Quantification of Fungal Gene Expression

The RNA extraction and construction of cDNA libraries was performed as described previously (He et al., 2021 (link); Wei et al., 2022 (link)). Conidia of F. oxysporum (1 × 10⁶ CFU) were added to PDB medium and incubated for 24 h. The mycelia were collected and ground to a powder in liquid nitrogen. Total RNA was extracted from the ground material using RNAiso Plus (TaKaRa, Japan). Real-time, quantitative PCR (RT-qPCR) analysis was performed with a SYBR Green master mix (Monad, Shanghai, China) and the ABI QuantStudio 3 PCR system (Applied Biosystems, Waltham, MA, USA). Relative expression levels of the genes were calculated using the threshold cycle (2^−ΔΔCT also known as 22DDCT) method (Livak and Schmittgen, 2001 (link)). Gene expression levels were normalized against the expression of the 18S rRNA housekeeping gene (Table 3). Details regarding the relevant primers are provided in Supplementary Table S3.