An integrated framework to elucidate mechanisms underlying host-branched broomrape infection

Abstract

Branched broomrape (Phelipanche ramosa) is an obligate root parasitic weed that threatens tomato production in many regions. Progress in understanding host resistance mechanisms has been hindered by the parasite’s subterranean life cycle and the technical limitations of traditional soil-based assays. Here, we introduce an integrated experimental framework that enables molecular, genetic, and cellular analysis of broomrape parasitism in tomato under controlled conditions. We implemented a transparent, soil-less co-cultivation system for non-destructive, real-time monitoring of broomrape development on tomato roots, and a dual-compartment in vitro co-culture system supporting parasite infection of transgenic hairy roots. This methodology enabled rapid functional testing of candidate host resistance genes, exemplified by CRISPR-edited mutants of the tomato transcription factor SCHIZORIZA (SlSCZ), which displayed localized lignin accumulation at the parasite entry site in the root. The observed lignification suggests a role for this gene in regulating inducible cell wall lignification against broomrape. Together, these tomato-focused integrated methods enable reproducible imaging, genetic perturbation, and high-resolution analysis of host–parasite interfaces. These provide a scalable platform for dissecting broomrape resistance and accelerating resistance gene discovery in tomato and a critical tool for combating the devastating consequences of this parasite on agriculture.

Introduction

Parasitic weeds such as broomrapes (Orobanche and Phelipanche spp.) are obligate root parasitic plants that complete most of their life cycle underground, attaching to host roots and extracting essential nutrients (Bennett and Mathews 2006, Joel 2009). Among them, branched broomrape (Phelipanche ramosa) poses severe threats to global agriculture by significantly reducing yields of economically important dicot crops such as tomato (Solanum lycopersicum) (Parker 2009). In heavily infested fields, tomato yield and quality can decline by up to 80% (Longo et al. 2010). Conventional management strategies have primarily relied on chemical control, including soil fumigation and pre- or post-emergence herbicide application, but these methods offer limited efficacy and raise environmental concerns. In addition, only a small number of tomato resistance loci have been reported. Limited attention to molecular and genetic approaches has left sustainable branched broomrape control methods relatively unexplored. These approaches offer opportunities to uncover and utilize molecular machinery that underlies host–parasite interactions as a viable long-term broomrape control strategy. The California Department of Food and Agriculture has classified branched broomrape as a category A pest, making research into this parasitic plant difficult outside of Contained Research facilities (Osipitan et al. 2021).

The broomrape life cycle has defined developmental stages. Seeds undergo a conditioning phase under moist, dark conditions in the rhizosphere during which imbibition and metabolic activation (e.g. gibberellin biosynthesis) relieve physiological dormancy. Germination is then triggered by host-derived strigolactones that are exuded from the host roots. This ensures that radicle emergence occurs only in proximity to a suitable host. The emergent radicle grows toward the host, differentiating at the apex into a haustorium that secretes cell wall-degrading enzymes to penetrate the host root cells. Following successful invasion, a tubercle forms, serving as a storage organ. Within the tubercle, xylem–xylem and phloem–phloem connections establish continuous pathways for the uptake of water, minerals, and nutrients from the host. (Joel and Losner-Goshen 1994, Xie et al. 2010). After tubercle maturation, aerial shoots emerge to flower and set seed. Each plant produces up to 500 000 seeds that disperse via water, wind, animals, and farm machinery and can persist in the soil seed bank for decades (Gevezova et al. 2012). Broomrape developmental processes largely take place beneath the soil surface, which limits access for research, leaving major features of parasite perception, attachment, and vascular integration insufficiently characterized.

Studying these interactions is further complicated by the genetic intractability of many broomrape host crops, such as sunflower and cowpea, which are recalcitrant to transformation, limiting reverse-genetic approaches (Somers et al. 2003, Darqui et al. 2021, Kausch et al. 2021). Tomato, in contrast, is genetically tractable and represents a promising model for dissecting host responses to broomrape infection. However, field-based assays and traditional soil systems lack the spatial resolution, reproducibility, and accessibility required for molecular-level analysis.

Host resistance mechanisms include both pre-attachment defenses, such as inhibiting parasite seed germination, and post-attachment strategies that block haustoria invasion or limit nutrient uptake by the parasite by blocking vascular connections (Zhang et al. 2022). Elucidation of post-attachment resistance mechanisms in field trials or soil experiments is difficult due to environmental heterogeneity of the rhizosphere, difficulty in imaging roots within soil, and the presence of other soil microbes and fungi, all of which can interfere with molecular-level analyses that utilize RNA sequencing and gene expression assays.

Controlled laboratory-based experimental methods, including transparent rhizotrons, semi-hydroponic setups, and sterile in vitro co-culture systems, have emerged as practical alternatives to traditional field studies (Losner-Goshen et al. 1996, Clarke et al. 2020, Eizenberg et al. 2003, Zhou et al. 2004). These platforms offer several advantages, including reproducible environmental conditions (temperature, humidity, and nutrients), consistent parasite inoculum application, and the ability for year-round experimentation. Rhizotrons filled with soil or sand permit real-time visualization of underground interactions, providing detailed insights into early stages of parasite infection, including germination, attachment, and development. Likewise, sterile in vitro co-culture systems allow precise manipulation of growth conditions and treatments, including nutrient concentrations, application of germination stimulants, inhibitors, or signaling molecules that coordinate various aspects of the parasite life cycle. Yet each approach has limitations: soil-based rhizotrons, while ideal for visualization, are space-consuming and can be laborious in their construction for imaging adult roots, while detached in vitro systems may not support later stages of parasite development. To overcome these constraints, transparent, soil-less rhizotron systems have emerged as an alternative to visualize below-ground parasite development, including germination, attachment, and early growth (Parker and Dixon 1983, Goldwasser et al. 1997; Goldwasser et al. 2002). Here, we developed an integrated tomato-focused experimental framework combining two complementary systems for studying broomrape infection in tomato building on previously described methods: (i) we implemented a transparent soil-less rhizotrons system for synchronized, high-throughput broomrape infection assays in tomato which is useful for comparative experiments, mutant screening, and high-throughput assessment of host responses under controlled environmental conditions. (ii) A dual-compartment in vitro hairy root culture system optimized for genetic manipulation and early-stage infection analysis. The co-cultivation pouch system is excellent for controlled experimentation and saves space. Multiple pouches can be simultaneously grown in larger tubs arranged in a hanging file folder as a compact array with a small footprint. The hairy root in vitro culture system employs Rhizobium rhizogenes-mediated “hairy root” transformation. Hairy roots are developmentally and functionally similar to wild-type roots and offer a fast and high-throughput system for genetic manipulation (Ron et al. 2014, Cheng et al. 2021, Ramasamy et al. 2023). Transgenic hairy roots can be established within weeks, bypassing the lengthy and technically challenging processes of generating stable transgenic plants. Hairy root cultures are grown independently of the shoot on nutrient media, enabling controlled co-culture with the parasite for high-throughput infection assays. We demonstrate that hairy root cultures can be used to perform genetic screens for defects in parasite attachment to the host root.

This study demonstrates the efficacy of this integrated experimental framework to investigate branched broomrape parasitism in tomato, combining soil-less co-culture systems and in vitro hairy root cultures. Together, these platforms offer complementary strengths for real-time monitoring of parasitism, genetic perturbations of the host, and functional validation across multiple biological scales from whole root phenotypes to specific gene targets for broomrape resistance.

Results

Soil-less co-cultivation pouch system enables monitoring of broomrape developmental stages

To characterize broomrape attachment in tomato, we used a transparent, soil-less co-cultivation system, similar to previously established soil-less rhizotron approaches with slight modifications. This system consistently supported tomato root growth and maintained adequate moisture and nutrient levels throughout the experimental period (Fig. 1a and b). Following preconditioning in the dark and germination stimulation with GR24 (a synthetic strigolactone analog), branched broomrape seeds exhibited synchronized germination within five days of co-culture. Germinated branched broomrape seeds with an emerging radicle were observed along the primary and lateral roots of tomato, and haustoria attachment was confirmed using stereomicroscopy (Fig. 1c). By eight days post-inoculation, early-stage tubercles (localized swellings ~0.5 mm) were consistently observed along tomato primary and lateral roots, indicating host penetration and parasite development. By Day 14, tubercles began transitioning to the “early spider” stage, characterized by radial expansion and lateral branching of parasite tissue (Fig. 1d). This approach proved reproducible and allowed for non-destructive monitoring of broomrape development across multiple stages. Quantification of broomrape attachment across four commercially available tomato cultivars (Heinz hybrids H9553, H9775, and HM Clause HM58841 and HM4885) demonstrated parasite attachment and statistically significant differences observed between cultivars (Fig. 1e). The transparent pouch assembly further enabled repeated imaging and facilitated downstream applications including sampling for transcriptomic profiling and spatially targeted imaging and analysis of host–parasite interfaces.

Figure 1.

Tomato–broomrape co-cultivation in a soil-less germination pouch system. (a) Overview of the transparent, soil-less pouch setup used to monitor tomato root-broomrape infections. Tomato seedlings were grown vertically along glass fiber sheets saturated with nutrient solution. (b) High-throughput germination pouch setup with tomato seedlings grown upright in a filing box for parallel screening of broomrape infection. (c) Close-up image showing germinated broomrape seeds attached to tomato roots during early infection stages. Haustorium and tubercle development is visible at the root interface. (d) Representative image of a later “spider” stage of broomrape infection, showing extensive parasite attachment and early developing tubercles along tomato roots. (e) Quantification of broomrape attachments across four commercially available tomato genotypes using the germination pouch system. Boxplots show the number of attachments per replicate. Each dot represents individual biological replicates colored by genotype. Letters indicate statistically significant groupings based on a one-way Analysis of Variance (ANOVA) followed by Tukey’s HSD (P < 0.05). n = 15, scale bar = 1 mm.

Tomato hairy root in vitro cultures support early-stage broomrape infection

To enable root-specific genetic and functional analysis in tomato during parasite infection, we evaluated the potential of hairy roots as a host system for branched broomrape. R. rhizogenes-induced tomato hairy roots were generated and maintained under sterile in vitro conditions. Media-based in vitro infections have precedent, including growing excised host roots on nutrient medium with parasite seeds presented on a low-nutrient surface, as well as callus-based infections on media, in which differentiated Orobanche calli were generated to infect the host rather than proceeding from parasite seeds (Losner-Goshen et al. 1996, Zhou et al. 2004). We retain the split-media logic but differ in three practical respects: (i) Host tissue: CRISPR-edited transgenic hairy roots enabling direct gene function analysis in tomato-broomrape interactions; (ii) Source of infection: infections initiated from preconditioned branched broomrape seeds to maintain the normal developmental sequence from seed to radicle to haustorium trajectory rather than callus-derived infections which is shown to lower infection rates; (iii) Microbial minimization: Earlier systems applied broad-spectrum antibiotic cocktails to the media to suppress contamination from broomrape seeds, here instead 0.06% Plant Preservative Mixture (PPM) was applied to the parasite (low-nutrient) compartment during preconditioning/placement. Higher PPM concentrations impaired broomrape germination and viability, confirming a narrow window for effective use. This localized PPM minimizes microbial growth yet preserves germination/attachment, and it keeps the host compartment antibiotic-free, reducing the risk of confounding effects on the infection process (Fig. 2a). Hairy roots grow and extend from the rich medium into the low-nutrient zone, where they encounter preconditioned broomrape seeds, which support parasite attachment to the host. This setup improved assay reproducibility and extended the duration of co-culture, allowing broomrape seedlings to attach successfully to tomato hairy roots within 10 days, with early-stage tubercles visible under stereomicroscopy (Fig. 2b and c). While this system effectively captured early-stage infection events, it did not support later-stage parasite development beyond the early spider stage, likely due to nutrient limitations in the split-media setup or a lack of photosynthetic tissue. Nonetheless, these results demonstrate that tomato hairy roots, cultured in a spatially partitioned media, provide a reliable system for dissecting host genetics in early-stage broomrape parasitism.

Figure 2.

In vitro co-cultivation system for broomrape infection on tomato hairy roots. (a) Overview of the co-culture setup: transgenic tomato hairy roots grown on MS medium in the middle, surrounded by agar containing no nutrients to support parasite seed placement and attachment. (b) Early-stage broomrape seedling attached to a tomato root, showing radicle swelling and haustorium formation. (c) Later-stage “early spider” broomrape infection and development.

To verify that observed haustoria attachments resulted in functional parasitism, we performed confocal laser scanning microscopy on transverse sections of broomrape infected wild-type hairy roots. Samples were stained with Direct Yellow 96 to visualize cell walls and Basic Fuchsin to detect lignin deposition. In infected wild-type roots, broomrape intrusive cells were observed penetrating the host cortex and reaching the central vascular cylinder (Fig. 3a). Xylem continuity between host and parasite was also observed, indicating the formation of functional xylem–xylem bridges (Fig. 3b). To preserve the spatial organization of host–parasite interactions that is often disrupted during sectioning, cleared whole-mount roots were examined using differential interference contrast (DIC) microscopy. Rapid Potassium Hydroxide (KOH) clearing followed by toluidine blue staining enabled clear visualization of xylem–xylem connections along the longitudinal axis of the interface, marked by characteristic secondary wall thickenings (Fig. 3c and d). These results validate that the hairy root in vitro system supports functional parasitism and is suitable for investigating host genetic responses to broomrape.

Figure 3.

Cellular resolution assessment of broomrape infection in tomato hairy roots. (a) Confocal microscopy of the tomato–broomrape interface. Transverse section of a wild type tomato hairy root infected by broomrape, showing haustorium intrusive cells penetrating the host cortex. Cell walls are visualized using fluorescent staining (cyan: cell walls; magenta: lignified structures). (b) Transverse section at a later stage of infection showing a fully developed xylem bridge between broomrape and tomato, indicating successful vascular connection. (c) Cleared tomato hairy root with an attached broomrape seedling. (d) Magnification of the xylem bridge at the attachment site in 3c. Lignin is visualized by Basic Fuchsin and cell wall staining is DY-96. Ep, epidermis; Ex, exodermis; Co, cortex; En, endodermis; Pe, pericycle; Xy, xylem; HR, host root; HX, host xylem; PH, parasite haustorium; PT, parasite tubercle.

CRISPR-edited mutants of SlSCZ generated using the hairy root in vitro culture system block broomrape vascular connection via increased lignin deposition

One plant defense response to broomrape infection is cell wall remodeling, a well-established mechanism that restricts parasite penetration and haustorial development. Lignification, suberization, and reinforcement of the cell wall at the host–parasite interface have been reported in resistant species, acting as physical and biochemical barriers to infection (Mutuku et al. 2019, Jhu et al. 2022, Kawa et al. 2024). Recent work shows that tomato roots have an exodermis layer that forms a polar lignin cap (PLC), an apoplastic diffusion barrier functionally analogous to an endodermal Casparian strip, yet genetically independent. Two transcription factors, SCHIZORIZA (SlSCZ) and SlEXO1, were shown to regulate PLC polarity and restrict lignification to the exodermis (Manzano et al. 2025). We hypothesized that loss of SlSCZ would further induce lignin deposition outside the exodermal PLC upon broomrape challenge. To test this, we used hairy root mutagenesis to analyze CRISPR-edited roots targeting SlSCZ (Manzano et al. 2025).

Wild-type hairy roots, when challenged with broomrape, did not trigger ectopic lignin deposition at the attachment site, and a xylem–xylem bridge was established (Fig. 4a). In uninfected slscz mutant alleles, ectopic lignin deposition is observed and restricted to a few cells in the inner cortex (Fig. 4b and c), whereas broomrape infection triggered ectopic lignin deposition across multiple cortical layers surrounding the invading haustorium, supporting that broad lignification is infection-induced (Fig. 4d and e). Lignin signal was clearly higher at sites of parasite attachment in the slscz mutant alleles compared to infected wild-type roots (Fig. 4f). Tubercles formed on slscz roots developed late compared to the wild-type roots and failed to grow beyond the initial swelling (Fig. 4g). This data suggests that SlSCZ disruption primes cortical cells for a lignin-based defense response upon broomrape invasion and that lignin accumulation at the interface may prevent or delay parasite access to host vasculature. These results provide a framework for dissecting post-attachment resistance in tomato and for testing additional genes involved in exodermal barrier formation in parasite susceptibility.

Figure 4.

Lignin-based resistance in slscz mutant tomato roots during broomrape infection. (a) Cross section of a wild type hairy root infected by broomrape. (b and c) Cross-sections of two uninfected slscz mutant alleles showing previously demonstrated ectopic lignification in the inner cortex cells (white arrowheads). (d and e) Cross-sections of infected slscz roots from two independent alleles, showing limited penetration of broomrape haustorium arrested near the cortex tissue (d) or epidermis (e), without formation of a xylem bridge. (f) Fluorescence from Basic Fuchsin staining was measured manually using ImageJ software. Letters indicate statistically significant groupings based on a one-way ANOVA followed by Tukey’s HSD (P < 0.05) (n = 6 sections per uninfected control line and 4 attachments/sections per each slscz line). (g) Representative images of branched broomrape tubercles on slscz hairy roots. Tubercles remain small, with arrested development. Lignin is visualized by Basic Fuchsin (magenta) and cell wall staining is Direct Yellow 96 (cyan). Ep, epidermis; Ex, exodermis; Co, cortex; En, endodermis; Pe, pericycle; Xy, xylem; HR, host root; PH, parasite haustorium; PT, parasite tubercle.

Discussion

Understanding broomrape parasitism in tomato and other host crops has remained a challenge due to the parasite's underground life cycle and the complexity of host–parasite interactions. The integrated experimental framework presented here addresses these challenges by combining a soilless co-culture pouch system in vitro hairy root cultures, each contributing complementary advantages in genetic accessibility and physiological fidelity.

The pouch system enabled synchronized, non-destructive monitoring of parasite development and quantification of infection dynamics across multiple tomato genotypes. Its simplicity and scalability make it suitable for comparative assays, environmental treatments, and downstream molecular sampling. The split-media hairy root system, on the other hand, offers a genetically tractable platform for functional studies of host genes involved in parasite recognition, attachment, and defense. The system supported early stages of broomrape infection and enabled in situ analysis of host responses using confocal microscopy.

Using this platform, we show that CRISPR-mediated disruption of SlSCZ, a transcription factor that represses cortical lignification, induced enhanced lignin deposition at broomrape infection sites, which results in failure to form xylem–xylem bridges (Fig. 4d and e). These observations suggest that SlSCZ acts as a negative regulator of lignin-based defenses and that its loss primes the host for post-attachment resistance. This mechanism aligns with previous reports of lignification blocking parasitic invasion in other species (Mutuku et al. 2019, Jhu et al. 2022) and highlights the utility of this system for identifying functional resistance genes in tomato.

While the hairy root system provides a powerful platform for studying early infection events, it does not support full parasite development. This limitation likely reflects the absence of whole-plant physiological processes required for sustained parasite growth, such as long-distance nutrient flow and systemic signaling. To address this, future work could incorporate composite plants, which retain transformed roots within a shoot-root system. Composite hosts have been shown to support complex biotic interactions, including nodulation, mycorrhization, and broomrape parasitism (Boisson-Dernier et al. 2001, Runo et al. 2012, Ho-Plágaro et al. 2018). Unlike detached root cultures, composite plants ensure the parasite encounters a photosynthetically active host with normal nutrient flow and signaling, which is critical for the parasite’s development beyond initial attachment and for assessing host physiological outcomes.

More broadly, this modular framework is not limited to tomatoes. It can be readily adapted to study other host species amenable to hairy root transformation. The combination of visual accessibility, genetic flexibility, and experimental scalability makes it well-suited for systematic screening of resistance traits and dissection of host–parasite interactions. As the need for sustainable alternatives to chemical control intensifies, such experimental models will be critical for translating genetic discoveries into applied agricultural solutions.

Materials and Methods

Germination pouch setup

Transparent germination pouches commercially supplied sterile (height: 30 cm, width: 25 cm—PhytoAB—SKU CYG-98LB) served as cultivation chambers, allowing direct visualization of parasitic developmental stages. Only the initial pouch assembly was carried out in the laminar flow hood to minimize contamination, and the system was not maintained under sterile conditions afterwards. A small front flap was cut with a razor to permit precise seed placement and repeated observation. GF/A glass-fiber sheets (Cytiva, 1820-915) and all nutrient solutions were autoclaved. Each pouch lined with a single autoclaved GF/A sheet was sealed with lab tape after plants were transferred, leaving only a minimal basal port for nutrient solution replenishment. This configuration maintained uniform moisture and supported robust tomato root growth without contamination for up to 3 weeks (adapted from Clarke et al. 2020 with modifications).

Broomrape seed sterilization and preconditioning

Branched broomrape seeds were collected from a broomrape-infested field in Yolo County, California, USA. Seeds were surface sterilized using chlorine gas. To generate chlorine gas, approximately 20 mL of household bleach (Clorox—8.25% sodium hypochlorite) was placed in a small glass beaker inside a desiccator positioned under a chemical fume hood. Then, 600 μL of concentrated hydrochloric acid (12N HCl) was carefully added to the bleach. The reaction released chlorine gas (Cl₂), and the desiccator was immediately sealed to contain the gas. Seeds were placed in open Eppendorf tubes held in a tube rack within the desiccator and exposed to chlorine gas for 3 h. After sterilization, the desiccator was opened under a fume hood to safely dissipate the gas. Seeds were then rinsed thoroughly using a 70 μm cell strainer (Sigma—CLS431751) with sterile distilled water to remove any residual chlorine before use. Sterilized seeds were preconditioned by spreading them on moistened filter paper (Whatman No. 1) in sterile Petri dishes (Sigma—PD20047S0). About 1 mL of 0.06% PPM (Plant Cell Technology) was added to each plate. Plates were sealed with Parafilm and incubated at 22°C in darkness for 7 days. Following preconditioning, seeds were rinsed with sterile water to remove excess PPM, and 1 mL of 10 μM GR24 (PhytoTech Labs, G3324) was applied to the dish to stimulate germination. Seeds were further incubated at 25°C in darkness for 24 h prior to co-cultivation with host tissue.

Co-cultivation of broomrape with tomato seedlings in the germination pouch

Tomato seeds (S. lycopersicum cv. M82; Heinz lines H9775, H9553, and HM Clause lines HM58841 and HM4885) were first germinated on half-strength Murashige and Skoog (MS) (Plant Cell Technology—Basal Salts) medium. At 7 days post-germination, seedlings were transferred into germination pouches and supplied with ¼ strength Hoagland solution containing 0.05 mM KH₂PO₄. Seedlings were grown in the pouches at 25°C (16/8 light cycle), with roots kept in darkness inside a filing folder hung inside a box while shoots were exposed to light. Once plants developed two true leaves, preconditioned seeds suspended in water were then applied with a paintbrush near the roots of tomato seedlings in the mid-section of germination pouches to promote host–parasite interactions. The pouches were sealed with tape and incubated vertically at 25°C. The bags were inspected every 2–3 days to monitor moisture levels. Beginning one week after transfer, 30 mL of ½ strength Hoagland solution (0.05 mM KH₂PO₄) was added as needed through a small opening at the base of each pouch to maintain moisture while minimizing parasite seed displacement.

Assessment of parasite development

Haustoria/tubercle formation was assessed under a stereomicroscope at 9 days post-inoculation. Late-stage tubercles were confirmed and quantified at 14 days post-inoculation.

Tomato hairy root induction and culture

Tomato (S. lycopersicum cv. M82) hairy roots were generated via R. rhizogenes (ATCC: strain 15834)–mediated transformation following a previously published protocol (Ron et al. 2014). Briefly, electrocompetent R. rhizogenes cells were electroporated with the desired binary vector and plated on nutrient agar (BD 247940) supplemented with spectinomycin (100 mg L⁻¹). Plates were incubated for 2–3 days at 28–30°C. Colonies that grew under selection were then inoculated into 10 mL of nutrient broth (BD 90002-660) containing the same antibiotic and cultured overnight at 30°C with shaking at 200 rpm. This overnight culture was used to infect tomato cotyledons harvested from 8–10-day-old sterile-grown M82 seedlings, prior to the emergence of true leaves. Tomato cotyledons were excised using a sterile scalpel and submerged for 20 min in the R. rhizogenes suspension adjusted to an OD₆₀₀ in 1× MS liquid medium. Following infection, cotyledons were placed (adaxial side down) onto solid MS medium (1× MS with vitamins (Plant Cell Technology), 3% sucrose, 1% agar) without antibiotic selection and incubated in the dark at 25°C for 3 days. They were then transferred to a selection medium containing cefotaxime (200 mg L⁻¹) and kanamycin (100 mg L⁻¹), and incubated again at 25°C. Typically, three to five transformed roots developed per cotyledon. Kanamycin-resistant roots were excised and subcultured on fresh selection medium. Typically for one construct, 15 independent transgenic roots were isolated for downstream imaging and genotyping analyses.

Generation of CRISPR/Cas9-edited SlSCZ mutant alleles

For detailed methodology, refer to the Methods section in Manzano et al. 2025. Briefly, guide RNAs were designed using CRISPR-PLANT to meet GC content and mismatch criteria. Gene sequences (ITAG3.2) were retrieved from Phytozome, and gene maps were constructed in SnapGene. Primers for genotyping were designed with Primer-BLAST and checked for specificity in S. lycopersicum. Single guide RNA (sgRNA) oligos were cloned into pYPQ131-3, assembled via Gateway cloning into p278, and then into pMR286 containing Cas9 and KanR. Final vectors were transformed into R. rhizogenes for hairy root transformation in S. lycopersicum (cv. M82, LA3475). Mutations were verified by Sanger sequencing. Two null slscz mutant alleles in hairy roots were infected with preconditioned broomrape seeds using the split-media system and analyzed 21 days post-inoculation for parasite attachment and cellular-level infection site characterization.

Tomato hairy root split-media co-cultivation system

A two-compartment split-media system was prepared using square Petri dishes 120 × 120 × 17 mm (Greiner BIO-One-688 161) divided into two zones. The middle portion was filled with a nutrient-rich medium (1× MS + 3% sucrose) using a sterile cookie cutter (2 inch—round) to support hairy root growth, while the rest of the plate was filled with a non-nutritive medium (1% agar; BD 214530) to promote broomrape seed germination. To reduce fungal and microbial contamination from broomrape seeds, 0.06% PPM was added to the low-nutrient medium. Hairy roots were positioned such that they extended from the nutrient-rich side into the low-nutrient compartment. Preconditioned branched broomrape seeds suspended in 0.1% agarose were applied with a pipette to the surface of the low-nutrient zone and positioned near the roots using a dissecting needle. Plates were sealed and incubated at 25°C in the dark.

Cellular resolution analysis of host–parasite interface

For rapid assessment of broomrape attachments, harvested roots were either cleared overnight in 10% KOH or quickly cleared by incubating in 10% KOH at 95°C for 5 min. Cleared root segments were gently rinsed three times with distilled water and incubated with 0.1% Toluidine Blue solution for 1 min and rinsed again gently with distilled water. Root segments were mounted on a slide in 50% glycerol, covered with a cover glass, and sealed around the edges with nail polish. Observations were recorded on a Leica compound microscope under DIC. Xylem strands are visible with secondary wall thickenings in spirals or networks.

Infected tomato root segments containing attached branched broomrape were excised and embedded in 4% (w/v) low-melting-point agarose. Embedded tissue blocks were sectioned at 100 μm thickness using a vibratome. Sections were transferred to ClearSee solution (Ursache et al. 2018) and incubated at room temperature for 1–2 days to enhance tissue clearing. Cleared sections were then stained with 0.01% (w/v) Basic Fuchsin for 30 min to visualize lignified xylem, followed by 15 min in 0.01% (w/v) Direct Yellow 96 to stain cell walls. After staining, sections were rinsed in ClearSee 3 times and mounted in the same solution on glass slides. Imaging was performed using a LSM 700 laser scanning microscope (Carl Zeiss) with the following settings: excitation/emission settings optimized for DY-96 (488 nm) and Fuchsin (561 nm). Attachment sites were examined for haustoria penetration, host cortex invasion, and formation of xylem–xylem connections between host and parasite.

Quantification of lignin fluorescence

For each image, the red channel was used to identify areas with magenta signal (Basic Fuchsin) based on visual inspection. Regions were selected using the freehand selection tool, and the mean intensity and area were measured using the Measure function. The total signal was then normalized to the root area to obtain an area-normalized fluorescence intensity for comparison across samples. All sections were processed as described above. In each experiment, laser power was calibrated on control samples and then kept constant for all subsequent imaging.

Conflict of Interest

None declared.

Funding

This work was supported by HHMI and NSF 2118017 to SMB, NSF 2119820 to SMB and NS, California Tomato Research Institute [CTRI, 2022;2025], the California Department of Food and Agriculture [CDFA, Grant No. 21–0433-46A-SF], and the Foundation for Food & Agriculture Research [FFAR, Grant ID 23–000823] to NS and SMB.

Data Availability

The data underlying this article are available in the article and in its online supplementary material.