CRISPR/Cas9-Mediated Mutagenesis of Four Putative Symbiosis Genes of the Tropical Tree Parasponia andersonii Reveals Novel Phenotypes

Abstract

<i>Parasponia</i> 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 <i>Parasponia</i> allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for <i>Parasponia</i>. Here, we present a fast and efficient protocol for <i>Agrobacterium tumefaciens</i>-mediated transformation and CRISPR/Cas9 mutagenesis of <i>Parasponia andersonii</i>. 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 - <i>PanHK4, PanEIN2, PanNSP1</i>, and <i>PanNSP2</i> - that control cytokinin, ethylene, or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in <i>Panhk4</i> and <i>Panein2</i> displayed developmental phenotypes, namely reduced procambium activity in <i>Panhk4</i> and disturbed sex differentiation in <i>Panein2</i> mutants. The symbiotic phenotypes of <i>Panhk4</i> and <i>Panein2</i> mutant lines differ from those in legumes. In contrast, <i>PanNSP1</i> and <i>PanNSP2</i> 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 <i>Parasponia</i> trees as a research model for reverse genetic studies.