Medicago genome. This experiments provides us with a digital in situ experiment for Medicago nodules. The data show that the genes from the Nod factor signaling cascade do not show the same expression pattern. The Nod factor receptor mRNAs of NFP and LYK3 are enriched in the meristem and distal infection zone. This is in line with our localization studies where the receptors accumulate at the border between meristem and infection zone. Most genes from the Nod factor signaling cascade show no differential expression at all (expressed equal in the entire nodule), where in situ or promoter GUS studies show enrichment of all these genes in the meristem/ infection zone. Further, some show remarkable behaviour. DMI1 and IPD3, for instance, are enriched in infected cells. These data could point to a new function for these proteins which is not related to the known signaling cascade. Also a survey on the differential expression of other LysM domain containing proteins show that there are more candidates that may have an important role in Nod factor perception. These LysM domain containing proteins are also enriched in the mersitem and/or infection zone and could thus function as a co-receptor for NFP or LYK3.

Nod factor perception is the key step in the progress of the rhizobium – legume symbiosis. Not only in the early steps in the root epidermis, but also in the nodule. Because of the developmental gradient in the indeterminate Medicago nodule this nodule is a perfect biological system to study the cell biology of the symbiosis. The differential expression analysis and the nodule fate map are important tools for these studies.

Transcriptional regulation of nodule development and senescence in Medicago truncatula
Karmarkar, V.M. - \ 2014
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - Wageningen : Wageningen University - ISBN 9789462570214 - 110
medicago truncatula - plantenontwikkeling - veroudering - wortelknolletjes - stikstoffixatie - genexpressie - symbiose - transcriptie - transcriptiefactoren - plant development - senescence - root nodules - nitrogen fixation - gene expression - symbiosis - transcription - transcription factors
Balancing the organic load and light supply in symbiotic microalgal–bacterial biofilm reactors treating synthetic municipal wastewater
Boelee, N.C. ; Temmink, B.G. ; Janssen, M. ; Buisman, C.J.N. ; Wijffels, R.H. - \ 2014
Ecological Engineering 64 (2014). - ISSN 0925-8574 - p. 213 - 221.
afvalwaterbehandeling - biofilms - symbiose - algen - bacteriën - heterotrofe micro-organismen - fotosynthese - acetaten - stikstof - fosfor - nitrificatie - denitrificatie - biologische waterzuiveringsinstallaties - biobased economy - waste water treatment - symbiosis - algae - bacteria - heterotrophic microorganisms - photosynthesis - acetates - nitrogen - phosphorus - nitrification - denitrification - biological water treatment plants - activated-sludge - nutrient removal - growth - phytoplankton - fluorescence - enhancement
Symbiotic microalgal–bacterial biofilms can be very attractive for municipal wastewater treatment. Microalgae remove nitrogen and phosphorus and simultaneously produce the oxygen that is required for the aerobic, heterotrophic degradation of organic pollutants. For the application of these biofilms in new wastewater treatment systems, the engineering aspects need to be investigated to obtain a balanced system where no additional oxygen is required. In this study symbiotic microalgal–bacterial biofilms were grown in flow cells with ammonium and phosphate, and with acetate as biodegradable organic pollutant at a hydraulic retention time of 4.5 h. The symbiotic biofilms removed acetate from 323 mg/L to 39 mg/L without an external oxygen or carbon dioxide supply at a removal rate of 43 g COD/m2/d. Ammonium and phosphate could not be completely removed, but removal rates of 3.2 g/m2/d and 0.41 g/m2/d were obtained, respectively. Further nitrogen removal may be obtained by nitrification and denitrification as the biofilm obtained a considerable heterotrophic denitrification capacity. The symbiotic relationship between microalgae and aerobic heterotrophs was proven by subsequently removing light and acetate. In both cases this resulted in the cessation of the symbiosis and in increasing effluent concentrations of both acetate and the nutrients ammonium and phosphate. Future research should investigate the dimensioning of an up-scaled symbiotic biofilm reactor, and the possibilities to obtain additional nitrogen and phosphorus removal under day–night cycles utilizing real wastewater.
Comparative and functional analysis of NODULATION SIGNALING PATHWAY 1 (NSP1) and NSP2 in rice and Medicago
Liu, W. - \ 2013
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - S.l. : s.n. - ISBN 9789461736369 - 147
oryza - medicago - knobbelvorming - symbiose - genen - rhizobium - wortelknolletjes - stikstoffixatie - nodulation - symbiosis - genes - root nodules - nitrogen fixation
Co-option of pre-existing pathways during Rhizobium-legume symbiosis evolution
Lillo, A. - \ 2012
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - S.l. : s.n. - ISBN 9789461733443 - 151
rhizobium - fabaceae - symbiose - evolutie - stikstoffixatie - wortels - fylogenetica - genomen - medicago - eerste wortels - symbiosis - evolution - nitrogen fixation - roots - phylogenetics - genomes - root primordia

Fixed nitrogen is one of the most limiting factors for plant growth. One of the most important nitrogen-fixing systems is the rhizobium root nodule symbiosis. In this Thesis I have studied the legume-rhizobium symbiosis, starting from the idea that part of pre-existing signalling pathways have been co-opted during evolution of this mutualistic interaction. Gene duplications -of which a whole genome duplication (WGD) is the most dramatic variant- are known as important driving forces in evolution of new traits. 56 to 65 million years ago an ancestral legume species within the Papilionoidae subfamily (Papilionoids) experienced a WGD event and subsequently gave rise to several major phylogenetic crowns. I hypothesize that among the orthologous gene pairs maintained are genes that are essential for nodulation. I adopted a phylogenetic strategy to identify new candidate genes involved in the legume-Rhizobium symbiosis
In a targeted approach, we focussed on the cytokinin phosphorelay pathway. This resulted in the identification of one gene pair encoding type-A Response Regulators (RRs) with a positive regulatory role for these proteins in root nodule formation. Yet the illustrated role for MtRR9 and MtRR11 in rhizobial symbiosis provides a proof of principle of this method to identify gene pairs involved in legume specific characters. An unbiased search for paralogous gene pairs revealed two conserved gene duplications in the NADPH oxidases gene family. NADPH oxidases are reactive oxygen species (ROS) producing enzymes. We identified two sets of duplicated genes that have been maintained after the Papilionoid specific WGD and we show that MtRBOHA and MtRBOHG are redundant, yet essential during symbiosis.
Moreover, although it is commonly believed that exclusively pericycle cells give rise to the lateral root primordium, similar as seen in Arabidopsis thaliana, we provide morphological evidence that in the studied legume species this is not the case. In both, Lotus and Medicago, also root cortical cell divisions occur during lateral root formation. Furthermore, we found a striking correlation in the cell layers that are recruited during lateral root and nodule primordium formation. This supports the hypothesis that at least parts of the lateral root developmental program have been recruited during evolution of symbiotic root nodules.

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