Genetic analysis of symbiosome formation

Endosymbiotic interactions form a fundament of life as we know it and are characterized by the formation of new specialized membrane compartments, in which the microbes are hosted inside living plant cells. A striking example is the symbiosis between legumes and nitrogen-fixing Rhizobium bacteria (rhizobia), which represents the most important source of biologically fixed nitrogen. The accommodation of rhizobia as novel nitrogen-fixing organelles, called symbiosomes, inside the cells of a novel organ, the root nodule, forms the heart of this ecologically and agriculturally important symbiosis. Understanding how these organelles are made will be keystone to exploit this symbiosis for sustainable agriculture in the future. In this thesis, we undertook a genetic approach to identity key components that control symbiosome formation especially in the genetically well-characterized garden pea (Pisum sativum) system. At the start of this thesis, the most extensive and morphologically best-characterized collection of mutants impaired in symbiosome formation was, and currently still is, available in pea. However, the cloning of the corresponding genes is severely hampered by its large genome size and recalcitrance to genetic transformation. Therefore, we used the model legume Medicago truncatula (Medicago) as reference genome to clone pea symbiosome mutants via a synteny-based cloning approach. We focused especially on three mutants in pea, named sym33, sym41 and sym31, which are affected most early in symbiosome formation: namely blocked in the release of bacteria from cell wall bound infection threads inside root nodule cells (sym33 and sym41) or induction of the subsequent differentiation of the symbiosomes (sym31). In Chapter 1, a general introduction is given on the process of symbiosome formation in legume root nodules. In this introduction, we focus on mechanisms by which these new nitrogen-fixing organelles are formed and address some of the recent insights, most of which were obtained after the start of this thesis, into plant components that control this process, which have been obtained from genetic studies in pea and the model legumes Medicago and Lotus japonicus (Lotus). Pea is part of the Papillionoid legume subfamily and closely related to the model legume Medicago. It has been shown that there is extensive synteny between the pea and Medicago genomes, which offers an efficient strategy to clone pea gene using Medicago as intergenomic cloning vehicle. In Chapter 2, we outline this synteny-based cloning approach and the molecular tools that we created to clone the pea genes required for symbiosome formation. In addition, we describe an efficient method to obtain transgenic roots via Agrobacterium rhizogenes mediated root transformation in pea that facilitates the functional analysis of pea genes in root endosymbioses. In Chapter 3, we report the cloning of the pea Sym33 and Medicago SYM1 genes those mutants are most strongly impaired in their ability to form symbiosomes, i.e. the release of rhizobia from the cell wall bound infection threads. Both pea Sym33 and Medicago SYM1 encode the interacting protein of DMI3, IPD3. IPD3 was shown to interact with DMI3/CCaMK, a calcium- and calmodulin-dependent kinase that is an essential component of the common symbiotic signalling pathway for both rhizobial symbiosis and arbuscular mycorrhiza. Our data reveal a novel, key role for IPD3 in symbiosome formation and development. Further, we show that MtIPD3 is required for the expression of a nodule-specific remorin MtSYMREM1, which is required for proper infection thread growth and essential for symbiosome formation. In Chapter 4, we report the synteny-based cloning of the pea sym41 mutant that is also impaired in the release of the bacteria from the infection threads. We show that Sym41 represents a weak allele of the common symbiotic signalling gene PsSym19/MtDMI2, a leucine-rich repeat domain containing receptor kinase that is essential for both rhizobial and mycorrhizal endosymbioses. Sym41 contains a splice-site mutation in intron 9, by which the formation of a functional transcript is reduced by ~90%. The implication of Sym19/DMI2 together with the identified role of Sym33/IPD3 in symbiosome formation (Chapter 3) strongly indicate that rhizobia have co-opted the signalling pathway from the ancient arbuscular mycorrhiza to be hosted as new organelles inside root nodule cells. In Chapter 5, we describe the synteny-based mapping of pea sym31, a mutant impaired in symbiosome differentiation. By making use of the synteny with Medicago, we fine mapped the Sym31 gene to a region of ~2.5 cM, which corresponds to a <450 kb region in Medicago. In this syntenic region, one gene MtN3.1, a putative sugar transporter stands out as prime candidate to control symbiosome differentiation. We describe and discuss our efforts to determine the role of this gene in symbiosome differentiation in pea and Medicago. In Chapter 6, we summarize and discuss our current insight into symbiosome formation and its relation to the arbuscular mycorrhiza and we give a perspective on the future of cloning the pea genes required for endosymbioses.