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

Micro-CT is a non-destructive tool that can analyze plant structures in three dimensions. The present protocol describes the sample preparation to leverage micro-CT to analyze parasitic plant structure and function. Different species are used to highlight the advantages of this method when coupled with specific preparations.

Micro-CT scanning has become an established tool in investigating plant structure and function. Its non-destructive nature, combined with the possibility of three-dimensional visualization and virtual sectioning, has allowed novel and increasingly detailed analysis of complex plant organs. Interactions among plants, including between parasitic plants and their hosts, can also be explored. However, sample preparation before scanning becomes crucial due to the interaction between these plants, which often differ in tissue organization and composition. Furthermore, the broad diversity of parasitic flowering plants, ranging from highly reduced vegetative bodies to trees, herbs, and shrubs, must be considered during the sampling, treatment, and preparation of parasite-host material. Here two different approaches are described for introducing contrast solutions into the parasite and/or host plants, focusing on analyzing the haustorium. This organ promotes connection and communication between the two plants. Following a simple approach, details of haustorium tissue organization can be explored three-dimensionally, as shown here for euphytoid, vine, and mistletoe parasitic species. Selecting specific contrasting agents and application approaches also allow detailed observation of endoparasite spread within the host body and detection of direct vessel-to-vessel connection between parasite and host, as shown here for an obligate root parasite. Thus, the protocol discussed here can be applied to the broad diversity of parasitic flowering plants to advance the understanding of their development, structure, and functioning.

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, micr....

1. Parasitic plant sample selection

1. Collect the entire parasitic plant haustorium, including the attached host stem/root and segments of both proximal and distal ends of the parasitized host organ; the ideal length of each segment is equivalent to double the diameter of the haustorium.
   NOTE: For lateral haustoria, include part of the parasite mother stem/root from which the haustorium was formed (Figure 1A,B,D). For endoparasites, collect .......

The haustorium of parasitic plants is a complex organ comprising different tissues and cell types that intertwine and connect with the tissues of another plant, used as a host²⁰. Micro-CT scanning can be leveraged to better understand this complex structure in a non-destructive and three-dimensional way when analyzing both small (Figure 1A-C) and large (Figure 1D,E) haustoria. To do .......

The use of heavy metal solutions to improve plant tissue contrast has become a crucial step in sample preparation for micro-CT analysis. Several compounds commonly available in plant micro-morphology laboratories have been tested by Staedler et al., who recommend using phosphotungstate as the most effective agent in penetrating samples and increasing contrast index⁸. Results obtained here in the analysis of the haustorium of P. pubera corroborate this recommendation. In terms of contrast .......

I would like to thank Dr. Simone Gomes Ferreira (Microtomography Laboratory, University of Sao Paulo, Brazil) and Dr. Greg Lin (Center for Nanoscale Systems, Harvard University, USA) for their paramount help and indispensable user training for different microtomography systems and data analysis software. I also thank the staff at the EEB Greenhouse at the University of Connecticut (USA), especially Clinton Morse and Matthew Opel for providing the specimens of Viscum minimum. Dr. John Wenzel provided the opportunity and great help for the sampling of Pyrularia pubera. MSc. Carolina Bastos, MSc. Yasmin Hirao, and Talitha Motta greatly helped with the s....