Sunflower HaWRKY29 dampens Orobanche cumana parasitism via transcriptional activation of HaLAC17 and lignin deposition

Orobanche cumana Wallr. is a root holoparasitic plant that mainly parasitizes and, therefore, threatens sunflower (Helianthus annuus L.) production. Compared with other pathogens, the molecular mechanisms underlying host resistance to parasitic plants remain largely elusive. Here, we used two strategies to identify and functionally characterize sunflower genes in response to invading broomrape. First, we established a transient overexpression system via seed-soak agroinfiltration (SSA), providing a convenient expression system for functional genomics in sunflower. Second, transcriptome sequencing of three sunflower cultivars following O. cumana infection identified 190 common differentially expressed genes (DEGs), among which WRKY family genes were highly enriched and were therefore functionally characterized. HaWRKY6 slightly facilitated O. cumana infection, while both WRKY transcription factor 29 (HaWRKY29) and WRKY transcription factor 53 (HaWRKY53) dampened O. cumana parasitism, with the former having the more pronounced effects. During the early stages of parasitism, HaWRKY29 induced lignin deposition at infection sites, blocked vascular connections between the parasite and host, and caused tubercle necrosis. Further investigations revealed that HaWRKY29 transcriptionally activates laccase 17 (HaLAC17) expression, a key gene in lignin biosynthesis, thereby increasing lignin content and establishing a physical barrier that impedes O. cumana infection. Moreover, mitogen-activated protein kinase 3-1 (HaMPK3-1) and mitogen-activated protein kinase 3-2 (HaMPK3-2) physically interacted with HaWRKY29 and enhanced its transcriptional activation on HaLAC17. Our study reveals the signaling module MPK3-WRKY29 activates host resistance to parasitic plants through upregulation of HaLAC17 expression and subsequent lignin deposition.