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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 .......

Discussion

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 .......

Materials

Name	Company	Catalog Number	Comments
3D X-ray microscope (XRM) system		Zeiss Versa 620	used to scan Pyrularia pubera
3D X-ray microscope + A2:D22	Zeiss	Versa 620	Used for scanning the species P. pubera
CT-Pro 3D software	Nikon	version XT 3.1.11	Used for three-dimensional reconstruction of scans
CT-Vox software	Bruker	version 3.3.1	Used for analyses and acquisition of images and videos
Dragonfly software	Object Research Systems - ORS	version	Used for analyses and acquisition of images and videos
Glass vials	Glass Vials Inc. SE	V2708C-FM-SP	Sold by VWR - USA; make sure that vials are able to withstand vacuum at ca. 10 psi
Inspect-X	Zeiss	version XT 3.1.11	Used for controlling the Nikon X-Tek HMXST225 system
Iodine solution 0.0282 N	WR Chemicals BDH	BDH7422-1	Sold by VWR - USA
Lead Nitrate II PA 500 g	Vetec	361.08	Sold by SPLab
Microtomography scanner	Bruker	Skyscan1176	Used for scanning the species C. americana, S. martianus, and S. fungiforme
Microtomography scanner	Nikon	X-Tek HMXST225	Used for scanning the species V. minimum
NRecon software	Bruker	version 1.0.0	Used for three-dimensional reconstruction
Phosphotungstic acid hydrate 3% in aqueous solution	Electron Microscopy Sciences	101410-756	Sold by VWR - USA
Plastic film (Parafilm)	Heathrow Scientific	PM996	Sold by VWR - USA
Plastic IV bag 500 mL	Taylor	3478	Sold by Fibra Cirurgica Produtos para Saude
PVC tubing 3/4''	Nalge Nunc International	SC63013-164	Sold by VWR - USA
Scanning system		Nikon X-Tek HMXST225	used to scan Viscum minimum
Scanning system		Bruker Skyscan 1176	used to scan C. americana
Scout-and-ScanTM software	Zeiss	version 16	Used for controlling the Zeiss Versa 620 system and for three-dimensional reconstruction of scans
Three-way valve	ToToT	DMTWVS-5	Sold by Amazon USA
Two-part syringe	HSW Henke-Ject	4850001000	Used without the plunger
Vacuum chamber	Binder	80080-434	Sold by VWR - USA; includes pump and connecting tubes
VG Studio Max software	Volume Graphics	version 3.0	Used for analyses and acquisition of images and videos