Plant material, growth conditions, and infection
N. dimidiatum was isolated and stored at Enhong Agricultural Technology Co., Ltd., Banqiao Town, Dongfang City, Hainan Province. The hyphae were collected from N. dimidiatum by culture on PDA medium at 15 °C, 28°C, and 37°C for 5 d, N. dimidiatum was cultured on PDA medium at 28 °C, and the hyphae were collected at 3 d, 5 d, and 10 d (Fig. 1).
Pitaya twigs with the same growth state were collected from the pitaya field, their surfaces were sterilized with 75% ethanol in the laboratory of the School of Tropical Crops, Hainan University, rinsed with tap water, and then dried. Spore suspension that had been cultured in PD medium for a week was collected. The spore suspension was evenly sprayed on the surface of pitaya, and the branches were kept moist at room temperature and 80% humidity, there were three replicates in each group. Lastly, pitaya–N. dimidiatum interaction samples were collected on 3 d, 4 d, and 5 d, frozen in liquid nitrogen, and stored at-80°C.
RNA extraction and cDNA synthesis
Total RNA from pitaya–N. dimidiatum interaction samples was extracted following the protocol of Xu et al. [2]. Harvested mycelia were ground to a fine powder in liquid nitrogen using a pre-cooled mortar and pestle. Total RNA of N. dimidiatum from the samples was extracted using TRI Reagent® RNA Isolation Reagent (Sigma, Shanghai, CHN) per the manufacturer’s instructions and then treated with RNase-free DNase I at 37 ℃ for 30 min. Purification with TRI Reagent® RNA Isolation Reagent was repeated to remove DNase I. RNA quality was analyzed by 2.0% agarose gel electrophoresis, and RNA integrity was assessed using a Nanophotometer (Implen, GER). Only RNA samples with an A260/A280 ratio of 1.8–2.2 and an A260/A230 ratio > 1.8 were used for further analysis. cDNA was synthesized from 1.0 μg of total RNA samples using a HiScript®Ⅲ cDNA Synthesis Kit per the manufacturer’s instructions (Vazyme).
Candidate gene selection and primer design
The expression of eight candidate reference genes (18S rRNA, Actin (1), Actin (2), Actin, Tubulin, GAPDH (1), GAPDH (2), and UBQ) was analyzed. The specific primers (Table 1) were designed using Primer Premier 5.0 with the following parameters: melting temperature (Tm), 48–61 °C, GC percent, 40–60%, primer lengths, 18–22 bp, and product lengths, 100–200 bp.
To confirm that the correct region was amplified by the selected primers, primer pairs were used to prime amplification in 100 ng of the first-strand cDNA in an end-point PCR reaction. For each genotype, one sample of synthesized first-strand cDNA was selected randomly and used for all primer tests. The master mix for this reaction was prepared using the HiScript®ⅠⅠⅠ One Step RT-qPCR SYBR® Green Kit (Vazyme Biotech Co., Ltd., Nanjing, China, Cat. No. R333) per the manufacturer’s instructions. The amplification was carried out using the following thermal cycling parameters: initial denaturation at 95 ℃ for 5 min, followed by 30 cycles of denaturation at 94℃ for 30 s, annealing for 30 s at the primer pair’s average annealing temperature, and an extension of 45 s at 72 ℃. After completion of the last cycle, a final extension was carried out at 72 ℃ for 5 min [20]. To confirm that a single product was amplified, the PCR products were loaded into a 1.5% agarose-TAE gel stained with ethidium bromide and run at 120 V for 15 min at room temperature. Gel images were obtained using Gel Doc™ XR+ (Bio-Rad, USA).
RT‑qPCR analyses
RT-qPCR assays were performed in a 20-μL reaction system using the ChamQ™ Universal SYBR® qPCR Master Mix kit (Q711–02/03, Vazyme Biotech Co., Ltd., Beijing, China) per the manufacturer’s protocol. Candidate genes were selected for RT-qPCR assays using an Applied Biosystems 7500 RT-qPCR System (Life Tech, 81 Wyman Street, Waltham, MA, 02454, USA). The thermal cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, 40 cycles at 95 °C for 15 s, 56 °C for 30 s, and 72 °C for 40 s. The melting curve was generated by heating the amplicon from 60 to 95 °C to confirm primer specificity. Three biological replicates were carried out for each PCR reaction. Relative fold changes in gene expression were calculated using the comparative 2-ΔΔCT method [21].
Analysis of gene expression stability
NormFinder, geNorm, and BestKeeper were used to evaluate the expression stability of the reference genes in N. dimidiatum at various development stages, at different temperatures, and during interaction with pitaya.
The geNorm program determines the stability and optimal number of genes required to calculate the M-value and pairwise variation Vn/Vn + 1 between two sequential normalization factors [22]. The optimal candidate reference genes were determined based on the average expression stability between samples and the results of variance analysis [23]. The BestKeeper program was used to identify stable reference genes, which calculates pairwise correlations based on values of standard deviation (SD) and percentage covariance (Cov) [24].
Validation of reference genes
To verify the reliability of the selected reference genes. Pectate lyase [25], an enzyme involved in the digestion of the cell wall of pitaya, was analyzed using RT-qPCR in the early stages of N. dimidiatum infection of pitaya. The specific primers used are shown in Table 1.