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>

<ol>
	<li>Haichar, F. e. Z., 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=Plant+host+habitat+and+root+exudates+shape+soil+bacterial+community+structure.">Plant host habitat and root exudates shape soil bacterial community structure.</a> The ISME Journal: Multidisciplinary Journal of Microbial Ecology. 2 (12), 1221-1230 (2008).</li><li>Badri, D. V., Vivanco, J. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corn+(Zea+mays+L.)+root+exudates+and+their+impact+on+14C-pyrene+mineralization.">Corn (Zea mays L.) root exudates and their impact on 14C-pyrene mineralization.</a> Soil Biology and Biochemistry. 33 (12), 1769-1776 (2001).</li><li>Lambert, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clover+root+exudate+produces+male-biased+sex+ratios+and+accelerates+male+metamorphic+timing+in+wood+frogs.">Clover root exudate produces male-biased sex ratios and accelerates male metamorphic timing in wood frogs.</a> Royal Society Open Science. 2 (12), (2015).</li><li>Tuason, M. M. S., Arocena, J. 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+organic+acid+exudates+and+properties+of+rhizosphere+soils+of+white+spruce+(Picea+glauca)+and+subalpine+fir+(Abies+lasiocarpa).">Root organic acid exudates and properties of rhizosphere soils of white spruce (Picea glauca) and subalpine fir (Abies lasiocarpa).</a> Canadian Journal of Soil Science. 89 (3), 287-300 (2009).</li><li>Grayston, S. 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 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 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 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

Nanyang Technological University

Research

JoVE Journal

Biology

12.4K 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

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

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 ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...