Single-Cell RNA-Sequencing Reveals the Generation of New Immune Response-Induced Cells with Novel Role in <i>Arabidopsis thaliana</i> Response to the Bacterial Flagellin Epitope flg22

Plant immune responses can inhibit the growth and development of seedlings and alter cell differentiation. The cell types most vulnerable to the immunity stress induced by plant defense responses and the extent to which cell differentiation is altered remain largely unknown. Here, we report on the exploration of single-cell RNA-sequencing approach to identify genes and regulatory processes underlying the fate and development of cotyledon cell types of wild-type (WT) and flagellin sensing 2 (fls2) mutant seedlings in response to the bacterial flagellin epitope flg22. After the treatment with flg22, two new immune response-induced cells (IMC) types were detected in WT but not in the fls2 seedlings. Immune-response marker genes were highly expressed in one IMC type, while photosynthesis-related genes in the other IMC type. Changes in cell differentiation were investigated using pseudotime trajectory analysis. Treatment of seedlings with flg22 affected both interactions and differentiation of various cell types of the cotyledon. Gene ontology analysis revealed that genes involved in hypoxia signaling were upregulated in almost all cell types in response to the activation of innate immunity. Subsequent analysis indicated that hypoxia signaling interacts with immune response signaling and plays a role in both seedling and IMC development. Transcription factor network analysis and subsequent loss-of-function analysis revealed that the APETALA2/ethylene-responsive transcription factor ERF13 plays an important role in regulating IMC differentiation both in innate immunity and under hypoxia stress. Results of our study provide new insights into how the interaction between innate immune response and hypoxia signaling regulates cell differentiation.