Single-cell transcriptomic analyses reveal cellular and molecular patterns of rubber tree response to early powdery...

As a perennial woody plant, the rubber tree (Hevea brasiliensis) must adapt to various environmental challenges through gene expression in multiple cell types. It is still unclear how genes in this species are expressed at the cellular level and the precise mechanisms by which cells respond transcriptionally to environmental stimuli, especially in the case of pathogen infection. Here, we characterized the transcriptomes in Hevea leaves during early powdery mildew infection using single-cell RNA sequencing. We identified 10 cell types and constructed the first single-cell atlas of Hevea leaves. Distinct gene expression patterns of the cell clusters were observed under powdery mildew infection, which was especially significant in the epidermal cells. Most of the genes involved in host-pathogen interactions in epidermal cells exhibited a pattern of dramatically increased expression with increasing pseudotime. Interestingly, we found that the HbCNL2 gene, encoding a nucleotide-binding leucine-rich repeat protein, positively modulated the defence of rubber leaves against powdery mildew. Overexpression of the HbCNL2 gene triggered a typical cell death phenotype in tobacco leaves and a higher level of reactive oxygen species in the protoplasts of Hevea leaves. The HbCNL2 protein was located in the cytomembrane and nucleus, and its leucine-rich repeat domain interacted with the histidine kinase-like ATPase domain of the molecular chaperone HbHSP90 in the nucleus. Collectively, our results provide the first observation of the cellular and molecular responses of Hevea leaves to biotrophic pathogen infection and can guide the identification of disease-resistance genes in this important tree species.