20 research outputs found
Parasponia: a missing piece of the evolutionary puzzle of nitrogen-fixing nodule symbiosis
Nitrogen-fixing root nodule symbiosis occurs in ten taxonomic lineages from four related orders -Fagales, Fabales, Rosales and Cucurbitales- that together are called the nitrogen-fixing clade (NFC). Nodulating plants within the NFC are scattered by non-nodulating species, as well as can interact either with rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. Despite a significant body of knowledge of the legume-rhizobium symbiosis, it remains elusive which signalling modules are shared between nodulating species in different taxonomic clades. Besides, it is generally assumed that nodulation evolved independently multiple times, though molecular genetic support for this hypothesis is lacking. Parasponia is the only non-legume plant which can establish nitrogen-fixing endosymbiosis with rhizobium, and it is the only nodulating plant within the Cannabaceae. The Parasponia lineage represents five species and phylogenetic analysis shows that this lineage is embedded within the non-nodulating Trema clade. As Parasponia and Trema are closely related, F1 hybrids could be created by crossing of the diploid Parasponia andersonii (2n=20) and the allotetraploid Trema tomentosa (2n=4X=40). Conceptually, P. andersonii x T. tomentosa F1 hybrid plants reflects a diploid T. tomentosa with a haploid genome of P. andersonii introduced. The F1 hybrid between diploid Parasponia andersonii and tetraploid Trema tomentosa can form nodules, whereas it is devoid of intracellular infection when inoculated with either Mesorhizobium plurifarium BOR2 or Bradyrhizobium elkanii WUR3. Based on its genetic composition and symbiotic phenotype, we argue that the F1 hybrid may mimic future engineer results. Therefore, we aimed to obtain a better understanding of the deviation in nodulation phenotype of wild type P. andersonii and F1 hybrid plants. To do so, we compared nodulation efficiencies and intracellular infection within nodule cells upon inoculation with a range of rhizobium strains, as Parasponia can interact with a wide range of rhizobia. This revealed that the host range of hybrid plants is narrower when compared to P. andersonii. We also show that the block in intracellular infection within hybrid nodules is consistent for all nodulating strains identified, cannot be overcome by increased LCO biosynthesis nor by mutating the type III or IV secretion systems of nodulating strains. The hybrid plants can establish arbuscular mycorrhization effectively, suggesting that the block of intracellular infection is rhizobium specific. Taken together, this indicates the occurrence of a yet unknown mechanism leading to an impaired host range and block of intracellular infection of hybrid plants. To answer evolution and genetic basis of nodulation, comparative genomic and transcriptomic analysis has been conducted using Parasponia species (Cannabaceae), the only non-legumes that can establish nitrogen-fixing nodules with rhizobium. 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 non-nodulating plant species show evidence of parallel loss in non-nodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION. Parallel loss of these symbiosis genes indicates that these non-nodulating lineages lost the potential to nodulate. By making use of the highly efficient Parasponia transformation platform, we conducted promoter:GUS expression analysis as well as CRISPR-Cas9 mutagenesis. 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 in non-elgume plant species. As Rosales, Fabales and Fagales diverged soon after the birth of the nodulation trait, we argue that NIN and NF-YA1 represent core transcriptional regulators in this symbiosis. Taken together, these results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ~100 million years ago 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
Phylogenetic analyses of Cannabaceae
Nucleotide alignments were generated using MAFFT version 7.017. The first phylogenetic reconstruction of Cannabaceae (MarkerData) was based on four plastid markers with five optimal partitions and models of sequence evolution: atpB-rbcL combined with trnL-F (GTR+I+G); first codon position of rbcL (GTR+I+G); second position of rbcL (SYM+I+G); third position of rbcL (GTR+G); rps16 (GTR+G). The second phylogenetic reconstruction of Cannabaceae (GenomeData) was based on whole chloroplast genomes with eight optimal partitions and models of sequence evolution: tRNA sequence (HKY+I), rRNA sequence (GTR+I), long single copy region (LSC) coding sequence (GTR+I+G), LSC non-coding sequence (GTR+G), short single copy region (SSC) coding sequence (GTR+G), SSC non-coding sequence (GTR+G), inverted repeat region (IR) coding sequence (GTR+G), and IR non-coding sequence (GTR+G)
Gene phylogenies based on Bayesian analysis
Phylogenetic analyses of genes of interest (EPR, HB, NFP, HCT, EPR, NIN, and RPG). Amino acid sequence alignments were generated using MAFFT version 7.017. Analyses were performed using MrBayes version 3.2.6 running 2.2 million generations, setting gamma-distributed rate variation and integrating over different models of amino acid sequence evolution (aamodelpr=mixed). For NFP analyses were based on the full-length sequences as well as separately on the kinase domain only
Draft genomes of Parasponia and Trema species
Draft genome assemblies of Parasponia rigida, Parasponia rugosa, Trema levigata, and Trema orientalis accession RG16 based on medium-coverage sequence data. Read data are available at GenBank under bioprojects PRJNA272486 (P. rigida), PRJNA272880 (P. rugosa) PRJNA38059 (T. levigata), and PRJNA272878 (T. orientalis RG16). Assembly was performed with the iterative de Bruijn graph assembler IDBA-UD version 1.1.1, iterating from 30-mers to 120-mers, with incremental steps of 20
Investigation of the Impact of CYP3A5 Polymorphism on Drug–Drug Interaction between Tacrolimus and Schisantherin A/Schisandrin A Based on Physiologically-Based Pharmacokinetic Modeling
Wuzhi capsule (WZC) is commonly prescribed with tacrolimus in China to ease drug-induced hepatotoxicity. Two abundant active ingredients, schisantherin A (STA) and schisandrin A (SIA) are known to inhibit CYP3A enzymes and increase tacrolimus’s exposure. Our previous study has quantitatively demonstrated the contribution of STA and SIA to tacrolimus pharmacokinetics based on physiologically-based pharmacokinetic (PBPK) modeling. In the current work, we performed reversible inhibition (RI) and time-dependent inhibition (TDI) assays with CYP3A5 genotyped human liver microsomes (HLMs), and further integrated the acquired parameters into the PBPK model to predict the drug–drug interaction (DDI) in patients with different CYP3A5 alleles. The results indicated STA was a time-dependent and reversible inhibitor of CYP3A4 while only a reversible inhibitor of CYP3A5; SIA inhibited CYP3A4 and 3A5 in a time-dependent manner but also reversibly inhibited CYP3A5. The predicted fold-increases of tacrolimus exposure were 2.70 and 2.41, respectively, after the multidose simulations of STA. SIA also increased tacrolimus’s exposure but to a smaller extent compared to STA. An optimized physiologically-based pharmacokinetic (PBPK) model integrated with CYP3A5 polymorphism was successfully established, providing more insights regarding the long-term DDI between tacrolimus and Wuzhi capsules in patients with different CYP3A5 genotypes
Reverse genetics using CRISPR-Cas9 in the tropical tree Parasponia andersonii revealed a promotive role for PanNODULE ROOT1 in stem secondary growth
NODULE ROOT (NOOT), BLADE-ON-PETIOLE (BOP), COCHLEATA (COCH)-LIKE (NBCL) are plant-specific developmental regulator participating in many developmental process of the primary growth. NBCL contribute to the meristem-to-organ-boundaries maintenance by inhibiting meristematic activities and promoting adjacent tissues initiation, development and determinacy. To determine if NBCL contribute to the regulation of tree secondary growth, we studied the impact of the PanNODULE ROOT1 (PanNOOT1) gene loss-of-function on the secondary growth of Parasponia andersonii (P. andersonii)
Examination of the Impact of CYP3A4/5 on Drug–Drug Interaction between Schizandrol A/Schizandrol B and Tacrolimus (FK-506): A Physiologically Based Pharmacokinetic Modeling Approach
Schizandrol A (SZA) and schizandrol B (SZB) are two active ingredients of Wuzhi capsule (WZC), a Chinese proprietary medicine commonly prescribed to alleviate tacrolimus (FK-506)-induced hepatoxicity in China. Due to their inhibitory effects on cytochrome P450 (CYP) 3A enzymes, SZA/SZB may display drug–drug interaction (DDI) with tacrolimus. To identify the extent of this DDI, the enzymes’ inhibitory profiles, including a 50% inhibitory concentration (IC50) shift, reversible inhibition (RI) and time-dependent inhibition (TDI) were examined with pooled human-liver microsomes (HLMs) and CYP3A5-genotyped HLMs. Subsequently, the acquired parameters were integrated into a physiologically based pharmacokinetic (PBPK) model to quantify the interactions between the SZA/SZB and the tacrolimus. The metabolic studies indicated that the SZB displayed both RI and TDI on CYP3A4 and CYP3A5, while the SZA only exhibited TDI on CYP3A4 to a limited extent. Moreover, our PBPK model predicted that multiple doses of SZB would increase tacrolimus exposure by 26% and 57% in CYP3A5 expressers and non-expressers, respectively. Clearly, PBPK modeling has emerged as a powerful approach to examine herb-involved DDI, and special attention should be paid to the combined use of WZC and tacrolimus in clinical practice
The BOP-type co-transcriptional regulator NODULE ROOT1 promotes stem secondary growth of the tropical Cannabaceae tree Parasponia andersonii
Tree stems undergo a massive secondary growth in which secondary xylem and phloem tissues arise from the vascular cambium. Vascular cambium activity is driven by endogenous developmental signalling cues and environmental stimuli. Current knowledge regarding the genetic regulation of cambium activity and secondary growth is still far from complete. The tropical Cannabaceae tree Parasponia andersonii is a non-legume research model of nitrogen-fixing root nodulation. Parasponia andersonii can be transformed efficiently, making it amenable for CRISPR-Cas9-mediated reverse genetics. We considered whether P. andersonii also could be used as a complementary research system to investigate tree-related traits, including secondary growth. We established a developmental map of stem secondary growth in P. andersonii plantlets. Subsequently, we showed that the expression of the co-transcriptional regulator PanNODULE ROOT1 (PanNOOT1) is essential for controlling this process. PanNOOT1 is orthologous to Arabidopsis thaliana BLADE-ON-PETIOLE1 (AtBOP1) and AtBOP2, which are involved in the meristem-to-organ-boundary maintenance. Moreover, in species forming nitrogen-fixing root nodules, NOOT1 is known to function as a key nodule identity gene. Parasponia andersonii CRISPR-Cas9 loss-of-function Pannoot1 mutants are altered in the development of the xylem and phloem tissues without apparent disturbance of the cambium organization and size. Transcriptomic analysis showed that the expression of key secondary growth-related genes is significantly down-regulated in Pannoot1 mutants. This allows us to conclude that PanNOOT1 positively contributes to the regulation of stem secondary growth. Our work also demonstrates that P. andersonii can serve as a tree research system
Reverse genetics using CRISPR-Cas9 in the tropical tree Parasponia andersonii revealed a promotive role for PanNODULE ROOT1 in stem secondary growth
NODULE ROOT (NOOT), BLADE-ON-PETIOLE (BOP), COCHLEATA (COCH)-LIKE (NBCL) are plant-specific developmental regulator participating in many developmental process of the primary growth. NBCL contribute to the meristem-to-organ-boundaries maintenance by inhibiting meristematic activities and promoting adjacent tissues initiation, development and determinacy. To determine if NBCL contribute to the regulation of tree secondary growth, we studied the impact of the PanNODULE ROOT1 (PanNOOT1) gene loss-of-function on the secondary growth of Parasponia andersonii (P. andersonii)
CRISPR/cas9-mediated mutagenesis of four putative symbiosis genes of the tropical tree parasponia andersonii reveals novel phenotypes
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