2D and 3D visualization of herbaceous plant–plant contact zones using high‐resolution X‐ray computed tomography (HRXCT)

Parasitic plants that deprive crops of water and nutrients are an increasingly concerning food security issue, affecting the livelihood of millions of subsistence, small‐ and mid‐scale farmers. An in‐depth understanding of parasite–host interactions is required to develop species‐specific and ecologically sustainable parasite management methods. The non‐invasive visualization of herbaceous contact zones, applicable to diverse parasite–host pathosystems presented in this study, brings methodological advance to the research of biotic interactions between crops and plant parasites belonging to the most devastating parasitic plant family (Orobanchaceae). This work also provides first insights into how the parasites' feeding organ displaces host tissue beyond the direct parasite–host interface.High‐resolution X‐ray computed tomography (HRXCT) enables sectioning‐free two‐dimensional imaging of biological structures and reconstruction of three‐dimensional objects. Although its application is common in many areas of biomedicine and despite its flexibility regarding resolution levels, the technology remains underutilized in the plant sciences. Here, we explored HRXCT for the study of parasitic plant–plant interactions by developing protocols to access soft‐tissue host–parasite contact zones at cell‐level resolution. We tested various sample preparation methods and contrast stains for their efficiency to improve the imaging of haustorium samples. In doing so, we achieved cellular resolution with the visible cellular organization of haustorial structures, especially of the vascular system. Fresh stained and dehydrated sample preparation of soft haustoria enables the highest spatial resolution with fine‐cellular discrimination of haustorium versus host cells. Application of cell‐level resolved HRXCT to five pathosystems: Alectra‐cowpea, Phelipanche‐tomato, Phtheirospermum‐tomato, Rhamphicarpa‐tomato, and Striga‐sorghum highlighted a life history‐specific organization and uncovered an as yet undescribed internal displacement of host tissue at parasite–host interfaces. Following image‐based training, our HRXCT approach could invoke AI‐based cell recognition for automated parasite cell–host cell differentiation. Superseding extensive microsectioning for 3D imaging, the newly established HRXCT protocol for 2D‐ and 3D‐visualization of herbaceous plant–plant contact zones and the first insights gained from it, is useful for mid‐throughput, comparative studies of parasitic plant–host interactions.