Introduction

Soil microorganisms form an important link between soil and plants, a link characterised by ecosystem processes that keep both the soil and plants healthy and productive. These processes are most pronounced in the region known as the rhizosphere, an ecological environment in close proximity to plant roots. Soil microorganisms are most abundant and diverse in this region, with two major groups being fungi and bacteria.

Rhizobium rhizogenes (formerly Agrobacterium rhizogenes) is a soil bacterium from the genus Rhizobium, which is known for its natural capability of trans-kingdom DNA transfer. The discovery of this natural phenomenon inspired to a whole new field of study dedicated to genetic modification of plants, and as a result, this genus is often referred to as natural genetic engineers.

Rhizobium rhizogenes alters the plant root architecture by transferring its DNA to plant cells inducing a hairy root system and the production of opines, which are compounds the bacterium uses as a food source. The historic origin of the species name ‘rhizogenes’ comes from the Greek words ‘rhiza’ meaning root and ‘gennao’ meaning to make, thus meaning a ‘root-producing’ bacteria (Reddy et al. 2019).

How to recognise Rhizobium rhizogenes

Rhizobium rhizogenes is associated with the hairy root phenomena, a growth change characterised by the extensive formation of adventitious roots at or near the root region of infection. The transformation process is a result of the transfer of DNA (T-DNA) fragments from the bacterium’s root-inducing plasmid (pRi) to the plant cell.

Rhizobium rhizogenes can be isolated from many plants species and can be visualised using a simple staining protocol (Gram-stain) and a microscope. It is a Gram-negative, rod-shaped (0.6–1.0 µm × 1.5–3.0 µm) bacterium, aerobic, non-spore forming and motile by 1–6 peritrichous flagella (Brenner et al. 2005).

Colony morphology shows convex, circular and smooth characteristics, while its colour becomes non-pigmented to light beige on a nutrient agar plate (Figure 1).

   
Figure 1: Characteristics of Rhizobium rhizogenes, A, A microscope slide of Rhizobium rhizogenes bacterium cells; B, Gram-stain of R. rhizogenes using bright field microscope; and C, Colony morphology of R. rhizogenes on a petri dish after 3–4 days incubation at 28°C (Gelvin et al. 2018).

Distribution and habitat

The host plant species of Rhizobium rhizogenes are distributed in moist to dry tropical and subtropical regions in South Africa such as Kwazulu-Natal, the Mpumalanga Escarpment and the Highveld. The host plants includes: apple, cucumber, tomato and melon with the microhabitat of these microorganisms being the rhizospheric zone of the above mention host plants.

Food

Rhizobium rhizogenes is an aerobic bacterium that uses oxygen for its growth needs (Brenner et al. 2005). The root-inducing plasmid (pRi) gives the bacterium the ability to induce the production and degradation of opine complex acids such as mannopine and agropinic acids as sole sources of nitrogen and carbon.

The conditions required for the successful culturing of R. rhizogenes under laboratory conditions are the following: a temperature between 25°C and 28°C, a pH level of 5–9 and biotin as a growth factor (Brenner et al. 2005).

Life cycle

Rhizobium rhizogenes multiply through the process of binary fission, where a bacterial cell (called a parent cell) makes a copy of its DNA and grows larger by doubling its cellular content. The parent cell then splits, pushing apart the duplicated cellular material and forming two identical ‘daughter’ cells. Generally, bacteria reproduce either via binary fission or budding, however, on both cases, the DNA found in parent cells and the offspring is exactly the same.

This, therefore, means that R. rhizogenes relies on other means to introduce variation into its genetic material, such as gene transfer using the plasmid. The resulting genetic variation ensures that the bacterium can adapt and survive amid changing surrounding environment and ensure that its host is prepared to tolerate changes as well. Root plant cells would release compounds, which are sensed by the bacterium in the soil, and which triggers it to be attracted to that area. The bacterium then transfers DNA from the plasmid into the host cell and integrates the T-DNA into the plant cell gene structure. After integration, the plant undergoes root architectural changes and produces an abundance of opines, which are beneficial for the growth of R. rhizogenes.

THE BIG PICTURE

Friends and foes

Agrocin-producing bacterial strains are able to compete with R. rhizogenes and ultimately inhibiting the process of transferring their DNA to plants.

Smart Strategies

Rhizobium rhizogenes causes hairy root growth in many plant species by transferring a specific DNA fragment from its root-inducing plasmid (pRi) to the host plant cells. As a result, rapidly growing and intensely branched (hairy) adventitious roots are developed, under laboratory conditions, these roots can grow for years.

The R. rhizogenes pRi plasmid induces the plant cells to produce and accumulate large quantities of opines (amino acids, α-keto acids and sugars), which are used by the bacterium as nitrogen, carbon and energy source for growth.

Some Rhizobium rhizogenes strains are known to secrete Agrocin 84, an antibiotic-like substance, which specifically inhibits many tumourigenic or Crown gall disease–causing Rhizobium species such as Rhizobium radiobacter.

Poorer world without me

The importance of this bacterial species in the ecosystem is shown by its ability to transfer part of its plasmid to a host plant where the plasmid can then modify the genome of the plant to express genes that aid to its survival under harsh conditions.

In nature, insertion of T-DNA in the plant genome and its subsequent transfer via sexual reproduction has been shown in several species in the genera Nicotiana, Linaria and Ipomoea. The presence of cellular T-DNA in these untransformed plants is indicative that Rhizobium rhizogenes infected these plant species during their evolution.

Contrary to other pathogenic strains of Rhizobium such as the ones containing the tumour-inducing plasmid (pTi), the pRi of R. rhizogenes is beneficial to the host plant as it induces rapidly growing hairy roots. This mutual association facilitates water and nutrient absorption, interaction with microorganisms and anchoring of the plant to the soil (Figure 2). In general, R. rhizogenes promotes healthy soil by preventing diseases, improve nutrient cycle and help reduce plant stress amid a changing environment.

Figure 2: Rhizobium rhizogenes–mediated transformation of the alfalfa plant showing A, upper part of the plant and B, root growth differences – the control plant is on the right on both pictures; the insert in the middle (C,D) show hairy root development on a transformed plant, with hairy roots most developed in the differential zone marked by the long arrow in D, compared to C, the elongation zone (marked by short arrows remains without hairy roots); orange line represents the scale; a close up of the differential zone of the root shown in E (Chen & Otten 2017).

People and I

The discovery of how the Rhizobium genus and specifically Rhizobium rhizogenes transfer part of its DNA to plant cells led to this natural phenomenon being used to introduce new DNA into plants. Rhizobium rhizogenes is the preferred candidate for the development of genetically modified plants as it is the only species in this genus capable of stably introducing its DNA in plants without harmful effects to the plant. In nature, although Rhizobium rhizogenes transforms specific to higher plant species, the experimental host range of the bacterium is very wide, more than 450 plant species. To date, this species can transform non-plants species such as algae, bacteria, fungi and even mammalian cells under laboratory conditions.

The ability of Rhizobium rhizogenes to induce hairy roots in the host plant has found use in the production of valuable chemicals of economic importance and those of medicinal use. Rhizobium rhizogenes-mediated transformation has several advantages including high growth rate and high root branching without added plant growth hormones. Recent research reported the use of hairy root induction protocol including tissue culture methodology for the high yield production of valuable alkaloid berberine (the active ingredient of berberine, which is useful in industry and medicine). If successful, this will be an effective way to reduce potential overharvesting of the endangered Berberis aristata and other plant species for their medicinal properties.

Agrocin 84 has been used successfully for more than ten years to control crown gall disease by pathogenic Rhizobium species and strains.

The Rhizobium-mediated transformation also forms a platform for modern basic plant research purposes such as studying gene function, root biology and plant interactions with other soil pathogenic microzoa (i.e. fungi, nematodes, etc.). Other practical application of Rhizobium rhizogenes through the use of hairy root cultures include; bioaccumulation (using plants to accumulate heavy metals), biopharmaceuticals (production of therapeutic proteins), biofortilication (raising of crop dietary substances) and phytoremediation (removal of environmental pollutants).

Threats

The most obvious threats to R. rhizogenes would be the same as those imposed on microorganisms that are the most important component of the soil. These include: ploughing, tilling, overuse of pesticides and fertilisers.

Relatives

Rhizobium rhizogenes is closely related to the better known Rhizobium radiobacter (formerly Agrobacterium tumefaciens). Rhizobium radiobacter forms parasitic association with plants due to the tumour-inducing plasmid (pTi) and has been identified as the etiological agent for Crown gall disease. R. radiobacter is by far the best-characterised species within the Rhizobium genera.

Official common name: Rhizobium rhizogenes

Scientific name and classification

Kingdom: Bacteria
Phylum: Proteobacteria
Class: Alphaproteobacteria
Order: Rhizobiales
Family: Rhizobiaceae
Genus: Rhizobium
Species: R. rhizogenes (Riker et al. 1930) Young et al. (2001)

References and further reading

  1. Brenner, D.J., Krieg, N.R. & Staley, J.T. 2005. Bergey’s Manual of Systematic Bacteriology Second Edition. Springer. 2: 340–345.
  2. Brijwal, L. & Tamta, S. 2015. Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC. Springer Plus 4 (1): 443.
  1. Rosenberg, E., & Zilber-Rosenberg, I. 2016. Microbes drive evolution of animals and plants: the hologenome concept. MBio7(2), e01395-15.
  2. Mehrotra, S. & Goyal, V. 2012. Agrobacterium-mediated gene transfer in plants and biosafety considerations, Applied Biochemistry and Biotechnology. 168 (7): 1953–
  3. Reddy, P.C.O., Raju, K.S., Sravani, K., Sekhar, A.C. & Reddy, M.K. 2019. Transgenic plants for remediation of radionuclides. In Transgenic Plant Technology for Remediation of Toxic Metals and Metalloids.Academic Press: 187–
  4. Taylor, C.G., Fuchs, B., Collier, R. & Lutke, W. K. 2006. Generation of composite plants using Agrobacterium rhizogenes. In Agrobacterium Protocols. Humana Press: 155–

Travella, S., Ross, S.M., Harden, J., Everett, C., Snape, J.W. & Harwood, W.A. 2005. A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Reports, 2 (12): 780–789.

Author: Mr Mduduzi Shinga
Email: M.Shinga@sanbi.org.za

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