R3 Non-inoculated + water deficit stress
The experiment was conducted with four treatments (non-inoculated + water deficit stress, non-inoculated + irrigated, NT33 + water deficit stress, NT33 + irrigated), and the treatments were replicated five times. All the treatments were irrigated with 500 ml of water per pot daily before inducing water stress. Three weeks after transplanting, irrigation was stopped for five days for the water deficit stress treatment, while control plants were irrigated daily. Leaf samples for the transcriptomic analysis were collected on the 5th day of irrigation withholding for the creation of water stress. For RNA extraction, the terminal leaflet from the fully expanded third leaf from the top was selected. Leaf samples were collected from five replications per treatment. Samples were frozen immediately in liquid nitrogen and stored at -80C until further use. RNA was extracted using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany), following the manufacturers instructions. During extraction, samples were treated with the RNAase free DNaseSet (Qiagen, Hilden, Germany) for the complete digestion of DNA during the RNA purification process by following the manufacturers protocol. The five biological replicates for each treatment were used for RNA sequencing. The RNA library was generated using the Illumina TruSeq standard mRNA Library Prep Kit (Illumina, San Diego, CA, USA) at the MCIC. The library was sequenced at OSU-The James Comprehensive Cancer Center Shared genomic resources in two lanes of the Illumina NovaSeq 6000 (SP-flow cell; 2 x 150 bp) instrument.