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Transformed Hairy Roots of Arachis hypogea: A Tool for Studying Root Nodule Symbiosis in a Non--Infection Thread Legume of the Aeschynomeneae Tribe

Arachis hypogea is a non--“infection thread” (IT) legume where rhizobial entry or dissemination in the nodules never involves IT. Rhizobia invade through epidermal “cracks” and directly access the cortical cells to develop the characteristic aeschynomenoid nodules. For investigating these nonclassical nodulation features in Arachis spp., we developed an efficient procedure for Agrobacterium rhizogenes R1000-mediated transformation of this plant. In this study, we optimized the induction of hairy roots and nodulation of composite Arachis hypogea plants in the presence of Bradyrhizobium sp. (Arachis) strain NC92. 35S promoter-driven green fluorescent protein and β-glucuronidase expression indicated transformation frequency to be above 80%. The transformed roots had the characteristic rosette-type root hairs and had normal level of expression of symbiosis-related genes SymRK and CCaMK. The transgenic nodules resembled the wild-type nodules with an exception of 2 to 3%, where they structurally deviated from the wild-type nodules to form nodular roots. A 16S rRNA profile of an infected-zone metagenome indicated that identical populations of bradyrhizobia invaded both composite wild-type plants grown in natural soil. Our results demonstrate that Arachis hairy root is an attractive system for undertaking investigations of the nonclassical features associated with its nitrogen-fixing symbiotic interactions.