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At the heart of endosymbiosis microbes are hosted inside living cells in specialized membrane compartments that from a host-microbe interface, where nutrients and signal are efficiently exchanged. Such symbiotic interfaces include arbuscules produced by arbuscular mycorrhiza (AM) and organelle-like symbiosomes formed during the rhizobium-legume symbiosis. Also during pathogenic interactions, microbes such as biotrophic fungi and oomycetes are hosted in specialized membrane compartments called haustoria. The formation of such new membrane compartments requires a major reorganization of the host endomembrane system, with a special role for the targeting of secretory/exocytotic vesicles and their cargo to the newly forming interfaces. In this thesis, I studied how exocytotic membrane traffic is regulated to facilitate the formation and maintenance of a host-microbe interface. Therefore, I especially focussed on the role of SNARE (Soluble NSF Attachment Protein Receptor) proteins, as key components of the exocytotic machinery, in symbiotic interface formation. In Chapter 1, I introduce the different symbioses in which host-microbe interfaces are formed, and the role of the host-microbe interface in these symbioses. Further, I introduce the evolutionary relationship between the different symbioses: AM symbiosis is the most ancient endosymbiosis in plants, which provided the blueprint for different symbioses that evolved later; other symbiotic microbes including rhizobia co-opted the signalling program and adaptations to membrane trafficking required for arbuscule formation, to be hosted inside cells. Finally, I will introduce the symbiosis dedicated SNAREs as key regulators of exocytosis to form a host-microbe interface. In Chapter 2, we tested the long-standing hypothesis that pathogens make use of the AM symbiotic program to allow the formation of haustoria. To test this, we set up a pathosystem using the biotrophic oomycete Phytophthora palmivora that is able to form haustoria in Medicago truncatula root cells. Using M. truncatula mutants impaired in AM and rhizobium symbioses, we demonstrated that neither the common symbiotic signalling genes, nor symbiosis dedicated regulators of vesicle trafficking are required for haustorium formation. This showed that biotrophic pathogens like P. palmivora, do not hijack the symbiotic program to be accommodated inside plant cells. In Chapter 3, we identified the t-SNARE SYP132α as a key regulator of both arbuscule and symbiosome formation. During vesicle fusion, a vesicle SNARE (v-SNARE) on the vesicle forms a complex with a target membrane SNAREs (t-SNAREs) on the target membrane. Previous work in our lab identified specific exocytotic v-SNAREs required for arbuscule and symbiosome formation. We identified the t-SNARE counterpart SYP132, and demonstrated that in most dicot plants SYP132 is spliced into two spliceforms; SYP132α and SYP132β. Interestingly, alternative splicing of SYP132 leading to the dominant use of a SYP132α-specific last exon coincides with the accommodation of AM fungi in arbuscule forming root cortex cells and rhizobium bacteria in nodule cells. Using a spliceform-specific RNAi construct, we showed that SYP132α is specifically required for the formation of a stable host-microbe interface in both AM symbiosis and rhizobium symbiosis. Furthermore, we showed that during arbuscular collapse, the two spliceforms localize differently to healthy and degrading arbuscule branches. These results indicated that alternative splicing of SYP132 allows plants to replace a t-SNARE involved in traffic to the plasma membrane with a t-SNARE that is more stringent in its localization to functional arbuscules. The evolutionary expansion of SNAREs in plants has been hypothesized to have allowed the adaptation of exocytosis to different biological processes. In Chapter 4, we studied what makes the symbiotic SNAREs so special in comparison to their non-symbiotic family members, of which many are also expressed in arbuscule cells. We hypothesized that symbiotic SNAREs define a distinct secretory pathway, that ensures specificity of protein delivery to the host-microbe interface. We show that all tested SYP1 family proteins, and most of the non-symbiotic VAMP72 members, were able to complement the defect in arbuscule formation upon knock-down of their symbiotic counterparts when expressed at sufficient levels. This functional redundancy is in line with the ability of all tested v- and t-SNARE combinations to form SNARE complexes at the peri-arbuscular membrane. This showed that the symbiotic SNAREs do not selectively interact to define a distinct vesicle trafficking pathway, but that their essential role in arbuscule formation can be largely explained by their dominant expression level. Interestingly, the symbiotic t-SNARE SYP132α appeared to occur less in SNARE complexes with v-SNAREs compared to the non-symbiotic syntaxins in the arbuscule cells, suggesting a more strict regulation of symbiotic SNARE complexes at the interface. Since the alternative splicing of SYP132 does not affect the total transcript levels, we hypothesized that there must be a functional difference between SYP132α and –β, potentially leading to subtle phenotypes that may have gone undetected in the Agrobacterium rhizogenes mediated complementation approach applied in Chapter 4. In Chapter 5, we therefore generated and characterized a stable mutant line in which all SYP132 transcripts are constitutively spliced into the non-symbiotic SYP132β form. Although this mutant is normally colonized by AM fungi, with no effects on arbuscule morphology, it has a severely reduced biomass after mycorrhization. This hints to a yet unknown role for SYP132α to control arbuscule functionality, and offers an explanation for the evolutionary conservation of the SYP132 alternative splicing in dicot plants. Finally, using fluorescent timer fusions to both SYP132 isoforms, we showed that the difference in localization of the two proteins during arbuscular collapse is the result of a different (endocytic) turnover of the two spliceforms at the healthy/functional arbuscule branches, possibly due to a difference in interactions with VAMPs. Together, our data show that, although both SYP132 isoforms can mediate arbuscule formation, SYP132α is functionally different from SYP132β, which may reveal new aspects of the control of nutrient exchange. In Chapter 6, I discuss the data generated during my thesis research in relation to additional symbiosis dedicated regulators of exocytosis, as well as in relation to other biological processes that depend on specific secretory SNAREs. Following our conclusion that the symbiotic SNAREs do not mark a separate exocytosis pathway, but are functionally different from non-symbiotic SNAREs, I will speculate on the possible scenarios in which symbiosis dedicated SNAREs are specialized for host-microbe interface functionality. |
