Single‐cell transcriptome atlas reveals a WRKY33 ‐ HSP81 regulatory module that orchestrates phenylpropanoid‐mediated defense against Sclerotium rolfsii in Dendrobium officinale

Plants employ cell-type-specific transcriptional programs to defend against pathogens, yet the cellular heterogeneity of immune responses in the medicinal orchid Dendrobium officinale Kimura et Migo remains largely unexplored. A single-cell transcriptomic atlas of the D. officinale stem base upon Sclerotium rolfsii infection was constructed, profiling over 20 000 cells across 12 and 24 h post-infection. A total of 21 cell clusters were resolved into 6 major cell types-epidermal, ground tissue parenchyma, proliferating, xylem, phloem, and vascular bundle parenchyma cells-with identities validated by fluorescence in situ hybridization. Cell-type-specific analyses revealed that epidermal and phloem cells mounted early defense responses, while xylem cells exhibited the most extensive transcriptional reprogramming at 24 hpi, coinciding with the activation of lignin biosynthesis pathways. WRKY33 was identified as a central transcriptional activator that directly binds W-box motifs in the promoters of lignin biosynthetic genes C4H and HCT, promoting lignin deposition and enhancing disease resistance. Conversely, the nuclear-localized chaperone HSP81 physically interacts with WRKY33 and functions as a broad regulator of multiple transcription factors via co-factor sequestration, thereby compromising defense when overexpressed. These findings uncover a WRKY33-HSP81 regulatory module that orchestrates phenylpropanoid-mediated defense in D. officinale, providing a high-resolution cellular framework for understanding plant immunity and offering potential targets for improving disease resistance in this economically valuable species.