|Identifying potential genes vital to the initiation and maintenance of symbiosis between Gunnera manicata and Nostoc Punctiforme
Gunnera manicata is the only genus of angiosperms known to form a nitrogen-fixing symbiosis with cyanobacteria1,2; furthermore, Gunnera represents the only group of land plants to host cyanobacteria intracellularly1. This genus of tropical plants is located primarily in the southern hemisphere and encompasses approximately 50 species1. They have the potential of growing anywhere from 6 to 8 feet in stature1; hence, symbiotic establishment with N2-fixing cyanobacteria promotes optimal development and plant maturity by fixing N2 into a form of nitrogen readily usable by plants, in exchange for sugar.
Within nature, Gunnera is known to form symbiotic relationships with cyanobacteria of the genus Nostoc1. Previous studies indicate that this symbiosis is limited to select isolates within the species of Nostoc2. It is the glandular cells of Gunnera, which only develop when the plant is deficient in nitrogen1, that allow it to host colonies of heterocystous Nostoc cells intracellularly2. Both the plant and bacteria exchange signals before and during glandular infection3. Once within the glandular cells, Nostoc cells loose their ability to fix carbon, but obtain an enhanced capacity to produce nitrogen compared to its free-living state1,2,3. Nostoc cells are able to fix nitrogen for the plant via specialized cells known as heterocysts3. The heterocyst differentiation among the cyanobiont is increased from two-fold to more than six-fold after symbiosis is established, allowing higher levels of nitrogen synthesis3.
Gunnera glands originate as tiny red spots on the hypocotyls beneath the point of cotyledon attachment1. Their presence correlates with each leaf of the plant: for every new leaf, a new gland develops at its base1,2. The gland is believed to be derived from a cluster of cells within the stem, which resume mitotic activities granted the right conditions1. Each cell within the group creates multicellular papilla which grows externally, breaking through the epidermal layer of the plant to form a circular pattern of papillae, which consists of a central papilla enclosed by five to eight others1. The organization of papillae establishes channels between the papillae that continue into the stem while preventing cell exposure to the external environment1. It is the cells lining these channels through which Nostoc enters thereby forming symbiosis; they move towards the interior of the glands through these intercellular channels to enter within host cells2. Moreover, the cell walls of some cells puzzlingly dissolve to allow Nostoc cells at the base of the channel entry3. Hence, it suffices that these reserved cells are responsible for creating the areas of symbiotic tissue within Gunnera, because they continue to divide after infection. It is therefore crucial for this elect group of cells to dominate the growth and behavior of Nostoc filaments with whom they form symbiosis.
In order to achieve successful symbioses between Gunnera and Nostoc, the cyanobacteria must have the ability to form motile filaments known as hormogonia1,3. This transformation in its morphology enables cyanobacteria to move towards and into the gland in addition to the ability to penetrate the inner cell walls of the gland2,3. Otherwise, symbiosis will not occur. Different environmental as well as plant signals may initiate hormogonia development. However, within 48 to 72 hours1, cyanobacteria usually return to its vegetative state in order to survive; according to (Meeks, 1998) prolonged time spent in this form may be lethal for the bacteria. Current investigations indicate that extended periods in such a state may furthermore prove deadly for the plant, since the motile filaments may spread into more cells than necessary, proliferating throughout the entire plant, hence destroying the plant (Wan-Ling, unpublished). The two changes in morphology, the establishment of hormogonia and the differentiation of heterocysts in order to produce and release nitrogen, indicate that Nostoc is responding to some external signals produced by its host3.
A vast abundance of research regarding plant-cyanobacteria symbiosis has been performed on Nostoc. Currently, we know nothing about the properties and/or mechanisms enabling host plants to attract and regulate its cyanobionts. Since both Gunnera and Nostoc possess the ability to thrive independently of one another, they both must have recognition mechanisms which they utilize to achieve symbiosis. It is known that very specific signals from the plant influence and manipulate the “symbiotically competent Nostoc species”, thereby activating different symbiotic-specific regulatory pathways3. With high hopes to discover all pathways endowing the host Gunnera the ability to regulate its endosymbiont, I will search for genes expressed explicitly within Gunnera’s gland. This goal may be achieved by analyzing the transcriptome of Gunnera’s gland. Gunnera’s genome, however, has never been fully sequenced. Due to the heavy restriction on the amount of genetic information known about Gunnera, I will obtain the information of interest utilizing a non-conventional method known as 454 pyrosequencing. This method has been proven to yield functional genomic tools for non-model organisms such as Gunnera6. Since we lack genomic sequence information, sequences obtained shall be organized based on de novo assembly.
Identification of regulatory genes implicated with symbiosis will serve as vital clues in determining the manner in which cells manage their cyanobiont in addition to how they utilize the fixed nitrogen provided for their usage. Such genes may represent the link to establishing successful nitrogen-fixing symbiosis among other crop plants.
In order to fulfill the goal of identifying potential host genes implicated with cyanobacterial-symbiosis, we have devised a plan to create two cDNA libraries from Gunnera’s gland, one normalized full-length cDNA library and one plasmid cDNA library. We will use the normalized cDNA library for 454 pyrosequencing, and the plasmid library will be stored as our reference. When we find genes of interest, we will have the ability to isolate and fully sequence them specifically for further analysis utilizing our plasmid cDNA library. The plasmid cDNA library will be sequenced using Sanger sequencing, which is less expensive while well-serving our purposes. Sequence products from both technologies will be analyzed respectfully with varying bioinformatics tools. The overall schematic for my experimental approach towards reaching our goal may be visualized in figure 2.
cDNA library construction:
We will isolate mRNA from stem gland tissue of Gunnera before and after infection with Nostoc, and at varying developmental stages. Genes expression data will be gathered by preparing cDNA libraries. This library is obtained by reverse transcription of the mRNA attained from the stem gland.
Extracted mRNA are harvested, cleaned of cellular debris, and converted into single-stranded cDNA utilizing reverse transcriptase. This single-strand is hybridized with a primer and its complimentary strand is produced via extension by DNA polymerase. Hence we have double stranded cDNA.
For plasmid cDNA libraries, the cDNA will be inserted within an appropriate plasmid or vector, most likely pGEM-TEasy in order to sustain the DNA. This plasmid can then be cloned into an appropriate organism, most likely E. coli in order to amplify and sequence the specific mRNA later for further analysis.
However, we wish to equalize mRNA concentrations within our cDNA library. Our first desire is to detect any new genes within Gunnera, which may not exist in related organisms, so we will invest in “cDNA Normalization”. This method will help increase our overall rate of novel gene discovery, by equalizing the various cDNA present in a sample. Therefore, genes more highly expressed will have approximate cDNA representations as those less expressed; there will be no over-representation of any one transcript in comparison to another4. This novel method known as Evrogen cDNA normalization utilizes a duplex-specific nuclease (DSN), an enzyme extracted from the hepatopancreas of the Kamchatka crab and that distinctively cleaves double-stranded DNA5. Moreover this method allows for the production of full-length cDNA sequences, which will help enhance the accuracy and efficiency of sequence assembly and annotation after sequencing.
cDNA normalization is based on the kinetics of cDNA reassociation, where more highly abundant transcripts will reassociate at faster rates, upon degradation and reassociation, due to their greater abundance in any particular sample than will less abundant transcripts4. Basically, denaturation and reassociation of cDNA is performed at the same temperature, which avoids nonspecific hybridizations of cDNA during DSN treatment4. The products after reassociation include both a normalized ss-fraction and a non-target fraction4. All ds-fractions, which represent the overabundant transcripts in the sample, will be cleaved and destroyed with DSN, which is active at 70 degrees Celsius. The remaining products, ss-fractions, will be amplified using PCR and our normalized full-length cDNA library is created. A schematic of this process is portrayed in figure 1.
454 sequencing and de Novo Assembly
In order to obtain unique sequences, we will use a single 454 GS20 sequencing run on our normalized cDNA library. Upon generation of 454 sequences, I will use several possible bioinformatics programs/tools to analyze them. Among many possibilities, it is likely that I will utilize databases such as NCBI to identify unique hits. I will probably use Biobike and Gene Mark to compare and further analyze unique sequences and genes of interest. After peculiar genes of interest have been targeted, we will employ various methods to discovery corresponding potential regulatory functions, such as gene knock-out and knock-down strategies. Such an analysis will assist in delineating specific products of and interactions between identified pathways of interest.
Possible Results and their Implications:
This project has the potential to provide valuable insight into the molecular characteristics which allow and promote plant-cyanobacteria symbiosis. As a matter of fact, it may offer valuable clues regarding the manner in which other angiosperms may participate in such a symbiosis. Once potential genes have been detected, we can gather a deeper insight into the various pathways utilized by the host in order to successfully regulate and maintain its endosymibont. Upon further analysis of these genes, such as their level of expression within the host, we may be able to characterize the symbiotic pathways expressed within Gunnera to host Nostoc which has never been achieved or perhaps even attempted prior to our research. The EST’s generated from our de novo assembly may be stored in databases explicitly for Gunnera as well as submitted to national databases, in order to reduce lack of information on this organism.
Of more significance, we may be able to induce a symbiotic relation between cyanobacteria and other crop plants by introducing these identified genes into other angiosperms, and if successful, possibly reduce the need for nitrogen fertilizers in the near future. Such an advancement has the capability of revolutionizing the world in terms of reducing the amount of pollution secreted to the environment via synthetic fertilizers, enhancing the food supply by utilizing a natural form of nitrogen to increase agricultural yield, and much more! Since no other plant model systems with a focus on plant-cyanobacterial symbiosis are currently available, our research will provide the foremost set of major and innovative discoveries within the field.
Chiu, Wan-Ling. et. al. (2005). Nitrogen Deprivation Stimulates Symbiotic Gland Development in Gunnera manicata. Plant Physiology. Vol. 139, pp. 224-230.
Bonnet, H.T. and Silvester, W. (1981). Specificity in the Gunnera-Nostoc Endosymbiosis. New Phytol. Vol. 89, pp. 121-128.
Meeks, John C. (1998). Symbiosis between Nitrogen-Fixing Cyanobacteria and Plants. Bioscience. Vol. 48, pp. 266-276.
Zhulidov, P.A. et. al. (2005). A Method for the Preparation of Normalized cDNA Libraries Enriched with Full-Length Sequences. Russian Journal of Bioorganic Chem. Vol. 31, pp. 170-177.
Evrogen JSC. DSN based normalization of full-length-enriched cDNA. Copyright 1999-2008. http://www.biocompare.com/technicalarticle/1604/DSN-based-Normalization-Of-Full-length-enriched-CDNA-from-Evrogen-JSC.html
Vera, J.C. et al. (2008). Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing. Molecular Ecology. Vol. 17, pp. 1636-1647.
Figure 1. (Zhulidov, P.A., 2005)
Figure 2. Overall schematic of my experimental approach this summer, 2008.