Thermal cycler dice tp800 system

Relative fungal growth was measured by quantitative real-time polymerase chain reaction (qPCR). All measurements were performed with three biological replicates, and each replicate consisted of five plants. Genomic DNA was extracted from plant tissues using MagExtractor (Toyobo, Osaka, Japan) following the manufacturer’s instructions. Real-time PCR was run on a Thermal Cycler Dice TP800 system (Takara Bio Inc., Otsu, Japan) using SYBR premix Ex Taq mixture (Takara) with cycles of 95°C for 5 s, 55°C for 20 s, and 72°C for 20 s. Three technical replicates were used for each biological replicate sample. The PCR primers used were as follows: (1) primers targeting the intergenic spacer region of the C. ilicicola rDNA, CiIGSF (forward) = 5′-TCCATTGCCTCTATTTATCCTGC-3′, and CiIGSR (reverse) = 5′-GCGTAAAGATTTTCCAACCCG-3′ (Ochi and Kuroda, 2021 (link)); (2) primers for soybean β-Actin gene (Gm-β-Actin; Glyma.15G050200), Gm-β-ActinF (forward) = 5′-GAGCTATGAATTGCCTGATGG-3′, and Gm-β-ActinR (reverse) = 5′-CGTTTCATGAATTCCAGTAGC-3′ (Sugano et al., 2013 (link)). Relative fungal growth was expressed as C. ilicicola rDNA amplification folds relative to the host actin gene amplification (Jiang et al., 2020 (link)).

Total RNA was isolated from rice leaves using Trizol reagent (Invitrogen, www.invitrogen.jp/) and purified with an RNeasy mini kit (Qiagen, http://www.qiagen.com). The RNAs were treated with DNase (Takara, www.takara-bio.co.jp/) and reverse-transcribed into cDNA using Revertra Ace (Takara) and oligo(dT)₂₃ primers (Sigma-Aldrich). qRT-PCR was performed with a Thermal Cycler Dice TP800 system (Takara) using a KAPA SYBR fast universal qPCR kit. Expression levels relative to the rice ubiquitin 1 (Rubq1, Os06g0681400) gene, whose expression was not affected by M. oryzae infection both in NB and WRKY45-ox rice (Fig. S5), were quantified using the delta–delta Ct method. Primers used in this study are listed in Table S1.

Rice seedlings at the four-leaf stage were blast-inoculated, and the inoculated fourth leaf blades of half the plants was cut off at 2 dpi. Sixth whole leaf was collected at 3 dpi, and leaf blades and leaf sheathes separately at 6 dpi. Three biological replicates were collected, four leaves in each replicate.
Real time-polymerase chain reaction (RT-PCR) was used to analyze the samples for expression of marker genes for JA (JAmyb and OMT), ABA (SalT and OsWsi18), auxin (ARF1 and IAA9), GA (OsGA2ox3 and OsGA20ox1) and SA (WRKY45 and OsNPR1), and PR genes OsPR1b and PBZ1. The genes and primer sequences used for qRT-PCR are listed in Supplementary Table 1.
Total RNA was isolated using the TRIzol reagent (Invitrogen) and reverse-transcribed by using ReverTra Ace (TOYOBO, Osaka, Japan) according to the manufacturer’s protocol. Quantitative RT-PCR (qRT-PCR) was run on a Thermal Cycler Dice TP800 system (Takara Bio) using SYBR premix ExTaq mixture (Takara Bio) as previously described (Shimono et al., 2007 (link)).