DECIPHERING GENETICS OF NITROGEN-FIXING SYMBIOSIS IN LOTUS JAPONICUS

As the world population approaches seven billion and is predicted to reach nine billion by the year 2040 it is essential to improve our agricultural methods in order to meet the growing demand for food. The challenge is to increase the yield without negatively affecting the environment. A new approach that would make use of beneficial plant-microbe interactions should be considered. One of these interactions is the establishment of nitrogen-fixing symbiosis between plants and soil bacterium, commonly known as Rhizobium. However the ability to interact symbiotically with Rhizobium is almost completely restricted to leguminous plants. Therefore, understanding how the legume-rhizobium symbiosis is established might allow us to improve or engineer new N2 acquiring plant-microbe associations. In recent years, we witnessed many breakthrough discoveries that improved our understanding of these interactions; however, significant gaps in our knowledge of this important biological process still remain. The research objective of my thesis has been, therefore, to enhance our understanding of the mechanisms governing the development of nitrogen-fixing root nodule symbiosis in a model legume, Lotusjaponicus. The key findings of this thesis are as follows: (1) the identification and characterization of many symbiosis-relevant loci in L. japonicus-, (2) discovery of an alternative mechanism for successful rhizobial colonization of legume roots; (3) the molecular cloning of a gene that is required for root hairs development in L. japonicus; this is in relation to the function of root hair as the primary sites for the initial physical contact and entry of the compatible nitrogen-fixing bacteria inside the host-plant root and last; (4) discovery of a key signaling element that is necessary and sufficient for nodule organogenesis. This breakthrough finding demonstrated that perception of the plant hormone cytokinin is crucial for development of the symbiotic root nodule

Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides

With only hundreds of genes contained within their genomes, mycoplasmas have become model organisms for precise understanding of cellular processes, as well as platform organisms for predictable engineering of microbial functions for mission-critical applications. Despite the availability of whole genome writing in Mycoplasma mycoides, some traditional methods for genetic engineering are underdeveloped in mycoplasmas. Here we demonstrate two facile transposon-mediated approaches for introducing genes into the synthetic cell based on M. mycoides. The marker-less approach involves preparing a fragment containing only a small genomic region of interest with flanking transposase-binding sites, followed by in vitro transposase loading and introduction into the cells. The marker-driven approach involves cloning an open reading frame (ORF) of interest into a vector containing a marker for mycoplasma transformation, as well as sites for transposase loading and random genomic integration. An innovative feature of this construct is to use a single promoter to express the transformation marker and the introduced ORF. The marker-driven approach can be conveniently applied to any exogenous or synthetic gene without any information on the effect of the gene on the strain, whereas the marker-less approach requires that the fragment has a recognizable effect. Using the marker-less method, we found that a region containing the nusG gene rescues a slow growth phenotype of a strain containing a larger deletion encompassing this gene. Using the marker-driven approach, we better defined this finding, thereby establishing that nusG is required for a normal growth rate in synthetic M. mycoides. These methods are suitable for complementation tests to identify genes responsible for assorted functions lacking in deletion mutants. These approaches are also expected to facilitate rapid testing of various natural and engineered genes or gene clusters from numerous sources in M. mycoides