Agrobacterium rhizogenes

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Classification

Figure 1: Tobacco Plant Showing Symptoms of Hairy Root Disease. Photo by Adriana M. Allippi, Facultad de Ciencias Agrarias y Forestales, Argentina

Higher order taxa

Bacteria (Domain); Proteobacteria (Phylum); Alphaproteobacteria (Class); Rhizobiales (Order); Rhizobiaceae (Family); Agrobacterium (Genus).

Species

NCBI: [2]

Agrobacterium rhizogenes

Description and Etymology

Figure 2: Agrobacterium Rhizogenes, Gram stain 1000X magnification, Brightfield Microscopy. From [1]. Photo by Simon Sublime

Agrobacterium rhizogenes is a gram-negative, rod-shaped (0.6-1.0 x 1.5-3.0 µm), aerobic, soil-borne bacteria that occurs singly or in pairs and is motile by 1-4 peritrichous flagella (1). Culturing techniques require the optimal temperature for growth of 25-28C. Colony morphology will show convex, circular and smooth characteristics, while color will be non-pigmented to light beige (1). The etymological origin of the species name "rhizogenes" originates from the Greek words "rhiza" meaning a root and "gennao" to make, thus resulting in a root-producing bacteria (2).

A. rhizogenes was first isolated by Riker et al in 1930 (1) and is commonly found in the nodules of leguminous plants (3). It is known for its ability to induce tumors and hairy roots in the event of wounding or infection which produces a condition called the Hairy Root Syndrome (2). This is characterized by the overabundant growth of the plant's root system (see Figure 2). A key characteristic of the hairy root system in plants is their ability to grow quickly in the absence of exogenous plant growth regulators (4).

The tumor induction phenotype is correlated with the presence of a large tumor inducing plasmid (Ti-plasmid), whereas the hairy root phenotype is associated with a Ri plasmid. A. rhizogenes has been a target of recent research due to its ability to use its Ri plasmid, containing t-DNA, to introduce its plasmid DNA into host plants. Due to this, it has been used for a variety of purposes such as, production of plant endangered species (4), recombinant protein production and genetic engineering (3).

Genome and genetics

A. rhizogenes forms part of the bacteria domain as its characteristics define it as a alphaproteobacteria (1). Within the Agrobacterium genus, A. tumefaciens, A. rubi and A. radiobacter are closely related to it. Whole genome shotgun sequencing of the A. rhizogenes strain ATCC 15834 was performed in 2014 (5) because of its emerging importance in the areas of genetic engineering and metabolite production for medicinal purposes. The researchers who conducted the project obtained the ATCC from a hairy root culture work with tomato (3). By following the DNA extraction and sequencing protocols, the researchers obtained data that showed the length of the A. rhizogenes genome, which contains 7,070,307 bp in 43 scaffolds with a GC content of 60% (5). Automated annotation was another tool that the researchers used to confirm the species to 54 other 16 sequences and construct a phylogenic tree with the FastTree 2 software. The whole-genome shotgun project was deposited at the DDBJ/EMBL/GenBank under the accession number JFZP00000000.

Nutrition and metabolism

A. rhizogenes is an aerobic bacteria that uses oxygen as its terminal electron acceptor (1). Its At plasmid, pArA4, is a catabolic plasmid which provides the bacteria wth the ability to utilize MOP, mannopinic acid, and agropinic acid as sole sources of carbon (6). The conditions required the successful culturing of A. rhizogenes under a laboratory setting are the following: an optimal growth temperature of 25-28C, a pH level of 5-9 and biotin as a growth factor. It is worth mentioning, that this bacteria will not grow at 35C, in a medium containing 2% NaCl and that it does not produce 3-ketolactose (1). The by-products produced by the action of this bacteria are opines, which are caused by the Ri plasmid and are an important source of nitrogen and energy (6).

Ecology / Pathology

A. rhizogenes is found in the rhizosphere of plants with as many as 10^6-10^7 cells/g soil. The impact of this bacteria within the rhizosphere is complex, as suggested by the number of populations that compete during the infection process (1), however, an important effect could be that of increasing the yield of important compounds in the rhizosphere due to the overabundant growth of the root system. The importance of this bacterial species in the ecosystem is shown by its ability to transfer its plasmid to a host plant as the plasmid can modify the genome of the plant to express genes that aid to its survival under harsh conditions and produce opine compounds that are used as an important source of nitrogen (6).

The mechanism by which A. rhizogenes infects wounded plant cells begins by the attraction of the bacterial cells to the wound site due to the production of phenolic compounds via chemotaxis. Once the bacteria reach the wound site, they infect the plant by integrating their pathogenic Ri plasmid into the plant genome which results in the development of hairy root disease (6). Under natural conditions, A. rhizogenes is restricted to a limited number of plant species, those being: apple, cucumber, tomato, and melon. However, under laboratory conditions, more than 450 plant species are susceptible to infection by this bacteria (6). One of the key methods by which researchers differentiate A. rhizogenes from other species within the Agrobacterium genus is the polarity of infection, which is possibly caused by the presence of a second T-DNA that encodes for genes that produce auxin in plants transformed by the agropine=type Ri plasmid. Strains that can compete with A. rhizogenes such as agrocin-producing strains are capable of inhibiting infection by this bacterial species (1).

Current Research

The ability that A. rhizogenes has in introducing its Ri pathogenic plasmid into host plant cells and induce overgrowth of root systems has been studied with the purpose of increasing yield of important metabolites. One study sought to devise a protocol for the successful induction of different strains of A. rhizogenes in the plant Fagopyrum tataricum, commonly known as tartary buckwheat with the goal of determining the highest growth rates, root numbers, transformation efficiency, and total anthocyanin and rutin content. Among the used strains of A. rhizogenes, R1000 was the most promising candidate as it showed the highest values for the above mentioned desired results. Fagopyrum tataricum has been a target of recent research due to the presence of pharmacologically important phenolic compounds, such as quercetin-3-glycoside, kaempferol-3-glycoside, chlorogenic acid, iso-orientin, orientin, rutin, vitexin, and quercitrin (7). The induction protocol consisted in dipping the plant seeds into suspensions of the different strains of A. rhizogenes for 20 minutes, followed by drying on a sterile tissue paper and incubation under dark conditions at 25°C on a half-strength solid MS medium for 2 das. After incubation, the emerged hairy roots were observed and analyzed through PCR analyses. Finally, the amount of the desired phenolic compounds was quantified via HPLC protocols, which isolated these compounds from the hairy root samples. Among the isolated phenolic compounds, rutin was the most abundant as it contributed an average of 92% of the total phenolic compounds (7). Rutin is a flavonol compound that has been used for a variety of therapies due to its pharmacological activities, including antioxidant, cytoprotective, vasoprotective, anticarcinogenic, neuroprotective and cardioprotective activities (8). Thus, the goal of this research was successful in inducing the hairy root phenotype on tartary buckweat plants, which in turn increased the yield of this important pharmacological metabolite.

Another study sought to use the root-inducing phenotype of pathogenic A. rhizogenes to enhance the growth rates of the endangered species Berberis aristata DC, commonly known as "Indian Berry" (4). The importance of endangered B. aristata lies in its medicinal applications as some of its metabolites like benzylisoquinoline (berberin), a natural alkaloid, have shown anticancer activity as well as aiding the treatment of diabetes and malarial fever (4). The study used two different strains of A. rhizogenes (MTCC 532 and 2364) obtained from IMTECH (Institute of Microbial Technology), Chandigarh, India. In the experiment, the researchers infected the leaves of this plant in order to observe the hairy root induction and calculate the metabolite yield. The goal of the study was to devise a protocol for the infection of plants with A. rhizogenes, in order to increase the production of berberin and reduce the overharvesting of this endangered species from its natural habitat (4).

References

1. Brenner, DJ., Krieg, NR., Staley, JT,. "Bergey's Manual of Systematic Bacteriology Second Edition". Vol.2. Springer. 2005. 340-345.

2. Nachimas, A.; Strobel, G.A. "Agrobacterium rhizogenes promotes the initial growth of bare root stock almond." Microbiology. 1985. 131: 1245–1249.

3. Ron M, Kajala K, Pauluzzi G, Wang D, Reynoso MA, Zumstein K, Garcha J,Winte S, Masson H, Inagaki S, Federici F, Sinha NR, Deal R, Bailey-Serres J, Brady SM. "Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model." Plant Physiol. 2014. 166:455–469.

4. Brijwal L, Tamta S. Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC. SpringerPlus. 2015;4:443. doi:10.1186/s40064-015-1222-1.

5. Kajala, Kaisa, David A. Coil, and Siobhan M. Brady. “Draft Genome Sequence of Rhizobium Rhizogenes Strain ATCC 15834.” Genome Announcements 2.5 (2014): e01108–14. PMC. Web. 30 Mar. 2017.

6. Veena Veena, Christopher G. Taylor, and Christian Walter. Agrobacterium rhizogenes: recent developments and promising applications. In Vitro Cellular and Developmental Biology - Plant 2007 43 (5), 383-403

7. Thwe, A., Valan Arasu, M., Li, X., Park, C. H., Kim, S. J., Al-Dhabi, N. A., & Park, S. U. (2016). Effect of Different Agrobacterium rhizogenes Strains on Hairy Root Induction and Phenylpropanoid Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum Gaertn). Frontiers in Microbiology, 7, 318. http://doi.org/10.3389/fmicb.2016.00318

8. Aditya Ganeshpurkar, Ajay K. Saluja, The Pharmacological Potential of Rutin, Saudi Pharmaceutical Journal, Volume 25, Issue 2, February 2017, Pages 149-164, ISSN 1319-0164, http://dx.doi.org/10.1016/j.jsps.2016.04.025.

Authored by [Isaac Tamez Salazar], a student of CJ Funk at John Brown University