We thank Jakob Viel for his helpful comments and suggestions. We also thank Anne de Jong for his help in the bioinformatics analysis. Yanglei Yi and Zhibo Li are supported by the China Scholarship Council (CSC). We thank NWO-TTW Perspectief Programma Back2Roots (TKI-AF-15510) for their financial support to OPK.

1. Germinating Potato in Sterilized Conditions
<ol>
	<li>Rinse the potato surface with sterile water. Bathe the potato in 70% ethanol and then in 3% sodium hypochlorite, for 5 min each. Rinse it again with sterile water to remove any remaining sodium hypochlorite.</li>
	<li>Prepare the materials needed for germinating and growing potato tubers; sterilize the plastic pots, engraftment baskets, vermiculite, and water by autoclaving them at 121 &#176;C for 20 min. 
	NOTE: Ensure that all materials used are autoclavab...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+function+of+root+exudates.">Regulation and function of root exudates.</a> Plant, Cell &amp; Environment. 32 (6), 666-681 (2009).</li><li>Rohrbacher, F., St-Arnaud, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+exudation:+the+ecological+driver+of+hydrocarbon+rhizoremediation.">Root exudation: the ecological driver of hydrocarbon rhizoremediation.</a> Agronomy Journal. 6 (1), 19 (2016).</li><li>Mark, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+bacterial+responses+to+root+exudates+identifies+genes+involved+in+microbe-plant+interactions.">Transcriptome profiling of bacterial responses to root exudates identifies genes involved in microbe-plant interactions.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (48), 17454-17459 (2005).</li><li>Fan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomic+profiling+of+Bacillus+amyloliquefaciens+FZB42+in+response+to+maize+root+exudates.">Transcriptomic profiling of Bacillus amyloliquefaciens FZB42 in response to maize root exudates.</a> BMC Microbiology. 12 (1), 116 (2012).</li><li>Yoshitomi, K. J., Shann, J. 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J., Vaughan, D., Jones, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhizosphere+carbon+flow+in+trees,+in+comparison+with+annual+plants:+the+importance+of+root+exudation+and+its+impact+on+microbial+activity+and+nutrient+availability.">Rhizosphere carbon flow in trees, in comparison with annual plants: the importance of root exudation and its impact on microbial activity and nutrient availability.</a> Applied Soil Ecology. 5 (1), 29-56 (1997).</li><li>Rovira, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+root+exudates.">Plant root exudates.</a> Botanical Review. 35 (1), 35-57 (1969).</li><li>Bargabus, R. L., Zidack, N. K., Sherwood, J. E., Jacobsen, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+systemic+resistance+in+sugar+beet+elicited+by+a+non-pathogenic,+phyllosphere-colonizing+Bacillus+mycoides,+biological+control+agent.">Characterisation of systemic resistance in sugar beet elicited by a non-pathogenic, phyllosphere-colonizing Bacillus mycoides, biological control agent.</a> Physiological and Molecular Plant Pathology. 61 (5), 289-298 (2002).</li><li>Peng, Y. -. 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The authors declare that they have no competing financial interests.

Plant-microbe interactions have been hypothesized to be determined by a finely tuned equilibrium between bacteria and plants. Such interactions are highly complex and difficult to study in a natural system, which comprises diverse microbial species, potentially acting as consortia. This paper describes a simplified protocol to study the bacterial response to root exudates under well-controlled conditions. The transcriptome profile of rhizobacteria, upon exposure to root exudates, provides detailed information on bacteria...

Plants may exudate up to 20% of the carbon fixed during photosynthesis through roots into the rhizosphere1, i.e., the narrow zone of soil near the roots. Due to the higher nutrient availability, the rhizosphere is a suitable habitat for diverse microorganisms, including plant-growth promoting bacteria. The root exudates contain a range of inorganic compounds like ions, inorganic acids, oxygen, and water. However, the majority of the root exudates is formed by organic materials, which can be divided into low molecular weight compounds and high molecular weight compounds. The low molecular weight compounds include amino acids, organic acids, sugars, phenolic compounds, fatty acids, and an array of secondary metabolites. The high molecular weight compounds consist of mucilage and proteins2,3. Rhizosphere microorganisms can use some of these compounds as an energy source for growth and development. The root exudates play an important role in shaping the rhizobacterial community since the plant-produced compounds in the exudates can influence the behavior of rhizosphere-associated bacteria by affecting the expression of specific genes.
Understanding the bacterial response to root exudates is a key step in deciphering plant-microbe interaction mechanisms. As the bacterial response to plant-microbe interactions is the product of differential gene expression, it can be studied by transcriptome analysis. Using this method, previous studies identified several important genes involved in plant-microbe interactions. In Pseudomonas aeruginosa, genes involved in metabolism, chemotaxis, and type II secretion were shown to respond to sugar beet root exudates4. Fan et al.5 studied the transcriptomic profiling of B. amyloliquefaciens FZB42 in response to maize root exudates. Their results show that, of the genes strongly induced by the root exudates, several groups are involved in metabolic pathways relating to nutrient utilization, chemotaxis, motility, and non-ribosomal synthesis of antimicrobial peptides and polyketides.
The accuracy of these studies relies on the collection of root exudates. Although several methods have described the collection of root exudates for different purposes, they either demand sophisticated instruments or are not performed in well-controlled conditions6,7,8. Moreover, rhizosphere-inhibiting microorganisms can influence root exudate composition by affecting plant cell membrane permeability and damaging the root tissues, particularly in the case of consortia of microorganisms9. When investigating the microbial response to root exudates, it is important to use well-defined conditions in order to avoid alteration of the compounds by other microorganisms10. Furthermore, high-quality RNA is required for RNA-seq based transcriptome studies. However, when dealing with non-model-bacterial strains, the standard protocols or commercial kits usually have a low efficiency due to unknown factors or special growth properties.
The protocol described here was verified using B. mycoides, which is a gram-positive, spore-forming bacterium of the Firmicute phylum. It is ubiquitous in the rhizosphere of various plant species. Several plant growth promoting properties have been reported for this species, including induction of systematic resistance (ISR) in sugar beet11, inhibition of the damping-off pathogen Pythium for cucumber12, as well as nitrogen fixation in the sunflower rhizosphere13. However, the molecular mechanisms of its interaction with a host plant are not well studied.
The objective of the experiments presented here is to study the transcriptomic response of endosphere-isolated B. mycoides to potato root exudates. In short, the protocol consists of the following steps: first, collect potato root exudates under sterile conditions. Then, extract high-quality RNA from bacterial cells treated with root exudates. The final step is data analysis using the web-based T-REx pipeline14. This protocol was used to identify B. mycoides genes that show a shift in expression levels upon contact with root-exudates and thus might play an important role in plant-microbe interactions.

<table border="0" cellpadding="0" cellspacing="0" width="987">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">sodium hypochlorite</td>
			<td style="width:236px;">Sigma</td>
			<td style="width:152px;">&#160;CAS: 7681-52-9&#160;</td>
			<td style="width:339px;">10-15%&#160; active chlorine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Luria-Bertani (LB) broth</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">incubater</td>
			<td style="width:236px;">New Brunswick Scientific</td>
			<td style="width:152px;">Innova 4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">spectrophotometer</td>
			<td style="width:236px;">Thermo Fisher Scientific</td>
			<td style="width:152px;">Genesys 20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">liquid nitrogen</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">glass beads</td>
			<td style="width:236px;">Sigma</td>
			<td style="width:152px;">G8893</td>
			<td>0.5 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">2.0 ml tube with screw cap</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>RNase free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">1.5 ml and 2.0 ml eppendorf tube</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>RNase free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bead mill homogenizer</td>
			<td>BioSpec</td>
			<td style="width:152px;">607</td>
			<td>Mini_beadbeater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">centrifuge</td>
			<td style="width:236px;">Eppendorf</td>
			<td style="width:152px;">5430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Diethyl pyrocarbonate (DEPC)</td>
			<td style="width:236px;">sigma</td>
			<td style="width:152px;">CAS: 1609-47-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:236px;">sigma</td>
			<td style="width:152px;">CAS: 151-21-3&#160;</td>
			<td>10% solution prepared with DEPC treated MQ water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">TE buffer</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>10 mM Tris-HCl; 1 mM EDTA, pH=8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">phenol</td>
			<td>Sigma</td>
			<td style="width:152px;"></td>
			<td>RNA grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">chloroform-isoamyl alcohol&#160;</td>
			<td style="width:236px;"></td>
			<td style="width:152px;"></td>
			<td>prepare 24:1 of chloroform:isoamyl alcohol, store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">High pure RNA isolation kit</td>
			<td style="width:236px;">Roche</td>
			<td style="width:152px;">11828665001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RNase Decontamination Solution</td>
			<td style="width:236px;">Invitrogen</td>
			<td style="width:152px;">AM9780</td>
			<td>RNase-Zap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Automated electrophoresis instrument</td>
			<td style="width:236px;">Agilent</td>
			<td style="width:152px;">2100</td>
			<td>Bioanalyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microvolume spectrophotometer</td>
			<td style="width:236px;">Thermo Fisher Scientific</td>
			<td style="width:152px;">Nanodrop ND-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RNA quality analysis kit</td>
			<td style="width:236px;">Agilent</td>
			<td style="width:152px;">RNA 6000 Nano kit&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RNase inhibitor</td>
			<td style="width:236px;">Thermo Fisher Scientific</td>
			<td style="width:152px;">RiboLock</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Directional RNA library Prep kit</td>
			<td style="width:236px;">NEB</td>
			<td style="width:152px;">Ultra</td>
			<td style="width:339px;">For Illumina</td>
		</tr>
	</tbody>
</table>

plant-microbe interaction: transcriptional response of bacillus mycoides to potato root exudates

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant growth-promoting rhizobacteria (PGPR) as biofertilizers or biocontrol agents has become an effective alternative to the use of conventional fertilizers and can increase crop productivity at low cost. Plant-microbe interactions depend upon host plant-secreted signals and a reaction hereon by their associated bacteria. However, the molecular mechanisms of how beneficial bacteria respond to their associated plant-derived signals are not fully understood. Assessing the transcriptomic response of bacteria to root exudates is a powerful approach to determine the bacterial gene expression and regulation under rhizospheric conditions. Such knowledge is necessary to understand the underlying mechanisms involved in plant-microbe interactions. This paper describes a detailed protocol to study the transcriptomic response of B. mycoides EC18, a strain isolated from the potato endosphere, to potato root exudates. With the help of recent high-throughput sequencing technology, this protocol can be performed in several weeks and produce massive datasets. First, we collect the root exudates under sterile conditions, after which they are added to B. mycoides cultures. The RNA from these cultures is isolated using a phenol/chloroform method combined with a commercial kit and subjected to quality control by an automated electrophoresis instrument. After sequencing, data analysis is performed with the web-based T-REx pipeline and a group of differentially expressed genes is identified. This method is a useful tool to facilitate new discoveries on the bacterial genes involved in plant-microbe interactions.

Plant-associated microorganisms can positively influence plant growth and health. However, the mechanisms of the complex interactions between plants and their microbial symbionts are not fully understood. Root exudates play an important role in regulating the rhizobacterial activity and behavior, and it is generally postulated that the microbial colonization of roots initiates with the attraction of microbes to root exudates. The aim of this work was to investigate the transcriptomic resp...

Watch this Scientific Journal Video about Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates at JoVE.com

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

The goal of the protocol presented here is to study the transcriptomic response of endosphere-isolated Bacillus mycoides to potato root exudates. This method facilitates the identification of important bacterial genes involved in plant-microbe interactions and is in principle applicable to other endophytes and plants, with minor adjustments.

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant ...

This method can help to answer key questions in the fields of plant-microbe interaction. You can, for instance, identify important genes from bacterial strains that were isolated from the endosphere or rhizosphere during the interaction of the bacteria with the plant. It's a versatile method that can also be used for studies on other bacteria with plants.The main advantage of this technique is that in a relatively short period a large number of bacteria genes that are important during plant-microbe interactions can be identified. To begin this procedure, rinse the potato surface with sterile water. Bathe the potato in 70%ethanol for five minutes and then in 3%sodium hypochlorite for another five minutes.Afterward, rinse it again with sterile water to remove any remaining sodium hypochlorite. Next, prepare the materials needed for germinating and growing potato tubers. Sterilize the plastic pots, engraftment baskets, vermiculite, and water by autoclaving them at 121 degrees Celsius for 20 minutes.Subsequently, put the surface-sterilized potato into the engraftment basket and place it in an autoclaved pot containing wet vermiculite. Use a large glass fiber box to cover the pot to avoid microbial contaminations. Keep the pot in a climate chamber at 24 degrees Celsius for three weeks, and set it on cycles of 16 hours of light and 8 hours of darkness.To collect the potato root exudates, when the shoots sprout, transfer the basket with the whole potato into a sterilized beaker filled with 150 milliliters of autoclaved deionized water with the potato tubers placed just above the water surface and the roots submerged in the water. Keep the seedling in the climate chamber, and use the same settings as before. From the second day on, collect the water containing the exudates, and refill the beaker with sterile, deionized water.Then store each sample separately at four degrees Celsius. For each sample, spread 100 microliters on a Luria-Bertani agar plate to check for microbial contaminations. Discard the contaminated samples.Combine the collected samples of one seedling. Then transfer the combined root exudates into 50 milliliter tubes. Freeze the tubes containing root exudates at minus 80 degrees Celsius.Concentrate them by freeze drying them at minus 40 degrees Celsius to a final volume of 150 milliliters. To grow the bacteria, streak a B.mycoides strain from a minus 80 degree Celsius glycerol stock onto an LB agar plate, and incubate the plate at 30 degrees Celsius overnight. Then inoculate the LB liquid medium with a single colony from the plate, and grow the culture in a shaking incubator at 200 rpm overnight at 30 degrees Celsius.To treat and sample the bacteria, dilute an overnight B.mycoides culture with 90 milliliters of pre-warmed LB medium to the initial OD600 of zero point zero five in a 300 milliliter flask. Subsequently, add 10 milliliters of root exudates to the culture, and use a 10 milliliter sterile, deionized water treatment as a control. Then incubate them at 30 degrees Celsius for one hour.After an hour, transfer the culture into 50 milliliter centrifuge tubes. Collect the cells from the culture by centrifugation at 9, 000 times G for two minutes at four degrees Celsius. Afterward, discard the supernatent, and immediately freeze the pellet in liquid nitrogen.Then store it at minus 80 degrees Celsius until use. To isolate RNA, thaw the cell pellets on ice, and suspend each of them in 400 microliters of TE buffer. Then transfer the suspensions into the two milliliter screw cap tubes.Add zero point five grams of glass beads into the tube. Then close the caps firmly and place the tubes in a bead mill homogenizer. Perform a 45-second pulse homogenization three times, with a one-minute interval on ice.Afterward, centrifuge the samples for 10 minutes at 11, 000 times G, and transfer the upper phase of each sample to a new tube. Add 500 microliters of chloroform:isoamyl alcohol to the upper phase, and centrifuge the mixture for five minutes at 11, 000 times G.Then transfer 500 microliters of the upper phase to a fresh tube. Add one milliliter of the lysis binding buffer, and mix it by pipetting up and down.Next, prepare one point five milliliter microcentrifuge tubes with 100 microliters of DNase buffer, 10 microliters of DNase I, and five microliters of an RNase inhibitor. Add the prepared solution mix on the filter of the filter tubes, and incubate them for 20 to 30 minutes at 15 to 25 degrees Celsius. Subsequently, perform the washing steps according to the manufacturer's instruction, and add 50 microliters of an elusion buffer to the tube.After centrifugation, elute the RNA. Transfer a few microliters of the sample to a one point five milliliter microcentrifuge tube to perform a quality check. Finally, cover the eluded RNA with a plastic paraffin film and keep it at minus 80 degrees Celsius.After collection, the root exudates were added to the B.mycoides culture at a 10%ratio, and the bacterial total RNA was isolated. The RNA was then subjected to a quality check by an automated electrophoresis instrument. These graphs represent the RNA isolated from B.mycoides treated with the root exudates, and these graphs represent the RNA isolated from the control group.All samples scored a RIN value above nine, with two clear bands corresponding to the 16S and 23S RNA subunits, demonstrating that high-quality RNA was obtained by this protocol. After library preparation, pair end reads were obtained with a high-throughput sequencing platform. The raw RNA-seq reads were trimmed from the adaptor sequences, and mapped against the reference genome sequence.The transcript home analysis was performed with the T-REx pipeline, and the ratio intensity plot of all the differentially expressed genes is shown here. After the potentially important gene's been identified, other in vivo methods like promoter GFP fusion, cue-pews-air, and fluorescent in situ hybridization can be performed in order to answer additional questions like time in reserved expression of certain genes during the interaction with host plant.

plant-microbe-interaction-transcriptional-response-bacillus-mycoides

Research

JoVE Journal

Biology

12.4K visualizações.  University of Groningen. This method can help to answer key questions in the fields of plant-microbe interaction. You can, for instance, identify important genes from bacterial strains that were isolated from the endosphere or rhizosphere during the interaction of the bacteria with the plant. It's a versatile method that can also be used for studies on other bacteria with plants.The main advantage of this technique is that in a relatively short period a large number of bacteria genes that are important during ...