Gene transcript analysis

For a complete list of nucleotide sequences of primers used in this study please refer to [3]. A. flavus was grown as described above and the total fungal RNAs were isolated using an RNeasy kit according to the manufacturer’s instructions (Qiagen, Germany). DNA-free RNAs samples were diluted to 50 ng L^− 1 using RNase-free water and stored at − 80 °C. Subsequently, the respective cDNAs were synthesized using M-MLV RT (Invitrogen) as described previously [2]. Gene transcripts were evaluated by RT-qPCR using an AriaMx Real-time PCR System (Agillent technologies, USA). The 25 μl reaction mixtures contained 0.5 μl of each of the target and reference gene primers, 12.5 μl of SYBR Green qRT-PCR mix (Bio-Rad, USA) and 2.5 μl of 10-fold diluted cDNA. RT-qPCR conditions were as previously described [2]. Each point was triplicated and the average of C_T was taken. The relative quantification RQ ₌ 2^{(−∆∆CT)} of the target gene was determined using the software of an Agillent AriaMx Real-time PCR System.