THE FORMATION OF ENDOSYMBIOTIC MEMBRANE COMPARTMENTS: MEMBRANE IDENTITY MARKERS AND THE REGULATION OF VESICLE TRAFFICKING Sergey Ivanov Thesis committee Thesis supervisor Prof. dr. T. Bisseling Professor of Molecular Biology Wageningen University Thesis co-supervisors Dr. E.E. Fedorova, Researcher, Laboratory of Molecular Biology, Wageningen University Dr. ir. E.H.M. Limpens Assistant professor, Laboratory of Molecular Biology, Wageningen University Other members Prof. dr. ir. J. Bakker, Wageningen University Prof. dr. ir. F.P.M. Govers, Wageningen University Dr. E.T. Kiers, VU University Amsterdam Prof. dr. F. Krajinski, Max Planck Institute of Molecular Plant Physiology, Potsdam, Ger- many This research was conducted under the auspices of the graduate School of Experimental Plant Sciences The formation of endosymbiotic membrane compartments: membrane identity markers and the regulation of vesicle trafficking Sergey Ivanov Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Thursday 6 September 2012 at 11 a.m. in the Aula Sergey Ivanov The formation of endosymbiotic membrane compartments: membrane identity markers and the regulation of vesicle trafficking, 124 pages PhD thesis, Wageningen University, Wageningen, NL (2012) With references, with summaries in Dutch and English ISBN 978-94-6173-343-6 Contents Outline...................................................................................................................................7 Chapter 1 Intracellular accommodation of symbiotic microorganisms: secretory pathways and the formation of perimicrobial compartments......................................................................11 Chapter 2 Medicago N2-fixing symbiosomes acquire the endocytic identity marker Rab7 but delay the acquisition of vacuolar identity.............................................................................21 Chapter 3 Rhizobium-legume symbiosis shares an exocytotic pathway required for arbuscule formation..............................................................................................................................53 Chapter 4 Multiple exocytotic markers accumulate at the sites of perifungal membrane biogenesis in arbuscular mycorrhizas...................................................................................77 Chapter 5 Concluding remarks .............................................................................................................97 Appendices Summary ............................................................................................................................114 Sumenvatting (summary in Dutch) ....................................................................................115 Acknowledgements............................................................................................................117 List of publications.............................................................................................................118 Curriculum Vitae................................................................................................................119 Education statement ...........................................................................................................121 Outline Plants as sessile organisms have limited access to vital elements like nitrogen and phospho- rus. The evolutionary pressure to improve access to such nutrients is well underlined by the success of certain endosymbiotic interactions. Plants acquired the ability to form a symbi- otic relation with certain fungal species like arbuscular mycorrizae (AM) fungi which fa- cilitate phosphorus uptake. The importance of this endosymbiosis is well illustrated by the fact that 80% of current higher plants have maintained this symbiosis that first evolved about 450 million years ago. The availability of a nitrogen source that can be used by plants especially depends on the process of biological nitrogen fixation by microbes. These mi- crobes have the ability to transform atmospheric nitrogen into ammonia. The endosymbio- sis of Rhizobium bacteria and plants of the legume family results in one of the most effi- cient biological nitrogen fixing systems. This symbiosis evolved about 60 million years ago shortly after the rise of legume plant family. In both symbiotic interactions the microbes are hosted in an intracellular membrane compartment made by the host. These membrane compartments form the heart of these en- dosymbiotic interactions as it controls the exchange of compounds between host and its mi- crosymbiont. In the model legume Medicago truncatula (Medicago) AM fungi form highly branched hyphae inside root cortical cells. These hyphae are called arbuscules and are sur- rounded by a periarbuscular membrane made by the host. Medicago can also establish a symbiosis with Rhizobium bacteria. In this symbiosis, bacteria are internalized into cells of new plant organ, root nodule. Root nodules are formed as a result of this plant-bacterium interaction. Intracellular bacteria are surrounded by a membrane, called symbiosome mem- brane and this membrane compartment and bacterium form a nitrogen-fixing organelle-like structure, which is named symbiosome. As Medicago can establish both symbiotic interac- tions, it is a good model system to study and compare these interactions. The main ques- tions I addressed in this thesis concerned the molecular and cellular mechanisms by which symbiotic (periarbuscular and symbiosome) membranes are formed. I have used identity markers of plant cellular endomembrane system to unravel molecular and cell biological mechanisms that are involved in symbiotic membrane formation. In chapter one, we describe the state of the art concerning the role of endo- and exo- cytotic-like processes in symbiosome formation. As the formation of symbiosomes primar- ily involves exocytosis, we discussed the role of secretory mechanisms in a broader per- spective comparing the Rhizobium symbiosis with plants interacting with symbiotic and pathogenic biotrophic fungi, respectively. To study the previously postulated endocytotic nature of symbiosome formation, we used in chapter two a set of molecular membrane identity markers. These markers are small GTPases belonging to the Rab family and SNARE (soluble N-ethylmaleimide sensitive fac- tor attachment protein receptor) proteins which prime and execute fusion of membranes, respectively. A default endocytotic pathway consists of several subsequent steps. It starts from engulfing molecules or larger particles by inward membrane budding. The formed membrane vesicles, that are named early endosomes, mature into late endosomes which in their turn ultimately fuse with vacuoles. To test whether the mechanism controlling release of rhizobia from the infection thread (symbiosome formation) is derived from endocytosis 7 Outline we studied whether an identity marker of early endosomes (SNARE MtSYP41) and one of late endosome (GTPase MtRab5) occur at symbiosome membranes. Neither of these two identity markers could at any stage of development be detected on symbiosomes. So our studies strongly suggest that symbiosome formation is not derived from the endocytotic pathway. However, symbiosomes are labeled by the vacuolar marker MtRab7 when they reach an elongated stage. As this indicates that symbiosomes might have a vacuolar nature, we tested whether vacuolar SNAREs are present on symbiosomes. This showed that neither SNAREs localized on the target membrane (t-SNARE: MtSYP22, MtSYP52, MtVTI11) nor those of transport vesicles (v-SNARE: MtVAMP711) are present on functional sym- biosomes. However, they do appear on symbiosome membranes at the onset of senescence. This explains that during senescence homotypic fusion of symbiosomes and fusion with vacuoles take place that triggers symbiosomes to be turned into a lytic compartment. In ad- dition, we tested an identity marker of the plasma membrane (t-SNARE MtSYP132). We showed that this SNARE is present on symbiosomes at all stages of development. Hence, we hypothesized that an exocytotic pathway is involved in symbiosome formation and this hypothesis was tested in chapter three. In chapter three, we described the function of SNAREs belonging to the VAMP72 group, which are involved in exocytosis to the plasma membrane via the trans-Golgi net- work compartment. We have identified six MtVAMP72 genes in Medicago. Functional analysis of the MtVAMP72 genes using an RNAi approach showed that two genes, MtVAMP72d and MtVAMP721e, are essential in establishing symbiosis. They are essential for release of the Rhizobium bacteria inside host cells as well as for symbiosome division. In the AM symbiosis they are also essential for intracellular accommodation of the fungus (arbuscule formation). Using GFP fusions as well as antibodies against these symbiotic MtVAMP72s we showed that MtVAMP-positive vesicles accumulate at the site of rhizo- bial release and periarbuscular membrane. So we identified an exocytotic pathway essential for symbiotic membrane formation in both symbioses and we postulated that this pathway was co-opted by the Rhizobium symbiosis from the more ancient AM fungal symbiosis. In chapter four, we have extended the studies on the function of the symbiotic MtVAMP72s to early steps of microbial infection of the root. We used real time confocal