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René Geurts - One of the best experts on this subject based on the ideXlab platform.
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Mutant analysis in the nonlegume Parasponia andersonii identifies NIN and NF‐YA1 transcription factors as a core genetic network in nitrogen‐fixing nodule symbioses
The New phytologist, 2020Co-Authors: Luuk Rutten, Arjan Van Zeijl, Ton Bisseling, Yuda Purwana Roswanjaya, Olga Kulikova, Marta Rodriguez-franco, Thomas Ott, René GeurtsAbstract:Nitrogen‐fixing nodulation occurs in ten taxonomic lineages, either with rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. In the legume‐rhizobium endosymbiosis, both processes are guarded by the transcription factor NODULE INCEPTION (NIN) and its downstream target genes of the NUCLEAR FACTOR Y (NF‐Y) complex. It is hypothesized that nodulation has a single evolutionary origin ~ 110 million years ago, followed by many independent losses. 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. We used Parasponia andersonii to investigate the role of NIN and NF‐YA genes in rhizobium nodulation in a non‐legume system. Consistent with legumes, P. andersonii PanNIN and PanNF‐YA1 are co‐expressed in nodules. By analyzing single, double and higher‐order CRISPR‐Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF‐YA1 are PanNIN‐dependent and that PanNF‐YA1 is specifically required for intracellular rhizobium infection. This demonstrates that NIN and NF‐YA1 commit conserved symbiotic functions. As Parasponia and legumes diverged soon after the birth of the nodulation trait, we argue that NIN and NF‐YA1 represent core transcriptional regulators in this symbiosis.
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crispr cas9 mediated mutagenesis of four putative symbiosis genes of the tropical tree Parasponia andersonii reveals novel phenotypes
Frontiers in Plant Science, 2018Co-Authors: Arjan Van Zeijl, Titis A. K. Wardhani, Maryam Seifi Kalhor, Luuk Rutten, Sidney Linders, Ton Bisseling, Wouter Kohlen, M.v. Hartog, Elena Fedorova, René GeurtsAbstract:Parasponia represents five fast-growing tropical tree species in the Cannabeaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knock-out mutants are obtained within three months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within six months after transformation. We mutated four genes - PanHK4, PanEIN2, PanNSP1, and PanNSP2 - that control cytokinin, ethylene or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.
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Parasponia: a novel system for studying mutualism stability.
Trends in plant science, 2014Co-Authors: Jocelyn E. Behm, René Geurts, E.t. KiersAbstract:Understanding how mutualistic interactions are stabilized in the presence of cheaters is a major question in evolutionary biology. The legume–rhizobia mutualism has become a model system for studying how plants control cheating partners. However, the generality and evolutionary origins of these control mechanisms are intensely debated. In this Opinion article, we argue that a novel system – the Parasponia–rhizobia mutualism – will significantly advance research in mutualism stability. Parasponia is the only non-legume lineage to have evolved a rhizobial symbiosis, which provides an evolutionary replicate to test how rhizobial exploitation is controlled. Evidence also suggests that this symbiosis is young. This allows studies at an earlier evolutionary stage in mutualisms, so the origin of control mechanisms can be better understood.
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Efficiency of Agrobacterium rhizogenes-mediated root transformation of Parasponia and Trema is temperature dependent
Plant Growth Regulation, 2012Co-Authors: Cao Qingqin, Rik Op Den Camp, Maryam Seifi Kalhor, Ton Bisseling, René GeurtsAbstract: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.
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Nonlegume Parasponia andersonii deploys a broad rhizobium host range strategy resulting in largely variable symbiotic effectiveness.
Molecular plant-microbe interactions : MPMI, 2012Co-Authors: Rik Op Den Camp, Elisa Polone, Ton Bisseling, Elena Fedorova, Wim Roelofsen, Andrea Squartini, Huub J. M. Op Den Camp, René GeurtsAbstract:The non-legume genus Parasponia has evolved the rhizobium symbiosis independent from legumes and has done so only recently. We aim to study the promiscuity of such newly evolved symbiotic engagement and determine the symbiotic effectiveness of infecting rhizobium species. It was found that Parasponia andersonii can be nodulated by a broad range of rhizobia belonging to four different genera, and therefore, we conclude that this non-legume is highly promiscuous for rhizobial engagement. A possible drawback of this high promiscuity is that low-efficient strains can infect nodules as well. The strains identified displayed a range in nitrogen-fixation effectiveness, including a very inefficient rhizobium species, Rhizobium tropici WUR1. Because this species is able to make effective nodules on two different legume species, it suggests that the ineffectiveness of P. andersonii nodules is the result of the incompatibility between both partners. In P. andersonii nodules, rhizobia of this 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 spp. Therefore, we argue that the Parasponia-rhizobium symbiosis is a delicate balance between mutual benefits and parasitic colonization.
Ton Bisseling - One of the best experts on this subject based on the ideXlab platform.
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Duplication of symbiotic lysin motif receptors predates the evolution of nitrogen-fixing nodule symbiosis
Plant physiology, 2020Co-Authors: Luuk Rutten, Arjan Van Zeijl, Sidney Linders, M.v. Hartog, Kana Miyata, Yuda Purwana Roswanjaya, Rik Huisman, Robin Van Velzen, Ton BisselingAbstract:Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2. These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.
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Mutant analysis in the nonlegume Parasponia andersonii identifies NIN and NF‐YA1 transcription factors as a core genetic network in nitrogen‐fixing nodule symbioses
The New phytologist, 2020Co-Authors: Luuk Rutten, Arjan Van Zeijl, Ton Bisseling, Yuda Purwana Roswanjaya, Olga Kulikova, Marta Rodriguez-franco, Thomas Ott, René GeurtsAbstract:Nitrogen‐fixing nodulation occurs in ten taxonomic lineages, either with rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. In the legume‐rhizobium endosymbiosis, both processes are guarded by the transcription factor NODULE INCEPTION (NIN) and its downstream target genes of the NUCLEAR FACTOR Y (NF‐Y) complex. It is hypothesized that nodulation has a single evolutionary origin ~ 110 million years ago, followed by many independent losses. 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. We used Parasponia andersonii to investigate the role of NIN and NF‐YA genes in rhizobium nodulation in a non‐legume system. Consistent with legumes, P. andersonii PanNIN and PanNF‐YA1 are co‐expressed in nodules. By analyzing single, double and higher‐order CRISPR‐Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF‐YA1 are PanNIN‐dependent and that PanNF‐YA1 is specifically required for intracellular rhizobium infection. This demonstrates that NIN and NF‐YA1 commit conserved symbiotic functions. As Parasponia and legumes diverged soon after the birth of the nodulation trait, we argue that NIN and NF‐YA1 represent core transcriptional regulators in this symbiosis.
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CRISPR/Cas9-Mediated Mutagenesis of Four Putative Symbiosis Genes of the Tropical Tree Parasponia andersonii Reveals Novel Phenotypes.
Frontiers in plant science, 2018Co-Authors: Arjan Van Zeijl, Titis A. K. Wardhani, Maryam Seifi Kalhor, Luuk Rutten, Sidney Linders, Ton Bisseling, M.v. Hartog, Elena Fedorova, Wouter KohlenAbstract:Parasponia represents five fast-growing tropical tree species in the Cannabeaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knock-out mutants are obtained within three months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within six months after transformation. We mutated four genes - PanHK4, PanEIN2, PanNSP1, and PanNSP2 - that control cytokinin, ethylene or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.
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crispr cas9 mediated mutagenesis of four putative symbiosis genes of the tropical tree Parasponia andersonii reveals novel phenotypes
Frontiers in Plant Science, 2018Co-Authors: Arjan Van Zeijl, Titis A. K. Wardhani, Maryam Seifi Kalhor, Luuk Rutten, Sidney Linders, Ton Bisseling, Wouter Kohlen, M.v. Hartog, Elena Fedorova, René GeurtsAbstract:Parasponia represents five fast-growing tropical tree species in the Cannabeaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knock-out mutants are obtained within three months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within six months after transformation. We mutated four genes - PanHK4, PanEIN2, PanNSP1, and PanNSP2 - that control cytokinin, ethylene or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.
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CRISPR/Cas9-Mediated Mutagenesis of Four Putative Symbiosis Genes of the Tropical Tree Parasponia andersonii Reveals Novel Phenotypes
Frontiers Media S.A., 2018Co-Authors: Arjan Van Zeijl, Titis A. K. Wardhani, Maryam Seifi Kalhor, Luuk Rutten, Marijke Hartog, Sidney Linders, Elena E. Fedorova, Ton Bisseling, Wouter KohlenAbstract:Parasponia represents five fast-growing tropical tree species in the Cannabaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knockout mutants are obtained within 3 months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within 6 months after transformation. We mutated four genes – PanHK4, PanEIN2, PanNSP1, and PanNSP2 – that control cytokinin, ethylene, or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies
Arjan Van Zeijl - One of the best experts on this subject based on the ideXlab platform.
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Duplication of symbiotic lysin motif receptors predates the evolution of nitrogen-fixing nodule symbiosis
Plant physiology, 2020Co-Authors: Luuk Rutten, Arjan Van Zeijl, Sidney Linders, M.v. Hartog, Kana Miyata, Yuda Purwana Roswanjaya, Rik Huisman, Robin Van Velzen, Ton BisselingAbstract:Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2. These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.
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Mutant analysis in the nonlegume Parasponia andersonii identifies NIN and NF‐YA1 transcription factors as a core genetic network in nitrogen‐fixing nodule symbioses
The New phytologist, 2020Co-Authors: Luuk Rutten, Arjan Van Zeijl, Ton Bisseling, Yuda Purwana Roswanjaya, Olga Kulikova, Marta Rodriguez-franco, Thomas Ott, René GeurtsAbstract:Nitrogen‐fixing nodulation occurs in ten taxonomic lineages, either with rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. In the legume‐rhizobium endosymbiosis, both processes are guarded by the transcription factor NODULE INCEPTION (NIN) and its downstream target genes of the NUCLEAR FACTOR Y (NF‐Y) complex. It is hypothesized that nodulation has a single evolutionary origin ~ 110 million years ago, followed by many independent losses. 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. We used Parasponia andersonii to investigate the role of NIN and NF‐YA genes in rhizobium nodulation in a non‐legume system. Consistent with legumes, P. andersonii PanNIN and PanNF‐YA1 are co‐expressed in nodules. By analyzing single, double and higher‐order CRISPR‐Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF‐YA1 are PanNIN‐dependent and that PanNF‐YA1 is specifically required for intracellular rhizobium infection. This demonstrates that NIN and NF‐YA1 commit conserved symbiotic functions. As Parasponia and legumes diverged soon after the birth of the nodulation trait, we argue that NIN and NF‐YA1 represent core transcriptional regulators in this symbiosis.
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Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses.
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Robin Van Velzen, Wei Liu, Arjan Van Zeijl, Luuk Rutten, Cao Qingqin, Rens Holmer, Luca Santuari, Trupti Sharma, Defeng ShenAbstract: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.
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CRISPR/Cas9-Mediated Mutagenesis of Four Putative Symbiosis Genes of the Tropical Tree Parasponia andersonii Reveals Novel Phenotypes.
Frontiers in plant science, 2018Co-Authors: Arjan Van Zeijl, Titis A. K. Wardhani, Maryam Seifi Kalhor, Luuk Rutten, Sidney Linders, Ton Bisseling, M.v. Hartog, Elena Fedorova, Wouter KohlenAbstract:Parasponia represents five fast-growing tropical tree species in the Cannabeaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knock-out mutants are obtained within three months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within six months after transformation. We mutated four genes - PanHK4, PanEIN2, PanNSP1, and PanNSP2 - that control cytokinin, ethylene or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.
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crispr cas9 mediated mutagenesis of four putative symbiosis genes of the tropical tree Parasponia andersonii reveals novel phenotypes
Frontiers in Plant Science, 2018Co-Authors: Arjan Van Zeijl, Titis A. K. Wardhani, Maryam Seifi Kalhor, Luuk Rutten, Sidney Linders, Ton Bisseling, Wouter Kohlen, M.v. Hartog, Elena Fedorova, René GeurtsAbstract:Parasponia represents five fast-growing tropical tree species in the Cannabeaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knock-out mutants are obtained within three months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within six months after transformation. We mutated four genes - PanHK4, PanEIN2, PanNSP1, and PanNSP2 - that control cytokinin, ethylene or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.
Luuk Rutten - One of the best experts on this subject based on the ideXlab platform.
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Duplication of symbiotic lysin motif receptors predates the evolution of nitrogen-fixing nodule symbiosis
Plant physiology, 2020Co-Authors: Luuk Rutten, Arjan Van Zeijl, Sidney Linders, M.v. Hartog, Kana Miyata, Yuda Purwana Roswanjaya, Rik Huisman, Robin Van Velzen, Ton BisselingAbstract:Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2. These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.
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Mutant analysis in the nonlegume Parasponia andersonii identifies NIN and NF‐YA1 transcription factors as a core genetic network in nitrogen‐fixing nodule symbioses
The New phytologist, 2020Co-Authors: Luuk Rutten, Arjan Van Zeijl, Ton Bisseling, Yuda Purwana Roswanjaya, Olga Kulikova, Marta Rodriguez-franco, Thomas Ott, René GeurtsAbstract:Nitrogen‐fixing nodulation occurs in ten taxonomic lineages, either with rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. In the legume‐rhizobium endosymbiosis, both processes are guarded by the transcription factor NODULE INCEPTION (NIN) and its downstream target genes of the NUCLEAR FACTOR Y (NF‐Y) complex. It is hypothesized that nodulation has a single evolutionary origin ~ 110 million years ago, followed by many independent losses. 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. We used Parasponia andersonii to investigate the role of NIN and NF‐YA genes in rhizobium nodulation in a non‐legume system. Consistent with legumes, P. andersonii PanNIN and PanNF‐YA1 are co‐expressed in nodules. By analyzing single, double and higher‐order CRISPR‐Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF‐YA1 are PanNIN‐dependent and that PanNF‐YA1 is specifically required for intracellular rhizobium infection. This demonstrates that NIN and NF‐YA1 commit conserved symbiotic functions. As Parasponia and legumes diverged soon after the birth of the nodulation trait, we argue that NIN and NF‐YA1 represent core transcriptional regulators in this symbiosis.
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Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses.
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Robin Van Velzen, Wei Liu, Arjan Van Zeijl, Luuk Rutten, Cao Qingqin, Rens Holmer, Luca Santuari, Trupti Sharma, Defeng ShenAbstract: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.
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CRISPR/Cas9-Mediated Mutagenesis of Four Putative Symbiosis Genes of the Tropical Tree Parasponia andersonii Reveals Novel Phenotypes.
Frontiers in plant science, 2018Co-Authors: Arjan Van Zeijl, Titis A. K. Wardhani, Maryam Seifi Kalhor, Luuk Rutten, Sidney Linders, Ton Bisseling, M.v. Hartog, Elena Fedorova, Wouter KohlenAbstract:Parasponia represents five fast-growing tropical tree species in the Cannabeaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knock-out mutants are obtained within three months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within six months after transformation. We mutated four genes - PanHK4, PanEIN2, PanNSP1, and PanNSP2 - that control cytokinin, ethylene or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.
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crispr cas9 mediated mutagenesis of four putative symbiosis genes of the tropical tree Parasponia andersonii reveals novel phenotypes
Frontiers in Plant Science, 2018Co-Authors: Arjan Van Zeijl, Titis A. K. Wardhani, Maryam Seifi Kalhor, Luuk Rutten, Sidney Linders, Ton Bisseling, Wouter Kohlen, M.v. Hartog, Elena Fedorova, René GeurtsAbstract:Parasponia represents five fast-growing tropical tree species in the Cannabeaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knock-out mutants are obtained within three months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within six months after transformation. We mutated four genes - PanHK4, PanEIN2, PanNSP1, and PanNSP2 - that control cytokinin, ethylene or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.
Wouter Kohlen - One of the best experts on this subject based on the ideXlab platform.
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CRISPR/Cas9-Mediated Mutagenesis of Four Putative Symbiosis Genes of the Tropical Tree Parasponia andersonii Reveals Novel Phenotypes.
Frontiers in plant science, 2018Co-Authors: Arjan Van Zeijl, Titis A. K. Wardhani, Maryam Seifi Kalhor, Luuk Rutten, Sidney Linders, Ton Bisseling, M.v. Hartog, Elena Fedorova, Wouter KohlenAbstract:Parasponia represents five fast-growing tropical tree species in the Cannabeaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knock-out mutants are obtained within three months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within six months after transformation. We mutated four genes - PanHK4, PanEIN2, PanNSP1, and PanNSP2 - that control cytokinin, ethylene or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.
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crispr cas9 mediated mutagenesis of four putative symbiosis genes of the tropical tree Parasponia andersonii reveals novel phenotypes
Frontiers in Plant Science, 2018Co-Authors: Arjan Van Zeijl, Titis A. K. Wardhani, Maryam Seifi Kalhor, Luuk Rutten, Sidney Linders, Ton Bisseling, Wouter Kohlen, M.v. Hartog, Elena Fedorova, René GeurtsAbstract:Parasponia represents five fast-growing tropical tree species in the Cannabeaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knock-out mutants are obtained within three months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within six months after transformation. We mutated four genes - PanHK4, PanEIN2, PanNSP1, and PanNSP2 - that control cytokinin, ethylene or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.
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CRISPR/Cas9-Mediated Mutagenesis of Four Putative Symbiosis Genes of the Tropical Tree Parasponia andersonii Reveals Novel Phenotypes
Frontiers Media S.A., 2018Co-Authors: Arjan Van Zeijl, Titis A. K. Wardhani, Maryam Seifi Kalhor, Luuk Rutten, Marijke Hartog, Sidney Linders, Elena E. Fedorova, Ton Bisseling, Wouter KohlenAbstract:Parasponia represents five fast-growing tropical tree species in the Cannabaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knockout mutants are obtained within 3 months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within 6 months after transformation. We mutated four genes – PanHK4, PanEIN2, PanNSP1, and PanNSP2 – that control cytokinin, ethylene, or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies
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Data_Sheet_1.PDF
2018Co-Authors: Arjan Van Zeijl, Titis A. K. Wardhani, Maryam Seifi Kalhor, Luuk Rutten, Marijke Hartog, Sidney Linders, Elena E. Fedorova, Ton Bisseling, Wouter KohlenAbstract:Parasponia represents five fast-growing tropical tree species in the Cannabaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knockout mutants are obtained within 3 months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within 6 months after transformation. We mutated four genes – PanHK4, PanEIN2, PanNSP1, and PanNSP2 – that control cytokinin, ethylene, or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.
