Leveraging Micro-CT Scanning to Analyze Parasitic Plant-Host Interactions

High-resolution x-ray microcomputed tomography (micro-CT) is an imaging method in which multiple radiographs (projections) of a sample are recorded from different viewing angles and later used to provide a virtual reconstruction of the sample¹. This virtual object can then be analyzed, manipulated, and segmented, allowing non-destructive exploration in three dimensions². Initially designed for medical analyses and later for industrial applications, micro-CT also offers the advantage of visualizing inner organs and tissues without the need for invasive procedures³. Like other forms of imaging, micro-CT works with a trade-off between the field of view and pixel size, which means that high-resolution imaging of large samples is nearly unattainable⁴. Advances in using high-energy X-ray sources (i.e., synchrotron) and secondary optical magnification are constantly being made, allowing the smallest resolution to reach under 100 nm^5,6. Nevertheless, longer scanning times are necessary for large samples, increasing the chance of artifacts due to sample movement or deformation inside the scanner. Furthermore, micro-CT is generally limited by natural density variations within the sample and how the sample interacts with X-rays. While a higher X-ray dose is best for penetrating denser samples, it is less efficient in capturing variations in density within and between the sample and its surrounding medium⁷. On the other hand, a lower X-ray dose offers less penetration power and often requires longer scanning times but more sensitivity in density detection⁷.

These restrictions have long hampered the use of microtomography for plant sciences, given that most plant tissues are composed of light (non-dense) tissue with low X-ray absorption⁸. The first applications of micro-CT were focused on mapping root networks within the soil matrix^9,10. Later, plant structures with more significant differences in tissue density, such as wood, began to be explored. This has allowed investigations of xylem functionality^11,12, development of complex tissue organizations^13,14, and interactions among plants^15,16,17. The analysis of soft and homogeneous tissue is becoming widespread due to contrast agents, which are now standard procedure in preparations for micro-CT scanning of plant samples. However, protocols for contrast introduction can have different results depending on sample volume, structural properties, and the type of solution used⁸. Ideally, the contrast agent should enhance distinction among different tissues, enable tissue/organ functionality evaluation, and/or provide biochemical information about a tissue¹⁸. Therefore, adequate sample treatment, preparation, and mounting before scanning become crucial for any micro-CT analysis.

Micro-CT of the parasitic plant haustorium
Parasitic flowering plants represent a distinct functional group of angiosperms characterized by an organ known as haustorium¹⁹. This multicellular organ, a developmental hybrid between a modified stem and a root, acts on the host's attachment, penetration, and contact by a parasite²⁰. For this reason, the haustorium is considered to "embody the very idea of parasitism among plants"²¹. A detailed understanding of this organ's development, structure, and functioning is crucial for parasitic plant ecology, evolution, and management studies. Nevertheless, parasitic plants' overall complexity and highly modified structure and haustoria often hinder detailed analysis and comparison. Haustorium connections are also usually extensive and not homogenous in tissue and cell distribution (Figure 1). In this context, while working with small tissue fragments allows easier manipulation and higher resolution, it can lead to erroneous conclusions about the three-dimensional architecture of complex structures, such as the parasitic plant haustorium.

Although there is a vast literature on haustorium anatomy and ultrastructure for most parasitic plant species, the three-dimensional organization and the spatial relationship between parasite and host tissues remains poorly explored¹⁷. In a recent work by Masumoto et al.²², over 300 serial semi-thin microtome sections were imaged and reconstructed into a three-dimensional virtual object representing the haustorium of two parasite species. This method's excellent level of detail provides unprecedented insights into the haustorium's cellular and anatomical 3-D structure. However, such a time-consuming technique would forbid a similar analysis in parasites with more extensive haustorium connections. The use of micro-CT emerges as an excellent tool for three-dimensional analysis of complex and often bulky haustoria of parasitic plants. Although not a substitute for detailed anatomical sectioning and other complementary forms of microscopy analyses^17,23, results obtained via micro-CT scanning, especially for large samples, can also serve as a guide to direct the sub-sampling of smaller segments, which can then be analyzed using other tools, such as confocal and electron microscopy, or re-analyzed with high-resolution micro-CT systems.

Figure 1: Parasitic plants of different functional groups used in this protocol. Euphytoid parasite Pyrularia pubera (A), endoparasite Viscum minimum (B) with green fruits (dashed black circle), parasitic vine Cuscuta americana (C), mistletoe Struthanthus martianus (D), obligate root parasite Scybalium fungiforme (E). Segments of the host root (Hr) or stem (Hs) facilitate the application of contrast into the parasite haustorium (P). The presence of parasite mother root/stem (arrows) in the sample allows analysis of haustorium vessel organization. Rectangles indicate segments of the sample used for analysis. Scale bars = 2 cm. Please click here to view a larger version of this figure.

As micro-CT becomes an increasingly popular technique in plant sciences, there are guides, protocols, and literature dealing with sample scanning, three-dimensional reconstruction, segmentation, and analysis^3,10,24. Thus, these steps will not be discussed here. As with any imagining technique, appropriate treatment and mounting of samples are a fundamental, albeit often being an overlooked procedure. For this reason, this protocol focuses on the preparation of haustorium samples for micro-CT scanning. More specifically, this protocol describes two approaches for introducing contrast agents into haustorium samples to improve visualization of different tissues and cell types in the haustorium, to facilitate the detection of parasitic tissue within the host root/stem, and to analyze parasite-host vascular connections in three dimensions. The preparations described here can also be adapted to the analysis of other plant structures.

Five species were used to better illustrate the convenience of the protocol described here. Each species represents one of the five functional groups of parasitic flowering plants, thus addressing specific points related to the functionality of each group. Pyrularia pubera (Santalaceae) was chosen to represent euphytoid parasites, which germinate in the ground and form multiple haustoria that connect the parasite to the roots of its hosts²⁵. The haustoria created by these plants are often tenuous and easily torn apart from the host²⁶ (Figure 1A), thus requiring a more delicate handling process. Endoparasites are represented here by Viscum minimum (Viscaceae). Species in this functional group are only visible outside the body of their hosts for short periods (Figure 1B) and live most of their life cycles as significantly reduced and mycelial-like strands of cells embedded within host tissues²⁵. A third functional group comprises parasitic vines, which germinate on the ground but form only rudimentary roots, relying on multiple haustoria that attach to the stems of host plants²⁵ (Figure 1C). Here, this functional group is represented by Cuscuta americana (Convolvulaceae). Contrary to parasitic vines, mistletoes germinate directly upon the branches of their host plants and develop either multiple or solitary haustoria²⁵. The species chosen to illustrate this functional group is Struthanthus martianus (Loranthaceae), which forms various connections with the host branch (Figure 1D). Analysis of solitary mistletoe haustoria using a combination of micro-CT and light microscopy can be found in Teixeira-Costa & Ceccantini¹⁷. Finally, obligate root parasites comprise species that germinate on the ground and penetrate the roots of host plants, upon which they are entirely dependent from the earliest growth stages²⁵. These plants are represented here by Scybalium fungiforme (Balanophoraceae), which produce large tuber-like haustoria (Figure 1E).

All plant samples used in this protocol were fixed in a 70% formalin acetic acid alcohol (FAA 70). The fixation upon sampling is crucial for preserving plant tissues, especially if subsequent anatomical analyses are needed. In the case of parasitic plant haustorium, fixation is also essential, as this organ is often primarily composed of non-lignified parenchyma cells²⁰. Detailed protocols for plant tissue fixation, including the preparation of fixative solutions, can be found elsewhere²⁷. On the other hand, to a greater or lesser degree, fixatives can cause alterations of a sample's physical and chemical properties, rendering it unsuitable for specific biomechanical and histochemical analyses. Thus, fresh samples, i.e., non-fixated material collected immediately before preparation, can also be used with this protocol. Details on how to handle fresh samples and troubleshooting suggestions for fixated material are provided in the discussion section.