Single-nucleus RNA-sequencing reveals the cellular programs driving nematode-induced giant cell formation in tomato

Abstract

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.

Acknowledgments

We thank S. Piya for advice on data analysis. This work was supported by funds from California League of Food Producers, University of Tennessee, Institute of Agriculture, and USDA-Agricultural Research Service (5070-21000-044-00D).

Author contributions

S.B.Z. performed most of the experiments, analyzed data, and wrote the first draft. P.L. and C.O. collected tissue samples for snRNA-seq libraries. M.S.S. and H.M.K.A. performed promoter activity assays. N.C., M.E., and J.H.R. contributed to nematode susceptibility assays. H.B.K. contributed to project conceptualization, data analysis, and manuscript editing. T.H. designed and supervised the experimental work, analyzed data, and wrote the final version. All authors contributed to the article and approved the submitted version.

Data availability

snRNA-seq data were submitted to the NCBI Gene Expression Omnibus (GEO) database under the accession number GSE289841.

Conflict of interest statement

The authors declare no competing interests.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Jones JT, Haegeman A, Danchin EGJ. et al. Top 10 plant-parasitic nematodes in molecular plant pathology. Mol Plant Pathol. 2013; 14:946-61

[2] Hewezi T, Baum TJ. Communication of sedentary plant-parasitic nematodes with their host plants. In: Becard G (ed.), Advance in Botanical Research, Vol. 82. 2017,305-24

[3] Escobar C, Barcala M, Cabrera J. et al. Overview of root-knot nematodes and giant cells. In: Escobar C, Fenoll C (eds.), Advances in Botanical Research, Vol. 73. 2015,1-32

[4] Jammes F, Lecomte P, de Almeida-Engler J. et al. Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsis. Plant J. 2005; 44:447-58

[5] Fuller VL, Lilley CJ, Atkinson HJ. et al. Differential gene expres-sion in Arabidopsis following infection by plant-parasitic nema-todes Meloidogyne incognita and Heterodera schachtii. Mol Plant Pathol. 2007; 8:595-609

[6] Shukla N, Yadav R, Kaur P. et al. Transcriptome analysis of root-knot nematode (Meloidogyne incognita)-infected tomato (Solanum lycopersicum) roots reveals complex gene expression profiles and metabolic networks of both host and nematode during suscep-tible and resistance responses. Mol Plant Pathol. 2018; 19:615-33

[7] Ozdemir S, Piya S, Lopes-Caitar VS. et al. Local and systemic transcriptome and spliceome reprogramming induced by the root-knot nematode Meloidogyne incognita in tomato. Hortic Res. 2024;11:uhae206

[8] Caillaud M-C, Dubreuil G, Quentin M. et al. Root-knot nematodes manipulate plant cell functions during a compatible interaction. J Plant Physiol. 2008; 165:104-13

[9] Domínguez-Figueroa J, Gómez-Rojas A, Escobar C. Functional studies of plant transcription factors and their relevance in the plant root-knot nematode interaction. Front Plant Sci. 2024; 15:1370532

[10] Atighi MR, Verstraeten B, De Meyer T. et al. Genome-wide shifts in histone modifications at early stage of rice infection with Meloidogyne graminicola. Mol Plant Pathol. 2021; 22:440-55

[11] Silva AC, Ruiz-Ferrer V, Müller SY. et al. The DNA methylation landscape of the root-knot nematode-induced pseudo-organ, the gall, in Arabidopsis, is dynamic, contrasting over time, and critically important for successful parasitism. New Phytol. 2022; 236:1888-907

[12] Bennett M, Piya S, Baum TJ. et al. miR778 mediates gene expres-sion, histone modification, and DNA methylation during cyst nematode parasitism. Plant Physiol. 2022; 189:2432-53

[13] Bennett M, Hawk TE, Lopes-Caitar VS. et al. Establishment and maintenance of DNA methylation in nematode feeding sites. Front Plant Sci. 2023; 13:1111623

[14] Ozdemir S, Zadegan SB, Sultana MS. et al. Regulation and func-tions of long non-coding RNAs during Meloidogyne incognita par-asitism of tomato. Mol Plant-Microbe Interact. 2024; 38:72-83

[15] Chavan SN, Degroote E, De Kock K. et al. ARGONAUTE4 and the DNA demethylase REPRESSOR OF SILENCING 1C mediate dehydroascorbate-induced intergenerational nematode resis-tance in rice. Plant Physiol. 2025;197:kiae598

[16] Hewezi T. Epigenetic mechanisms in nematode-plant interac-tions. Annu Rev Phytopathol. 2020; 58:119-38

[17] Fosu-nyarko JOHN, Jones MG, Wang Z. Functional character-ization of transcripts expressed in early-stage Meloidogyne javanica-induced giant cells isolated by laser microdissection. Mol Plant Pathol. 2009; 10:237-48

[18] Barcala M, García A, Cabrera J. et al. Early transcriptomic events in microdissected Arabidopsis nematode-induced giant cells. Plant J. 2010; 61:698-712

[19] Damiani I, Baldacci-Cresp F, Hopkins J. et al. Plant genes involved in harbouring symbiotic rhizobia or pathogenic nematodes. New Phytol. 2012; 194:511-22

[20] Ji H, Gheysen G, Denil S. et al. Transcriptional analysis through RNA sequencing of giant cells induced by Meloidogyne graminicola in rice roots. JExp Bot. 2013; 64:3885-98

[21] Portillo M, Cabrera J, Lindsey K. et al. Distinct and con-served transcriptomic changes during nematode-induced giant cell development in tomato compared with Arabidopsis: a functional role for gene repression. New Phytol. 2013; 197: 1276-90

[22] Jean-Baptiste K, McFaline-Figueroa JL, Alexandre CM. et al. Dynamics of gene expression in single root cells of Arabidopsis thaliana. Plant Cell. 2019; 31:993-1011

[23] Gulati GS, Sikandar SS, Wesche DJ. et al. Single-cell transcrip-tional diversity is a hallmark of developmental potential. Science. 2020; 367:405-11

[24] Dorrity MW, Alexandre CM, Hamm MO. et al. The regulatory landscape of Arabidopsis thaliana roots at single-cell resolution. Nat Commun. 2021; 12:3334

[25] Frank M, Fechete LI, Tedeschi F. et al. Single-cell analysis iden-tifies genes facilitating rhizobium infection in Lotus japonicus. Nat Commun. 2023; 14:7171

[26] Li C, Zhang S, Yan X. et al. Single-nucleus sequencing deciphers developmental trajectories in rice pistils. Dev Cell. 2023; 58:694-708.e4

[27] Liew LC, You Y, Auroux L. et al. Establishment of single-cell tran-scriptional states during seed germination. Nat Plants. 2024; 10: 1418-34

[28] Yang L, Yang Z, Liu M. et al. Integrated single-cell transcrip-tomics and spatial metabolomics unveil cellular differentiation and ginsenosides biosynthesis in Panax root tips. Hortic Res. 2025;uhaf202

[29] de Almeida Engler J, Kyndt T, Vieira P. et al. CCS52 and DEL1 genes are key components of the endocycle in nematode-induced feeding sites. Plant J. 2012; 72:185-98

[30] de Almeida Engler J, de Vleesschauwer V, Burssens S. et al. Molec-ular markers and cell cycle inhibitors show the importance of cell cycle progression in nematode-induced galls and syncytia. Plant Cell. 1999; 11:793-807

[31] Caillaud M-C, Lecomte P, Jammes F. et al. MAP65-3 microtubule-associated protein is essential for nematode-induced giant cell ontogenesis in Arabidopsis. Plant Cell. 2008; 20:423-37

[32] de Almeida Engler J, van Poucke K, Karimi M. et al. Dynamic cytoskeleton rearrangements in giant cells and syncytia of nematode-infected roots. Plant J. 2004; 38:12-26

[33] Favery B, Chelysheva LA, Lebris M. et al. Arabidopsis formin AtFH6 is a plasma membrane-associated protein upregulated in giant cells induced by parasitic nematodes. Plant Cell. 2004; 16: 2529-40

[34] Wieczorek K, Elashry A, Quentin M. et al. A distinct role of pectate lyases in the formation of feeding structures induced by cyst and root-knot nematodes. Mol Plant-Microbe Interact. 2014; 27:901-12

[35] Mitchum MG, Sukno S, Wang X. et al. The promoter of the Ara-bidopsis thaliana Cel1 endo-1, 4-β glucanase gene is differentially expressed in plant feeding cells induced by root-knot and cyst nematodes. Mol Plant Pathol. 2004; 5:175-81

[36] Kyndt T, Goverse A, Haegeman A. et al. Redirection of auxin flow in Arabidopsis thaliana roots after infection by root-knot nematodes. JExp Bot. 2016; 67:4559-70

[37] de Almeida Engler J, Gheysen G. Nematode-induced endoredu-plication in plant host cells: why and how. Mol Plant-Microbe Interact. 2013; 26:17-24

[38] de Almeida Engler J, Vieira P, Rodiuc N. et al. The plant cell cycle machinery:usurped and modulated by plant-parasitic nematodes. In: Escobar C, Fenoll C (eds.), Advances in Botanical Research, Vol. 73. 2015,91-118

[39] Favery B, Quentin M, Jaubert-Possamai S. et al. Gall-forming root-knot nematodes hijack key plant cellular functions to induce multinucleate and hypertrophied feeding cells. J Insect Physiol. 2016; 84:60-9

[40] Abad P, Castagnone-Sereno P, Rosso M-N. et al. Invasion, feeding and development. In: Perry RN, Moens M, Starr JL (eds.), Root-Knot Nematodes. CABI: UK, 2009,163-81

[41] Aniento F, de Medina S, Hernández V. et al. Molecular mecha-nisms of endomembrane trafficking in plants. Plant Cell. 2022; 34: 146-73

[42] Kraus M, Pleskot R, Van Damme D. Structural and evolution-ary aspects of plant endocytosis. Annu Rev Plant Biol. 2024; 75: 521-50

[43] Schatlowski N, Stahl Y, Hohenstatt ML. et al. The CURLY LEAF interacting protein BLISTER controls expression of polycomb-group target genes and cellular differentiation of Arabidopsis thaliana. Plant Cell. 2010; 22:2291-305

[44] Salem MA, Li Y, Bajdzienko K. et al. RAPTOR controls devel-opmental growth transitions by altering the hormonal and metabolic balance. Plant Physiol. 2018; 177:565-93

[45] Jamsheer KM, Awasthi P, Laxmi A. The social network of target of rapamycin complex 1 in plants. JExp Bot. 2022; 73:7026-40

[46] Wang S, Chang Y, Ellis B. Overview of OVATE FAMILY PROTEINS, a novel class of plant-specific growth regulators. Front Plant Sci. 2016; 7:417

[47] Chevalier D, Batoux M, Fulton L. et al. STRUBBELIG defines a receptor kinase-mediated signaling pathway regulating organ development in Arabidopsis. Proc Natl Acad Sci. 2005; 102: 9074-9

[48] Vaddepalli P, Fulton L, Batoux M. et al. Structure-function analy-sis of STRUBBELIG, an Arabidopsis atypical receptor-like kinase involved in tissue morphogenesis. PLoS One. 2011; 6:e19730

[49] Hammes UZ, Schachtman DP, Berg RH. et al. Nematode-induced changes of transporter gene expression in Arabidopsis roots. Mol Plant-Microbe Interact. 2005; 18:1247-57

[50] Bartlem DG, Jones MGK, Hammes UZ. Vascularization and nutri-ent delivery at root-knot nematode feeding sites in host roots. JExp Bot. 2014; 65:1789-98

[51] Rodiuc N, Vieira P, Banora MY. et al. On the track of transfer cell formation by specialized plant-parasitic nematodes. Front Plant Sci. 2014; 5:160

[52] Siddique S, Grundler FM. Parasitic nematodes manipulate plant development to establish feeding sites. Curr Opin Microbiol. 2018; 46:102-8

[53] Siddique S, Grundler FMW. Metabolism in nematode feeding sites. In: Escobar C, Fenoll C (eds.), Advances in Botanical Research, Vol. 73. 2015,119-38

[54] Smant G, Helder J, Goverse A. Parallel adaptations and common host cell responses enabling feeding of obligate and facultative plant parasitic nematodes. Plant J. 2018; 93:686-702

[55] Hewezi T. Phytopathogens reprogram host alternative mRNA splicing. Annu Rev Phytopathol. 2024; 62:173-92

[56] Zhang T-Q, Chen Y, Liu Y. et al. Single-cell transcriptome atlas and chromatin accessibility landscape reveal differen-tiation trajectories in the rice root. Nat Commun. 2021; 12: 2053

[57] Lee TA, Nobori T, Illouz-Eliaz N. et al. A single-nucleus atlas of seed-to-seed development in Arabidopsis. 2023;bioRxiv

[58] Wolf FA, Angerer P, Theis FJ. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 2018; 19:1-5

[59] McGinnis CS, Murrow LM, Gartner ZJ. DoubletFinder: doublet detection in single-cell RNA sequencing data using artificial nearest neighbors. Cell Syst. 2019; 8:329-337.e4

[60] Young MD, Behjati S. SoupX removes ambient RNA contam-ination from droplet-based single-cell RNA sequencing data. Gigascience. 2020;9:giaa151

[61] Lun ATL, McCarthy DJ, Marioni JC.A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconduc-tor.F1000Res. 2016; 5:2122

[62] Street, Townes FW, Risso D. et al. Scry: small-count analysis methods for high-dimensional data. R Packag version 1. 2021. https://bioconductor.org/packages/scry.html

[63] Kajala K, Gouran M, Shaar-Moshe L. et al. Innovation, conserva-tion, and repurposing of gene function in root cell type develop-ment. Cell. 2021; 184:3333-3348.e19

[64] Farmer A, Thibivilliers S, Ryu KH. et al. Single-nucleus RNA and ATAC sequencing reveals the impact of chromatin accessibility on gene expression in Arabidopsis roots at the single-cell level. Mol Plant. 2021; 14:372-83

[65] Denyer T, Ma X, Klesen S. et al. Spatiotemporal develop-mental trajectories in the Arabidopsis root revealed using high-throughput single-cell RNA sequencing. Dev Cell. 2019; 48: 840-852.e5

[66] Ryu KH, Huang L, Kang HM. et al. Single-cell RNA sequencing resolves molecular relationships among individual plant cells. Plant Physiol. 2019; 179:1444-56

[67] Shahan R, Hsu CW, Nolan TM. et al. A single-cell Arabidopsis root atlas reveals developmental trajectories in wild-type and cell identity mutants. Dev Cell. 2022; 57:543-560.e9

[68] Setty M, Kiseliovas V, Levine J. et al. Characterization of cell fate probabilities in single-cell data with Palantir. Nat Biotechnol. 2019; 37:451-60

[69] Waskom ML. Seaborn: statistical data visualization. JOpenSource Softw. 2021; 6:3021

[70] Kolde R. R pheatmap: pretty heatmaps. R package v1.0.12. 2019. https://CRAN.R-project.org/package=pheatmap

[71] Hadley W. Ggplot2:Elegrant Graphics for Data Analysis.Cham, Switzerland: Springer, 2016

[72] Neuwirth E. RColorBrewer: Colorbrewer Palettes. 2014. Available online at: https://CRAN.Rproject.org/package=RColorBrewer

[73] Petrova V, Niu M, Vierbuchen T. et al. Studying cis-regulatory heterogeneity in single-cells at allelic resolution. 2025; bioRxiv

[74] Hafemeister C, Halbritter F. Single-cell RNA-seq differential expression tests within a sample should use pseudo-bulk data of pseudo-replicates. 2023; bioRxiv

[75] Muzellec B, Teleńczuk M, Cabeli V. et al. PyDESeq2: a python package for bulk RNA-seq differential expression analysis. Bioin-formatics. 2023;39:btad547

[76] Sultana MS, Niyikiza D, Hawk TE. et al. Differential transcrip-tome reprogramming induced by the soybean cyst nematode Type 0 and Type 1.2.5.7 during resistant and susceptible inter-actions. Mol Plant-Microbe Interact. 2024; 37:828-40