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

Summary

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.

Abstract

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.

Introduction

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 ac....

Protocol

1. Germinating Potato in Sterilized Conditions

1. 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.
2. Prepare the materials needed for germinating and growing potato tubers; sterilize the plastic pots, engraftment baskets, vermiculite, and water by autoclaving them at 121 °C for 20 min.
   NOTE: Ensure that all materials used are autoclavable. Otherwise, use other sterilizing methods.
3. Put the surface-sterilized potato into the engraftment basket, and place this in an autoclaved....

Results

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

Discussion

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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