Soybean Hairy Root Transformation for the Analysis of Gene Function

Summary

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

Abstract

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

Introduction

Soybean (Glycine max) is among the most valuable crops in agriculture. It has thousands of commercial and industrial uses, such as food, animal feed, oil, and as a source of raw materials for manufacturing¹. Its ability to form a symbiotic relationship with nitrogen-fixing soil microorganisms, namely rhizobia, further elevates the importance of studying soybean genetics². For instance, fine-tuning nitrogen fixation properties in soybean roots can lead to the reduction of carbon emissions and greatly reduce requirements for nitrogen fertilizer³. Thus, understanding the genetics that controls aspects of soybean root biology, in particular, has wide applications in agriculture and industry. Considering these benefits, it is important to have a reliable protocol to analyze the function of soybean genes.

Agrobacterium tumefaciens is perhaps the most commonly used tool for plant genetic transformation as it has the capability of integrating transfer DNA (T-DNA) into the nuclear genome of many plant species. When Agrobacterium infects a plant, it transfers the tumor-inducing (Ti) plasmid into the host chromosome, leading to the formation of a tumor at the infection site. The Agrobacterium-mediated transformation has been widely used for decades for gene functional analysis and in order to modify crop traits⁴. Although any gene of interest can be readily transferred into host plant cells through A. tumefaciens-mediated transformation, this method has several drawbacks; it is time-consuming, expensive, and requires extensive expertise for many plant species, such as soybean. Although a few varieties of soybean can be transformed by the cotyledonary node approach using A. tumefaciens, the inefficiency of this approach necessitates the need for an alternative genetic transformation technology that is rapid and highly efficient^4,5. Even a non-expert can use this Agrobacterium rhizogenes-mediated hairy root (HR) transformation method to overcome these disadvantages.

HR transformation is a relatively fast tool, not only for analyzing gene function, but also for biotechnological applications, such as the production of specialized metabolites and fine chemicals, and complex bioactive glycoproteins⁶. The production of soybean HRs does not require extensive expertise, as they can be generated by wounding the surfaces of cotyledons, followed by inoculation with Agrobacterium rhizogenes⁷. A. rhizogenes expresses virulence (Vir) genes encoded by its Ti plasmid that transfer, carry, and integrate its T-DNA segment into the genome of plant cells while simultaneously stimulating ectopic root growth⁸.

Compared to other soybean gene expression systems, such as biolistics or A. tumefaciens-based transformation of tissue, cell, and organ culture, the HR expression system exhibits several advantages. Firstly, HRs are genetically stable and produced quickly on hormone-free media^1,9,10. Additionally, HRs can produce specialized metabolites in amounts equivalent to or greater than native roots^11,12. These advantages make HRs a desirable biotechnological tool for plant species that are incompatible with A. tumefaciens or that require special tissue culture conditions to form compatible tissues. The HR method is an efficient approach for analyzing protein-protein interactions, protein subcellular localization, recombinant protein production, phytoremediation, mutagenesis, and genome wide effects using RNA sequencing^13,14,15. It can also be used to study the production of specialized metabolites that have value in the industry, including glyceollins, which medicate soybean's defense against the important microbial pathogen Phytophthora sojae and have impressive anticancer and neuroprotective activities in humans^16,17.

This report demonstrates an easy, efficient protocol to produce soybean HRs. Compared to previous HR transformation methods, this protocol provides a significant (33%-50%) improvement in the rate of HR formation by prescreening A. rhizogenes transformants for the presence of the Ti plasmid prior to inoculating soybean cotyledons. We demonstrate the applicability of this protocol by transforming several binary vectors that overexpress or silence soybean transcription factor genes.

Protocol

NOTE: It is recommended that all of the proceeding steps be conducted under sterile conditions.

1. Soybean seed sterilization

1. In a biosafety cabinet, place 16-20 round Williams 82 soybean seeds in pristine condition (i.e., no cracks or blemishes) in a 50 mL centrifuge tube.
2. Add 30 mL of 70% isopropyl alcohol, gently shake for 30 s, and then decant the alcohol.
3. Gently shake the seeds with 30 mL of 10% bleach for 10 s and allow the seeds to sit in the solution for 5 min at room temperature (RT, 25 °C). After 5 min, drain the bleach.
4. Repeat the shaking three times with 30 mL of sterile ultrapure H₂O for 1 min per rinse and discard the H₂O between each rinse.
5. Place the sterilized seeds on filter paper saturated with 5 mL of germination and co-cultivation (GC) medium (autoclave half-strength liquid Murashige and Skoog [MS] medium with 1% sucrose [pH = 5.8] and then add 2.5 mL/L vitamins) in sterile Petri dishes.
6. Place the plates in the dark at RT (25 °C) for 3 days before transferring the plates at 22 °C under 16 h cool-white T5 fluorescent lights (100 µE m^-2·s^-1) for 4 days to allow the seeds to germinate.
   ​NOTE: Discard seeds that are wrinkled or cracked. The best seeds are those that are large and uniformly yellow. Sterilize at least double the number of seeds required for the experiment to select seeds of good quality, as some seeds may not be optimal due to damage, diseases, or failure to germinate.

2. Infection of cotyledons with K599

NOTE: Use pGWB series vectors, as their dual selection ensures genomic integration of the entire T-DNA cassette. Electroporation was used to transform a binary vector harboring the gene of interest into A. rhizogenes pRi2659¹⁸.

1. Based on the sequence of the A. rhizogenes pRi2659 plasmid¹⁸, design primers to detect the Ti plasmid gene VirD2 (VirD2 forward: 5'-CCCGATCGAGCTCAAGTTAT-3'; VirD2 reverse: 5'-TCGTCTGGCTGACTTTCGT-3'; expected amplification size: 221 bp). Following transformation, test the Agrobacterium colonies for the retention of VirD2 by polymerase chain reaction (PCR) using a PCR Kit (see Table of Materials). PCR cycle: 94 °C for 3 min; 35 cycles (94 °C for 1 min, 58 °C for 30 sec, 72 °C for 1 min); and 72 °C for 10 min.
2. Following the selection of the A. rhizogenes colony that contains both VirD2 and the gene of interest (GOI), streak out some onto Luria-Bertani (LB) medium plates containing the appropriate antibiotics (50 mg/L) for the plasmid of interest and incubate overnight at 30 °C. If using spectinomycin, add a concentration of 100 mg/L.
3. Use a P200 pipette tip to scrape off a ~1.5 cm length of the K599 Agrobacterium from the LB plates and resuspend the cells in 1 mL of phosphate buffer (PB; 0.01 M Na₂HPO₄, 0.15 M NaCl, pH 7.5).
4. Dilute the Agrobacterium in sterilized, ultrapure H₂O (v/v = 1:1) and acetosyringone (AS; 100 mM stock in dimethyl sulfoxide [DMSO], v/v = 1:1000). Measure the absorbance using a cuvette tube at an optical density of 600 nm (OD600). The expected optimal range is between 0.5 and 0.8.
5. In a biosafety cabinet, dip a sterilized scalpel in the K599 Agrobacterium solution and make several 1 mm deep cuts along the inner surface (adaxial, flat side) of the cotyledon. During the inoculation, use sterilized forceps to stabilize the cotyledon.
6. Place about 6-8 cotyledons cut side-down on a Petri dish containing filter paper saturated with GC medium with AS.
   NOTE: To prepare 6 plates, 50 mL of GC medium and 50 µL of 100 mM AS to achieve a final concentration of 100 µM is sufficient.
7. Incubate the plates at RT (25 °C) for 3 days under a 16 h photoperiod (~65 µE).
8. Transfer the infected cotyledons to hairy root growth (HRG) plates (autoclave half-strength MS with 3% sucrose [pH = 5.8] and 2.6 g/L Gelzan, then add 2.5 mL/L vitamins mixture and 500 mg/L timentin).
   NOTE: There were some issues with timentin from some alternative vendors, of which the K599 was not eliminated. It is recommended to use taller Petri dish plates (100 mm x 25 mm) for HRG medium.
9. Incubate the HRG plates at 22 °C in a growth chamber with the parameters set to 100 µE light on a 16 h photoperiod until primary roots with secondary roots 2-3 cm in length are observed (~3-4 weeks).

3. Selection and harvesting of HRs

1. Harvest primary roots (5-7 cm in length) that grow from the callus and contain secondary roots (2-3 cm in length) using a sterile scalpel and forceps. Transfer to selection HRG plates containing the appropriate antibiotics. Let the HRs grow for 5 more days on the selection HRG plates.
   NOTE: Kanamycin (50 mg/L), hygromycin (50 mg/L), or phosphinothricin (10 mg/L) are typically used for selection. For expression vectors, pGWB2 is used for overexpression, pANDA35HK is used for RNAi silencing, pGWB6 is used for subcellular localization, and pMDC7 is used for inducible expression.
2. On day 5, harvest transgenic HRs with secondary roots 3-6 cm in length. If observing fluorescent proteins, the secondary roots have little autofluorescence. If performing elicitor or chemical treatments, cut the secondary roots into 1 cm pieces and place ~100 mg on HRG agar in a pile. Then, saturate the piles with 80 µL of the appropriate treatment and allow the plates to incubate at RT (25 °C).
3. After the desired treatment time, proceed with RNA or metabolite extractions.
4. For RNA, rapidly dab the roots dry on a sterilized paper towel and harvest them directly into a 2 mL microcentrifuge tube.
5. Immediately seal the top of the tube using parafilm, make two small holes using pointed forceps, and submerge the tubes in liquid nitrogen. Lyophilize for 3 days, then store the samples at -80 °C.
   NOTE: It is essential to select HRs that are white. After 5 days of growth on the selective plate, transgenic HRs will remain white, but non-transgenic roots will turn brown.

Representative Results

The representative results are from the published data^19,20. The colony PCR (cPCR) results of the transformed K599 Agrobacterium are shown in Figure 1. As indicated by the positive colonies in Figure 1, the gene of interest was detected by cPCR (Figure 1A). However, one-third to one-half of the colonies were negative for the VirD2 gene screening (Figure 1B), indicating the loss of the Ti plasmid, and would be incapable of generating callus or hairy roots. Figure 2 illustrates the overall preparation procedure of soybean HRs and gene expression analysis. Figure 3 demonstrates the subcellular localization of GFP-GmJAZ1-6. Figure 4 is a gene expression analysis showing overexpression of the glyceollin transcription factor GmHSF6-1 and RNAi-silencing of GmMYB29A2 in Williams 82 hairy roots. Similar results have been obtained in several recent reports^20,21.

Figure 1: Colony PCR (cPCR) of the K599 Agrobacterium using colony PCR primers of the gene of interest or VirD2. (A) Gene of interest cPCR. (B) VirD2 cPCR. Abbreviations: C = colony; +ve = positive control; -ve = negative control. Please click here to view a larger version of this figure.

Figure 2: Overview of the soybean hairy root (HR) culture and gene function analysis procedure. Calluses formed on the wounding site 2 weeks after K599 Agrobacterium infection. Cell differentiation occurred after 1 week, then 1 week elapsed for HR elongation. This was followed by HR harvesting and wall glucan elicitor (WGE)/mock treatment for 24 h. The HRs were subjected to metabolite extraction for ultra performance liquid chromatography (UPLC) analysis and RNA isolation for gene expression analysis, respectively. WGE is the wall glucan elicitor from Phytophthora sojae. Please click here to view a larger version of this figure.

Figure 3: Fluorescence microscopy of GmJAZ1-6 translationally fused to a green fluorescent protein (GFP) in transgenic Williams 82 hairy roots. (A) Green channel (GFP). (B) Blue channel (DAPI). (C) Merged green and blue channels. All images were collected using a Zeiss confocal microscope. DAPI (6 µg/mL) images indicate nuclear staining. Scale bars are 5 µm. Please click here to view a larger version of this figure.

Figure 4: Gene expression analysis. (A) Gene expression in overexpressing GmHSF6-1¹⁹ Williams 82 soybean HRs under 24 h mock treatment or elicited for 24 h with WGE. (B) Gene expression in RNAi-GmMYB29A2²⁰ Williams 82 soybean HRs elicited for 24 h with WGE. WGE is the wall glucan elicitor from Phytophthora sojae. ^aSignificantly greater and ^bsignificantly less than control, paired students t-test (p < 0.01). Error bars represent SE (n ≥ 3 biological replicates). Secondary roots collected from one primary root indicated one biological replicate. This figure has been modified with permission from Lin et al.¹⁹ and Jahan et al.²⁰. Please click here to view a larger version of this figure.

Discussion

During the past decade, the soybean HR method has been developed as a powerful tool to study genes involved in nitrogen fixation^22,23, biotic and abiotic stress tolerance^24,25, and metabolite biosynthetic pathways^26,27. The knowledge of how plants produce metabolites has a plethora of benefits for agricultural production and the pharmaceutical industry, as it can be used to study gene networks that are involved in mediating biochemical defenses against pathogens²⁸.

To prioritize productivity and cost efficiency, this protocol simplifies the procedure. For instance, instead of using more expensive solid media containing a gelling agent, soybean seeds are germinated under sterile conditions using industrial-grade paper towels saturated with liquid growth medium. Aseptic lab techniques and maintaining sterile work conditions are essential for HR transformation experiments, since a wide range of microorganisms, such as fungi and yeast, can cause contamination of in vitro cultures.

Additionally, using the appropriate intensity of light and photoperiod is crucial for HR culture. The growth and development process of plants in in vitro cultures is significantly affected by the quality of light intensity and photoperiod, as observed in various previous studies. If the light intensity is either too low or too high, it can slow down the process of root induction. Similarly, if the photoperiod is inappropriate, it may lead to the failure of callus formation and differentiated cell development²⁹. Moreover, based on our past experiments, non-transgenic HRs are resistant to kanamycin (50 mg/L) when used as the sole antibiotic. For this reason, we typically use vectors encoding dual resistance for hygromycin and kanamycin. Under this stringent selection, we are able to get a positive transgenic HR rate of 15%-30% of the total roots, yet ~80% of those HRs have significantly overexpressed/silenced genes as encoded by the vectors.

To ensure the success of HR transformation experiments, it is crucial to test if the A. rhizogenes strain used in the experiment retains the Ti plasmid that encodes virulence genes. The presence of the Ti plasmid is necessary for the successful integration of the T-DNA into the plant genome⁴. The use of cPCR to detect the Ti plasmid has become an essential step in this protocol. In the past, we found that 33%-50% of our transformations would fail to produce calli and roots. Unknowingly, this was due to the loss of the Ti plasmid during the transformation or subsequent culturing of A. rhizogenes. Now, by PCR analysis of the Agrobacterium colonies following their transformation, we ensure that the Ti plasmid is present and that 100% of the transformation experiments produce roots. The use of cPCR in this protocol has proven to be a valuable addition to the standard HR transformation procedure. It has reduced the number of failed experiments, thereby saving both time and resources. The cPCR step has also allowed us to confirm that the transformation process works as intended, ensuring that the experiments' results are reliable and reproducible.

Notwithstanding, this simplified method has some limitations. For example, this protocol can answer basic cell biological questions about gene functions in transgenic HRs. However, questions pertaining to otherplant tissues, such as shoots and leaves, may not be testable in HRs. It is always important to confirm that the process being studied is not affected by ectopic hormone levels or other factors introduced by A. rhizogenes. Researchers should take note of its limitations and carefully design experiments to ensure accurate and meaningful results.

In summary, the protocol demonstrated here is a highly efficient method for investigating gene function in soybean roots. We have recently demonstrated its value in studying multigene engineering approaches and understanding gene networks involved in regulating soybean biochemical defenses¹⁹. The relative efficiency of the approach renders it ideal for answering complex questions in plant biology that require the investigation of numerous genes.

Materials

Name	Company	Catalog Number	Comments
Acetosyringone	Cayman	23224
Bleach	lavo	21124
DMSO	Fisher bioreagents	195679
Gelzan	Phytotech	HYY3251089A
Hygromycin	Phytotech	HHA0397050B
Isopropyl alcohol	Fisher chemical	206462
Kanamycin	Phytotech	SQS0378007G
LB powder	Fisher bioreagents	200318
MS powder	Caisson labs	2210001
Na2HPO4	Fisher bioreagents	194171
NaCl	Fisher chemical	192946
Petri dishes	Fisherbrand	08-757-11	100 mm x 25 mm
Phosphinothricin	Cedarlane	P034-250MG
REDExtract-N-Amp PCR Kit	Sigma	R4775
Sucrose	Bioshop	2D76475
Timentin	Caisson labs	12222002
Vitamins	Caisson labs	2211010