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Legume-rhizobium symbiosis results in nitrogen-fixing root nodules. This symbiosis allow legumes to grow in nitrogen-poor soil. NIN (NODULE INCEPTION) is a transcription factor that essential for multiple processes of nodule formation. However, how it can play a key role in these processes, which occur in different times and locations, remain elusive. Here, we found that during nodule initiation, the regulatory sequences sufficient for the epidermal infection process are located within a -5 kb region in Medicago truncatula. Furthermore, we identified a remote upstream regulatory region, which is required for the expression of NIN in the pericycle to initiate nodule organogenesis. In mature nodule, we identified late stage Nod factor (NF) signaling controlled set of genes (including NIN). These genes turned to be markedly different from the genes activated in roots upon NF application. Moreover, by analyzing two nin weak allele mutants, nin-13/16, we found that the level of NIN in mature nodule is essential for the transition from infection to fixation zone in establishing a functional symbiosis. Besides, we showed that NIN is not functionally equivalent with its orthologues outside the nitrogen fixation clade (NFC). Further, we identified difference between NIN and its orthologues outside of the NFC. These changes are important for the function of NIN in nodule formation. In conclusion, essential changes has been introduced to NIN, allowing it plays a key role in all stages of nodule development, and its versatility depends on its spatial–temporal concentration level.

PTEN hamartoma tumour syndrome is a diverse multi-system disorder predisposing to the development of hamartomatous growths, increasing risk of breast, thyroid, renal cancer, and possibly increasing risk of endometrial cancer, colorectal cancer and melanoma. There is no international consensus on cancer surveillance in PHTS and all current guidelines are based on expert opinion. A comprehensive literature review was undertaken and guidelines were developed by clinicians with expertise from clinical genetics, gynaecology, endocrinology, dermatology, radiology, gastroenterology and general surgery, together with affected individuals and their representatives. Recommendations were put forward for surveillance for breast, thyroid and renal cancers. Limited recommendations were developed for other sites including endometrial, colon and skin. The proposed cancer surveillance recommendations for PHTS require a coordinated multidisciplinary approach and significant patient commitment. The evidence base for cancer surveillance in this guideline are limited, emphasising the need for prospective evaluation of the effectiveness of surveillance in the PHTS population.

Nitrogen-fixing root nodule symbiosis occurs in ten taxonomic lineages from four related orders -Fagales, Fabales, Rosales and Cucurbitales- that together are called the nitrogen-fixing clade (NFC). Nodulating plants within the NFC are scattered by non-nodulating species, as well as can interact either with rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. Despite a significant body of knowledge of the legume-rhizobium symbiosis, it remains elusive which signalling modules are shared between nodulating species in different taxonomic clades. Besides, it is generally assumed that nodulation evolved independently multiple times, though molecular genetic support for this hypothesis is lacking.Parasponia is the only non-legume plant which can establish nitrogen-fixing endosymbiosis with rhizobium, and it is the only nodulating plant within the Cannabaceae. The Parasponia lineage represents five species and phylogenetic analysis shows that this lineage is embedded within the non-nodulating Trema clade. As Parasponia and Trema are closely related, F1 hybrids could be created by crossing of the diploid Parasponia andersonii (2n=20) and the allotetraploid Trema tomentosa (2n=4X=40). Conceptually, P. andersonii x T. tomentosa F1 hybrid plants reflects a diploid T. tomentosa with a haploid genome of  P. andersonii introduced. The F1 hybrid between diploid Parasponia andersonii and tetraploid Trema tomentosa can form nodules, whereas it is devoid of intracellular infection when inoculated with either Mesorhizobium plurifarium BOR2 or Bradyrhizobium elkanii WUR3. Based on its genetic composition and symbiotic phenotype, we argue that the F1 hybrid may mimic future engineer results. Therefore, we aimed to obtain a better understanding of the deviation in nodulation phenotype of wild type P. andersonii and F1 hybrid plants. To do so, we compared nodulation efficiencies and

Nitrogen-fixing symbioses provide a beneficial platform for plants to sustain the growth and development under the nitrogen-limiting environment. The nitrogen-fixing symbioses occur exclusively in a subset of genera of 10 taxonomic families. These nodulating plant genera form a paraphylitic clade in the orders Fabales, Fagales, Cucurbitales and Fagales, which collectively are known as ‘Nitrogen Fixing Clade’. Nodulating species of eight families establish the symbiotic interaction with the filamentous bacteria Frankia, and therefore are classified as actinorhizal plants. Nodulating species of two other families interact with the nitrogen-fixing rhizobium; namely legumes (Fabaceae, Fabales) and Parasponia (Cannabaceae, Rosales). Both nitrogen-fixing microbes induce the formation of a niche organ, so-called root nodules. Despite varieties in nodule morphology across nodulating species, the root nodules serve a general purpose to accommodate the bacteria intracellularly and to provide a suitable physiological environment for the bacteria to convert the atmospheric nitrogen (N2) into ammonium. A major aim in nitrogen-fixing nodulation research is to unravel the signalling pathways required for a successful mutualism. Recently, phylogenomic comparisons of nodulating and non-nodulating species postulated that the nodulation trait likely originated from a common ancestor of the Nitrogen-Fixing Clade. This implies that the nodulation trait encountered massive parallel loss during the divergence of this clade. Considering the contribution of nitrogen-fixing symbioses to sustainable plant growth under low nitrogen input farming, it leads to a question whether it is possible to extend the host range of nitrogen-fixing symbionts to non-nodulating crops (such as wheat, maize, or rice). To this end, it is important to identify the key genetic adaptations underlying these successful symbioses. To date, a massive body of knowledge on nodulation has been generated in molecular genet

Nitrogen-fixing root nodule symbiosis can sustain the development of the host plants under nitrogen limiting conditions. Such symbiosis only occurs in a clade of angiosperms known as the nitrogen-fixing clade (NFC). It has long been proposed that root nodule symbiosis evolved several times (in parallel) in the NFC. There are two main arguments supporting this parallel hypothesis. First, the occurrence of root nodule symbiosis is scattered across the NFC, hypothesizing a single origin is evolutionarily unparsimonious as this would require massive loss of the trait. Second, legume-type and actinorhizal-type nodules are fundamentally different. Two recent phylogenomic studies compared the genomes of nodulating and related non-nodulating species across the four orders of the NFC and found that genes essential for nodule formation are lost or pseudogenized in the non-nodulating species. As these symbiosis genes are specifically involved in the symbiotic interaction, it means that the presence of pseudogenes and the loss of symbiosis genes strongly suggest that their ancestor that still had functional genes most likely formed the nodule symbiosis. Therefore, the first argument supporting the parallel hypothesis is disproved. Actinorhizal-type nodules have long been regarded as modified lateral roots, originating from root pericycle cells, with newly formed cortical cells being infected by bacteria. Here, we compared the lateral root and nodule development in two plant species forming actinorhizal-type nodules (Parasponia andersonii (Parasponia) and Alnus glutinosa (Alnus)). We showed that in Parasponia and Alnus, lateral roots are mainly originated from pericycle-derived cells, endodermis cells only form the outmost layer of the lateral root cap. During the formation of Parasponia and Alnus nodules, pericycle-derived cells only form the nodule vasculature, instead of a lateral root (as was previously reported for these species). In both cases, parental root cortical cells

Most land plants (>80%) rely on the symbiosis with arbuscular mycorrhizal (AM) fungi belonging to the Glomeromycotina subphylum to obtain sufficient nutrients from the soil. How AM fungi manage to colonize and cooperate with such an extremely broad range of plants is a key question in AM biology. AM fungi must have evolved efficient ways to deal with the plant immune system and be able to adapt to their different host environments to ensure a beneficial interaction. However, the underlying mechanisms are still largely unknown. In this thesis, I studied cell-type transcriptome of both the fungus and the plant at different stages of the interaction and the fungal responses in diverse host plants, with a focus on effector biology and reciprocal nutrient transfer. My results suggests that AM fungi can adjust their transcriptome and secretome to colonize evolutionary distantly related hosts, namely Medicago truncatula, Nicotiana benthamiana and Allium schoenoprasum. By applying laser microdissection coupled with RNA sequencing in Medicago, I dissected the expression pattern of AM genes particularly AM effectors into different stages, including arbuscules, intraradical mycelium, extraradical mycelium and germinating spores. These approaches collectively resulted in an expression atlas of the fungal transcriptome and secretome and allowed me to pinpoint potential key genes in AM symbiosis. In addition, I performed detailed functional analyses on one of the highest intraradically expressed effector, encoding a LysM domain-containing effector (RiSLM). This showed that RiSLM can bind chitin (oligomers), protect fungal cell walls from plant chitinases and suppress chitin-triggered immune responses, therefore play a positive role in the symbiosis. These results revealed a conserved role for LysM effectors in both pathogenic as well as symbiotic fungal interactions with plants. Although sugars have long been proposed to be transferred from the host to AM fungi, the mechanisms by

We selected the legume Indigofera argentea for our research. It was collected in the desert of Jizan in Saudi Arabia. We selected this species for two reasons. It is well adapted to heat and drought and therefore it has the potential to study, in the future, mechanisms that confer tolerance to these abiotic stresses. Further, Indigofera represents an early branching lineage within the indigoferoid/milletioid clade. Therefore, Indigofera is a key genus in studying the evolution of nodulation within the Papilionoideae subfamily. In Chapter 1, a general introduction is given on nitrogen fixing symbiosis of legumes and rhizobia. In this introduction, we focus on the process of nodule initiation and organogenesis. Two main nodule types, determinate and indeterminate nodule, are introduced based on the knowledge of the few well-studied legumes species. Further, terminal differentiation of rhizobia that is induced by NCR peptides of the host is introduced. In Chapter 2, we characterized the desert legume I. argentea and developed a platform by which future studies on mechanisms controlling abiotic stress become available. We developed an Agrobacterium rhizogenes-mediated root transformation procedure and did a de novo transcriptome assembly using RNA of various organs. In Chapter 3, about 60 rhizobium strains have been isolated from nodulated I. argentea plants that were collected in the desert. The strains were characterized by 16S sequencing and their nodulation abilities were studied. One of the efficient nodulating Bradyrhizobium strains was selected for further studies. In Chapter 4, nodule development of Indigofera and Tephrosia species was analysed and described in detail. Species from both genera have an indeterminate growth. However, it was shown that this is not due to a meristem that is formed at the primordium stage, which is the basis of indeterminate growth of IRLC species like Medicago. The indeterminate growth was shown to be due to secondary clusters of di

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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

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 signal

Legumes don’t need fertiliser, because they can get their nitrogen from the air instead of the soil. They do this with the help of rhizobium bacteria, but how does this work? Ton Bisseling will tell you the story of how legumes can tame bacteria in order to get nature’s nitrogen.

Background and Aims: The aim of this study was to determine the yield of endoscopic screening in first-degree relatives (FDRs) of CDH1-negative hereditary diffuse-type gastric cancer (HDGC) patients. Methods: In this retrospective observational cohort study, in 2 expert centers in the Netherlands data were collected on FDRs from families fulfilling the international HDGC criteria that underwent endoscopic screening. Extensive inspection of the stomach was performed by gastroscopy, taking random and/or targeted stomach biopsy specimens to identify diffuse-type gastric cancer. Results: Between 2004 and 2016, 90 persons (40% men; mean age, 48 years) from 40 families were offered endoscopic screening. The mean number of endoscopies per person was 3. The mean follow-up time was 46 months and mean endoscopic interval 20 months. Signet ring cell carcinoma foci restricted to the mucosa (pT1a) were identified in 4 persons (4%) from 1 family, which afterward was diagnosed with a germline CTNNA1 mutation. Advanced poorly cohesive gastric carcinoma was diagnosed in 1 person from another family. Intestinal metaplasia was diagnosed in 38 persons (42%) and low-grade dysplasia in 4 persons (4%). Additionally, in 40 persons (44%) scar tissue was observed in the gastric mucosa, which can hinder the endoscopic detection of small white lesions typical for HDGC. Conclusions: Endoscopic screening in HDGC families without a pathogenic CDH1 mutation may be reasonable, as we detected signet ring cell carcinomas in 6% of persons screened. However, the criteria and frequency of screening may have to be reconsidered.

Background: Second primary malignancies are a major cause of excess morbidity and mortality in cancer survivors. Hodgkin lymphoma survivors who were treated with infradiaphragmatic radiotherapy and/or high-dose procarbazine have an increased risk to develop colorectal cancer. Colonoscopy surveillance plays an important role in colorectal cancer prevention by removal of the precursor lesions (adenomas) and early detection of cancer, resulting in improved survival rates. Therefore, Hodgkin lymphoma survivors treated with infradiaphragmatic radiotherapy and/or high-dose procarbazine could benefit from colonoscopy, or other surveillance modalities, which are expected to reduce colorectal cancer incidence and mortality. Current knowledge on clinicopathological and molecular characteristics of therapy-related colorectal cancer is limited. The pathogenesis of such colorectal cancers might be different from the pathogenesis in the general population and therefore these patients might require a different clinical approach. We designed a study with the primary aim to assess the diagnostic yield of a first surveillance colonoscopy among Hodgkin lymphoma survivors at increased risk of colorectal cancer and to compare these results with different screening modalities in the general population. Secondary aims include assessment of the test characteristics of stool tests and evaluation of burden, acceptance and satisfaction of CRC surveillance through two questionnaires. Methods/Design: This prospective multicenter cohort study will include Hodgkin lymphoma survivors who survived =8years after treatment with infradiaphragmatic radiotherapy and/or procarbazine (planned inclusion of 259 participants). Study procedures will consist of a surveillance colonoscopy with removal of precursor lesions (adenomas) and 6-8 normal colonic tissue biopsies, a fecal immunochemical test and a stool DNA test. All neoplastic lesions encountered will be classified using relevant histomorphological, im

Control of plant growth is an important aspect of crop productivity and yield in agriculture. Overexpression of the AtCHR12/23 genes in Arabidopsis thaliana reduced growth habit without other morphological changes. These two genes encode Snf2 chromatin remodelling ATPases. Here, we translate this approach to the horticultural crop tomato (Solanum lycopersicum). We identified and cloned the single tomato ortholog of the two Arabidopsis Snf2 genes, designated SlCHR1. Transgenic tomato plants (cv. Micro-Tom) that constitutively overexpress the coding sequence of SlCHR1 show reduced growth in all developmental stages of tomato. This confirms that SlCHR1 combines the functions of both Arabidopsis genes in tomato. Compared to the wild type, the transgenic seedlings of tomato have significantly shorter roots, hypocotyls and reduced cotyledon size. Transgenic plants have a much more compact growth habit with markedly reduced plant height, severely compacted reproductive structures with smaller flowers and smaller fruits. The results indicate that either GMO-based or non-GMO-based approaches to modulate the expression of chromatin remodelling ATPase genes could develop into methods to control plant growth, for example to replace the use of chemical growth retardants. This approach is likely to be applicable and attractive for any crop for which growth habit reduction has added value.

Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides (LCOs). Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation. Several studies have implicated a function for cytokinin in this process. However, whether cytokinin accumulation and subsequent signaling are an integral part of rhizobium LCO signaling remains elusive. Here, we show that cytokinin signaling is required for the majority of transcriptional changes induced by rhizobium LCOs. In addition, we demonstrate that several cytokinins accumulate in the root susceptible zone 3 h after rhizobium LCO application, including the biologically most active cytokinins, trans-zeatin and isopentenyl adenine. These responses are dependent on calcium- and calmodulin-dependent protein kinase (CCaMK), a key protein in rhizobial LCO-induced signaling. Analysis of the ethylene-insensitive Mtein2/Mtsickle mutant showed that LCO-induced cytokinin accumulation is negatively regulated by ethylene. Together with transcriptional induction of ethylene biosynthesis genes, it suggests a feedback loop negatively regulating LCO signaling and subsequent cytokinin accumulation. We argue that cytokinin accumulation is a key step in the pathway leading to nodule organogenesis and that this is tightly controlled by feedback loops.

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Legume rhizobium symbiosis is initiated upon perception of bacterial secreted lipo-chitooligosaccharides (LCOs). Perception of these signals by the plant initiates a signaling cascade that leads to nodule formation. Several studies have implicated a function for cytokinin in this process. However, whether cytokinin accumulation and subsequent signaling are an integral part of rhizobium LCO signaling remains elusive. Here, we show that cytokinin signaling is required for the majority of transcriptional changes induced by rhizobium LCOs. In addition, we demonstrate that several cytokinins accumulate in the root susceptible zone 3 h after rhizobium LCO application, including the biologically most active cytokinins, trans-zeatin and isopentenyl adenine. These responses are dependent on calcium- and calmodulin-dependent protein kinase (CCaMK), a key protein in rhizobial LCO-induced signaling. Analysis of the ethylene-insensitive Mtein2/Mtsickle mutant showed that LCO-induced cytokinin accumulation is negatively regulated by ethylene. Together with transcriptional induction of ethylene biosynthesis genes, it suggests a feedback loop negatively regulating LCO signaling and subsequent cytokinin accumulation. We argue that cytokinin accumulation is a key step in the pathway leading to nodule organogenesis and that this is tightly controlled by feedback loops.

Nodules on the roots of legume plants host nitrogen-fixing Rhizobium bacteria. Several lines of evidence indicate that nodules are evolutionarily related to roots. We determined whether developmental control of the Medicago truncatula nodule meristem bears resemblance to that in root meristems through analyses of root meristem-expressed PLETHORA genes. In nodules, MtPLETHORA 1 and 2 are preferentially expressed in cells positioned at the periphery of the meristem abutting nodule vascular bundles. Their expression overlaps with an auxin response maximum and MtWOX5, which is a marker for the root quiescent center. Strikingly, the cells in the central part of the nodule meristem have a high level of cytokinin and display MtPLETHORA 3 and 4 gene expression. Nodule-specific knockdown of MtPLETHORA genes results in a reduced number of nodules and/or in nodules in which meristem activity has ceased. Our nodule gene expression map indicates that the nodule meristem is composed of two distinct domains in which different MtPLETHORA gene subsets are expressed. Our mutant studies show that MtPLETHORA genes function redundantly in nodule meristem maintenance. This indicates that Rhizobium has recruited root developmental programs for nodule formation.

The nitrogen-fixing rhizobia in the symbiotic infected cells of root nodules are kept in membrane compartments derived from the host cell plasma membrane, forming what are known as symbiosomes. These are maintained as individual units, with mature symbiosomes having a specific radial position in the host cell cytoplasm. The mechanisms that adapt the host cell architecture to accommodate intracellular bacteria are not clear. The intracellular organization of any cell depends heavily on the actin cytoskeleton. Dynamic rearrangement of the actin cytoskeleton is crucial for cytoplasm organization and intracellular trafficking of vesicles and organelles. A key component of the actin cytoskeleton rearrangement is the ARP2/3 complex, which nucleates new actin filaments and forms branched actin networks. To clarify the role of the ARP2/3 complex in the development of infected cells and symbiosomes, we analyzed the pattern of actin microfilaments and the functional role of ARP3 in Medicago truncatula root nodules. In infected cells, ARP3 protein and actin were spatially associated with maturing symbiosomes. Partial ARP3 silencing causes defects in symbiosome development; in particular, ARP3 silencing disrupts the final differentiation steps in functional maturation into nitrogen-fixing units.

Interaction of key regulators of exocytosis with potassium channels enhances both secretion and K+ uptake, making these processes intertwined and jointly coordinated.

Background Strigolactones are a class of plant hormones whose biosynthesis is activated in response to phosphate starvation. This involves several enzymes, including the carotenoid cleavage dioxygenases 7 (CCD7) and CCD8 and the carotenoid isomerase DWARF27 (D27). D27 expression is known to be responsive to phosphate starvation. In Medicago truncatula and rice (Oryza sativa) this transcriptional response requires the GRAS-type proteins NSP1 and NSP2; both proteins are essential for rhizobium induced root nodule formation in legumes. In line with this, we questioned whether MtNSP1-MtNSP2 dependent MtD27 regulation is co-opted in rhizobium symbiosis. Results We provide evidence that MtD27 is involved in strigolactone biosynthesis in M. truncatula roots upon phosphate stress. Spatiotemporal expression studies revealed that this gene is also highly expressed in nodule primordia and subsequently becomes restricted to the meristem and distal infection zone of a mature nodules. A similar expression pattern was found for MtCCD7 and MtCCD8. Rhizobium lipo-chitooligosaccharide (LCO) application experiments revealed that of these genes MtD27 is most responsive in an MtNSP1 and MtNSP2 dependent manner. Symbiotic expression of MtD27 requires components of the symbiosis signaling pathway; including MtDMI1, MtDMI2, MtDMI3/MtCCaMK and in part MtERN1. This in contrast to MtD27 expression upon phosphate starvation, which only requires MtNSP1 and MtNSP2. Conclusion Our data show that the phosphate-starvation responsive strigolactone biosynthesis gene MtD27 is also rapidly induced by rhizobium LCO signals in an MtNSP1 and MtNSP2-dependent manner. Additionally, we show that MtD27 is co-expressed with MtCCD7 and MtCCD8 in nodule primordia and in the infection zone of mature nodules.