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Short resume:

Parasponia represents five fast-growing tropical tree species in the Cannabeaceae, which are known as the only species besides legumes that can establish nitrogen-fixing nodules with rhizobium. We establish Parasponia as a research model to allow genetic dissection of the nitrogen fixation trait. Comparative genomics of Parapsonia and legumes uncovered a conserved gene set for nodulation. To investigate the function of these genes in Parasponia we established an efficient and relatively fast stable transformation protocol. In combination with CRISPR/Cas9 mutagenesis, biallelic mutant lines can be obtained in a period of three-to-four months. Due to efficient in vitro micro-propagation, mutant plantlets can be studied in T0 generation, allowing phenotypic evaluation within six months after transformation. We mutated 20 conserved nodulation genes to study their function in the Parasponia-rhizobium nitrogen-fixing symbiosis. This provided novel insights into evolution the nitrogen fixation trait.