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

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 allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for . Here, we present a fast and efficient protocol for -mediated transformation and CRISPR/Cas9 mutagenesis of . Using this protocol, knockout mutants are obtained within 3 months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T generation, allowing phenotypic evaluation within 6 months after transformation. We mutated four genes - , and - that control cytokinin, ethylene, or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in and displayed developmental phenotypes, namely reduced procambium activity in and disturbed sex differentiation in mutants. The symbiotic phenotypes of and mutant lines differ from those in legumes. In contrast, and 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 trees as a research model for reverse genetic studies.