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.
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.
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.
1. Germinating Potato in Sterilized Conditions
2. Collecting Potato Root Exudates
3. Growing Bacteria
4. Treatment and Sampling of Bacteria
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.
6. RNA Quality Check and Sequencing
7. Data Analysis Using the Web-Based Pipeline T-REx
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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Gene tag | Strand | Fold change | Annotation |
BG05_RS09165 | + | 3.3 | multidrug efflux protein |
BG05_RS20930 | + | 25.3 | membrane protein |
BG05_RS10935 | + | 23.3 | stage III sporulation protein AD |
BG05_RS08990 | – | 11.8 | sporulation protein |
BG05_RS16405 | + | 3.9 | IclR family transcriptional regulator |
BG05_RS24905 | – | 3 | tryptophan synthase subunit alpha |
BG05_RS24920 | – | 2.3 | indole-3-glycerol phosphate synthase |
BG05_RS22255 | – | 27 | acetolactate synthase |
BG05_RS22250 | – | 6.5 | ketol-acid reductoisomerase |
BG05_RS22265 | – | 26.4 | branched-chain amino acid aminotransferase |
BG05_RS18715 | – | 2.8 | pullulanase |
BG05_RS18040 | + | -9.1 | germination protein YpeB |
BG05_RS16930 | + | -4.2 | sugar ABC transporter ATP-binding protein |
BG05_RS27345 | + | -2.9 | MFS transporter |
BG05_RS19555 | – | -3.3 | PTS cellobiose transporter subunit IIB |
BG05_RS24345 | – | -2.7 | putrescine importer |
BG05_RS22525 | – | -12.4 | cardiolipin synthase |
BG05_RS15225 | – | -3.3 | membrane protein |
BG05_RS18475 | + | -5.7 | membrane protein |
BG05_RS19095 | + | -2.1 | germination protein |
Table 1: List of differentially expressed genes of a root exudates-treated group in comparison with a control.
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.
The authors have nothing to disclose.
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.
sodium hypochlorite | Sigma | CAS: 7681-52-9 | 10-15% active chlorine |
Luria-Bertani (LB) broth | |||
incubater | New Brunswick Scientific | Innova 4000 | |
spectrophotometer | Thermo Fisher Scientific | Genesys 20 | |
liquid nitrogen | |||
glass beads | Sigma | G8893 | 0.5 µm |
2.0 ml tube with screw cap | RNase free | ||
1.5 ml and 2.0 ml eppendorf tube | RNase free | ||
Bead mill homogenizer | BioSpec | 607 | Mini_beadbeater |
centrifuge | Eppendorf | 5430 | |
Diethyl pyrocarbonate (DEPC) | sigma | CAS: 1609-47-8 | |
Sodium Dodecyl Sulfate (SDS) | sigma | CAS: 151-21-3 | 10% solution prepared with DEPC treated MQ water |
TE buffer | 10 mM Tris-HCl; 1 mM EDTA, pH=8 | ||
phenol | Sigma | RNA grade | |
chloroform-isoamyl alcohol | prepare 24:1 of chloroform:isoamyl alcohol, store at room temperature | ||
High pure RNA isolation kit | Roche | 11828665001 | |
RNase Decontamination Solution | Invitrogen | AM9780 | RNase-Zap |
Automated electrophoresis instrument | Agilent | 2100 | Bioanalyzer |
Microvolume spectrophotometer | Thermo Fisher Scientific | Nanodrop ND-1000 | |
RNA quality analysis kit | Agilent | RNA 6000 Nano kit | |
RNase inhibitor | Thermo Fisher Scientific | RiboLock | |
Directional RNA library Prep kit | NEB | Ultra | For Illumina |