GNPS - Transient expression of Arabidopsis UDP-dependent glycosyltransferase candidates in Nicotiana benthamiana

Experimental Goal: To determine a UDP-dependent glycosyltransferase from Arabidopsis capable of glycosylating N-hydroxypipecolic acid, a small molecule signal in plant systemic acquired resistance.

Methods: For screening of candidate UGTs, Agrobacteria harboring Arabidopsis ALD1, FMO1, and respective UGT genes were combined in equal proportions with each at an OD600 of 0.1. In all cases, Agrobacteria harboring GFP was added to ensure an equal final OD600 of 0.6. These solutions were infiltrated into leaves of 4-week-old N. benthamiana plants using needleless syringes. Plants were incubated for 72 h on growth shelves on a 16-h light/8-h dark cycle prior to sample harvest. Plant tissue was harvested, lyophilized to dryness, and homogenized using a ball mill (Retsch MM 400) at 25 Hz for 2 min. Samples were resuspended in 20 ul of 80:20 MeOH:H2O per mg dry tissue and incubated at 4C for 10 min. The liquid fraction of each sample was filtered using a 0.45 um filter and analyzed using previously published methods on an Agilent 1260 HPLC coupled to an Agilent 6520 quadrupole time-of-flight electrospray ionization (Q-TOF ESI) mass spectrometer (Chen et al., 2018. PNAS. E4920-E4929). Samples were analyzed for the production of NHP-Glc (m/z = 308.134).