Maize Total RNA Isolation and qRT-PCR Analysis

The maize tissue total RNAs were isolated by using the method was described by Yu et al.^{61 (link)}, and first-strand cDNAs were synthesized using a PrimeScript First Strand cDNA Synthesis kit (TaKaRa, Japan). The cDNAs were combined with SYBR master mix (TIANGEN, China), and an ABI7300 system (Applied Biosystems, USA) was used to monitor the kinetics of the PCR products for qRT-PCR (consist of 94 °C for 3minutes, and then 40 cycles of 94 °C for 30 s, 60 °C for 15 s, 72 °C for 34 s). The maize actin gene (GRMZM2G126190) was used as an internal control for normalization of the template cDNA. The amount of accumulated transcript of the ZmWRKY65 gene normalized to the internal actin control gene was determined using the 2^−ΔΔCT method. The qRT-PCR primers of ZmWRKY65 were provided in Table S2. Each sample PCR was repeated three to four biological replicates.