RNA-Seq and data analysis

According to the reported method⁷⁰. RNA-Seq and data analysis were carried out. The treatments used for transcriptome sequencing were as follows: 7 mM lactic acid treatment for 24 h (LA24h) or DW treatment for 24 h (DW24h), after spraying with 7 mM lactic acid, and inoculated with P. nicotianae for 24 h (LA_Pn) or mock inoculation for 24 h (LA_Mo), after spraying with DW, and inoculated with P. nicotianae for 24 h (DW_Pn) or mock inoculation for 24 h (DW_Mo). Leaves from the same part of three plants were collected and immediately frozen using liquid nitrogen and stored at − 80 °C for transcriptome sequencing.

Total RNA was extracted using TRIzol reagent (Invitrogen), and each sample was purified with Plant RNA Purification Reagent (Invitrogen) according to the manufacturer’s instructions. The samples were analyzed by 2100 Agilent Technologies for RNA size quantification and quality control. Each transcriptome library consists of 1 μg total RNA. According to the manufacturer’s instructions, the TruSeqTM RNA sample preparation kit (Illumina, Inc., San Diego, CA) was used to generate a sequencing library. The library preparation and high-throughput RNA sequencing were completed using HiSeq 4000 equipment (Illumina) operated by Major Genome Center, Shanghai, China.

RawData were obtained by sequencing on an Illumina high-throughput sequencing platform, and CleanData were obtained by removing linker sequences and low-quality reads. CleanData and tobacco reference genome (https://www.ncbi.nlm.nih.gov/genome/425?Genome_assembly_id=274804) to obtain MappedData. RSEM software was used to analyze the differential expression of transcripts. Then, selected DEGs were annotated using the GO function and enriched using the KEGG pathway. DEGs were screened using DESeq2, and p < 0.01 and a multiple change (FC) ≥ 2 was selected as the threshold for significant differentially expressed genes (SDEGs). Based on the GO and KEGG databases, the functional enrichment analysis of all SDEGs was carried out using GoTools (github.com/Tang Haibao/GoTools) and Kobas software (kobas.cbi.pku.edu.cn/home.do). In Mapman software, absolute log2 times change data are used, and a heat map is constructed using the gene cluster method.