Endophyte DNA was isolated following previously reported methodologies (Araujo et al 2002, Conn and Franco 2004, Miles et al 2012). Briefly, the plant tissue was surface-sterilized by washing with sterile H2O to remove dirt, placed in NAP buffer (124mM Na2HPO4.H2O) and vortexed for 1min to dislodge epiphytes. Leaves were then shaved to remove the pubescence on their surface, which facilitates the subsequent sterilization process (Miles et al 2012), washed with sterile H2O, submerged in 90% ethanol (60s), 5.25% sodium hypochlorite solution (6min), and 70% ethanol (30s), and finally rinsed with sterile distilled water. Sterilization was checked by taking an imprint of the leaf on Malt Extract Medium (Miles et al 2012) and incubating at 25ºC. 1g of the previously treated material was cut into 0.1-0.5mm sections, placed in a 1.5mL Eppendorf tube containing 1g of sterile 0.1mm-diameter glass beads and 1mL TE (10mM Tris, 10mM EDTA, pH 8.0) and homogenized in a mini-bead beater (BioSpec Products) for 5min. DNA was extracted using the PowerSoil DNA Isolation Kit (MOBIO Laboratories, Carlsbad, CA; USA), following the manufacturer’s instructions. We obtained epiphyte DNA by first releasing bacteria from the surface of leaves by submerging 10-20g of healthy plant tissue in 100mL of Release buffer (0.1M Potassium Phosphate, 0.1% Glycerol, 0.15% Tween 80, pH 7.0), and vortexing for 7 minutes (Bodenhausen et al 2013, Izhaki et al 2013, Zhang et al 2010). The remaining bacteria were dislodged from the leaves with the help of a sterile swab and the buffer was then filtered through a 0.2µm-pore filter. DNA was extracted using the PowerSoil DNA Isolation Kit. Combined epiphyte and endophyte DNA was extracted from root and necromass samples by cutting the tissue into 0.5-1cm fragments that were placed in 25mL of Release buffer in a 50mL tube and homogenized by vortexing for 10 minutes. The buffer was filtered through a 0.2µm-pore filter and the filters were used for DNA extraction using the PowerSoil DNA Isolation. All DNA extractions were quantified using Qubit® 2.0 Fluorometer (Life Technologies Corporation, Carlsbad, CA; USA). In total, we obtained six epiphyte and six endophyte DNA extractions, corresponding to the upper and mid tiers from three plant replicates, three DNA extractions for the necromass tier, one for each replicate, and two for the roots.
Label
Cy3
Label protocol
Samples were labelled according to the protocol described previously (He et al 2010, Yan et al 2015) (Glomics (Norman, OK; USA).
Hybridization protocol
Samples were hybridized according to the protocol described previously (He et al 2010, Yan et al 2015) (Glomics (Norman, OK; USA).
Scan protocol
Samples were scanned according to the protocol described previously (He et al 2010, Yan et al 2015) (Glomics (Norman, OK; USA).
Data processing
(i) remove the poor quality spots, which were flagged as 1 or 3 by ImaGene or with a signal to noise ratio (SNR) of less than 2.0; (ii) normalize the signal intensity of each spot by dividing the signal intensity by the total intensity of the microarray followed by multiplying by a constant; (iii) transform the data to the natural logarithmic form;
