Nanodrop 1000c spectrophotometer

The types of P. ramorum strains and the isolates of fungal, oomycete, and Bursaphelenchus species that were examined in this study are listed in Table 1. The P. ramorum isolates were obtained from the Animal, Plant, and Food Inspection Center of Nanjing and Shanghai Customs. The other Phytophthora species and fungal isolates used in this study are kept in a collection at the Department of Plant Pathology, Nanjing Forestry University (NFU) in China. All isolates were identified using both morphological and molecular biology methods. Fungal isolates were grown on potato dextrose agar (PDA) at a temperature of 25°C in the dark for 3 to 5 days. Isolates of oomycete species were cultured on 10% clarified V8 juice agar at a temperature of 18 to 25°C in the dark (Xiao et al., 2023 (link)). Bursaphelchus xylophilus and B. mucronatus were propagated for one generation using the mycelia of Botrytis cinerea at 25°C for 4 to 5 days. Genomic DNA (gDNA) was extracted from all the isolates using the DNA secure Plant Kit (Tiangen Biotech, Beijing, China). The extracted gDNA was quantified using a NanoDrop 1000c spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) and diluted accordingly. All DNA samples were stored at a temperature of -20°C until use.

Total RNA was isolated from the tissues of T. chinensis using the EASYspin plant total RNA isolation kit (Aidlab RN38, Beijing, China). RNA integrity and quantity were determined by 1.2% agarose gel electrophoresis and NanoDrop 1000C spectro-photometer (Thermo Scientific, Waltham, MA, USA). Reverse transcription was performed using the FastKing RT Kit (with gDNase) (TIANGEN, Beijing, China). The qRT-PCR are performed with SYBR^® rapid quantitative PCR Kit (KAPA KK4601, Pleasanton, CA, USA). Primers used for qRT-PCR are listed in Table S1. Tcactin was used as a reference gene as described previously [16 ]. Gene expression levels were calculated according to the 2^-∆∆Ct method for the different tissues and aged xylem samples [17 (link),18 (link)].

Total RNA was isolated from the tissues preserved in liquid nitrogen using the RNA extraction kit RN38 (Aidlab Biotech, China). RNA integrity was identifed with a 1.2% agarose gel, and RNA quantity and quality were determined by NanoDrop 1000C Spectro-photometer (Thermo Scientific, USA). The cDNA was obtained by reverse transcription of total RNA using the FastKing RT Kit (With gDNase) KR116 (TIANGEN, China) and used for qRT-PCR by SYBR® rapid quantitative PCR Kit (KAPA KK4601, USA). Gene-specific primers are listed in Table S2 with products lengths between 100 bp and 300 bp. Tcactin was used as a reference gene. Relative abundance of TcMYBs were calculated according to the comparative Cq method described in previous study (Li & Lu, 2014 (link); Ma et al., 2012 (link)). The expression levels of miR159, miR828 and miR858 were analyzed using the method as described previously (Shi & Chiang, 2005 (link)). One-way ANOVA was calculated using IBM SPSS 19 software. P < 0.01 was considered statistically significant and was represented by asterisks. The heat maps of differential expression of TcMYB genes were performed using HemI 1.0 with gradient bar (Clustering Method is Average linkage and Similarity Metric is Pearson distance (default)) (Deng et al., 2014 (link)).