Primer Design and qRT-PCR Analysis

According to the research on A. solani, we selected a relatively stable housekeeping gene "Actin" as the internal reference gene [27 (link), 28 (link)]. Specific primers for Actin and AsCEP50 were designed using the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). PP2A was selected as a housekeeping gene in N. benthamiana [29 (link)]. The SEN4, SAG12 and DHAR1 sequences were obtained from Uniport (https://www.uniprot.org/), and their specific primers were designed using Primer 3 Plus (https://www.primer3plus.com/). The cDNAs of the hyphae and growing on potato and N. benthamiana leaves were serial diluted to detect primer specificity. The RNAs were extracted using an Easy Pure Plant RNA Kit and cDNA were synthesized using a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix. The cDNAs were obtained using an Easy Pure Plant RNA Kit and a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix. All the primers used in qPCR were tested for amplification efficiency by serial dilution. The qRT-PCR was performed using the Bio-Rad CFX384 Touch Real-Time System (Bio-Rad), and the total reaction volumn was 20 μL containing 2 μL gene specific primers, 6 μL of cDNA and 10 μL of 2×Magic SYBR Mixture (CWBIO, Jiangsu, China). The qRT-PCR was performed under the following conditions: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 59°C for 30 s. The threshold cycle (CT) values were used to determine the fold change in transcript accumulation with the 2^−ΔΔCt method [30 (link)]. Six biological and three technical replicates were performed for each sample in all the qRT-PCR reactions.