Name: measuring bacterial colonization on arabidopsis thaliana roots in hydroponic condition
Uploaded: 2024-03-01T14:10:00.000+00:00
Duration: 5 min 37 s
Description: Watch this Scientific Journal Video about Author Spotlight: Enhancing Rhizobacteria Colonization on Plant Roots for Improved Microbial Fertilizer Efficiency at JoVE.com

This work was funded by the National Natural Science Foundation of China (32370135), the Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-CSAL-202302), the Science and Technology Project of Jiangsu Vocational College of Agriculture and Forestry (2021kj29).

1. Sterile hydroponic A. thaliana cultivation
<ol>
	<li>Prepare A. thaliana seedlings.
	<ol>
		<li>Prepare culture A. thaliana seedlings medium, which consists of 1/2 MS medium (Murashige and Skoog) with 2% (wt/vol) sucrose and 0.9% (wt/vol) agar.</li>
		<li>Pour the prepared sterilization medium into sterile square Petri dishes (13 cm x 13 cm) before solidification. Avoid air drying to maintain humidity.</li>
		<li>Immerse A. thaliana seeds in a 2 mL microcentrifuge tube filled with 1 mL of sterile water at 4 &#176;C for over 12 h, and then sterilize the seeds with 1 mL of 2% (vol/vol) NaClO for 2 min.</li>
		<li>Thoroughly wash the seeds with sterile water six times to remove the NaClO solution. Sow the seeds on Petri dishes with MS agar medium with a sterile 1 mL tip.</li>
		<li>Seal the Petri dishes with breathable medical tape and place them vertically in a light incubator to grow for 1 week. Set the light incubator day/night ratio to 16 h/8 h, maintain the temperature at 23 &#176;C and humidity at 40%-60%.</li>
	</ol>
	</li>
	<li>Transplant A. thaliana.
	<ol>
		<li>Cut 5 mm holes on 40 &#181;m pore size cell stainer and sterile them.</li>
		<li>Transplant three 7-day-old A. thaliana plants through the holes of a cell strainer and put them into a 6-well plate containing 3 mL of sterile liquid 1/2 MS medium with 0.5% sucrose.</li>
		<li>Place the 6-well plates in a light incubator (Figure 1A) and incubate for 10 days.</li>
	</ol>
	</li>
</ol>
2. Bacteria cultivation and inoculation
<ol>
	<li>Prepare bacterial suspension for inoculation.
	<ol>
		<li>For Bacillus velezensis, inoculate bacterial cells into sterile LB medium and incubate at 37 &#176;C with shaking at 170 rpm until it reaches OD600 of 0.8-1.2.</li>
		<li>Collect the bacterial cells by centrifugation at 6000 x g for 2 min and resuspend the cells in PBS buffer (2 mM KH2PO4,&#160;8 mM Na2HPO4, 136 mM NaCl, 2.6 mM KCl). Repeat the centrifuging and resuspending steps 3 times to remove the LB medium and the bacterial metabolites.</li>
		<li>Suspend the bacteria cells in a fresh 1/2 MS medium at a final concentration of OD600 0.01. Inoculate the bacteria suspensions to the 6-well plates that grow A. thaliana seedlings, place the 6-well plates in the light incubator, and cultivate them for 2 days.</li>
	</ol>
	</li>
</ol>
3. Measuring bacteria colonized on roots
<ol>
	<li>Harvest the root tissue and plate the colonized cells. 
	NOTE: All steps should be conducted under gnotobiotic condition
	<ol>
		<li>Weigh the 2 mL tubes and record the weight as W0.</li>
		<li>Harvest the co-cultured A. thaliana root tissues and wash them in sterile water 3 times. Place the root on filter paper to remove the excess water.</li>
		<li>Put the root into the weighed microcentrifuge tube, weigh the roots together with the tube, and record it as W1.</li>
		<li>Add 1 mL of PBS to each tube and vortex the tubes for 8 min at the maximum speed.</li>
		<li>Dilute the suspensions with the collected root-colonized bacterial cells to 1 x 10-1-1 x 10-4&#160;(according to the bacteria colonization ability). Spread the diluted bacterial suspensions on plates containing LB agar medium and incubate at 37 &#176;C for 10 h.</li>
	</ol>
	</li>
	<li>Count the colonies and normalize the data.
	<ol>
		<li>Count the bacterial colonies on LB plates.</li>
		<li>Calculate the CFUs according to the corresponding dilution and normalize the data with root fresh weight (W1-W0). The final results indicate the count of bacterial cells colonized per gram of root at 2 days post-inoculation (Figure 1B).</li>
	</ol>
	</li>
</ol>

Rhizobacterial Colonization Detection on Arabidopsis thaliana

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The authors declare that they have no conflicts of interest with the contents of this article.

To achieve good reproducibility, there are four critical steps for the colonization detection process of this protocol. First, it is necessary to ensure that the number of inoculated bacteria cells is exactly the same in each experiment. Second, controlling the uncolonized bacteria cleaning intensity with sterile water is also necessary. Third, every sample dilution process needs to be vortexed before being performed to let the sample be in a complete mixing state to avoid the absorption errors due to the characteristics...

There are quantitative and qualitative methods for detecting rhizosphere colonization by a single bacterial strain. For the qualitative method, a strain that constitutively expresses fluorescence should be used, and the fluorescence distribution and intensity should be examined under fluorescence microscopy or laser confocal instruments1,2. Those strategies can well reflect bacterial colonization in situ3, but they are not as accurate as traditional plate counting methods in quantification. Besides, due to the limitation of only displaying partial root zones under the microscope, sometimes it can be influenced by subjective bias. 
Here, we describe a quantitative method, which includes collecting the colonized bacterial cells and counting the bacterial CFUs on a plate. This method is based on dilution and plating by which the colonized strains that were stripped from plant roots can be counted, and the total colonized bacteria number on the root can be calculated4,5. 
First, A. thaliana was cultured in hydroponic conditions, and then bacterial cells were inoculated in the rhizosphere at a final concentration of 0.01 OD600. The infected root tissues were harvested 2 days post-inoculation and washed in sterile water to remove the uncolonized bacterial cells. Further, bacterial cells colonized on the root were collected, diluted in phosphate-buffered saline (PBS) buffer, and plated onto a Luria-Bertani (LB) agar medium. After incubation at 37 &#176;C for 10 h, single colonies on LB plates were counted and normalized to determine the bacterial cells colonized on roots.
This method is highly applicable, has good repeatability, and is more suitable for accurate rhizosphere bacterial colonization determination.

<table><tbody><tr><td>6-well plate</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>Filter cell stainer</td><td>Solarbio</td><td>F8200-40&amp;micro;m</td><td></td></tr><tr><td>Microplate reader&amp;nbsp;</td><td>Tecan</td><td>Infinite M200 PRO</td><td></td></tr><tr><td>Murashige and Skoog medium</td><td>Hopebio</td><td>HB8469-5</td><td></td></tr><tr><td>NaClO</td><td>Alfa</td><td>L14709</td><td></td></tr><tr><td>Phytagel</td><td>Sigma-Aldrich</td><td>P8169</td><td></td></tr><tr><td>Square petri dish</td><td>Ruiai Zhengte</td><td>PYM-130</td><td></td></tr><tr><td>Vortex Genie2</td><td>Scientific Industries</td><td>G560E</td><td></td></tr></tbody></table>

measuring bacterial colonization on arabidopsis thaliana roots in hydroponic condition

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized method for measuring bacterial colonization in the rhizosphere is necessary to improve reproducibility. We first cultured sterile A.thaliana in hydroponic conditions and then inoculated the bacterial cells in the rhizosphere at a final concentration of OD600 of 0.01. At 2 days post-inoculation, the root tissue was harvested and washed three times in sterile water to remove the uncolonized bacterial cells. The roots were then weighed, and the bacterial cells colonized on the root were collected by vortex. The cell suspension was diluted in a gradient with a phosphate-buffered saline (PBS) buffer, followed by plating onto a Luria-Bertani (LB) agar medium. The plates were incubated at 37 &#176;C for 10 h, and then, the single colonies on LB plates were counted and normalized to indicate the bacterial cells colonized on roots. This method is used to detect bacterial colonization in the rhizosphere in mono-interaction conditions, with good reproducibility.

To test the accuracy of the bacteria colonization ability detected by this method in the A. thaliana&#160;rhizosphere, we inoculated Bacillus velezensis SQR9 WT and a derived mutant &#916;8mcp into A. thaliana rhizosphere separately. The &#916;8mcp is a mutant that lacks all the chemoreceptor encoding genes, and it has a significantly decreased colonization6. We measured their colonization at 2 days post-inoculation by the present root colonization assa...

Watch this Scientific Journal Video about Author Spotlight: Enhancing Rhizobacteria Colonization on Plant Roots for Improved Microbial Fertilizer Efficiency at JoVE.com

Author Spotlight: Enhancing Rhizobacteria Colonization on Plant Roots for Improved Microbial Fertilizer Efficiency

Colonization of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere is essential for its growth-promoting effect. It is necessary to standardize the method of detection of bacterial rhizosphere colonization. Here, we describe a reproducible method for quantifying bacterial colonization on the root surface.

Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized ...

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Immunology and Infection

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Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Biology

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...

Quantification of Arbuscular Mycorrhizal Fungi Colonization Rate in the Study of Invasive Alien Plants

Arbuscular mycorrhizal fungi (AMF) are widely distributed soil fungi in ecosystems and can form symbiotic associations (mycorrhizae) with the roots of most terrestrial plants. Plants provide carbon sources to AMF through mycorrhizal associations, while AMF hyphae can expand the range of nutrient absorption by roots and promote plant nutrient uptake. There are many different species of AMF, and the symbiotic relationships between different species of AMF and different plants vary. Invasive plants can enrich AMF species with better symbiotic capabilities through root exudates, promoting their growth and thereby increasing their colonization in invasive plant roots. At the same time, invasive plants can also disrupt the symbiotic relationship between AMF and native plants, affecting the local plant community, which is one of the mechanisms for successful plant invasion. The colonization rate of AMF in the roots of invasive and native plants indirectly reflects the role of AMF in the process of invasive plant invasion. In this method, collected plant roots can be processed directly or saved in a fixative for later batch processing. Through decolorization, acidification, staining, and destaining treatment of roots, the hyphae, spores, and arbuscular structures of AMF in the root system can be clearly observed. This method can be completed in a basic laboratory to observe and calculate the colonization rate of AMF in the root systems of invasive plants.

This protocol provides a simple and easy-to-use approach for determining the colonization rate of Arbuscular mycorrhizal fungi (AMF) in the roots of invasive plants.

Arbuscular mycorrhizal fungi (AMF) are widely distributed soil fungi in ecosystems and can form symbiotic associations (mycorrhizae) with the roots of ...

Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

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