MEDICAGO GENETICS GROUP

The Medicago Genetics Group in the Institute of Genetics has extensive experience in plant molecular biology, symbiotic nitrogen fixation, classical plant genetics and genetic mapping. The main trend of our work is to use genetics, genomics and molecular biology resources for structural, comparative and functional genomics of Medicago species and to study plant genes involved in symbiotic nodule development and nitrogen fixation. Besides the forward genetic approach, the phenotype-driven map-based cloning strategy we use modern genomic techniques to find candidate genes and verify them by reverse genetics. Genes identified are then subjected to detailed investigation to reveal their structure, regulation as well as their location and the function of their product.

Molecular background of the nitrogen fixing symbiosis

Legumes are essential sources of protein, carbohydrates and minerals for animal and human nutrition. The high protein content of legume plants is made possible by the establishment of a root symbiosis with rhizobia to reduce atmospheric nitrogen. This symbiotic capacity is indispensable for the global nitrogen cycle and allows farmers to reduce nitrogen (fertilizer) input and its associated cost and pollution and will be essential for the development of a sustainable agriculture. In addition, legumes contain unique bioactive compounds such as iso-flavonoids that can improve human and animal health (health promoting compounds). Although legume crops are currently grown only on about 5% of cultivated land in Europe, it is envisaged that they will play an increasingly significant role due to their importance for sustainable agriculture, through reducing the use of costly and polluting nitrogenous fertilizers. Therefore this symbiosis is of high importance to agronomy and the environment, in which biotechnology is being used in environmentally friendly technologies to improve legume growth.

Nitrogen-fixing symbiosis is initiated and maintained by exchanges of signaling molecules between the host plant and the microsymbionts – controlled by special genes in both organisms. The formation of nitrogen-fixing nodules on legumes’ roots requires an integration of infection by rhizobia at the root epidermis and the initiation of cell division in the cortex, several cell layers away from the sites of infection. The main focus of our team has been the identification and function of plant symbiotic genes, especially using forward genetics or recently other genomic tools. We have identified the first symbiotic receptor gene from Medicago by map-based cloning approach (Endre et al., Nature, 2002). Since then, several symbiotic genes have been identified and more are on the way in different laboratories all over the world. Our aim is to improve our knowledge about the genes and their protein products, as well as their interaction during this important plant-bacterium symbiosis for further possible exploitation of this biological process. To this end, besides classical and molecular genetic work our laboratory uses a wide variety of techniques of molecular biology, and our research experience covers structural genomics, forward genetics, plant tissue culture, transformations, yeast two hybrid system, protein expression and purification.

Medicago genomics

Model organisms proved to be useful in answering biological questions that are more difficult to study directly in cultivated and often genetically recalcitrant species because of their disadvantageous characteristics such as large genome, long life-cycle and lack of techniques for genetic manipulation. In recent years, Medicago truncatula has been recognized as an excellent legume model in view of its advantageous characteristics (a small, diploid genome, self-fertility and a short life cycle) and various genomic and genetic tools have been developed. Different genomic and cDNA libraries are available, and sequencing projects have resulted in large EST datasets and long stretches of genomic sequences of the gene-rich parts of the genome. DNA chips and diverse mutant and ecotype collections have also been developed. Thereby M. truncatula has become suitable for identifying genes in this plant and, subsequently, in other legumes when feasible.

Our group has a long-standing research activity on different legume genetic systems such as alfalfa (Medicago sativa) and the model plant Medicago truncatula. After the pioneer work of the development of genetic maps, the group was involved in comparative genetic studies among different legume plants, and in the BAC end sequencing project in the structural genomics of M. truncatula. Recently, functional genomic studies have also been initiated in our laboratory. Ongoing projects include DNA chip hybridizations and further investigation of the resulting candidate genes and their protein products. Resources are also available for the ultimate validation of these candidate genes by a reverse genetic approach. A recently finished EU FP6 Grain Legumes Integrated Project has contributed significantly to this resource with a large-scale mutagenesis program of M. truncatula. We participated in this program and currently hold part of the seed collection of the insertional mutant lines. Since the model legume M. truncatula is a close relative of several plants cultivated in Europe (alfalfa, pea, clovers) and comparative genomic studies suggest good synteny with these species, we can also expect to be able to use the results obtained on the model plant in the improvement of the cultivated legume species.

TOXIN-ANTITOXIN MODULES IN SYMBIOTIC NITROGEN-FIXING SOIL BACTERIA

One of the major limiting nutrients for plant growth is utilizable nitrogen in the environment. Acquisition and assimilation of nitrogen is therefore second in importance only to photosynthesis. Soil bacteria belonging to Rhizobiaceae are able to form a symbiotic relationship with leguminous plants, and in new plant organs, the root nodules, reduce the most abundant nitrogen source, the atmospheric nitrogen to ammonia. Host plants utilize the fixed nitrogen and in turn, provide carbon source and energy for nitrogen fixing bacteroids.
We investigate the presence and role of toxin-antitoxin (TA) systems in rhizobia under conditions of free-living state and during symbiosis with host plants. TA modules may participate in the adjustment of bacterial metabolism to varying environmental conditions. Moreover, the drastic physiological changes during the transition from free-living to symbiotic state may also require the active contribution of TA modules to metabolic regulation.

Toxin-antitoxin (TA) modules consisting of two partially overlapping genes are ubiquitous among bacteria and archaea. TA systems encode proteins that form a complex acting as repressors for the TA operons. The mechanism of action of TA modules is based on the different stabilities of toxin and antitoxin proteins. Signals triggered by various stress factors lead to a decrease in the amount of labile antitoxin, thus the free stable toxin exerts its effect on various cellular targets. The physiological function of chromosomally located toxins is still controversial. Certain toxins, when activated by stress conditions induce significant loss of viability leading to programmed cell death. Other observations demonstrated that ectopic expression of toxins resulted in bacteriostasis rather than bactericidal effect. TA loci were also shown to participate in bacterial persistence and biofilm formation.

The proposed role of TA loci as general stress managers adjusting the metabolic rates under varying environmental stimuli may be of special importance during the adaptation of soil bacteria to oligotrophic conditions. In addition, symbiotic nitrogen fixing soil bacteria, which develop an intimate interaction with leguminous plants also have the ability to adapt and function within the plant host cells during symbiosis.

Our aim is to investigate the presence and role of TA systems in Sinorhizobium meliloti and Bradyrhizobium japonicum, the microsymbionts of two agriculturally important crops, alfalfa and soybean, respectively. We have shown that the ntrPR operon of Sinorhizobium meliloti represents a vapBC-type TA system, which is the most abundant group of the seven typical TA gene families. Insertion of transposon Tn5 into the ntrR gene resulted in increased transcription of both nodulation genes (responsible for the production of bacterial nodulation signals the Nod factors) and nitrogen fixation genes determining the enzyme nitrogenase. Moreover, the dicarboxylate transport system providing carbon source for bacteroids in nodules during symbiosis was also expressed at an increased level. As a result, alfalfa plants inoculated by this mutant strain had increased nitrogen content and biomass production as compared to those of the plants inoculated by the wild-type strain.

TA modules are surprisingly abundant in bacterial genomes. They are present in the chromosomes of almost all prokaryotes, often in very high numbers. Interestingly, obligate intracellular organisms have no functional TA loci, suggesting that these organisms which multiply under constant environmental conditions do not require TA modules as stress response mechanisms. Sinorhizobium meliloti also carries multiple copies of TA modules representing different TA families. These loci are distributed on the chromosome as well as on the megaplasmids of Sinorhizobium meliloti. Some of them show high sequence homology to the ntrPR operon; however, our preliminary data suggest that these additional copies are involved in the control of metabolic functions other than the regulation of nodulation and nitrogen fixation gene expression. Our aim is to determine the importance and role of TA copies, their possible cooperation, and their target sites in the cell. We investigate the molecular mechanism of the toxin molecules and the effect of TA systems on the stress tolerance of bacteria under free-living and symbiotic conditions. Understanding their function may help to improve the efficiency of the symbiotic nitrogen fixation interaction with host plants as was already demonstrated for the ntrPR operon in Sinorhizobium meliloti.