Name: leveraging micro-ct scanning to analyze parasitic plant-host interactions
Uploaded: 2022-01-12T15:00:00
Description: Watch this Scientific Journal Video about Leveraging Micro-CT Scanning to Analyze Parasitic Plant-Host Interactions at JoVE.com

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 sampling of Scybalium fungiforme. MSc. Ariadne Furtado, and Drs. Fernanda Oliveira and Maria Aline Neves provided the reference for the use of phloxine B for the analysis of endophytic fungi. Video recording at the Vrije Universiteit Brussel was made possible through the help of Dr. Philippe Claeys, Dr. Christophe Snoeck, MSc. Jake Griffith, Dr. Barabara Veselka, and Dr. Harry Olde Venterink.&#160;Funding was provided by the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil) and the Harvard University Herbaria (USA).

1. Parasitic plant sample selection
<ol>
	<li>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 ...

<ol>
	<li>Stock, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microcomputed tomography: Methodology and applications. , (2020).</li><li>Hounsfield, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computerized+transverse+axial+scanning+(tomography):+I.+Description+of+system.">Computerized transverse axial scanning (tomography): I. Description of system.</a> British Journal of Radiology. 46 (552), 1016-1022 (1973).</li><li>Dutilleul, P., Lafond, J. 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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 ...

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 nm5,6. 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 matrix9,10. Later, plant structures with more significant differences in tissue density, such as wood, began to be explored. This has allowed investigations of xylem functionality11,12, development of complex tissue organizations13,14, and interactions among plants15,16,17. The analysis of soft and homogeneous tissue is becoming widespread due to contrast agents, which are now standard procedure&#160;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&#39;s attachment, penetration, and contact by a parasite20. For this reason, the haustorium is considered to &#34;embody the very idea of parasitism among plants&#34;21. A detailed understanding of this organ&#39;s development, structure, and functioning is crucial for parasitic plant ecology, evolution, and management studies. Nevertheless, parasitic plants&#39; 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&#39;s excellent level of detail provides unprecedented insights into the haustorium&#39;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 analyses17,23, 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63423/63423fig01.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/63423/63423fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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 analysis3,10,24. 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&#160;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 &#38; 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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D X-ray microscope (XRM) system</td>
			<td></td>
			<td>Zeiss Versa 620</td>
			<td>used to scan Pyrularia pubera</td>
		</tr>
		<tr>
			<td>3D X-ray microscope + A2:D22</td>
			<td>Zeiss</td>
			<td>Versa 620</td>
			<td>Used for scanning the species P. pubera</td>
		</tr>
		<tr>
			<td>CT-Pro 3D software</td>
			<td>Nikon</td>
			<td>version XT 3.1.11</td>
			<td>Used for three-dimensional reconstruction of scans</td>
		</tr>
		<tr>
			<td>CT-Vox software</td>
			<td>Bruker</td>
			<td>version 3.3.1</td>
			<td>Used for analyses and acquisition of images and videos</td>
		</tr>
		<tr>
			<td>Dragonfly software</td>
			<td>Object Research Systems - ORS</td>
			<td>version</td>
			<td>Used for analyses and acquisition of images and videos</td>
		</tr>
		<tr>
			<td>Glass vials</td>
			<td>Glass Vials Inc. SE</td>
			<td>V2708C-FM-SP</td>
			<td>Sold by VWR - USA; make sure that vials are able to withstand vacuum at ca. 10 psi</td>
		</tr>
		<tr>
			<td>Inspect-X</td>
			<td>Zeiss</td>
			<td>version XT 3.1.11</td>
			<td>Used for controlling the Nikon X-Tek HMXST225 system</td>
		</tr>
		<tr>
			<td>Iodine solution 0.0282 N</td>
			<td>WR Chemicals BDH</td>
			<td>BDH7422-1</td>
			<td>Sold by VWR - USA</td>
		</tr>
		<tr>
			<td>Lead Nitrate II PA 500 g</td>
			<td>Vetec</td>
			<td>361.08</td>
			<td>Sold by SPLab</td>
		</tr>
		<tr>
			<td>Microtomography scanner</td>
			<td>Bruker</td>
			<td>Skyscan1176</td>
			<td>Used for scanning the species C. americana, S. martianus, and S. fungiforme</td>
		</tr>
		<tr>
			<td>Microtomography scanner</td>
			<td>Nikon</td>
			<td>X-Tek HMXST225</td>
			<td>Used for scanning the species V. minimum</td>
		</tr>
		<tr>
			<td>NRecon software</td>
			<td>Bruker</td>
			<td>version 1.0.0</td>
			<td>Used for three-dimensional reconstruction</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid hydrate 3% in aqueous solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>101410-756</td>
			<td>Sold by VWR - USA</td>
		</tr>
		<tr>
			<td>Plastic film (Parafilm)</td>
			<td>Heathrow Scientific</td>
			<td>PM996</td>
			<td>Sold by VWR - USA</td>
		</tr>
		<tr>
			<td>Plastic IV bag 500 mL</td>
			<td>Taylor</td>
			<td>3478</td>
			<td>Sold by Fibra Cirurgica Produtos para Saude</td>
		</tr>
		<tr>
			<td>PVC tubing 3/4&#39;&#39;</td>
			<td>Nalge Nunc International</td>
			<td>SC63013-164</td>
			<td>Sold by VWR - USA</td>
		</tr>
		<tr>
			<td>Scanning system</td>
			<td></td>
			<td>Nikon X-Tek HMXST225</td>
			<td>used to scan Viscum minimum</td>
		</tr>
		<tr>
			<td>Scanning system</td>
			<td></td>
			<td>Bruker Skyscan 1176</td>
			<td>used to scan C. americana</td>
		</tr>
		<tr>
			<td>Scout-and-ScanTM software</td>
			<td>Zeiss</td>
			<td>version 16</td>
			<td>Used for controlling the Zeiss Versa 620 system and for three-dimensional reconstruction of scans</td>
		</tr>
		<tr>
			<td>Three-way valve</td>
			<td>ToToT</td>
			<td>DMTWVS-5</td>
			<td>Sold by Amazon USA</td>
		</tr>
		<tr>
			<td>Two-part syringe</td>
			<td>HSW Henke-Ject</td>
			<td>4850001000</td>
			<td>Used without the plunger</td>
		</tr>
		<tr>
			<td>Vacuum chamber</td>
			<td>Binder</td>
			<td>80080-434</td>
			<td>Sold by VWR - USA; includes pump and connecting tubes</td>
		</tr>
		<tr>
			<td>VG Studio Max software</td>
			<td>Volume Graphics</td>
			<td>version 3.0</td>
			<td>Used for analyses and acquisition of images and videos</td>
		</tr>
	</tbody>
</table>

leveraging micro-ct scanning to analyze parasitic plant-host interactions

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&#160;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.

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 ...

Watch this Scientific Journal Video about Leveraging Micro-CT Scanning to Analyze Parasitic Plant-Host Interactions at JoVE.com

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 ...

This protocol improves considerably the contrast between the tissues of the parasitic plant and the host in samples that will be analyzed using microtomography. The technique described here speeds up the process of contrast perfusion through the sample, allowing the analysis of specific tissues and structures formed between the parasitic plant and the host. The vacuum approach is very straightforward and should offer no difficulties.The perfusion apparatus, on the other hand, might be a bit challenging to set up. But practice beforehand could help. For small non-woody samples, the vacuum method can be used for application.Whereas the profusion method should be used for samples that are larger and woody, including a segment of the host stem or root. If using the vacuum method, place the sample in the vial containing the contrasting solution. Then, place the vial in a vacuum chamber or desiccator connected to a vacuum pump.Remove the lid from the vial and close the vacuum chamber or desiccator. Check that there are no cracks on the vacuum chamber or desiccator. Open the exhaustion valve of the chamber or desiccator to force the air out.Turn on the pump and wait until the pressure reaches approximately 20 inches of mercury, or 10 psi. Close the chamber or desiccator exhaust valve to prevent air from reentering. Then quickly turn off the pump.Leave the sample under vacuum for at least two hours. If using the profusion method, select the supply tank according to the sample size. For small samples, a 50 milliliter syringe without a needle can be used as a tank.Connect one end of a transparent plastic tubing to the supply tank. Then connect the other end to a two-way or three-way valve. Connect a second tubing to a different outlet in the valve.Secure the supply tank at an elevated position without disassembling the apparatus set up in the previous step. Close the three-way or two-way valve to prevent liquid from exiting the tubing system and pour the contrasting solution into the supply tank. Ensure there are no large air bubbles in the tubing system and close the valve again, leaving the apparatus in place.To prepare the sample for profusion of the contrast solution, keep it submerged in liquid and cut off the tip of the proximal end of the host stem or root. Carefully open the valve to allow the contrasting solution to flow slowly and fill the plastic tubing connected to the tank while holding the open end of the system at a slightly elevated position to prevent the contrasting solution from spilling. Connect the proximal end of the host stem or root in the sample to the open end of the tubing system.Let the solution perfuse the sample for at least two hours or until the solution accumulates inside the container. Close the valve and carefully disconnect the sample from the apparatus. Wash the sample by submerging it in water for two minutes.Place the sample in a paper towel at room temperature to allow excess water to evaporate for two to five minutes without allowing the sample to dry out completely. Wrap the sample in a paraffin film and avoid folding the film on top of the sample. Micro-CT scanning can be leveraged to better understand the complex structures of parasitic plants and their interaction with hosts in a non-destructive, three-dimensional way.The protocols described here work for different micro-CT systems. However, the settings and parameters depend on the system and the samples. 3D X-ray microscope images were as effective as anatomical sections observed under a light microscope to analyze tissue organization and topology at the parasite host interface.Based on the bright white and dark gray color difference due to differential absorption of the contrast solution, it is possible to observe the abundance of parenchyma surrounding the vascular core of the haustorium. The vascular core is easily observed in cross-sections as two vascular strands are separated by parenchyma. The endoparasite Viscum minimum, growing inside a succulent host plant, showed parenchyma cells that store carbohydrates in the form of starch.The difference in iodine absorption allowed the detection of the intricate web of cortical strands formed by the endoparasite within the host body. Results obtained for Cuscuta Americana and Struthantus marcianus help illustrate the convenience of the microtomography approach for small and larger samples respectively. The virtual serial sectioning Sibelium fungiform showed that the vessels in the host root bifurcate into the parasite tuber, and that the xylem continuity between the two plants is formed by vessel-to-vessel connection via perforation plates.The procedure described here can be combined with other techniques, such as virtual segmentation, to provide new insights into the three dimensional structure of the connection between parasitic plants and their hosts. This technique has paved the way for analyzing different aspects of the biology of parasitic plants, including the development of endoparasites and the functionality of hyperparasitic connections.

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