Sybr green 1 master mix

Total RNA was extracted from frozen tissues using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Contaminating DNA was removed using RNase-free DNase (Qiagen). The RNA (0.5 μg) was reverse transcribed using a High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. The sequences of four Zmtat and 12 Zmyuc genes were obtained from the MaizeGDB database (http://www.maizegdb.org/) (Supplementary Table S1 at JXB online). Primers specific for the Zmtat, Zmyuc, and Zmubiquitin genes (Supplementary Table S2; Nishimura et al., 2014 ) were used to amplify gene transcripts. The KOD FX DNA polymerase (Toyobo, Osaka, Japan) was used for the RT–PCR. The resulting amplified products were analysed by 3% agarose gel electrophoresis and visualized by ethidium bromide staining. qPCR was completed using a LightCycler 480 system (Roche, Basel, Switzerland) with the SYBR Green I Master Mix (Applied Biosystems). Zmubiquitin was used as the reference gene to normalize transcription levels. All qPCR experiments were performed using three biological replicates and two technical replicates. Melting and standard curves were prepared for each qPCR experiment.

All primer pairs used in PCR, outward PCR, and quantitative real time PCR are listed in Table 2. PCR was performed to determine the presence of transducing phage particles harboring MGEs using primers specific to MGEs associated with strain RF122 including: νSaα (the set gene), νSaβ (the lukE gene), and νSaγ (the hla gene), SaPIbov1 (the tst gene), SaPI122 (the mdr gene), and ϕSaBov (the int gene). To estimate the frequency of transducing phage particles harboring MGEs, the absolute copy number of each MGE per nanogram of phage DNA was determined using quantitative real time PCR. Briefly, PCR products specific to MGEs (above) were cloned into pCR™4-TOPO® TA Vector (Life Technologies). Plasmids were quantified using a Nanodrop (Thermo Scientific) and the number of DNA copies/ng was calculated as described previously [19 (link)]. Quantitative real time PCR reaction was performed using SYBR green I master mix (Applied Biosystems) and a serial dilution of plasmid templates. Standard curves were generated by linear regression analysis calculating the slope, intercept, and correlation coefficient (R²) using Microcal OriginPro (Microcal origin, Version 7.5). Quantification of MGEs was calculated by interpolation the C_T from the standard curve.

Expression levels of miRNAs and miRNA* were examined by quantitative real-time PCR following the protocol in previous works (Varkonyi-Gasic et al., 2007 (link); Turner et al., 2013 (link)). Total RNAs were prepared from control plants or different transformants and treated with DNase before being subjected to cDNA synthesis using Superscript III reverse transcriptase (Invitrogen) primed by miRNA/miRNA*/U6-Specific primers (Supplementary file 1G). Quantitative real-time RT-PCR was performed in 384-well plates with an ABI 7900HT real-time PCR system using the SYBR Green I master mix (Applied Biosystems) in a volume of 10 μl. PCR conditions included one cycle at 50°C for 2 min, one cycle at 95°C for 10 min and 50 cycles of 96°C for 10 s followed by 60°C for 1 min. The U6 gene (Turner et al., 2013 (link)) was included as an internal control for normalization. Three biological replicates were performed, and the reactions were performed in triplicate for each run. The comparative C_T method was used to evaluate the relative quantities of each amplified product in the samples. The threshold cycle (C_T) was automatically determined for each reaction by the system.