Use of Crispr/Cas genome editing demonstrates a critical role for uricase and xanthine dehydrogenase in soybean nitrogen fixation and nodule development.
C. NGUYEN (1), M. Stacey (2) (1) Divisions of Plant Sciences and Biochemistry, National Center for Soybean Biotechnology University of Missouri, U.S.A.; (2) Divisions of Plant Sciences and Biochemistry, National Center for Soybean Biotechnology University of Missouri, U.S.A.

Previous biochemical studies suggested that de novo purine biosynthesis is required for the incorporation of fixed nitrogen in ureide exporting nodules, as formed on soybean roots.  However, in many cases, the enzymes involved in this pathway have been deduced strictly from genome annotations with little direct genetic evidence, such as mutant studies, to confirm their biochemical function or importance to nodule development.  While efforts to develop large mutant collections of soybean are underway, research on this plant is still hampered by the inability to obtain mutations in any specific gene of interest. However, this situation is now significantly changed through the ability to apply the methods of CRISPR/Cas9 genome editing using Agrobacterium rhizogenes-mediated hairy root transformation. Using this approach, we were able to generate homozygous mutant roots lacking either uricase (UOX) or xanthine dehydrogenase (XDH) activity.  The uox knock out soybean mutants were unable to fix nitrogen, as exemplified by their internal greenish appearance reflecting a lack of leghemoglobin production. As added confirmation, a uox knock-out mutation generated through fast neutron mutagenesis displayed a similar phenotype. Similarly, a knock out XDH mutant, generated with the Crispr/Cas system, also displayed a fix- phenotype. These studies demonstrate the great utility of the Crispr/Cas system for studying root associated gene traits when coupled with hairy root transformation. Furthermore, these genetic studies confirm the critical role of the de novo purine biosynthetic pathway, not in incorporation of fixed nitrogen but in the successful development of a functional, nitrogen fixing nodule.

Abstract Number: C1-6, P2-60
Session Type: Concurrent