Abstract Clubroot disease caused by the infection of Plasmodiophora brassicae is widespread in China, and significantly reduces the yield of Chinese cabbage (Brassica rapa L. ssp. pekinensis). However, the resistance mechanism of Chinese cabbage against clubroot disease is still unclear. It is important to exploit the key genes that response to early infection of P. brassicae. In this study, it was found that zoospores were firstly invaded hair roots on the 8th day after inoculating with 1 × 10^7 spores/mL P. brassicae. Transcriptome analysis found that the early interaction between Chinese cabbage and P. brassicae caused the significant expression change of some defense genes, such as NBS-LRRs and pathogenesis-related genes, etc. The above results were verified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Otherwise, peroxidase (POD) salicylic acid (SA) and jasmonic acid (JA) were also found to be important signal molecules in the resistance to clubroot disease in Chinese cabbage. This study provides important clues for understanding the resistance mechanism of Chinese cabbage against clubroot disease. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-024-76634-0. Subject terms: Plant sciences, Transcriptomics Introduction Chinese cabbage (Brassica rapa L. ssp. pekinensis) is Brassica plant in the Cruciferae family^[38]1. Clubroot disease caused by Plasmodiophora brassicae Woronin (P. brassicae) is a worldwide soil-borne disease of the Brassicaceae, which can lead to plant wilting, weak growth, low absorption of water and nutrients to influent the production of Chinese cabbage^[39]2,[40]3. The life cycle of P. brassicae is divided into two main stages: the primary infection of root hair and the secondary infection of the cortex tissue^[41]4,[42]5. In the primary infection period of root hair, each resting spore germinates to a primary zoospore. These zoospores swim to and infect root hairs by penetrating the cell wall. Within the root hairs, primary plasmodia develop quickly and cleave into zoosporangia, each containing 4–16 secondary zoospores. Secondary infection is followed by the development of secondary plasmodia within the root cortex, which results in the production of clubroot symptoms^[43]6. Each secondary plasmodium was cleaved into large numbers of resting spores within the clubbed root. As the root tissues disintegrate, the resting spores are released into the soil and remain in the soil for at least 10 years^[44]7, making it difficult to control in the field once soil is contaminated^[45]8,[46]9. The virulence of populations of P. brassicae contained a range of pathotypes^[47]10. P. brassicae usually exists as a mixture of several pathotypes, which has hampered the research on resistance mechanisms of cruciferous crops against P. brassicae^[48]11. The pathotypes of P. brassicae were commonly assessed using the Williams classification^[49]12, the European Clubroot Differential (ECD) set^[50]13, and Sinitic clubroot differential (SCD) set^[51]14,[52]15. The Williams classification is still widely used because the small number of host lines are easier to manage, requiring less space and time than the other systems. Lv et al. (2021) developed a stable method for isolation and production of single-spore brassicas using Williams system, and revealed pathotype 4 was the most prevalent in China^[53]11. So pathotype 4 was used as pathogen in this study. To P. brassicae populations, when the inoculation concentration was 1 × 10^7 spores/mL, infection rate of root hairs was the highest, and plasmodia spores were found in the root hairs on the 4th day after inoculation^[54]16. Therefore, previous researchers used the P. brassicae populations with a concentration of 1 × 10^7 to infect Chinese cabbage seedlings^[55]17,[56]18. To use pathotype 4 as pathogen for subsequent studies, it is necessary to find its optimal infection concentration and the earliest infection period. Plants are attacked by a variety of microbial pathogens throughout their life cycle. Unlike animals, plants have no adaptive immune system, but they can make use of innate immunity of their own cells to generate systemic acquired resistance (SAR) or induced systemic resistance (ISR)^[57]19. Of these, the salicylic acid (SA) signaling pathway results in the activation of SAR, while the jasmonic acid (JA)/ethylene (ET) signaling pathway is involved in ISR^[58]20. SA signaling generally regulates plant defense against biotrophic pathogens, and JA/ET-dependent signaling pathways are required for resistance against necrotrophic pathogens^[59]21. As an endogenous signal molecule of SAR signal transduction pathway, SA has been demonstrated in tobacco, cucumber and Arabidopsis^[60]22. JA is an important endogenous regulator in plants and a major regulator of induction defense signaling network^[61]23,[62]24. POD is one of the most stable plant enzymes, converts hydrogen peroxide into water^[63]25. Increasing the content of POD in plants can improve the antioxidant capacity of plants, and then improve the stress resistance of plants^[64]26. In recent years, an increasing number of studies have been employed to examine the interactions between hosts and P. brassicae by transcriptome. RNA-seq technology is frequently used and created favorable conditions for us to explore the molecular mechanisms between plants and P. brassicae^[65]3,[66]9,[67]27,[68]28,]. It was predicted the possible regulatory mechanisms among plant genes or proteins after P. brassicae infection of roots and stems through transcriptome analysis. Transcriptome analysis of Arabidopsis at the beginning of P. brassicae infection (24 and 48 h) showed that flavonoids and the lignin synthesis pathways were enhanced, glucosinolates, terpenoids, and proanthocyanidins accumulated and many hormonal and receptor-kinase related genes were expressed^[69]29. In the early stage of infection by P. brassicae, the differences between the R line and S line of Chinese cabbage had been already shown along with the host’s defense against P. brassicae was induced. Among them, the activity of defense enzymes was elevated, and some genes involved in DNA repair were expressed^[70]8,[71]30. Therefore, studying early response genes in the host-pathogen interactions may provide a considerable discovery. In this study, the optimal concentration and time for P. brassicae spore suspension for infecting seedlings of Chinese cabbage were studied; RNA-seq analysis was performed on the roots infected by P. brassicae at the initial stage; The content levels of some key signal molecules were detected. Finally, the earliest regulatory genes were tried to be explored. These results will provide new ideas for understanding the interaction mechanism between Chinese cabbage and P. brassicae in the early infection stage. Materials The materials used in the experiment were susceptible variety‘SN742’of Chinese cabbage and physiological race no.4 of P. brassicae. Both were preserved by Liaoning Key Laboratory of Genetics and Breeding for Cruciferous Vegetable Crops, College of Horticulture, Shenyang Agricultural University. Methods Preparation of P. brassicae spore suspension The pathogen used in this study was NO.4 physiological race of P. brassicae, which was isolated and reproduced with Chinese cabbage in previous work^[72]11. 200 g clubbed roots with NO.4 physiological race of P. brassicae were rinsed three times with sterile water, homogenized in 500 mL of sterile water with a blender (JYL-C022E, Joyoung, China), and then filtered through eight layers of cotton gauze, and the filtrate was clarified by centrifugation twice (BiofugeStratos, Thermo Scientific, Waltham, MA, USA) at 3000 rpm for 9 min and 4000 rpm for 12 min. The concentration of resting spores was measured using a hemocytometer and adjusted to 1 × 10^5, 1 × 10^6, 1 × 10^7, 1 × 10^8 (spores/mL) respectively with Hoagland nutrient solution. Preparation of plant materials Seeds of Chinese cabbage variety ‘SN742’ were reproduced by self-pollination. The seeds were surface-sterilized with 70% ethanol for 1 min, washed twice with sterile water, placed in a petri dish lined with wet filter paper, and cultured in the dark at about 25℃ for about 2 days until the root hairs grew out. The seedlings were divided into two groups: one group was inoculated with P. brassicae (Treatment group, T) and the other group was control group (CK). For treated group, a drop of agar solution (80 µL, 1.0%) was pipetted on a sterilized glass slide. After solidification,1 mL of the different concentration of P. brassicae spore suspension was added to the surface of agarose block and then placed on the root hairs of germinated seeds such that the spore side was touching the roots. After 3 days of cultivation in the dark at 25℃, the seedlings of treated and control group were transferred to a 2 mL uncovered centrifuge tube and cultured with Hoagland nutrient solution in an incubator (16 h light/8 h dark, 25℃, 60% humidity). Three randomly selected plants were observed under a microscope (Nikon Eclipse 80i, Japan) every 24 h until root hair infection was confirmed for sampling. Five plants were randomly selected from T or CK groups to form two sample pools. Three biological replicates were performed for each Pool. The roots were cleaned with distilled water, dried on a paper towel, wrapped in tin foil, quickly frozen in liquid nitrogen, and stored at -80℃. The cytological observation methods for the infection process were as described in a previous study^[73]11. RNA extraction Frozen roots were placed in a mortar, followed by grinding with a pre-cooled pestle in the presence of liquid nitrogen. Total RNA was extracted with the RNA extraction kit (Sangon, Shanghai, China) following the manufacturer’s instructions. The extracted RNA samples were assessed by 1% agarose gel electrophoresis to avoid RNA degradation or impurities. RNA-seq The qualified samples were sent to BGI Shenzhen Co., Ltd. for RNA-seq. Total RNA is extracted from the samples, and the long-stranded RNA is split into short fragments, which are then reverse-transcribed into cDNA. cDNA libraries for sequencing are constructed through steps such as joining joints (adapters) and PCR amplification. The cDNA libraries are sequenced using high-throughput sequencing platforms (e.g. Illumina, Ion Torrent, etc.). Finally, data analysis is performed. The raw reads were cleaned by removing the adapter sequence, N-sequence, and low-quality reads. Differentially expressed genes (DEGs) between two groups were screened by NOISeq method and according to the following: foldchange ≥ 2, false discovery rate (P value) < 0.05, and diverge probability ≥ 0.8. Bioinformatics analysis Gene Ontology (GO) ([74]https://www.geneontology.org) was used to enrich analyze the genes with significant differential expression, and obtained GO annotation of each differentially expressed gene. Kyoto Encyclopedia of Genes and Genomes (KEGG) public database ([75]https://www.kegg.jp/ or [76]https://www.genome.jp/kegg/) was used for pathway enrichment analysis of DEGs. The cDNA sequences of the differential genes were obtained from the Brassica Database ([77]http://brassicadb.cn/#/). Search the National Center for Biotechnology Information (NCBI) database ([78]https://www.ncbi.nlm.nih.gov/) for detailed functions of some key genes. The above references to previous methods^[79]31,[80]32.