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 autoclavable. Otherwise, use other sterilizing methods.</li>
	<li>Put the surface-sterilized potato into the engraftment basket, and place this in an autoclaved pot containing wet vermiculite.</li>
	<li>Keep the pot in a climate chamber at 24 &#176;C for three weeks. Set it on cycles of 16 h of light (120 &#181;mol m-2s-1) and 8 h of darkness. To avoid microbial contaminations, use a large glass fiber box to cover the pot.</li>
</ol>
2. Collecting Potato Root Exudates
<ol>
	<li>When the shoots sprout, transfer the basket with the whole potato into a sterilized beaker filled with 150 mL of autoclaved deionized water, with the potato tubers placed just above the water surface and the roots submerged in the water (see Figure 1). Keep the seedling in the climate chamber, and use the same settings as before [24 &#176;C; cycles of 16 h of light (120 &#181;mol m-2s-1), 8 h of darkness].</li>
	<li>From the second day on, collect the water containing the exudates, and refill the beaker with sterile deionized water. Perform the sampling every day until the seventh day after transferring the seedling.</li>
	<li>Store each sample separately at 4 &#176;C. For each sample, spread 100 &#181;L on a Luria-Bertani (LB; 1% tryptone, 0.5% yeast extract, 0.5% NaCl) agar plate to check for microbial contaminations. Discard the contaminated samples.</li>
	<li>Combine the collected samples of one seedling, and concentrate them by freeze-drying them at -40 &#176;C to a final volume of 150 mL.</li>
</ol>
3. Growing Bacteria
<ol>
	<li>Streak a B. mycoides strain from a -80 &#176;C glycerol stock onto an LB agar plate, and incubate the plate at 30 &#176;C overnight.</li>
	<li>Inoculate an LB liquid medium with a single colony from the plate, and grow the culture in a shaking incubator at 200 rpm overnight at 30 &#176;C.</li>
</ol>
4. Treatment and Sampling of Bacteria
<ol>
	<li>To compare the growth curve of the bacteria treated with root exudates to a negative control, prepare a series of flasks containing 50 mL of LB liquid medium. Add 0.5 mL of the overnight bacterial culture to all flasks, and add either 0%, 5%, 10%, or 15% (v/v) root exudate or sterile deionized water to the medium, respectively.</li>
	<li>Use a culture with deionized water instead of root exudates as a control. Measure the optical density at 600 nm (OD600) every 1 h afterward. Generate a growth curve by plotting the OD600 values versus time. 
	NOTE: We saw that 10% of the root exudates did not affect the growth of B. mycoides (see Figure 2); this ratio was used for RNA-seq experiments.</li>
	<li>Dilute an overnight B. mycoides culture with 90 mL of pre-warmed LB medium to the initial OD600 of ~ 0.05 in a 300 mL flask. Add 10 mL of root exudates to the culture and incubate it at 30 &#176;C for 1 h.</li>
	<li>Use a 10 mL sterile deionized water treatment as a control. Collect cells from the culture by centrifugation at 9,000 x g for 2 min at 4 &#176;C. Discard the supernatant, and immediately freeze the pellet in liquid nitrogen, then store it at -80 &#176;C until use.</li>
</ol>
5. RNA Isolation
NOTE: Before starting the isolation, prepare the workbench, racks, and pipettes by cleaning them with an RNase decontamination solution (see Table of Materials). Wear gloves at all time, and make sure all tubes, tips, and solutions are RNase-free. Always keep the samples on ice when possible.
<ol>
	<li>Add 0.1% (v/v) of diethyl pyrocarbonate (DEPC) to ultrapure water (100 &#181;L of DEPC per 100 mL solution), mix it well, and keep it at room temperature overnight. Autoclave the mixture to inactivate the DEPC after overnight treatment. Use this DEPC-treated ultrapure water to prepare the TE buffer [10 mM Tris-HCl (pH = 8); 1 mM EDTA] and a 10% SDS (w/v) solution.</li>
	<li>Thaw the cell pellets on the ice and suspend each in 400 &#181;L of TE (DEPC) buffer. Transfer the suspensions into 2 mL screw cap tubes.</li>
	<li>Premix 300 &#181;L of chloroform-isoamyl alcohol (24:1 v/v) and 300 &#181;L of phenol (acid phenol, RNA grade), and allow the mixture to stand for 5 min. Take 500 &#181;L of the organic upper phase to add to the resuspended cells. Then add 50 &#181;L of 10% SDS and 0.5 g of glass beads (0.5 &#181;m) into the tube.</li>
	<li>Close the cap firmly and place the tube in a bead-mill homogenizer (see Table of Materials). Perform a 3&#215; 45 s pulse homogenization with a 1 min interval on ice.</li>
	<li>Centrifuge the samples for 10 min at 11,000 x g (4 &#176;C), and transfer the upper phase to a new tube.</li>
	<li>Add 500 &#181;L of chloroform-isoamyl alcohol (24:1) to the upper phase from step 5.5 and centrifuge the mixture for 5 min at 11,000 x g (4 &#176;C). 
	NOTE: The following steps were modified from the manufacturer&#39;s instruction of a commercial glass fiber filter-based RNA isolation kit (see Table of Materials).</li>
	<li>Transfer 500 &#181;L of the upper phase to a fresh tube, add 2 volumes (1 mL) of a lysis/binding buffer and mix it by pipetting up and down.</li>
	<li>Combine the filter and collection tubes (from the RNA isolation kit) and pipette the mixture from step 5.7 to the filter tube. Centrifuge the tubes for 15 s at 8,000 x g and discard the flow-through.</li>
	<li>Prepare 1.5 mL microcentrifuge tubes with 100 &#181;L of DNase buffer, 10 &#181;L of DNase I, and 5 &#181;L of an RNase inhibitor (see Table of Materials). Add the prepared solution mix on the filter of the filter tube, and incubate it for 20 - 30 min at 15-25 &#176;C.</li>
	<li>Perform the washing steps according to the manufacturer&#39;s instruction. Use 50 &#181;L of an elution buffer to elute the RNA.</li>
	<li>Save the eluted RNA at -80 &#176;C, and put a plastic paraffin film around it. At the same time, transfer a few microliters of the sample to a 1.5 mL microcentrifuge tube to perform a quality check.</li>
</ol>
6. RNA Quality Check and Sequencing
<ol>
	<li>Check the RNA with a microvolume spectrophotometer. 
	NOTE: Absorbance ratios at 260/280 should be above 1.8, and ratios at 260/230 should be above at least 1.8, preferably above 2.0.</li>
	<li>Run the purified RNA in an automated electrophoresis instrument using the recommend RNA analysis kit (see Table of Materials); the RNA integrity number (RIN) must be at least above 7 to proceed.</li>
	<li>Use a total amount of 3 &#181;g of RNA per sample to generate libraries with an RNA-Seq library preparation kit (see Table of Materials) following the manufacturer&#39;s recommendations. 
	NOTE: The library preparations were sequenced on a high-throughput sequencing platform and paired-end reads were generated.</li>
</ol>
7. Data Analysis Using the Web-Based Pipeline T-REx
<ol>
	<li>Perform a quality filtering using the FASTX-Toolkit version 0.0.13 (Phred quality scores of &#62; 20). Trim the raw RNA-Seq reads from the adapter sequences with the Trimmomatic tool. 
	NOTE: All the downstream analyses were based on clean data with high quality.</li>
	<li>Download the B. mycoides ATCC 6462 NCBI genome database (NCBI accession No.: CP009692.1) and use it as a reference genome for mapping the clean reads using Bowtie2/2.2.3.</li>
	<li>Use HTSeq v0.6.1 to count the reads numbers mapped to each gene and the reads per kilobase of transcript per million mapped reads (RPKM) of each gene based on the length of the gene and reads count mapped to this gene.</li>
	<li>For the analysis of the data by the T-REx pipeline, use the RPKM table as input, as well as three descriptive files: (i) a factors file to describe the experiment in factors, (ii) a contrasts file to define which factors will be compared for differential gene expression, and (iii) a class file to describe groups of genes of interest. 
	NOTE: The descriptive files are in.txt format. Examples can be found on the webpage of T-REx (http://genome2d.molgenrug.nl/). A detailed instruction for data analysis using T-REx can be found in a previous publication by de Jong et al.14.</li>
</ol>

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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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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 bacterial adaptation to the rhizosphere niche. This root exudates collection protocol does not require complicated procedures and specialized equipment. However, all procedures must be carried out under strictly sterile conditions and a sterility control should be included. We recommend growing several potato tubers in parallel and discarding the contaminated ones. Modifications can be made to this protocol if other plant species are being studied. It is important to use an appropriate method to sterilize any seeds/tubers because they may vary in tolerance to the disinfectants applied.
Once the root exudates are obtained, high-quality RNA must be isolated from the bacterial cells. Various reagents and standard protocols that are primarily based on the phenol/chloroform or TRIzol method are time-consuming16. The commercial kits are mostly designed for model organisms but are less applicable to others. This RNA isolation protocol that combines the phenol/chloroform method and an RNA isolation kit overcomes the disadvantages of these methods. Moreover, a bead-beating step is included to homogenize B. mycoides cells that typically aggregate in the planktonic culture. Potential DNA contamination is removed by an extra incubation with DNase prior to elution. The high RIN number implies the isolation of intact RNA. Thus, this protocol is especially efficient and time-saving for environmental bacterial strains.
After RNA sequencing, data analysis is performed with T-REx, a web-based statistical analysis pipeline for RNA-seq gene expression data14. This pipeline is user-friendly, especially for biologists without extensive bioinformatics knowledge. The input file is the raw RNA expression level data, such as RPKM, fragments per kilobase per million mapped reads (FPKM), counts per million mapped reads (CPM), or other gene expression units. Such gene expression value files can be generated by available tools including SAMtools17, BEDtools18, and NGS-Trex19. In order to run the RNA-seq analysis, three other input files are needed. These files are used to define the factors that describe the experiments and the replicates, the comparisons between the various experimental conditions, and the groups of genes of interest. When the input files are uploaded, the analysis process will only take a couple of minutes.
Several differentially expressed genes of B. mycoides EC18, when treated with root exudates, are listed in Table 1. Among them, the expression of several genes encoding membrane proteins is altered. Genes related to the sporulation or germination process are differentially expressed. The expression of genes involved in sporulation also changes in B. subtilis when co-cultured with rice seedlings, because root exudates supply the energy required for the dynamic growth of bacterial cells18. The expression of the IclR transcriptional regulator, which is related to multidrug resistance and the degradation of aromatic compounds in soil bacteria, is upregulated. The IclR deletion strain of rhizobacteria Klebsiella pneumoniae has decreased the mineral phosphate solubilization, compared with the wild-type strain19. Several genes related to amino acids metabolism and synthesis are stimulated, while genes involved in sugar transport including a cellobiose PTS transporter are downregulated. This suggests that strain EC18 may have a metabolic preference for amino acids over sugars in the rhizosphere. The function of the altered genes can be further studied in situ by making knockout or overexpression mutants. In summary, the protocol described here enables a quick identification of a large number of potentially important bacterial genes involved in plant-microbe interactions.

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><tbody><tr><td>sodium hypochlorite</td><td>Sigma</td><td>&amp;nbsp;CAS: 7681-52-9&amp;nbsp;</td><td>10-15%&amp;nbsp; active chlorine</td></tr><tr><td>Luria-Bertani (LB) broth</td><td></td><td></td><td></td></tr><tr><td>incubater</td><td>New Brunswick Scientific</td><td>Innova 4000</td><td></td></tr><tr><td>spectrophotometer</td><td>Thermo Fisher Scientific</td><td>Genesys 20</td><td></td></tr><tr><td>liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>glass beads</td><td>Sigma</td><td>G8893</td><td>0.5 &amp;micro;m</td></tr><tr><td>2.0 ml tube with screw cap</td><td></td><td></td><td>RNase free</td></tr><tr><td>1.5 ml and 2.0 ml eppendorf tube</td><td></td><td></td><td>RNase free</td></tr><tr><td>Bead mill homogenizer</td><td>BioSpec</td><td>607</td><td>Mini_beadbeater</td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td>5430</td><td></td></tr><tr><td>Diethyl pyrocarbonate (DEPC)</td><td>sigma</td><td>CAS: 1609-47-8</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>sigma</td><td>CAS: 151-21-3&amp;nbsp;</td><td>10% solution prepared with DEPC treated MQ water</td></tr><tr><td>TE buffer</td><td></td><td></td><td>10 mM Tris-HCl; 1 mM EDTA, pH=8</td></tr><tr><td>phenol</td><td>Sigma</td><td></td><td>RNA grade</td></tr><tr><td>chloroform-isoamyl alcohol&amp;nbsp;</td><td></td><td></td><td>prepare 24:1 of chloroform:isoamyl alcohol, store at room temperature</td></tr><tr><td>High pure RNA isolation kit</td><td>Roche</td><td>11828665001</td><td></td></tr><tr><td>RNase Decontamination Solution</td><td>Invitrogen</td><td>AM9780</td><td>RNase-Zap</td></tr><tr><td>Automated electrophoresis instrument</td><td>Agilent</td><td>2100</td><td>Bioanalyzer</td></tr><tr><td>Microvolume spectrophotometer</td><td>Thermo Fisher Scientific</td><td>Nanodrop ND-1000</td><td></td></tr><tr><td>RNA quality analysis kit</td><td>Agilent</td><td>RNA 6000 Nano kit&amp;nbsp;</td><td></td></tr><tr><td>RNase inhibitor</td><td>Thermo Fisher Scientific</td><td>RiboLock</td><td></td></tr><tr><td>Directional RNA library Prep kit</td><td>NEB</td><td>Ultra</td><td>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 response of rhizobacterial B. mycoides to potato root exudates. To fulfill this, potato tubers were surface sterilized and germinated in autoclaved vermiculite. Then the root exudates were collected as shown in Figure 1. In order to rule out the possibility of the root exudates affecting the bacterial growth, up to 15% of the root exudates were added to the B. mycoides culture, and no change in growth was detected during the measuring time (Figure 2). Thus, the gene expression changes observed in this study were not likely caused by growth-related effects.
After collection, the root exudates were added to the B. mycoides culture at a 10% ratio (v/v), and the bacterial total RNA was isolated as previously described. The RNA was then subjected to a quality check by an automated electrophoresis instrument of which the results are shown in Figure 3. Figure 3A and 3B represent the RNA isolated from B. mycoides treated with the root exudates, and Figure 3C and 3D represent the RNA isolated from the control group. All samples scored a RIN value above 9 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 adapter sequences and mapped against the reference genome sequence. Of the resulting data, the RPKM table was generated. The transcriptome analysis was performed with the T-REx pipeline. The ratio intensity plot of all the differentially expressed genes is shown in Figure 4. As compared with a control, the addition of potato root exudates induced 715 genes to be differentially expressed. Of those, 408 genes were upregulated, and 307 genes were downregulated15. The relative change of some of the genes with altered expression is listed in Table 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57606/57606fig1.jpg" /> 
Figure 1: Process scheme of the collection of potato root exudates. The materials used are autoclaved and the germination is performed in a climate chamber. Wash the potato tuber with sterilized water, and bath it in 2-3% NaOCl for 5 min. Bath it in 75% ethanol for another 5 min. Place the potato into an autoclaved basket and put it into a pot containing wet vermiculite. Grow the potato in a climate chamber for 3 - 4 weeks, and then transfer the basket with the potato seedling to a beaker. Collect the root exudates every day and refill the beaker with sterilized water. <a href="/files/ftp_upload/57606/57606fig1large.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/57606/57606fig2.jpg" /> 
Figure 2: The growth curve of B. mycoides with different concentrations of potato root exudates. The strain EC18 was grown in a liquid LB medium with the addition of potato root exudates or sterile H2O. OD600 was measured every 1 h and plotted versus time. All the groups show a similar growth pattern, indicating that up to 15% of the root exudates addition has no significant effects on B. mycoides growth. <a href="/files/ftp_upload/57606/57606fig2large.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/57606/57606fig3.jpg" /> 
Figure 3: RNA quality check by the automated electrophoresis instrument. A and B represent the RNA isolated from the B. mycoides treated with root exudates and C and D represent the RNA isolated from the control group. All the RNA samples show two clear bands corresponding to 23S and 16S rRNA and a weak 5S rRNA band. <a href="/files/ftp_upload/57606/57606fig3large.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/57606/57606fig4.jpg" /> 
Figure 4: Ratio intensity plot for visualizing differential gene expression of RNA-seq samples of B. mycoides in response to potato root exudates. The figure is automatically generated by T-REx. The X-axis represents the gene expression level, and the Y-axis represents the log2-transformed fold change. The genes being up- and downregulated have positive and negative log2 ratio values. The dots in the striped area indicate genes that are not significantly over- or under-expressed. <a href="/files/ftp_upload/57606/57606fig4large.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>Gene tag</td>
			<td>Strand</td>
			<td>Fold change</td>
			<td>Annotation</td>
		</tr>
		<tr>
			<td>BG05_RS09165</td>
			<td>+</td>
			<td>3.3</td>
			<td>multidrug efflux protein</td>
		</tr>
		<tr>
			<td>BG05_RS20930</td>
			<td>+</td>
			<td>25.3</td>
			<td>membrane protein</td>
		</tr>
		<tr>
			<td>BG05_RS10935</td>
			<td>+</td>
			<td>23.3</td>
			<td>stage III sporulation protein AD</td>
		</tr>
		<tr>
			<td>BG05_RS08990</td>
			<td>-</td>
			<td>11.8</td>
			<td>sporulation protein</td>
		</tr>
		<tr>
			<td>BG05_RS16405</td>
			<td>+</td>
			<td>3.9</td>
			<td>IclR family transcriptional regulator</td>
		</tr>
		<tr>
			<td>BG05_RS24905</td>
			<td>-</td>
			<td>3</td>
			<td>tryptophan synthase subunit alpha</td>
		</tr>
		<tr>
			<td>BG05_RS24920</td>
			<td>-</td>
			<td>2.3</td>
			<td>indole-3-glycerol phosphate synthase</td>
		</tr>
		<tr>
			<td>BG05_RS22255</td>
			<td>-</td>
			<td>27</td>
			<td>acetolactate synthase</td>
		</tr>
		<tr>
			<td>BG05_RS22250</td>
			<td>-</td>
			<td>6.5</td>
			<td>ketol-acid reductoisomerase</td>
		</tr>
		<tr>
			<td>BG05_RS22265</td>
			<td>-</td>
			<td>26.4</td>
			<td>branched-chain amino acid aminotransferase</td>
		</tr>
		<tr>
			<td>BG05_RS18715</td>
			<td>-</td>
			<td>2.8</td>
			<td>pullulanase</td>
		</tr>
		<tr>
			<td>BG05_RS18040</td>
			<td>+</td>
			<td>-9.1</td>
			<td>germination protein YpeB</td>
		</tr>
		<tr>
			<td>BG05_RS16930</td>
			<td>+</td>
			<td>-4.2</td>
			<td>sugar ABC transporter ATP-binding protein</td>
		</tr>
		<tr>
			<td>BG05_RS27345</td>
			<td>+</td>
			<td>-2.9</td>
			<td>MFS transporter</td>
		</tr>
		<tr>
			<td>BG05_RS19555</td>
			<td>-</td>
			<td>-3.3</td>
			<td>PTS cellobiose transporter subunit IIB</td>
		</tr>
		<tr>
			<td>BG05_RS24345</td>
			<td>-</td>
			<td>-2.7</td>
			<td>putrescine importer</td>
		</tr>
		<tr>
			<td>BG05_RS22525</td>
			<td>-</td>
			<td>-12.4</td>
			<td>cardiolipin synthase</td>
		</tr>
		<tr>
			<td>BG05_RS15225</td>
			<td>-</td>
			<td>-3.3</td>
			<td>membrane protein</td>
		</tr>
		<tr>
			<td>BG05_RS18475</td>
			<td>+</td>
			<td>-5.7</td>
			<td>membrane protein</td>
		</tr>
		<tr>
			<td>BG05_RS19095</td>
			<td>+</td>
			<td>-2.1</td>
			<td>germination protein</td>
		</tr>
	</tbody>
</table>
Table 1: List of differentially expressed genes of a root exudates-treated group in comparison with a control.

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Biology

12.5K Views.  University of Groningen. 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.

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

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

Environment

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

Potato Virus X-Based microRNA Silencing VbMS In Potato.

We present a detailed protocol for potato virus X (PVX)-based microRNA silencing (VbMS) system to functionally characterize endogenous microRNAs (miRNAs) in potato. Target mimic (TM) molecules of miRNA of interest are integrated into the PVX vector and transiently expressed in potato to silence the target miRNA or miRNA family.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...

Rhizobacterial Colonization Detection on Arabidopsis thaliana

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.

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 root is one of the most frequent experiments in plant-microbe interaction studies. A standardized ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Immunology and Infection

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

Bioengineering

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

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. ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Semihydroponic Glass Jar System for Exudate Analysis in Multiple Plant Species

A Versatile Glass Jar System for Semihydroponic Root Exudate Profiling

Root exudates shape the plant-soil interface, are involved in nutrient cycling&#160;and modulate interactions with soil organisms. Root exudates are dynamic and shaped by biological, environmental, and experimental conditions. Due to their wide diversity and low concentrations, accurate exudate profiles are challenging to determine, even more so in natural environments where other organisms are present, turning over plant-derived compounds and producing additional compounds themselves. The semihydroponic glass jar experimental system introduced here allows control over biological, environmental, and experimental factors. It allows the growth of various phylogenetically distinct plant species for up to several months with or without microbes, in a variety of different growth media. The glass-based design offers a low metabolite background for high sensitivity and low environmental impact as it can be reused. Exudates can be sampled nondestructively, and conditions can be altered over the course of an experiment if desired. The setup is compatible with mass spectrometry analytics and other downstream analytical procedures. In summary, we present a versatile growth system suited for sensitive root exudate analysis in a variety of conditions.

Author Spotlight: Exploring Plant-Microbe Interactions Through Root Exudates in a Novel Growth System 

We present a protocol for a glass-based, semihydroponic experimental system supporting the growth of a variety of phylogenetically distinct plants with or without microbes. The system is compatible with different growth media and permits nondestructive root exudate sampling for downstream analysis.

Root exudates shape the plant-soil interface, are involved in nutrient cycling&#160;and modulate interactions with soil organisms. Root exudates are ...

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

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