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 2 strategies to identify and functionally characterize sunflower genes in response to invading broomrape. First, we established a transient overexpression system via seed-soak agroinfiltration, providing a convenient expression system for functional genomics in sunflower. Second, transcriptome sequencing of 3 sunflower cultivars following O. cumana infection identified 190 common differentially expressed genes, 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.

Plants adapt to abiotic stresses by a variety of physiological and molecular mechanisms, among which the root plays important roles via responding to underground and soilborne signals. Fructan is a polysaccharide that is related to energy metabolism and diverse adverse adaptations. Orobanche cumana is a holo-parasitic plant which mainly attaches to the root of host sunflower (Helianthus annuus). Here we characterized a O. cumana fructan 6-exohydrolase, Oc6-FEH, which was involved in both fructan metabolism and flooding responses. Expression of Oc6-FEH is induced by flooding and IAA treatment. Oc6-FEH possesses fructan catabolism activity and is associated with fructose release. Overexpression of Oc6-FEH in host sunflower reduces the production of MDA and H2O2, boosts the activation of antioxidant enzymes such as POD and SOD, and also elevates the photosynthetic activities. Expression level of Oc6-FEH is positively associated with flooding tolerance of invading O. cumana which is connected to host root. Furthermore, IAA treatment also improves flooding tolerance of O. cumana. In summary, the metabolism of fructan and the activity of Oc6-FEH are demonstrated to ameliorate waterlogging stress. Oc6-FEH provides a promising genetic target for improvement of flooding tolerance in crops.

Plants adapt to abiotic stresses by a variety of physiological and molecular mechanisms, among which the root plays important roles via responding to underground and soilborne signals. Fructan is a polysaccharide involved in energy metabolism and stress adaptation. Orobanche cumana is a holo-parasitic plant that mainly attaches to the root of the host sunflower (Helianthus annuus). Oc6-FEH, a fructan 6-exohydrolase from O. cumana, is involved in both fructan metabolism and flooding responses. Expression of Oc6-FEH is induced by flooding and indole-3-acetic acid (IAA). Oc6-FEH possesses fructan catabolism activity and is associated with fructose release. Overexpression of Oc6-FEH in the host sunflower reduces malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) accumulation, boosts the activities of antioxidant enzymes, including peroxidase (POD) and superoxide dismutase (SOD), and enhances photosynthetic performance. The expression level of Oc6-FEH was found to be positively associated with the flooding tolerance of invading O. cumana, which is connected to the host root. Furthermore, IAA treatment also improved the flooding tolerance of O. cumana. In summary, the metabolism of fructan and the activity of Oc6-FEH were demonstrated to ameliorate waterlogging stress. Oc6-FEH provides a promising genetic target for the improvement of flooding tolerance in crops.

Plant viral diseases cause severe yield losses in global crop production. SRC7, a novel soybean TIR-NBS-BSP-type resistance (R) protein, confers broad-spectrum antiviral immunity against multiple viruses including soybean mosaic virus (SMV), tobacco mosaic virus (TMV), potato virus Y (PVY), and plum pox virus (PPV). Its TIR-NBS domain (SRC7^TN) mediates full antiviral resistance, making it an ideal bait for screening immune-related proximal candidate proteins. In this study, TurboID-based proximity labeling (PL) was applied to profile the SRC7^TN proximal candidate proteins in N. benthamiana. We identified 262 high-confidence proximal candidate proteins, which were mainly enriched in plant immune signaling, redox regulation, protein modification and nuclear-related pathways. Among them, 21 candidates were functionally characterized, revealing multiple host factors contributing to SRC7-dependent antiviral immunity. Eleven interactions were confirmed via bimolecular fluorescence complementation (BiFC), and two nucleoside/nucleotide hydrolase-family proteins MZN14.5 and RIHA were subjected to further interaction-domain analysis. Collectively, this work constructs a high-confidence SRC7^TN proximal-interaction network and highlights critical host factors in SRC7-mediated broad-spectrum antiviral immunity. It also validates TurboID’s utility for screening for NLR-proximal candidate proteins in heterologous plant systems and provides insights into R-protein-governed plant antiviral defense.