2.3 Construction of RNA-Seq library and RNA sequencing
Four-week-old A. thaliana were treated with AR156 by root irrigation and the same volume of 0.85% NaCl was used as control. Arabidopsis leaves from different treatments were gathered after 3 d, with three replicates of each treatment. The total RNA extraction method was referenced to the RNA simple Total RNA Kit (TIANGEN, Cat. No. DP419) instructions. RNA-seq experiments were performed at BGI Genomics (Shenzhen, China). The RNA-seq results were uploaded to the NCBI database (BioProject accession number: PRJNA879188).
2.4 RNA extraction and RT-qPCR About 100 mg of treatedArabidopsis leaves were harvested in a 2 mL centrifuge tube, ground to a fine powder with liquid nitrogen, and mixed with 1 mL of TRIZOL followed by 200 μL of chloroform. Total RNA samples were reverse transcribed using the HiScript Q Select RT Super Mix kit (Vazyme, Cat. No. R323). Real-time quantitative PCR was performed using the ChamQ SYBR Color qPCR Master Mix kit (Vazyme, Cat. No. Q711) and the ABI 7500 system. Primers used for RT-qPCR are shown in Supplementary Table S1.2.5 Trypan blue staining To evaluate Arabidopsis leaf lesion infected with P. capsic i. The trypan blue stock solution (a mixture of 10 g phenol, 10 mL glycerol, 10 mL lactic acid, 10 mL deionized water, and 0.02 g trypan blue) was mixed with 96% ethanol at a dilution ratio of 1:2 (v/v). Arabidopsis leaves were collected to the staining solution, boiled in a water bath for 2 min, and stained overnight in the dark. The leaves were decolorized with chloral hydrate solution, and then the lesion area was measured while the results were photographed.2.6 Camalexin analysis The detection of camalexin is referred to the previous description. At a certain time point, 0.1 g of Arabidopsis leaves inoculated with pathogens were taken. The leaves were ground to powder in liquid nitrogen. Add 1 mL extraction buffer to the sample and shake tubes at 4℃ (30 min, 100 rpm). Add another 2 mL dimethyl sulfoxide to the sample, followed by a shaking tube for the same time and conditions. The samples were centrifuged at 4℃ and 5000 g for 10 min. The lower phase was pipetted and the sample was dried in a nitrogen evaporator. The samples were dissolved with 100 μL methanol and filtered through a 0.45 μm filter membrane. The content of camalexin was determined by UFLC-MS/MS system.