Dissecting hormonal pathways in nitrogen-fixing rhizobium symbioses
Zeijl, Arjan van - \ 2017
Wageningen University. Promotor(en): T. Bisseling, co-promotor(en): R. Geurts. - Wageningen : Wageningen University - ISBN 9789463436311 - 231
plants - root nodules - rhizobium - symbiosis - cytokinins - plant-microbe interactions - biosynthesis - mutagenesis - genes - nodulation - planten - wortelknolletjes - rhizobium - 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.
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
parasponia - rhizobium - root nodules - rhizobium rhizogenes - temperature - nitrates - symbiosis - genetics - parasponia - rhizobium - wortelknolletjes - rhizobium rhizogenes - 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.
Stikstofbinding voor kleine boeren in Afrika
Giller, K.E. - \ 2015
Vork 2 (2015)3. - ISSN 2352-2925 - p. 16 - 21.
tuinbouw - kleine landbouwbedrijven - afrika - stikstofbindende bacteriën - rhizobium - bodemvruchtbaarheid - inkomen van landbouwers - agrarische bedrijfsvoering - peulgewassen - sojabonen - voedselproductie - projecten - teeltsystemen - horticulture - small farms - africa - nitrogen fixing bacteria - rhizobium - soil fertility - farmers' income - farm management - legumes - soyabeans - food production - projects - cropping systems
Het project N2Africa is onlangs de tweede fase ingegaan met als doel dat in 2020 een half miljoen kleine boeren in Afrika, ten zuiden van de Sahara, stikstofbinding hebben geïntegreerd in hun bedrijfsvoering. Op een manier die hen past, zegt Ken Giller. Stikstofbinding verbetert de bodemvruchtbaarheid, terwijl de teelt van bonen, die samen met bacteriën de stikstof vastleggen, een belangrijke aanvulling vormt op het menu en op het inkomen van de boer.
Intracellular accommodation of rhizobia in legume host cell: the fine-tuning of the endomembrane system
Gavrin, A.Y. - \ 2015
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): E. Federova. - Wageningen : Wageningen University - ISBN 9789462574182 - 160
peulgewassen - rhizobium - bodembacteriën - endosymbiose - wortelknolletjes - membranen - waardplanten - legumes - rhizobium - soil bacteria - endosymbiosis - root nodules - membranes - host plants
The symbiosis of legumes with rhizobia leads to the formation of root nodules. Rhizobia which are hosted inside specialized infected cells are surrounded by hostderived membranes, forming symbiosomes. Although it is known that symbiosome formation involves proliferation of membranes and changing of host cell architecture the mechanisms involved in these processes remain largely uncovered.
In this thesis, I studied in more detail the adaptation of the endomembrane system of infected cells to intracellular rhizobia. I have shown that in the first cell layer of the nitrogen-fixing zone, the vacuole of the infected cells shrinks, creating space for the expanding symbiosomes. Here the expression of homotypic fusion and vacuole protein sorting complex (HOPS) genes VPS11 and VPS39 are switched off, whereas tonoplast proteins, like the vacuolar aquaporin TIP1g, are targeted to the symbiosome membrane. These observations suggest that tonoplast-targeted traffic in infected cells is altered. This retargeting is essential for the maturation of symbiosomes.
Accommodation of intracellular rhizobia requires also the reorganization of the actin cytoskeleton. I have shown that during symbiosome development the symbiosomes become surrounded by a dense actin network and in this way, the actin configuration in infected cells is changed markedly. The actin nucleating factor ARP3 is operational in the rearrangement of actin around the symbiosome.
It is known that the plasma membrane is inelastic; its capacity to stretch is only around 1-3%. Exocytosis of new membrane material is therefore involved in changes in the size of the membrane surface and in repair of damaged membrane loci. Membrane tension may create a vector for the fusion of membrane vesicles. To test this, the localization of proteins from the group of synaptotamin calcium sensors involved in membrane fusion, was studied. I have shown that the Medicago synaptotamins, MtSyt2 and MtSyt3, are localised on protrusions of the host plasma membrane created by expanding rhizobia (infection threads, cell wall-free unwalled droplets). Hence, at these sites of contact between symbionts membrane tension may create a vector for exocytosis.
It is known that the host cell wall is modified during the development of infected cells. This process is mediated by the exocytotic pathway employing vesicle-associated membrane proteins (VAMPs) from the VAMP721 family. Previously it was shown in Medicago nodules, that cell-wall free interface membrane formation during bacterial release is dependent on these proteins. I have shown that the pectin modifying enzyme pectate lyase is delivered to the site of bacterial release in soybean nodules by VAMP721-positive vesicles.
My study uncovered new mechanisms involved in the adaptation of host cells to intracellular rhizobia: defunctionalization of the vacuole, actin cytoskeleton rearrangement and the retargeting of host cell proteins to the interface membrane.
Optimalisatie N-bemesting soja
Timmer, R.D. ; Visser, C.L.M. de - \ 2014
Lelystad : PPO AGV - 15
akkerbouw - sojabonen - peulvruchten - veevoeding - bemesting - stikstofmeststoffen - opbrengst - duurzaamheid (sustainability) - rhizobium - arable farming - soyabeans - grain legumes - livestock feeding - fertilizer application - nitrogen fertilizers - outturn - sustainability - rhizobium
Soja is weliswaar een vlinderbloemig gewas dat (via de vorming van stikstofknolletjes aan de wortels) in z’n eigen stikstofbehoefte kan voorzien, de ervaring leert echter dat het gewas in Nederland vaak een erg lichte bladkleur heeft gedurende het seizoen wat kan duiden op N-gebrek. Voor de vorming van de stikstofknolletjes aan de wortels is een goed geslaagde enting nodig met Rhizobium bacteriën. Over hoeveel knolletjes er per plant nodig zijn om in de N-behoefte te kunnen voorzien bestaat nog grote onduidelijkheid. Ook is niet duidelijk of een N-bemesting bij soja kan leiden tot hogere en stabielere opbrengsten.
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 - medicago truncatula - root nodules - nodules - rhizobium - 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.
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 - oryza - medicago - nodulation - symbiosis - genes - rhizobium - root nodules - nitrogen fixation
Efficiency of Agrobacterium rhizogenes-mediated root transformation of Parasponia and Trema is temperature dependent
Cao, Q. ; Camp, R. Op den; Seifi Kalhor, M. ; Bisseling, T. ; Geurts, R. - \ 2012
Plant Growth Regulation 68 (2012)3. - ISSN 0167-6903 - p. 459 - 465.
medicago-truncatula - gene-transfer - non-legume - plants - nitrogen - andersonii - nodulation - rhizobium - phaseolus - sequences
Parasponia trees are the only non-legume species that form nitrogen-fixing root nodules with rhizobium. Based on its taxonomic position in relation to legumes (Fabaceae), it is most likely that both lineages have gained this symbiotic capacity independently. Therefore, Parasponia forms a bridging species to understand the evolutionary constraints underlying this symbiosis. However, absence of key technologies to genetically modify Parasponia seriously impeded studies on these species. We employed Agrobacterium rhizogenes to create composite Parasponia andersonii plants that harbour transgenic roots. Here, we provide an optimized protocol to infect P. andersonii as well as its non-symbiotic sister species Trema tomentosa with A. rhizogenes. We show that the transformation efficiency is temperature dependent. Whereas the optimal growth temperature for both these species is 28 °C, the transformation is most efficient when co-cultivation with A. rhizogenes occurs at 21 °C. Using this optimized protocol up to 80 % transformation efficiency can be obtained. These robust transformation platforms will provide a strong tool to unravel the Parasponia–rhizobium symbiosis
Genetic analysis of symbiosome formation
Ovchinnikova, E. - \ 2012
Wageningen University. Promotor(en): Ton Bisseling; I.A. Tikhonovich, co-promotor(en): Erik Limpens. - S.l. : s.n. - ISBN 9789461733610 - 147
rhizobium - fabaceae - endosymbiose - wortelknolletjes - genregulatie - moleculaire genetica - moleculaire biologie - plant-microbe interacties - rhizobium - fabaceae - endosymbiosis - root nodules - gene regulation - molecular genetics - molecular biology - plant-microbe interactions
Endosymbiotic interactions form a fundament of life as we know it and are characterized by the formation of new specialized membrane compartments, in which the microbes are hosted inside living plant cells. A striking example is the symbiosis between legumes and nitrogen-fixing Rhizobium bacteria (rhizobia), which represents the most important source of biologically fixed nitrogen. The accommodation of rhizobia as novel nitrogen-fixing organelles, called symbiosomes, inside the cells of a novel organ, the root nodule, forms the heart of this ecologically and agriculturally important symbiosis. Understanding how these organelles are made will be keystone to exploit this symbiosis for sustainable agriculture in the future. In this thesis, we undertook a genetic approach to identity key components that control symbiosome formation especially in the genetically well-characterized garden pea (Pisum sativum) system. At the start of this thesis, the most extensive and morphologically best-characterized collection of mutants impaired in symbiosome formation was, and currently still is, available in pea. However, the cloning of the corresponding genes is severely hampered by its large genome size and recalcitrance to genetic transformation. Therefore, we used the model legume Medicago truncatula (Medicago) as reference genome to clone pea symbiosome mutants via a synteny-based cloning approach. We focused especially on three mutants in pea, named sym33, sym41 and sym31, which are affected most early in symbiosome formation: namely blocked in the release of bacteria from cell wall bound infection threads inside root nodule cells (sym33 and sym41) or induction of the subsequent differentiation of the symbiosomes (sym31).
The formation of endosymbiotic membrane compartments: membrane identity markers and the regulation of vesicle trafficking
Ivanov, S. - \ 2012
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Elena Fedorova; Erik Limpens. - S.l. : s.n. - ISBN 9789461733436 - 121
planten - rhizobium - stikstof - stikstoffixatie - medicago - endosymbiose - celmembranen - blaasjes - biochemische omzettingen - moleculaire biologie - wortels - mycorrhizae - plants - rhizobium - nitrogen - nitrogen fixation - medicago - endosymbiosis - cell membranes - vesicles - biochemical pathways - molecular biology - roots - mycorrhizas
In symbiosis of plants and arbuscular mycorrhizal fungi as well as in rhizobium-legume symbiosis the microbes are hosted intracellularly, inside specialized membrane compartments of the host. These membrane compartments are morphologically different but similar in function, since they control the exchange of compounds between host and its microsymbiont thus forming a highly specialized symbiotic interface. These are the arbuscules, containing highly branched fungal hyphae, and organelle-like symbiosomes containing rhizobium bacteria. Recent studies have markedly extended our insight in the evolution of the signaling mechanism underlying the formation of these symbiotic interfaces. These studies strongly suggest that rhizobium co-opted the complete signaling mechanism (including lipo-oligosaccharides signal molecules) from the more ancient AM fungi symbiosis. Further, in plant species (Parasponia) where rhizobium nodulation evolved rather recent and independent from legumes, the same lipo-oligosaccharide receptor is essential for the formation of the rhizobium symbiotic interface as well as arbuscules. Therefore it seems likely that rhizobium also co-opted the cellular mechanism controlling arbuscule formation to form a rhizobium symbiotic interface. This would imply that even after co-evolution in legumes the key regulators involved in the formation of these interfaces are similar or even identical.
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 - rhizobium - fabaceae - symbiosis - evolution - nitrogen fixation - roots - phylogenetics - genomes - medicago - 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
Evolution of rhizobium symbiosis
Camp, R.H.M. Op den - \ 2012
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - S.l. : s.n. - ISBN 9789461731982 - 136
papilionoideae - rhizobium - symbiose - genomen - parasponia - mycorrhizae - evolutie - moleculaire biologie - papilionoideae - rhizobium - symbiosis - genomes - parasponia - mycorrhizas - evolution - molecular biology
The evolution of rhizobium symbiosis is studied from several points of view in this thesis. The ultimate goal of the combined approaches is to unravel the genetic constrains of the symbiotic interaction. To this end the legume rhizobium symbiosis is studied in model plant species from the Papilionoideae subfamily such as Medicago truncatula and Lotus japonicus. In these model plants the genetic signaling cascade used for rhizobium symbiosis has been largely unraveled. The cascade is triggered by lipo-chitooligosaccharade-based signal molecules excreted by rhizobia, called Nod factors.
In chapter 2 we make use of a whole genome duplication that has occurred at the root of the legume Papilionoideae subfamily to identify maintained paralogous gene pairs. We hypothesized that a substantial fraction of gene pairs which are maintained in distinct Papilionoideae lineages that split roughly 54 million years ago fulfill legume specific functions, among which is rhizobium symbiosis. Furthermore we argue that such approach could identify novel genes as it can also identify genes pairs that are (partially) redundant in function. With applying this approach specifically to the cytokinin phosphorelay pathway we identified a pair of type-A cytokinin Response Regulators that are involved in rhizobium symbiosis. This study provides a proof-of-principle for this strategy.
It is known for over fifty years that cytokinin plays an important role in the symbiotic interaction between rhizobia and legume hosts. External application of cytokinin can even result in nodule formation. Only, never had cytokinin levels been quantified in legume root extracts upon symbiotic interaction. In chapter 3 we describe a method for extraction of both cytokinins and auxin from Medicago truncatula roots. We show that cytokinins accumulate in the root zone susceptible to symbiotic interaction upon Nod factor exposure and that this response is dependent on CCaMK; a key gene of the Nod factor signaling cascade. Furthermore, it was found that ethylene signaling has a negative effect on Nod factor induced cytokinin accumulation. The method set up to measure cytokinin as well as auxin provides a tool to further study hormone interactions in rhizobium symbiosis.
Parasponia,the only non-legume that can engage the rhizobium symbiosis is also subject of study in this thesis. The genetics of the Parasponia-rhizobium symbiosis had not been studied before. It was therefore unknown whether this independently evolved rhizobium symbiosis makes use of the same symbiotic signaling cascade as legumes. In chapter 4 we provide first evidence that Parasponia indeed makes use of the same signaling cascade as found in legumes. Furthermore, we show that in Parasponia a single Nod factor-like receptor is indispensable for two symbiotic interactions; rhizobium and mycorrhiza, respectively. Therefore we conclude that the rhizobium Nod factor perception mechanism is recruited from the widespread endomycorrhizal symbiosis.
Parallel to our studies in Parasponia (Chapter 4), the research team of Jean Dénarié of the French National Institute for Agricultural Research (INRA) published the structure of the signal molecule of the arbuscular endomycorrhizae; theMyc factor (Maillet et al., 2011, Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature, 469, 58-63). It appeared that Myc factors and Nod factors are structurally very similar. In chapter 5 we discuss these findings and present a more thorough phylogenetic analysis of the NFP-like LysM-type receptor kinases. Together, these results suggest that non-legumes that can engage an arbuscular endomycorrhizaesymbiosis can recognize Nod factor-like molecules as well.
The last chapter is about a study on the promiscuity and effectiveness of the Parasponia-rhizobium symbiosis. Parasponia uses a single receptor to control entry of rhizobium as well as arbuscular endomycorrhizal fungi and has evolved the rhizobium symbiosis only recently. This made us to hypothesize that Parasponia Nod factor receptors did not coevolve yet with rhizobia and therefore did not diverge from mycorrhizal recognition to develop specificity for the Nod factor. This implies that Parasponia could be a very promiscuous host for rhizobium species. In chapter 6 we describe that Parasponia andersonii can be nodulated by a broad range of rhizobia belonging to 4 different genera, and therefore it is concluded that Parasponia is highly promiscuous for rhizobial engagement. There is a drawback to this high symbiotic promiscuity. Among the strains identified to nodulate Parasponia, a very inefficient rhizobium species, Rhizobium tropici WUR1, was characterized. As this species is able to make effective nodules on two different legume species it suggests that the ineffectiveness of Parasponia andersonii nodules is the result of the incompatibility between both partners. In Parasponia andersonii nodules rhizobia of the ineffective strain become embedded in a dense matrix, but remain vital. This suggests that sanctions or genetic control against underperforming microsymbionts may not be effective in Parasponia. Therefore we argue that the Parasponia-Rhizobium symbiosis is a delicate balance between mutual benefits and parasitic colonization.
Parasponiahas been given little attention in the rhizobium symbiosis field over the past two decades but with our efforts renewed interest has been established. We believe that in the end, the comparison of Parasponia to its closest related non-symbiotic sister species Trema, will result in the determination of the genetic constrains of rhizobium symbiosis.
De kracht van de stikstofbinders
Giller, K.E. ; Bisseling, T. - \ 2012
WageningenWorld 2012 (2012)1. - ISSN 2210-7908 - p. 30 - 37.
peulgewassen - stikstoffixatie - wortelknolletjes - stikstofbindende bacteriën - rhizobium - veldgewassen - afrika - legumes - nitrogen fixation - root nodules - nitrogen fixing bacteria - rhizobium - field crops - africa
Hoogleraar Ken Giller propageert onder Afrikaanse boeren het gebruik van peulvruchten. Die hebben dankzij hulp van bacteriën geen stikstofmeststof nodig. In Wageningen onderzoekt hoogleraar Ton Bisseling de finesses van deze symbiose.
Translational genomics from model species Medicago truncatula to crop legume Trifolium pratense
Lang Chunting, Chunting - \ 2012
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - [S.l.] : s.n. - ISBN 9789461731975 - 147
trifolium pratense - medicago truncatula - genomica - cytogenetica - rhizobium - symbiose - genetische kartering - chromosomen - retrotransposons - trifolium pratense - medicago truncatula - genomics - cytogenetics - rhizobium - symbiosis - genetic mapping - chromosomes - retrotransposons
The legume Trifolium pratense (red clover) is an important fodder crop and produces important secondary metabolites. This makes red clover an interesting species. In this thesis, the red clover genome is compared to the legume model species Medicago truncatula, of which the genome sequence is presented. We describe the red clover genome structure and compare it to the Medicago sequence. Thus is shown that although red clover and Medicago are closely related species, their genomes have diverged widely. Further analysis shows that much of the divergence is unique to red clover, not occurring in other clover species. By zooming in on a single rearrangement, a transposable element is found that occurs within the breakpoint region and is widely distributed in the red clover genome, but not in related clover species. Therefore we predict that this transposable element has been involved in the red clover genome rearrangement.
Agrobacterium-mediated transformation of Mycosphaerella fijiensis, the devastating Black Sigatoka pathogen of bananas
Díaz-Trujillo, C. ; Adibon, H. ; Kobayashi, K. ; Zwiers, L.H. ; Souza, M.T. ; Kema, G.H.J. - \ 2010
Gewasbescherming 41 (2010)3. - ISSN 0166-6495 - p. 151 - 151.
mycosphaerella fijiensis - fungiciden - bananen - genotypen - fenotypen - rhizobium - genetische transformatie - genoomanalyse - mycosphaerella fijiensis - fungicides - bananas - genotypes - phenotypes - rhizobium - genetic transformation - genome analysis
Mycosphaerella fijiensis, M. musicola en M. eumusae veroorzaken de Sigatoka-ziekte in banaan. Op dit moment is de toepassing van fungiciden de enige optie om deze ziekte te bestrijden. Het PRPB (Pesticide Reduction Program for Banana) investeert in de ontwikkeling van technieken voor de genotype- en fenotypebepaling van M. fijiensis. Hierbij wordt gebruikt gemaakt van ATMT (Agrobacterium tumefaciens-mediated transformation).
Genetic analysis of breeding-related traits in Brassica rapa
Bagheri, H. - \ 2009
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - [S.l. : S.n. - ISBN 9789085855491 - 130
brassica campestris - brassica - genetische analyse - loci voor kwantitatief kenmerk - kwantitatieve kenmerken - kenmerken - rhizobium - zaadkenmerken - zaden - genetische transformatie - plantenveredeling - brassica campestris - brassica - genetic analysis - quantitative trait loci - quantitative traits - traits - rhizobium - seed characteristics - seeds - genetic transformation - plant breeding
Brassica rapa is an important crop with a variety of forms, and a wide distribution in the world. It is used as oil seed and vegetable crop and a valuable source of diverse health-promoting metabolites. It also can serve as a model for genetic and molecular analysis in the Brassica genus, to which all rapes, kales and cabbages belong, as it has the smallest genome size and some genotypes with a rapid life cycle.
Insertional mutagenesis using heterologous maize transposons has been a valuable tool for the identification and isolation of genes in Arabidopsis. Transposon-based activation tagging systems use a construct with constitutive enhancer elements that can cause transcriptional activation of flanking plant genes, which can result in dominant mutant phenotypes and subsequent isolation of the genes involved. Chapter 2 describes the action of an En/I activation tagging construct in B. rapa through Agrobacterium rhizogenes–mediated hairy root transformation. Successful transformation of this construct to B. rapa ssp. by A. tumefaciens was not achieved, probably due to the combination of an inefficient plant transformation and regeneration system, the length of the construct and most importantly the presence of the SU1 gene in the construct that appears to inhibit the regeneration of transformed shoots.
As an alternative to the insertional mutagenesis approach to identify genetic loci that impact traits, there is a genetic approach based on quantitative trait locus (QTL) analysis. Segregating populations are needed to map QTLs for traits of interest. Chapter 3 describes the analysis of an F2 population derived from a cross between two distinct, but early flowering and self compatible, B. rapa genotypes, L58 and R-o-18. Amplified fragment length polymorphism (AFLP) markers together with simple sequence repeat (SSR) markers were used to genotype this F2 population and anchor the linkage map to the reference genetic map of B. rapa. Highly significant QTLs associated with the production of adventitious roots and the transformation competence to A. rhizogenes were detected, which will allow the selection of lines that are more efficient in transformation experiments. The analysis detected a strong QTL associated with seed coat color as well as QTLs for various morphological traits.
To fix the recombination events as much as possible and to obtain an “immortal” mapping population, a recombinant inbred line (RIL) population was developed from this F2 population. Chapter 4 describes development of this RIL population, for which a genetic linkage map was constructed using the Illumina® BeadXpressTM genotyping platform of Keygene NV and additional SSR markers. Analysis revealed an additional QTL for seed coat colour as well QTL for pod shattering, carpel number, cuticular wax and seed vivipary. Chapter 5 describes the detection of QTLs related to primary and secondary metabolites in this RIL population. The two parental lines show clear differences in metabolite profile, which allowed the finding of QTLs for glucosinolates, phenylpropanoids, glucose, glutamate and amino acids after analysis with H1- NMR. HPLC analysis of tocopherols revealed four QTLs controlling the levels of this important antioxidant.
The information on the genetic control of health related compounds indicates the potential to improve nutritional quality in classical crop breeding programs.
Planten presteren beter bij koud dankzij bacterie: 'gunstige' micro-organismen kunnen de plantengroei bevorderen (interview met Joeke Postma)
Arkesteijn, M. ; Postma, J. - \ 2008
Onder Glas 5 (2008)3. - p. 12 - 13.
kassen - tuinbouw - teelt onder bescherming - enten - tomaten - cucumis sativus - plantmateriaal - micro-organismen - groeibevorderaars - rhizobium - glastuinbouw - groenten - greenhouses - horticulture - protected cultivation - scions - tomatoes - cucumis sativus - planting stock - microorganisms - growth promoters - rhizobium - greenhouse horticulture - vegetables
Bacteriën kunnen rondom de wortels een handje helpen bij tal van processen. De Wageningse plantenziektekundige Joeke Postma zocht naar micro-organismen die een groeiverbetering laten zien bij tomaten en komkommers, geteeld bij een lagere temperatuur. Ze isoleerde circa 200 bacteriën, afkomstig van wortels uit de biologische grondteelt en screende ze via diverse selectiestappen. Er bleven drie isolaten over, waarbij Rhizobiumstam T5.3 er als beste afkwam. De geselecteerde stam gaf een groeistimulatie van 5 tot 10% bij een suboptimale temperatuur
Bodemtoets voor wortelknobbels gereed
Meijer, H. ; Dinkla, I. ; Meekes, E. - \ 2008
De Boomkwekerij 21 (2008)46. - ISSN 0923-2443 - p. 12 - 13.
boomkwekerijen - houtachtige planten - vruchtbomen - straatbomen - houtachtige planten als sierplanten - rozen - rhizobium - wortelknobbel - bodemonderzoek - forest nurseries - woody plants - fruit trees - street trees - ornamental woody plants - roses - rhizobium - crown gall - soil testing
Agrobacterium, de veroorzaker van wortelknobbels, vormt een groot probleem in de boomkwekerij. Er is nu een mogelijkheid om de grond vóór de teelt te toetsen op Agrobacterium
Nod factor signaling and infection in Rhizobium-legume symbiosis
Smit, P.E.J. - \ 2007
Wageningen University. Promotor(en): Ton Bisseling. - [S.l.] : S.n. - ISBN 9789085047940 - 94
rhizobium - peulgewassen - symbiose - signaaltransductie - genen - moleculaire biologie - infectie - rhizobium - legumes - symbiosis - signal transduction - genes - molecular biology - infection
Plants require nutrients in order to grow. Most of these are readily available, but a few, like the macronutrients nitrogen and phosphorous, are often limiting growth due to presence in low concentrations or in complexes that cannot be taken up by the plant root. To acquire these macronutrients plants can engage in a mutualistic symbiosis with other organisms. Most plants can interact with arbuscular mycorrhizal fungi that are able to solubilize complexed phosphorous. A subset of these plants, the legumes, can also interact with bacteria that can reduce atmospheric nitrogen into compounds plants can assimilate. Both interactions are so-called endosymbioses because the microsymbiont enters host cells. A symbiosis requires a good balance between costs and benefits. Therefore, infection is only initiated when the specific nutrient is scarce. Further, the host plant requires that the mutualistic microsymbionts produce signal molecules to identify themselves, and thereby prevents other (pathogenic) microorganisms to enter by similar mechanisms. The signal molecule(s) involved in mycorrhizal symbioses have not yet been identified. However, the rhizobial signal molecule, the so-called nodulation (Nod) factor has been identified and extensively studied. It has been intensively studied how Nod factors are perceived and how they induce Nod factor specific responses, including the formation of a completely new organ, the root nodule. In chapter one, we describe the state of the art (in 2005) regarding Nod factor signal transduction. Several approaches have been used to unravel Nod factor signaling in legumes, but the molecular-genetic approach has been most successful. More than 10 years after the first characterization of a Nod factor structure most genes essential for Nod factor signal transduction have been cloned and characterized. Additionally, the identification of these genes provides insight in how the Rhizobium-legume symbiosis evolved, because some of the identified Nod factor signaling genes are also essential to the arbuscular mycorrhizal symbiosis. The DMI genes, encoding a LRR receptor kinase, an ion channel, and a calcium/calmodulin dependent protein kinase, are required for both symbioses and are essential for most responses. The genes encoding the Nod factor receptors (NFR1, NFR5, LYK3, NFP), Nod factor response factors (NSP1, NSP2) and Nod factor responsive factors (NIN) show that besides the DMI genes additional genes are required to induce Nod factor specific responses. Therefore it is likely that the Rhizobium-legume symbiosis evolved from the more ancient mycorrhizal symbioses, but during evolution the nodulation specific genes have been recruited to induce Nod factor specific responses. In chapter two, we elaborate on two of the genes, NSP1 and DMI3, we introduced in chapter 1. We show in that chapter that NSP1 is a primary transcriptional regulator essential for the regulation of all Nod factor regulated genes, NSP1 is: a member of the GRAS transcription factor family; constitutively expressed; localized in the nucleus where also DMI3, acting upstream of NSP1, is being localized; essential for regulation of all known Nod factor regulated genes. NSP1 is a conserved member of the GRAS type protein family in the plant kingdom. Therefore, it is likely that NSP1, besides its normal function, gained a function in Nod factor signaling. In chapter three, we describe the identification of HCL, a gene required for root hair curling and infection, but not essential for other Nod factor-induced responses. We show by isolation of the weak hcl-4 mutant allele that HCL is also involved in infection thread formation. HCL encodes the previously identified Nod factor receptor LYK3. In hcl-4 infection thread growth, but not root hair curling, is dependent on the Nod factor structure excreted by the Rhizobium bacteria; only if the bacteria excrete the wild type Nod factor they are able to nodulate hcl-4 mutants. LYK3 is also essential for the regulation of a subset of Nod factor induced genes. However, NIN, a Nod factor regulated gene that is essential for both infection and nodule primordium formation, is not regulated by LYK3. The identification and characterization of hcl/lyk3 mutants is important as from previous work it was not clear whether LYK3 acts as a signaling or entry receptor. In this chapter, we clearly show that LYK3 acts as an infection receptor that regulates infection in a Nod factor structure dependent manner. In chapter three, we have shown that the infection receptor LYK3 is essential for specificity during infection. This receptor regulates bacterial growth in the infection thread. However, what mechanism the plant uses to prevent that other incompatible bacteria do grow along during infection? In chapter four we study bacterial growth in infection threads and show, using the fluorescent timer protein DsRED1-E5, that probably only at the tip of infection threads bacteria do grow. Therefore, during infection thread growth, most likely a continual selection of Nod factor producing Rhizobium bacteria takes place that will ultimately infect the nodule primordium established in the root cortex. This thesis is finalized (chapter five) by discussing new data obtained during the last couple of years, and draw new or readjust old conclusions. We discuss the following themes: how is the Nod factor signal transduced from the plasmamembrane into the nucleus; how did Nod factor signaling genes evolve; are there any new Nod factor signaling genes identified; is it useful to conduct more classical genetic screens for genes involved in root nodule symbiosis; and finally how Nod factor signal transduction plays a role in infection thread formation.
Mogelijkheden biologische bestrijding van Agrobacterium in Aster
Wubben, J.P. ; Wolf, J.M. van der - \ 2006
Wageningen : PPO, Glastuinbouw (Rapporten PPO Glastuinbouw ) - 20
asteraceae - aster - plantenziekteverwekkende bacteriën - rhizobium - asteraceae - aster - plant pathogenic bacteria - rhizobium