title: CH3-hp-c-water-root
organism: Populus tremula x Populus alba
description: Protocols: Plant leaves and roots were collected and directly frozen in liquid nitrogen Populus×canescens were multiplied by micropropogation and grown on half-strength Murashige and Skoog medium for around 3 weeks in glass jars with controlled room conditions of 24 °C, 16h/8h light/dark cycle with 75 μE m−2⋅s−1 photosynthetically active radiation (PAR). Two different ectomycorrhizal fungal strains, Paxillus involutus (MAJ) and Cenococcum geophilum Fr. were pre-grown on the cellophane membranes with modiﬁed Melin–Norkrans (MMN) mediumfor around 10 days at 20 °C in the darkness. For the co-cultivation of poplar with different ectomycorrhizal fungal isolates, we used the sandwich system as described by Müller et al (2013).The co-culture system of poplar and fungi was incubated in Percival for 3 weeks at 23 °C with a 16h/8h light/dark cycle and 60% relative air humidity. Then each clean plantlet with or without ectomycorrhizal colonization was transferred to the greenhouse and potted into the 3L pot containing a mixture of coarse sand (Ø 0.71-1.25 mm), fine sand (Ø 0.4-0.8 mm), and peat containing no nutrition with the ratio of 8:2:2 by volume.The plants were maintained in a greenhouse chamber under the conditions of 23 °C, 16h light/8h dark with 180 μE m−2⋅s−1 PAR, and 50% relative air humidity. After 16 weeks of normal irrigation, the plants of each fungi group were exposed to well-watered, drought, and re-watered conditions for 4 weeks and harvested. 3 weeks-old poplar were co-cultured with or without different ectomycorrhizal fungi, Paxillus involutus (MAJ) and Cenococcum geophilum Fr. for 3 weeks in Petri dishes, then transferred to the greenhouse. Before drought treatment, plants were well irrigated daily with 200 ml distilled water and 50 ml modified Long Ashton nutrient solution containing (500 μM KNO3, 900μM Ca(NO3)2, 300.2 μM MgSO4, 59.99μM KH2PO4, 4.13μM K2HPO4, 10μM H3BO3, 2μM MnSO4,7μM Na2MoO4, 50 nM CoSO4·7H2O, 200 nM ZnSO4·7H2O, 200 nM CuSO4·5H2O, 10μM EDTA-Fe). After 16 weeks of normal irrigation, each fungi group was divided into 3 subgroups: well-watered, drought, and re-watered. Plants under the well-watered treatment were irrigated with the same amount of water and nutrient solution as before per day, while plants under the drought treatments were only irrigated with 50 ml nutrient solution. For the re-watered treatment, plants were first exposed to drought condition, only irrigated daily with 50 ml nutrient solution for 3 weeks, then rewatered with 200 ml distilled water and 50 ml nutrient solution at the last week. Plant leaves and roots with 3 biological replicates per treatment were used for RNA isolation. About 300 mg frozen samples were grind into powder with a metal ball mixer mill and RNA was extracted with CTAB method as described by Chang et al. (1993). The RNA purification of DNA removal was conducted with the TURBO DNA-freeTM kit. Libraries were constructed using the TruSeq mRNA Sample Prep kit v2 (Illumina) according to the manufacturer's instructions