Plant-parasitic root-knot nematodes (Meloidogyne species) are highly polyphagous parasites that alter cellular identity of terminally differentiated root cells to induce the formation of giant cells and knot-like structures known as galls, whose ontogeny remains largely unknown. In this study, we generated single-nucleus RNA-seq data of galls and neighboring root tissues at two distinct stages of Meloidogyne incognita infection of tomato (Solanum lycopersicum) plants. Analysis of 35 393 high-quality nuclei resulted in the identification of three stele-associated cell clusters that captured young and more differentiated giant cells, where 772 genes were preferentially expressed. Giant cell-specific expression patterns of a set of these genes were validated using promoter activity assays. We used pseudotime analysis to trace how gene activity changes as giant cells develop. Developmental trajectory analysis revealed a gradual activation of more complex gene regulatory networks as young giant cells adopt specific fates and become more differentiated. Functional assays using gene silencing confirmed the functional importance of giant cell-expressed genes in mediating plant susceptibility to M. incognita. Cell type-specific gene expression analysis revealed that xylem, phloem, stele, endodermal, and protophloem cells undergo extensive transcriptome reprograming, which facilitates coordinated cellular responses to nematode infection, including immune signaling, structural support, and metabolic adjustments. Together, our analyses represent the first single-nucleus transcriptomic map of nematode-induced giant cells and provide novel insights into the molecular events leading to the formation of a new plant organ and feeding cells orchestrated by an animal parasite.

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