Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Plant Growth-Promoting Bacteria
Nodulation

Competition among nodulating organisms

One of the primary goals of agricultural biotechnology is to use Introduction/32.html">Genetic Engineering to develop Rhizobium strains that can increase crop yields more effectively than indigenous strains. Many commercially available inoculant strains—which are excellent nitrogen fixers—were developed through mutagenesis and subsequent Selection; however, they do not sufficiently stimulate nodulation on host plant roots when competing with indigenous Rhizobium strains already present in the soil. Conversely, many indigenous strains successfully outcompete laboratory strains but are inefficient at Nitrogen Fixation. Therefore, for commercial inoculant strains to be practically useful, it is necessary either to enhance their nodulation competitiveness or to eliminate indigenous Rhizobium strains.

Research has been conducted to determine the GENETIC BASIS OF the "competitiveness" of indigenous strains, with the ultimate goal of introducing the corresponding genes into inoculant strains.

Manipulation of nodulation genes

To identify nodulation genes (nod genes), genetic complementation was used once again. A nodulation-deficient (Nod^-) mutant strain of R. meliloti was transformed with a chromosomal DNA clone library of wild-type R. meliloti, and colonies that regained The ability to form nodules on alfalfa roots were isolated (Fig. 14.6). The strategy was as follows.

1. A clone library of wild-type (Nod⁺) R. meliloti chromosomal DNA was constructed by partially digesting R. meliloti DNA with the restriction enzyme EcoRI and inserting fragments of up to 40 kb into the unique EcoRI site of the broad-host-range cosmid pLAFR1.

2. Recombinant Plasmids were packaged into phage λ particles, introduced into E. coli, and then transferred into Cells of the Nod^- strain of R. meliloti by conjugation. The vector contained a tetracycline resistance Gene, which could be used as a selectable marker in both E. coli and R. meliloti.

3. Following conjugation, Suspensions containing 200 to 300 transformed R. meliloti cells were tested for their ability to initiate nodulation in sterile alfalfa plants. It was expected that only those transformants carrying and expressing the gene that complements the nodulation defect in R. meliloti cells would possess this ability.

Class="center">

Fig. 14.6. Identification of R. meliloti nodulation genes. Wild-type R. meliloti DNA is inserted into the broad-host-range cosmid pLAFR1, packaged into phage λ particles, and introduced into E. coli. The clone library is transferred from E. coli to a Nod^- strain of R. meliloti via conjugation. Alfalfa plants are inoculated with the transformed Nod^- R. meliloti; plants that form ROOT nodules are infected with R. meliloti Nod⁺ cells, which presumably carry the complementing nodulation gene within the cosmid vector. Transformed Nod⁺ R. meliloti cells are then isolated from the root nodules.

4. Nodulating Bacteria were isolated from the nodules, and the vector carrying the complementing gene was extracted from these bacteria. The large insert containing this gene was subcloned and analyzed further.

5. The identified nodulation gene was used as a probe to detect flanking regions of R. meliloti chromosomal DNA in a genomic library.

As a result of these highly labor-intensive experiments, the entire set of R. meliloti nodulation genes was characterized. Detailed biochemical and genetic studies revealed that nodulation and its regulation are complex processes involving the products of A large number of genes (approximately 20; Table 14.6). Some of these genes are highly conserved (identical in all nodulating microorganisms), while others are species-specific. They can be grouped into three distinct classes: conserved, species-specific, and the regulatory gene nodD. For instance, the nodABC genes are identical in all Rhizobium species and are structurally interchangeable; in most species, they form a single Operon. The nodulation process has been shown to involve several steps. First, the product of the constitutively expressed nodD gene binds to a flavonoid molecule secreted by the root cells of the host plant. Flavonoids are plant Phenolic Compounds structurally based on two aromatic rings joined by a three-carbon bridge. They perform various Functions in plants, including pigmentation and defense against Fungi and insects. The binding of flavonoids to the NodD protein is a key factor in host plant recognition, as each Rhizobium species recognizes only a limited number of flavonoid structures, and each plant species synthesizes its own specific set of these molecules. Some strains, such as R. leguminosarum biovar (bv.) trifolii, have a very narrow host range because they recognize only a few types of flavonoids, whereas other strains, such as Rhizobium sp. NGR234, have a very broad host range.

Table 14.6. Some Proteins encoded by Rhizobium nodulation genes and their proposed functions

¹⁾ Where biochemical or genetic data regarding protein function are unavailable, functions are assigned based on Homology to proteins with similar Amino acid sequences. Different Rhizobium strains contain different sets of these proteins. The term "conserved" indicates that the protein performs the same function in all Rhizobium species.

The binding of flavonoid molecules activates the NodD protein product, apparently by inducing a conformational change. The flavonoid–NodD complex then binds to a promoter region of the nodulation genes known as the "nod box." This region is located upstream of all nodulation genes except nodD and initiates their METABOLISM/31.html">Transcription. The nodABC genes encode proteins that cause root Hair Swelling and curling, which is considered the first step of bacterial infection of the plant root. Together, the plant and bacteria synthesize an oligosaccharide factor that is modified by the NodH gene product, and possibly by NodQ and NodP products as well. This factor, designated NodRm-1 (Fig. 14.7), induces a specific response in the host plant, including root curling and deformation.

Fig. 14.7. Proposed Structure OF THE oligosaccharide factor NodRm-1. This compound induces a specific response in the host plant, including root curling and deformation.

Depending on the Rhizobium or Bradyrhizobium strain, approximately 20 additional nod gene products are eventually synthesized. Together with certain plant-encoded proteins, they participate in nodule development.

To elucidate The Role of each identified nod gene, further research is required; additionally, it is possible that new nod genes will be discovered over time. For example, DNA Sequencing and computer analysis have shown that in a slow-growing form of Bradyrhizobium sp., the DNA region between the nodD and nodABC genes contains an Open Reading Frame, whereas this sequence is absent in the fast-growing form. This open reading frame was designated nodK. When plants are inoculated with a Bradyrhizobium sp. strain containing a mutant nodK gene (NodK), nodules begin to form 5 days earlier than in plants infected with the wild-type strain; furthermore, the number of nodules doubles, and plant yield increases by 120%.

To date, no simple genetic approaches have been developed to utilize nod genes to enhance the competitiveness of Rhizobium inoculant strains. However, host Specificity can be altered by transferring the nodD gene from a broad-host-range Rhizobium strain into a narrow-host-range strain. In any case, it is clear that nodulation is a highly complex process, and further enhancement of Rhizobium strain competitiveness will require comprehensive research using genetic engineering techniques.