The authors like to acknowledge Mark Youles, Sophien Kamoun and Sylvestre Marillonnet for making Golden Gate cloning parts available through the Addgene database. Additionally, we would like to thank E. James, P. Hadobas, and T. J. Higgens for P. andersonii seeds. This work was supported by The Netherlands Organization for Scientific Research (NWO-VICI grant 865.13.001; NWO-Open Competition grant 819.01.007) and The Ministry of Research, Technology and Higher Education of the Republic of Indonesia (RISET-PRO grant 8245-ID).

<ol>
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The authors have nothing to disclose.

Legumes and the distantly-related Cannabaceae genus Parasponia represent the only two clades of plant species able to establish an endosymbiotic relationship with nitrogen-fixing rhizobia and form root nodules. Comparative studies between species of both clades are highly relevant to provide insights into the core genetic networks allowing this symbiosis. Currently, genetic studies are mainly done in legumes; especially the two model species M. truncatula and L. japonicus. To provide an additional experimental platform and facilitate comparative studies with a nodulating non-legume, we describe here a detailed protocol for stable transformation and reverse genetic analyses in P. andersonii. The presented protocol uses in vitro propagation of T0 transgenic P. andersonii lines, allowing phenotypic analysis to be initiated within 4 months after A. tumefaciens co-cultivation. This is substantially faster than current protocols that have been established for stable transformation of legumes33. This makes P. andersonii an attractive research model.
The protocol described here contains several critical steps. The first of which concerns seed germination. To prepare P. andersonii seeds for germination, seeds need to be isolated from the berries. This is done by rubbing the berries on a piece of tissue paper or against the inside of a tea sieve. This procedure needs to be performed gently in order to prevent damage to the seed coat. If the seed coat gets damaged, bleach could enter the seed during sterilization, which reduces seed viability. To break seed dormancy, seeds are subjected to a 10-day temperature cycle. However, despite this treatment, germination is not entirely synchronized. Generally, the first seeds show radicle emergence after 7 days, but others might take several days longer to germinate.
Critical points in the transformation procedure concern the choice of the starting material and the duration of the co-cultivation step. To reach efficient transformation, it is best to use healthy and young stems or petioles of non-sterile greenhouse-grown plants as the starting material. In order to induce the growth of young branches, it is advisable to trim Parasponia trees every 2-3 months and refresh trees once a year. Additionally, the co-cultivation step needs to be performed for 2 days only. Prolonged co-cultivation promotes over-colonization of tissue explants by A. tumefaciens and generally reduces transformation efficiency. To prevent over-colonization by A. tumefaciens it is also important to regularly refresh the plates on which the explants are cultivated. In case over-colonization does occur, tissue explants could be washed (see Section 3.8) to remove A. tumefaciens cells. We advise adding bleach to the SH-10 solution used for washing (final concentration: ~2% hypochlorite). It is important to note that this additional washing step might not work on heavily-infected explants (Figure 2B). In case a transformation with a CRISPR/Cas9 construct yields only a limited number of putatively-transformed shoots or if mutagenesis of a particular gene is expected to cause problems in regeneration, it is advisable to include an empty vector control construct as the positive control. Lastly, it is important to ensure that all transgenic lines that are selected are resulting from independent T-DNA integration events. Therefore, we instruct to take only a single putatively-transgenic shoot from each side of an explant. However, we realize that this reduces the potential number of independent lines. If many lines are required, researchers could decide to separate putatively-transformed calli from the original explants when these calli are &#8805;2 mm in size and culture these calli independently. In this way, multiple lines could be isolated from each explant, which raises the number of potential transgenic lines.
In the current protocol, transgenic lines of P. andersonii are propagated vegetatively through in vitro propagation. The advantage of this is that many transgenic plantlets can be generated in a relatively short time period. However, this method also has several limitations. Firstly, the maintenance of T0 transgenic lines through in vitro propagation is labor intensive and could result in unwanted genetic or epigenetic alterations34,35. Secondly, T0 lines still contain a copy of the T-DNA, including the antibiotic resistance cassette. This limits the number of possible re-transformations, as different selection markers are required for each re-transformation. Currently, we have only tested transformation using kanamycin or hygromycin selection (data not shown). Furthermore, the presence of the Cas9-encoding sequence and sgRNAs in the T0 transgenic lines complicates complementation studies. Complementation assays are possible but require the sgRNA target site(s) to be mutated as such that gene-editing of the complementation construct is prevented. Thirdly, a disadvantage of working with T0 lines is that CRISPR/Cas9 mutants might be chimeric. To prevent phenotypic analysis of chimeric mutant lines, we recommend repeating the genotyping analysis after in vitro propagation on at least 3 different shoots. Although, the number of chimeric mutants obtained using the protocol described here is limited, they are occasionally observed10. To overcome the limitations of working with T0 lines, P. andersonii mutant lines could be propagated generatively. P. andersonii trees are dioecious and wind-pollinated2. This means that each transgenic line needs to be manipulated as such that male and female flowers are produced on a single individual, and subsequently grown as such that cross pollination does not occur. As P. andersonii is a fast-growing tree it requires a substantial amount of space in a tropical greenhouse (28 &#176;C, ~85% relative humidity). Therefore, although technically possible, generative propagation of P. andersonii transgenic lines is logistically challenging.
In the protocol section, we described 3 methods for nodulation of P. andersonii. The advantage of the plate and pouch systems is that the roots are easily accessible, which may allow spot-inoculation of bacteria and following nodule formation over time. However, the plate system is quite labor intensive, which makes it less suited for large-scale nodulation experiments. A disadvantage of the pouch system is that it is difficult to prevent fungal contamination. Pouches are not sterile, and therefore fungal growth is often observed on the top half of the pouch. However, this does not affect P. andersonii growth, and therefore does not interfere with nodulation assays. Additionally, the pouch system is only suitable for seedlings. Despite several attempts, we have been unable to grow plantlets obtained through in vitro propagation in pouches.
The P. andersonii reverse genetics pipeline described here offers a substantial improvement compared to the existing A. rhizogenes-based root transformation method11. Using the described procedures, stable transgenic lines can be generated efficiently and can be maintained via in vitro propagation. In contrast, A. rhizogenes transformation is transient and only results in the formation of transgenic roots. Because each transgenic root results from an independent transformation, A. rhizogenes&#173; transformation-based assays suffer from substantial phenotypic variation. This variation is much less in case of stable lines, although in vitro propagation also creates some level of variation. Because of this reduced variation and the fact that multiple plantlets could be phenotyped for each stable line, stable lines are more suited for quantitative assays compared to A. rhizogenes-transformed roots. Additionally, the stable transformation does not depend on the introduction of the A. rhizogenes root inducing locus (rol) that affects the endogenous hormone balance15. Therefore, stable lines are better suited for reverse genetic analysis of genes involved in hormone homeostasis compared to A. rhizogenes-transformed roots. A more general advantage of P. andersonii as research model is that it did not experience a recent whole genome duplication (WGD). The legume Papilionoideae subfamily, which includes the model legumes M. truncatula and L. japonicus, as well as the Salicaceae (order Malpighiales) that includes the model tree Populus trichocarpa experienced WGDs ~65 million years ago36,37. Many paralogous gene copies resulting from these WGDs are retained in the genomes of M. truncatula, L. japonicus and P. trichocarpa37,38,39, which creates redundancy that might complicate reverse genetic analyses. As P. andersonii did not experience a recent WGD, reverse genetic analyses on P. andersonii might be less affected by redundant functioning of paralogous gene copies.
Taken together, we provide a detailed protocol for reverse genetic analysis in P. andersonii. Using this protocol, single mutant lines can be efficiently generated in a timeframe of 2-3 months10. This protocol can be extended to create higher order mutants through multiplexing of sgRNAs targeting different genes simultaneously, as shown for other plant species40,41,42. Additionally, the stable transformation procedure described here is not limited to CRISPR/Cas9 gene-targeting but could also be used to introduce other types of constructs (e.g., for promoter-reporter assays, ectopic expression or trans-complementation). We established P. andersonii as a comparative research model to study mutualistic symbioses with nitrogen-fixing rhizobia or endomycorrhizal fungi. However, the protocols described here also provide tools to study other aspects of the biology of this tropical tree, such as wood formation, the development of bi-sexual flowers or the biosynthesis of Cannabaceae-specific secondary metabolites.

Parasponia andersonii is a tropical tree belonging to the Cannabis family (Cannabaceae) and is native to Papua New Guinea and several Pacific Islands1,2,3. Together with 4 additional Parasponia species, it represents the only non-legume lineage that can establish a nitrogen-fixing nodule symbiosis with rhizobia. This symbiosis is well studied in the legume (Fabaceae) models Medicago truncatula and Lotus japonicus, which has resulted in acquiring detailed knowledge of the molecular genetic nature of nodule formation and functioning4. Additionally, it was demonstrated that the root nodule symbiosis in legumes is founded on the much older, and widespread arbuscular mycorrhizal symbiosis5. Phylogenomic comparisons suggest that the nitrogen-fixing nodule symbioses of legumes, Parasponia, as well as, the so-called actinorhizal plant species that host diazotrophic Frankia bacteria, have a shared evolutionary origin6,7,8. To determine whether the genes identified to be involved in the legume nodule formation are the part of a conserved genetic basis, studies on non-legume species are essential. To this end, we propose to use P. andersonii as a comparative research model, alongside legumes, to identify the core genetic networks underlying root nodule formation and functioning.
P. andersonii is a pioneer that can be found on the slopes of volcanic hills. It can meet growth speeds of 45 cm per month and reach lengths of up to 10 meters9. P. andersonii trees are wind-pollinated, which is facilitated by the formation of separate male and female flowers3,10. We recently sequenced and annotated the diploid genome (2n = 20; 560 Mb/1C) of P. andersonii, and assembled draft genome sequences of 2 additional Parasponia species; P. rigida and P. rugosa6. This revealed ~35,000 P. andersonii gene models that can be clustered in &#62;20,000 orthogroups together with genes from M. truncatula, soybean (Glycine max), Arabidopsis thaliana, woodland strawberry (Fragaria vesca), Trema orientalis, black cotton poplar (Populus trichocarpa) and eucalypt (Eucalyptus grandis)6. Additionally, transcriptome comparisons between M. truncatula and P. andersonii identified a set of 290 putative orthologues that display a nodule-enhanced expression pattern in both species6. This provides an excellent resource for comparative studies.
To study the gene function in P. andersonii roots and nodules, a protocol for Agrobacterium rhizogenes-mediated root transformation has been established11. Using this protocol, compound plants bearing transgenic roots can be generated in a relatively short time frame. This method is, also, widely applied in the legume-symbiosis research12,13,14. However, the disadvantage of this method is that only roots are transformed and that each transgenic root represents an independent transformation event, resulting in substantial variation. Also, the transformation is transient and transgenic lines cannot be maintained. This makes A. rhizogenes-based root transformation less suited for CRISPR/Cas9-mediated genome editing. Additionally, A. rhizogenes transfers its root inducing locus (rol) genes to the plant genome, which once expressed interfere with hormone homeostasis15. This makes studying the role of plant hormones in A. rhizogenes-transformed roots challenging. To overcome these limitations, we recently developed a protocol for Agrobacterium tumefaciens-based transformation and CRISPR/Cas9-mediated mutagenesis of P. andersonii10.
Here, we provide a detailed description of the A. tumefaciens-based transformation procedure and reverse genetics pipeline developed for P. andersonii. Additionally, we provide protocols for the downstream handling of transgenic plantlets, including assays to study symbiotic interactions. Using the protocol described here, multiple transgenic lines can be generated in a 2-3 months period. In combination with CRISPR/Cas9-mediated mutagenesis, this allows efficient generation of knockout mutant lines. These mutant lines can be vegetatively propagated in vitro10,16,17, which allows sufficient material to be generated to start phenotypic characterization at 4 months after the transformation procedure has been initiated10. Together, this set of procedures should allow any lab to adopt P. andersonii as a research model for studies aimed at understanding rhizobial and mycorrhizal associations, as well as potentially other aspects of the biology of this tropical tree.

<table><tbody><tr><td>Sigma-Aldrich</td><td>N0640</td><td>NAA</td><td></td></tr><tr><td>Duchefa Biochemie</td><td>M1503.0250</td><td>MES</td><td></td></tr><tr><td>Sigma-Aldrich</td><td>D134406</td><td>Acetosyringone</td><td></td></tr><tr><td>Duchefa Biochemie</td><td>X1402.1000</td><td>X-Gal</td><td></td></tr><tr><td>Merck</td><td>101236</td><td>For nucleic acid electrophoresis gel</td><td></td></tr><tr><td>-</td><td>-</td><td>Pouches box material, hangers</td><td></td></tr><tr><td>Merck</td><td>101188</td><td>NH&lt;sub&gt;4&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;</td><td></td></tr><tr><td>Sigma-Aldrich</td><td>B3408-1G</td><td>BAP</td><td></td></tr><tr><td>Merck</td><td>100156</td><td>H&lt;sub&gt;3&lt;/sub&gt;BO&lt;sub&gt;3&lt;/sub&gt;</td><td></td></tr><tr><td>Thermo-Fisher</td><td>ER1011</td><td>Used as restriction enzyme in Golden Gate cloning assembly</td><td></td></tr><tr><td>Thermo-Fisher</td><td>15561020</td><td>Used in Golden Gate cloning assembly</td><td></td></tr><tr><td>Merck</td><td>137101</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td></tr><tr><td>Duchefa Biochemie</td><td>C0111.0025</td><td>C&lt;sub&gt;16&lt;/sub&gt;H&lt;sub&gt;16&lt;/sub&gt;N&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;S&lt;sub&gt;2&lt;/sub&gt;Na</td><td></td></tr><tr><td>Thermo-Fisher</td><td>K1231</td><td>Used for cloning the blunt-ended PCR amplicons in genotyping procedure</td><td></td></tr><tr><td>Agronutrition</td><td>AP2011</td><td>Containing Rhizophagus irregularis DAOM 197198 (1,000 spores/mL), used for mychorrization assay</td><td></td></tr><tr><td>Merck</td><td>102790</td><td>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;5H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td></tr><tr><td>Duchefa Biochemie</td><td>D1004.1000</td><td>Used for plant tissue culture agar-based medium</td><td></td></tr><tr><td>Merck</td><td>105101</td><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td></td></tr><tr><td>VWR Chemicals</td><td>20302.293</td><td>Na&lt;sub&gt;2&lt;/sub&gt;&amp;middot;EDTA</td><td></td></tr><tr><td>Duchefa Biochemie</td><td>M0803.1000</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;14&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;</td><td></td></tr><tr><td>Thermo-Fisher</td><td>ER0291</td><td>Used as restriction enzyme in Golden Gate cloning assembly</td><td></td></tr><tr><td>Merck</td><td>100983</td><td>C&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;OH</td><td></td></tr><tr><td>VWR Chemicals</td><td>BDH9232-500G</td><td>EDTA</td><td></td></tr><tr><td>Sigma-Aldrich</td><td>Z377600-1PAK</td><td>Cellophane membrane</td><td></td></tr><tr><td>Biomatters, Ltd.</td><td>R9 or higher</td><td>Bioinformatics software for in silico cloning and designing of sgRNAs</td><td></td></tr><tr><td>Mega International</td><td>-</td><td>Technical information at https://mega-international.com/tech-info/</td><td></td></tr><tr><td>Sigma-Aldrich</td><td>65882</td><td>Used for fixating nodule tissues</td><td></td></tr><tr><td>VWR Chemicals</td><td>24385.295</td><td>-</td><td></td></tr><tr><td>Vink</td><td>219341</td><td>Pouches box material, bottom part</td><td></td></tr><tr><td>Leica Biosystems</td><td>14702218311</td><td>Used as a template for plastic embedding</td><td></td></tr><tr><td>Merck</td><td>100317</td><td>HCl</td><td></td></tr><tr><td>Sigma-Aldrich</td><td>I5386-1G</td><td>IBA</td><td></td></tr><tr><td>Merck</td><td>103862</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;FeO&lt;sub&gt;7&lt;/sub&gt;</td><td></td></tr><tr><td>Merck</td><td>103965</td><td>FeSO&lt;sub&gt;4&lt;/sub&gt;O&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td></tr><tr><td>Duchefa Biochemie</td><td>I1401.0005</td><td>IPTG</td><td></td></tr><tr><td>Duchefa Biochemie</td><td>K0126.0010</td><td>&amp;nbsp;</td><td></td></tr><tr><td>Sigma-Aldrich</td><td>L2000</td><td>&amp;nbsp;</td><td></td></tr><tr><td>Merck</td><td>105886</td><td>MgSO&lt;sub&gt;4&lt;/sub&gt;O&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td></tr><tr><td>Merck</td><td>105934</td><td>MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td></tr><tr><td>Merck</td><td>102786</td><td>MnSO&lt;sub&gt;4&lt;/sub&gt;O</td><td></td></tr><tr><td>Duchefa Biochemie</td><td>M1002.1000</td><td>Used for bacterial culture agar-based medium</td><td></td></tr><tr><td>Manutan</td><td>92007687</td><td>Pouches material</td><td></td></tr><tr><td>Paraxisdienst</td><td>130774</td><td>Elastic sealing foil</td><td></td></tr><tr><td>Pull Rhenen</td><td>Agra-Perlite No.3</td><td>Used as growing substrate in pots for nodulation assay</td><td></td></tr><tr><td>VWR Chemicals</td><td>391-0581</td><td>Used as container for cellophane membranes</td><td></td></tr><tr><td>Thermo-Fisher</td><td>F130WH</td><td>For genotyping transgenic lines</td><td></td></tr><tr><td>Addgene </td><td>50337</td><td>Level 0 terminator, 3&amp;rsquo;UTR, 35s (Cauliflower Mosaic Virus)</td><td></td></tr><tr><td>Addgene </td><td>48017</td><td>End-link 2 for assembling 2 level one part into a level 2 acceptor</td><td></td></tr><tr><td>Addgene </td><td>48018</td><td>End-link 3 for assembling 3 level one part into a level 2 acceptor</td><td></td></tr><tr><td>Addgene </td><td>48001</td><td>Level 1 acceptor. Position 5. Forward orientation</td><td></td></tr><tr><td>Addgene </td><td>48007</td><td>Level 1 Acceptor. Position 1. Reverse orientation</td><td></td></tr><tr><td>Addgene </td><td>50268</td><td>Level 0 promoter (0.4 kb), 35s (Cauliflower Mosaic Virus) + 5&amp;rsquo;UTR, &amp;Omega; (Tobacco Mosaic Virus)</td><td></td></tr><tr><td>Addgene </td><td>46966</td><td>Used for designing CRISPR/Cas9 module</td><td></td></tr><tr><td>Addgene </td><td>46968</td><td>Used for designing CRISPR/Cas9 module</td><td></td></tr><tr><td>Addgene </td><td>50334</td><td>Level 0 Kanamycin/Neomycin/Paromomycin resistance cassette</td><td></td></tr><tr><td>Topzeven</td><td>-</td><td>Used as filters for washing spore suspension</td><td></td></tr><tr><td>Sigma-Aldrich</td><td>8.17003</td><td>PEG400</td><td></td></tr><tr><td>Duchefa Biochemie</td><td>E1674.0001</td><td>Pots to grow &lt;em&gt;Parasponia&lt;/em&gt; plantlets/seedlings</td><td></td></tr><tr><td>Merck</td><td>104871</td><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td></tr><tr><td>Merck</td><td>105033</td><td>KOH</td><td></td></tr><tr><td>Merck</td><td>105153</td><td>K&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;O</td><td></td></tr><tr><td>Van Leusden b.v.</td><td>-</td><td>Used as growing substrate for mychorrhization assay</td><td></td></tr><tr><td>Duchefa Biochemie</td><td>S0225.0050</td><td>SH-basal salt medium</td><td></td></tr><tr><td>Duchefa Biochemie</td><td>S0411.0250</td><td>SH-vitamin mixture</td><td></td></tr><tr><td>Lehle Seeds</td><td>VIS-02</td><td>Used as non-ionic surfactant in the washing step of stable transformation</td><td></td></tr><tr><td>Merck</td><td>137017</td><td>NaCl</td><td></td></tr><tr><td>VWR Chemicals</td><td>89230-072</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;11&lt;/sub&gt;NaO&lt;sub&gt;7&lt;/sub&gt;</td><td></td></tr><tr><td>Merck</td><td>106521</td><td>Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td></tr><tr><td>Merck</td><td>106574</td><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td></tr><tr><td>Merck</td><td>567549</td><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td></tr><tr><td>Sigma-Aldrich</td><td>239313</td><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;O</td><td></td></tr><tr><td>Duchefa Biochemie</td><td>S0809.5000</td><td>C&lt;sub&gt;12&lt;/sub&gt;H&lt;sub&gt;22&lt;/sub&gt;O&lt;sub&gt;11&lt;/sub&gt;</td><td></td></tr><tr><td>Thermo-Fisher</td><td>B69</td><td>Used in Golden Gate cloning assembly</td><td></td></tr><tr><td>Thermo-Fisher</td><td>EL0013</td><td>Used in Golden Gate cloning assembly</td><td></td></tr><tr><td>Kulzer-Mitsui Chemicals Group</td><td>64708806</td><td>Methyl methacrylate-based resin powder</td><td></td></tr><tr><td>Kulzer-Mitsui Chemicals Group</td><td>64709003</td><td>HEMA (2-hydroxyethyl methacrylate)-based resin solution</td><td></td></tr><tr><td>Kulzer-Mitsui Chemicals Group</td><td>66022678</td><td>Methyl methacrylate-based resin solution</td><td></td></tr><tr><td>Merck</td><td>1159300025</td><td>&amp;nbsp;</td><td></td></tr><tr><td>Acros</td><td>189350250</td><td>&amp;nbsp;</td><td></td></tr><tr><td>VWR Chemicals</td><td>663684B</td><td>Polysorbate 20</td><td></td></tr><tr><td>Stout Perspex</td><td>-</td><td>pouches box material, lid</td><td></td></tr><tr><td>Duchefa Biochemie</td><td>Y1333.1000</td><td>&amp;nbsp;</td><td></td></tr><tr><td>Merck</td><td>108816</td><td>ZnCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td></tr><tr><td>Alfa Aesar</td><td>33399</td><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;O&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td></tr></tbody></table>

transforming, genome editing and phenotyping the nitrogen-fixing tropical cannabaceae tree parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Watch this Scientific Journal Video about Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii at JoVE.com

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...

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12.9K Views.  Wageningen University & Research. Parasponia is een tropische boom uit de cannabisfamilie die in staat is om een stikstofbindende symbiose met rhizobium vast te stellen. Vergelijkende studies tussen Parasponia en peulvruchten kunnen inzicht geven in de kerngeneeskundige netwerken die ten grondslag liggen aan rhizobiumsympiose. Met dit protocol kunnen Parasponia andersonii stabiele transgene mutantlijnen worden gegenereerd in een periode van twee tot drie maanden.Deze lijnen kunnen efficiënt worden gepropageerd door mi...

Video: Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic resources available to study how plants adapt to environmental stresses, its value as a functional genomics model and tool has been limited by the lack of a feasible transformation system. In this protocol, we report how to generate stable transgenic S. parvula lines using an Agrobacterium-mediated floral-dip method. We modified the transformation protocol used for A. thaliana to account for unique traits of S. parvula, such as an indeterminate flowering habit and a high epicuticular wax content on leaves. Briefly, S. parvula seeds were stratified at 4 &#176;C for five days before planting. Plants were grown at a photoperiod of a 14 h light and 10 h dark and a 130 &#181;mol m-2s-1 light intensity, at 22 &#176;C to 24 &#176;C. Eight to nine week-old plants with multiple inflorescences were selected for transformation. These inflorescences were dipped in an infiltration solution of Agrobacterium tumefaciens GV3101 carrying the pMP90RK plasmid. We performed two rounds of flower dipping with an interval of three to four weeks to increase the transformation efficiency. The T1 seeds were collected and dried for four weeks in a container with desiccants before germination to screen for candidate transformed lines. Resistance to BASTA was used to screen T1 plants. We sprayed the BASTA solution three times with an interval of three days starting at two week-old plants to reduce false positives. A BASTA drop test was performed on surviving individual plants to identify true positive transformants. The transformation efficiency was 0.033%, yielding 3&#8211;4 transgenic plants per 10,000 T1 seeds propagated.

Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula

Agrobacterium-mediated transformation using a floral-dip method can be successfully employed to create stable transgenic lines of the extremophyte model Schrenkiella parvula. We present a protocol modified from that for Arabidopsis thaliana, considering different growth habits and physiological characteristics of the extremophyte.

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Developmental Biology

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is an easy-to-use genome editor that has been used in model and non-model species. Here we present a protein-based version of this system that was used to introduce a premature stop codon into a mating gene of a non-model filamentous ascomycete fungus.

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species ...

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.

Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to ...

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.&#160;stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.&#160;ratti is closely related to S.&#160;stercoralis and is often used as a laboratory model for S.&#160;stercoralis. Both S.&#160;stercoralis and S.&#160;ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique.
Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S.&#160;stercoralis and S.&#160;ratti.

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

Immunology and Infection

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs.  Towards this end, much recent ...

Biology

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase).
In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Successful germline transformation in the fall armyworm, Spodoptera frugiperda, was achieved using mRNA of hyperactive piggyBac transposase.

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

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