Name: combining clearing and fluorescence microscopy for visualising changes in gene expression and physiological responses to plasmodiophora brassicae
Uploaded: 2022-08-05T15:00:00.000+00:00
Duration: 6 min 58 s
Description: Watch this Scientific Journal Video about Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae at JoVE.com

The work was supported by the National Science Centre Poland OPUS17 grant No. 2019/33/B/NZ9/00751 &#34;Long distance Vascular Coordination in Plants Infected by Plasmodiophora brassicae&#34;. We thank Prof. Yrj&#246; Helariutta (Sainsbury Laboratory, University of Cambridge) for sharing the proHCA2::erRFP line.

1. Plant growth conditions
<ol>
	<li>Grow Arabidopsis thaliana plants in a short-day light regime with 9 h of light at 22 &#176;C and 15 h of dark at 20 &#176;C and an irradiance of 120 &#181;mol&#183;m&#8722;2s&#8722;1 (measured as the photosynthetically active radiation, PAR at the canopy level, see Table of Materials).</li>
</ol>
2. Preparation of spore inoculum
NOTE: For details, see Fuchs et al.14.
<ol>
	<li>Homogenize two to three frozen galls from Chinese cabbage plants (Brassica rapa var. pekinensis cultivar &#34;Granaat&#34;) in a blender containing 300 mL of autoclaved distilled water and filter through four layers of sterile gauze.</li>
	<li>Centrifuge the filtrate (at 6,000 x g, for 5 min, and at 4 &#176;C) and mechanically remove the starch layer from the spore pellet using a spatula. Repeat this process until most of the starch is removed.</li>
	<li>Determine the spore concentration using a hemocytometer13 by counting the number of spores in 20 different field areas.</li>
	<li>Ensure that the final concentration of spore suspension used for inoculating is 1 x 106 spores&#183;mL&#8722;1 for Arabidopsis thaliana. Inoculate each plant 20 days after germination with 2 mL of calibrated spore suspension.</li>
</ol>
3. Tissue preparation and fixation
<ol>
	<li>Prepare the plant tissue.
	<ol>
		<li>Carefully remove soil from the root systems of clubroot-infected and non-infected plants. Clean thoroughly with water and collect hypocotyls and galls (length between 0.5-2 cm) into microcentrifuge tubes.</li>
	</ol>
	</li>
	<li>Perform PFA fixation (4% paraformaldehyde, PFA in 1x PBS + 0.01% Triton X-100) following the steps below.
	<ol>
		<li>Add 4 g of PFA powder (see Table of Materials) to 100 mL of PBS (phosphate-buffered saline, pH 7.4, Table 1) by stirring at 65 &#176;C (do not boil). Use KOH (10 M and 1M) dropwise to obtain a clear solution with a pH of around 11.</li>
		<li>Adjust the pH with H2SO4 to bring it down to 6.9 and add 0.01% (v/v) Triton X-100 to improve fixation. Aliquot the solution to store at &#8722;20 &#176;C (do not refreeze) or store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Perform tissue fixation.
	<ol>
		<li>Fix the samples in 200-500 &#181;L of PFA fixative for 1 h at room temperature by applying a constant vacuum (700 mbar) using a vacuum pump. Ensure that the object is completely immersed in the PFA solution. 
		&#8203;NOTE: Samples can be stored for a few weeks at 4 &#176;C (fridge) and used later for sectioning.</li>
	</ol>
	</li>
</ol>
4. Tissue embedding and sectioning
<ol>
	<li>Use 4% w/v agarose for agarose embedding of non-infected hypocotyls and infected galls. Boil the solution to dissolve agarose and pour it when it cools down while it is still viscous.</li>
	<li>Dry the object slightly on a tissue for a few seconds to remove excess PFA.</li>
	<li>Use toothpicks/forceps to carefully embed and orient the plant object in agarose. To prevent oblique sections, ensure that the object is oriented and molded correctly so that its axis is perpendicular to the plane of the vibratome blade (for radial sections).</li>
	<li>To accelerate agarose solidifying, place the&#160;Petri or multi-culture plate at 4 &#176;C for 10 min. 
	&#8203;NOTE: If the object is too large (as in the case of larger galls of Arabidopsis at 21 days post-inoculation [DPI]), the object can be sectioned even without embedding in agarose.</li>
</ol>
5. Vibratome sectioning
NOTE: The vibratome is equipped with a vibrating razor blade to cut through plant organs/tissue. The vibration speed, amplitude, angle of the blade, and section thickness are all parameters that can be adjusted (see Table of Materials).
<ol>
	<li>Using a blade, carefully cut and mold the object within an agarose block, noting the preferred orientation for sectioning.</li>
	<li>Stick the agarose block/large gall to properly mount it on the specimen holder using cyanoacrylate/instant glue and masking tape (see Table of Materials).</li>
	<li>For Arabidopsis hypocotyls and galls (early stages), keep the thickness of sections between 30-40 &#181;m for obtaining good quality images. 
	NOTE: For analyzing the later stages of gall progression, the thickness of sections can be between 50-80 &#181;m.</li>
	<li>Refrain from cutting thinner sections as it can destroy the gall sample due to the vibrations of the moving blade.</li>
	<li>Adjust the thickness for sections, speed, and vibration amplitude according to the gall&#39;s thickness and size (40 &#181;m or 60 &#181;m).</li>
	<li>Add distilled water to the water bath and begin with sectioning.</li>
	<li>Carefully collect sections using forceps or brush and transfer them into a microcentrifuge tube containing 1mL of 1x PBS buffer (pH 7.4). 
	&#8203;NOTE: Typically, the higher the vibration amplitude, the better the sectioning quality. For non-infected hypocotyls or galls that do not contain mature resting spores, the vibration amplitude can be kept at 1.2 mm with 0.60 mm&#183;s&#8722;1 speed. In case of large galls with partial loss of cellular integrity and visible signs of spore maturation, the vibration amplitude should be decreased to 0.55 mm with 0.45 mm&#183;s&#8722;1 speed.</li>
</ol>
6. Specimen clearing
<ol>
	<li>Remove 1x PBS from the microcentrifuge tube and add 200-500 &#181;L of clearing solution (Table 1).</li>
	<li>From now on, store the samples at room temperature in the dark. Ensure that the objects are always submerged in the clearing solution at all times.</li>
	<li>Replace the clearing solution with the new one if its color changes after some time during sample processing. 
	&#8203;NOTE: The color of the clearing solution can turn yellowish due to sample processing. In such a case, replacing the solution is recommended to improve the clearing of tissues.</li>
</ol>
7. Staining procedure
<ol>
	<li>For Calcofluor white staining for the cell walls (for outlaying plant cells), prepare 5% v/v of Calcofluor white stain (see Table of Materials) in the clearing solution. Stain for at least 5 min in the dark.</li>
	<li>For staining lipids with Nile Red (for staining resting spores and oil droplets in infected cells), prepare 1 mg/mL of the stock (see Table of Materials) in clearing solution. Dilute it further to obtain 1:99. Stain for at least 10 min in the dark.</li>
	<li>For Basic Fuchsin lignin staining, prepare 0.2% w/v of the stain (see Table of Materials) in clearing solution. Stain for at least 10 min in the dark. 
	&#8203;NOTE: During double staining, samples are first stained with Nile Red for 10 min before Calcofluor white application. The excess staining solutions were removed after each step. If the object seems overstained, remove the excess stain by washing with the clearing solution. Dilute stains using the clearing solution, if required. Always stain the sample with Nile Red/Basic Fuchsin before Calcofluor staining. Staining first with Calcofluor can prevent proper staining of the spores by Nile Red.</li>
</ol>
8. Microscopy
<ol>
	<li>Mount cleared sections on the microscopy slide and observe under an epifluorescence or a confocal microscope (see Table of Materials). Use clearing solution as the mounting medium to prevent drying of the sample.</li>
	<li>Use multiple acquisition modes for imaging more than one fluorescence spectrum simultaneously. 
	NOTE: The excitation/emission spectra used in the presented protocol are as follows: for Nile Red 553/636 nm, for xylem autofluorescence 380/475 nm, for Basic Fuchsin 561/650 nm, for Calcofluor white 405/475 nm, for erRFP 585/608 nm, and for GFP 488/509 nm (Table 2).</li>
</ol>

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M. M., Ludwig-M&#252;ller, J., Sandbichler, A. M., Neuhauser, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demystifying+biotrophs:+FISHing+for+mRNAs+to+decipher+plant+and+algal+pathogen-host+interaction+at+the+single+cell+level.">Demystifying biotrophs: FISHing for mRNAs to decipher plant and algal pathogen-host interaction at the single cell level.</a> Scientific Reports. 10, 14269 (2020).</li><li>Kurihara, D., Mizuta, Y., Sato, Y., Higashiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ClearSee:+A+rapid+optical+clearing+reagent+for+whole-plant+fluorescence+imaging.">ClearSee: A rapid optical clearing reagent for whole-plant fluorescence imaging.</a> Development. 142 (23), 4168-4179 (2015).</li><li>Ursache, R., Andersen, T. 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The authors have nothing to disclose.

Applying the clearing solution on vibratome-cut sections of galls surely enhances the ability to study the biotrophic interaction between P. brassicae and the host plant. Although the clearing protocol applies even to hand sections, it works better with vibratome sections. Fixing samples in PFA fixative acts as a critical step in the protocol as samples can then be stored at 4 &#176;C for a few days before proceeding with sectioning. This provides flexibility to store samples for a limited period without compromising the expression and preservation of fluorescent proteins during fixation.
Nile Red (in DMSO or methanol) is incompatible with resin sections due to its hydrophobicity, which dissolves resin and destroys resin-embedded sections17. Thus, vibratome sections prove instrumental for studying the pathogen distribution and its life-cycle within developing galls, wherein Nile Red staining can be easily used.
The clearing solution used in this protocol is highly versatile12, allowing a variety of fluorescence stains to be used in different combinations to stain various biomolecules/components of the cell walls (suberin, lignin, cellulose, and chitin in fungal interactions). It is also possible to counterstain sections of fluorescent GFP marker lines and thereby correlate the promoter activity or protein accumulation pattern with the presence of the pathogen in particular cells or regions of the gall. However, background autofluorescence from xylem and pathogen-filled giant cells could not be eliminated even after the clearing protocol. This presents a limitation for looking at fluorescent markers during the later stages of gall formation, especially when using an epifluorescence microscope and imaging weak signals.
Due to the low levels of expression/accumulation of fluorescent signals, transcription factors are difficult to detect, but, with this technique, it is possible to obtain satisfactory images for them. Overall, combining vibratome sectioning with the tissue-clearing approach expands the toolkit for histological observations of complex gall tissues. The flexibility of this protocol eases the process of tissue fixation and reduces the time required for cutting and imaging fresh tissue samples to observe fluorescent proteins and promoter activities. With further improvements and by utilizing other fluorescent dyes specific for various biomolecules, this method will mark greater advances in histological studies and image analysis of dense, opaque tissues with complex tissue organization. In recent times, the presented tissue clearing method has emerged as a popular and widely used protocol to combine and enable the simultaneous acquisition of different fluorescent signals11,12,13. Future development and modifications in such techniques will greatly improve the image resolution for observing plant-pathogen interactions at the cellular level.

Plants affected by pathogens or insects may develop abnormal structures (organ deformations or galls), which allow the invader to ingest nutrients and reproduce1. Here, an efficient histopathological approach was undertaken to study the changes taking place in galls that develop on the underground parts of plants infected with the protist Plasmodiophora brassicae (Figure 1). The minor thread associated with this pathogen emerges from the fact that P. brassicae resting spores can retain their ability to invade plants for many years. In the case of large-scale cultivation of oilseed rape (Brassica napus), this is a serious problem since economic factors restrict crop rotation, leading to resting spore accumulation in the soil2. Oilseed rape&#39;s resistance to clubroot disease caused by P. brassicae is genetically determined. Sadly, the pathogen often outsmarts resistance because of its biology and the narrow genetic pool from which oilseed rape originated. Therefore, it has become relevant to study the post-infection responses in host plants and their capability to slow disease progression or prevent certain symptoms from developing.
In clubroot disease, severity is generally evaluated based on the development of galls and the degree of root system damage. This is known as the Disease Index-DI3. However, it does not fully capture the true appraisal of this plant-pathogen interaction. In particular, it does not address how the pathogen is distributed within the roots and if the plant can restrain P. brassicae movement within its tissues. Further, it is not easy to anticipate to what extent P. brassicae reprograms underground organ anatomy. Studies on the model plant Arabidopsis thaliana have shown that P. brassicae infection leads to the inhibition of xylogenesis (both the initiation and maturation steps) and the enhancement of phloem differentiation within galls4,5. Moreover, in roots and hypocotyls of infected plants, cambial cell progeny does not quit the mitotic state and proliferates longer than in healthy plants6. This process governs the final size of the gall and determines the number of pathogen resting spores produced within the infected plant. P. brassicae-driven developmental, metabolic, and physiological reprogramming in the host is very complex7; therefore, the application of tools allowing the inspection of internal changes within galls is crucial for properly assessing this interaction. The life cycle progression of P. brassicae is accompanied by reprogramming of the host cell metabolism, which can be observed as starch or lipid deposition7,8. The main obstacle to the successful microscopy of galls comes from their low transparency. Due to this, the majority of histological specimens presenting clubroot-driven changes within galls originate from fixation-embedding (wax or resin) techniques followed by microtome sectioning. Such approaches were successfully used for locating the promoter activity for numerous genes active in clubroot galls4,5 or various staining techniques facilitating the observation of P. brassicae life-cycle progression9. However, it must be noted that the fixing and embedding stages are time-consuming and result in partial or complete washing out of important biomolecules (e.g., lipids), significantly hindering certain observations. Recently, P. brassicae life cycle progression in the host was visualized with the help of the Fluorescent In Situ Hybridization (FISH), in which a SABATH-type methyltransferase&#160;(PbBSMT) gene-specific probe was used to mark resting spore formation10. A good alternative is the use of other fluorescence-based methods where the autofluorescence of some cellular components, the activity of 5&#39;- upstream regulatory regions of genes fused to fluorescent protein markers, and the accumulation of particular fluorescently tagged proteins can be seen. However, in addition to the low transparency of the samples, a major drawback associated with such objects is working with unfixed specimens, which significantly decreases the time in which good quality images can be documented. In 2015, Kurihara et al.11 developed a clearing reagent, which allows the preservation of fluorescent proteins and increases the transparency of plant tissue specimens. Additionally, it is compatible with numerous histological stains. Recently, the same technique was successfully applied to visualize different cell wall components in plant tissues12,13. Here, this protocol has been used to analyze various clubroot gall development aspects. The workflow begins with the fixation of galls, vibratome sectioning, tissue clearing, staining, and fluorescence imaging. Depending on one&#39;s needs, directly or after particular staining, the resulting sections can be subjected to inspection under an epifluorescence or confocal microscope. This method provides an effective solution to study local changes in gene expression and physiological responses, including phytohormone balance and signaling. Disease progression can be tracked by looking at the distribution pattern of resting spores and maturation dynamics. Furthermore, the protocol can be easily applied for imaging characteristic changes within P. brassicae infected plants, including the inhibition of xylogenesis or the host plant defense responses visible as local lignification in resistant genotypes. Examples in this protocol come from imaging conducted on the Arabidopsis thaliana model; however, the protocol can also be applied to other crop species belonging to the Brassicaceae family. The method described below will facilitate future detailed studies of cellular structures and molecular changes accompanying gall formation in P. brassicae-infected plants.
The general workflow of the protocol is quite straightforward, and all stages of gall development can be easily imaged and characterized (Figure 2). Since P. brassicae is a soilborne pathogen, all experiments must be carried out in soil-based systems. The pathogen prefers acidic conditions; therefore, non-lime-treated soil substrates must be used. Although P. brassicae does not pose a threat to humans, it is strictly a plant pathogen that can spread through soil and water. Therefore, all parts of the infected plant, as well as the soil, need to be destroyed after the experiment by autoclaving or by treatment with bleach.

<table><tbody><tr><td>2N Sulfuric acid (H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Roth</td><td>UN2796</td><td>pH adjustment</td></tr><tr><td>Agarose</td><td>PRONA</td><td>BGQT100</td><td>Embedding</td></tr><tr><td>Basic Fuchsin</td><td>BIOSHOP</td><td>BSF410.5</td><td>Fluorescent dye</td></tr><tr><td>Calcofluor White</td><td>Sigma Aldrich</td><td>18909-100ML-F</td><td>Fluorescent dye</td></tr><tr><td>Commercial Bleach</td><td>Domestos</td><td></td><td></td></tr><tr><td>Cyanoacrylate/ Instant glue</td><td>Kropelka</td><td></td><td>Adhesive</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>BIOSHOP</td><td>DMS555.500</td><td>Solvent</td></tr><tr><td>Epifluorescence microscope</td><td>Carl Zeiss M2 automated epifluorescence microscope with Colibri LED system</td><td>Carl Zeiss M2</td><td>Carl Zeiss Filter Set&lt;br/&gt; filter set 38, 43, 49 used</td></tr><tr><td>Fully automated Vibratome</td><td>Leica</td><td>VT1200 S</td><td></td></tr><tr><td>Lightmeter /Photometer</td><td>LI-COR Biosciences</td><td>LI-250A + LI-190R quantum sensor</td><td>For measuring&amp;nbsp; light intensity within the 400-700nm (PAR) waveband</td></tr><tr><td>Masking tape</td><td></td><td></td><td>For sticking agarose block on mould</td></tr><tr><td>Murashige &amp;amp; Skoog Medium (MS Medium)</td><td>Duchefa Biochemie</td><td>MO222.0050</td><td>Plant Growth Medium</td></tr><tr><td>Nile Red</td><td>Sigma Aldrich</td><td>N3013-100MG</td><td>Fluorescent dye</td></tr><tr><td>Paraformaldehyde PFA</td><td>Sigma Aldrich</td><td>158127-100G</td><td>Fixative</td></tr><tr><td>Potassium Chloride (KCl)</td><td>POCH</td><td>739740114</td><td>PBS component</td></tr><tr><td>Potassium Hydroxide (KOH)</td><td>Sigma Aldrich</td><td>P1767-250G</td><td>pH adjustment</td></tr><tr><td>Potassium Phosphate Monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>BIOSHOP</td><td>PPM302.500</td><td>PBS component</td></tr><tr><td>Sodium chloride (NaCl)</td><td>BIOSHOP</td><td>SOD001.1</td><td>PBS component</td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma Aldrich</td><td>D6750-25G</td><td>Clearing Solution</td></tr><tr><td>Sodium Phosphate Dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>POCH</td><td>799490116</td><td>PBS component</td></tr><tr><td>Triton X-100</td><td>BIOSHOP</td><td>TRX506.100</td><td>Fixative</td></tr><tr><td>Urea</td><td>Sigma Aldrich</td><td>U5378-100G</td><td>Clearing Solution</td></tr><tr><td>Vacuum/Pressure pump and Dessicator</td><td>Welch by Gardner Denver</td><td>2522C-02</td><td>For Vacuum Infilteration</td></tr><tr><td>Xylitol</td><td>Sigma Aldrich</td><td>X3375-25G</td><td>Clearing Solution (componenet)</td></tr></tbody></table>

combining clearing and fluorescence microscopy for visualising changes in gene expression and physiological responses to plasmodiophora brassicae

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

With Nile Red that stains lipids and suberin, it is possible to view the pathogen resting spores containing lipids (Figure 3A,B). Hence, using double staining, crisp images can be obtained to look at the pattern of pathogen distribution within the galls. Counterstaining with Calcofluor white creates contrast and helps to simultaneously track xylem development with P. brassicae maturation (Figure 3B).
Xylem formation and development could also be checked by observing autofluorescence in unstained samples (Figure 4A) or by using stains such as Basic Fuchsin that enable fluorescence-based imaging of lignin (Figure 4B).
By using this method, one could track gene expression changes or responses to growth regulators. A perfect example is where Arabidopsis plants harboring pHCA2:erRFP construct were utilized to visualize HIGH CAMBIAL ACTIVITY 2 (HCA2) gene expression in phloem tissue within clubroot galls. HCA2 gene activity has been previously found in meristematicaly active cambium and phloem-lineage cells15. Here, it co-localizes with the phloem in the late stages of P. brassicae-driven gall development, and its activity reflects how P. brassicae increases phloem complexity (Figure 5). The resulting image shows phloem proliferation at the late stage of gall development when the cambium gets fragmented. Figure 5A shows an uncleared hand section of the gall, whereas Figure 5B shows more crisp and localized fluorescent signals obtained by vibratome sectioning followed by tissue clearing. The objects were counterstained with Calcofluor white. Figure 6 compares this image (Figure 6B) with a similar region depicted in resin-embedded and microtome-sectioned galls (Figure 6A) at 21 DPI. Differential cytokinin responses between infected and non-infected plants were assessed by checking the expression of the TCS:GFP (Two Component Signalling) marker16 in developing galls (Figure 7). While imaging weak GFP signals in galls and tissues with secondary thickenings, it is important to note that additional background signal due to autofluorescence of mature xylem cells is also captured during imaging.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64297/64297fig01.jpg" /> 
Figure 1: Clubroot disease symptoms on oilseed rape (B. napus) and Arabidopsis thaliana (Columbia-0) at 26 DPI with Plasmodiophora brassicae spores. During the course of disease, large galls develop on the entire root system, making it extremely brittle. It concludes by releasing spores into the surrounding soil to promote future infections.&#160;The upper parts of the plant body also show signs of poor growth and development. Finally, the infected plants succumb to the devastating effects on growth metabolism and development once the root system gets completely damaged and the plant can no longer cope with the disease. The scale bar represents 1 cm. The -INF stands for mock-inoculated, whereas +INF stands for P. brassicae-inoculated plants. On this occasion, a picture of oilseed rape plants is provided before the soil removal to present healthy root systems. After washing, only the hypocotyl and upper part of the root are collected. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64297/64297fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64297/64297fig02.jpg" /> 
Figure 2: The general workflow. Washed Arabidopsis root systems are (A) dissected, (B) fixed, (C) agarose embedded, (D) mounted, and (E) sectioned on the vibratome. The resulting objects are subjected to tissue clearing (3 days to several weeks at RT in the dark, depending on the tissue type and thickness). (F) Cleared objects can then be stained and inspected under the microscope. (G) Summary of the workflow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64297/64297fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64297/64297fig03.jpg" /> 
Figure 3: Marking pathogen spores with Nile Red stain. (A,B) Nile Red stains lipids in resting spores, which works perfectly to track P. brassicae maturation. (A) Enlarged cells colonized by P. brassicae and filled with pathogen spores. (B) Mature xylem cells also get stained by Nile Red. The section has been counterstained with Calcofluor white to see the outlay of host cells (A: Objective lens = 20x and section thickness = 60 &#181;m; B: Objective lens = 5x and section thickness = 60 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64297/64297fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64297/64297fig04.jpg" /> 
Figure 4: Tracking the extent of xylem development and maturation. (A) Lignin gives strong autofluorescence upon excitation with UV; therefore, mature xylem can be discriminated relatively easily. (B) Double staining with Basic Fuchsin and Calcofluor gives better results since all cells get stained by the latter dye, while mature xylem is distinctly stained with Basic Fuchsin. In this way, double staining provides images with improved contrast depicting noticeable inhibition of xylogenesis (A: Objective lens = 10x and section thickness = 60 &#181;m; B: Objective lens = 10x and section thickness = 60 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64297/64297fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64297/64297fig05.jpg" /> 
Figure 5: Phloem-specific signal for the HCA2 gene in hypocotyls of P. brassicae-infected plants at 21 DPI. Promoter activity for proHCA2::erRFP harboring transgenic Arabidopsis thaliana can be seen in (A) and (B). Differences can be observed between a non-cleared hand section in panel (A) where the erRFP signal appears diffused due to superimposition and overlapping cell layers, especially in uneven hand sections. On the other hand, (B) shows a vibratome section post-tissue-clearing where the erRFP signal precisely marks the phloem cells in a mature vascular bundle (Objective lens = 20x and section thickness = 60 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64297/64297fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64297/64297fig06v2.jpg" /> 
Figure 6: Comparison between TB, resin-embedded, and microtome-sectioned galls with the help of fluorescence. (A) A comparison between images of Toluidine Blue (TB), resin-embedded, and microtome-sectioned galls, and (B) a representative object (also presented in Figure 5B) acquired with the help of fluorescence. Xylem cells are labeled with yellow asterisks, the cambial area with vivid green brackets, phloem with cyan asterisks, and Plasmodiophora brassicae-colonized cells with a white PB symbol. Resin-embedded sections (A) provide a good resolution for studying the distribution of resting spores in hypertrophied organs, degree of disease progression, and other processes such as local lignification in resistant plants. However, the protocol described here (B) enables the sensitive observation of gene expression or protein accumulation and the visualization of other physiological changes and important molecules such as lipids (in spores). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64297/64297fig06largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64297/64297fig07.jpg" /> 
Figure 7: Tracking cytokinin signaling responses in hypocotyls of non-infected (-INF) and P. brassicae-infected (+INF) Arabidopsis plants at 16 DPI. TCS::GFP marker was used for characterizing in planta cytokinin responses. Vibratome sections were subjected to clearing treatment followed by staining with Calcofluor white. Based on the image, at 16 DPI, cytokinin responses appear to be largely diminished in infected galls (right panel), while they remain strong, especially in the phloem pool (cells that will eventually differentiate to form phloem tissue), in non-infected plants (left panel) (Objective lens = 5x and thickness = 30 &#181;m). Certain levels of xylem autofluorescence can also be visible. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64297/64297fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Clearing Solution (toxic)</td>
			<td></td>
		</tr>
		<tr>
			<td>Components</td>
			<td>Percentage (%)</td>
			<td>in 100 mL distilled water</td>
		</tr>
		<tr>
			<td>Xylitol</td>
			<td>10%</td>
			<td>10g</td>
		</tr>
		<tr>
			<td>Sodium Deoxycholate</td>
			<td>15%</td>
			<td>15g</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>25%</td>
			<td>25g</td>
		</tr>
		<tr>
			<td colspan="2">10x PBS (Phosphate buffered saline)</td>
			<td>1x PBS (100 mL)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>8 g</td>
			<td>10 mL 10x PBS + 90 mL distilled water</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>0.2 g</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>0.24 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 &#183; 2H2O</td>
			<td>1.81 g</td>
			<td></td>
		</tr>
		<tr>
			<td>distilled water</td>
			<td>100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>pH</td>
			<td>pH adjusted to 7.4 using HCl</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Autoclave and store at 4 &#176;C.</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Composition of the Clearing solution and Phosphate-buffered saline (PBS).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fluorescent stain/ Tag</td>
			<td>Excitation/Emission Wavelengths</td>
			<td>Microscope filter set used</td>
		</tr>
		<tr>
			<td>Nile Red</td>
			<td>553/636 nm</td>
			<td>filter set 43</td>
		</tr>
		<tr>
			<td>Xylem Autofluorescence</td>
			<td>380/475 nm</td>
			<td>filter set 49</td>
		</tr>
		<tr>
			<td>Calcofluor White</td>
			<td>405/475 nm</td>
			<td>filter set 49</td>
		</tr>
		<tr>
			<td>Basic Fuchsin</td>
			<td>561/650 nm</td>
			<td>filter set 43</td>
		</tr>
		<tr>
			<td>erRFP</td>
			<td>585/608 nm</td>
			<td>filter set 43</td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>488/509 nm</td>
			<td>filter set 38</td>
		</tr>
	</tbody>
</table>
Table 2: Excitation/emission spectra selected for the present study.

Watch this Scientific Journal Video about Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae at JoVE.com

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

The present protocol describes an optimized method for the histological observation of galls induced by Plasmodiophora brassicae. Vibratome sections of hypocotyls are cleared before fluorescence imaging to study the involvement of transcription factors and phytohormones during disease progression. This protocol overcomes resin embedding limitations, enabling in planta visualization of fluorescent proteins.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls ...

combining-clearing-fluorescence-microscopy-for-visualising-changes

Nanyang Technological University

Research

JoVE Journal

Biology

2.6K Views.  Institute of Plant Genetics Polish Academy of Sciences. Plasmodiophora brassicae is de door de bodem overgedragen verplichte biotrofe ziekteverwekker die planten uit de Brassicaceae-familie aanvalt. Bij infectie veroorzaakt pathogeen de ontwikkeling van gallen op de ondergrondse delen van de plant, vandaar de naam van de ziekte clubroot. De ontwikkeling van gallen gaat gepaard met zeer complexe herprogrammering.Daarom is de mogelijkheid om interne veranderingen tijdens ziekte te volgen cruciaal voor het begrijpen van deze plant-pathogeen in...

Video: Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Developmental Biology

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Chemistry

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Deep Fluorescence Observation in Rice Shoots via Clearing Technology

The recently developed clearing technology that eliminates refractive index mismatches and diminishes auto-fluorescent material has made it possible to observe plant tissues in three dimensions (3D) while preserving their internal structures. In rice (Oryza sativa L.), a monocot model plant and a globally important crop, clearing technology has been reported in organs that are relatively easy to observe, such as the roots and leaves. Applications of clearing technology in shoot apical meristem (SAM) and stems have also been reported, but only to a limited degree because of the poor penetration of the clearing solution (CS) in these tissues. The limited efficiency of the clearing solutions in these tissues has been attributed to auto-fluorescence, thickening, and hardening of the tissues in the stem as the vascular bundles and epidermis develop and layering of the SAM with water-repellent leaves. The present protocol reports the optimization of a clearing approach for continuous and 3D observation of gene expression from the SAM/young panicle to the base of the shoots during development. Fixed tissue samples expressing a fluorescent protein reporter were trimmed into sections using a vibrating micro-slicer. When an appropriate thickness was achieved, the CS was applied. By specifically targeting the central tissue, the penetration rate and uniformity of the CS increased, and the time required to make the tissue transparent decreased. Additionally, clearing of the trimmed sections enabled the observation of the internal structure of the whole shoot from a macro perspective. This method has potential applications in deep imaging of tissues of other plant species that are difficult to clear.

Deep Fluorescence Observation in Rice Shoots via Clearing Technology

The present protocol describes a clearing technique for rice shoots, which are difficult to prepare for internal structural observations owing to the hard, thick, or layered nature of the tissues. This method facilitates continuous and deep fluorescence observations, even in adult rice plants.

The recently developed clearing technology that eliminates refractive index mismatches and diminishes auto-fluorescent material has made it possible to ...

Visualization of Cellular Interactions Between the Maize Sheaths and Fungi

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 &#181;L drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 &#176;C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.

Author Spotlight: Investigating Fungal Pathogenicity Mechanisms in Maize 

This manuscript details an optimized inoculation protocol that uses detached maize leaf sheaths for reproducible cytological, physiological, and molecular studies of maize interactions with fungal plant pathogens. The leaf sheaths facilitate real-time observation of cellular interactions between the living plant and fungus in unfixed tissues.

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one ...

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

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