Integration of single nucleus RNA-seq and bulk RNA-seq reveals gene regulatory networks for vascular connection between parasitic plants and host plants.

The facultative parasitic plant Phtheirospermum japonicum forms a specialized organ, the haustorium, to invade its host, Arabidopsis thaliana, establishing a vascular connection via the formation of a xylem bridge. This connection depends on coordinated interactions between the vascular systems of both plants, yet the molecular dynamics of these interactions within the haustorium and the host roots remain elusive. This study aimed to unravel the transcriptomic heterogeneity of haustoria and gene regulatory networks involved in this process by integrating single nucleus RNA sequencing (snRNA-seq) and bulk RNA sequencing (bulk RNA-seq). snRNA-seq identified a total of 7 P. japonicum cell clusters and 4 A. thaliana cell clusters, each with cluster-specific marker genes, allowing for a distinct characterization of vascular cells within the haustorium. Differential gene expression analyses revealed up-regulation of genes associated with xylem formation and auxin transport in both parasites and hosts, suggesting the presence of shared molecular mechanisms facilitating vascular connection. Further gene network analysis combining snRNA-seq and bulk RNA-seq identified conserved homologous genes across both species, indicating potential molecular interactions of vascular-related genes from hosts and parasites. Our study reveals the high heterogeneity of haustorium cells, characterizing the expression profiles of each cell type in haustoria and host roots during haustorium development at single-cell resolution. These findings provide insights into the molecular interactions between parasitic plants and hosts, presenting potential targets for disrupting these interactions to manage parasitic plant infestations in crops.